UNIVERSITY OF CINCINNATI
Date:August 31, 2006______
I, Allison Nichole Krentz______, hereby submit this work as part of the requirements for the degree of: Master of Science in: Chemistry It is entitled: Investigation on the Chemical Association of Important Elements in Seaweed Using SEC-ICP-MS
This work and its defense approved by:
Chair: _Joeseph Caruso ______Patrick Limbach______Bruce Ault______
Investigation on the Chemical Association of Important Elements in Seaweed Using SEC-
ICP-MS
A thesis submitted to the
Graduate School
of the University of Cincinnati
In partial fulfillment of the
Requirements for the degree of
MASTER OF SCIENCE
In the Department of Chemistry
of the College of Arts and Sciences
By
ALLISON NICHOLE KRENTZ
B.S., Chemical Technology
University of Cincinnati, Cincinnati, Ohio, June 2004
Committee Chair: Dr. Joseph Caruso
ii Abstract
Seaweeds are found in oceans throughout the world and have been consumed by humans
for centuries. There are many commercially available varieties of edible seaweed known for
their medicinal properties and food values. However, seaweed can concentrate exceptionally
high amounts of heavy elements available from sea, some of which are toxic. Seaweeds have
also been used as bioindicators to monitor environmental pollution and for decontamination of
some toxic elements from wastewaters [1].
In this study, multielemental speciation analysis of essential and toxic elements in
commercially available brown seaweeds such as hiziki and wakame is performed using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry in order to examine the association of trace elements and metals with different molecular weight extracts of seaweed. Total elemental determination using ICP-MS revealed that these seaweeds have high amounts of elements such as As, Sr, Hg, and Zn. Preliminary speciation studies using SEC-ICP-
MS reveal that major type of biomolecules responsible for association of these elements in seaweeds are polysaccharides such as alginic acid and fucans. Other biological macromolecules such as proteins and polyphenols also contribute to some extent towards accumulation of trace elements in seaweeds. Other studies have examined trace element concentrations in various extracts of seaweeds but none have analyzed the different trace elements and metals associated with different molecular weight extracts.
iii iv Acknowledgements
I would like to thank the Department of Chemistry for the opportunity to study chemistry and grow as a scientist and a person. I would like to thank my advisor and mentor Dr. Joseph
Caruso for welcoming me into his research group. The amount of support and encouragement I received from Dr. Caruso was more than I could have ever wished for. Dr. Caruso is not just an intelligent and renowned professor, but he is also a kind and caring individual. I would also like to thank my other committee members Dr. Bruce Ault and Dr. Pat Limbach for their input and support.
I would also like to thank all of my groupmates, especially Dr. Monika Shah. Monika was a wonderful mentor to me. My groupmates have become great friends who were always there when I needed to vent, a shoulder to cry on, or just help figuring out why something was not working.
I would like to thank Dr. Rajiv Soman who was a mentor to me during my undergraduate years at the University of Cincinnati College of Applied Science. Dr. Soman always pushed me to do my best and he encouraged me to attend graduate school.
I would also like to thank my family. My husband Mike and my son Evan have been very supportive of me throughout my college education. Without Mike’s support, I never would have graduated with an undergraduate degree, never less a master’s degree. I’m appreciative of
Evan for understanding that sometimes Mommy had work to do on the computer, and playing quietly in his room until I finished. I also want to thank Evan for not crying the first time I dropped him off at daycare when I started graduate school (even though I was bawling my eyes out). I want to thank my parents Jim and Debbie Bruning for instilling a good work ethic in me and teaching me the importance of education. I would like to thank my parents and my in laws
v Steve and Ann Krentz for babysitting when Mike was out of town so I could study or work late.
vi Table of Contents
Abstract………………………………………………………………………………………….ii
List of Tables……………………………………………………………………………………vi
List of Figures…………………………………………………………………………………..vii
Introduction……………………………………………………………………………………....1
Experimental……………………………………………………………………………………..9
Instrumentation………………………………………………………………………….9
Reagents and Standards………………………………………………………………..10
Total Element Analysis…………………………………………………………………11
Extraction Procedures………………………………………………………………….12
Chromatographic Conditions………………………………………………………….14
Results and Discussion………………………………………………………………………….15
Total element determination…………………………………………………………...15
Chromatographic Speciation Analysis………………………………………………...16
Conclusions and Future Work…………………………………………………………………28
References……………………………………………………………………………………….31
vii List of Tables
Table Page
1. Chromatographic Conditions………………………………………………………………...9
2. Instrumental Parameters and Conditions………………………………………………….10
3. Microwave digestion parameters……………………………………………………………11
4. Total elemental concentrations in µg g -1…………………………………………………...16
5. Approximate Retention times of inorganic elements bound with higher molecular weight
species……………………………………………………………………………………19
6. Crude total elemental concentration in µg g-1 of proteinase K …………………………...21
7. Total elemental concentrations of various hiziki extracts in µg g-1……………………….25
8. Total elemental concentrations of various wakame extracts in µg g-1……………………26
viii List of Figures
Figure Page
1. Chemical structure of alginate……………………………………………………………….2
2. Chemical structure of disaccharide repeating unit extracted from A. nodosum………….3
3. Polyphloroglucinol containing biphenyl and ether linkages……………………………….4
4. SEC-ICP profiles of various elements in protein extracts………………………………...18
5. Fe chromatogram for protein extracts and proteinase K…………………………………20
6. SEC-ICP profiles of various elements in polyphenol extracts…………………………….22
7. Strontium chromatogram for Hiziki alginate extracts…………………………………….23
8. SEC-ICP profiles of various elements in fucoidan extracts……………………………….24
ix Introduction
Seaweeds are found in oceans throughout the world. Seaweeds are extensively used as
food by people throughout the world, particularly Japan and Korea, and are also consumed in the form of dietary supplements. Extracts of seaweed are also used throughout the world in food production and can be found in foods such as meat products, baked goods, dairy items, and salad dressings [2].
