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Determination of Xanthine and Uric Acid in Xanthinuric Urine and Extracellular Fluid of Porcine Endothelial Cells of the...

Permanent Link: http://ufdc.ufl.edu/UFE0021781/00001

Material Information

Title: Determination of Xanthine and Uric Acid in Xanthinuric Urine and Extracellular Fluid of Porcine Endothelial Cells of the Pulmonary Artery by High Performance Liquid Chromatography
Physical Description: 1 online resource (107 p.)
Language: english
Creator: Affum, Andrews O
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: analytical, biofluid, buffer, calibration, catabolic, cells, clinical, detection, endothelial, extracellular, hplc, linear, lod, methanol, pathway, phosphate, physiological, potassium, pump, purine, purines, selectivity, sensitivity, ultraviolet, uric, urine, water, wavelength, xanthine, xanthinuric
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A reverse phase high performance liquid chromatography method with ultraviolet light detection was developed to determine the concentrations of xanthine and uric acid in the extracellular fluid of endothelial cells of porcine pulmonary arteries and in normal and xanthinuric urine. Normal urine samples were collected randomly and filtered through a 0.45 ?m nylon filter before analysis. Xanthine and uric acid concentrations were determined by a constant volume standard addition method. The mobile phase was 20 mM KH2PO4 at pH 5.1 and the detection wavelength 270 nm (xanthine) and 293 nm (uric acid). The injection volume was 20.0 ?L and attenuation was 0.01 absorbance unit full scale (AUFS). Retention times for xanthine and uric acid standards were respectively 13 ? 0.1 minutes and 7.0 ? 0.1 minutes. The linear correlation coefficient for xanthine and uric acid working curves were 0.995 and 0.998 respectively. The linear dynamic range, at the low concentration limits of xanthine and uric acid, was 5 ?M to 40 ?M, and 2 ?M to 20 ?M, respectively. The sensitivity of xanthine in 31 mM Na2HPO4/NaH2PO4 at pH 7.4 was 0.08 AU/?M, whilst that of uric acid in the same physiological buffer was 0.21 AU/?M. The limit of detection (LOD) for xanthine and uric acid were respectively 5.1 ?M, (S/N = 2) and 1.6 ?M (S/N = 3). Xanthine in xanthinuric urine was 2.8 ? 0.1 mM; while uric acid in normal urine was 5.7 ? 0.1 mM. In the extracellular fluid, the oxypurine peaks were identified as uric acid, hypoxanthine and xanthine.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Andrews O Affum.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Brajter-Toth, Anna F.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021781:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021781/00001

Material Information

Title: Determination of Xanthine and Uric Acid in Xanthinuric Urine and Extracellular Fluid of Porcine Endothelial Cells of the Pulmonary Artery by High Performance Liquid Chromatography
Physical Description: 1 online resource (107 p.)
Language: english
Creator: Affum, Andrews O
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: analytical, biofluid, buffer, calibration, catabolic, cells, clinical, detection, endothelial, extracellular, hplc, linear, lod, methanol, pathway, phosphate, physiological, potassium, pump, purine, purines, selectivity, sensitivity, ultraviolet, uric, urine, water, wavelength, xanthine, xanthinuric
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A reverse phase high performance liquid chromatography method with ultraviolet light detection was developed to determine the concentrations of xanthine and uric acid in the extracellular fluid of endothelial cells of porcine pulmonary arteries and in normal and xanthinuric urine. Normal urine samples were collected randomly and filtered through a 0.45 ?m nylon filter before analysis. Xanthine and uric acid concentrations were determined by a constant volume standard addition method. The mobile phase was 20 mM KH2PO4 at pH 5.1 and the detection wavelength 270 nm (xanthine) and 293 nm (uric acid). The injection volume was 20.0 ?L and attenuation was 0.01 absorbance unit full scale (AUFS). Retention times for xanthine and uric acid standards were respectively 13 ? 0.1 minutes and 7.0 ? 0.1 minutes. The linear correlation coefficient for xanthine and uric acid working curves were 0.995 and 0.998 respectively. The linear dynamic range, at the low concentration limits of xanthine and uric acid, was 5 ?M to 40 ?M, and 2 ?M to 20 ?M, respectively. The sensitivity of xanthine in 31 mM Na2HPO4/NaH2PO4 at pH 7.4 was 0.08 AU/?M, whilst that of uric acid in the same physiological buffer was 0.21 AU/?M. The limit of detection (LOD) for xanthine and uric acid were respectively 5.1 ?M, (S/N = 2) and 1.6 ?M (S/N = 3). Xanthine in xanthinuric urine was 2.8 ? 0.1 mM; while uric acid in normal urine was 5.7 ? 0.1 mM. In the extracellular fluid, the oxypurine peaks were identified as uric acid, hypoxanthine and xanthine.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Andrews O Affum.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Brajter-Toth, Anna F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021781:00001


