1

Isolation of Urinary Epithelial Cell DNA for the Analysis of

4-Aminobiphenyl DNA Adducts

by nano-LC/ESI-MS/MS

by

Samantha J. Sokup

to The Department of Chemistry and Chemical Biology

In partial fulfillment of the requirements for the degree of Master of Science

in the field of

Chemistry

Northeastern University Boston, Massachusetts

December 2011

2

Isolation of Urinary Epithelial Cell DNA for the Analysis of

4-Aminobiphenyl DNA Adducts

by nano-LC/ESI-MS/MS

by

Samantha J. Sokup

ABSTRACT OF THESIS

Submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry

in the Graduate School of Northeastern University December, 2011

3

ABSTRACT

4-aminobiphenyl (4-ABP) is a known tobacco smoke carcinogen that has been shown to

form DNA adducts in the bladder epithelium, which may lead to cancer development. Our

laboratory has developed and implemented a highly sensitive and quantitative nano-flow liquid

chromatography electrospray ionization mass spectrometry (nano-LC/ESI-MS/MS) method for

the analysis of 4-ABP DNA adducts in vitro from liver and bladder tissue. In collaboration with

Roswell Park Cancer Institute we sought to apply our method for the analysis of 4-ABP DNA

adducts from bladder epithelial cells excreted in urine during the course of a smoking cessation

study. However, significant challenges were presented when applying this method to exfoliated

human urinary epithelial cells. One such challenge is that DNA yields from urine samples are

often low and extremely variable, due to factors such as gender of the donor, sample storage

conditions, extent of bacterial contamination, and release of nucleases from hydrolyzed cells.

The major impediment in this study was the sediment that forms in the samples as a result of

storage at -80 °C. Most of this sediment is due to the formation of urinary crystals, such as

calcium oxalate, magnesium ammonium phosphate, and uric acid. Treatment of the urine

samples with a 5 mM solution of EDTA (ethylenediaminetetraacetic acid) has proven to

significantly reduce these crystalline precipitates, and has allowed for successful isolation of

urinary epithelial cell DNA from frozen urine samples. Our current nano-LC/ESI-MS/MS

method is ideal for urinary epithelial cell DNA isolation, in which DNA yields are often limited,

for it requires only 2 µg of DNA, which is digested into mononucleosides for analysis. 4-ABP

adducts are detectable at attomole levels (LOD 140 amol, LOQ 560 amol). Preliminary results

from a small blind subset of patient samples show no detection of 4-ABP DNA adducts.

Analysis of remaining patient samples is in progress.

4

ACKNOWLEDGEMENTS

I owe a debt of gratitude to Dr. Paul Vouros, my research advisor, who gave me the

opportunity to be a part of his laboratory, and this project. It has been a privilege to work on a

project that has such practical application as a potential method of biomarker monitoring. I have

gained much knowledge, experience, and creativity during my time in this lab. Dr. Vouros has

provided me valuable research and career advice, for which I am forever grateful.

I would also like to thank our collaborators, Dr. Richard O’Connor and Dr. Yuesheng

Zhang, and their labs at Roswell Park Cancer Institute in Buffalo, NY, where the smoking

cessation study sample urine collections took place. Without their expertise and vision, our

analysis of clinical human urine samples would not have been possible.

Special thanks goes to those on my thesis committee: Dr. Penny Beuning, Dr. Geoffrey

Davies, and Dr. Paul Vouros.

I’d like to acknowledge all of the members of the Vouros Mass Spectrometry lab, both

past and present, including Johnny Capece, Dr. Steve Coy, Rose Gathungu, Dr. Jim Glick, Adam

Hall, Katelyn Hardy, Amol Kafle, Dr. Ravi Kc, Dr. Roger Kautz, Dr. Kristen Randall, Vaneet

Sharma, with special thanks to Josh Klaene, who served as a mentor and project partner

throughout my time spent in the group.

5

I would also like to thank the Department of Chemistry and Chemical Biology and

Barnett Institute at Northeastern University, for the education and opportunities provided to me

over the years. Thank you to Andrew Bean, Kathleen Cameron, Jean Harris, Alex Henriksen,

Graham Jones, Rich Pumphrey, and Cara Shockley. I owe a debt of gratitude to Jordan Swift,

my academic and co-op advisor, who was always willing to go above and beyond to provide

guidance in my coursework and career choices.

Finally, I would like to express my sincerest gratitude to my family and friends, who

have provided support and encouragement along the way. The highest honor goes to my

parentsAngela and Harold, without whom none of my success would have been possible. They

have given me more than I could ever hope to repay, in providing me the opportunity to attend

Northeastern University and in their unfailing love and support in everything I’ve done. I would

also like to thank my grandparents, whose encouragement over the years has been instrumental

to my accomplishments. Also, thank you to my close friends Sandra Rago, Sara Evarts, Rachel

Regonini, Rebecca Thibault, Paul Ippoliti, Rachel Hubbard, Arielle Smitt, and Katherine Hoarn,

for unwavering support and for making my college experience one that I treasure.

6

TABLE OF CONTENTS

Abstract 3 Acknowledgements 4 Table of Contents 6 List of Figures 7 List of Abbreviations 10 1. Introduction: Bladder Cancer and 4-Aminobiphenyl DNA Adducts 12

1.1 Roswell Park Cancer Institute Smoking Cessation Study 16

2. Materials 17

3. Urinary Epithelial Cell DNA Isolation Optimization 18

3.1 Urinary Epithelial Cell DNA Isolation Protocol 26

3.2 DNA Digestion and Preparation for Analysis by nano-LC/ESI-MS/MS 28

3.3 Preparation of dG-C8-4-ABP Standard Calibration Curves 28

4. nano-LC/ESI-MS/MS Analysis of dG-C8-4-ABP 29

5. Results and Discussion 33

5.1 Urinary Epithelial Cell DNA Isolation from Roswell Park Samples 33

5.2 Calibration Curve, Limit of Detection, and Limit of Quantitation 34

5.3 Preliminary Results from Roswell Park Samples 39

6. Conclusions 40

7. Appendix 1- LC/MS Troubleshooting 41

8. References 49

7

LIST OF FIGURES

Section 1

Figure 1.1 Metabolism of 4-aminobiphenyl 14

Figure 1.2 Structures of caffeine and its metabolites, in relation to CYP 1A2 15 and NAT2 activity

Section 3

Figure 3.1 Microscopic image of transitional/urothelial cells 19

Figure 3.2 Microscopic image of squamous cells 19

Figure 3.3 Microscopic image of a cluster of renal tubular cells 19

Figure 3.4 Calcium carbonate crystals 20

Figure 3.5 Magnesium ammonium phosphate (MgNH4PO4) crystals 20

Figure 3.6 Ammonium biurate crystals 20

Figure 3.7 Uric acid crystals, in common diamond shape 21

Figure 3.8 Uric acid crystals, in barrel and cube shape 21

Figure 3.9 Calcium oxalate crystals 21

Figure 3.10 Cystine crystals 21

Figure 3.11 Bilirubin crystals 21

Figure 3.12 Total urinary epithelial DNA isolated versus the initial volume of urine 23

Figure 3.13 Table of average yields of urinary epithelial DNA, comparing

male versus female samples 23 Figure 3.14 Treatment of urine samples with 5 mM EDTA was found to

reduce crystalline precipitates observed in cell pellets following centrifugation 25

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Section 4

Figure 4.1 Agilent microfluidics chip employed for online sample enrichment and analytical separation 31

Figure 4.2 MS/MS fragmentations for dG-C8-4-ABP (435319) and internal

standard dG-C8-4-ABP-d9 (444328) 32 Figure 4.3 Extracted ion chromatograms for dG- C8-4-ABP (1.1 fmol) and

internal standard dG-C8-4-ABP-d9 (3 fmol) 33

Section 5

Figure 5.1 Table of DNA yields from a subset of Roswell Park Cancer Institute smoking cessation study samples 34