The elemental content of the seaweed depends on the type of seaweed and the environment in which they are found. The iodine enrichment factor in Laminaria japonica
(Kombu) reaches 106 [3]. Strontium, calcium, potassium, magnesium, and sodium have been reported as major elements present in various brown seaweeds[4]. High arsenic content has been reported in various brown seaweeds, particularly Hizikia fusiforme [4-6]. In one study the daily dietary intake values for arsenic, iodine, mercury, and cadmium could be exceeded based on the
Japanese algae consumption estimates[6]. There are many studies that have been published on the determination of trace elements in seaweeds[1, 3-14]. The occurrence of high metal content in seaweed is of considerable importance, because there may be a possible toxicological hazard.
Certain elements when concentrated in living organisms, are regarded as human toxins.
Although few have extracted the different constituents of seaweed for analysis, and none have analyzed the different trace elements and metals associated different molecular weight extracts.
Since bioavailability and properties (beneficial and toxic ) of different elements depend upon the physiochemical form in which they are found, it is desirable to determine the chemical form in which they are present.
1 There are three types of seaweeds that are classified by the pigments present, red, brown,
and green[15]. Brown seaweeds readily accumulate metals and other trace elements from their
environment. There are many important chemical constituents of brown seaweed. Some
components of brown seaweed which may play an important role in the accumulation of trace
elements are proteins, polyphenols, and anionic polysaccharides such as alginate and fucoidan.
Alginate or alginic acid is a major constituent in all brown seaweeds that have been
investigated. Alginate is a polysaccharide made up of only uronic acids. Alginate is a
heteropolymer that contains two different units, D-mannuronic acid and L-glucuronic acid[15].
Alginates have the unique property of thickening and gelling and in water they thicken and swell,
increasing its viscosity. Aliginic acids have the ability to jellify, thicken, emulsify, and stabilize
various food and industrial products. For these reasons the main fields were alginates are used
are the food, textile, cosmetic, paper, and pharmaceutical industries.[16] The chemical structure
for alginate is shown in Figure 1. The M represents the mannuronic acid units and the G
represents glucuronic acid units.
Figure 1. Chemical structure of alginate taken from http://www.genialab.de/inventory/alginate.htm.
The carboxyl groups of the uronic acids are thought to participate in ion exchange with metal ions. Although the term ion exchange is often used to describe the biosorption of heavy metals and other cations in seaweed, it is not meant to explicitly identify the binding mechanism.
2 Ion exchange is used as a term to describe previous experimental observations in which most
alginate is either protonated or containing light metal ions such as K+, Na+, and Mg2+, which are
released upon binding of a heavy metal cation[17]. The actual binding mechanisms may range
from electrostatic or London- van der Waals forces to ionic or covalent binding. The salts of
alginic acid with monovalent ions are soluble, whereas those with divalent or polyvalent metal
ions with the exception of Mg2+ and the acid itself are insoluble[17].
Fucoidans or sulphated fucans are polymers that are formed by sulfated α-L- fucose that are present in the cell wall of brown algae. Fucoidan polymers also may contain small proportions of other sugars such as galactose and mannose[18]. Fucoidans have not been found
in other algae or plants[19]. It is thought that metals and other elements may interact with the
sulfate groups of the fucoidan. Figure 2 shows the disaccharide repeating unit extracted from a
fraction of brown algae.
.
Figure 2. Chemical structure of disaccharide repeating unit extracted from A. nodosum taken from Berteau and Mulloy[19].
Polyphenols occur in seaweeds as phlorotannins, specifically phloroglucinols[15]. These
phloroglucinols occur as a mixture of phenyl and ester linkages. In seaweed, they are found in a wide range of molecular weights from 126Da to 650kDa[7]. The aromatic rings of the
3 polyphenols may chelate metals and other trace elements found in the plants[7]. Figure 3 shows an example of a chemical structure of polyphloroglucinol with both biphenyl and ether linkages.
Figure 3. Polyphloroglucinol containing biphenyl and ether linkages taken from Lobban and Wynne[15].
Proteins consist of amino acids and provide structural support to the plant. There are more than a thousand proteins found in seaweeds[8]. These proteins may contain trace elements.
Munilla et al. analyzed several types of seaweeds that are consumed as food for total determination of various elements by ICP-AES. High levels of strontium were found in three of the four seaweeds analyzed with concentrations of those seaweeds ranging from 1230-4370
µg/g[4].
The zinc and cadmium concentrations in one brown seaweed was studied in Sepetiba Bay in Brazil by Amado et al. to contribute to monitoring heavy metal contamination. The seaweeds from the contaminated area were compared to seaweeds from a non-contaminated area. The concentrations of Zn and Cd in seaweed from the contaminated area were 25 and 4 times higher in than the seaweed from the non-contaminated area. The samples were also analyzed by transmission electron microscopy, and dense granules were found near the cell walls. These granules were found to be zinc. The sulfated fucans and alginates were thought to act as an ionic filter protecting cells from high concentrations of heavy metals by precipitating the metals in the cell wall[1].
4 Hou used various chemical separation techniques to remove constituents of the plants in order to analyze them for various metals. Protein, pigment, polyphenol, alginate, and fucoidan extractions were carried out and the material extracted was analyzed by neutron activation analysis for several metals. The concentrations of alkaline earth metals were higher in the alginate extracts than those in the original algae[8]. The concentrations of Fe, Zn, and Cr in the protein extracts were much higher than those of the original algae[8].
Shah et al. studied the speciation of iodine in brown seaweeds. Extractions were carried out on the samples, and several chromatographic techniques including size exclusion chromatography, ion chromatography, and reverse phase chromatography, coupled to ICP-MS was carried out in order to speciate the iodine in the seaweed[9].
