Article pubs.acs.org/JAFC
Changes in the Content of Free and Conjugated Polyamines during Lettuce (Lactuca sativa) Growth Edgar Pinto* and Isabel M. P. L. V. O. Ferreira REQUIMTE/Department of Chemical Sciences, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto. R Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal ABSTRACT: Polyamines (PAs) in plant foods are relevant due to the association of these bioactive nutrients with health and disease. The scope of the present study was to monitor the content of free, conjugated, and total (free + conjugated) putrescine (Put), spermidine (Spd), and spermine (Spm) at five stages of lettuce growth in three different greenhouses. The daily intake of PAs from lettuce consumption was estimated since its consumption represents about 7.2% of vegetables intake. Results showed that the content of free Put, Spd, and Spm decreased during plant growth, while the content of conjugated Put, Spd, and Spm increased. Nevertheless, the total PA content remained fairly constant. Significant differences were observed in the PAs content in lettuces grown in different greenhouses. The conjugated fraction of PAs in mature lettuces has an important contribution to the total PAs and will certainly influence the bioavailability and/or bioactivity of dietary polyamines. KEYWORDS: lettuce, polyamines, growth, daily intake, human health
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converts Put into Δ1-pyrroline and generates NH3 and H2O2 as byproducts.3 The amount of PAs present at a certain time within the human body is the result of three sources: (i) endogenous supply, (ii) through intestinal microorganisms, and (iii) exogenous supply through the diet.6 The exogenous dietary source provides a much larger quantity of PAs than the other two sources. Thus, diet plays a key role controlling the body pool of these compounds and, consequently, in the regulation of several physiological mechanisms within the human body.2 The dietary intake of PAs influences human health.7 It has been suggested that a PA-rich diet could have several health benefits such as decreasing age-associated pathologies and increasing longevity.8 However, a reduced intake of exogenous PAs through diet has also been proposed as an efficient nutritional strategy for several human conditions.9−11 In humans, PAs are involved in the regulation of many basic cellular processes, such as DNA replication, transcription, translation, cell proliferation, modulation of enzyme activities, cellular cation−anion balance, and membrane stability.4,5 This occurs due to the inherent physicochemical properties of PAs that enable them to interact with negatively charged macromolecules such as nucleic acids, phospholipids, and proteins.6 The importance of PAs in cellular growth and proliferation is well-established and has been related to their role in stabilizing the negative charges of DNA and of the chromatin structure, the regulation of several transcriptional factors, and protein synthesis.7 PAs do not trigger cancer, but their increased availability enhances cell growth. However, it has been also documented that excessive accumulation of PAs induces apoptosis.2 PAs play a vital role in the maintenance of intestinal
INTRODUCTION
Putrescine (Put), spermidine (Spd), and spermine (Spm) form a group of low molecular and aliphatic nitrogen polycations known as polyamines (PAs). These are commonly found in higher plants in free, soluble conjugated, and insoluble bound forms. Soluble conjugated PAs are mainly conjugated to small molecules such as phenolic compounds, whereas insoluble bound PAs are bound to macromolecules such as nucleic acids and proteins.1 Put, Spd, and Spm usually have been included within the group of biogenic amines. However, due to their specific biological roles in plants they have become a peculiar group. In plants, PAs have been found in both the cytoplasm and other organelles (e.g., vacuoles, mitochondria, and chloroplasts).2 The biosynthetic pathways of PAs is highly conserved through evolution and starts from the two amino acid precursor molecules, L-arginine and L-methionine.3 Two alternative biosynthetic pathways have been characterized. Put is synthesized through either arginine decarboxylase (ADC, EC 4.1.1.19) via agmatine (Agm) or ornithine decarboxylase (ODC, EC 4.1.1.17). Conversion of Agm into Put requires two distinct enzymes: N-carbamoylputrescine amidohydrolase (CPA, EC 3.5.1.53) and agmatine iminohydrolase (AIH, EC 3.5.3.12). Spd is synthesized by spermidine synthase (SPDS, EC 2.5.1.16) through the addition of an aminopropyl group, transferred from the decarboxylated S-adenosylmethionine (dcSAM), to Put. Spd can be aminopropylated at either Spm or thermospermine by the action of spermine synthase (SPMS, EC 2.5.1.22).4,5 With regard to PAs catabolism, both Spd and Spm can be converted back to Put. Spermine oxidase (SMO, EC 1.5.3.16) mediates the back-conversion of Spm to Spd. Moreover, the enzyme polyamine oxidase (PAO, EC 1.5.3.11) catalyzes the conversion of Spd and Spm to 4-aminobutanal and N-(3-aminopropyl)-4-aminobutanal, respectively. Put is catabolized by diamine oxidases (DAOs) in a reaction that © 2014 American Chemical Society
Received: Revised: Accepted: Published: 440
September 19, 2014 December 24, 2014 December 24, 2014 December 24, 2014 DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
Article
Journal of Agricultural and Food Chemistry