Figure 5.2 Neat curve for dG-C8-4-ABP—ratio of standard dG-C8-

4-ABP to internal standard versus fmol standard on column 35 Figure 5.3 Low range (lowest 5 points) for the standard curve in Figure 5.2 35 Figure 5.4 Calibration curve prepared in calf thymus DNA digest matrix—

ratio of standard dG-C8-4-ABP to internal standard versus fmol standard on column 37

Figure 5.5 Low range (lowest 5 points) for the calibration curve in Figure 5.4 37 Figure 5.6 Selection of calibration points prepared in urinary epithelial cell

DNA digest matrix—ratio of standard dG-C8-4-ABP to internal standard versus fmol standard on column 38

Figure 5.7 Low range (lowest 3 points) for the curve in Figure 5.6 38 Figure 5.8 Extracted Ion Chromatogram for Roswell Park Cancer Institute, preliminary patient sample—with IS 39 Figure 5.9 Extracted Ion Chromatogram for Roswell Park Cancer Institute sample—with standard dG-C8-4-ABP and IS 39

9

Section 7

Figure 7.1 Full LC/MS system schematic, including dual capillary/nano pump, Chip Cube, and autosampler (with focus on 6-port valve configuration) 42

Figure 7.2 Diagrams of the two operational modes of the autosampler valve,

bypass and mainpass modes 43 Figure 7.3 Unusual capillary pump pressure profile observed prior

to troubleshooting 44 Figure 7.4 Extracted ion chromatogram for benzo(a)pyrene standard

when system is in full functionality 45 Figure 7.5 Evidence that the capillary pump pressure issues negatively

affected the chromatography of the analyte 45 Figure 7.6 Correlation to the seven solvent vial draws that occur during

the needle wash 46 Figure 7.7 Capillary pump pressure profile when method runs without the

use of the needle wash 46 Figure 7.8 Capillary pump pressure profile, following replacement of injector

valve assembly 47 Figure 7.9 Improved and stable capillary pump pressure profile following

replacement of the waste line capillary and PEEK fitting in Port 4 48 Figure 7.10 Improved chromatography for analytes following replacement of

the waste line capillary and PEEK fitting in Port 4 48

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LIST OF ABBREVIATIONS

4-ABP 4-aminobiphenyl

ACN Acetonitrile

amol Attomole

ctDNA Calf thymus DNA

CYP 1A2 Cytochrome P450

dG-C8-4-ABP N-(deoxyguanos-8-yl)-4-aminobiphenyl

DEAE Diethylaminoethyl

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

DNaseI Deoxyribonuclease I

EDTA Ethylenediaminetetraacetic acid

EIC Extracted ion chromatogram

ESI Electrospray ionization

Ethos Ethanol

fmol Femtomole

GC Gas chromatography

HPLC High performance liquid chromatography

IARC International Agency for Research on Cancer

i.d. Internal diameter

IS Internal Standard (in this case dG-C8-4-ABP-d9)

LC Liquid chromatography

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LOD Limit of detection

LOQ Limit of quantitation

m/z Mass to charge ratio

mM Millimolar

MS/MS Tandem mass spectrometry

NAT1 N-acetyltransferase 1

NAT2 N-acetyltransferase 2

NCI National Cancer Institute

ng Monogram

N-OH-4-ABP N-hydroxy-4-aminobiphenyl

PAH Polyaromatic hydrocarbon

PBS Phosphate buffered saline

RSD Relative standard deviation

S/N Signal to noise ratio

TE buffer 10 mM Tris-HCl / 1 mM EDTA

µg Microgram

µL Microliter

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1. INTRODUCTION: BLADDER CANCER AND 4-AMINOBIPHENYL DNA ADDUCTS

According to the National Cancer Institute (NCI), the prevalence of bladder cancer in the

United States is still a widespread issue, with 69,250 new cases and 14,990 deaths recorded to

date in 2011. The most common treatment to prolong survival for those with superficial cancer

is transurethral resection (TUR), sometimes coupled with intravesical chemotherapy. There is no

cure for invasive tumors, but standard treatments in North America include radical cystectomy

and urinary diversion.1 These treatment procedures are expensive, drastic, and highly invasive

for patients. Thus, it is necessary to increase understanding of the causes and mechanisms of

bladder cancer progression, as well as improve methods for early detection and prevention.

A variety of factors for increased risk of bladder cancer have been cited, among these are

family history, certain cancer treatments, and exposure to polycyclic aromatic hydrocarbons

(PAHs) and arylamines.1-4 Sources of exposure to such arylamines are found in industries

(rubber, plastics, cable, and wood manufacturing),4,5 household items (colorants, pesticides, and

hair dyes,6,7 and heated fuel emissions), but the primary source is tobacco smoke.5,6 It has been

reported that smoking accounts for 40-70% cases of bladder cancer. 8-10 Of the arylamines found

in tobacco smoke, many have been classified by the International Agency for Research on

Cancer (IARC) as carcinogenic to humans. 4-Aminobiphenyl (4-ABP) is one such arylamine,

and it is now a well-known human bladder carcinogen.11

Metabolism of 4-ABP in the body consists of a series of competing activating and

deactivating chemical derivitizations.10 Active 4-ABP metabolites react with DNA to form

adducts which may cause gene mutations and ultimately cancer.11-13 Activating reactions create

4-ABP metabolites that are eventually able to form adducts, while deactivating reactions

13

detoxify the intermediates, which are excreted as non-toxic 4-ABP metabolites. Activation of 4-

ABP begins in the liver, where hepatic enzymes such as P450 1A2 (CYP1A2) facilitate N-

hydroxylation, to form N-hydroxy-4-ABP (N-OH-4-ABP).10,14 Once it is transferred from the

liver to the bladder, N-OH-4-ABP is N-acetylated by N-acetyltransferase 1 (NAT1), forming an

N-acetoxy ester that, if introduced to acidic conditions, will form the electrophilic nitrenium ion

that reacts with DNA to form mutagenic adducts.10,15 The most common of these adducts is N-

(deoxyguanos-8-yl)-4-aminobiphenyl (dG-C8-4-ABP).10-12,16,17

This series of activation reactions is always in competition with a number of detoxifying

reactions. The liver serves as an early detoxifying source, where 4-ABP can undergo ring-

hydroxylation, O-esterification, N-acetylation, and N-glucuronidation to form 4-ABP

metabolites that cannot be converted to N-OH-4-ABP, in which case they are excreted in the

urine. If N-OH-4-ABP is formed in the liver, it can serve as a substrate to UDP-

glucuronosyltransferase, which gives an N-glucuronide conjugate that is then transported to the

bladder, where it can be excreted or converted back to an active metabolite.10 N-acetylation of 4-

ABP by N-acetyltransferase-2 (NAT2) yields an arylacetamide that is not a good substrate for

the CYP1A2 enzyme, and therefore serves as a mechanism for detoxification. Thus, N-

acetylation of the parent amine has been deemed a deactivation reaction, which is always in

competition with activation mechanisms described above.10 The metabolism and pathway of 4-

ABP in the liver and bladder is represented in Figure 1.1.

