An Extensive Survey of Rumex crispus for anti-tumor compounds - DOC

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					  A Survey of Rumex crispus for antitumor compounds.




A proposal submitted in partial fulfillment of the degree of Master of Science




                                    by
                         Eric Jeffrey Lawrence




                      To Graduate Committee
                    Department of Biotechnology
                  Stephen F. Austin State University
                            July 2, 2002
Introduction

       The purpose of this study is to determine the presence of anti-tumor

compounds in Rumex crispus. R. crispus is a common weed that is present on

                            every continent.(1) R. crispus is known to contain

                            several compounds with anti-tumor properties, but no

                            in vitro studies have been performed using this

                            plant.(2) R. crispus is commonly used as herbal

                            remedy to treat anemia, as well as hepatic and

                            dermatological disorders.(3,4) R. crispus was used in

                            several anti-cancer formulas during the 19th

                            century.(5) Very little scientific research has been
            (50)
 Figure 1
 R. crispus a common        performed with this plant. This study hopes to
 weed found throughout
 the world. The plant       achieve one of two outcomes: 1.) Identify a new anti-
 turns brown when it is
 mature in late spring.
                            cancer compound, 2.) Be able to utilize this plant as a

source of known natural anti-cancer compounds.

       This project takes a novel approach to obtaining a dose-response curve

from plant extracts. Upon finding a sample that has a positive response, the

unknown “reactor” (compound) will already be classified. Samples to be tested

are first separated by plant-part: rhizome, flower, root, leaf, or stem. Each plant

part is then separated by solvent extraction: ethanol, methanol, or chloroform.

Then each of these samples is further separated by solid phase extraction (SPE)

in thirteen sorbents that separate compounds into general chemical

classifications. Samples from each SPE column are then exposed to colon-




                                        -1-
cancer cells (DLD-1) as well as a normal colon cell-line (CCD-112Co) as a

control. A cell proliferation assay will then be performed in 96-well plates using

an MTS colorimetric assay to measure cell viability. Data obtained from these

assays will be used to construct a dose-response curve and LD50 for samples

yielding positive results. Any samples yielding positive results will then have a

time-assay performed and a dose vs. time plot for positive samples can be

constructed. A time assay will also provide direction into the mode of action for

positive samples, whether the anti-tumor compound’s action is during the

stationary, or log phase of the cell cycle. These results would conclude this

portion of the study, but this project will be continued by performing HPLC, GC,

TLC, IR, NMR, and other chromatographic techniques on the unknown

compound(s) for identification and concurrently determine the compounds’ mode

of action.




                                        -2-
Objectives

       The final objective of this project is to obtain dose-response activity from

extracts of R. crispus and to characterize the class of possible compound(s). To

accomplish this goal the following objectives must be met:



Objective 1: Obtain R. crispus, separate plant parts, and prepare for extraction.

       The whole plant(s) will be obtained from a known source and the plant will

be separated into five parts: leaf, flower, stem, root, and rhizome. The fresh

plant parts will then be ground to powder in a mortar and pestle with liquid N 2.



Objective 2: Extraction of each plant part using solvents of different polarity.

       Each ground plant part will be extracted in the Accelerated Solvent

Extractor (ASE) from Dionex. Three extractions will be done on each plant part,

each with a different extraction solvent. The solvents methanol, ethanol, and

chloroform will be used to extract the samples. The samples will then be

evaporated by spin drying them in a vacuum at 40 oC.



Objective 3: Solid Phase Extraction of samples.

       Each extraction sample will be further extracted using solid phase

extraction. Approximately 13 different chromatography columns will be used to

separate the extraction samples into specific classes of compounds. Evaporated

ASE extracts will be resuspended in a solvent appropriate for the SPE column to




                                        -3-
be used and following extraction from SPE the samples will evaporated by

centrifugal evaporation.



Objective 4: Tissue culture of DLD-1’s and CCD-112Co cells.

       The colon adenocarcinoma DLD-1 cell line and the normal colon CCD-

112Co cell line purchased from American Type Culture Collection will be

subcultured to several passages. Enough cells will be cultured to perform the

cell proliferation assay on DLD-1 cells and the control CCD-112Co cells.



Objective 5: Cell proliferation assay with extract titrations.