acid (10% v/v) and deionized water, and kept at 4 °C on the way to the laboratory. Soil was also sampled from the same locations using a stainless steel auger. In order to generate a representative sample, individual samples (n = 25) were combined to generate a composite sample. At the laboratory, plant samples were cleaned and washed with deionized water to remove soil contamination. Afterward, inedible leaves were removed, and the remaining lettuce leaves were frozen at −80 °C and then freeze-dried. The dried samples were homogenized by grinding in a blender and sieved through a nylon sieve of 150 μm mesh size. Soil samples were spread in plastic trays, oven-dried for 24 h at 40 °C, crushed and sieved through a 2 mm nylon sieve, and stored at 4 °C until analysis. Chemicals and Standard Solutions. Putrescine dihydrochloride, spermidine trihydrochloride, 1,7-diaminoheptane (internal standard, IS), spermine tetrahydrochloride, hydrochloric acid, acetonitrile, and 3,5-dinitrobenzoyl chloride (DNBZ-Cl) were purchased from Sigma− Aldrich (St. Louis, MO). Boric acid, sodium hydroxide, potassium chloride, ammonium nitrate and sodium chloride were from VWR (Radnor, PA). Perchloric acid (HClO4) and carbon tetrachloride were both obtained from Merck (Darmstadt, Germany). Standard stock solutions of PAs were prepared by dissolving each standard in 0.1 M HCl to provide a final concentration of 1000 mg/L. Working standard solutions (in the 0.5 to 10 mg/L interval range) were prepared daily from the stock solutions by adequate dilution with 0.1 M HCl. Sodium borate buffer was prepared from H3BO3 and KCl in water (0.5 M each) followed by titration with NaOH (0.5 M) to pH 10. DNBZ-Cl (56 mM) was prepared by dissolving a proper amount of the solid compound in acetonitrile. Multielement calibration standards were prepared from 1000 mg/L single-element standard solutions (Sigma, MO) of Ca2+, Mg2+, K+, and Na + for AAS and Cl − , NO 3 − , PO 4 3− , and SO 4 2− for ion chromatography. Instrumentation and Chromatographic Conditions. A Telstar (Terrassa, Spain) Cryodos-80 freeze-dry system was used to lyophilize lettuce samples. The moisture content was determined using HR73 Moisture Analyzer from Mettler Toledo (Greifensee, Switzerland). Water pH, electrical conductivity, and total dissolved solids were measured in situ by the IQ170 waterproof portable pH/conductivity/ TDS meter (IQ Scientific Instruments, Carlsbad, CA). A PerkinElmer (Ü berlingen, Germany) 3100 flame (air-acetylene) atomic absorption spectrometer (AAS) was used for the determination of Ca2+, Mg2+, K+, and Na+. A PU-2089Plus pump (Jasco, Tokyo, Japan) was used in conjunction with an IC-PAK anion-exchange column (10 μm, 50 × 4.6 mm) for Cl−, NO3−, PO43−, and SO42− determination. Detection was done by a Waters Model 431 conductivity detector. The eluent was a 1.3-mM sodium gluconate/1.3-mM borax buffer solution adjusted to pH 8.5. Chromatographic analysis of PAs was performed with an analytical HPLC unit (Jasco, Tokyo, Japan) equipped with Jasco PU-2080 HPLC pumps, Column Heater (Model 7981; Jones Chromatography, Hengoed, UK), and an MD-2010 Plus multiwavelength detector. The column was reversed-phase Ultracarb ODS (30) C18 (5 μm, 250 × 4.6 mm) (Phenomenex, Milford, MA). Borwin PDA Controller Software (JMBS Developments, Le Fontanil, France) was used for system control and data acquisition. Chromatographic separation was carried out by gradient elution with a mixture of two solvents and at a 1 mL/ min flow rate. Solvent A consisted of 10 mM aqueous sodium formate/formic acid buffer at pH 3, and solvent B consisted of acetonitrile. The linear gradient program was as follows: 0−5 min, 40% B; 5−11 min, 58% B; 11−15 min, 58% B; 15−20 min, 65% B; 20−23 min, 65% B; 23−28 min, 95% B; 28−30 min, 95% B; 30−40 min, column rinse and re-equilibration. Column temperature was set at 35 °C. Detection was carried out at 250 nm. Soil Analysis. Soil pH was measured in the supernatant of a 1:5 (w/v) suspension made up with ultrapure water. Organic matter content was determined based on its oxidation with a potassium dichromate (0.27 M) and sulfuric acid (98% v/v) mixture at 135 °C. CEC and particle size distribution were determined according to ISO methods.20,21
function and gut maturation. When damaged, the intestinal mucosa is able to repair itself, and PAs are essential for these repair processes. Moreover, PAs are widely described as essential growth factors for small intestinal and colonic mucosal growth and maturation.6,7 PAs also show antioxidant activity and seem to be engaged in the reduction of cell membranes and DNA damage. At physiological levels, PAs can act as potent scavengers of reactive oxygen species (ROS).2 Although dietary PAs have been known as an important factor of health and disease, data on PA contents in foods are limited. Put, Spd, and Spm are present in variable amounts in various foods.6,12 Regarding plant foods, very low levels, ranging from a few milligrams to less than a 100 mg/kg, are usually found.2 Some work has been done in quantifying the PAs content in several plant foods.13,14 However, no studies have addressed the PA content during the plant food growth period. Moreover, information concerning the content of conjugated PAs in food is scarce. Since in the last years an increased number of papers have been published encouraging the consumption of plant foods at their initial stages of growth (known as microgreens),15,16 information regarding the PAs content of plant foods during their growth period is of great importance for consumers. Lettuce is known to be an important source of mineral nutrients and phytochemicals that benefit human nutrition.17 Additionally, it is one of the most consumed vegetables, accounting with a mean daily consumption in Europe of 23.4 g, which represents about 7.2% of the total dietary intake of vegetables.18 Its global production was around 25 million tons in 2011.19 The aim of this study was to monitor the free and conjugated Put, Spd, and Spm content of a test plant food (Lactuca sativa) at five growth stages produced in three greenhouses as well as to estimate the daily intake of these compounds from lettuce consumption.