14

Figure 1.1 Metabolism of 4-ABP to DNA adduct dG-C8-4-ABP, plus detoxifying reactions N-acetylation, ring hydroxylation, and N-glucuronidation of 4-ABP.10

Acetylation has been shown to be one of the major contributing factors that results in

either the formation of a carcinogenic DNA adduct of 4-ABP or the excretion of non-toxic

metabolites, and it is the rate of acetylation that determines this outcome. Humans can be

classified as phenotypically fast or slow acetylators, which is determined based on caffeine

metabolism in the body.18,19 At a fixed time point after a dose of caffeine has been administered,

a urine sample is collected and the caffeine metabolites are quantitated. Ratios of certain

caffeine metabolites (Figure 1.2) to unmodified caffeine are used to give a basis for enzyme

activity. The metabolism of caffeine by CYP1A2 yields 1,7-dimethyl uric acid and 1,7-

dimethylxanthine, which are compared to the level of unmodified caffeine in the sample to

measure CYP1A2 activity. The metabolite 5-acetylamino-6-formamylamino-3-methyluracil is

formed via acetylation of 1-methylxanthine (a further demethylated caffeine intermediate) by

15

NAT2. Individuals with molar ratios of 5-acetylamino-6-formylamino-3-methyluracil to 1-

methylxanthine of < 0.6 are considered phenotypically slow acetylators.19 Studies have shown

that those who exhibit slow acetylation via NAT2 tend to have higher levels of adducted

DNA.10,20

Figure 1.2 Structures for caffeine and four of its metabolites quantitated in studies to determine CYP1A2 and NAT2 enzyme activity. The ratio of 1,7-dimethylxanthine and 1,7-dimethyluric acid to caffeine is used to measure CYP1A2 activity, and the ratio of 5-acetylamino-6-formylamino-3-methyluracil to 1-methylxanthine is used to measure NAT2 activity.19

Primary methods for analysis of 4-ABP adducts were limited to 32P-postlabelling assays,

immunoassays, and gas chromatography/mass spectrometry (GC/MS) of adducts in liver and

bladder biopsy samples or, more recently, in exfoliated urinary epithelial cell DNA.11,21,22, 27

Although 32P-postlabelling and immunochemical methods are sensitive and selective, with

reported detection limits of 1 adduct in 109 normal nucleosides, they exhibit specific limitations

that can be improved upon with liquid chromatography, coupled with tandem mass spectrometry

(LC/MS-MS).21 32P-postlabelling analysis is limited by the specific activity of the isotope used,

as well as the yield obtained from the labeling process.21 Fluorescence-based techniques, such as

CYP1A2

NAT2

16

HPLC coupled with fluorescence detection, though sensitive for select compounds, are limited

by lack of fluorescent properties for others.21 It is here that mass spectrometry advances small

molecule analysis, with its applications for chemical structure elucidation based on mass, charge,

and common fragmentation patterns exhibited by the compounds. The modified base dG-C8-4-

ABP gives a distinct fragmentation pattern of [M+H-116]+, for the loss of deoxyribose upon

collisional activation; therefore, it is easy to monitor the loss [435319] with MS-MS.23,27 The

advantage of LC/MS-MS is the structural information, and quantitation via peak integration in

the extracted ion chromatogram. The levels of 4-ABP DNA adducts in bladder and liver samples

have been successfully measured with the use of chromatographic separation and mass

spectrometry methods recently developed in our lab.27 The next objective was to establish a

method for the detection and quantitation of 4-ABP DNA adducts found in bladder epithelial

cells exfoliated in urine, which would be a much less invasive alternative to analysis of tissue

biopsies.

1.1 Roswell Park Cancer Institute Smoking Cessation Study

Prior studies24,25 have revealed potential correlation of increased levels of 4-ABP DNA

adducts to exposure to cigarette and tobacco smoke. Through the efforts of Roswell Park Cancer

Institute in Buffalo, NY, urine samples were collected from patients to be analyzed for levels of

4-ABP DNA adducts within parameters of a clinical smoking cessation study. These patients

donated urine samples on the date of cessation from smoking, as well as various other time

points surrounding that date (4, 2, and 1 week before, and 2, 5, 12, and 17 weeks after). Past

studies24-27 have cited limitations when working with DNA from exfoliated urinary epithelial

17

cells due to low and very inconsistent DNA yields, as well as complexities acquired from the

sample matrix. Inconsistency in DNA yield from cells found in urine is correlated to urine

storage conditions, gender of the donor, extent of bacterial contamination, and release of

nucleases from hydrolyzed cells in the sample.28 In addition, a considerable amount of

precipitate formation was observed following storage of urine samples at -80oC, which made

DNA isolation almost impossible in some cases. Therefore, prior to the commencement of this

clinical sample study it was necessary to make improvements to the current DNA isolation

procedure to address new obstacles due to urine sample complexity. A second objective of this

project was to ensure that the established nano-LC/ESI-MS/MS method used for tissues samples

is amenable to a urinary epithelial cell DNA sample matrix.

2. MATERIALS

A subset of 20 patient urine samples was provided by Roswell Park Cancer Institute

(Buffalo, NY). Calf thymus DNA, Nuclease P1 from Penicillium citrinium, deoxyribonuclease I

(DNase I) type 2 from bovine pancreas, alkaline phosphatase from Escherichia coli (type IIIs),

ethanol, magnesium chloride, zinc chloride, 1M Trizma® hydrochloride solution (Tris-HCl),

ethylenediaminetetraacetic acid (EDTA), dimethylsulfoxide (DMSO), 4-aminobiphenyl (4-ABP)

were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). Phosphodiesterase I

(Crotalus adamanteous venom) was purchased from USB Corporation (Cleveland, OH).

Sodium hydroxide pellets were purchased from Fisher (Pittsburgh, PA). Quant-ItTM double

strand (ds) DNA BR assay kit and a Qubit fluorometer were purchased from Invitrogen

Corporation (Carlsbad, CA). Glacial acetic acid, 99.99+% was obtained from Aldrich Chemical

18

Co. (Milwaukee, WI). HPLC grade solvents (methanol, water, acetonitrile) used for analysis

were purchased from Thermo Fisher Scientific (Pittsburgh, PA). N-(2-deoxyguanosin-8-yl)-4-

aminobiphenyl (dG-C8-4-ABP) was obtained from Toronto Research Chemicals (North York,

ON). The deuterium labeled form of dG-C8-4-ABP (dG-C8-4-ABP-d9) was previously

synthesized in our labs according to a reported synthesis protocol.29

3. URINARY EPITHELIAL DNA ISOLATION OPTIMIZATION

It has been observed that voided urine, although it is 96% water,30 is comprised of

complex mixtures of components, which can differ greatly between samples. These components

include different types of cells, organic and inorganic crystals, salts, pigments, mucus, yeast,

bacteria, and a variety of other possible contaminants that may interfere with DNA isolation

procedures. For purposes of this study, the most important constituent to focus on for analysis

are the cells from which the DNA is to be extracted. For a given urine sample, any and all of

these cells may be present: red blood cells, white blood cells, and exfoliated epithelial cells

(including renal tubular, transitional (urothelial), and squamous cells), shown in Figures 3.1-3.3.

The difference between the types of epithelial cells lies in the organ from which they originate.

Renal tubular cells exist in the kidneys, which are responsible for the creation of urine.

Transitional (urothelial) cells are found in the renal calyces, renal pelvis, ureter, bladder, and

distal portion of the male urethra, and few are typically present in urine samples.30,31 Squamous

cells, the largest and most common of these epithelial cells, line the entire length of the urethra in

females, and exclusively the distal portion of the urethra in males. It is very important to note

that in previous and current studies dealing with urothelial DNA isolation, a method to separate

19

the types of cells from one another has not been utilized. Therefore, any “urothelial” DNA

isolated from urine samples may have originated from any of these urinary epithelial cells.27,31

(3.1) (3.2)

Figure 3.1 shows a squamous cell (lower left) & transitional epithelial cell (upper right) and Figure 3.2 shows two squamous cells covered in bacterial contaminants31

(3.3)

Figure 3.3 shows a cluster of renal tubular cells31

Another class of urine constituents is organic and inorganic salt crystals that form in

cooled or frozen samples. Crystalline precipitates can also form as a result of urinary pH,

concentration of solutes in the urine, and restricted flow of urine through the urinary system.31 In

alkaline urinary pH conditions, commonly present crystals include calcium carbonate (Figure

3.4), amorphous phosphate, magnesium ammonium phosphate (MgNH4PO4) (Figure 3.5),

calcium phosphate, and ammonium biurate (Figure 3.6). When urinary pH is acidic, the most

X-Ray Contrast Media Crystals

EPITHELIAL CELtS

Figure 61 Two squamous epithelial cells covered with bacteria,

known as clue cells, and a single typical or normal squamous

epithelial cell. ln urine that has been contaminated with vaginal

secretions, clue cells may be observed. This is not a common

occurrence.

