ter applying DPASV revealed that two different cad-
mium species were present in the voltammograms
obtained. By UV-irradiation experiments, an inor-
ganic and organic cadmium form could be detected.
Water column samples exhibited an enrichment of inorganic cadmium by depth [6].
Electrochemical detection was also used to spec-
ify Cd2+ and monomethyl-Cd+ ions in Atlantic
Ocean water. The concentrations measured resulted
in the range of about 0.5 µg L−1 for monomethyl-
cadmium ions as represented in Figure 2.5.1.1.
Thereby, humic acids did not influence the
voltammetric determination of monomethylcad-
mium. It was also discovered that biomethyla-
tion was the most probable formation process for
this methylated cadmium species [7]. This resultwas confirmed by another study of Pongratz and
Heumann who showed that monomethylcadmium
in ocean waters had a maximum concentration
C d 2 +
( − 0
. 8 7 1 V )
M e
C d + ( −
0 . 7
5 9 V )
P b 2 +
( − 0
. 6 3 0 V )
1500
1000
500
0
−1.0 −0.8 −0.6
Potential [V]
C u r r e n
t [ n A ]
Figure 2.5.1.1. DPASV (differential pulse anodic strippingvoltammogram) of a seawater sample from the AtlanticOcean with a determined MeCd+ concentration of 492 pg L−1.(Reproduced from Reference [7] by permission of AmericanChemical Society.)
in water depths up to 50 m, often correlating
well with the chlorophyll-a content represented in
Figure 2.5.1.2 [8]. In depths of about 200 m, sig-
nificant concentrations of methylated metal com-
pounds could be detected by using a voltam-
metric method, in which no chlorophyll-a waspresent. The authors concluded that marine bacte-
ria predominantly contributed to methylated met-
als at deeper water levels [8]. An overview of
trace element speciation techniques in waters con-
cerning the relationship between aquatic toxic-
ity and bioaccumulation of dissolved metal com-
pounds like those of cadmium, zinc, and other trace
elements was given by Florence, Morrison, and
Stauber [9].
The speciation analysis of Cd in biomatrices
is mainly applied to microorganisms [10– 12],animals [13–17], and phytosystems [18–31].
1.2 Microorganisms
In bacteria, a cadmium-binding form was ana-
lyzed by high-performance gel permeation liquid
chromatography combined with inductively cou-
pled plasma mass spectrometry. Cadmium was
bound to a metallothionein (MT)-like protein with
a molecular mass of about 10,000 Da [10]. A com-
bined procedure consisting of extraction, gel per-
meation chromatography (GPC), anion-exchange
chromatography, high performance liquid chro-
matography (HPLC), and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE)
was applied to purify a cadmium-binding protein
with a molecular mass of 23,000 Da in bacilli [11].
In bacteria cells of Escherichia coli , a cadmium-
binding protein with a molecular mass of about
39,000 Da was detected by using Sephadex G-100,
metal chelate affinity chromatography, and disc gel
electrophoresis in the purification procedure. Cad-mium levels were estimated by atomic absorption
trophoresis [23, 24]. Guenther and Umland inves-tigated the cadmium species existing in leaves
from unpolluted rape. Two cadmium elution ranges
were detected in the cytoplasmic fraction, one cad-
mium species greater than 80,000 Da and two cad-
mium species with a molecular mass of 4400 Da.
No relationship of one of the low-molecular mass
cadmium species to the classes of phytochelatins
or MTs could be stated [25]. Further cadmium-
binding components were detected in pea [26] and
soybean plants [27].
In the process of Rhizobium–faba bean symbio-sis, two cadmium-binding protein complexes with
molecular masses of 200,000 Da and 67,000 Da
were formed by the nodules at higher levels of cad-
mium. This result was thought to be a mechanism
by which Rhizobium– faba bean elevated resis-
tance to cadmium toxicity [28]. By conducting gel
chromatographic separation at pH 7.5, a cadmium-
binding protein in algae was analyzed. This protein
was suggested to be a dimer with a molecular
mass of 6500 Da [29]. Roots of grass produced
a MT-like protein when they were exposed tocadmium for seven days. To purify this protein
with a molecular mass of 3700 Da, a combina-
tion of anion exchanger and gel filtration was
used [30].
