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ISSN 1756-5901

www.rsc.org/metallomics Volume 2 | Number 12 | December 2010 | Pages 781–832

MetallomicsIntegrated biometal science

PAPERSzpunar et al.A comparative study of element concentrations and binding in transgenic and non-transgenic soybean seeds

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800 Metallomics, 2010, 2, 800–805 This journal is c The Royal Society of Chemistry 2010

A comparative study of element concentrations and binding in transgenic

and non-transgenic soybean seeds

Lidiane Raquel Verola Mataveli,abc Pawel Pohl,ad Sandra Mounicou,a

Marco Aurelio Zezzi Arrudabc

and Joanna Szpunar*a

Received 23rd August 2010, Accepted 1st October 2010

DOI: 10.1039/c0mt00040j

Transgenic and non-transgenic soybean seeds were compared in terms of total element

concentrations, behavior of elements during sequential extraction fractionation and element

bioaccessibility using an in vitro simulated gastrointestinal digestion. The analysis were carried out

by ICP-sector field-MS or size-exclusion ICP-MS (25 elements in concentrations varying from

ng g�1 to the % level). It seems that transgenic and non-transgenic soybean seeds exhibit

statistically significant differences in concentrations of Cu, Fe and Sr, which are also reflected by

element contents in water extracts and residues. Additionally, contributions of bioaccessible

fractions of Cu, Fe and other elements (Mn, S, Zn) for transgenic soybean seeds appear to be

larger than those found in non-transgenic soybean seeds.

Introduction

Soybean (Glycine max (L) Merrill) is a cultivar of great interest

in the world economy, the total crop value in 2009 exceeding in

the USA 31.7 billion dollars.1 It represents a significant source

of fatty acids and proteins for human and animal nutrition,

and is also applied for non-edible uses, including industrial

feed-stocks and production of bio-diesel.2 The major source of

this commodities is the seed, rich in proteins (B40%), oil

(B20%) and carbohydrates (B35%).3,4

Soybean seeds are also known to be rich in elements, such as

Ca, Cu, Fe, Mg, Mn, P and Zn. Calcium has several important

functions in human body, such as bone and teeth formations.

An adequate Ca intake for healthy adults is 1000–1300 mg day�1.

Copper is a component of enzymes required for Fe

metabolism. The recommended dietary allowance (RDA) of

Cu for adults is about 0.9 mg day�1.5 Iron is a constituent of

hemoglobin and various enzymes and its RDA for men and

women are 8 and 18 mg day�1, respectively. The bioaccessibility

of this element is affected mainly by the presence of phytate

and fibers.6 Magnesium is a co-factor for enzymatic systems

and its RDA varies from 240 to 420 mg day�1. Manganese is

involved in bone formation and enzymes responsible for

amino acids, cholesterol and carbohydrate metabolism, its

RDA value varies from 1.6 to 2.3 mg day�1. Phosphorus is

responsible for maintenance of pH, storage and transfer of

energy and nucleotide synthesis. The RDA for this element is

about 700 mg day�1. Finally, Zn is a component of multiple

enzymes and proteins. The RDA for this element is

8–11 mg day�1. An accurate determination of these elements in

soybean seeds can provide access to their nutritional quality.

Infestation of soybean cultures with weeds can cause

prejudice in productivity index, product quality and crop

yield. As consumption of herbicides has a high impact on

productivity costs,7 areas cultivated with transgenic soybeans

tolerant to herbicides are growing rapidly. Roundup Readys

soybean seeds represent more than a half of cultivated areas of

genetically modified organisms in the world.8 Roundup

Readys

crop lines contain a gene derived from Agrobacterium

sp. strain CP4, encoding glyphosate-tolerant enzyme so-called

CP4 EPSP synthase. Expression of CP4 EPSP synthase results

in glyphosate-tolerant crops, enabling a more effective weed

control by allowing post-emergent herbicide application.7

Effects of genetic modification are known to greatly change

the proteome in general,8 but its effects on the elemental

composition (metallome) are largely unknown. The goal of

this research was the investigation of the effects of the genetic

modification on elemental composition in non-transgenic and

Roundup Readys

transgenic soybean seeds. The differences in

multielement concentrations were addressed on the total

element level, sequential extraction fractionation and during

simulated gastrointestinal digestion. The molecular weight

distribution of elements in soybean seed water extracts was

examined as well.