Figure 63 A squamous epithelial cell (lower left cell) and a

transitional epithelial cell (upper right cell). Note the similarity in

the size of their nuclei yet the difference in the amount ofcy"toplasm, that i1 different nucleus-to-cytoplasm ratios. Several

large rof shaped bacteria are also present.

Figure 60 X-ray contrast media, that is, diatrizoate meglumine(Renografin) crystals.

Figure 62 Three squamous epithelial cells and a

blood cell. Note the similarity in size between the whibcell and the nuclei of these epithelial cells.

Figure 64 A typical transitional epithelial cell and a

cast.

X-Ray Contrast Media Crystals

EPITHELIAL CELtS

Figure 61 Two squamous epithelial cells covered with bacteria,

known as clue cells, and a single typical or normal squamous

epithelial cell. ln urine that has been contaminated with vaginal

secretions, clue cells may be observed. This is not a common

occurrence.

Figure 63 A squamous epithelial cell (lower left cell) and a

transitional epithelial cell (upper right cell). Note the similarity in

the size of their nuclei yet the difference in the amount ofcy"toplasm, that i1 different nucleus-to-cytoplasm ratios. Several

large rof shaped bacteria are also present.

Figure 60 X-ray contrast media, that is, diatrizoate meglumine(Renografin) crystals.

Figure 62 Three squamous epithelial cells and a

blood cell. Note the similarity in size between the whibcell and the nuclei of these epithelial cells.

Figure 64 A typical transitional epithelial cell and a

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acid (an oxalate precursor), which is found in vegetables and citrus fruits. Oxalate ions associate

with calcium ions in the renal tubules to form calcium oxalate, which is excreted in urine. Some

of these crystals, such as uric acid, bilirubin, and ammonium biurate, exhibit an intrinsic yellow-

brown color, while some, such as amorphous urates, adopt their color from pigment deposits.

Uroerythrin pigments can form deposits on the surfaces of the crystals, giving them a pink-

orange color, which was observed in a number of the samples processed in this study.31

Figure 3.4 Calcium carbonate crystals31 Figure 3.5 Ammonium biurate crystals31

Figure 3.6 Magnesium ammonium phosphate crystals31

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(3.7) (3.8)

Figure 3.7 shows uric acid crystals in common diamond shape and Figure 3.8 shows uric acid crystals in cube or barrel form31

(3.9) (3.10)

Figure 3.9 shows a single calcium oxalate dihydrate Figure 3.10 shows cystine crystals, clustered and crystal (top left) and numerous calcium oxalate stacked31 monohydrate crystals31

Figure 3.11 Bilirubin crystals31

Casts, another possible urine sediment, are formed in the distal and collecting tubules of

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22

forms fibrils that attach to lumen cells after it is secreted. Uromodulin then incorporates other

substances, such as cellular inclusions, crystals, fatty materials, or pigments, into its matrix,

which creates its distinctive cylindrical shape. Other possible contaminants in a given urine

sample include yeast, fat, bacteria, fecal matter, food, starch, fibers, oil droplets, and

parasites.30,31

The current methods of DNA isolation from exfoliated urinary epithelial cells have

presented numerous issues, including low and inconsistent yields26 of DNA, formation of

crystalline precipitates following storage of urine samples at -80oC, clogging of Qiagen DNA

purification columns, and poor solubility of urothelial DNA in various digestion buffers. One

previous study32 reports a DNA yield at concentrations of 2-96 µg/L urine (0.002-0.096 µg/mL).

These claims have been corroborated in this study, as an average of 2.11 µg DNA could be

isolated from 100-900 mL urine, and in some cases, DNA could not be recovered at all. Also,

experimental data show no correlation between the volume of urine collected and the DNA yield

(Figure 3.12). Another observation made was in regards to DNA recoveries from male versus

female sources. As shown in Figure 3.13, female urine samples have a tendency to give higher

yields than male samples, which was affirmed in previous studies.32,33 In a small study of 26

samples (11 female, 15 male), the female samples gave yields of 0-14 µg DNA (average yield of

4.155 µg), and the male samples gave yields of 0-0.281 µg (average yield of 0.033 µg). A

possible explanation for this is the area of origin for squamous cells (the most common type of

urinary epithelial cell). Since squamous cells line the entire urethra in females, but only the

distal portion in males,30,31 the area of squamous cell origin is larger for females. Therefore,

there is a larger possibility for the presence of squamous cells in female urine specimens. These

are significant issues because the current analysis requires 2 µg DNA for digestion and analysis

23

by nano-LC/ESI-MS/MS. Therefore, efforts to optimize DNA yield were made wherever

possible.

Figure 3.12 There was no observable correlation between the volume of urine sample and the amount of DNA recovered.

Female Samples Male Samples

Volume Urine (mL) DNA Yield (µg) Volume Urine (mL) DNA Yield (µg) 226 0 516 0 100 1.860 480 0 100 1.880 580 0.043 250 3.613 280 0 510 4.886 280 0 240 0.031 280 0 310 2.740 280 0 183 2.210 530 0 155 5.260 350 0.281 212 9.220 285 0.057 314 14.000 120 0

420 0 450 0.035 560 0 210 0.085

Average yield: 4.155 Average Yield: 0.033

Figure 3.13 shows DNA recovery results for a small subset of male vs female urine samples. Recovery for female samples ranged from 0-14 µg (average 4.155 µg), and recovery for male samples ranged from 0-0.281 µg (average 0.033 µg). There was no observable correlation between the volume of urine sample and the amount of DNA recovered.

051015202530354045

0 200 400 600 800 1000

DN

A is

olat

ed (u

g)

Volume Urine (mL)

Total DNA (ug) vs Volume Urine (mL)

24

It was discovered that samples stored at -80oC were forming crystalline precipitates,

which may be damaging to the cells26 upon initial centrifugation of urine, the most common of

which are calcium oxalate, uric acid, and magnesium ammonium phosphate (MgNH4PO4).24,26

In addition to the possibility of damaging the cells, these precipitates were observed to cause

clogging of the Qiagen purification columns. Therefore, changes to the protocol were made to

dissolve crystals prior to centrifugation.

The initial effort to remedy this problem was filtering34 the urine through filter paper

(Fisherbrand Q5, 1-5 µm particle retention), capturing the cells on the filter, and releasing them

with PBS buffer. This unfortunately did not produce any difference between filtered and

unfiltered samples, which is in agreement with the findings of an earlier publication.32 Previous

studies have reported the use of sucrose buffers24, 26,34 and the use of 5-250 mM EDTA buffers35-

38 to wash cells after centrifugation. EDTA has been shown to exhibit chelating properties with

heavy metals,39 giving it many uses in removing impurities due to metal ions. In this case,

electron-rich groups of EDTA chelate with positively charged ions involved in the precipitating

salts. For example, calcium oxalate salts are not readily soluble in water, but adding EDTA can

remove the calcium from the salt, aiding in solublization of the oxalate ion.37 It was

experimentally observed (Figure 3.14) that treatment with a 5 mM EDTA and 5 mM EDTA/125

mM sucrose buffer significantly reduced the amount of crystalline precipitate in the cell pellets

and, although this reduced clogging of purification columns, it was not found to increase the

yield of DNA. It was also found that there was no difference between using 5 mM EDTA and 5

mM EDTA/125 mM sucrose. Therefore 5 mM EDTA was used for the duration of the study.