       A cell proliferation assay will be performed with sample dilutions from 10 0

– 10-9 concentration of the evaporated SPE extracts resuspended in

dimethylsulfoxide (DMSO). The assay will be performed in 96-well plates and

detection of proliferation will be done using an MTS tetrazolium compound from

Promega. The assay will be performed in triplicate with positive and negative

controls.



Objective 6: Measure cell proliferation in a time assay using active samples.

       A follow-up cell proliferation assay will be performed using samples that

tested positive for antitumor activity. The time assay will measure cell

proliferation of active samples at 12, 24, 48, and 72 hours. Assay will be

performed in duplicate with positive and negative controls.




                                        -4-
Objective 7: Dose-response curve for active samples.

       A dose-response curve will be constructed for samples that are positive

for antitumor activity with colon cancer cells. A dose-response curve will be used

to determine the LD50 of the unknown compounds and the dose-response curve

will be compared for all active compounds to determine the most promising

extract to further pursue study on.




                                       -5-
Literature Review

       R. crispus is a common weed of the Polygonacease family.(1) It grows

between 30 – 160 cm. high with narrow leaves that have curly edges.(1,42) The

tap root is large and fleshy and typically grows to 165 cm. deep.(1) Flowers are

yellow to green and form clusters, and the seeds are winged and triangular(42) R.

crispus has invaded every continent and the genus seems to follow the migratory

patterns of man throughout the world.(1) It is a perennial that can be found in

pastures, fields, and wastelands, but prefers a moist soil habitat.(1,42)

       Since R. crispus is almost ubiquitous it has been utilized for a wide variety

of medicinal usage and is quite popular as a naturopathic treatment. R. crispus

is well known for its treatment of skin ailments, as a poultice or taken internally.

The leaves are used to treat skin ailments, such as: boils, hives, ringworm, itch,

jaundice, acne, scabies, psoriasis, eczema, and other skin disorders.(3,42,43,44,45)

The root is used for its well documented laxative properties, purportedly due to its

high content of anthraquinones.(42,44,46,47) It has also been used widely as an

alterative, that is, a remedy for syphilis or as a “blood cleanser”.(44,45,46,47)

Alteratives are also used to treat anemia (iron deficiency in blood), R. crispus

contains a high iron content.(3,47) The naturopathic use of R. crispus to treat

cancer or as an antitumor agent is rare, but the root has been documented in

several anticancer formulas from the 19th century, such as, Scudders cancer

alterative, Kloss’ anticancer herbal, and Eli Jones Formula.(5) According to Dr.

James Duke,(2,3) the plant can be used to treat cancer, and Table 1 lists some of

the compounds that he has discovered in R. crispus to have proven antitumor




                                           -6-
     Table 1(2)
       Compounds in R. crispus that have proven antitumor acitivity.
      ( * = no quantitative results have been determined for this compound)
                                                  Plant
       Compound           Antitumor activity                    Amounts
                                                  Part
      Ascorbic Acid        Antitumor (lung)       Leaf       300-4,054 ppm

     Beta-carotene            Antitumor           Leaf       10.38-140 ppm

         Emodin           Antitumor (breast)      Root              *

       Erucic Acid            Antitumor           Root     9,000-121,612 ppm

        Quercetin      Anticarcinomic (breast)    Leaf              *
                            IC50 = 1.5 uM
                        Antitumor (bladder)       Leaf              *

                          Antitumor (breast)      Leaf              *

                          Antitumor (colon)       Leaf              *

                          Antitumor (ovary)       Leaf              *

        Quercitrin            Antitumor           Leaf              *

          Rutin               Antitumor           Leaf              *

         Tannin               Antitumor           Root     30,000-60,000 ppm.




properties.(2) A number of sources have identified medically active compounds

that account for the uses described above. R. crispus has a high amount of

anthraquinones with sources quoting between 3-4%, among the anthraquinones

identified, include: nepodin, chrysophanol, physcion, emodin, chrysophanic acid,

and rhein.(3,44,45,46,47) The flavonoid class of compounds has also been identified,

including, quercitrin and quercetin.(44,45) Tannins and oxalates, which can be

lethal in high doses are present in R. crispus, as well as several of their



                                        -7-
derivatives.(3,44,45,47) Several naphthalene derivatives, napodin and lapodin, have

also been identified.(44,45) A recent scientific study showed R. crispus to have a

high degree of antioxidant activity and antimicrobial activity against Gram-

positive bacteria.(48) The study aconcluded that there was a relationship between

the antioxidant activity of extracts and the amount of phenolic compounds

detected.(48)