Experimental

Instrumentation

An Element (Thermo Fisher Scientific, Bremen, Germany) XR

ICP-SF-MS, equipped with a demountable Fassel-type torch

shielded with a Pt ground electrode and a quartz bonnet, was

used throughout. Two different sample introduction systems

were used. Aqueous solutions were introduced with a MicroMist

glass concentric nebulizer (Glass Expansion, Australia) fitted

a CNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique etEnvironnement, UMR 5254, Helioparc, 2, Av. Pr. Angot,F-64053 Pau, France. E-mail: joanna.szpunar@univ-pau.fr;Fax: +33-5-59-40-7782; Tel: +33-5-59-40-7755

b Spectrometry, Sample Preparation and Mechanization Group –GEPAM, Institute of Chemistry, University of Campinas –UNICAMP, P.O. Box 6154, 13083-970, Campinas, SP, Brazil

c National Institute of Science and Technology for Bioanalytics,Institute of Chemistry, University of Campinas – UNICAMP,P.O. Box 6154, 13083-970, Campinas, SP, Brazil

dWroclaw University of Technology, Faculty of Chemistry,Division of Analytical Chemistry, Wybrzeze StanislawaWyspianskiego 27, 50-370 Wroclaw, Poland

PAPER www.rsc.org/metallomics | Metallomics

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This journal is c The Royal Society of Chemistry 2010 Metallomics, 2010, 2, 800–805 801

to a double-pass quartz spray chamber. A standard 1.75 mm

ID quartz injector and Pt sample (1.10 mm orifice diameter)

and skimmer (0.8 mm orifice diameter) cones were used.

Sample and standard solutions were delivered using a CETAC

(Omaha, NE) ASX-520 autosampler.

For the analysis of organic solutions, a CETAC modified

total consumption micro-flow nebulizer DS-5 attached to a

laboratory-made low-dead volume (8 mL) single-pass drain-

free glass spray chamber was used. The spray chamber was

jacketed and heated to 80 1C with a water–glycol mixture

circulating through a NesLab RTE-111 thermostat (Thermo

Fisher Scientific, Waltham, MA). A 1.0-mm ID quartz injector

for organic matrices and Pt sampler (1.1 mm orifice diameter)

and skimmer (0.8 mm orifice diameter) cones were used. O2

was added to the sample Ar flow through an additional mass

flow controller of the spectrometer. Sample and standard

solutions were introduced in a micro-flow injection mode

(mFI) with tetrahydrofuran (THF) as a carrier. mFI was carriedout using a Dionex (Amsterdam, Holland) HPLC system,

comprising of a UltiMate 3000 pump, a UltiMate 3000

autosampler and a low port-to-port dead volume mFI valve.

Reagents, solutions and materials

Deionized water from a Millipore ELIX 3 water purification

system (Molsheim, France) was used throughout. ACS grade

THF and n-hexane, CHROMASOLV LC-MS iso-propanol

and methanol, NaOH pellets and reagents used for gastro-

intestinal digestion were purchased from Sigma-Aldrich.

CHROMASOLV chloroform was supplied by Riedel-de-Haen

(Seelze, Germany). INSTRA-ANALYZED HNO3 (69–70%)

and H2O2 (30%) were obtained from J. T. Baker (Deventer,

Holland). A SPEX CertiPrep (Matuchen, NJ, USA) Claritas

PPT multi-element standard solution (10 mg g�1 of Au, Hf, Ir,

Pd, Pt, Rh, Ru, Sb and Te), a Sigma-Aldrich TraceCERT

multi-element standard solution (10 mg g�1 of Be, Cd, Co and

Mn, 20 mg g�1 of Cr, Cu, and Ni, 40 mg g�1 of Al, As, Ba, Pb

and V, and 100 mg g�1 of B, Fe, Se, Tl and Zn) and SCP

Science (Clark Graham, QC, Canada) PlasmaCAL single-

element standards (1000 mg g�1 of Ag, Bi, Ca, Hg, K, Li,

Mg, Mo, P, S, Si, Sr and Ti) were used to prepare multi-

element standard solutions in 5.0% (w/v) HNO3 for external

calibration (0, 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1000 ng g�1)

and for standard additions. A 1 mg L�1 SPEX CertiPrep

Claritas PPT standard solution (Ba, B, Co, Fe, Ga, In, Li,

Lu, Na, Rh, Sc, Tl, U, Y and K) was used to tune the

ICP-SF-MS instrument combined with aqueous solutions

introduction.

A SCP Science Conostan S-21 multi-element oil-based

standard containing 100 mg g�1 of Ag, Al, B, Ba, Ca, Cd,

Cr, Cu, Fe, Mg, Mn, Mo, Na, Ni, P, Pb, Si, Sn, Ti, V and Zn,

and Conostan 1000 mg g�1 single-element oil-based standards

of As, Bi, Li, S, Co, Hg, Sb and Sr were used to prepare multi-

element working standard solutions at 10, 20, 50, 100, 200 and

500 ng g�1 in hexane, hexane/iso-propanol (1 : 1) and chloroform–

methanol (1 : 1). A 1 ng g�1 tuning solution was prepared by

diluting SCP Science PlasmaCAL 1000 mg g�1 mono-element

standards of Ba, B, Co, Fe, Ga, In, Li, Lu, Na, Rh, Sc, Tl, U,

Y and K in THF.