25

Figure 3.14 represents a thawed 200 mL urine sample, split equally by volume—one untreated (left), and one treated with 5 mM EDTA (right), prior to centrifugation. EDTA treatment has been shown to significantly reduce the amount of crystalline precipitate in the resulting cell pellet.

Another option available for method development is the size of Qiagen purification

columns. They are available in three sizes for a variety of DNA isolation capacities. The

suggested isolation ranges for 500/G, 100/G, and 20/G columns are 80-500 µg, 10-100 µg, and

1-20 µg, respectively. Although most of the samples in this study would be producing <1 µg -

20 µg DNA, the 100/G column was chosen, to accommodate all of the extra components that

may be present in the samples, and to reduce column clogging. Each of these columns was

tested in parallel to determine the optimal column size for this study. One sample was split into

three cell pellets, which were then lysed, digested, and purified in buffer volumes corresponding

to each column size.40

Another area that warranted optimization is the buffer used to resolublize the DNA once

it has been isolated. Previously, DNA was dissolved in the 5 mM Tris-HCl/10 mM ZnCl2 buffer

used for DNA digestion, but resuspending in this buffer proved to be difficult. It was discovered

26

that TE buffer (10 mM Tris-Cl/1 mM EDTA) solubilized the isolated DNA with much success,

and therefore was used for the duration of the study.

3.1 Urinary Epithelial Cell DNA Isolation Protocol

Urine samples were collected and stored in bulk at -80 oC until analysis. Samples were

thawed at room temperature or in a room temperature water bath, and pipetted in 25 mL volumes

to 50 mL Falcon tubes with 25 mL 5 mM EDTA, to solublize crystalline precipitates formed

during storage. (For samples that were clear upon thawing, 10 mL 5 mM EDTA with 40 mL

urine was sufficient to solublize precipitates.) Samples were centrifuged at 5,000 x g for 10

minutes at 4 oC (Thermo Scientific, Sorvall RT-1). The supernatant was decanted, and the

process was repeated until all urine was centrifuged. Cell pellets were washed 1x with 10 mL

cold PBS (1x phosphate buffered saline), centrifuged at 5,000 x g for 10 minutes at 4 oC, and

resuspended in 2 mL PBS for cell lysis.

From this point, protocols for cell lysis and DNA isolation were followed as outlined in

the Qiagen Blood and Cell Culture DNA Isolation Midi-kit.40 To each cell pellet was added 2

mL C1 Buffer (Qiagen DNA Isolation kit—1.28 M sucrose, 40 mM Tris-Cl, pH 7.5; 20 mM

MgCl2, 4% Triton X-100) and 6 mL deionized water, which were both kept on ice for the

duration of the lysis protocol. Tubes were inverted 10-15 times to mix, incubated on ice for 10

minutes, and centrifuged at 1,300 x g for 15 minutes at 4 oC. Nuclear pellets were washed with 1

mL C1 buffer and 3 mL deionized water, and centrifuged at 1,300 x g for 15 minutes at 4 oC.

Nuclear pellets were resuspended in 5 mL G2 buffer (Qiagen DNA Isolation kit—800

mM guanidine HCl, 30 mM Tris-Cl, pH 8.0; 30 mM EDTA, pH 8.0; 5% Tween-20, 0.5% Triton

27

X-100), and vortexed for 10-30 seconds. Qiagen Protease (95 µL) was added, and samples were

incubated at 55 oC for 1 hour.

Purification was carried out under positive pressure to streamline the process. The

Qiagen anion exchange columns contain positively charged diethylaminoethyl (DEAE) resin,

which captures the negatively charged phosphate groups of DNA. Impurities were washed

away, and DNA was eluted with an increase in salt content and pH of the buffers. Columns were

first equilibrated with 4 mL QBT buffer (Qiagen—750 mM NaCl, 50 mM MOPS, pH 7.0; 15%

isopropanol, 0.15% Triton X-100). QBT buffer (5mL) was added to samples to further reduce

column clogging, samples were vortexed at maximum setting for 10 seconds, and added directly

to the Qiagen columns. Once the sample was loaded, columns were washed twice with 7.5 mL

QC buffer (Qiagen—1.0 M NaCl, 50 mM MOPS, pH 7.0; 15% isopropanol). DNA was eluted

with 5 mL QF buffer at 50 oC (Qiagen—1.25 M NaCl, 50 mM MOPS, pH 8.5; 15%

isopropanol), which was collected in 50mL Falcon tubes.

Room temperature isopropanol (3.5mL) was added to the eluent to precipitate DNA.

Tubes were centrifuged at maximum speed (10,000 x g) for 15 minutes at 4 oC to pellet DNA.

DNA pellets were transferred to 1.5 mL Eppendorf tubes with 1.4 mL cold 70% EtOH.

Eppendorf tubes were centrifuged at maximum speed (14,600 x g) for 15 minutes at 4 oC to re-

pellet DNA. Supernatant was carefully removed with a pipette and discarded. Isolated DNA

was resuspended in 20 µL TE buffer (10 mM Tris-HCl / 1 mM EDTA, pH 8.0) for enzymatic

digestion.

DNA samples were quantified with a Quant-ItTM double strand (ds) DNA BR assay kit

with a Qubit fluorometer (Invitrogen Corporation, Carlsbad, CA).

28

3.2 DNA Digestion and Preparation for Analysis by nano-LC/ESI-MS/MS

The DNA digestion procedure utilizes four different enzymes—three nucleases to digest

the DNA into phosphonucleotides, and alkaline phosphatase to remove the phosphate groups.

DNase I (endonuclease) and Phosphodiesterase I (exonuclease) yield 5’-phosphonucleotides

from DNA. Nuclease P1 is also a nuclease that forms 5’-phosphonucleotides, but it will digest

RNA as well as single-stranded DNA.41

Urinary epithelial cell DNA (2 µg) was taken from each sample for enzymatic digestion.

An equal volume of 20 mM ZnCl2 was added to each sample, because both Nuclease P1 and

alkaline phosphatase require Zn2+ for activation.42 To each sample was added 2 µg DNase1 and

2 µg Nuclease P1, followed by incubation at 37 oC for 5 hours. Phosphodiesterase I (0.01 units)

and alkaline phosphatase (0.0024 units) were added, and the samples were incubated for 18

hours at 37 oC. Prior to protein precipitation, 12 fmol internal standard (dG-C8-4-ABP-d9) was

added to each sample. Proteins were precipitated with 5 volumes of ice cold 100% ethanol,

followed by centrifugation at 7,500 x g for 15 minutes at 4 oC. Supernatant was transferred to a

new 1.5 mL Axygen low-bind Eppendorf tube, and samples were dried under vacuum (Savant

Speed Vac, SC110). Samples were reconstituted in 20 µL 15% methanol just prior to analysis.

Samples not analyzed directly following digestion were stored dry at -80 oC.

3.3 Preparation of dG-C8-4-ABP Standard Calibration Curves

Calibration curves were prepared both (neat) and in calf thymus DNA matrix. The neat

calibration curve was prepared with 12 fmol internal standard (dG-C8-4-ABP-d9), along with

29

standard dG-C8-4-ABP calibration solutions (final sample concentrations of 0.028, 0.056, 0.11,

0.22, 0.45, 0.9, 1.8, 3.6, 7.2, and 14 fmol/µL). Calf thymus DNA was digested in bulk and

aliquotted in 2 µg per sample for the calibration curve to be used for quantitation. Protein

precipitation was carried out as described above, and samples were reconstituted in 20 µL 15%

methanol just prior to analysis. Since samples were analyzed in 5 µL volumes, this would

correspond to 0.14, 0.28, 0.56, 1.1, 2.2, 4.5, 9, 18, 36, and 72 fmol dG-C8-4-ABP, and 3 fmol

dG-C8-4-ABP-d9 on column for analysis. The calf thymus DNA calibration curve was prepared

in triplicate for precision.