Tobacco plants were exposed to nonphytotoxic
levels of cadmium to examine the role of the
cadmium-binding peptides (CdBPs) in the tobacco
leaves. For that, the protoplasts and vacuoles
were isolated from leaves of cadmium-exposed
seedlings to directly determine the localization
of Cd and CdBPs. Thereby, it turned out thatthe purified vacuoles contained all of the CdBPs
and cadmium found in protoplasts. Probably, the
CdBPs were synthesized extravacuolarly [31]. A
chromatogram of purified CdBPs is presented in
Figure 2.5.1.3 and the amino acid composition of
this compound is found in Table 2.5.1.1. Amino
acid analysis showed that the main components
were γ -(Glu-Cys)3-Gly and γ -(Glu-Cys)4-Gly.
1.0
4
2
4′5
6
3
0.5
0.0
0.0
A 2 1 4
10.0 20.0
Retention time (min)
Figure 2.5.1.3. HPLC chromatogram of purified CdBPs (cad-mium-binding peptides) from tobacco leaves after acidicextraction, gel filtration on Sephadex G-50, and lyophiliza-tion. Detection was in the UV range at 214 nm. Amino acidanalysis represented in Table 2.5.1.1 showed that the predom-inant components in the system, peaks 2 and 3, consistedonly of three amino acids, cysteine, glutamate/glutamine, andglycine. Peaks 2 and 3 were identified as γ -(Glu-Cys)3-Glyand γ -(Glu-Cys)4-Gly, respectively. (Reproduced from Ref-erence [31] by permission of American Society of PlantBiologists.)
Table 2.5.1.1. Amino acid composition of purified cad-mium-binding peptides from tobacco leaves.
The investigation of the phytoavailability of cad-
mium in soils is very important to understand the
mechanisms of trace metal uptake and transport
by the plant root. The speciation of cadmium in
soils was performed by using sequential extraction
methods for dividing the particulate-bound cad-
mium into several fractions. Statistical data showed
that the two fractions “exchangeable cadmium”
and the “metal-fulvic acid-complex-bound cad-
mium” represented the plant-available cadmium
fractions to a great extent [32]. Sequential chemi-
cal extraction procedures were applied to the spe-
ciation of cadmium in different soils and sedi-
ments [33– 38]. Thereby, the respective matrices
were incubated with special aqueous solutions, likemagnesium nitrate solution for simply exchange-
able cadmium ions or sodium acetate solution for
carbonate-bound cadmium ions under determined
conditions. The respectively extracted part of cad-
mium in the single solutions was analyzed.
In soil solutions, methods based on dialysis and
ion exchange as well as special computer pro-
grams (e.g. GEOCHEM) to the elucidation of the
cadmium-binding proportions were applied [39].
Different chromatographic methods such as exclu-
sion and reversed-phase HPLC as well as ion-exchange chromatography were used to identify
the various species of cadmium in the same
matrices. Using ion-exchange chromatography,
cadmium was found to be mainly in the form of
inorganic cationic species, including the free ionic
form, Cd2+. Also, some organic and inorganic neu-
tral species were detected, especially in soils of
higher pH [40].
The activity of free Cd2+, Zn2+, and other ionsin other soil solutions was determined by using a
combination of the Donnan equilibrium and graphite
furnace AAS method. The principal species of Cd
and Zn in these matrices were free metal ions
and hydrolyzed ions [41]. In contaminated soils
containing between 0.1 and 38 mg Cd kg−1, free
Cd2+ activity in solution was determined by the
use of DPASV, assuming DPASV was sensitive
to easily dissociated inorganic ion-pairs and free
Cd2+ ions while excluding organic complexes [42].
Otto, Carper, and Larive found that Cd2+ ions werepredominantly bound to the oxygen-containing
functional groups of the fulvic acids investigated
using cadmium-113 nuclear magnetic resonance
spectroscopy. These results are environmentally
important because soil and aquatic fulvic acids
affect the bioavailability and transport of metal
ions [43]. In Figure 2.5.1.4, a well-characterized
soil fulvic acid is represented. It contains a mixture
of phenol-carboxylate polyelectrolytes and has a
molecular mass of about 1000 Da. Light scattering
data emphasized the polydisperse character of thismixture. The equilibrium behavior of metal ion
binding by this matrix was examined theoretically
and experimentally [44].
COOH
COOH
COOHO
OCH3
C
COOH COOH
COOH
OH
OH
OH
O
O
CH
OH
HO
CH2
CH2CH2C
O
O
O
CH2
CH2
Figure 2.5.1.4. A speculative sketch of a plausible component of a soil fulvic acid mixture. (Reproduced from Reference [44]by permission of Wiley-VCH.)