A Standard reference material of non-fat milk powder

(SRM 1549) was purchased from National Institute of

Standards and Technology (NIST, Gaithersburg, MD).

Measurements

Isotopes of 25 elements (27Al, 75As, 137Ba, 209Bi, 44Ca, 111Cd,59Co, 52Cr, 63Cu, 56Fe, 202Hg, 7Li, 24Mg, 55Mn, 95Mo, 60Ni,31P, 208Pb, 32S, 121Sb, 78Se, 118Sn, 88Sr, 125Te, 66Zn) were

measured by ICP-SF-MS in medium resolution (R = 4000)

in order to resolve them from plasma-based polyatomic inter-

fering ions, especially in the case of Al, As, Ca, Cr, Cu,

Fe, Mg, Mn, Ni, S, Se, P and Zn isotopes. ICP-SF-MS

instrumental settings were optimized daily using adequate

tuning solutions (Table 1). The highest stable and reproducible

(relative standard deviations o3%) signals obtained for 7Li,115In and 238U isotopes, and the lowest 138Ba16O/138Ba

intensity ratios were considered for best analytical performance.

Mass calibration was carried out at resolutions of 300 and

4000. Mass off-set values were evaluated each time and

implemented into data acquisition methods in order to

compensate mass drifts related to magnet hysteresis. A

quantification mode was used for determining concentrations

of elements in aqueous solutions. A time-resolved mode was

Table 1 Relevant operating conditions of ICP-SF-MS with introductionof aqueous (A) and organic (B) solutions

A B

Inductively coupled plasma mass spectrometryTorch position/mm +2.20 (X), +1.10 (Y), �3.50 (Z)Forward power, W 1200 1500Plasma Ar flow rate, L min�1 16.00 16.00Auxiliary Ar flow rate, L min�1 1.00 0.90Sample Ar flow rate, L min�1 1.10 0.65O2 flow rate, L min�1 — 0.12Sample flow rate, mL min�1 300 15Injection volume, mL — 5Drain flow rate, mL min�1 450 —Ion transmissionExtraction lens, V �2000 �2000Focus lens, V �1150 �1100X-deflection lens, V +0.30 +1.15Y-deflection lens, V �2.40 �2.80Shape lens, V +130 +125SEM +2800SEM deflection +570Data acquisitionIsotopes measured 7Li, 24Mg, 27Al, 31P, 32S, 44Ca, 52Cr,

55Mn, 56Fe, 59Co, 60Ni, 63Cu, 66Zn,75As, 78Se, 88Sr, 95Mo, 111Cd, 115In,118Sn, 121Sn, 125Te, 137Ba, 202Hg,208Pb, 209Bi

Mass window (%) 125Integration window (%) 60Settling time, ms 1 (Al, Bi, Co, Cu, Fe, In, Mn, Mo,

Pb, S, Se, Sn, Te, Zn), 34 (Ni),35 (Cr), 37 (P), 39 (As), 42 (Ba, Cd),43 (Ca), 60 (Hg), 71 (Mg),300 (Li, Sr)

Sampling time, ms 20 (As, Ba, Bi, Cd, Hg, In, Li, Mo, Pb,Sb, Se, Sn, Sr, Te), 50 (Al, Ca, Co, Cr,Cu, Fe, Mg, Mn, Ni, P, S, Zn)

Number of samples per peak 20Detection mode TripleScan type EScanIntegration type AverageResolution 4000 (medium)

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802 Metallomics, 2010, 2, 800–805 This journal is c The Royal Society of Chemistry 2010

used for analysis of organic solvents with mFI-ICP-SF-MS.

Built-in Element XR ICP-MS software was used for acquisition

and integration of measured signal.

Samples and procedures

Soybean seeds. Laboratory grown transgenic (variety

MSOY 7575 RR) and non-transgenic soybean seeds

(variety MSOY 7501) were donated by Prof. Siu Mui Tsai

(CENA/USP, Piracicaba, SP).9 These seeds were originally

obtained from Monsanto, Brazil.