A smaller set of calibration points was prepared in urinary epithelial cell DNA matrix for

comparison and to establish sensitivity. Isolated urothelial DNA (8 µg) was digested following

the procedure described above, and was aliquotted into four 2 µg samples. Internal standard dG-

C8-4-ABP-d9 (12 fmol) was added to each sample, along with 0.028, 0.11, 0.44, and 1.8 fmol/µL

dG-C8-4-ABP calibration points (0.14, 0.56, 2.2, and 9 fmol on column). These samples were

analyzed and compared to corresponding calf thymus DNA calibration points.

4. NANO-LC/ESI-MS/MS ANALYSIS OF dG-C8-4-ABP

Liquid chromatography was carried out via ChemStation for LC3D Systems on an

Agilent 1100 Series system (Agilent Technologies, Wilmington, DE), equipped with nano pump,

capillary pump, 1200 Series micro well-plate autosampler, and Chip Cube interface. Liquid

chromatography separations were carried out on an Agilent Technologies small molecule

microfluidic chip with a 40 nL trap and 75 µm i.d. x 43 mm analytical column of reversed phase

ZorbaxSB-C18 (5 µm particles). Samples (5 µL) were loaded via capillary pump at a flow rate

30

of 4 µL/min, followed by a 4 minute wash with 93% mobile phase A (3% ACN, 0.1% acetic acid

in water)/ 7% mobile phase B (0.1% acetic acid in methanol). Analytical separations were

facilitated by the nano pump, which held a flow rate of 300 nL/min. The gradient was held at

90% A (0.1% acetic acid in water) / 10% B (0.1% acetic acid in methanol) for 4.21 minutes. It

was increased on a linear scale to 90% B over 2 minutes, held at 90% B for 2 minutes, and

stepped back down to 10% B for 5 minutes for a post-run equilibration period. While the nano

pump carries out analytical separations, the system switches to bypass mode, and employs a

needle wash program, and is then held at 90% B (0.1% acetic acid in methanol) for 3 minutes to

wash the autosampler components in preparation for the next injection.

Since these samples in DNA matrix are very complex, additional precautions27 were

taken to reduce sample carryover. First, a 0.5 µm inline pre-column filter between the needle

seat and Chip Cube interface was utilized. Additionally, 3 blank samples (1x 100% methanol

and 2x 15% methanol) were run between each sample to wash the column to reduce carryover.

Also, the method implements a needle wash protocol that washes the needle seat, needle seat

capillary, and rotor seal sequentially with acetonitrile, methanol, and water. Finally, the Agilent

microfluidics chip (Figure 4.1) employs a dual column for enrichment and subsequent analysis

for online sample cleanup.

31

Figure 4.1 The Agilent microfluidics chip used for nano-LC/ESI-MS/MS analysis (courtesy of Agilent Technologies). The diagram shows the chip in enrichment mode, in which the sample is loaded from the micro well plate autosampler, and salts are washed through to waste. In analysis mode, the nano pump facilitates the solvent gradient through the analytical column, which is connected to the mass spectrometer.

Mass spectrometry was carried out via an Agilent 6330 Ion Trap mass spectrometer

(Santa Clara, CA), which was controlled by 6300 Series Ion Trap HPLC-MS Software (version

6.1) and 6300 Series Ion Trap Control Software (version 6.1) acquired from Bruker Daltonik

GmbH (Bremen, Germany). N2 drying gas was set to 3.0 L/min at a temperature of 325 oC for

analyses. The capillary voltage was set at -1950 V with an end-plate offset of -500 V. Ion optics

and trap parameters were as outlined here: skimmer 1: 40.0 V, capillary exit: 200 V, trap drive:

46.5 V, ultra scan mode (24,000 m/z per scan), and positive ion mode. Ion charge control

parameters were as follows: maximum accumulation of 50 ms or 500,000 ions and 3 spectral

averages per scan. [CID pressure was 3mT He and voltage ramped from 0.45-3 V.] MS/MS

spectra were collected within a 290-475 m/z scan window, with precursor ions 435.4 ± 1.0 and

444.0 ± 1.5 Da isolated and fragmented with fragmentation cutoff of 27% of precursor ion mass.

Data processing was completed with Quant Analysis (version 1.8) and Data Analysis

(version 3.4) for 6300 Series Ion Trap LC/MS (Bruker Daltonik GmbH). For each sample, the

extracted ion chromatograms were used to monitor the loss of deoxyribose [M+H-116]+. For the

Please cite this article in press as: K.L. Randall, et al., J. Chromatogr. A (2009), doi:10.1016/j.chroma.2009.11.006

ARTICLE IN PRESSG Model

CHROMA-350552; No. of Pages 9

4 K.L. Randall et al. / J. Chromatogr. A xxx (2009) xxx–xxx

Fig. 1. HPLC–MS of dG-C8-4-ABP standards. (A) The chemical structure of the dG-C8-4-ABP adduct is shown. The dG-C8-4-ABP-d9 internal standard is isotopically labeledwith deuterium around the biphenyl moiety. The extracted ion chromatograms (435 ! 319 and 444 ! 328 for the analyte and IS respectively) of 1.89 fmol dG-C8-4-ABP and1.74 fmol IS are shown above. Retention time for the standard and the internal standard is 8.9 min. (B) MS/MS spectrum of dG-C8-4-ABP. (C) MS/MS spectrum of internalstandard.

characteristic loss of deoxyribose, [M+H]+ ! [M+H"116]+. Follow-ing infusion, both the dG-C8-4-ABP standard and dG-C8-4-ABP-d9internal standard were injected on column and the retention timewas determined to be 8.9 min. The chemical structures, extractedion chromatograms and MS/MS spectra of 1.89 fmol standard and1.74 fmol internal standard are displayed in Fig. 1.

3.2. Online adduct enrichment

Sample preparation can have a substantial impact on sensitiv-ity in the quantification of DNA adducts by mass spectrometry[17]. The online sample clean-up method presented here elimi-nates the need for solid-phase extraction (SPE), reducing samplepreparation time, sample handling, and analyte loss as well aseliminating the introduction of SPE related artifacts which cancause ion suppression. The microfluidic chip (Fig. 2) used in this

method incorporates both a trap and analytical column, enablingthe online adduct enrichment step. The sample was loaded ontothe 40 nL trap column and salts and unmodified nucleosides werewashed off for 4 min before placing the trap column inline withthe analytical column during analysis mode. The analyte was thenwashed from the trap column to the analytical column for separa-tion.

3.3. dG-C8-4-ABP stability assessment during heat treatment

Samples are subject to heat treatment during the digestion pro-cedure to denature the DNA for efficient hydrolysis by NucleaseP1. Therefore, to determine the stability of 4-ABP adducts towardsheat treatment at 98 #C for 3 min, we compared standards that wereexposed to heat treatment with standards that were not. Four setsof triplicate mixtures were evaluated in which 8.4 fmol of dG-C8-4-

Fig. 2. A detailed view of the Agilent microfluidic chip used in our online sample enrichment method (courtesy of Agilent Technologies, Inc.). The chip integrates a 40 nLtrap column, a 75 !m i.d. $ 43 !m analytical column of reverse phase Zorbax SB-C18 and 5 !m particle size, and a nanospray emitter. The chip is shown in enrichment modeduring which the sample was washed with 7% methanol, removing unwanted salts and excess unmodified nucleosides. In analysis mode, the analyte was moved from thetrap column to the analytical column for separation.