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DETERMINATION OF XANTHINE AND URIC ACID IN XANTHINURIC URINE AND
EXTRACELLULAR FLUID OF PORCINE ENDOTHELIAL CELLS OF THE PULMONARY
ARTERY BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY




















By

ANDREWS OBENG AFFUM


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA

2007




































2007 Andrews Obeng Affum




























To my parents Mr. Francis Obeng Affum and Mary Afful, my siblings, and Kakra Biritwum who
through their encouragement have made it possible for me to complete an MSc degree in
chemistry, University of Florida.









ACKNOWLEDGMENTS

I express my sincere gratitude to my heavenly father for making it possible for me to

complete my MSc degree successfully.

I extend my heart-felt appreciation to Dr. Brajter-Anna Toth for her supervision and

encouragement, without which, this MSc wouldn't have been possible. I thank Dr. Ben Smith,

Mrs Lori Clark, Dr. Thomas Lyons, laboratory colleagues (Mehjabin Kathiwala and Alpheus

Mautjana), supporting staff, machine shop staff, electronic shop staff (Mr. Harry and Mr.

Stephen), and Computer Desk staff in the Chemistry Department of the University of Florida for

their assistance.

I also wish to express my sincere gratitude to Dr. John Toth for donating an HPLC pump

for my research work. I thank my committee members for their time and support in my research

work at the Chemistry Department, University of Florida.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF TABLES ......... ........... .............................................. 8

LIST OF FIGURES .................................. .. ..... ..... ................. .9

L IST O F A B B R E V IA T IO N S ..... .... ................................ ............................... ... .................. 11

A B S T R A C T ................................ ............................................................ 13

CHAPTER

1 IN TR OD U CTION .......................................................................... .. ... ... .. 15

General Introduction......... ............ ....................... ........... ............. 15
Analytical Methods for Measurement of Purines...........................................................16
Application of HPLC in the Analysis of Bio-fluids for Purine Metabolites...................17
C clinical M ethod for U ric A cid ............................................................. .....................2 1

2 PURINE METABOLITES AND RELATED ENZYMES IN MAN.............................. 22

Endothelial Cells of Pulm onary A rteries .................................................. ........ .............22
Xanthinuria .........................................22
Types of Xanthinuria .................. .................................. ..... .. .......... ....23
Enzym es Involved in X anthinuria.............................................................................. ...24
X anthine O xidoreductase ........................................................................ .................. 24
A ldehyde O xidase ........................ .. ........................ .. .... ........ ........ 26

3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY.................... .................28

C h ro m ato g ra p h y ............................................................................................................... 2 8
Instrum entation ............. ....................................... ............................ ............... 28
Reverse Phase High Performance Liquid Chromatography (RPLC).............................30
P rin cip le o f O p eratio n ....... .... .................................................................. .......... ........ 1
Column Selection and Efficiency ........... .. ......... .................... 33
M obile phase selection ............................... ..... ........ ..... .. .... .. ........ .... 36
F low rate selection ............................................ ................... .. ......36

4 BIOLOGICAL FLUIDS WITH URIC ACID AND XANTHINE...................................46

U r in e ....................................................................................................................................... 4 6
Com position of U rine ....... .. ....................... ........ .. .. .............. .............. .. 46
U ric A cid (2, 6, 8 trihydroxypurine) ........................................ ........................ 46









Xanthine (2, 6 dihydroxypurine) ........................................................................... 47
Solubility of xanthine and uric acid....................................................................... .... 48