Total element content analysis. Samples were dried in an

oven at 60 1C to constant mass. Then, 0.2 g of dried samples

were digested in EasyPrep vessels using 6.3 mL of concen-

trated HNO3 and 0.75 mL of 30% (w/v) H2O2. Vessels were

closed and subjected to digestion at 160 1C for 25 min in a

CEM (Matthews, NC, USA) MARS microwave digestion

system. In the case of NIST SRMs, 0.2 g samples were digested

in 50 mL polypropylene (PP) tubes with 1.2 mL of concen-

trated HNO3 and 0.3 mL of 30% (w/v) H2O2 at 80 1C for 3 h

using a SPC Science DigiPrep MS heating block with time and

temperature controller. Resulting digests were cooled, diluted

to 25 mL (SRMs) or 50 mL (soybean seeds) with water and

analyzed on the total content of elements by ICP-SF-MS with

3 standard additions. Three independent replicates were made;

respective blanks were considered in final results.

Sequential extraction. In the first step, performed to separate

the water-insoluble fraction, 0.1 g of soybean samples were

sonicated in a Branson (Danbury, CT, USA) ultrasonic bath

with 2 mL of a hexane/iso-propanol mixture (1 : 1 v/v) for

30 min. This was repeated 3 times; then, supernatants were

pooled and analyzed by ICP-SF-MS using external standard

solutions prepared in the hexane/iso-porpanol mixture for

calibration. For the second step, residues from the previous

step were dried in a sample concentrator DB-3 system (Bibby

Scientific, Staffordshire, UK) to remove organic solvents and

next sonicated with 2.5 mL of water for 2 min using a Bioblock

Scientific (Illkirch, France) Vibracell 75115 ultrasonic device

at 20% of its maximum power and in a pulse mode (1-s pulses

interrupted by 1-s breaks). Supernatants were recovered using

a Hettich (Tuttligen, Germany) Universal 16 centrifuge at

3000 rpm for 10 min. This procedure was repeated two

times, and the supernatants were pooled and analyzed by

ICP-SF-MS with 3 standard additions. Finally, sample residues

were digested with 1.2 mL of concentrated HNO3 and 0.3 mL

of 30% (w/v) H2O2 at 80 1C for 3 h using 50 mL PP tubes in a

SCP ScienceDigiPrep MS heating block. Final solutions were

diluted with water to 15 mL and analyzed by ICP-SF-MS with

3 standard additions.

Three independent replicates were made for each digestion and

extraction. Respective blanks were considered in the final results.

SEC-ICP-SF-MS analysis. Chromatographic separations

were carried out using an Agilent 1100 (Wilmington, DE)

high performance liquid chromatography (HPLC) system,

comprising an HPLC pump, a degasser, an autosampler and

an UV detector, and a Superdex 75 HR 10/30 (Amersham

Pharmacia Biotech, Uppsala, Sweden) size exclusion column

(10 � 300 mm � 5 mm), with optimum fractionation range of

3–70 kDa. 100 mL aliquots of water extracts were injected into

the SEC column and chromatographic run was isocratically

made using 100 mmol L�1 ammonium acetate (pH 7.4) buffer

as eluent at 0.7 mL min�1. The eluent from the column was

directly fed into ICP-SF-MS.

Bioaccessibility test. The protocol used was described

elsewhere.10 Briefly, for gastric extraction, 5 mL of a gastric

solution (50 mg of pepsin with 5 mL of 150 mmol L�1 NaCl,

pH 2.5) was added to 0.3 g of soybean samples and incubated

at 37 1C for 4 h. For simulating intestinal extraction, pH of

sample solutions was adjusted to 7.4 with concentrated NaOH

solution and then, 10 mL of an intestinal solution, containing

solutions of 3.0% (w/v) pancreatin, 1.0% (w/v) amylase and

1.5 g L�1 bile salts, were added and incubated at 37 1C for 4 h.

Resulting supernatants were centrifuged in an Universal

16 centrifuge at 3000 rpm for 15 min and measured to

determine concentrations of elements using ICP-SF-MS with

3 standard additions. Additionally, sample residues were

digested and analyzed to provide the mass balance. Three

independent replicates were made, and respective blanks were

considered in final results.

Results and discussions

Multielement total analysis

Instrumental detection limits (DL) of studied elements

obtained with ICP-SF-MS were assessed according to 3scriterion (3 � SD of 10 repeated measures of blanks divided

by slopes). These DLs can be divided into 3 groups: from

0.0003 to 0.001 mg L�1 (As, Ba, Bi, Cd, Co, Hg, Li, Mo, Pb,

Sb, Sr and Te); from 0.02 to 1 mg L�1 (Al, Cr, Cu, Mn, Ni, S,

Se, Sn and Zn) and higher than 1 mg L�1 (Ca, Fe, Mg and P).

Precision, expressed as the relative standard deviation

(RSD) of 3 repeated measurements of multi-element standard

solutions was within 1.1 (P, Cr) to 21% (Mo).