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MATERIALS AND METHODS
Lettuce Cultivation, Sampling, and Sample Preparation. Lettuce (Lactuca sativa) plants (n = 100) with 2 weeks of growth were transferred on the same day to three different greenhouses: A1 (41° 26.991 N, 8° 46.335 W), A2 (41° 25.249 N, 8° 44.936 W), and A3 (41° 27.435 N, 8° 45.377 W) located in NW Portugal, in a small region between Esposende and Vila do Conde. These three greenhouses were chosen because they were located in an intensive agriculture area, which is known to be the main supplier of vegetables to the city of Porto, Portugal. The experiments were conducted from December to February. All plants were exposed to the same conditions: light intensity, a total of 334 h of sunshine duration; photoperiod, on average 11.16 h; and temperature, average minimum temperature, 7.6 °C and average maximum temperature, 20.5 °C. Inorganic fertilizers were dissolved in 50 L water tanks and injected by an electric pump in long dripper pipes, from where the plants were fertigated. The nutrient elements provided (per plant and per fertigation) were as follows: 0.126 g N, 0.042 g K, 0.03 g P, 0.21 mg Fe, 0.051 mg Mn, and 0.013 mg Zn. The irrigation water used had the following characteristics: pH 6.91, electrical conductivity (EC) 401 mS/cm (at 25 °C), total dissolved solids (TDS) 201 mg/L, Ca2+ 70.2 mg/L, Mg2+ 19.2 mg/L, K+ 5.2 mg/L, Na+ 25.2 mg/L, Cl− 83.4 mg/L, NO3− 37.4 mg/L, and SO42− 66.8 mg/L. Samples collection was always performed during the same period of the day (9−12 am). One kilogram (or at least 10 units) of lettuce was randomly harvested at five growth stages (2, 4, 6, 8, and 10 weeks hereinafter referred to as T1, T2, T3, T4, and T5). After 10 weeks, lettuces reach their commercial maturity stage (T5). Plant samples were placed into plastic containers, previously rinsed with diluted nitric 441
DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
Article
Journal of Agricultural and Food Chemistry The extraction of Ca2+, Mg2+, K+, and Na+ from soil samples was performed following ISO 19730:2008.22 Ten grams of air-dried soil and 25 mL of 1 M NH4NO3 solution were added to a 50 mL centrifuge tube and shaken for 2 h at 20 °C. Afterward, the solution was centrifuged at 5000g for 10 min and analyzed by AAS. The extraction of Cl−, NO3−, PO43−, and SO42− from soil samples was conducted following ISO/TS 14256−1:200323 with some modifications. Briefly, soil samples were extracted with ultrapure water at a ratio 1:5 (w/v) at 20 °C. After 1 h of extraction, the solution was centrifuged at 5000g for 10 min. The above-mentioned anions were determined in the supernatant by ion chromatography. Extraction and Analysis Of PAs. Lyophilized lettuce samples (100 mg) were homogenized in 2.5 mL of 5% (v/v) cold HClO4 and incubated at 4 °C for 1 h. After centrifugation at 17000g for 30 min, the supernatant was collected and used as the sample extract to determine the content of free PAs. In order to extract HClO4-soluble conjugated PAs, a suitable portion of the previous extract was mixed with 12 M HCl (1:1, v/v) for 18 h at room temperature in centrifuge tubes as proposed by Fontaniella et al.24 After acid hydrolysis, NaOH (10 M) was added to the centrifuge tubes to neutralize the HCl in excess, and the volume was made up to 2 mL with ultrapure water. This solution was used as a source of HClO4-soluble conjugated PAs and free PAs. PAs recovered from the nonhydrolyzed supernatant (free PAs) and the hydrolyzed supernatant (free + conjugated PAs) were quantified, after derivatization with DNBZ-Cl, by HPLC-DAD according to Pinto, Melo, and Ferreira.14 Representative chromatograms of free and conjugated PAs are shown in Figures 1 and 2, respectively. Results are presented on a fresh weight (fw) basis. The detection limit (LOD) for Put, Spd, and Spm was 0.018, 0.034, and 0.042 μg/g, respectively, while the quantification limit (LOQ) was 0.062, 0.115, and 0.141 μg/g for Put, Spd, and Spm, respectively.
