Chemical form of selenium in naturally selenium-rich lentils (Lens culinaris L.) from Saskatchewan

Article abstract of DOI:10.1021/jf070681i

Lentils (Lens culinaris L) are a source of many essential dietary components and trace elements for human health. In this study we show that lentils grown in the Canadian prairies are additionally enriched in selenium, an essential micronutrient needed for general well-being, including a healthy immune system and protection against cancer. Selenium K near-edge X-ray absorption spectroscopy (XAS) has been used to examine the selenium biochemistry of two lentil cultivars grown in various locations in Saskatchewan, Canada. We observe significant variations in total selenium concentration with geographic location and cultivar; however, almost all the selenium (86-95%) in these field-grown lentils is present as organic selenium modeled as selenomethionine with a small component (5-14%) as selenate. As the toxicities of certain forms of arsenic and selenium are antagonistic, selenium-rich lentils may have a pivotal role to play in alleviating the chronic arsenic poisoning in Bangladesh.

Full text of DOI:10.1021/jf070681i

J. Agric. Food Chem. 2007, 55, 7337−7341 7337
Chemical Form of Selenium in Naturally Selenium-Rich Lentils
(Lens culinaris L.) from Saskatchewan
DIL THAVARAJAH,† ALBERT VANDENBERG,‡ GRAHAM N. GEORGE,† AND
INGRID J. PICKERING*,†
Department of Geological Sciences, University of Saskatchewan, 114 Science Place, Saskatoon,
Saskatchewan, Canada S7N 5E2, and Crop Development Centre, College of Agriculture, University of
Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8
Lentils (Lens culinaris L.) are a source of many essential dietary components and trace elements for
human health. In this study we show that lentils grown in the Canadian prairies are additionally enriched
in selenium, an essential micronutrient needed for general well-being, including a healthy immune
system and protection against cancer. Selenium K near-edge X-ray absorption spectroscopy (XAS)
has been used to examine the selenium biochemistry of two lentil cultivars grown in various locations
in Saskatchewan, Canada. We observe significant variations in total selenium concentration with
geographic location and cultivar; however, almost all the selenium (86-95%) in these field-grown
lentils is present as organic selenium modeled as selenomethionine with a small component (5-
14%) as selenate. As the toxicities of certain forms of arsenic and selenium are antagonistic, selenium-
rich lentils may have a pivotal role to play in alleviating the chronic arsenic poisoning in Bangladesh.
KEYWORDS: X-ray absorption spectroscopy (XAS); selenium; selenomethionine; selenate; lentils
INTRODUCTION
Selenium is an essential micronutrient for mammals and birds
with known functions in a variety of systems. It is required for
both intra- and extracellular glutathione peroxidases, iodothy-
ronine deiodinase, and thioredoxin reductase, as well as a
number of selenoproteins with less well-defined physiological
roles (e.g., selenoproteins P and W) (1). In all of these cases
the selenium is present as the amino acid selenocysteine, which,
where its role is understood, forms part of the catalytic active
site of the enzyme. Selenium is also said to provide protection
against various cancers including prostate cancer (2), colorectal
cancer (3), lung cancer (4), and various gastrointenstinal cancers
(5). Significantly, selenium can also protect against heavy metal
poisoning by elements such as arsenic (6, 7), mercury (8), and
cadmium (9). With arsenic, the mechanism of this protective
effect is the formation of a novel arsenic-selenium compound,
the selenobis(S-glutathionyl)arsinium ion (Figure 1) (6), which
is formed in erythrocytes (7) and excreted in bile (10). The
arsenic-selenium species is thought to be a mechanism by
which the body eliminates arsenic that necessarily involves the
loss of one atom of essential selenium for every atom of arsenic
excreted. This finding has significance for the widespread
poisoning of rural communities in Bangladesh and surrounding
areas. Here, tens of millions of people are affected by a chronic
low-level arsenic poisoning (arsenicosis) due to exposure to
Figure 1. Schematic of the structure of the selenobis(S-glutathionyl)-
arsinium ion.
toxicologically low but nutritionally high levels (up to 1-2 ppm)
of arsenic in drinking water (11). We have previously suggested
that chronic low-level arsenicosis might in fact be an arsenic-
induced selenium deficiency (6) caused by the formation and
excretion of the selenobis(S-glutathionyl)arsinium ion. More-
over, the diets of affected communities in Bangladesh are
naturally low in selenium (12), and selenium supplements have
been suggested as a treatment (6). Two independent clinical
trials involving selenite (13) and selenomethionine (14) are
currently under way. Both of these trials involve administering
selenium as a supplement in tablet form. Because any change
in daily routine tends to be opposed in rural third-world
communities, augmenting dietary selenium in currently accepted
foodstuffs might be a more acceptable method of providing such
supplements.
