Folate Biofortification in Hydroponically Cultivated Spinach by the

May 26, 2017 - Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. ‡ Japan Society...
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Folate Biofortification in Hydroponically Cultivated Spinach by the Addition of Phenylalanine Sho Watanabe,† Yuta Ohtani,† Yohei Tatsukami,†,‡ Wataru Aoki,†,§ Takashi Amemiya,∥ Yasunori Sukekiyo,∥ Seiichi Kubokawa,∥ and Mitsuyoshi Ueda*,†,§ †

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan Japan Society for the Promotion of Science, Sakyo-ku, Kyoto 606-8502, Japan § Kyoto Integrated Science & Technology Bio-Analysis Center, Shimogyo-ku, Kyoto 600-8813, Japan ∥ Mitsubishi Plastic, Inc., Chiyoda-ku, Tokyo 100-8252, Japan ‡

S Supporting Information *

ABSTRACT: Folate is an important vitamin mainly ingested from vegetables, and folate deficiency causes various health problems. Recently, several studies demonstrated folate biofortification in plants or food crops by metabolic engineering through genetic modifications. However, the production and sales of genetically modified foods are under strict regulation. Here, we developed a new approach to achieve folate biofortification in spinach (Spinacia oleracea) without genetic modification. We hydroponically cultivated spinach with the addition of three candidate compounds expected to fortify folate. As a result of liquid chromatography tandem mass spectrometry analysis, we found that the addition of phenylalanine increased the folate content up to 2.0-fold (306 μg in 100 g of fresh spinach), representing 76.5% of the recommended daily allowance for adults. By measuring the intermediates of folate biosynthesis, we revealed that phenylalanine activated folate biosynthesis in spinach by increasing the levels of pteridine and p-aminobenzoic acid. Our approach is a promising and practical approach to cultivate nutrient-enriched vegetables. KEYWORDS: Spinacia oleracea, folate, hydroponics, biofortification, LC-MS/MS

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INTRODUCTION Folate is a water-soluble vitamin (vitamin B9) first isolated from Spinacia oleracea1 and is mainly found in vegetables. The chemical structure of folic acid (pteroylmonoglutamate), a fully oxidized folate, comprises pteridine, p-aminobenzoic acid (PABA), and glutamate moieties (Figure 1a).2 Folate forms various chemical structures such as tetrahydrofolic acid (THF), 5-methyltetrahydrofolic acid (5-Me-THF), and 5-formyltetrahydrofolic acid (5-CHO-THF) (Figure S1). These folates play many roles in our bodies, such as the production of erythrocytes, biosynthesis of DNA and RNA, biosynthesis of proteins, differentiation and proliferation of somatic cells, and consumption of carbohydrates and amino acids.3,4 In various studies, the importance of folate intake has been reported. For example, folate supplementation leads to a decrease in the frequency of fetal neural-tube defects5 and prevents obstructive vascular disorder induced by increases in the homocysteine concentration in blood.6 Therefore, to prevent these symptoms, the U.S. Public Health Service recommends an intake of 400 μg of folate per day7 for adults. Considering the additional benefit of folate intake along with other vitamins,8 it is ideal to consume dietary folates from various foods containing abundant folates. Several approaches have been performed to attain folate biofortification in vegetables. To enhance the folate content in plants, two enzymes were overproduced, GTP cyclohydrolase I (GCHI) and aminodeoxychorismate synthase (ADCS), using metabolic engineering. GCHI is the rate-limiting enzyme of pteridine biosynthesis, and ADCS is the key enzyme of PABA © 2017 American Chemical Society

biosynthesis (Figure 1b). Hossain et al. achieved 2- to 4-fold folate biofortification in Arabidopsis thaliana by overproduction of GCHI derived from Escherichia coli.9 In another study, Diaz de la Garza et al. achieved 25-fold folate biofortification in tomato by the overproduction of GCHI.10 Storozhenko et al. also achieved 100-fold folate biofortification in rice by cooverproduction of GCHI and ADCS.11 Furthermore, the same approach has also been used to achieve high folate content in other crops.12−14 However, all these reports on folate biofortification used genetic modification techniques. The production and sales of genetically modified crops are regulated or restricted in many countries.15 Therefore, folate biofortification in vegetables without genetic modification is meaningful. The purpose of this study was to enhance the folate content in spinach without genetic modifications. To increase folate content in spinach, we added the following three compounds into the liquid fertilizer: glutamic acid, magnesium, and phenylalanine, which were expected to increase the folate content during hydroponic cultivation. Glutamic acid is a precursor in folate biosynthesis16 (Figure 1b), and the supply of glutamic acid was expected to activate folate synthesis. Phenylalanine is known to induce the feedback inhibition of chorismate mutase or arogenate dehydratase (Figure 1b), enzymes for phenylalanine synthesis,17 and the supply of Received: Revised: Accepted: Published: 4605