Accuracy of ICP-SF-MS was verified by analyzing NIST

SRM 1549 (non-fat milk powder). Results of the analysis are

given in Table 2. Recoveries of measured elements were within

84–113%, indicating a good agreement between certified and

determined concentrations. Precision as RSD for analysis of

3 digested SRM samples varied from 0.4 to 10%.

Total concentrations of elements in transgenic (T) and

non-transgenic (NT) soybean seeds obtained after micro-

wave-assisted digestion are given in Table 2. According to

t-test (p = 0.005, n = 4), statistically significant differences

between T and NT soybean seeds were found for Co, Cu, Fe

and Sr. Concentrations of Co, Cu and Fe in T soybean seeds

are higher by 39, 40 and 20%, respectively, than corresponding

concentrations of these elements in NT soybean seeds. For Sr,

a higher concentration was found in NT soybean seeds; and

the difference between concentrations was of 34%. Differences

in Cu and Fe concentrations between T and NT soybean seeds

were lately established by Sussulini et al.8 when protein spots

cut from a 2D electrophoretic gel were analyzed by ETAAS. It

is important to note that elemental concentrations in

soybean seeds are dependent on various factors, including soil

characteristics, water source composition, that can affect plant

development.11 Because the majority of these factors were

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This journal is c The Royal Society of Chemistry 2010 Metallomics, 2010, 2, 800–805 803

controlled during the growth of T and NT soybean seeds

investigated here, it could be expected that differences in

concentrations that were found for Co, Cu, Fe and Sr should

be related to genetic modification.

Fractionation analysis

A two-step solvent extraction procedure was carried out to get

to know how Co, Cu, Fe, Sr and other elements (Ca, Mg, Mn,

Ni, S, Zn) measured in both soybean seeds would be distributed

between hydrophobic (by ultrasound assisted extraction with a

mixture of hexane/iso-propanol) and water soluble species (by

ultrasound assisted extraction with water). Residues left were

digested to get mass balance information. Results of this

analysis are shown in Table 3 for T and NT soybean seeds.

It can be seen that sums of element concentrations in

separated fractions and in digested residues are in good

agreement with total concentrations determined. Accordingly,

recoveries of elements varied from 80% in the case of Fe in NT

soybean seeds to 122% in the case of Zn in T soybean seeds.

Only P could be quantified in the organic fraction (B6% in

reference to total concentrations in both samples), probably

due to the presence of corresponding phospholipids.

The water fraction, mostly related to the presence of

proteins,12 contributed from 36 (Ca) to 104% (Zn) to total

concentrations of elements in T soybean seeds. For NT

soybean seeds, water soluble element species accounted for

35 (Ca) to 100% (Zn) in reference to total concentration of

these elements. In the case of Sr, it was found that only 5% of

Table 2 Results of NIST SRM 1549 analysis and total analysis from transgenic (T) and non-transgenic (NT) soybean seeds by ICP-SF-MS afteroxidative digestion (average values, n = 6 � standard deviations)

Element

SRM, mg g�1 Soybean seeds, mg g�1

Found Certified T NT

Al 1.90 � 0.18 2 o0.24a o0.24a

As o0.0009a o0.0029 o0.0009a o0.0009a

Ba — — 0.104 � 0.004 o0.002b

Bi — — o0.001a o0.001a

Ca 1.44 � 0.07c 1.3c 998 � 92 924 � 96Cd o0.0006b o0.0007 o0.0006b o0.0006b

Co — — 0.046 � 0.002 0.028 � 0.001Cr o0.15a 0.0023 � 0.0004 o0.15a o0.15a

Cu 0.61 � 0.02 0.7 9.8 � 1.0 5.9 � 0.9Fe 1.75 � 0.14 1.78 81.1 � 1.7 65.9 � 2.5Hg o0.0015a o0.0008 o0.0015a o0.0015a

Li — — o0.006a o0.006a

Mg 0.13 � 0.01c 0.12c 3979 � 407 3693 � 474Mn 0.24 � 0.01 0.26 27.9 � 0.5 24.8 � 1.7Mo 0.34 � 0.02 0.34 0.32 � 0.02 0.35 � 0.05Ni — — 0.50 � 0.06 0.34 � 0.07P 1.13 � 0.05c 1.06c 6589 � 308 6366 � 595Pb 0.019 � 0.003 0.0181 � 0.0010 0.006 � 0.004 0.009 � 0.003S 0.33 � 0.02c 0.35c 3083 � 135 2801 � 200Sb o0.0003a o0.006 0.002 � 0.001 0.002 � 0.001Se 0.011 � 0.01 0.119 � 0.007 0.11 � 0.04 o0.02b

Sn o0.031a o0.0016 o0.031a o0.031a

Sr — — 19.9 � 3.0 30.1 � 3.5Te — — o0.0003a o0.0003a

Zn 45.0 � 2.4 46.1 38.8 � 5.2 37.5 � 4.7

a Detection limit. b Quantitation limit. c Concentration in %.