5 EXPERIMENTAL ............... ........................................................... 50

M materials and C hem icals.............................................................................. .....................50
Instrumentation ................................. ........................... 50
Biological Sample Collection and Treatment........................................51
X an th in u ria U rin e ...................................................................................................... 5 1
N orm al U rin e .................51..............................................
C e ll C u ltu re ..............................................................................5 1
E xtracellular F luid ......................................................................................... ....... 51
H P L C S olv ents .................................................................52
Filtration of HPLC Solvents ..... .......... ......... ....... ........52
D egassing of H PLC solvent ................................................ ............... 52
Conditioning of HPLC Colum n...................................................... 52
B e fo re A n aly sis ............................................................................................................... 5 2
A after A nalysis................................................... 53
C alib ration C u rv e ................................................................53
X anthine Calibration Curve ............................................... ..............................53
U ric A cid C alibration C urv e ..................................................................................... 53
C hrom atography C conditions ..................................................................................... 54
Selection of Mobile Phase ................ ................. .. ............... 54
UV-Absorbance Maximum for Xanthine. ......................................... .........55
UV-Absorbance M aximum for Uric Acid .................. ..............................................55
Selection of Optimum Flow Rate .......................................... ............... ......55
Selection of HPLC Column ...... .................... ....... .........56
General Chromatographic Conditions ...................................... 56
Determination of Void Volume ............... .............................. 57
H P L C C olum n V alidation ......................................................................................... 57
Standard A addition M ethod ...........................................................58
X anthinuric U rine ....................................................... 58
N o rm al U rin e .................................... ..............................................................................5 9
Qualitative Analysis of Extracellular Fluid ...................................... ........ 59

6 RESULTS AND DISCUSSION ...................................... .........60

Xanthine and Uric Acid Ultra-violet Absorption Spectra ...................................... 60
X anthine C alibration C urv e .............................................................................................. 62
U ric A cid C alibration C urve............................................................................................. 63
Xanthine Concentration in Xanthinuric Urine.......................... ... ................ ......... 64
Uric Acid Concentration in N orm al Urine ....................................................................... 65
Extracellular Fluid from Endothelial Cells....................................................................... 65

7 SUMMARY AND CONCLUSION ......... ...... ................ ........ 83









APPENDIX

A UV-ABSORPTION SPECTRUM OF XANTHINE AND URIC ACID ...............................86

B NORMAL URINE CHROMATOGRAM .............................................................................90

C LIMIT OF DETECTION OF XANTHINE AND URIC ACID............... ........... ..........91

D SIG N A L T O N O ISE R A T IO ...................................................................... .....................92

E H PLC C O LU M N EFFICIEN C Y .......................................... ............................................93

F PREPARATION OF SOLUTIONS .............................................. ............................. 95

G CALCULATION ............................................. ... ........................ 97

R E F E R E N C E S .........................................................................100

B IO G R A PH IC A L SK E T C H ......................................................................... ............................ 107

































7









LIST OF TABLES


Table page

6-1 Peak height and signal to noise ratio of xanthine in physiological buffer.........................69

6-2 Peak height, and signal to noise ratio of uric acid in physiological Buffer ...................72

6-3 Standard addition determination of xanthine in xanthinuric urine. ..................................74

6-4 Standard addition determination of uric acid in normal urine. .........................................78

A-5 Limit of detection of xanthine and uric acid.......................................... ....... ........ 91

E -6 H PL C column n validation. ........................................................................ ....................93

E-7 M manufacture's HPLC column validation. ........................................ ....................... 94









LIST OF FIGURES


Figure page

2-1 Schematic of metabolic pathway in purine nucleotide metabolism in man..................27

3-2 Piston and check valve of a reciprocating pump .................................... ............... 40

3-3 A n internal loop valve w ith four pots ........................................ .......................... 41

3-4 A schematic of the self integrated HPLC system ..................................42

3-5 A typical aklylated silica surface for reverse phase stationary phase............................43

3-6 Typical retention time versus pH graph for a cationic species ............................... 44

3-7 The figure above represents the theoretical Van Demeter plot for uric acid.................45

4-8 Acid dissociation of uric acid and pKa values.................................. .............49

4-9 Acid dissociation of xanthine, pKa values ................................................49

6-10 Ultra-violet absorption spectra of xanthine and uric acid in mobile phase 67

6-11 UV-absorption spectra of xanthine and uric acid in physiological buffer.........................68

6-12 C alibration curve of xanthine............................................................................. ....... 70

6-13 Peak shape of 50.0 gM xanthine prepared in physiological buffer ................................. 71

6-14 Calibration curve of uric acid .................................................. .............................. 73

6-15 Constant volume standard addition curve of xanthinuric urine............... ...................75

6-16 Unspiked xanthine in xanthinuric urine................................ ................... ...... ........ 76

6-17 Spiked xanthine in xanthinuric urine ...................................................... ..................77

6-18 Constant volume standard addition of normal urine ......................................................79