March 27, 2017 May 25, 2017 May 26, 2017 May 26, 2017 DOI: 10.1021/acs.jafc.7b01375 J. Agric. Food Chem. 2017, 65, 4605−4610

Article

Journal of Agricultural and Food Chemistry

Hydroponic Cultivation. S. oleracea NPL-8 (Mitsubishi Plastic Agri Dream) was the spinach strain that was used for this study. All spinach samples were cultivated using NAPPER LAND (Mitsubishi Plastic Agri Dream). NAPPER LAND is an indoor hydroponic cultivation system (Mitsubishi Plastic Agri Dream) (Figure S2). Spinach seeds were germinated and grown for 3 days at 22 °C on granular rock wool, and young seedlings were grown under the following conditions: 22 °C, 12 h light/19 °C, 12 h dark cycle under artificial light for 8 days. Grown seedlings were then planted in a hydroponic cultivation system using High Tempo Ar, High Tempo Cu, and potassium nitrate solution in a ratio of 3:9:10 as the initial nutrient solution. In addition, electrical conductivity was set to 3.0 mS/cm, and the pH was adjusted in the range 5.5−6.5. During cultivation, the liquid temperature was set at 20 °C, and the fertilizer was replaced with water 2 days before harvesting the phenylalanineand Mg2+-added samples. In cultivation of the phenylalanine-added sample, white turbidity was observed in the fertilizer. The turbidity was derived from growth of some microorganisms. Then, octcloth, a silvercoated cloth pesticide (Mishima Kosan, Kitakyushu, Japan), was added to the nutrient solution tank to restrict the growth of microorganisms. We did not observe the turbidity in the fertilizer with glutamic acid or magnesium. In compound-added cultivation experiments, glutamic acid was added in 1.4 mM increments 4 times (on days 0, 3, 6, and 10), for a final concentration of 5.6 mM. Mg2+ was added in increments of 0.5 mM Mg2+ twice (days 10 and 14), for a final concentration of 1 mM. Phenylalanine was added in increments of 1 mM twice (days 10 and 14), for a final concentration of 2 mM. Glutamic-acid-added samples were harvested mid-day on day 13, and the fresh weight was measured. Other samples were harvested at midday on day 15, and the fresh weights were measured. The differences in the harvesting date or replacement of fertilizer were due to limitations of our cultivation scheduling. We did not find statistically significant differences between these control samples. Hence, we assumed that the differences did not largely influence our results. Harvested samples were immediately stored at −80 °C. Extraction. We extracted folates according to the method optimized by Tamura.19 For the extraction of folates, 0.3 g of spinach was homogenized to a fine powder in liquid nitrogen. The powder was transferred to a 2.0 mL tube, and 1.5 mL of extraction buffer (20 mM ammonium acetate, 10 mM ascorbic acid, and 0.2 mM 2mercaptoethanol, pH 8.0; freshly prepared) was added. After mixing, the capped tube was placed at 100 °C for 30 min and flash-cooled on ice. For the digestion of carbohydrate and protein matrices where folates are possibly trapped, α-amylase and protease treatments were performed, respectively.20 α-Amylase (10 mg/mL) was added, and the sample was incubated for 2 h at 37 °C. Next, 10 mg/mL of protease was added, and the sample was incubated for 2 h at 37 °C. Immediately after the protease reaction, samples were placed at 100 °C for 30 min and flash-cooled on ice. The resultant solution was centrifuged at 14 000g for 30 min, and 0.5 mL of supernatant was transferred to a 1.5 mL tube. Then, 100 μL of rat serum was added to the supernatant,19 for the deconjugation of polyglutamylated folates, and the sample was incubated at 37 °C for 3 h. To quantify only the folates in spinach, the rat serum was mixed with activated charcoal, and the freed folate was adsorbed before use for folate deconjugation.21 An additional treatment of 10 min at 100 °C was carried out, again followed by cooling on ice. A final centrifugation was performed at 14 000g for 15 min. The final supernatant was ready for LC-MS/MS analysis. During the sample preparation, all manipulations were carried out under subdued light. The experiments were performed in triplicate. For the extraction of pteridine and PABA, 0.5 g of spinach was homogenized to a fine powder in liquid nitrogen.22 The powder was transferred to a 15 mL tube, and 10 mL of extraction buffer (cold methanol at −8 °C containing 10 mM ascorbic acid and 200 μM 2mercaptoethanol, pH 8.0; freshly prepared) was added. The capped tube was rotated for 20 min at 4 °C. Samples were centrifuged at 3000g for 30 min, and the supernatants were transferred to 2 mL tubes. The methanolic layer was dried completely using a vacuum centrifuge. A 0.2 mL buffer (20 mM ammonium acetate, 10 mM L-