In this study, the extraction procedures developed by Hou et al. are followed and the extracts are analyzed to understand the molecular weight distribution of various toxic and essential elements in the commercially available seaweeds. Also, an attempt is made to prove the association of these elements with the various biological macromolecules using molecule specific enzymes. For this purpose, size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry is employed. A brief description of both techniques is as follows.
Size exclusion chromatography is the variation of high performance liquid chromatography in which separation takes place on the basis of the size or the hydrodynamic radius of the molecules. The role of size exclusion chromatography in speciation studies is to provide preliminary information about the distribution of elements in a sample. It gives the size estimate of metallospecies by separating them on the basis of their molecular weight. Molecules that are larger than the pore size of the packing take short paths through the stationary phase and
5 are consequently eluted first. Molecules having diameters that are smaller than the pores elute last because they are able to penetrate into the pores, and are retained for the greatest amount of time. Since the separation is based on the physical impedance of the analytes rather than chemical interactions, interactions between the analyte and the stationary phase are undesirable.
Size exclusion chromatography has been used in many speciation studies to understand the molecular weight distribution of elements with inorganic and organic forms[9, 20]. Due to its limited peak capacity complete separation of compounds in complex biological matrices cannot be obtained by size exclusion chromatography, however preliminary information obtained is useful in terms of choosing other kinds of chromatography to obtain the complete information on individual size-exclusion peak.
Coupling of size-exclusion chromatography with element specific detector such as inductively coupled plasma mass spectrometry is relatively simple and straightforward. The outlet of the UV detector was connected online to the liquid sample inlet of the ICP-MS nebulizer using 300 mm long by 0.25 mm id PEEK tubing. Nebulization of liquid sample into fine aerosol takes place at sample introduction system of the ICP-MS and is transported to through the ICP torch to the plasma. The torch consists of three concentric quartz tubes through which argon gas flows. The flowing argon is ionized by a spark from a Tesla coil. The end of the torch is surrounded by an induction coil that is powered by a 27 MHz radio frequency generator. The argon ions and free electrons collide as a result of the oscillating high-energy field produced by the induction coil forming a stable plasma. As a result of the high temperatures and energies present inside the plasma a sample is rapidly vaporized, atomized, and ionized after being transported into the central channel of the plasma.
6 A series of ion lenses directs the ions into the intermediate stages of the instrument. The
instrument may contain a collision/reaction cell, where spectral interferences can be removed. A
collision or reaction gas is passed through the cell where the gas collides/reacts with polyatomic species. In the collision mode helium gas is used and polyatomic interferences are removed based on their physical size, not on a specific reaction with a reaction gas. Polyatomic interferences will collide with the helium cell gas atoms more frequently than the analyte ions
since all polyatomic interferences are larger than the analytes they interfere with. As a result, the polyatomic ions will lose more energy and will be prevented from entering the mass analyzer by a positive discrimination voltage.
From the intermediate stage the ions then pass into the analyzer vacuum stage where they are separated according on their mass to charge ratio. The most common type of mass analyzer utilized in ICP-MS is the quadrupole. Other types of mass analyzers that are used are the sector field and the time-of- flight. The quadrupole consists of four parallel cylindrical rods that serve as electrodes. The opposite rods are connected electrically with one pair being attached to the positive side of a dc source and the other pair being attached to a negative terminal. Variable radio-frequency ac potentials that are 180 degrees out of phase are also applied to each pair of rods. The mass spectrum is obtained by applying a potential and accelerating the ions into the space between the rods. The ac and dc voltages are increased simultaneously while maintaining a constant ratio allowing different masses to be selectively allowed to pass through to the filter.
These voltages can be scanned very rapidly, allowing the elemental mass range to be scanned very quickly.
The goals of this study are to gain a greater understanding of the molecular weight distribution of various toxic and essential elements in extracts of commercially available
7 seaweeds. Also, attempts are made to suggest the association of these elements with the various biological macromolecules using molecule specific enzymes. For these purposes, size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry is employed. Beyond this preliminary study, a more specific look could be taken at different components of seaweed and the specific nature of binding with the elements of interest.
8 Experimental
Instrumentation
Chromatographic separations were performed using an Agilent 1100 liquid chromatographic system (Agilent Technologies, Palo Alto CA, USA) equipped with an HPLC binary pump, an auto sampler, vacuum degasser, a thermostated column compartment and a diode array detector. The chromatographic column used was a Superdex 75 HR (10mm x
300mm x 13um particle size) column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) for size-exclusion chromatography. Chromatographic conditions are summarized in Table 1.
Table 1. Chromatographic Conditions
Mobile phase 30mM Tris-HCl buffer pH 7.5 Column Superdex 75 HR 10/30 UV Wavelengths monitored 280 nm, 254 nm, and 240 nm Flow rate 0.6 mL min-1 Injection Volume 100 µL
An Agilent 7500ce ICP-MS (Agilent Technologies, Tokyo, Japan) equipped with a micromist nebulizer and a Peltier cooled spray chamber (2oC) and a shielded torch system was used for elemental detection. The outlet of the UV detector was connected online to the liquid sample inlet of the ICP-MS nebulizer using a 300 mm long by 0.25 mm PEEK tubing. The instrumental operating conditions are summarized in Table 2.