6-19 U nspiked uric acid in norm al urine......................................................... ............... 80

6-20 Spiked uric acid in norm al urine............................................... ................................... 81

6-21 The oxypurine profile of extracellular fluid from normoxic endothelial cells .................82









A-22 UV-absorption spectrum of uric acid in physiological buffer .........................................86

A-23 UV-absorption spectrum uric acid in mobile phase................................ ...............87

A-24 UV-absorption spectrum of xanthine in physiological buffer .........................................88

A-25 UV-absorption spectrum of xanthine in mobile phase ..................................................89

A -26 A 1 in 5 dilution of norm al urine .......................................................................................90

D -27 Signal to noise ratio .......................... ........................... .... ........ ........ 92









LIST OF ABBREVIATIONS

S/N Signal to noise ratio

Nm Nanometers

[iM Micromolar

XOR Xanthine oxidoreductase

XDH Xanthine dehydrogenase

NADH Nicotinamide adenine dinucleotide

FAD Flavin adenine dinucleotide

PEEK Polyetheretherkitone

k' Capacity factor

tr Retention time (minutes)

to Void time (minutes)

V Retention volume (milliliters)

Vo Void volume (milliliter)

N Number of theoretical plates

L Column length (cm)

H Plate height (cm)

dp Diameter of packing material

Rs Resolution

h Reduced plate height (cm)

AUF Absorbance unit full scale

a Selectivity factor

Wi Base width length of peak 1

tl Retention time of peak 1

Dm Diffusion coefficient in stationary phase









Ds Diffusion coefficient in mobile phase

x Column parking factor

z Tourtosity factor

f (k') Function of capacity factor









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

DETERMINATION OF XANTHINE AND URIC ACID IN XANTHINURIC URINE AND
EXTRACELLULAR FLUID OF PORCINE ENDOTHELIAL CELLS OF THE PULMONARY
ARTERY BY HIGH PERFORMANCE LIQUID CHROMATOGRAPY

By

Andrews Obeng Affum

December 2007

Chair: Brajter-Anna Toth
Major: Chemistry

A reverse phase high performance liquid chromatography method with ultraviolet light

detection was developed to determine the concentrations of xanthine and uric acid in the

extracellular fluid of endothelial cells of porcine pulmonary arteries and in normal and

xanthinuric urine. Normal urine samples were collected randomly and filtered through a 0.45 tpm

nylon filter before analysis.

Xanthine and uric acid concentrations were determined by a constant volume standard

addition method. The mobile phase was 20 mM KH2PO4 at pH 5.1 and the detection wavelength

270 nm (xanthine) and 293 nm (uric acid). The injection volume was 20.0 pL and attenuation

was 0.01 absorbance unit full scale (AUFS). Retention times for xanthine and uric acid standards

were respectively 13 0.1 minutes and 7.0 + 0.1 minutes. The linear correlation coefficient for

xanthine and uric acid working curves were 0.995 and 0.998 respectively

The linear dynamic range, at the low concentration limits of xanthine and uric acid, was 5

pM to 40 pM, and 2 iM to 20 pM, respectively. The sensitivity of xanthine in 31 mM

Na2HPO4/NaH2PO4 at pH 7.4 was 0.08 AU/pM, whilst that of uric acid in the same

physiological buffer was 0.21 AU/iM. The limit of detection (LOD) for xanthine and uric acid

13









were respectively 5.1 riM, (S/N = 2) and 1.6 iM (S/N= 3). Xanthine in xanthinuric urine was

2.8 + 0.1 mM; while uric acid in normal urine was 5.7 + 0.1 mM. In the extracellular fluid, the

oxypurine peaks were identified as uric acid, hypoxanthine and xanthine.









CHAPTER 1
INTRODUCTION

General Introduction

The need to develop an analytical technique to selectively detect and quantitate purine

metabolites such as xanthine and uric acid biomarkers in human urine and extracellular fluids is

of interest to the analytical chemist and the clinician in the diagnosis of diseases. A suitable

analytical method for detection and quantitation of xanthine and uric acid must be fast, robust,

less expensive, and reproducible. In addition, it is important that the developed method must be

selective, for xanthine and uric acid detection and quantitation. Developing a suitable analytical

method for these analyte is important because this method could be adapted and used to separate

other analytes that have similar physical and chemical properties.

Xanthine and uric acid are known to be involved in many clinically important diseases and

metabolic disorders that are known to be genetically related, such as xanthinuria and Lesch

Nyhan syndrome. In addition, endothelial cells of the pulmonary arteries under oxidative stress

conditions generate xanthine and uric acid metabolites.