Figure 1. Chemical structure of folic acid and the common folate pathway in plants. (a) Folic acid (pteroylglutamate) consists of pteridine, p-aminobenzoate, and glutamate moieties. Pteroylmonoglutamate (n = 1) has one glutamine chain, and pterolypolyglutamate (n = 2−7) has γ-linked polyglutamyl tails.35 (b) A common folate pathway in plants. GTP, guanosine triphosphate; DHN, dihydroneopterin; DHM, dihydromonapterin; DHN-PPP, dihydroneopterin triphosphate; HMDHP, hydroxymethyldihydropterine; HMDHP-PP, hydroxymethyldihydropterinediphosphate; DHP, dihydropteroate; DHF, dihydrofolic acid; THF, tetrahydrofolic acid; folate derivatives, other folate-compounds indicated in Figure S1; PABA, p-aminobenzoic acid; ADCS, aminodeoxychorismate; CM, chorismate mutase; ADT, arogenate dehydratase; and Glu, glutamic acid.

phenylalanine was expected to increase the synthesis of PABA (Figure 1b). Magnesium ion was expected to increase the activity of enzymes such as GCHI, involved in folic acid biosynthesis.18 As a result of liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, we achieved 2.0-fold increase of the folate content in spinach when phenylalanine was added. Because our method used only inexpensive compounds and not genetic modification, it will be a practical method for producing folate-abundant vegetables.

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MATERIALS AND METHODS

Reagents. Folic acid (pteroylglutamic acid (PteGlu)), p-aminobenzoic acid (PABA), L-ascorbic acid, 2-mercaptoethanol, ammonium acetate, acetate, sodium hydroxide, normal rat serum, acetonitrile, ultrapure water, formic acid, L-phenylalanine, L-glutamic acid, and activated carbon were purchased from Wako Pure Chemical Industries (Osaka, Japan). Dihydrofolic acid (DHF), formylfolic acid (10formylfolic acid; 10-CHO-FA), 5-CHO-THF calcium salt hydrate, 5Me-THF disodium salt, α-amylase from Aspergillus oryzae, and protease from Streptomyces griseus were purchased from Sigma− Aldrich (St. Louis, MO, USA). Dihydroneopterin (DHN) was purchased from Schircks Laboratories (Bauma, Switzerland). High Tempo Ar and High Tempo Cu, liquid fertilizers containing metal components to activate plant growth, were purchased from Mitsubishi Plastic Agri Dream (Tokyo, Japan). Ethylenediaminetetraacetic acid disodium magnesium salt 4-hydrate that was added into the liquid fertilizer to provide Mg2+ was purchased from Chubu Chelest (Mie, Japan). 4606