9 Table 2. Instrumental parameters and conditions
Forward power 1500W
Plasma gas flow rate 15.0 l min-1
Auxiliary gas flow rate 1.21 l min-1
Carrier gas flow rate 0.96 l min-1
Dwell time 0.1 s per isotope
Isotopes monitored 24Mg, 53Cr, 57Fe, 59Co, 63Cu, 66Zn,
75As, 82Se, 88Sr, 95Mo, 107Ag, 116Cd,
202Hg, 208Pb
Collision Cell (He) flow rate 2.0 ml min-1
Reagents and Standards
All reagents used were analytical grade reagents and presence of iodine was not detected in the working range. All solutions were prepared in 18 MΩ cm doubly deionized water generated by a NanoPure treatment system (Barnstead, Boston, MA, USA). The following reagents were purchased from Sigma (Sigma-Aldrich Co, St-Louis, Mo, USA): tris
(hydroxymethyl) aminomethane (TRIS) and sodium dodecyl sulfate (SDS). Hydrochloric acid was obtained from Fisher (Fairlawn, NJ, USA). HPLC grade methanol, ethanol, and acetone
(Fisher Scientific, Pittsburgh, PA, USA) were used throughout. For the determination of total elemental concentrations, working solutions were prepared daily by appropriate dilution of the
1000 µg ml-1 silver, 1000 µg ml-1 arsenic, 1000 µg ml-1 chromium, 1000 µg ml-1 cobalt, 1000 µg
10 ml-1 copper, 1000 µg ml-1 iron, 1000 µg ml-1 mercury, 1000 µg ml-1 magnesium, 1000 µg ml-1 molybdenum, 1000 µg ml-1 lead, 1000 µg ml-1 selenium, 1000 µg ml-1 zinc standard solutions
obtained from High Purity Standards (Charleston, SC, USA) and 10 µg ml-1 cadmium and 10 µg
ml-1 strontium standard solutions from SPEX CertiPrep, Inc. (Metuchen, NJ, USA). Nitric acid
(Suprapure) 68% from Pharmaco and hydrogen peroxide 30% from Fisher Scientific were used
for sample digestion.
Total Element Analysis
Dried samples of Hizikia fusiforme (hiziki) and Undaria pinnatifida (wakame) were obtained from local Asian food stores. The samples were ground in a coffee grinder. Samples analyzed for totals were digested in a CEM Discover microwave system (Matthews, NC) by placing 50 mg of sample in a microwave vial along with 500 µL of concentrated nitric acid, 500
µL doubly deionized water, and 500 µL 30% hydrogen peroxide. The microwave parameters used are listed in Table 3. The digested samples were diluted to 5 mL total volume before being analyzed by ICP-MS.
Table 3. Microwave digestion parameters.
Power 150W
Ramp 2:00
Hold 5:00
Temperature 150ºC
Pressure 250 psi
11 Extraction Procedures
Protein Extraction
The procedures used for protein extractions were adapted from Hou [8]. Seventy-five mg
of each sample was leached several times with acetone to leach pigments from the matrix. The
residue was then treated with a solution of 0.5% caffeine and 1% CaCl2, to precipitate
polyphenols and alginates from the solution. The residue was then treated with a 30 mM tris
solution adjusted to pH 7.5 with HCl, containing 0.5% caffeine, 1% CaCl2, 1% SDS, 0.5% NaN3,
and 50 mg PVPP. The 1% SDS was added in order to solubilize water insoluble proteins. The
NaN3 was used to prevent microbial growth in the solution. The PVPP was used to bind with polyphenols in the solution[8]. After stirring for 48 hours in the solution, the mixture was centrifuged and the supernatant was removed. Proteins were precipitated by the addition of acetone until the solution was 80% acetone. The solution was centrifuged and the supernatant was removed and the protein residue was redissolved in tris-HCl solution.
High M.W. species (most likely proteins) were digested by employing enzymatic hydrolysis with
Proteinase K from Sigma-Aldrich (St. Louis, MO, USA).[21] About 1 ml of Proteinase K
solution (50 mg ml-1) was added to 1 ml of protein extract. The solution was kept at constant temperature of 50oC with continuous stirring for 12 h. The final solution was filtered with .45µm
filters. A volume of 100µl of the resulting solution was injected into SEC-ICP-MS system.
Polyphenol Extraction
Polyphenolic compounds were extracted from the ground sample by first leaching with
ethanol several times and collecting the extract. The procedures followed were adapted from Hou
[8]. Then the ethanol was evaporated on a warm hot plate under a stream of nitrogen until a
12 small volume remained (about 300 µL). The polyphenolic extract was then placed onto a PVPP
column. Polyphenols should be absorbed by the PVPP, allowing for the pigments also present in
the extract to be eluted with acetone. Then polyphenols were eluted from the column with
methanol. The methanol was evaporated on a warm hotplate under a stream of nitrogen, then the
extract was redissolved in 1mL of doubly deionized water.
Alginate and Fucoidan Extraction
The procedures followed for alginate and fuicoidan extractions were adapted from Hou
[8]. Alginate and fucoidan were extracted using hot water and stirring for several hours. The
still warm solution was centrifuged and the supernatant was removed. Ethanol was added to
30% of the total volume to precipitate the alginate. The solution was centrifuged and the
supernatant was removed. The remaining residue was redissolved in 1 mL of doubly deionized
water. The supernatant that was collected was treated several times with chloroform to 20% of
the total volume several times to remove proteins. Each time the solution was shaken,
centrifuged, and then the supernatant was removed. After the protein was removed, the solution
was treated with ethanol to 70% to precipitate fucoidan. The solution was centrifuged and the
supernatant was removed. The residue was then redissolved in 1 mL of doubly deionized water.
High M.W. species thought to be alginate were digested by employing enzymatic hydrolysis
with Alginate Lyase from flavobacterium sp. from Sigma-Aldrich (St. Louis, MO, USA). About
0.15 ml of alginate lyase solution (1 mg ml-1) was added to 0.5 ml of alginate extract. The solution was kept at constant temperature of 30oC with for 30 minutes. The reaction was terminated by the addition of 0.2 ml of 2.25M NaOH. The final solution was filtered with .45µm
filters. A volume of 100µl of the resulting solution was injected into SEC-ICP-MS system.