Table 3 Ca, Co, Cu, Fe, Mg, Mn, Ni, S, Sr and Zn concentrations in each extracted fraction, in residues, and respective mass balances fortransgenic (T) and non-transgenic (NT) soybean seeds samples (average values, n = 6 � standard deviations)

Element

Concentration, mg g�1

Mass balance (%)Organic fraction Water fraction Residue

T NT T NT T NT T NT

Ca o23a o23a 358 � 32 325 � 31 500 � 67 485 � 45 84.7 � 1.67 88.2 � 10.5Co o0.1a o0.1a 0.038 � 0.003 0.023 � 0.002 0.007 � 0.001 0.006 � 0.001 97.6 � 7.4 103.9 � 10.5Cu o3.4a o3.4a 6.20 � 0.74 3.11 � 0.39 2.37 � 0.40 1.84 � 0.23 88.1 � 14.1 82.7 � 8.6Fe o46a o46a 41.2 � 4.3 29.3 � 5.6 32.7 � 7.2 26.9 � 7.7 92.3 � 8.0 80.0 � 6.8Mg o0.4a o0.4a 2873 � 220 2564 � 232 1058 � 192 1144 � 306 105.1 � 0.3 98.6 � 17.1Mn o0.4a o0.4a 15.2 � 1.3 11.8 � 0.7 8.35 � 1.58 9.80 � 0.35 87.3 � 11.7 86.6 � 1.4Ni o2.3a o2.3a 0.40 � 0.03 0.27 � 0.03 0.036 � 0.009 0.037 � 0.008 90.8 � 5.9 94.1 � 7.4S o2.4a o2.4a 2742 � 352 2429 � 204 778 � 245 855 � 115 121.3 � 7.7 114.1 � 1.7Sr o0.9a o0.9a 2.41 � 0.35 1.47 � 0.28 17.1 � 1.8 24.1 � 0.9 90.4 � 10.4 89.4 � 1.9Zn o4.0a o4.0a 40.5 � 2.5 37.7 � 3.5 6.48 � 1.34 9.15 � 0.34 122.4 � 8.6 121.6 � 9.8

a Quantitation limit.

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804 Metallomics, 2010, 2, 800–805 This journal is c The Royal Society of Chemistry 2010

this element can be found in water extracts of both soybean

seeds. An overwhelming part of Sr was established to be

associated with residues. These results are consistent with data

reported by Koplık et al.,13 who also found high extractabilities

of different elements, i.e., from 37% for Ca to 82% for Mo,

from soybean flour leached with a Tris-HCl buffer.

Considering concentrations of Co, Cu and Fe determined in

water extracts and Sr in residues, a statistically significant

difference between T and NT soybean seeds was assessed, as in

the case of total concentrations of these elements. Concentrations

of Co, Cu and Fe in T soybean seeds water extracts were

higher by 39, 50 and 29% as compared to respective concen-

trations found in water extracts of NT soybean seeds. In the

case of Sr, its concentration in the NT soybean seed residue

was higher by 34% as compared to the respective one for T

soybean seeds.

For further experiments, only Ca, Cu, Fe, Mg, Mn, S and

Zn, for which concentrations higher than 2 mg g�1 were

determined in water extracts, were considered.

SEC-ICP-SF-MS of water extracts

Molecular weight (MW) distribution of Cu and Fe and other

elements (Ca, Mg, Mn, S and Zn) in water extracts was

examined by SEC with UV and element specific (ICP-SF-MS)

detections. The summary of SEC-ICP-SF-MS analysis on the

Superdex 75 column is given in Table 4.

Apexes of major peaks of Cu, Mn, S and Zn were found at

22.5–23.0 min which corresponds to lowmolecular weight (LMW)

species (B2 kDa, below the column lower calibration limit).