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Journal of Agricultural and Food Chemistry ascorbic acid, 200 μM 2-mercaptoethanol, pH 8.0; freshly prepared) was added followed by sonication for 5 min. For the release of conjugated pterins, 25 μL of 2 M hydrochloric acid was added. The capped tube was incubated at 80 °C for 1 h. After cooling on ice, 25 μL of 2 M sodium hydroxide was added for neutralization. Finally, the samples were centrifuged at 14 000g for 15 min, and the supernatants were used for LC-MS/MS analysis. The experiments were performed in triplicate. Analysis. The quantification of compounds was performed by LC (Nexera System, Shimadzu, Kyoto, Japan)-triple quadrupole mass spectrometry (LC-TQMS; LCMS-8050, Shimadzu). Samples were injected and separated by an InertSustain AQ-C18 column (150 mm × 2.1 mm i.d. and 1.9 μm particle size from GL Sciences, Osaka, Japan) at a flow rate of 400 μL/min. The gradient was provided by changing the mixing ratio of the two eluents: A, 0.1% (v/v) formic acid; and B, acetonitrile containing 0.1% (v/v) formic acid. The gradient was initiated with 5% B with a 3 min hold, increased to 40% B for 12.5 min, and then increased immediately to 100% with a 3.5 min hold. Finally, the mobile phase was immediately adjusted to its initial composition and held for 4 min in order to re-equilibrate the column. The sample injection volume was 5 μL. The column temperature was set at 40 °C. The autosampler (kept at 4 °C) of the Nexera system was equipped with a black door, which prevented light exposure of the samples. Data acquisition was performed in the multiple reaction monitoring (MRM) mode. Under these conditions, the retention times of DHF, 5-Me-THF, 10-CHO-FA, 5-CHO-THF, PteGlu, DHN, and PABA were 7.15, 5.95, 6.95, 7.10, 7.25, 1.25, and 6.75 min, respectively. All compounds showed much better sensitivity in positive mode than in negative mode. Thus, the positive mode was chosen for all analyses. The parameters for MRM are listed in Table 1. These

Figure 2. Addition of candidate compounds for folate biofortification. (a) Fresh weight in the control and each compound-added treatment. (b) Total folate content in the control and each compound-added treatment. Bars indicate mean ± SD (three biological replicates). Statistical significance was determined by the Student’s t-test (* p < 0.05). Glu, glutamic-acid-added treatment; Mg2+, Mg2+-added treatment; Phe, phenylalanine-added treatment. The glutamic-acid-added treatment had increments of 1.4 mM glutamic acid added 4 times (0, 3, 6, and 10 days), for a final concentration of 5.6 mM. The Mg2+added treatment had increments of 0.5 mM Mg2+ added 2 times (10 and 14 days), for a final concentration of 1 mM. The phenylalanineadded treatment had increments of 1 mM phenylalanine added 2 times (10 and 14 days), for a final concentration of 2 mM.

Table 1. Parameters for Multiple Reaction Monitoring of Folates and Intermediatesa

PteGlu DHF 5-CHOTHF 10-CHOFA 5-MeTHF DHN PABA a

precursor ion (m/z)

product ion (m/z)

Q1 pre bias (V)

Q3 pre bias (V)

CE (V)

442.4 444.4 474.5

295.1 178.1 327.1

−21 −17 −18

−13 −18 −14

−20 −17 −20

470.4

295.0

−23

−14

−18

460.5

313.2

−17

−14

−22

256.1 138.2

165.1 65.1

−19 −20

−30 −20

−23 −25

increase folate synthesis. Phenylalanine is known to induce the feedback inhibition of chorismate mutase or arogenate dehydratase, enzymes for phenylalanine synthesis,17 and the supply of phenylalanine was expected to increase the synthesis of PABA (Figure 1b). Magnesium ion was expected to increase the activity of enzymes such as GCHI that catalyzes a limiting step of folic acid biosynthesis.18 These three compounds were added to the liquid fertilizer of spinach, as described in the Materials and Methods, and subsequently, the growth and amount of folate were measured. Spinach cultivated under the glutamic-acid-added treatment showed growth disorder compared to the control sample. Spinach cultivated under Mg2+- or phenylalanine-added treatments showed growth similar to the control samples (Figure 2a). Folate quantification showed that the total folate content in the phenylalanine-added sample was significantly increased by 1.8-fold, and that of the glutamic-acid-added sample was significantly increased by 1.4-fold compared to that of the control sample (Figure 2b). In the magnesium-added samples, there was no change in the amount of folate (Figure 2b). In addition, we found that 80% or more of the total folate content was in the form of 5-Me-THF in all samples based on LC-MS/ MS analysis (Figure 2b), indicating that 5-Me-THF is the most abundant folate derivative in hydroponic-cultured spinach. Optimized Conditions for the Addition of Phenylalanine. In the previous section, we described that phenylalanine was a promising compound to increase folate content in spinach. To optimize the culture conditions, spinach was cultivated under several patterns of phenylalanine addition, and the growth and amount of folate were subsequently quantified