Figure 2. Representative chromatograms of conjugated PAs: (A) mixed standard solution of 5 mg/L and (B) lettuce. Peaks: Put, putrescine; Cad, cadaverine; Spd, spermidine; IS, internal standard; and Spm, spermine. Estimated Daily Intake of PAs from Lettuce. The estimated daily intake (EDI) of Put, Spd, and Spm resulting from lettuce consumption was calculated. The EDI, which is dependent on both the PAs content of the edible part of the plants (Cpolyamine; μg/g fw basis) and the average daily intake of lettuce (DIlettuce), was calculated using the following formula:
EDI = DIlettuce × C polyamine where EDI is expressed as μmol/day, and DI was assumed to be 23.4 g/day for adults.18 Statistical Analysis. Data exploration, descriptive statistics calculation, and ANOVA were performed with IBM SPSS Statistics for Windows, version 22.0 (IBM Corp, Armonk, NY). Statistical significance was assumed for p < 0.05.
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RESULTS AND DISCUSSION Lettuce Leaf Biomass and Moisture Content. Lettuce leaf biomass at the 5 growth stages in the three greenhouses (A1, A2, and A3) is shown in Table 1. A similar increase of leaf biomass was observed during lettuce growth in A1, A2, and A3 greenhouses. No significant differences were observed between lettuce leaf biomass at T1 and T2. However, the increase of lettuce biomass was significant among T3, T4, and T5 growth stages. Only at T5 did A2 lettuces show significantly higher biomass content compared to that of A1 and A3 lettuces.
Figure 1. Representative chromatograms of free PAs: (A) mixed standard solution of 5 mg/L and (B) lettuce. Peaks: Put, putrescine; Cad, cadaverine; Spd, spermidine; IS, internal standard; and Spm, spermine. 442
DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
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Journal of Agricultural and Food Chemistry
3). Given these results, it is hypothesized that the higher bioavailability of Na+ and Cl− in A1 and A3 soils is indicative of higher soil salinity. Since PAs are involved in salinity stress, these results can explain the highest amount of PAs (mainly in the free form) in A1 and A3 lettuces. The highest NO3− content in A2 soil may have also influenced the PA content of lettuces, limiting the accumulation of Put in A2 lettuces. Houdusse et al.26 found that NO3− nutrition limited Put accumulation in wheat and pepper. The same authors also showed that the effect of salinity on PA content is highly dependent on nitrogen nutrition. Since the environmental conditions light intensity, temperature, photoperiod, and drought that can greatly influence the PAs content in plants were controlled in the present study, it seems that mineral nutrition and salinity were the factors responsible for the variation observed in the PA content among lettuces grown in different greenhouses. Temporal Variation of Free and Conjugated PA Content in Lettuce Leaves. The free, conjugated, and total (free + conjugated) Put, Spd, and Spm contents at the five growth stages of lettuce (T1, T2, T3, T4, and T5) in the three greenhouses (A1, A2, and A3) are shown in Figures 3, 4, and 5. Regardless of the PA, a general trend was observed in all greenhouses: the content of free Put, Spd, and Spm decreased during plant growth (Figures 3A, 4A, and 5A), while the content of conjugated Put, Spd, and Spm increased (Figures 3B, 4B, and 5B). Despite the fluctuations observed in the content of free and conjugated PAs, total Put, Spd, and Spm content showed lower differences between different growth stages (Figure 5). In plant foods, higher levels of free PAs are usually found in actively growing tissues.5 Therefore, it is understandable that, in our study, higher levels of free Put, Spd, and Spm were obtained in the initial growth stages of all lettuces. Moreover, in plants, PAs are present not only as free molecules but also in the form of conjugates.1 Similar to the free forms of PAs, conjugated PAs play key roles in plant survival, including cell division, flowering, and response to biotic and abiotic stresses. However, evidence suggests that these conjugates serve mainly as a reserve of PAs that can become available.1,27 In the present study, higher levels of conjugated PAs were observed in the later stages of lettuce growth. This may be the reflex of a steady reduction of cell division during the lettuce growth period that results in an increase of the conjugated PA pool in plants. Sood and Nagar28 also found an increase of conjugated Put, Spd, and
Table 1. Lettuce Leaf Biomass (g FW/Plant) during the Five Stages of Lettuce Growth in the Three Greenhouses (A1, A2, and A3)a lettuce ID growth stage T1 T2 T3 T4 T5
A1 1.91 6.69 24.42 56.68 173.96
± ± ± ± ±
A2 0.04 0.25 1.45 3.53 6.94
a a b c d
1.85 6.84 24.08 58.39 186.85
± ± ± ± ±
A3 0.03 a 0.31 a 1.96 b 4.36 c 12.31 d
2.13 8.23 23.7 59.54 169.28
± ± ± ± ±
0.09 0.53 1.75 4.31 8.17
a a b c d
Data are presented as the mean ± SD (n = 5). In a column, different letters (a, b, c, and d) indicate significant differences between growth stages at p < 0.05.