* To whom correspondence should be addressed. Phone: (306) 966-
5706. Fax: (306) 966-8593. E-mail: ingrid.pickering@usask.ca.
† Department of Geological Sciences.
Pulses, mainly lentil (Lens culinaris L.), chickpea (Cicer
arietinum L.), and yellow pea (Pisum satiVum L.), together with
‡ College of Agriculture.
10.1021/jf070681i CCC: $37.00
© 2007 American Chemical Society
Published on Web 08/09/2007

7338 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Thavarajah et al.
rice (Oryza satiVa L.), are staple Bangladeshi foods. Thus, Se-
enriched pulses could provide a culturally acceptable method
for increasing dietary selenium intake. Soils of the lentil-growing
region of western Canada (mainly south Saskatchewan) are
naturally rich in selenium (>1 ppm) (15), and thus, Canadian
pulses stand to be higher in selenium than their counterparts
from other regions. Here we present data on the selenium content
and chemical form of the selenium in two red lentil cultivars,
CDC Redberry (16) and CDC Robin (17), from different parts
of Saskatchewan, showing them to be an excellent source of
selenium. As the chemical form of selenium in the lentils
governs both bioavailability and projected health benefits, we
use synchrotron X-ray absorption spectroscopy (XAS) to
determine the chemical form of selenium in the whole lentil
seeds. To our knowledge, this is the first report of Se near-
edge XAS analysis in lentil seed and its chemical species at
low selenium concentration (<0.29 ppm or 3.7 µmol of Se
kg-1). The dual objectives of this study were to measure the
total selenium concentration for two red lentil cultivars (CDC
Redberry, CDC Robin) from different locations in Saskatchewan
and to identify the chemical species of selenium in the lentils.
Table 1. Total Se Concentrations for Field-Grown Saskatchewan
Lentilsa
total Se concn, ppm (value
± SD)
location
CDC Robin CDC Redberry
mean
Saskatoon
Rouleau
Davidson
Wilkie
Swift Current
mean
0.72
0.45
0.27
0.22
0.16
±
±
±
±
±
0.03
0.03
0.02
0.01
0.01
0.33
0.21
0.29
0.16
0.20
±
±
±
±
±
0.03
0.01
0.01
0.01
0.01
0.52 a
0.33 b
0.28 c
0.19 d
0.18 d
0.36 a
0.24 b
a Means within a column (total Se concentration for locations) or a row (mean
total Se concentration for cultivars) followed by different letters are significantly
different at P < 0.05. Four replicate samples were analyzed for each location and
cultivar.
ssrl.slac.stanford.edu/exafspak.html). Quantitative determination of the
different chemical forms of selenium present was carried out by
principal component analysis and least-squares fitting (20). Least-
squares fitting methods using X-ray absorption spectroscopy are now
widely accepted to be valid speciation methods for complex samples.
In early work, results from these methods were found to be in excellent
agreement with the results obtained from hydride generation atomic
absorption spectroscopy (HG-AAS) (20).
MATERIALS AND METHODS
Sample Preparation. Lentil seeds were obtained from breeding trials
conducted in 2005 by the Crop Development Centre (CDC), University
of Saskatchewan, Canada. The selected locations were Davidson, Swift
Current, Wilkie, Rouleau, and Saskatoon, covering the major lentil
growing areas in Saskatchewan. Cultivars CDC Redberry and CDC
Robin are widely grown commercially in Saskatchewan because of their
early maturity, disease resistance, high yield, and consumer acceptance.