       Accelerated solvent extraction (ASE) was chosen as the solvent extraction

method for the ground plant material. ASE uses high temperature and pressure

to increase the speed and efficiency of extraction.(6) Results from ASE provide

extractions comparable to conventional extraction methods when compared with

analysis by UV-Vis spectroscopy and HPLC.(6) Increased temperature is used for

extraction because as the temperature increases the solvent’s viscosity

decreases, therefore, giving the solvent a greater ability to wet the matrix and

solubilize analytes.(8) Elevated temperature also breaks bonds between the

analyte and the matrix, and it encourages analytes to diffuse to the surface of the

matrix.(8) Pressure is increased in ASE to move the mobile phase through the

system rapidly, and to maintain the solvents in liquid phase while they are above

their atmospheric boiling points.(8) Up to ten grams of sample can be extracted in

less than twenty minutes using ASE.(6) Solvents used in the ASE were chosen

based on their polarity and based on documentation of solvent extractions

performed on other plant extracts.(6,7) Solvents of varying polarity can be used to

extract a broad-range class of compounds.(8) Methanol, ethanol, and chloroform

were chosen as solvents for ASE of the plant samples because of their solvent




                                        -8-
properties. Methanol has high polarity and is aqueous, ethanol is_______ and

aqueous, and chloroform is nonpolar and immiscible in water.(9)

       There are many advantages to using solid phase extraction (SPE) as a

second step to separating compounds from the initial solvent extraction. A wide

variety of sorbents, chemistries, and sizes of SPE columns are available. When

combined with liquid extraction, solid-phase extraction concentrates specific

phase extractions without sacrificing quantitative recoveries.(9) Further, SPE is

rapid, easy to perform, can be automated, and solvent usage is minimal. (9) SPE

method involves passing the sample (dissolved in appropriate solvent) through a

conditioned matrix in which specific analytes are bound.(10) Interference

compounds are washed from the matrix with a weak solvent, then the sample is

eluted form the matrix with a small volume of strong solvent.(10) This produces a

clean, concentrated, sample that is ready for a cell proliferation assay.(10) SPE

uses the same analyte/matrix interactions that is used in the high performance

liquid chromatography (HPLC) separation technique.(11) The SPE columns are

silica gel-based, bonded phase packing that selectively retains specific classes of

chemical compounds from within the given matrix.(11) Bonded silica sorbents are

capable of having a variety of different functional groups readily bonded to the

silica surface.(11) A description of the type of functional groups and what is

retained by each sorbent to be used is in Figure 2.(9) Bonded silica also has a

high surface area due to porosity, it’s a rigid support that will not shrink or swell,

and it’s stable under a wide range of aqueous and organic solvent conditions. (11)

Supelco silica-based packing has 40 μm particles with 60 Å pores.(9) The 3 mL




                                         -9-
INSERT FIGURE 2 HERE: SPE PHASE TYPES




                - 10 -
columns are polypropylene columns which contain 500 mg of sorbent and is

flanked by polyethylene frits.(10)

       Efficiency of SPE is particularly due to the vacuum manifold in which the

SPE column resides in during extraction. The Visiprep vacuum manifold from

Supelco enables up to 24 samples to be prepared simultaneously and utilizes a

pressure gradient to pull volumes through the sorbent efficiently.(10) Disposable

liners in the vacuum manifold eliminates the possibility of cross-contamination,

and precise flow control is capable through each SPE tube by flow control

valves.(10)

       SPE achieves clean-up, concentration, and solvent exchange of the

analytes in four basic steps:



       1.) Conditioning: The sorbent is prepared by solvating it with an

              appropriate volume of solvent that is similar in nature to the sample

              matrix.(11) Conditioning solvents depend on the tube packing and the

              application.(12)

       2.) Load Sample: The dried sample that is dissolved in appropriate

              solvent is passed through the sorbent and analytes are retained by

              specific chemical interactions.(11) Some interference compounds may

              also be retained by weak chemical interactions.(11)

       3.) Wash: Weakly retained materials are removed using a solvent with

              weak eluting strength, while the analyte is undisturbed in the sorbent

              bed.(12)