a
No significant differences were found for the mean moisture content (94.3 ± 0.1%) of lettuces grown in the A1, A2, and A3 greenhouses at the five growth stages. Spatial Variation of Free and Conjugated PA Content in Lettuce Leaves. Significant differences were observed depending on the greenhouse where the lettuce was grown. A1 lettuces showed significantly higher levels of all free PAs, while A2 lettuces showed the lowest levels (Figures 3A, 4A, and 5A). For the conjugated PAs, this difference was not so clear for Put and Spd. However, A2 lettuces showed a significantly higher level of conjugated Spm (Figures 3B, 4B, and 5B). For the total PAs, A1 lettuces showed significantly higher levels of Put and Spd at all growth stages, whereas A2 lettuces showed the lower levels of these PAs (Figures 3C and 4C). A great body of evidence supports that abiotic stresses, such as metal toxicity, oxidative stress, drought, low temperature, and salinity, modulate the production of PAs in plants.5 In order to understand the influence of soil properties in the PA content of lettuces, the analysis of soils from the three greenhouses was performed. A2 soil showed a significantly higher pH, CEC, OM, and EC than the other two soils (A1 and A3). Moreover, A2 soil was classified as a loamy sand soil, while A1 and A3 soils were sandy soils (Table 2). These characteristics are very important with regard to the bioavailability of essential and toxic elements to plants.25 In Table 3 is shown the extractable content of Ca2+, Mg2+, K+, Na+, Cl−, NO3−, PO43−, and SO42− of each of the three greenhouse soils. A2 soil had the highest extractable content of Ca2+, Mg2+, K+, NO3−, PO43−, and SO42− and the lowest extractable content of Na+ and Cl−. On the contrary, A1 soil had the highest extractable content of Na+ and Cl− (Table
Figure 3. Free (A), conjugated (B), and total (C) putrescine (Put) content at five stages of lettuce growth (T1, T2, T3, T4, and T5) in the three greenhouses (A1, A2, and A3). Data are presented as the mean ± SD (n = 5). At a growth stage, different symbols (*, §, and £) indicate significant differences between greenhouses at p < 0.05. For a greenhouse, different letters (a, b, c, d, and e) indicate significant differences between growth stages at p < 0.05. 443
DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
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Journal of Agricultural and Food Chemistry
Figure 4. Free (A), conjugated (B), and total (C) spermidine (Spd) content at five stages of lettuce growth (T1, T2, T3, T4, and T5) in the three greenhouses (A1, A2, and A3). Data are presented as the mean ± SD (n = 5). At a growth stage, different symbols (*, §, and £) indicate significant differences between greenhouses at p < 0.05. For a greenhouse, different letters (a, b, c, d, and e) indicate significant differences between growth stages at p < 0.05.
Figure 5. Free (A), conjugated (B), and total (C) spermine (Spm) content at five stages of lettuce growth (T1, T2, T3, T4, and T5) in the three greenhouses (A1, A2, and A3). Data are presented as the mean ± SD (n = 5). At a growth stage, different symbols (*, §, and £) indicate significant differences between greenhouse at p < 0.05. For a greenhouse, different letters (a, b, c, d, and e) indicate significant differences between growth stages at p < 0.05.