CE, collision energy; V, volt.

methods were obtained from Zhang et al.23 To calculate the contents of folate in spinach, we used the external standard method, in which the concentrations of the compounds were calculated from the calibration curve. Statistical Analysis. The results were presented as mean values from triplicates ± standard deviation (SD). Statistical analyses in Figure 2 were performed with the Student’s t-test (* p < 0.05). Statistical analyses in Figure 3 were performed with the Tukey− Kramer method (* p < 0.05). Statistical analyses in Figure 4 were performed with the Student’s t-test (* p < 0.05).

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RESULTS Evaluation of Candidate Compounds for Folate Biofortification in Spinach. For folate biofortification in spinach, candidate compounds that were expected to increase the amount of folate were added into the liquid fertilizer during the hydroponic culture of spinach. We tested the following three compounds: glutamic acid, magnesium, and phenylalanine. Glutamic acid is a precursor in folate biosynthesis16 (Figure 1b), and the supply of glutamic acid was expected to 4607

DOI: 10.1021/acs.jafc.7b01375 J. Agric. Food Chem. 2017, 65, 4605−4610

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Figure 3. Addition of phenylalanine at three conditions. (a) Fresh weight of the control and each phenylalanine-added sample. (b) Total folate content of spinach. Bars indicate mean ± SD (three biological replicates). Statistical significance was determined by the Tukey−Kramer method (* p < 0.05). The Phe (5) sample had increments of 1 mM phenylalanine added 5 times during cultivation, for a final concentration of 5 mM. The Phe (2) sample had increments of 0.5 mM phenylalanine added twice, and the Phe (1) sample received a single dose of 1 mM phenylalanine, for a final concentration of 1.0 mM.

derived from microbial overgrowth, and it may have prevented the sufficient growth of spinach. In all conditions of phenylalanine addition, the folate content increased by about 2.0-fold in comparison to the control, and no differences were observed between the three conditions (Figure 3b). The results also showed that 5-Me-THF was the main contributor to the increase of total folate content. Quantification of the Intermediates of Folate Biosynthesis. To evaluate the metabolic state of folate biosynthesis in the phenylalanine-added spinach sample, we quantified the intermediates of folate biosynthesis. We hypothesized that the increase of the folate content was activated by the increase in the folate synthesis in the phenylalanine-added samples (Figure 1b). Therefore, we attempted to support this hypothesis by the quantification of the folate intermediates DHN and PABA. DHN in the phenylalanine-added sample was increased by 1.6fold, and PABA was significantly increased by 1.4-fold (Figure 4). The results indicate a correlation between the increase in folate content and intermediate accumulation. We also found that the amount of folate intermediates in the glutamic-acid- or magnesium-added samples did not increase compared with the control (Figure 4). The reason why glutamic acid increased the folate content but not folate intermediates was that glutamic acid was needed at downstream steps of folate synthesis (Figure 1).

Figure 4. Quantification of intermediates of folate synthesis. Analysis was performed for the control and phenylalanine-added samples cultured following the schedule of Figure 2. Bars indicate mean ± SD (three biological replicates). Statistical significance was determined by the Student’s t-test (* p < 0.05). DHN, dihydroneopterin; PABA, paminobenzoic acid.