Analytical method which can separate and detect uric acid and xanthine in biological fluids

is relevant to biomedical research and in the prognosis and diagnosis of the diseases known to

cause imbalance in xanthine and uric acid concentrations. In addition, xanthine and uric could

serve as biomarkers for diseases. The common analytical methods, which have been used to

detect purines in biological fluids, include enzymatic assays, colorimetry, and chromatography.

In these established methods, analyte detection used is important in biological analysis.

The general objective of this project was to develop a comprehensive HPLC method with

ultraviolet detection to determine xanthine and uric acid concentration in biological fluids. The

specific objectives are: (1) to set-up an HPLC instrument; (2) to determine the sensitivity of the









HPLC method; (3) to determine the limit of detection (LOD) for xanthine and uric acid. and (3)

measure the concentration of xanthine and uric acid in (a) xanthinuric urine and (b) in the

extracellular fluid of porcine endothelial cells of the pulmonary arteries.

Although popular, isocratic elution methods are less common for xanthine and uric acid

separation in biological fluids. Nevertheless, an isocratic elution method was used to achieve a

separation of uric acid and xanthine in xanthinuric and normal urine, as well as in hypoxic and

normoxic extracellular fluid from porcine endothelial cells of the pulmonary arteries, which,

have been oxidatively stressed for forty-eight hours.

Analytical Methods for Measurement of Purines

A number of analytical methods have been utilized to separate purines (adenine, guanine,

xanthine, hypoxanthine and uric acid) from biological fluids. For example, purines (oxypurines)

were separated and detected from urine (Boulieu, R. et al., 1982; Boulieu, R. et al., 1983;

Gonnet, C. et al; 1983), plasma (Boulieu, R. etal., 1982; Boulieu, R. etal., 1983; Gonnet, C. et

al; 1983), serum (Castilo, J.R et al., 2001) cerebro spinal fluids (Michal, K. et al., 2005) and

saliva (Nakazawa, H. et al., 2003). In addition, purines have also been extracted and separated

from biological tissues such as animal heart (Mei, D.A. et al., 1996; Yacoub, M.H. et al., 1990)

and placenta (Westermeryer, F.A. et al., 1986). To achieve a suitable separation and quantitation

of purine metabolites in biological fluids, sample preparation and detection, which are dependent

on the physical and chemical properties of uric acid and xanthine must be considered.

Analytical methods that have been used to achieve oxypurine separation in biological

fluids include: (a) chromatography methods such as ion-exchange chromatography (Katz, S. et

al., 1983; Iwase, H. et al., 1975); gas-liquid chromatography coupled to mass spectrometry

(Iwase, H. et al.,Chadard, J.L. et al., 1980) and reverse phase high performance liquid









chromatography coupled to an ultra-violet light, electrochemical or mass spectrometry detection

(Baltassat, P. et al., 1984; Baltassat, P. et al.,1982; Gonnet, C. et al., 1983; Hassoun, P.M. et al.,

1992; Boulieu, R. et al., 1983; Boulieu, R. et al., 1983; Katz S. et al., 1983; Machoy, Z. and

Safranow, K. 2005; Mei, D.A. et al., 1996; Nakaminami, T. et al., 1999;Yacoub, M.H. et al

1990. (b) enzymatic methods which involve measurements of hydrogen peroxide levels from

xanthine oxidase reaction with xanthine or uric acid (Davis, J. et al., 2005; Hart, J.M. et al.,

1943).

Application of HPLC in the Analysis of Bio-fluids for Purine Metabolites

In a reverse phase chromatography separation of purines from urinary calculi, Zygmunt, M

and Safranow, K. 2005 used a gradient elution method and varied methanol concentration as well

as pH to separate 16 oxypurines, which included uric acid and xanthine. In this method the

average retention times of uric acid and xanthine were 3 and 5.5 minutes respectively.

Although, the retention time was much shorter than that reported in other HPLC methods,

the ternary mobile phase composition (solvent A: 50 mM KH2PO4 pH 4.6; solvent B: 50 mM

KH2PO4/K2HPO4 at pH 6.4; solvent C: methanol) was highly complex to use in routine

laboratory purine analysis. Moreover, the high ionic strength of the phosphate buffer (100 mM

K2HPO4/KH2PO4) was not best because it could easily precipitate in the mobile phase containing

methanol used in the separation as an organic modifier.