32

dG-C8-4-ABP analyte, fragmentation was 435319, and for the deuterium labeled internal

standard, 444328, as shown in the MS/MS spectra in Figure 4.2. Peaks were smoothed with

Gaussian function (peak width set at 1.29 seconds), manually integrated, and the ratios of 4-ABP

analyte to internal standard were calculated (Figure 4.3).

Figure 4.2 Fragmentation of dG-C8-4-ABP standard (435319), and deuterated dG-C8-4-ABP-d9 internal standard (444328), respectively. Both exhibit loss of 116, for the loss of the deoxyribose moiety.

31

dG-C8-4-ABP analyte, fragmentation was 435!319, and for the deuterium labeled internal

standard, 444!328, as shown in the MS/MS spectra in Figure 4.2. Peaks were smoothed with

Gaussian function (peak width set at 1.29 seconds), manually integrated, and the ratios of 4-ABP

analyte to internal standard were calculated (Figure 4.3).

Figure 4.2 Fragmentation of dG-C8-4-ABP standard (435!319), and deuterated dG-C8-4-ABP-d9 internal standard

(444!328), respectively. Both exhibit loss of 116, for the loss of the deoxyribose moiety.

33

Figure 4.3 Extracted ion chromatograms for dG-C8-4-ABP standard (435319) and internal standard (444328), both exhibiting a retention time of ~8.9 minutes. This is the neat system suitability point, with 1.1 fmol dG-C8-4-ABP standard and 3 fmol dG-C8-4-ABP-d9 on column.

5. RESULTS AND DISCUSSION

5.1 Urinary Epithelial Cell DNA Isolation from Roswell Park Samples

Figure 5.1 shows the total DNA yield from the subset of 20 blind clinical samples sent

from Roswell Park Cancer Institute. These 20 samples represent urine collections from 10

patients, with 2 time points each. Other than this basic identification, no other information

regarding these samples was provided. As shown, 10 of the 20 samples gave yields above the 2

ng detection limit, and of these, 5 (highlighted in red) were viable for DNA digestion and

analysis for dG-C8-4-ABP DNA adducts.

34

Figure 5.1 Urinary epithelial cell DNA yields for a subset of Roswell Park Cancer Institute samples. Ten of the 20 samples gave yields above the detection limit of 2 ng (by Qubit fluorometer), and 5 of these (in red) gave sufficient yields for 2 µg DNA digestion and analysis.

5.2 Calibration Curves, Limit of Detection, and Limit of Quantitation

The neat curve for the amount of dG-C8-4-ABP (0.140-71.7 fmol) versus the mean

analyte to internal standard peak area ratio is shown in Figure 5.2. (Figure 5.3 shows the lower

range of detection, from 0.140 fmol to 2.24 fmol). Microsoft Excel was used to generate a linear

regression analysis for a least squares fit to the line y=mx+b, the calculated values are as follows:

slope= 0.3289, y-intercept= 0.1701, and correlation coefficient R2= 0.9913. The limit of

detection (LOD) for this study is 0.140 fmol (~9 adduct in 108 normal nucleosides) with a signal

to noise ratio of 3, and the limit of quantitation (LOQ) is 0.560 fmol (37 adducts in 108 normal

nucleosides), with a signal to noise ratio of 10. All of the points in the neat curve have %RSD

below 35%.

Patient Collection #1 Collection #6

Urine Vol (mL) DNA Yield (ug) Urine Volume (mL) DNA Yield (ug)

A 160 - 222 -

B 240 0.0286 122 -

C 240 8.02 145 17.6

D 134 7.66 137 1.25

E 152 - 185 0.0328

F 342 - 158 -

G 167 0.184 213 0.039

H 240 - 214 -

I 96 0.0512 80 -

J 240 32.96 259 5.86

35

36

The standard curve for the amount of dG-C8-4-ABP (0.140 – 71.7 fmol) versus the mean

analyte to internal standard peak area ratio in calf thymus DNA digest matrix is shown in Figure

5.4. (Figure 5.5 shows the lower range of detection, from 0.140 to 2.24 fmol.) Again, Microsoft

Excel was used to generate a linear regression analysis for a least squares fit to the line y=mx+b,

and error ranges based on the standard deviation are shown. The values derived from the

standard curve are as follows: slope= 0.9364, y-intercept= -0.573, and correlation coefficient R2=

0.9931. The limit of detection (LOD) for this study is 0.140 fmol (~9 adduct in 108 normal

nucleosides) with a signal to noise ratio of 3, and the limit of quantitation (LOQ) is 0.560 fmol

(37 adducts in 108 normal nucleosides), with a signal to noise ratio of 10. All points in the calf

thymus DNA digest calibration curve have %RSD below 22%.

To demonstrate the feasibility of the detection of dG-C8-4-ABP adducts in human

urinary epithelial cell DNA, four selected points (0.028, 0.11, 0.44, and 1.8 fmol/µL) of the

calibration curve were spiked into isolated DNA from human samples, along with 12 fmol

internal standard. Results, shown in Figure 5.6 and Figure 5.7, were comparable to the calf

thymus DNA calibration curve. The LOD and LOQ were retained—0.140 fmol (9 adducts in

108 normal nucleosides), and 0.560 fmol (37 adducts in 108 normal nucleosides), respectively.

These points also exhibit comparable linearity and precision (under 22% RSD).

37

38

39

5.3 Preliminary Results from Roswell Park Cancer Institute Samples

Five of the clinical samples from Roswell Park Cancer Institute were viable for 2 µg

DNA digestion and analysis using this method. Preliminary results from these samples show no

evidence of dG-C8-4-ABP DNA adducts (Figure 5.8). Since this first subset of samples from

Roswell Park Cancer institute was a blind study for our lab, it was not known if DNA adduct

detection was expected. It is not inconceivable that these may have been control samples. To

further validate the detection method, 1.1 fmol of standard dG-C8-4-ABP was spiked into the

same clinical sample DNA digest, and it was detected at the expected level (Figure 5.9).

Figure 5.8 Extracted ion chromatogram for a 0.5 µg injection of urinary epithelial DNA digest from a Roswell Park Cancer Institute clinical sample (Patient J, Collection 1), with 3 fmol internal standard. A strong internal standard peak (red) is detected, and dG-C8-4-ABP DNA adduct (blue) was not detected.

Figure 5.9 Extracted ion chromatogram for a 0.5 µg injection of urinary epithelial DNA digest from a Roswell Park Cancer Institute clinical sample (Patient J, Collection 1), with 3 fmol internal standard (red) and 1.1 fmol standard dG-C8-4-ABP (blue).

40

6. CONCLUSION

In summary, this study has produced an optimized sample preparation method for the

isolation of urinary epithelial cell DNA for the analysis of 4-ABP DNA adducts by nano-

LC/ESI-MS/MS. It has been shown that the observed crystalline precipitates formed due to

frozen storage conditions were significantly reduced with treatment of thawed urine samples

with 5 mM EDTA. This, in turn, reduced clogging of the Qiagen DNA isolation columns, and

allowed DNA to be isolated from urine samples in which it was previously unobtainable. This

study has also shown that the detection and quantitation of dG-C8-4-ABP in digested tissue

DNA matrix by the previously established27 nano-LC/ESI-MS/MS method is transferrable to

urinary epithelial cell DNA matrix. DNA isolation from Roswell Park Cancer Institute samples

has commenced, and initial analysis of these samples shows no detection of dG-C8-4-ABP

adducts. Analysis of the remaining patient DNA samples for adducts is currently in progress.