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DISCUSSION In this study, phenylalanine was the most effective additive, increasing folate content by 2.0-fold in spinach (Figure 3b). We assume that the addition of phenylalanine induces folate enrichment for the following reasons. First, the addition of phenylalanine could indirectly activate PABA synthesis. Both PABA and phenylalanine are synthesized from chorismate through the shikimate pathway. Excess phenylalanine induces the feedback inhibition of chorismate mutase or arogenate dehydratase, used for phenylalanine synthesis.17 This biochemical evidence suggests that the flux in the shikimate pathway was redirected to PABA (Figure 4). Second, the addition of phenylalanine could increase the demand of total folate. THF is involved as a coenzyme in the metabolic reaction from phenylalanine to tyrosine in S. oleracea.24 Moreover, the

(Figure 3). The Phe (5) sample had 1 mM phenylalanine increments added 5 times during cultivation for a final concentration of 5 mM. The Phe (2) sample had 0.5 mM phenylalanine incrementally added twice, and the Phe (1) sample had a single dose of 1 mM phenylalanine, for a final concentration of 1.0 mM. By comparing Phe (2) and Phe (1), the result showed that the frequency of addition did not affect the growth of spinach. However, high amounts of phenylalanine had a negative effect on growth, as observed in the Phe (5) sample. In the Phe (5) treatment, turbidity was observed in the liquid fertilizer (Figure 3a). The turbidity may have been 4608

DOI: 10.1021/acs.jafc.7b01375 J. Agric. Food Chem. 2017, 65, 4605−4610

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(Figure 3b), whereas 100 g of spinach cultured by the addition of phenylalanine contained 306 μg of folate, representing 76.5% of the recommended daily allowance for adults. Therefore, folate biofortification in spinach is an important approach to achieve the amount of recommended folate intake more easily and would be a valuable and cost-effective intervention to address the problem of folate deficiency worldwide.

activity of the metabolic enzyme that converts phenylalanine to tyrosine depends on folate compounds in gymnosperms.25 These facts suggest that the addition of phenylalanine activated folate synthesis to supply folate compounds as cofactors involved in phenylalanine metabolism. This suggestion was supported by the increase of pteridines, intermediates of folate metabolism (Figure 4). These results for the intermediates suggest that the synthesis of folates in the phenylalanine-added sample would be activated by the addition of phenylalanine. Although we propose that the combined effect of these factors results in the increase of folate content in spinach, we have not yet uncovered which factors contribute the most. The mechanisms of phenylalanine metabolism and folate biosynthesis are assumed to be similar in almost all plants. Therefore, an approach of phenylalanine addition could be applied to other plants that can be hydroponically cultivated. Glutamic acid was the second effective additive, increasing folate content by 1.4-fold in spinach. The reason why glutamic acid was not as effective as phenylalanine might be that the rate-determining step for folate biosynthesis was not the concentration of glutamic acid as a precursor of folates. In addition, the folate content did not increase in magnesium-added spinach probably because magnesium did not activate enzymes that are involved in the synthesis of folate in our experimental conditions. Our results of folate quantification by LC-MS/MS revealed the subcomposition of folate compounds. 5-Me-THF accounted for the majority of the total folate content. Previous research indicated 5-Me-THF was the most abundant vitamin in tomato (>90%).26 Also, 5-Me-THF was found to be the predominant form of folate (>95%) in several types of berries by LC.27 Our results for 5-Me-THF are similar, with it being the predominant form of folate (>80%). 5-Me-THF is known to be the physiologically active form of folate for bioavailability in vivo.28 In human cells, PteGlu is converted to 5-Me-THF by the following steps. Folate in vegetables is mainly stored as pteroylpolyglutamate (Figure 1a) in which glutamates are linked in a chain.2 Pteroylpolyglutamate ingested from vegetables is first degraded into PteGlu by intestinal folate conjugase.29 After that, PteGlu bound to a folate-binding protein passes through the small intestine membrane, where PteGlu is converted to THF in the small intestinal membrane epithelial cells.30 THF is further methylated into 5-Me-THF, and this methylation allows it to penetrate the cell membrane of the small intestine.31 Further, 5Me-THF can penetrate the cell membrane of each tissue from blood.32 Within a cell, 5-Me-THF is the starting material for folate metabolism.33 Therefore, conversion to 5-Me-THF is important for absorption by our body. However, it is reported that the deficiency of methylenetetrahydrofolate reductase that is needed to synthesize 5-Me-THF is the most common inborn error of folate metabolism.34 Therefore, it is suggested that direct ingestion and incorporation of 5-Me-THF into our body is meaningful. Because the spinach cultured by addition of phenylalanine mainly increases the amount of 5-Me-THF, such spinach is of value to improve folate metabolism in our body. Our strategic approach to increase folate content, especially 5-Me-THF, in spinach during hydroponic cultivation could be a significant first step to produce biofortified plant foods without genetic modification. In general, the amount of recommended folate intake for adults is 400 μg/day (National Institutes of Health, Office of Dietary Supplements: http://ods.od.nih.gov/ factsheets/Folate-HealthProfessional/). In this research, 100 g of control spinach contained only about 140 μg of folate