In addition, the extended use of highly aqueous mobile phase could cause a stationary

phase collapse (poor wettability of stationary phase) and can adversely affect analyte peak shape,

retention time, and column life span. In another gradient method developed by Mei, D.A. et al.,

1998, a microbore column (ODS-2, C18, 5 rim, 250 mm x 1.0 mm) was used to achieve a









separation for xanthine, hypoxanthine, and uric acid. The retention times for this separation were

10.3 and 6.9 minutes for xanthine and uric acid respectively.

The problem associated with Mei, D.A. et al., 1998 method is the high back pressure that

is known to be associated with columns of reduced internal diameter. The retention times (uric

acid 7.5 and xanthine 10 minutes) were relatively similar compared to previously established

chromatography methods. Microbore columns have an advantage of improved sensitivity and

less mobile phase consumption because of the reduced internal diameter of the column.

Further, microbore columns are expensive if compared to the standard HPLC columns.

The gradient elution method as mentioned above requires a careful equilibration of the column

between samples to ensure reproducible retention time. Purines have been separated by reverse-

phase HPLC with organic modifier in the aqueous mobile phase, but, this method is time

consuming because of longer equilibration time.

In an isocratic HPLC method previously used by Boulieu, R. et a.,l 1982, and modified by

Hassoun, P.M. et al., 1992, a mobile phase composition of 20 mM KH2PO4 at pH 3.60 was

utilized to achieve xanthine, uric acid, and hypoxanthine separation on a C-18 column. The

retention times of uric acid and xanthine were 9 and 14 minutes respectively. These retention

times were longer than those achieved by gradient methods reported by Mei, D. A. et al., 1998.

However, the limit of detection for xanthine obtained in this isocratic method was lower (0.05

tM) compared with the gradient method (8 tM) of Mei, D.A. et al., 1998.

In a reverse phase method reported by Nakasawa, H. et al., 2003, with an amperometric

detection (+0.6V) of uric acid in saliva, the limit of detection of uric acid was 3 nM at a flow rate

of 0.2 mL/min with 74 mM potassium phosphate buffer at pH 3.0 as mobile phase. In this









method, the low flow rate and high ionic strength of the mobile phase could cause phosphate to

precipitate in the head of the HPLC pump and stainless tubing.

To dissolve the phosphate deposit in the HPLC system, it is necessary to flush with water

for a long time. The method of Nakasawa, H. et al 2003 is therefore time consuming because of

extensive column flushing time. In addition, analytes that have similar oxidation potentials tend

to suppress uric acid detection. In an isocratic elution method by Gonnet, C. et al., 1983, a

shortest run time was obtained for uric acid which eluted at 4.1 minutes and xanthine at 5.3

minutes. Although retention time was improved, Gonnet, C. et al., 2003 did not achieve a

baseline separation of oxypurines. In addition, uric acid and xanthine peaks appeared to nearly

co-elute at 1.5 ml/min flow rate. Furthermore, the low pH of the mobile phase (0.02M KH2PO4

at pH 3.65) is not suitable for the stainless tubing in the HPLC system (Gonnet, C. et al., 1983;

Chadard, J.L. et al., 1980).

Ion pair chromatography method for determination of uric acid in human brain dialysis

fluid was reported by Marklund, N. et al., 2000. At a flow rate of 0.8 mL/min, and with a 5.0

mM H3P04 at pH 2.4 as mobile phase, the limit of detection for uric acid was 0.25 aM. This

method requires control of column temperature (28C) for biological fluid HPLC purine

separation. A regular HPLC instrument without temperature controlling equipment is therefore

not sufficient. In addition, the ion pairing agents (tetrabutylammonium hydrogensulphate)

present in the mobile phase makes regeneration of HPLC column difficult, and may compromise

the performance and shorten the shelf life of the HPLC column. Also changing HPLC column

regularly could affect the reproducibility of retention time.

In addition to HPLC chromatography techniques utilized in the separation of uric acid and

xanthine from biological fluids, other analytical methods such as enzyme assays (Telefoncu, A.









et al., 2004; Korf, J. et al., 1995), colorimetry (Miller, P. and Oberhozer, V. 1990; Carrol, J.J. et

al., 1971) and nuclear magnetic resonance have been used to detect and quantitate either uric

acid or xanthine in biological fluids. In most of these methods only uric acid was analyzed;

however it is a generally believed that this method is applicable to xanthine.