Figure 2.5.1.5. Percentual cadmium in the cytosols (supernatants) of commercial vegetable foodstuffs after ultra-turrax treatmentin buffer and centrifugation of the homogenates obtained. The soluble parts are now available to a further speciation analysis.(Reproduced from References [53, 55] by permission of Springer Verlag GmbH & Co KG.)
species of the two different vegetable foodstuffs
have a very similar chemical structure [46]. It is
very interesting that in the model plant Arabidop-sis thaliana , cadmium proteins of a similar size
range, compared to the high molecular mass cad-
mium species available in spinach and radish, were
detected by using a combination of GPC, PNC-
PAGE, and electrothermal atomic absorption spec-
trometry (ET-AAS) [56].
In native lettuce, the majority of cadmium
was bound to a low molecular mass protein of
about 3200 Da and to a high molecular mass
protein of more than 75,000 Da by using a
combination of ultra-turrax homogenization inbuffer and subsequent ultracentrifugation and gel
filtration of the cytosol [57, 58].
2.2 Legumes and rice
In soybeans harvested in the region where the well-
Figure 2.5.1.6. Distribution of the cadmium recovered on low molecular mass (<5000 Da) and high molecular mass(>30,000 Da) species in the cytosols of vegetable foodstuffs after cell breakdown, centrifugation, and gel permeationchromatography on Sephadex G-50. (Reproduced from Reference [53] by permission of Forschungszentrum Juelich GmbH.)
and served as a basis for the toxicological evalu-
ation of the element in food [59–61]. The speci-
ation analysis of cadmium was carried out after
extraction by the coupling of liquid chromato-graphic methods with element analytical proce-
dures in the on-line-mode. In other soybeans har-
vested from soil treated with cadmium-containing
sewage sludge, cadmium species of more than
50,000 Da were analyzed [62]. Another study con-
cerning the bioavailability of metal compounds in
food revealed that cadmium in rice grains existed
as an insoluble complex with phytic acid and
protein by using gel filtration chromatography [63].
After an extraction of polished rice grains grown
in cadmium-contaminated rice fields, cadmium
was mostly bound to glutelin in soluble fractions.This compound was analyzed by HPLC with on-
line detection by inductively coupled plasma mass
spectrometry [64]. Bean fruits were investigated
by a combined ultra- and diafiltration technique
for use in speciation analysis of protein-bound cad-
Figure 2.5.1.7. The Cd distributions in uncontaminated (1) and contaminated (2–5) spinach cytosols are shown. Plant groups 2to 5 were each contaminated with four different amounts of cadmium. The cytosols were separated by Sephacryl S-400 S gel
permeation chromatography. The most important Cd-binding form in the cytosols of all spinach plants examined was found tobe high molecular weight Cd species (fractions 32–40). The Cd elution maxima were detected in the range of about 200 kDa(fraction 37). (Reproduced from Reference [46] by permission of Springer Verlag GmbH & Co KG.)
2.3 Cereals
A major dietary source of cadmium are cere-
als [45]. In wheat, three to four cadmium-containing
substances were eluted in the range of 4000 to
>100,000 Da using GPC [66, 67]. General con-
siderations on cadmium species and other metal-
binding forms in corn and cereals were published
by Brueggemann and Ocker [68, 69].
2.4 Yeast
In brewers’ yeast, Saccharomyces cerevisiae , a
cadmium-binding protein of 9000 Da, was ana-
lyzed. The characteristics of MTs for this pro-
tein were proved, and thus these molecules were
expected to play a role in cadmium-resistance. The
cadmium compound was purified by gel perme-
ation and subsequent ion-exchange column chro-matography [70]. Another cadmium-binding pro-
tein with a molecular mass of 8000 Da was found
in the same organism [71].
2.5 Mushrooms
In the mushroom Agaricus bisporus, no MT-
like components were detected by applying a
combination of gel filtration, ion-exchange, and
affinity chromatography [72]. In a cadmium-accu-
mulating mushroom, Agaricus macrosporus , a Cd-
binding phosphoglycoprotein with a molecular
mass of 12,000 Da was isolated. This compound
was proposed to bind cadmium by its phospherine
groups and furthermore, it was not related to
MT [73]. Kruse and Lommel found two cadmium-
containing protein fractions in the mushroom
Agaricus arvensis Schff. ex Fr. with molecular
masses of 2000 and 15,000 to 20,000 Da [74].
2.6 Shellfish
In marine animals, mainly the common mussel
Mytilus edulis was investigated [75– 77]. Mussel
cytosols were purified by GPC [75– 77], reveal-
ing cadmium-binding proteins with two molec-
ular masses [75, 76]. Each of the two proteinswas further resolved into four subcomponents
by ion-exchange chromatography. As a result,
small cytosolic cadmium amounts were bound to
macromolecules of more than 50,000 Da [75, 76].