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01375. Figure S1, chemical structures of folate compounds; Figure S2, indoor hydroponic cultivation system (NAPPER LAND) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone/fax: +81-75-7536112. ORCID

Mitsuyoshi Ueda: 0000-0003-2268-2391 Author Contributions

S.W., Y.O., Y.T., W.A., A.T., and M.U. designed the experiment. T.A., Y.S., and S.K. prepared all spinach samples. S.W. and Y.O. performed the experiments and analyzed the data. S.W., Y.O., Y.T., A.W., and M.U. wrote the manuscript. All authors read and approved the final manuscript. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Hoffbrand, A. V.; Weir, D. G. The history of folic acid. Br. J. Haematol. 2001, 113, 579−589. (2) Hanson, A. D.; Gregory, J. F., III Synthesis and turnover of folates in plants. Curr. Opin. Plant Biol. 2002, 5, 244−249. (3) Goh, Y. I.; Koren, G. Folic acid in pregnancy and fetal outcomes. J. Obstet. Gynaecol. 2008, 28, 3−13. (4) Bailey, L. B.; Gregory, J. F., III Folate metabolism and requirements. J. Nutr. 1999, 129, 779−782. (5) MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 1991, 338, 131−137. (6) Boushey, C. J.; Beresford, S. A.; Omenn, G. S.; Motulsky, A. G. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995, 274, 1049−1057. (7) Centers for Disease Control. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. MMWR 1992, 41, 1−7. (8) Wilcox, A. J.; Lie, R. T.; Solvoll, K.; Taylor, J.; McConnaughey, D. R.; Abyholm, F.; Vindenes, H.; Vollset, S. E.; Drevon, C. A. Folic acid supplements and risk of facial clefts: national population based casecontrol study. BMJ. 2007, 334, 464. (9) Hossain, T.; Rosenberg, I.; Selhub, J.; Kishore, G.; Beachy, R.; Schubert, K. Enhancement of folates in plants through metabolic engineering. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 5158−5163. (10) Diaz de la Garza, R.; Quinlivan, E. P.; Klaus, S. M.; Basset, G. J.; Gregory, J. F., III; Hanson, A. D. Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 13720−13725. (11) Storozhenko, S.; De Brouwer, V.; Volckaert, M.; Navarrete, O.; Blancquaert, D.; Zhang, G. F.; Lambert, W.; Van Der Straeten, D.

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DOI: 10.1021/acs.jafc.7b01375 J. Agric. Food Chem. 2017, 65, 4605−4610