Bangladeshi consumers tend to show a preference for red cotyledon,
extrasmall seed size (30 mg or less) cultivars such as CDC Robin. CDC
Redberry is preferred in countries where consumers desire larger red
lentils (45 mg or greater). Dry lentil seed samples (14% moisture, 20%
protein) from Davidson, Swift Current, and Wilkie were used for Se
near-edge XAS measurements. Samples of between 5 and 10 g of dry
lentil seeds were collected from each location for this study. Lentil
seeds were collected from each location with four replicates, then the
samples were homogenized, and one composite sample from each
location was used for near-edge XAS measurements.
X-ray Absorption Spectroscopy. The seeds were ground into a fine
powder (<0.5 mm sieve). Samples of between 0.2 and 0.4 mg of
homogenized seeds were carefully packed into 2 mm path length Lucite
sample holders with Mylar windows and then frozen in liquid nitrogen
prior to data collection. X-ray absorption spectroscopy experiments were
performed at the Stanford Synchrotron Radiation Laboratory (SSRL),
with the SPEAR-3 storage ring containing 90-100 mA at 3.0 GeV,
using the Structural Molecular Biology XAS beamline 9-3, which is
equipped with a Si(220) double-crystal monochromator. Harmonic
rejection was achieved by using the beamline upstream, vertically
collimating, mirror, and downstream, refocusing mirror, both Rh-coated.
The incident X-ray intensity was monitored using a N2-filled ionization
chamber, and the absorption spectrum was collected by monitoring the
Se KR fluorescence using a Canberra 30-element germanium detector
equipped with arsenic filters and Soller slits (18). Samples were
maintained between 5 and 10 K in an Oxford instruments liquid helium
flow cryostat, and the X-ray energy was calibrated with reference to
the lowest energy inflection point of hexagonal elemental Se, which
was assumed to be 12658.0 eV. Two replicate data sets were collected,
taking approximately 40 min of total time per sample. Standards
(sodium selenate, sodium selenite, selenomethionine, and Se-methylse-
lenocysteine) were purchased in powder form from Sigma-Aldrich and
were of the highest quality available. Trimethylselenonium iodide was
synthesized by treating dimethyl selenide (Strem Chemicals, Inc.) with
stoichiometric methyl iodide (Sigma-Aldrich) (19). Standards were
measured as frozen dilute aqueous solutions (5-10 mM with 30% (v/
v) glycerol, in 50 mM HEPES buffer, pH 7.0).
Total Se Concentration. For each replicate from each location, the
total Se concentration in the lentil seeds was measured following a
HNO3-H2O2 digestion (21), modified as described below. All the
chemicals used were of analytical grade or higher purity. Dried lentil
seeds were ground into a fine powder (<0.5 mm sieve), and ap-
proximately 200 mg was placed in a digestion tube with 3 mL of
concentrated HNO3. The tubes were heated to 70-80 °C to complete
the digestion, and then 0.5 mL of 30% H2O2 was added and the resulting
solution vortexed for 2 min. After digestion, the sample solution was
diluted with Millipore water to a known volume. Then all the samples
were reduced with 6 M HCl at 70 °C for at least 10-15 min in a water
bath. The total Se concentration was determined using an atomic
absorption spectrophotometer equipped with a flow-injection hydride
generator (AA220, Varian Inc., Palo Alto, CA). Measurements of total
selenium levels using this modified digestion method were validated
using NIST standard reference material 1573a (tomato leaves; [Se] )
0.054 ( 0.003 mg kg-1). Astragalus bisulcatus shoots (hydroponically
grown with 10 µM K2SeO4 solution; total [Se] ) 45 mg kg-1) were
used as a laboratory reference material (LRM) measured periodically
to ensure consistency in the methods. Additionally, the modified
digestion method was compared for randomly selected samples with
pressure digestion with a HNO3/H2O2 mixture, and both were deter-
mined using HG-AAS, yielding identical values within the errors of
the techniques. In independent measurements, ICP-MS and HG-AAS
on the same biological samples gave identical results. Finally, the edge
jump of the XAS data, which gives an estimate of total selenium, was
compared. In all cases, the results were consistent with our total
selenium analysis.
Statistical Analysis. The experiment design for total Se concentration
was a randomized complete block design with four replicates, at five
locations with two cultivars. The data from each location for two
cultivars were combined and analyzed after error variances were tested
for homogeneity. Analysis of variance was done using the general linear
model procedure (Proc GLM) of SAS version 8.2 (22). The means
were separated by Fisher’s protected LSD at P < 0.05.