Table 2. Main Physicochemical Properties of the Three Greenhouse Soils (A1, A2, and A3)a pH CEC (cmol/kg) OM (%) EC (μS/cm) clay (%) silt (%) sand (%) soil classification
A1
A2
A3
6.5 ± 0.2 a 8.0 ± 0.5 a 53.1 ± 4.1 a 297 ± 49 a 1.9 ± 0.3 a 2.7 ± 0.4 a 94.9 ± 2.3 a sandy soil
6.8 ± 0.1 b 18.1 ± 0.8 b 73.2 ± 2.8 b 645 ± 84 b 12.6 ± 0.4 b 12.8 ± 0.2 b 74.2 ± 2.0 b loamy sand soil
6.6 ± 0.1 a 7.4 ± 0.6 a 21.3 ± 1.9 c 162 ± 19 c 2.3 ± 0.5 a 1.8 ± 0.3 a 95.4 ± 4.2 a sandy soil
Table 3. Extractable Content (mg/kg) of Chemical Species in the Three Greenhouses Soils (A1, A2, and A3)a A1 Ca2+ Mg2+ K+ Na+ Cl− NO3− PO43− SO42−
784.0 85.2 167.1 194.4 335.7 174.7 50.9 188.2
± ± ± ± ± ± ± ±
111.1 a 11.9 a 19.3 a 22.9 a 50.9 a 44.0 a 3.0 a 27.7 a
A2 1285.3 199.8 397.7 40.3 16.2 411.0 101.4 485.3
± ± ± ± ± ± ± ±
A3 161.3 b 10.1 b 35.7 b 13.7 b 3.8 b 63.3 b 3.1 b 95.8 b
885.9 115 109.8 76.5 126.9 102.1 63.5 217.1
± ± ± ± ± ± ± ±
99.5 c 20.2 c 21.0 c 7.9 c 27.4 c 31.5 c 4.5 c 64.6 a
a
Data are presented as the mean ± SD (n = 15, i.e., triplicate analysis in each of the 5 sampling time points). Differences were tested according to ANOVA followed by Tukey’s test. In a line, different letters (a, b, and c) indicate significant differences (p < 0.05) between the greenhouse soils.
Data are presented as the mean ± SD (n = 15, i.e., triplicate analysis in each of the 5 sampling time points). Differences were tested according to ANOVA followed by Tukey’s test. In a line, different letters (a, b, and c) indicate significant differences (p < 0.05) between greenhouses.
Spm during flower development of two species of rose (Rosa damascena and Rosa bourboniana). Regardless of the greenhouse where lettuces were grown, free Put was always the most abundant PA, while free Spd was the least abundant. In particular, at T5, free Put content was 6.4, 3.8, and 5.9 μg/g in A1, A2, and A3 lettuces, respectively. These values are in close agreement with the 4 to 7.9 μg/g interval range described in the literature for lettuce.2,13 For free Spd, the obtained levels in A1, A2, and A3 lettuces at T5 were 0.81, 0.38, and 0.66 μg/g, respectively. These values are significantly lower compared to the ones described by Ali et al.13 in lettuces (6.2 to 9.1 μg/g). For free Spm, the obtained content at T5 was 1.5,
0.7, and 0.9 in A1, A2, and A3 lettuces, respectively, which are very similar to those reported in the literature (0.8 to 1.2 μg/g) for lettuce.2,13 Similar to free Put, conjugated Put was the most abundant conjugated PA (9.1, 6.3, and 8.4 μg/g at T5 in A1, A2, and A3 lettuces, respectively), while conjugated Spd and Spm showed very similar contents. Since most studies carried out to date only determine the content of free Put, Spd, and Spm, data comparison between the content of conjugated PAs in our lettuces and other plant foods was impossible to perform. Estimation of the PA Daily Intake from Lettuce Leaves. The EDI values of free, conjugated, and total (free + conjugated) PAs were calculated at each of the five growth
a
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DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
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Journal of Agricultural and Food Chemistry
during lettuce growth in three different greenhouses. Free PA content is significantly higher at the initial stages of lettuce growth, while at later growth stages conjugated PAs predominate. Despite these fluctuations, the total (free + conjugated) PA content remained fairly constant during the studied period. Data described here should be included in food databases to enable a reliable estimation of PA intake and how this intake can affect human health. The form in which a certain PA is present in foods will certainly influence its toxicokinetics. Thus, more studies in this field are required.
stages of A1, A2, and A3 lettuces. Overall, the EDI values of free PAs were the highest in the first growth stage of lettuce (T1), decreasing until the last growth stage (T5). With regard to conjugated PAs, the inverse was observed: the first growth stage of lettuce (T1) showed the lowest content, steadily increasing until the last growth stage (T5). Regarding the relative contribution of each PA for the dietary intake, Put was the most abundant PA (70% of the total PA fraction), followed by Spm (19%) and Spd (11%). Nishibori et al.29 determined the content of PAs in several foods groups (e.g., vegetables, fruits, meat, cheese, cereals, nuts, eggs, and dairy) and estimated the daily intake of PAs from each food group. These authors found that vegetables are the main source of Put and Spd and that Put was responsible for almost half of the total PA intake considering all food groups. Other studies have also addressed the importance of vegetables as an important source of PAs in the