Metallothionein-like proteins of the common mus-
sel from natural populations were characterized
and partially purified. For that purpose, different
Figure 2.5.1.8. HPLC – ICP mass spectra of the cadmiumspecies from (a) uncooked and (b) cooked pig kidney;(c) equine renal MT (metallothionein). 1, 2, and 3 arehigh molecular, medium molecular and low molecular mass114 Cd-containing peaks, respectively. (Reproduced from Refer-ence [82] by permission of Royal Society of Chemistry.)
coupled plasma mass spectrometry (ICP-MS)
and fast protein liquid chromatography were
applied [77]. Oyster extracts were also separated
by GPC. In the resulted fractions corresponding
to a high molecular mass protein that was heat
labile, cadmium and zinc could be detected [78].In another study, the Cd-binding proteins in Amer-
ican oysters were investigated after exposing these
animals to Cd concentrations in a flowing seawater
system [79].
2.7 Meat
Predominantly liver and kidney are the critical
target organs for cadmium toxicity and accumu-
lation in organisms. Consequently, some methodsfor characterizing chemical forms of trace metals
in animal liver were provided. Metal speciation
was applied by a combination of supercritical fluid
extraction with on-line detection by AAS [80]. On
simulated gastric digestion of cooked pig kidney,
all of the cadmium present in solution eluted as
one peak that corresponded to ionic cadmium. A
subsequent simulated intestinal digestion of the
gastric digest under neutral conditions proved that
the retention time for the whole cadmium corre-
sponded to that of MT. The authors concluded thatthe metal had dissociated from the MT binding
sites under the acidic conditions and was rebound
at pH 7 [81].
In a study of Crews, Dean, Ebdon, and Massey,
cooked and uncooked pig kidney was compared
0
0
1.0
2.0
C d [ n g
]
10 40 50 60 70
RT [min]
Figure 2.5.1.9. Concentrations of Cd in HPLC fractionsof breast milk after sample pretreatment. The collected“MT-fractions” (56–59 min) were run on isoelectric focusingas shown in Figure 2.5.1.10. (Reproduced from Reference [83]by permission of Walter de Gruyter GmbH & Co KG.)
Figure 2.5.1.10. Distribution profile of MT (metallothionein)-standard, accepted MT-fraction (56– 59 min, Figure 2.5.1.9)obtained from human milk and citrate in an isoelectric focusing cell. In the resulted electropherogram, good correspondencewas obtained by comparing the MT-fraction from human milk with that of the MT-standard. Therefore, cadmium occurring inbreast milk is predominantly present as cadmium-metallothionein species. (Reproduced from Reference [83] by permission of
Walter de Gruyter GmbH & Co KG.)
with respect to the investigation of cadmium
species in these matrices. By a combination of
HPLC and inductively coupled plasma mass spec-
trometry, it was found that the majority of soluble
cadmium in retail pig kidney was bound to a MT-
like protein that was heat stable and that survived
in vitro gastrointestinal digestion. In uncooked
kidney, three peaks corresponding to molecular
masses of 1,200,000 Da, 70,000 Da and 6000
to 9000 Da were detected after aqueous extrac-tion [82]. The small amount of the high molecular
and the medium molecular mass cadmium species
from this matrix was presumably rendered insolu-
ble during cooking. In Figure 2.5.1.8, mass spectra
of the cadmium species of uncooked and cooked
pig kidney and equine renal MT are presented.
In all cases, the same retention time (24 min) for
the low molecular mass cadmium species (6000 to
9000 Da) were measured [82].
2.8 Milk
For the elucidation of the cadmium-binding forms
in human milk, a combination of high performance
liquid chromatography (HPLC) and voltammetry
was applied. The determination of the cadmium in
the single HPLC fractions followed after a nitric
acid digestion by DPASV [83]. The cadmium elu-
tion time was in accordance with the retention of
the MT-standard (57 min) verified by this method.
In Figure 2.5.1.9, the elution profile of cadmium
from human milk is shown. The resulted fraction
with the highest cadmium content (57 min), an
MT-standard and citrate, were further separated by
isoelectric focusing and the following distribution
profiles of standards and sample were compared to
each other [83]. As a result, in Figure 2.5.1.10 it
is shown that good correspondence was achieved
between the MT-standard and the accepted “MT-fraction” from human breast milk. Michalke and
Schramel concluded that cadmium occurring in
human milk was mainly available as MT. Further-
more, as represented in Figure 2.5.1.10, it could be
shown that cadmium was not associated with the
known zinc binding factor citrate although there
is a chemical relationship between Cd and Zn.
Both elements are bound to completely different
biomolecules in human milk [83] and also in the
most vegetable food [53].
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