13
Chromatographic Conditions
The various extracts were analyzed by size exclusion high performance liquid
chromatography coupled to inductively coupled mass spectrometry. The size exclusion column
used is a Superdex 75 HR 10/30 with a molecular weight range from 70 kDa to 3 kDa. The size-
exclusion column was calibrated using the following protein standards: albumin (66 kDa);
myoglobin (17.6 kDa); aprotinin (6.5 kDa); and Vitamin B12 (1.35 kDa). The HPLC was operated at a flow rate of 0.6 mL/minute and the UV absorbance was monitored at the following wavelengths: 280 nm, 254 nm, and 240 nm. The mobile phase used was a 30mM Tris-HCl buffer with a pH of 7.5. The injection volume was 100 µL in each case.
14 Results and Discussion
Total element determination:
Instrumental conditions were optimized to permit proper determination of the elemental
distribution. The ICP-MS instrument was tuned by using a multielemental standard solution
containing Li, Y, Mg, Co, and Tl at a concentration of 10 µg L-1 of each element. The collision cell gas was optimized by choosing a flow rate which reduced the baselines of a 5% nitric acid solution without diminishing the response from a 50 ng L-1 multielement standard.
The total elemental analysis the results are shown in Table 3. Both the hiziki and wakame had high concentrations of Mg, 3,500 ppm and 4,160 ppm, respectively. This was not surprising due to the fact that sea water has been found to contain 1,290 ppm magnesium in one study[22]. Strontium, the other alkaline earth metal which was monitored, also had high concentrations of 1,230 ppm and 689 ppm for hizki and wakame, respectively. Strontium is less abundant in seawater, with concentrations found to be 8 ppm in the same study[22]. The arsenic concentrations in hiziki and wakame were found to be 128 ppm and 34.5 ppm respectively.
Some arsenic concentrations have been found to be between 2.1 ppm and 3.2 ppm in a separate report of trace element concentrations of seawater samples[10]. The high arsenic concentrations in seaweed are not that surprising because hiziki is known to contain relatively large amounts of inorganic arsenic[11]. Since the elemental concentration of the seawater in which the samples were grown is unknown, it is difficult to draw further conclusions from the total elemental analysis alone. The results presented in table 3 are consistent with the elemental concentrations reported in brown algae in the study reported by Munilla et al.[4].
15 Table 4. Total elemental concentrations in µg g -1
Wakame Hiziki Mg 4,160 3,500 Cr 1.1 2.15 Fe 279 292 Co 0.182 0.367 Cu 3.65 2.35 Zn 24.3 8.3 As 34.5 128 Se 11.9 5.43 Sr 689 1,230 Mo 0.212 0.414 Ag 0.952 0.072 Cd 1.28 0.564 Hg 2.33 1.42
Pb 1.07 7.61
Chromatographic Speciation Analysis: Association of various elements with biological macromolecules
Different seaweed components such as proteins, polyphenols, alginates and fucoidan were selectively extracted from two seaweed types and analyzed by size-exclusion chromatography coupled to ICP-MS in order to better understand their association with various elements. All of the above mentioned elements were monitored, but only those which exhibited interesting profiles are mentioned in the speciation analysis. The chromatograms obtained from the injection of the protein extracts are shown in figure 4. In both the seaweeds lead (208Pb) is distributed throughout a wide range of molecular weights in the protein extract as shown in figure 4. On the other hand, elution profile of arsenic (75As) in the hiziki protein extract constitutes a large sharp peak around 1350 seconds. Since hiziki is known to contain high
16 amounts of inorganic arsenic, an elemental arsenic standard was injected into the system. The
retention time of this peak matched with the retention time of the elemental arsenic standard on the column, which was close to the retention time of the aprotinin protein standard peak eluted, which has a molecular weight of 6.5 kDa. This would suggest that the inorganic arsenic species interacts in some way with the column that causes it to elute faster than lower molecular weight species. Other inorganic elements also elute around the same retention before expected. A table that shows the approximate retention times of various inorganic elements is shown in Table 4. In both hiziki and wakame extracts, 57Fe and 88Sr were found in the high molecular weight species, presumably proteins. In wakame Ag and Hg were also found in high molecular weight species of the protein extracts.
17 Hiziki Protein Hiziki Protein 57Fe 100000 16kDa 1.3kDa 75 88 70kDa 6.5kDa 75 Sr inorganic As As 16kDa 1.3kDa 208 70kDa 6.5kDa 80000 Pb
1000 60000 s t n Counts Cou 40000
20000
0 0 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec)
Wakame Protein Wakame Protein 1200 16kDa 1.3kDa 14000 70kDa 6.5kDa 16kDa 1.3kDa 70kDa 6.5kDa 57 1000 Fe 12000 88 208 Sr Pb 107 10000 800 Ag
ts 202 8000 Hg un 600 75 Co Counts As 6000 400 4000
200 2000
0 0 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec)
Figure 4. SEC-ICP profiles of various elements in protein extracts.
18 Table 5. Approximate Retention times of inorganic elements bound with higher molecular
weight species.
Element Retention Time (sec) Mg ~2500 Cr ~1750 Fe ~1400 Co ~1300 Cu ~1250 Zn ~1750 As ~1350 Se ~1500 Sr ~1750 Ag ~2750 Cd ~1750 Hg ~2750 Pb ~1750
To attempt to break down the protein extracts to learn more about the association of these metals with the protein, a proteinase K digestion was attempted. When the proteinase K digestion product was injected into the SEC-HPLC-ICP-MS system the response for all the elements of interest in the protein extracts increased over a wide molecular weight range. This is demonstrated in figure 5, which shows the Fe chromatograms obtained for the proteinase K, the hiziki protein extract, and the hiziki protein extract treated with proteinase K. From the figure it is evident that proteinase K contains a high concentration of Fe. Proteinase K has a molecular weight of 28.9 kDa, but Fe is seen over a large molecular weight range. Similar trends were seen for all the other elements of interest in protein extracts. Contaminants are present in the proteinase K over a wide molecular weight range. To verify the presence of these elements in the proteinase K a digestion was carried out using the same procedure that was used for total elemental analysis of the seaweed samples. A crude one point calibration analysis was carried out for an estimate of the elemental concentrations with the results shown in Table 5. Due to the
19 presence of these elements of interest in the proteinase K, it was not possible to utilize proteinase
K in order to break down proteins in this study.