                                          - 11 -
       4.) Elution: A small volume of strong eluting solvent is passed through the

          sorbent which disrupts analyte-sorbent chemical interactions yielding

          selective elution of a particular class of compounds.(11)



       The classes of analytes extracted for each sorbent type are listed on

Figure 2. Separation retention occurs by three methods, reversed phase

extraction (LC-8, LC-18, LC-Ph, LC-CN), normal phase extraction (LC-Diol, LC-

CN, LC-NH2, LC-Si, LC-Florisil), and ion-exchange (LC-SAX, LC-SCX, LC-WCX,

LC- NH2). Reversed phase separation uses hydrophobic interactions. The

retention of analytes from a polar matrix is due to the attractive forces between

the carbon-hydrogen bonds in the analyte and the functional groups bonded to

the silica surface.(9) Nonpolar solvents disrupt the binding forces and are used to

elute the adsorbed compounds.(9) Normal phase extraction utilizes hydrophilic

interactions for separation.(9) Polar groups in the analyte interact with polar

groups on the sorbent surface and are retained.(9) Solvents of higher polarity

than the matrix disrupt the binding mechanism and elute the analytes.(9) Ion-

exchange is used to separate compounds that are charged when in solution. (9)

The retention mechanism for ion-exchange is based on electrostatic attractions

and elution of retained analytes is performed by displacement of the adsorbed

compounds using a solution with a high ionic strength.(9)

       By separating the ASE samples into 13 different classes of compounds by

SPE then the active anti-tumor compound(s) can be easily classified into a

particular class of compounds. SPE seems to be the method to perform this




                                        - 12 -
separation, due to its reliability, precision, and efficiency. SPE has been used to

separate a variety of drugs and organic compounds with excellent analytical

results.(13) When compared to liquid-liquid extraction SPE has proven to yield the

cleanest and best extraction results when separating 15 different organic

compounds.(14)

       Colorectal cancer is the fourth most common form of cancer in men and it

represents 8.9% of all new cancers per year worldwide and surgery is the only

option available for treatment of patients with colorectal cancer.(15) The human

cell-line chosen for this study is the DLD-1 colorectal adenocarcinoma. The

DLD-1 cell-line was isolated in 1979 by D.L. Dexter and is available through the

American Type Culture Collection (ATCC).(16,18) The DLD-1 cell-line is well-

characterized in studies of the effects of polar solvents.(17) Doubling time for

DLD-1 cells was determined to be 20 hours, and they are usually plated at 3 x

104 cells per 60 mm. dish.(16) Culturing methods of DLD-1’s are well

documented, they are cultured in DMEM supplemented with 10% fetal calf serum

(FCS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 oC and 5% CO2 in

aiR.(15,19,20) DLD-1 cells are generally subcultured at confluency and are plated

with a subculture ratio of 1:5 – 1:6.(18)

       CCD-112Co is a normal colon cell-line that is used as a control for this

experiment. This normal colon cell-line is also available from the ATCC.(21)

CCD-112Co is an adherent, fibroblast, that very little studies have been

performed on, although information was obtained from other CCD colon cell-lines

as reference for culturing CCD-112Co.(21) The recommended growth medium by




                                            - 13 -
the ATCC for the CCD-112Co cell-line is minimum essential medium (MEM)

Eagle with 2 mM L-glutamine and Earle’s BSS adjusted to contain 1.5 g/L sodium

bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate,

with 10% fetal bovine serum.(21) It is recommended that normal cell-lines need to

be subcultured at log growth phase just prior to confluency.(24) These normal

cells are recommended to be split with a subculture ratio of 1:3 – 1:4.(21) Opti-

MEM reduced serum medium has shown in the lab to be able to culture the

CCD-112Co cell-line. Opti-MEM is a modification of MEM (Eagle’s) that claims

versatility and the ability to maintain equivalent growth rate of fibroblast cells in 2

– 4 % FBS supplementation as to basal media at 10% FBS. (25) Opti-MEM

contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine,

thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and

phenol red reduced to 1.1 mg/L.(25)

       Characterization of the cell lines can be performed using a number of

methods. The common method is by isoenzyme analysis, but cell-lines can be

characterized using genetic analyses or fluorescent markers. Characterization

can also be contracted out by companies that perform automated and reliable

characterizations.(26,27)