Unfortunately, these methods besides chromatography have major deficiencies such as

poor sensitivity and selectivity, which makes them a poor choice for xanthine and uric acid

detection in the urine of man and extracellular fluid of porcine endothelial cells in the pulmonary

arteries. For instance, in the uricase assay used to determine uric acid in serum, the redox

conversion of phosphotungsten to tungsten, which is used as a measure of uric acid

concentration, is affected by matrix components of the reaction medium. Visible light absorption

by the phosphotungsten complex or the amount of hydrogen peroxide produced is used as a

measure of the concentration of uric acid in the serum.

Despite the suitability of the uricase method, phosphotungsten or hydrogen peroxide

measurements have not been successful for uric acid detection and quantitation in biological

samples because of turbidity of the reaction medium from proteins and other possible

interference that accompany the analyte of interest. A recent shift from ultraviolet detection

mode for enzyme assay to fluorescence detection has had a limited success, because of possible

interference from other fluorescent substances in the sample (Castillo, J.R. et al., 2001).

One popular enzyme assay that has been used to determine uric acid concentration in

biological fluids is the xanthine oxidase assay. In this method, the alkalinity (pH 9) of the

reaction medium showed a deleterious effect on the structure of xanthine oxidase (Castilo, J.R. et

al., 2001). In another enzyme assay, xanthine oxidase modified glassy carbon paste electrode

developed by Telefoncu, A. et al., 2004, was used to determine xanthine concentrations in blood









plasma. This method may not show the true uric acid concentration because it does not include

any prior sample treatment, thus fluorescent interference may mask the uric acid concentration

through signal cross talk. Furthermore, this method has low reproducibility and is time

consuming.

Clinical Method for Uric Acid

Clinical methods that have been used to detect uric acid and xanthine includes

spectrophotometry, enzymatic assays, and electrochemical assays. In the spectrophotometry

methods, ultra-violet light at 293 nm is used to determine the concentration of uric acid in

biofluids. However, this method is not specific because of interference from endogenous

components which seem to absorb ultra-violet light at same wavelength as uric acid. With

enzymatic assay, uricase method is used to determine the concentration of uric acid in biofluids.

Uricase converts uric acid to allantoin and the decrease in absorbance of uric acid at 293 nm is

measured as a function of uric acid concentration (Skoug, J.W. et al., 1986; Roland, E. et al.,

1979). Electrochemical sensors have been used to detect and measure uric acid in biofluids. For

example, carbon fibre sensors were used by Davis, J. et al., 2006 to detect uric acid directly from

serum (Davis, J et al., 2006). In a xanthine oxidase modified glassy carbon paste electrode to

detect and quantitate uric acid, an oxidation potential of 0.6 Volts was applied to this electrode to

produce a current which was proportional to uric acid concentration present in biofluid

(Telefoncu, A. et al., 2004). The only problem associated with this technique is the cross signal

talk from other analytes such as ascorbic acid which have similar oxidation potential as uric acid.









CHAPTER 2
PURINE METABOLITES AND RELATED ENZYMES IN MAN

Endothelial Cells of Pulmonary Arteries

The pulmonary artery is lined with smooth monolayer of cells called endothelial cells.

Endothelial cells form a barrier between the blood and vascular tissue of the pulmonary artery. In

addition, endothelial cells facilitate a two way transport of biomolecules between the blood and

the vascular tissue. Pulmonary artery is known to be affected by diseases such as pulmonary

edema: a condition in which fluid accumulates in the lungs usually because the heart left

ventricle does not pump adequately (Seki, T. et al., 2007), and pulmonary thromboembolism: a

blockage of the pulmonary artery by a blood clot (Steiner, I. 2007; Scalea, M.T. et al., 2007).

These diseases present low oxygen concentration (< 3% 02) below the physiological oxygen

concentration which is exposed to endothelial cells. At such low oxygen tensions, purine

catabolism in endothelial cells can be enhanced, which result in the generation of uric acid and

xanthine from the purine catabolic pathway (Hassoun, P.M. et al., 1992). These biomolecules are

excreted into the extracellular space of the endothelial cells. The ability to detect uric acid and

xanthine in normoxic endothelial cells will serve as a gold standard for detecting these

biomolecules in a diseased pulmonary artery.

