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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: [email protected];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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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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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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