       Mycoplasma contamination in eukaryotic cell-lines is the major concern of

contamination for these cells. Antibiotics are generally added to media to

prevent bacterial contamination and any other form of contamination besides

mycoplasma virus would be visible, thus easily detectable. ATCC claim that cell

samples purchased from them are mycoplasma-free, nevertheless, cultures can




                                         - 14 -
be contaminated anytime they are worked with. One method for mycoplasma

detection is by growing a culture on agar plates with the Hayflick formula. (28)

Mycoplasma can then be stained with Dienes stain and observed at 100x

magnification.(28)

       Cells should also be maintained by making a working cell bank, that is, to

freeze several cryovials of cells per passage as a precautionary measure. If the

cell line were to be contaminated or die off for some reason, then they could

easily be replaced from a preserved sample. This is more practical for the

cancer cells since they grow quite readily, nevertheless, it is also recommended

for the normal cell-line, too.

       A cell proliferation assay is the best means to determine activity of a

suspected anticancer compound using an in vitro culture. Several methods of

cell proliferation assays are available and used readily, including Trypan blue

exclusion dye, MTT tetrazolium salt assay, and the [3H] thymidine incorporation

assay. This experiment will use a fairly new method, the MTS assay, which is

very similar to the MTT assay.

       Promega offers a Cell Titer 96® AQueous One Solution Cell Proliferation

Assay which is used for microassays in 96-well plates, it is non-radioactive, and

requires fewer steps than an MTT assay.(29) MTS is a tetrazolium compound [3-

(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-

tetrazolium] that is reduced by active cells to a colored formazan product and is

soluble in tissue culture medium (Figure 4).(29,30) After addition of the reagent,

cells are cultured for 1 – 4 hours and then absorbance is recorded at 490 nm with




                                        - 15 -
a 96-well plate reader(29) When running an assay it is necessary to prepare a

triplicate set of control wells with just culture medium and the MTS reagent as a

standard to measure background absorbance.(29)

       Although the mechanism by which MTS is reduced is not fully understood,

it is thought to occur as a result of the action of dehydrogenase enzymes

generating reducing equivalents such as NADH or NADPH,(31) see Figure 4. The

One Solution reagent contains MTS as well as phenozine ethosulfate (PES)

which is the electron transfer reagent (ETR).(31) The amount of formazan

produced and recorded at 490 nm is directly proportional to the amount of viable

cells based on cellular metabolism. The MTS assay has been proven reliable for

a variety of methods, such as: measuring cell proliferation, cytotoxicity, viability

and apoptosis, cell adhesion, and chemotaxis, all using a variety of cultured

mammalian cells.(30) The use of tetrazolium reagents is extensive and has been

proven to be a reliable method.(30)

       The use of the DLD-1 cell-line with cell-proliferation assays is extensively

reported on in the current scientific literature. Determination of several factors

such as: DMSO percentage to dissolve plant extract in, number of cells to seed

per well, whether or not to pre-incubate cells before addition of extract, volume

per well, length of time for assay, all vary accordingly in the literature. To allow

sufficient time for drug interaction with cells, a 72 hour assay will be employed

and the total volume per well will be 100 uL to minimize reagent usage and

coincide with Cell Proliferation Assay Kit (Promega) recommendations.(29)

Evaporated samples from SPE are dissolved in DMSO and added to cell culture




                                        - 16 -
media at a 1:100 dilution.(7) Cells are then dispensed into 96-well plates at 5,000

cells per well.(29,32,33) A passage rate between P5 – P8 for DLD-1 and CCD-

112Co is commonly used for these cell types to perform the cell proliferation

assay.( ) Promega claims that the incubation time after addition of MTS reagent

varies with cell-line, but according to several papers using the MTT tetrazolium

reagent for DLD-1 cell proliferation assays the incubation time was four

hours.(15,32,33) Addition of the extract was also performed after a 24 hour pre-

incubation period after the cells are added to the 96-well plates, according to

several sources.(7,19,20,32,34)

       A positive control for tumor cell cytotoxicity was also considered to be

used to compare effects of the test samples to a known antitumor compound.