RESULTS AND DISCUSSION
Total Se Concentration. We examined the total selenium
in the seeds of two lentil cultivars from five locations in
Saskatchewan. Elemental analysis indicates that the total Se
concentration is in the range 0.1-0.7 ppm, which is equivalent
to 2-9 µmol of Se kg-1 (Table 1). Significant effects of both
the cultivar and location on the total Se concentration were
observed. The interaction between the cultivar and location
X-ray absorption spectroscopy data reduction and analysis was
performed using the EXAFSPAK suite of computer programs (http://

Naturally Selenium-Rich Lentils
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7339
Figure 2. Se K near-edge X-ray absorption spectra of selected Se
aqueous standards: (a) selenate; (b) selenite; (c) selenomethionine.
shows that most of the variation in the lentil total Se concentra-
tion may be due to available soil Se. Between the two cultivars,
CDC Robin showed a significantly higher total Se concentration
than CDC Redberry (Table 1). Lentil seeds from Saskatoon
show the greatest mean total Se concentration (0.52 ppm, 6.6
µmol of Se kg-1), and those from Swift Current show the lowest
(0.18 ppm; 2.3 µmol of Se kg-1).
Selenium Near-Edge XAS. Synchrotron XAS is unique as
a tool that can provide molecular information essentially without
sample pretreatment (which can change the chemical form).
X-ray absorption spectroscopy provides information on the local
physical and electronic structure of a particular absorbing atom
(in this case selenium). The XAS spectrum can be divided into
two regions, the near-edge also known as the X-ray absorption
near-edge structure (XANES) and the extended X-ray absorption
fine structure (EXAFS). The near-edge of selenium is a unique
tool in the speciation of metals and metalloids in biological
tissues including plants. The near-edge spectrum can provide a
“fingerprint” of the type of chemical species present (23), and
quantitative information can be obtained by least-squares curve
fitting (20). Selenium shows rich variations in its near-edge
spectra as a function of chemical form (23) and has been used
to distinguish selenium chemical forms in samples such as soils
(20), bacteria (24, 25) plants (26-28), and insects (29). Figure
2 shows the Se K near-edge spectra of aqueous solutions of
selenomethionine, selenite, and selenate. The spectra of sele-
nomethionine and Se-methylselenocysteine (data not shown) are
very similar due to the close similarity of the local structure
around the selenium (H3CSeCH2R in both cases). A variety of
selenium speciation tools have been applied to investigate
selenium in biological systems including food crops (see, for
example, ref 30 and references therein), but essentially all of
these require digestion or other preprocessing, during which it
is possible that the speciation is modified or not all of the
selenium is extracted. Se K-edge XAS identifies the chemical
type rather than specific molecules and contrasts with conven-
tional methods both in requiring no sample digestion and in
probing all states (e.g., solid, solution, gaseous) of the selenium
in the sample.
Figure 3. Se K near-edge X-ray absorption spectra of (a) CDC Redberry
lentils from Davidson and (b) CDC Robin lentils from Saskatoon
(Saskatchewan, Canada). Total selenium contents are (a) 290 and (b)
-
720
µ
g kg 1. Compositions determined from the least-squares fits are
(a) 5
± 2% selenate and (b) 10 ± 2% selenate, with the balance modeled
as selenomethionine. The figure shows background-subtracted and
normalized data as filled circles, the best fit from the least-squares fit as
a solid black line, and the fit residual as a dotted line offset below.
Components of the fit, selenomethionine (dotted−dashed line) and selenate
(dashed line), are scaled according to their contributions to the fit.
Table 2. Percentage Composition of Selenium Species in Field-Grown
Saskatchewan Lentils
CDC Robin
percentage of Sea as
organic
CDC Redberry
percentage of Sea as
organic
location
selenideb
selenate
selenideb
selenate
Saskatoon
Davidson
Wilkie
90
94
90
95
±
±
±
±
2
1
2
2
10
6
10
5
±
±
±
±
2
1
2
2
90
95
90
86
±
±
±
±
2
2
1
2
10
5
10
14
±
±
±
±
2
2
1
2
Swift Current
a Selenium speciation as determined using XAS near-edge least-squares fitting.
Errors are 3 times the estimated standard deviation derived from the variance
−
covariance matrix. b Organic selenide (CH3SeCH2
selenomethionine.