Hiziki Protein 57Fe Chromatograms 25000
70kDa 16kDa 6.5kDa
20000 protein protein treated with 15000 proteinase K S proteinase K
CP 10000
5000
0 0 500 1000 1500 2000 2500 3000 Time (sec)
Figure 5. Fe chromatograms for protein extracts and proteinase K.
20 Table 6. Crude total elemental concentration in µg g-1 of proteinase K.
Mg 276
Cr 27.6
Fe 117
Cu 10.0
Zn 32.4
As 3.61
Se 221
Sr 3.59
Cd 0.20
Hg 84.1
Pb 1.73
Polyphenols:
The element specific chromatograms obtained from the SEC-ICP-MS analysis of the
polyphenol extracts are shown in figure 6. Polyphenol extracts obtained from both the hiziki and
wakame seaweed contained 107Ag and 202Hg in the high molecular weight region. Unlike proteins, 75As in the polyphenol is mainly found in the high molecular weight species in wakame, and a peak is not seen at the retention time that the elemental arsenic eluted. On the other hand, 80Se in the wakame polyphenol extracts is present only in the lower molecular weight region.
21 The polyphenol extract of hiziki seaweed contained Fe in mainly the high molecular weight range while other elements such as Pb and Zn has a wide distribution ranging from high to low molecular weight region.
Hiziki Polyphenol Hiziki Polyphenol 16kDa 1.3kDa 16kDa 1.3kDa 107 12000 70kDa 6.5kDa 70kDa 6.5kDa Ag 202 Hg 57 400 10000 Fe 66 Zn 8000 208
ts Pb
6000
200 Coun Counts 4000
2000
0 0 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec)
Wakame Polyphenol Wakame Polyphenol 2200 300 16kDa 1.3kDa 80 2000 70kDa 6.5kDa Se 16kDa 1.3kDa
70kDa 6.5kDa 75 1800 250 As 107 1600 Ag 202 1400 200 Hg ts 1200 ts
150 1000 un
Coun 800 Co 100 600
400 50 200
0 0 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec)
Figure 6. SEC-ICP profiles of various elements in polyphenol extracts
Alginates and Fucoidans:
The main element found in the alginate extract of both the seaweeds is strontium. This is not to say that only strontium was present in the alginate in the sample, but possibly the strontium alginate was more easily solublized in boiling water than alginates that were complexed with other elements. To verify the presence of alginate in the extract, the alginate
22 extract was treated with an alginate lyase obtained from Sigma-Aldrich. Figure 7 shows the 88Sr chromatogram obtained for the hiziki alginate extract and the alginate extract after treatment with the alginate lyase. Before treatment with alginate lyase the alginate extract showed a large strontium peak in the high molecular weight range. After treatment with the alginate lyase, the strontium peak was shifted to a lower molecular weight range.
Hiziki Alginate Extract 180000 88Sr before treatment with alginate lyase 88 160000 Sr after treatment with alginate lyase
140000
120000
100000 S
80000 CP
60000
40000
20000
0
0 500 1000 1500 2000 2500 3000 Time (sec)
Figure 7. Strontium chromatogram for Hiziki alginate extracts.
The elution profile of various elements in the fucoidan extracts of both seaweeds is
depicted in Figure 8. As in the protein extracts, Pb and Fe were found in the high molecular
weight range for the fucoidan extracts. Although the sample was treated with chloroform to
remove protein that was possibly extracted during the alginate and fucoidan extraction, it is
possible that some protein remained in the fucoidan extract. That would explain the similarity of
Pb and Fe profiles in protein and fucoidan. Additional investigation is needed into these extracts
to determine if protein is present in the fucoidan extracts. Silver was also found in the high
23 molecular weight fucoidan extracts of both seaweeds. In the wakame, As and Cu were also found in the high molecular weight extracts.
57 Fe 600 Hiziki Fucoidan Hiziki Fucoidan 88 16kDa 1.3kDa 16kDa 1.3kDa Sr 15000 70kDa 6.5kDa 70kDa 6.5kDa 107 208 500 Ag 14000 Pb
13000 400 12000 s 11000 300 unts 10000 Count Co 200 9000
8000 100 7000
6000 0 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec)
Wakame Fucoidan 800 70kDa Wakame Fucoidan 16kDa 1.3kDa 12000 6.5kDa 16kDa 1.3kDa 57Fe 70kDa 6.5kDa 208 10000 Pb 600 75 As 63Cu 107Ag 8000 s 400 unts unt 6000 Co Co
200 4000
2000 0 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec)
Figure 8. SEC-ICP profiles of various elements in fucoidan extracts.
In order to find the distribution of the various elements between the extracts obtained through the various extraction procedures, the total elemental analysis was carried out on each of the extracts. The same digestion procedure was used as described in the experimental section.