Doxorubicin is an antitumor compound readily available from Sigma

Chemicals(35) and has shown strong antitumor activity in studies done on mice.(36)

Doxorubicin is also used as a positive control for DLD-1 cell-line tumor assays by

MDS Pharma at an IC50 of 0.35 uM.(37) VWR claims that doxorubicin has an IC50

equal to 0.1 uM. Doxorubicin’s activity is based upon inhibiting topoisomerase II

and also intercalating into the DNA double helix, thus inhibiting nucleic acid

synthesis.(38)

       The effect being measured in this experiment is cell death versus

concentration (dose) and cell death versus time for the LD50 of the effective

samples. First, a dose response curve will be generated by plotting the

absorbance at 490 nm on the Y-axis versus the serial dilution of concentration of

the sample on the X-axis.(39) As agonist (sample that causes a response)




                                       - 17 -
concentration increases on a dose response curve the curve should go

downhill.(39) Doxorubicin as a standard is the full agonist (produces a full

response) and will be a good comparison to agonist samples. When a dose-

response curve is generated for cell death versus concentration four parameters

are defined on the curve. The baseline response is the bottom of the curve

where the slope is equal to zero, and the maximum response is the top of the

curve where the slope is equal to zero.(39) Both of these must be defined to

define the LD50 (lethal dose) which is the response that occurs halfway between

the baseline and maximum.(39) The fourth parameter to define is the slope of the

curve, an example dose-response curve is Figure 5.1. For samples that yield an

LD50, a graph of cell death versus time will be made based on the time assay, an

example of a time assay graph is Figure 6.1. The toxicity of an antitumor drug, or

any drug, is based on two factors, first, the concentration of the drug, and

second, the amount of exposure to the drug.(40) A time assay of the effective

samples also aids in determining the mode of action of the drug, for example,

what stage of tumor growth the drug produces the greatest cytotoxicity.

       Dose response is an integral part of drug development, as it is important

for determining the safety and effectiveness of a drug.(41) When determining

dose response, the studies should be well-controlled and use accepted

techniques to minimize bias.(41) A time assay has many benefits, and is easy to

perform following up a dosage assay. There is a tight interaction between dose-

response and time, as in drug treatments the choice of the size of an individual

dose is intertwined with the frequency of dosing.(41)




                                       - 18 -
Justification




Proposed Method of Study



Preparation of the cell-line will be the bottle-neck of this experiment. Enough

cells are going to be needed to perform assays in over 400 96-well plates. Cell-

lines must also continuously be kept free of contamination, and they also need to

be characterized by karyotyping to insure there is no cross-contamination of cell-

lines.



Cell proliferation assays will be performed with serial dilutions of the SPE sample

from 1 – 10-7 of the 1:100 sample (resuspended in DMSO) in culture medium.(7)

After a 24 hour pre-incubation period the extract dilutions will be added to the test

well. After a 68 hour incubation the MTS reagent will be added to each well and

incubated. The MTS tetrazolium salt will be allowed to react for 4 hours, then

absorbance will be recorded at 490 nm in a 96-well plate readeR. Test samples

that perform with positive results in the serial dilution assay will then be submitted

to a time assay, in which the LD50 of the positive samples will be added to 96-well

plates as before and cell proliferation will be measured at intervals of 12, 24, and

48 hours. Data from each of these tests will be recorded and graphed.




                                        - 19 -
- 20 -
                                                          100




         10-9
                10-8
                       10-6
                              10-5
                                     10-4
                                            10-3
                                                   10-1
                                                                DLD-1 w/ sample




         O
         O
         O
         O
         O
         O
         O
         O
                                                                 (duplicate)




         O
         O
         O
         O
         O
         O
         O
         O                                                      DLD-1 w/out sample




         O
         O
         O
         O
         O
         O
         O
         O
                                                                 (duplicate)




         O
         O
         O
         O
         O
         O
         O
         O

                                                                DLD-1 w/ (+ control)




- 21 -
         O
         O
         O
         O
         O
         O
         O
         O



                                                                 (duplicate)




         O
         O
         O
         O
         O
         O
         O
         O




                                                                CCD-18 w/ sample




         O
         O
         O
         O
         O
         O
         O

         O



                                                                 (duplicate)


         O
         O
         O
         O
         O
         O
         O

         O




                                                                CCD-18 w/ out sample
         O
         O
         O
         O
         O
         O
         O

         O




                                                                 (duplicate)
         O
         O
         O
         O
         O
         O
         O

         O




         O
         O
         O
         O
         O
         O
         O




                                                                CCD-18 w/ (+ control)
         O




         O
         O
         O
         O
         O
         O
         O

         O




                                                                 (duplicate)