human diet.11,30,31 Both free and conjugated PA content should be considered in the dietary intake estimation; thus, the total (free + conjugated) Put, Spd, and Spm mean content was calculated and discussed. A general trend was observed: A1 lettuces contributed to the highest daily intake of PAs (3.81, 0.36, and 0.44 μmol/day for Put, Spd, and Spm, respectively), while A2 lettuces contributed to the lowest daily intake of PAs (2.36, 0.22, and 0.32 μmol/day for Put, Spd, and Spm, respectively). Several studies have estimated the daily intake of PAs; however, great variation is described depending on the type of food as well as on regional dietary habits. Among these studies, the lowest total PAs intake was obtained in Japan (200 μmol/ day)29 and the highest in European countries (316 μmol/ day).32 Considering this low and high total PA intake, it was observed that lettuce only contributes between 1.5 to 2.3% of the low total PA intake (200 μmol/day) and between 0.9 to 1.5% of the high total PA intake (316 μmol/day). It should be highlighted that most studies that deal with dietary PA intake consider only the free form of PAs present in foods.2,13,31 Until now, it is not clear if the form in which PAs are present in foods influences their bioavailability and/or bioactivity. However, evidence suggests that this aspect is of major importance when studies are carried out with the aim of assessing the potential implications of dietary PA intake in human health since it is hypothesized that the toxicokinetics (absorption, distribution, metabolism, and excretion) of free, conjugated, and bound PAs will greatly differ among them.6 In recent years, increased consumption of plant foods at their initial growth stages (known as microgreens or baby leaf vegetables) has been observed due to their higher nutritional value, intense flavors, attractive colors, and tender textures.16,17 According to our results, lettuce at the initial growth stages showed a higher content of free Put, Spd, and Spm than the conjugated form of these PAs. However, when lettuces are consumed in their mature stage (T5), a higher contribution of conjugated Put, Spd, and Spm is observed. This information is very important considering the differences that are likely to exist between the bioavailability and/or bioactivity of free and conjugated PAs. Thus, it emphasized that due to the high contribution of the conjugated form of Put, Spd, Spm in the last stage of lettuces growth (maturity stage), conjugated PAs must be considered in studies evaluating the daily intake of PAs. Until now, studies monitoring the PA content in plant foods have not detailed the form in which these compounds are present. This study describes relevant information about the evolution of free and conjugated Put, Spd, and Spm content
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AUTHOR INFORMATION
Corresponding Author
*Tel: +351 220428642. Fax: +351 226093390. E-mail: ecp@ estsp.ipp.pt. Funding
Edgar Pinto thanks FCT (Portuguese Foundation for Science and Technology) for his Ph.D. grant (SFRH/BD/67042/ 2009). This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT) through project Pest-C/EQB/LA0006/2013. This work also received financial support from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0124-FEDER-000069. To all financing sources, we are greatly indebted. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Takahashi, T.; Kakehi, J. I. Polyamines: ubiquitous polycations with unique roles in growth and stress responses. Ann. Bot. (Oxford, U. K.) 2010, 105, 1−6. (2) Kalač, P. Health effects and occurrence of dietary polyamines: A review for the period 2005−mid 2013. Food Chem. 2014, 161, 27−39. (3) Fuell, C.; Elliott, K. A.; Hanfrey, C. C.; Franceschetti, M.; Michael, A. J. Polyamine biosynthetic diversity in plants and algae. Plant Physiol. Biochem. 2010, 48, 513−520. (4) Kusano, T.; Berberich, T.; Tateda, C.; Takahashi, Y. Polyamines: essential factors for growth and survival. Planta 2008, 228, 367−381. (5) Hussain, S. S.; Ali, M.; Ahmad, M.; Siddique, K. H. M. Polyamines: natural and engineered abiotic and biotic stress tolerance in plants. Biotechnol. Adv. 2011, 29, 300−311. (6) Larque, E.; Sabater-Molina, M.; Zamora, S. Biological significance of dietary polyamines. Nutrition 2007, 23, 87−95. (7) Moinard, C.; Cynober, L.; de Bandt, J. P. Polyamines: metabolism and implications in human diseases. Clin. Nutr. 2005, 24, 184−197. (8) Soda, K. Polyamine intake, dietary pattern, and cardiovascular disease. Med. Hypotheses 2010, 75, 299−301. (9) Rivat, C.; Richebe, P.; Laboureyras, E.; Laulin, J. P.; Havouis, R.; Noble, F.; Moulinoux, J. P.; Simonnet, G. Polyamine deficient diet to relieve pain hypersensitivity. Pain 2008, 137, 125−137. (10) Bell, R. F.; Borzan, J.; Kalso, E.; Simonnet, G. Food, pain, and drugs: does it matter what pain patients eat? Pain 2012, 153, 1993− 1996. (11) Cipolla, B. G.; Havouis, R.; Moulinoux, J. P. Polyamine contents in current foods: a basis for polyamine reduced diet and a study of its long term observance and tolerance in prostate carcinoma patients. Amino Acids 2007, 33, 203−212. (12) Kozova, M.; Kalac, P.; Pelikanova, T. Contents of biologically active polyamines in chicken meat, liver, heart and skin after slaughter and their changes during meat storage and cooking. Food Chem. 2009, 116, 419−425. (13) Ali, M. A.; Poortvliet, E.; Stromberg, R.; Yngve, A. Polyamines in foods: development of a food database. Food Nutr Res. 2011, 55. DOI: 10.3402/fnr.v55i0.5572. 445
DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
Article
Journal of Agricultural and Food Chemistry (14) Pinto, E.; Melo, A.; Ferreira, I. M. P. L. V. O. Sensitive quantitation of polyamines in plant foods by ultrasound-assisted benzoylation and dispersive liquid-liquid microextraction with the aid of experimental designs. J. Agric. Food Chem. 2014, 62, 4276−4284. (15) Sun, J. H.; Xiao, Z. L.; Lin, L. Z.; Lester, G. E.; Wang, Q.; Harnly, J. M.; Chen, P. Profiling polyphenols in five Brassica species microgreens by UHPLC-PDA-ESI/HRMSn. J. Agric. Food Chem. 2013, 61, 10960−10970. (16) Xiao, Z. L.; Lester, G. E.; Luo, Y. G.; Wang, Q. Assessment of vitamin and carotenoid concentrations of emerging food products: edible microgreens. J. Agric. Food Chem. 2012, 60, 7644−7651. (17) Pinto, E.; Almeida, A. A.; Aguiar, A. A. R. M.; Ferreira, I. M. P. L. V. O. Changes in macrominerals, trace elements and pigments content during lettuce (Lactuca sativa L.) growth: influence of soil composition. Food Chem. 2014, 152, 603−611. (18) WHO. GEMS/Food Cluster Diets; WHO: Geneva, Switzerland, 2013. (19) FAOSTAT. The FAO (Food and Agriculture Organization of the United Nations) Statistical Database; FAO: Rome, Italy, 2011. (20) ISO. ISO 11260:1994: Soil Quality - Determination of Effective Cation Exchange Capacity and Base Saturation Level Using Barium Chloride Solution; International Organization for Standardization: Geneva, Switzerland, 1994; p 10. (21) ISO. ISO 11277:1998: Soil Quality - Determination of Particle Size Distribution in Mineral Soil Material - Method by Sieving and Sedimentation; International Organization for Standardization: Geneva, Switzerland, 1998; p 30. (22) ISO. ISO 19730:2008 - Soil Quality - Extraction of Trace Elements from Soil Using Ammonium Nitrate Solution; International Organization for Standardization: Geneva, Switzerland, 2008; p 10. (23) ISO/TS 14256−1:2003. Soil Quality - Determination of Nitrate, Nitrite and Ammonium in Field-Moist Soils by Extraction with Potassium Chloride Solution - Part 1: Manual Method; International Organization for Standardization: Geneva, Switzerland, 2003; pp 14. (24) Fontaniella, B.; Mateos, J. L.; Vicente, C.; Legaz, M. E. Improvement of the analysis of dansylated derivatives of polyamines and their conjugates by high-performance liquid chromatography. J. Chromatogr., A 2001, 919, 283−288. (25) Pinto, E.; Aguiar, A. A. R. M.; Ferreira, I. M. P. L. V. O. Influence of soil chemistry and plant physiology in the phytoremediation of Cu, Mn, and Zn. Crit. Rev. Plant Sci. 2014, 33, 351−373. (26) Houdusse, F.; Garnica, M.; Zamarreno, A. M.; Yvin, J. C.; Garcia-Mina, J. Possible mechanism of the nitrate action regulating free-putrescine accumulation in ammonium fed plants. Plant Sci. 2008, 175, 731−739. (27) Luo, J.; Fuell, C.; Parr, A.; Hill, L.; Bailey, P.; Elliott, K.; Fairhurst, S. A.; Martin, C.; Michael, A. J. A novel polyamine acyltransferase responsible for the accumulation of spermidine conjugates in Arabidopsis seed. Plant Cell 2009, 21, 318−333. (28) Sood, S.; Nagar, P. K. Changes in endogenous polyamines during flower development in two diverse species of rose. Plant Growth Regul. 2004, 44, 117−123. (29) Nishibori, N.; Fujihara, S.; Akatuki, T. Amounts of polyamines in foods in Japan and intake by Japanese. Food Chem. 2007, 100, 491− 497. (30) Bardocz, S.; Duguid, T. J.; Brown, D. S.; Grant, G.; Pusztai, A.; White, A.; Ralph, A. The importance of dietary polyamines in cell regeneration and growth. Br. J. Nutr. 1995, 73, 819−828. (31) Zoumas-Morse, C.; Rock, C. L.; Quintana, E. L.; Neuhouser, M. L.; Gerner, E. W.; Meyskens, F. L. Development of a polyamine database for assessing dietary intake. J. Am. Diet Assoc. 2007, 107, 1024−1027. (32) Ali, M. A.; Poortvliet, E.; Stromberg, R.; Yngve, A. Polyamines: total daily intake in adolescents compared to the intake estimated from the Swedish Nutrition Recommendations Objectified (SNO). Food Nutr. Res. 2011, 55. DOI: 10.3402/fnr.v55i0.5455
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DOI: 10.1021/jf505453s J. Agric. Food Chem. 2015, 63, 440−446