The total element concentrations of each of the extracts from hiziki and wakame are shown in
Table 6 and 7, respectively. The concentration of magnesium and copper were highest in the
24 fucoidan extracts from both seaweeds. The protein extracts contained higher concentrations of
iron and arsenic. The alginate extract of the wakame contained a higher concentration of
strontium than the other extracts as expected from the chromatographic results. In the case of
hiziki, the highest amount of strontium was found to be present in the protein extract. These
results are somewhat surprising because in the Hou’s study, strontium was only found in the alginate extracts of the seaweeds[8]. In this study, strontium was found in the protein, alginate, and fucoidan extracts. Although this is interesting, these seaweeds analyzed, although also species of brown algae, they are not the same type of seaweed that was analyzed by Hou. The seaweeds that were analyzed in this study, also were grown in conditions that are unknown. The conditions in which the algae were grown may also play some role in which parts of the plant the strontium is accumulated.
Table 7. Total elemental concentrations of various hiziki extracts in µg g-1
Protein Polyphenol Alginate Fucoidan Mg 58.2 ND 82.4 160 Cr ND ND ND ND Fe 30.6 0.316 19.4 3.12 Co 0.142 0.002 0.041 0.084 Cu 0.204 0.052 0.273 0.855 Zn 0.301 0.308 0.377 3.73 As 26.8 0.017 1.75 3.11 Se ND ND ND ND Sr 23.3 0.007 5.85 3.32 Ag 0.065 ND ND ND Cd 0.217 0.066 0.158 0.247 Hg 0.666 0.268 0.499 0.490 Pb 0.054 0.045 0.031 0.066
25 Table 8. Total elemental concentrations of various wakame extracts in µg g-1
Protein Polyphenol Alginate Fucoidan Mg 46.3 ND 82.4 146 Cr ND ND ND ND Fe 20.9 0.389 4.29 2.31 Co ND ND ND ND Cu 0.752 0.021 0.192 1.39 Zn 1.36 0.204 0.323 1.04 As 1.66 0.006 0.332 0.170 Se ND ND ND ND Sr 9.84 0.001 10.8 0.526 Ag 0.966 ND ND ND Cd 0.21 0.070 0.230 0.199 Hg ND 0.343 0.820 0.556 Pb 0.049 ND 0.161 0.008
In the case of Mg, Fe, Cu, Zn, Hg, and Pb the protein extract element concentration was
significantly less than the elemental concentration of the proteinase K. Proteinase K had a Mg
concentration of 276 µg g-1 while the protein extract of hiziki had a Mg concentration of 58.2 µg
g-1 and the protein extract of wakame had a Mg concentration of 46.3µg g-1. Proteinase K had an
Fe concentration of 117 µg g-1 while the protein extract of hiziki had an Fe concentration of
58.30.6 µg g-1 and the protein extract of wakame had an Fe concentration of 20.9µg g-1.
Proteinase K had a Cu concentration of 10 µg g-1 while the protein extract of hiziki had a Cu
concentration of 0.204 µg g-1 and the protein extract of wakame had a Cu concentration of 0.752
µg g-1. This once again suggests that proteinase K should not be used to break down proteins when the association of these elements with different molecular weight fractions is of interest.
Highly pure HCl is one possible alternative to proteinase K for breaking down proteins when the metalloprofile of the protein is of interest. High purity HCl will not introduce metal contaminants into the sample, but offer less control over where the protein is cleaved.
26 Other elements besides Sr were found in the alginate extracts, but only Sr was seen in the high molecular weight range of the elemental profiles. It is known from previous research that the salts of alginic acid with divalent or polyvalent metal ions are insoluble in water[17]. It is possible that during the extraction procedure some of the elements of interest were released from the alginate.
27 Conclusions and Future Work
High molecular weight protein extracts from both seaweeds were found to contain lead, iron, and strontium. The high molecular weight protein extracts of wakame were also found to contain silver and mercury. The attempt to break down the protein in the protein extract in order to learn more about the association of with proteinase K failed. In the future those working in the field of metalloproteomics should not employ proteinase K and possibly other enzymes to break down proteins due to the possibility of metal contamination. It may be more beneficial to use a highly pure reagent such as HCl to break down proteins for further analysis.
Higher concentrations of iron and arsenic were found in the protein extracts than the other extracts. In the case of hiziki, arsenic existed mainly as inorganic arsenic compounds, while peaks arsenic peaks associated with the high molecular weight range and inorganic arsenic were found in wakame protein extracts.
High molecular weight polyphenol extracts of both seaweeds were found to contain silver and mercury. Arsenic was also found in the high molecular weight polyphenol extract of wakame as it was in the protein extract. Iron, zinc, and lead were found in the polyphenol extracts from hiziki.
Strontium was the main element associated with the high molecular weight extracts of both seaweeds. The strontium peak was shifted to the lower molecular weight range by treating the alginate extract with alginate lyase. It is known that magnesium alginate is a common component of alginate, although the magnesium chromatograms only showed a peak in the low molecular weight range associated mainly with inorganic magnesium. This suggests that magnesium alginate was not soluble in the hot water used for the extraction of alginate or possibly that during the extraction process magnesium was released from the magnesium
28 alginate. Other extraction techniques should be investigated that could possibly extract alginate from the algae while minimizing the release of elements of interest from the alginate.
High molecular weight fucoidan extracts of both seaweeds were found to contain iron and lead as in the protein extracts. Silver was also found in the high molecular weight fucoidan extracts of both seaweeds. The wakame fucoidan extracts were found to contain arsenic and copper in the high molecular weight range.
Before future work is carried out on this study, further investigation is needed into the interaction of the Superdex 75 with inorganic compounds. It is possible that it may be advantageous to use a different size exclusion column, if one exists that doesn’t interact unfavorably with inorganic compounds. Other future work includes confirming the presence of protein in the protein extract. This may be accomplished by employing gel electrophoresis.
The seaweeds that were analyzed in this study grew in conditions that are unknown. It would be interesting to grow seaweed in a controlled environment and supplement the seaweed with various elements. This would allow the opportunity to study how the chemical association of seaweed changes with supplementation.