Previous studies devoted to soybean flours13,14 showed a

similar elution behavior of Cu and Zn in water extracts. As

reported before, both elements were established to be bound

by corresponding LMW compounds. Traces of Cu and Zn

were also found to be present in medium and high molecular

weight (MMW, HMW) ranges. No HMW species of Mn were

detected in water extracts of T and NT soybean seeds as

previously reported in the literature,15 however, it was already

assessed that MW distribution of Mn among LMW, MMW,

and HMW fractions alters during aging of extracts, and that

HMW Mn related species are relatively unstable.13 In the case

of Fe, major peaks found in water extracts of soybean seeds

were eluted around 13.0 min, which corresponds to 160 kDa

(above upper column calibration limit) species. These could

be some specific metalloproteins or protein non-specific Fe

chelators of molecular weight4100 kDa, that are able to form

very stable Fe complexes in soybeans.13,16

Although morphology of chromatograms obtained with

ICP-SF-MS detection (Fig. 1) were similar for analyzed water

extracts of both soybean seeds, areas of the most abundant

peaks for Cu and Fe in T soybean seeds were 3- and 2-fold

higher, respectively, than those found in NT samples.

This could lead to the conclusion that Cu and Fe are

associated with compounds more expressed in T soybean

seeds. These results corroborate as well to the ones described

by Brandao et al.,17 who studied soybean proteome and

provided identification of 10 protein spots with different

expressions between T and NT soybean seeds.

Bioaccessibility of elements in soybean seeds

Soybeans are considered to be a great source of proteins

(B40% of their dried mass), fat (B20%) and carbohydrates

(B35%) for human and animal feeds. They are also recognized

to contain relatively high amounts of minerals that can cover

Table 4 Summary of SEC-ICP-SF-MS analysis on superdex 75column

Element

Transgenic soybean seeds Non-transgenic soybean seeds

MW, kDa Peak (%) MW, kDa Peak (%)

Ca 1.9 0.17 2.1a 0.190.01 100 8.7a,b 100

Cu 148 0.89 155c 1.154 3.2 55 6.729 1.9 29 1.113 0.89 13 0.282 93 2.1a 91

Fe 162 87 158c 8162 7.9 62 104.1 5 4.1 8.5

Mg 155 0.004 162c 0.00468 0.06 68 0.050.62 100 0.62a 100

Mn 74 14 74c 142.2 86 2.2a 86

S 57 18 62 1819 13 19 136.6 13 6.6 92.1 52 2.1a 550.08 4.1 0.07a 4.3

Zn 151 0.11 155c 0.0456 2.8 56 2.526 0.12 26 0.092.2 97 2.2a 97

a Estimation below lower limit (3 kDa) of the calibration range given

by the manufacturer. b Value expressed in Da. c Estimation above

upper limit (70 kDa) of the calibration range given by the

manufacturer.

Fig. 1 Chromatograms of Cu and Fe compounds in water extracts of

transgenic (T) and non-transgenic (NT) soybean seeds samples.

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This journal is c The Royal Society of Chemistry 2010 Metallomics, 2010, 2, 800–805 805

element deficiency in the diet.4 For that reason, it is important

to quantify and compare bioaccessible fraction of elements in

T and NT soybean seeds.

A gastrointestinal digestion approach was applied for this

purpose. Results obtained for Ca, Cu, Fe, Mg, Mn, S and Zn

are shown in Table 5 for T and NT soybean seeds. In reference

to total concentrations, contributions of elements in the

bioaccessible fraction were found to vary from 95 (Ca) to

9.2% (Fe) in NT soybean seeds; and from 88 (Ca) to 35% (Cu)

for T soybean seeds. Except for Ca and Mg, extractabilities of

elements achieved for gastrointestinal digestion are lower if

compared to respective ones obtained for water extracts. One

of the reasons for this could be the presence of fibers and

phytates in soybean seeds,18 typically in amounts of 7 and 2%

(w/v), respectively, that are well know to form insoluble

complexes with positively charged proteins and cations of

Ca, Cu, Fe, Mg, Zn in intestinal pH. In a consequence,

absorption of these elements after gastrointestinal digestion

can be decreased. As already reported,19 Fe absorption in

soybeans is relatively low. Even after removal of phytates,

which slightly enhance absorption of this element, soy proteins

themselves are still inhibitory to Fe absorption, likely due to

the presence of some Fe-binding peptides.20

Concentrations of bioaccessible fractions for Cu, Fe, Mn, S

and Zn were established to be different for both soybean seeds.

In the case of Cu, Mg, Mn and Zn, bioaccessible fractions of

these elements could be related to LMW species, such as some

organic acids (ascorbic, succinic, mallic). These species have

already been recognized to increase bioavailability of metals in

soybean and soy products.18

Conclusions

Transgenic and non-transgenic soybean seeds show differences

in concentrations of Co, Cu, Fe and Sr which are also

reflected by element contents in water extracts and residues.

Although element distribution profiles in water extracts of

T and NT soybean seeds are similar, Cu and Fe in transgenic

soybean seeds exhibit higher areas of major size exclusion

chromatographic peaks. Bioaccessible fractions of such

elements as Cu, Fe, Mn, S and Zn are larger in transgenic

soybean seeds.