Moving forward, there are a few points to take into consideration for future studies

involving urinary epithelial cell DNA. First, although the issue of formation of crystalline

precipitates has been alleviated in this study, DNA yields from urinary epithelial cells remain

quite variable. Each individual sample contains a variety of urine components outlined in

Section 3, levels of which are heavily dependent on the urine donor. Some samples may not

contain any (or enough) cellular DNA for extraction, and therefore cannot be analyzed.

Additionally, bacterial contamination and storage conditions of the samples are causes for

concern.

Second, if a specific organ is the desired target for biomarker monitoring, one must take

into the consideration the origin of the cells pelleted from urine samples. There are three types

of epithelial cells found in urine, all of which originate from different areas of the urinary

41

system. In this study, though the bladder epithelium is the target, any 4-ABP DNA adducts

found in kidney and urethra epithelial cells may also be detected. Therefore, separating these

epithelial cells may be the next worthy challenge for these clinical studies.

7. APPENDIX – LC/MS SYSTEM TROUBLESHOOTING

As with any analytical study, progress is largely dependent on the performance of the

analytical system being utilized. The analytical portion of this project was somewhat hindered

by various instances of LC/MS system maintenance and repairs. This section will outline steps

taken to remedy a particular system pressure issue in regards to the dual pump system

configuration employed in this study.

The HPLC System

In the current method, liquid chromatography is carried out on an Agilent 1100 Series

system, with a capillary pump, nano pump, 1200 Series autosampler, and chip cube interface, as

shown in Figure 7.1. (Refer to Section 4 for specifications regarding the Agilent microfluidics

chip and solvent gradients.) For this section, the focus is directed to the dual pump system

configuration, and how it played a role in troubleshooting an abnormal system pressure issue.

42

Figure 7.1 Full LC/MS system schematic, including the autosampler (with focus on the 6-port valve arrangement), dual capillary/nano pump system, and Chip Cube configuration. Sample injections travel from the needle seat (Port 5), to the Chip Cube (through Port 6) for enrichment/separation, followed by mass spectrometric analysis (via Ion Trap system).

Solvent flow from the capillary pump is facilitated through the 6-port injection valve

located in the autosampler, which has two operational modes—bypass and mainpass (Figure

7.2). When it is in mainpass mode, solvent flows from the capillary pump (Port 1) to the

metering device (through Port 2), and sample from the needle seat (Port 5) flows through Port 6,

to the Chip Cube. When in bypass mode, solvent flows from the capillary pump (Port 1) to the

Chip Cube through Port 6, and waste from the needle seat flows through Port 5, and out through

the waste line in Port 4. While the system is in bypass mode, a programmed needle wash is

implemented while the nano pump independently carries out analytical separation on the C18

column.

43

(Mainpass)

(Bypass) Figure 7.2 Autosampler 6-port injection valve configurations, showing both mainpass (top) and bypass (bottom) flow paths. When in mainpass, solvent flows from the capillary pump (Port 1) to the metering device (through Port 2), and sample from the needle seat (Port 5) flows through Port 6, to the Chip Cube. When in bypass, solvent flows from the capillary pump (Port 1) to the Chip Cube through Port 6, and waste from the needle seat flows through Port 5, and out to the waste line in Port 4. (Images courtesy of Agilent Technologies 1200 Series High Performance Autosamplers and Micro Well Plate Autosampler manual)

44

The HPLC Problem The system began to experience unexpected automatic shutdowns due to “unstable

column flow” errors. It was at this time that the capillary pump pressure trace began to exhibit a

very unique reproducible pressure profile (Figure 7.3), with both blanks and sample injections.

The system recognized the sharp increases and decreases in pressure, causing it to abort the

sample run. If a queue was set up, the system randomly aborted the run at some point during the

queue, in no observable pattern with respect to the number of samples completed before the shut

down. The chromatography in these runs was negatively affected, as the sample peaks were

obscured, or not detected at all. (Figures 7.4 and 7.5 show extracted ion chromatograms for a

benzo(a)pyrene standard, from the system in normal functionality and when it was experiencing

these pressure issues, respectively).

Figure 7.3 Capillary Pump pressure profile (red), which caused the “unstable column flow” error

45

Figure 7.4 Extracted ion chromatogram of benzo(a)pyrene standard—when system was in normal functionality

Figure 7.5 Extracted ion chromatogram of benzo(a)pyrene standard, negatively affected by pressure issue

Troubleshooting The initial hypothesis for the cause of the unusual pressures was clogging of the capillary

sample lines or chromatography column. The Agilent microfluidics chip was exchanged, along

with the sample capillaries, and the system was backflushed in the attempt to remove any

residual particulates clogging the system, but no change was observed. Over the next three

months various hardware components, including the EMPV valve, in-line filter, flow sensor,

filter assembly, metering device, rotor seal, needle seat, seat capillary, and sample loop were

replaced, but it did not lead to lasting improvement.

It was not until all of the autosampler motions were meticulously studied, in relation to

the pressure profile, that a connection was made to the needle wash program that is unique to the

system. Once the sample is injected onto the chip column and the 4-minute enrichment period

concludes, the needle wash program is initialized and runs simultaneously as the sample is

analyzed. The specific needle wash program includes drawing solvent from three separate wash

46

solvent vials (100% acetonitrile, 100% methanol, and 100% water), multiple times (seven

washes total). After each draw into the sample loop, the needle ejects the solvent into the needle

seat, where it travels to a separate waste line (Port 4). It was observed that each time the solvent

was ejected into the needle seat, the pressure sharply increased, and as it drew new solvent from

each vial, the pressure dropped (Figure 7.6). To test the influence of the needle wash program, a

blank sample was run without the needle wash. The disappearance of the pressure spikes in the

capillary pump pressure profile proves that the wash program was involved somehow, either

directly or indirectly (Figure 7.7).

Figure 7.6 Pressure spikes are consistent with solvent draws and ejections during needle wash program.

Figure 7.7 Capillary pump pressure profile (red) observed when system ran a blank sample without the needle wash program. The characteristic pressure spikes disappear when the needle wash is not used.

!"#$%&%'#()%

!"#$%*%'#()%

!"#$%+%'#()%

,#-.$/%"01/2340%

47

This observation prompted the inspection of the valve assembly for malfunctions. It was

suggested that the switching between bypass and mainpass modes was not occurring properly,

which prompted the replacement of the entire internal injector valve assembly, except the 6-port

face. This replacement seemed to remedy the capillary pressure profile problem, so sample

analysis resumed. Unfortunately, over the next few days, the seven characteristic pressure peaks

gradually returned (Figure 7.8).

Figure 7.8 After the injector valve assembly replacement, the capillary pump pressure (blue) seemed to improve, but it gradually declined into its previous unusual pressure profile. The reappearance of the same pressure profile was a cue to reconsider anything that could

be contributing to the pressure readings, including, but not limited to, the autosampler assembly,

EMPV unit, sample lines, solvent lines, and waste lines. All of these had recently been replaced

or serviced except for the needle seat waste line in Port 4. A clogged needle seat waste line

48

would, in theory, correlate to the pressure spikes observed as the needle tried to eject needle

wash solvent into the waste line. Upon attempting to exchange the capillary in Port 4, the PEEK

fitting broke off, and half remained jammed, in the stator face. All of the current fittings and

capillaries were transferred to a gently used stator face, and a new PEEK fitting and waste line

capillary were fitted into Port 4. The results from this change were significant improvements to

the pressure profile (Figure 7.9). An improvement in sample chromatography was also observed

(Figure 7.10).

Figure 7.9 Improved capillary pump pressure profile (blue). The sharp increases from the needle wash solvent ejections disappeared. The pressure profile is reproducible, with an increased overall stability.

Figure 7.10 Improved sample chromatography after Port 4 waste line capillary and PEEK fitting replacements.

49

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