This study was a preliminary study to see how elements were associated over a molecular weight range. In the future further focus could also be on specific components of seaweed, such as polyphenols and their interaction with elements of interest. The peaks from the size exclusion chromatographic run could be collected and further analysis could be carried out to further elucidate the specific nature of the interaction that takes place between the polyphenol and the elements of interest.
A large number of elements were observed in this study. In the future further focus should be on fewer elements and a more in depth study could be carried out on the association of
29 those elements with the different components of seaweed. Arsenic was found in high molecular
weight extracts of protein, polyphenol, and fucoidan of wakame. Further investigation of the
association of arsenic with these compounds is of interest, due to the fact that arsenic is such a toxic element. Fractions of the protein extracts containing arsenic could be collected and further separated using gel electrophoresis. More information about the specific size of these proteins could be gained from this type of separation. Similarly the same type of analysis could be carried out for fractions of the protein extracts containing lead because the lead that was found in the high molecular weight extracts of the protein extracts of wakame.
30 References
1. Amado, G.M., et al., Brown algae species as biomonitors of Zn and Cd at Sepetiba Bay, Rio de Janeiro, Brazil. Marine Environmental Research, 1999. 48(3): p. 213-224. 2. Abbott, Ethnobotany of seaweeds: clues to uses of seaweeds. Hydrobiologia, 1996. 326- 327 (1): p. 15. 3. Hou, X., et al., Determination of chemical species of iodine in some seaweeds (I). Science of the Total Environment, 1997. 204(3): p. 215-222. 4. Munilla, M.A., et al., Determination of metals in seaweeds used as food by inductively coupled atomic-emission spectrometry. Analusis, 1995. 23(9): p. 463-466. 5. Laparra, J.M., et al., Bioaccessibility of inorganic arsenic species in raw and cooked Hizikia fusiforme seaweed. Applied Organometallic Chemistry, 2004. 18(12): p. 662-669. 6. van Netten, C., et al., Elemental and radioactive analysis of commercially available seaweed. The Science of the total environment, 2000. 255(1-3): p. 169-75. 7. Karez, C.S. and R.C. Pereira, Metal Contents In Polyphenolic Fractions Extracted From The Brown Alga Padina-Gymnospora. Botanica Marina, 1995. 38(2): p. 151-155. 8. Hou, X., Study on chemical species of inorganic elements in some marine algae by neutron activation analysis combined with chemical and biochemical separation techniques. Journal of Radioanalytical and Nuclear Chemistry, 1999. 242(1): p. 49-61. 9. Shah, M., et al., Iodine speciation studies in commercially available seaweed by coupling different chromatographic techniques with UV and ICP-MS detection. Journal Of Analytical Atomic Spectrometry, 2005. 20(3): p. 176-182. 10. Liang, J., Q. Wang, and B. Huang, Concentrations of hazardous heavy metals in environmental samples collected in Xiamen, China, as determined by vapor generation non-dispersive atomic fluorescence spectrometry. Analytical sciences: the international journal of the Japan Society for Analytical Chemistry, 2004. 20(1): p. 85-8. 11. Raab, A., P. Fecher, and J. Feldmann, Determination of Arsenic in Algae - Results of an Interlaboratory Trial: Determination of Arsenic Species in the Water-Soluble Fraction. Microchimica Acta, 2005. 151(3-4): p. 153-166. 12. Hou, X.L., X.J. Yan, and C.F. Chai, Chemical species of iodine in some seaweeds II. Iodine-bound biological macromolecules. Journal Of Radioanalytical And Nuclear Chemistry, 2000. 245(3): p. 461-467. 13. Ji, S., et al., Fractional Distributions of Major-to-Ultratrace Elements in Coastal Seawater and Their Partitionings in Laboratory-Made Salts as Investigated by Inductively Coupled Plasma Atomic Emission Spectrometry and Inductively Coupled Plasma Mass Spectrometry with Aid of Membrane- and Ultra-filtration Techniques. Bulletin of the Chemical Society of Japan, 2000. 73(5): p. 1179-1186. 14. Sauter, L., D. Van der Ben, and R. Van Grieken, Trace analysis of estuarine brown algae by energy-dispersive x-ray fluorescence. X-Ray Spectrometry, 1979. 8(4): p. 159-63. 15. Lobban, C.S., M.J. Wynne, and Editors, Botanical Monographs, Vol. 17: The Biology of Seaweeds. 1981. 786 pp. 16. Khotimchenko, Y.S., et al., Physical-chemical properties, physiological activity, and usage of alginates, the polysaccharides of brown algae. Russian Journal of Marine Biology (Translation of Biologiya Morya (Vladivostok, Russian Federation)), 2001. 27(Suppl. 1): p. S53-S64.
31 17. Davis, T.A., B. Volesky, and A. Mucci, A review of the biochemistry of heavy metal biosorption by brown algae. Water Research, 2003. 37(18): p. 4311-4330. 18. Andrade, L.R., et al., Ultrastructure of acidic polysaccharides from the cell walls of brown algae. Journal Of Structural Biology, 2004. 145(3): p. 216-225. 19. Berteau, O. and B. Mulloy, Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiology, 2003. 13(6): p. 29R-40R. 20. Fernandez Sanchez, L. and J. Szpunar, Speciation analysis for iodine in milk by size- exclusion chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICP MS). Journal of Analytical Atomic Spectrometry, 1999. 14(11): p. 1697-1702. 21. Wrobel, K., S.S. Kannamkumarath, and J.A. Caruso, Hydrolysis of proteins with methanesulfonic acid for improved HPLC-ICP-MS determination of seleno-methionine in yeast and nuts. Analytical and Bioanalytical Chemistry, 2003. 375(1): p. 133-138. 22. Azparren, J.E., et al., Blood strontium concentration related to the length of the agonal period in seawater drowning cases. Forensic Science International, 2000. 108(1): p. 51- 60.
32