Acknowledgements

Lidiane R. V. Mataveli and Marco A. Z. Arruda are grateful

to Coordenacao de Aperfeicoamento de Pessoal de Nıvel

Superior (CAPES) and Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo (FAPESP) for fellowship and financial

support. The authors are thankful to Marcelo A. O. da Silva

for helping with the cover artwork.

Notes and references

1 SoyStats, The American Soybean Association, available athttp://www.soystats.com.

2 J. M. Saz and M. L. Marina, J. Sep. Sci., 2007, 30, 431–451.3 M. Hajduch, A. Ganapathy, J. W. Stein and J. J. Thelen, PlantPhysiol., 2005, 137, 1397–1419.

4 Y. Yip, K. Chan, P. Cheung, K. Poon and W. Sham, Food Chem.,2009, 112, 1065–1071.

5 Food and Nutrition Board of the Institute of Medicine, StandingCommittee on the Scientific Evaluation of Dietary ReferenceIntakes, Subcommittee on Interpretation and Uses of DietaryReference Intakes, DRI Dietary Reference Intakes. Application inDietary Assessment, National Academy Press, Washington, DC,2000.

6 A. G. Gharib, S. G. Mohseni, M. Mohajer and M. Gharib,Radiochem. Radioanal. Letters, 2006, 207, 209–215.

7 T. Funke, H. Han, M. L. H. Fried, M. Fischer and E. Schonbrunn,Proc. Natl. Acad. Sci. U. S. A., 2006, 35, 13010–13015.

8 A. Sussulini, G. H. M. F. Souza, M. N. Eberlin andM. A. Z. Arruda, J. Anal. At. Spectrom., 2007, 22, 1501–1506.

9 C. A. Moldes, L. O. Medici, O. S. Abrahao, S. M. Tsai andR. A. Azevedo, Acta Physiol. Plant., 2008, 30, 469–479.

10 S. Mounicou, J. Szpunar, R. Lobinski, D. Andrey and C. J. Blake,J. Anal. At. Spectrom., 2002, 17, 880–886.

11 J. Naosuka and P. V. Oliveira, J. Braz. Chem. Soc., 2007, 18,1547–1553.

12 A. Sussulini, J. S. Garcia, M. F. Mesko, D. P. Moraes, E. M.M. Flores, C. A. Perez and M. A. Z. Arruda, Microchim. Acta,2007, 158, 173–180.

13 R. Koplık, H. Pavelkova, O. Mestek, F. Kvasnicka andM. Suchanek, J. Chromatogr., B: Anal. Technol. Biomed. LifeSci., 2002, 770, 261–273.

14 J. Schoppenthau, J. Nolte and L. Dunemann, Analyst, 1996, 121,845–852.

15 S. Yoshida, Agric. Biol. Chem., 1988, 52, 2149–2153.16 S. Yoshida, Agric. Biol. Chem., 1989, 53, 1071–1075.17 A. Brandao, H. S. Barbosa and M. A. Z. Arruda, J. Proteomics,

2010, 73, 1433–1440.18 M. A. Garcia, M. Torre, M. L. Marina and F. Laborda, Crit. Rev.

Food Sci. Nutr., 1997, 37, 361–391.19 A. S. Sandberg, Br. J. Nutr., 2002, 88, S281–S285.20 S. Ambe, J. Agric. Food Chem., 1994, 42, 262–273.

Table 5 Ca, Cu, Fe, Mg, Mn, S and Zn concentrations in bioaccessible fractions, residues, and recoveries related to total concentrations oftransgenic (T) and non-transgenic (NT) soybean seeds samples (average values, n = 3 � standard deviations)

Element

Concentration, mg g�1

Recovery (%)Bioaccessible fraction Residue

T NT T NT T NT

Ca 824 � 114 817 � 72 160 � 17 193 � 8 105 � 13.2 117 � 12.7Cu 3.52 � 0.13 1.85 � 0.02 4.77 � 0.57 3.39 � 0.20 80 � 8.1 85 � 8.6Fe 29.1 � 2.4 5.99 � 0.26 52.6 � 3.8 59.0 � 9.1 101 � 6.3 100 � 6.8Mg 1690 � 248 1318 � 78 492 � 53 511 � 20 91 � 11.9 75 � 7.6Mn 12.2 � 0.1 10.5 � 0.3 7.67 � 0.90 7.77 � 0.33 71 � 9.0 73 � 10.0S 1322 � 56 1961 � 76 958 � 64 1476 � 68 73 � 8.8 107 � 9.6Zn 22.1 � 0.1 19.6 � 0.4 15.7 � 0.6 22.6 � 0.8 92 � 2.3 107 � 7.6

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