−) is modeled as aqueous
transformation was used to test whether a particular standard
spectrum was a component of a spectrum of a mixture and
showed that aqueous selenate and organic selenium modeled
as selenomethionine are the major chemical forms of selenium
present in field-grown lentils from Saskatchewan.
The relative content of selenium chemical forms in the seeds
was determined by fitting the seed near-edge spectra to the
spectra of selected selenium standard species (as Figure 2).
Numerical results are shown in Table 2, and Figure 3 shows
the results of the least-squares fitting of two of the lentil samples
showing different selenate contents. Our results clearly indicated
that the field-grown lentils contain predominately (86-95%)
organic selenium similar to selenomethionine or Se-methylse-
lenocysteine with a variably minor component of selenate (5-
14%); other forms such as selenite and trimethylselenonium
Se K near-edge spectra were collected on two lentil cultivars
from four locations, and two of the spectra are shown in Figure
3. The low selenium concentrations (below 300 ppb) in the
lentils are generally considered to be challenging concentrations
for XAS measurements. Nevertheless, the Se near-edge spectrum
is clearly identified in data averaged from two sweeps collected
over approximately 40 min. The spectra of the lentil samples
show a maximum intensity at 12662.8 eV, corresponding to
organic selenium and an additional, higher energy peak whose
intensity varies in different lentil samples. Principal component
analysis indicated that two components are needed to fit the
set of spectra from each location (data not shown). Target

7340 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Thavarajah et al.
were not significant. While variations in the percentage of
selenate are observed among the measured samples, within our
limited data set these small variations do not appear to correlate
with the cultivar, location, or total selenium content.
studies. Future work will investigate the biochemistry and
cellular location of the selenium chemical form in lentils by
using XAS imaging techniques (1) as well as a determination
of the exact form of organic selenium in the lentil seeds.
In summary, our results show that lentils grown in Saskatche-
wan fields are a naturally rich source of organic selenium. Since
certain forms of selenium have an antagonistic effect on the
toxicity of arsenic, and since selenium accumulation is dem-
onstrated in lentil cultivars favored by the Bangladesh market,
it is possible that Se-rich lentils could be a whole-food solution
to the arsenicosis in Bangladesh.
Whole-Food Solution. Selenium is an essential micronutrient
which has protection against several cancers. Lentils are well-
known for their health benefits in providing several essential
dietary components (31). Our results show that lentils grown
in the Canadian prairies are additionally enriched in selenium.
There is some variation in the total selenium concentration of
the lentil seed, probably reflecting variability in available soil
selenium concentrations in Canadian prairies (Table 1).
Saskatchewan soils are naturally rich in selenium (>1 ppm),
and there is a unique potential for Se-rich pulses to be grown
in Canada even without soil supplementation, although in
selenium-deficient growing regions the concentration of Se in
the seed can be increased by application of selenium to the
growing crop or to the soil (32). The amount of selenium found
in the whole Saskatchewan lentil seed (0.16-0.72 ppm) would
be equivalent to 16-72 µg of selenium in a 100 g portion of
dry lentils, providing 29-130% of the recommended daily
amount (55 µg per day) for an adult (33). Prior to consumption,
most red lentils are decorticated and are then prepared in food
dishes in whole or split form. The decortication process would
likely enrich the selenium content of the consumed lentils by a
further ca. 10-15% by removal of the seed coat, which is mostly
cellulose fiber (34).
ACKNOWLEDGMENT
We thank B. Barlow for providing the seeds from the Crop
Development Centre, members of the Pickering/George research
group for assistance with XAS data collection, J. Ruszkowski
for synthesis of the trimethylselenonium iodide standard, and
R. Prince for reviewing the manuscript.
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Received for review March 8, 2007. Revised manuscript received June
7, 2007. Accepted June 9, 2007. Portions of this research were carried
out at the Stanford Synchrotron Radiation Laboratory (SSRL), a
national user facility operated by Stanford University on behalf of the
U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL
Structural Molecular Biology Program is supported by the Department
of Energy, Office of Biological and Environmental Research, and by
the National Institutes of Health, National Center for Research
Resources, Biomedical Technology Program. Support for this work is
provided by the Canada Research Chairs Program (I.J.P., G.N.G.), the
Province of Saskatchewan, and NSERC Canada.
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