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Journal of Agricultural and Food Chemistry Folate fortification of rice by metabolic engineering. Nat. Biotechnol. 2007, 25, 1277−1279. (12) Blancquaert, D.; De Steur, H.; Gellynck, X.; Van Der Straeten, D. Present and future of folate biofortification of crop plants. J. Exp. Bot. 2014, 65, 895−906. (13) Farré, G.; Naqvi, S.; Zorrilla-López, U.; Sanahuja, G.; Berman, J.; Sandmann, G.; Ros, G.; López-Nicolás, R.; Twyman, R. M.; Christou, P.; et al. Transgenic Multivitamin Biofortified Corn: Science, Regulation, and Politics. In Handbook of Food Fortification and Health; Preedy, V. R., Srirajaskanthan, R., Patel, V. B., Eds.; Springer: New York, 2013; Vol. 1, pp 335−347. (14) Nunes, A. C.; Kalkmann, D. C.; Aragao, F. J. Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene. Transgenic Res. 2009, 18, 661−667. (15) James, C. Global Status of Commercialized Biotech/GM Crops: 2014. In ISAAA Brief 49; International Service for the Acquisition of Agri-Biotech Applications: Ithaca, NY, 2014; Vol. 49, pp 1−270. (16) Shane, B. Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism. Vitam. Horm. 1989, 45, 263− 335. (17) Tzin, V.; Galili, G. The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids in Arabidopsis thaliana. Arabidopsis Book 2010, 8, e0132. (18) Schoedon, G.; Redweik, U.; Frank, G.; Cotton, R. G.; Blau, N. Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli. Eur. J. Biochem. 1992, 210, 561−568. (19) Tamura, T. Determination of food folate. J. Nutr. Biochem. 1998, 9, 285−293. (20) Aiso, K.; Tamura, T. Trienzyme treatment for food folate analysis: optimal pH and incubation time for alpha-amylase and protease treatment. J. Nutr. Sci. Vitaminol. 1998, 44, 361−370. (21) Kamen, B. A.; Caston, J. D. Purification of folate binding factor in normal umbilical cord serum. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 4261−4264. (22) Zhang, G. F.; Mortier, K. A.; Storozhenko, S.; Van De Steene, J.; Van Der Straeten, D.; Lambert, W. E. Free and total paraaminobenzoic acid analysis in plants with high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2005, 19, 963−969. (23) Zhang, G. F.; Storozhenko, S.; Van Der Straeten, D.; Lambert, W. E. Investigation of the extraction behavior of the main monoglutamate folates from spinach by liquid chromatographyelectrospray ionization tandem mass spectrometry. J. Chromatogr A 2005, 1078, 59−66. (24) Nair, P. M.; Vining, L. C. Phenylalanine hydroxylase from spinach leaves. Phytochemistry 1965, 4, 401−411. (25) Pribat, A.; Noiriel, A.; Morse, A. M.; Davis, J. M.; Fouquet, R.; Loizeau, K.; Ravanel, S.; Frank, W.; Haas, R.; Reski, R.; Bedair, M.; Sumner, L. W.; Hanson, A. D. Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts. Plant Cell 2010, 22, 3410−3422. (26) Iniesta, M. D.; Perez-Conesa, D.; Garcia-Alonso, J.; Ros, G.; Periago, M. J. Folate content in tomato (Lycopersicon esculentum). influence of cultivar, ripeness, year of harvest, and pasteurization and storage temperatures. J. Agric. Food Chem. 2009, 57, 4739−4745. (27) Stralsjo, L. M.; Witthoft, C. M.; Sjoholm, I. M.; Jagerstad, M. I. Folate content in strawberries (Fragaria x ananassa): effects of cultivar, ripeness, year of harvest, storage, and commercial processing. J. Agric. Food Chem. 2003, 51, 128−133. (28) Verhaar, M. C.; Wever, R. M.; Kastelein, J. J.; van Dam, T.; Koomans, H. A.; Rabelink, T. J. 5-methyltetrahydrofolate, the active form of folic acid, restores endothelial function in familial hypercholesterolemia. Circulation 1998, 97, 237−241. (29) Wang, T. T.; Reisenauer, A. M.; Halsted, C. H. Comparison of folate conjugase activities in human, pig, rat and monkey intestine. J. Nutr. 1985, 115, 814−819. (30) Sirotnak, F. M.; Tolner, B. Carrier-mediated membrane transport of folates in mammalian cells. Annu. Rev. Nutr. 1999, 19, 91−122.

(31) Selhub, J.; Powell, G. M.; Rosenberg, I. H. Intestinal transport of 5-methyltetrahydrofolate. Am. J. Physiol. 1984, 246, G515−520. (32) Sorrell, M. F.; Frank, O.; Aquino, H.; Thomson, A. D.; Baker, H. In vivo and in vitro penetration of vitamins into human red blood cells. Am. J. Clin. Nutr. 1971, 24, 924−929. (33) Thaler, C. J. Folate Metabolism and Human Reproduction. Geburtshilfe Frauenheilkd. 2014, 74, 845−851. (34) Fattal-Valevski, A.; Bassan, H.; Korman, S. H.; Lerman-Sagie, T.; Gutman, A.; Harel, S. Methylenetetrahydrofolate reductase deficiency: importance of early diagnosis. J. Child Neurol 2000, 15, 539−543. (35) Hawkes, J. G.; Villota, R. Folates in foods: reactivity, stability during processing, and nutritional implications. Crit Rev. Food Sci. Nutr 1989, 28, 439−538.

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DOI: 10.1021/acs.jafc.7b01375 J. Agric. Food Chem. 2017, 65, 4605−4610