Xanthinuria

In the metabolic pathways of purine, many clinical defects can occur which may lead to

diseases. Changes in purine concentrations in biological fluids such as urine could be used as a

diagnostic measure of these defects and an indicator of the effectiveness of the associated

enzymes in the purine catabolic pathway. One well known and a rare clinical defect is

xanthinuria. Xanthinuria is a purine metabolic disorder, which is usually clinically mild and









asymptomatic. As an inherited autosomal recessive disorder, it is known to be associated with

low uric acid and high xanthine concentrations in the plasma and urine.

The physiological plasma xanthine level in a healthy person is less than 5 [iM while in

xanthinuric person is over 10 [iM (Scriver, B.V. et al., 2001). Boulieu, R. et al., 1983 found by

HPLC method that the mean normal xanthine concentration in plasma is 1.4 0.7[iM; clinical

range: < 0.5- 2.5 [iM while the urinary xanthine is 68 42 itM; clinical range: 41 161 [iM.

Xanthinuric individuals have high xanthine and moderate hypoxanthine concentrations in urine

and in blood, because hypoxanthine is salvaged to inosine monophosphate by hypoxanthine

guanine phosphoribosyl phosphate (HGPT) in the purine salvage pathway. High xanthine

concentrations can cause xanthine deposits in the urinary tract, and is known to result in

hematuria or renal colic, and acute renal failure or chronic complications related to urolithiasis

(http://teaching.shu.ac.uk/hwb/chemistry/tutotrials/ mo Ispec/uvvisabl.html).

In addition, xanthine could also be deposited in the muscles, which is normally associated

with severe pain. The main source of xanthine is through the guanine nucleotide catabolic

pathway. The schematic of nucleotide catabolic pathway is clearly shown in Figure 2 1.

Types of Xanthinuria

The two major sub-types of xanthinuria, which have been reported, are xanthinuria type I

and xanthinuria type II. These subtypes are a result of the effect of mutations in the molybdenum

cofactor sulfurase on the active sites of xanthine dehydrogenase/xanthine oxidase and aldehyde

oxidase. Mutations that have been determined in the molybdenun cofactor sulfurase are a G to C

in nucleotide 466,GCC (Ala) to CCC (Pro) in exon 2 and C to T in nucleotide 1255, CGA (Arg)

to TGA (Ter),(Hada et al., 2003). A deficiency in molybdenum cofactor sulfurase causes

xanthine dehydrogenase/xanthine oxidase and aldehyde oxidase to remain desulfonated and









deficient. Xanthinuria type I therefore results from a genetic deficiency of xanthine

dehydrogenase, and aldehyde oxidase, while xanthinuria type I is known to be involved with a

deficiency in xanthine oxidoreductase. A mutational analysis by Topalogue, R. et al., 2003, on

the XDH gene at exon 20 identified an A to T base change in nucleotide 1264, AAG (Lys) to

TAG (Tyr). This mutation distinguishes xanthinuria type I from xanthinuria type II. The main

physiological substrates of xanthine oxidoreductase are hypoxanthine and xanthine, which are

derived from the breakdown of the major purines in the cell: adenine and guanine. It has been

established that xanthine oxidoreductase is the last enzyme in the purine catabolic pathway in

humans and higher apes. In other animals such as birds and reptiles, uric acid is converted to

allantoin by uricase.

Enzymes Involved in Xanthinuria

Biological enzymes that have been known to be associated with xanthinuria include

xanthine oxidoreductase, aldehyde oxidase and sulfite oxidase. These enzymes belong to

molybdenum iron-sulfur flavin hydroxylases (Hada et al., 2003). Molybdenum is essential in

their catalytic activities. They work in synergy to effect a particular sub-type of xanthinuria.

Xanthine Oxidoreductase

Under physiological conditions, xanthine oxidoreductase (XOR) exists in two convertible

forms as xanthine oxidase (XO) and xanthine dehydrogenase (XDH). Xanthine oxidoreductase

(XOR) is found both in the cytoplasm and on cell membranes (Hare, M. J. and Berry, C.E.

2004). It has been established that proteolysis (protein treated with proteases, such as trypsin,

chymotrypsin, and pancreatin) and cysteine oxidation (Cyst 535 and Cyst 992) of XOR results in

an irreversible conversion of XDH to XO. In xanthine dehydrogenase the electron acceptor is

nicotinamide adenine dinucleotide (NAD) while in xanthine oxidase it is molecular oxygen (02).









However, it has been established that under some physiological conditions such as hypoxia,

molecular oxygen could also serve as electron acceptor for XDH (Hare, M.J. and Berry, E.C.

2004).

In the catalysis of xanthine oxidase and xanthine dehydrogenase with xanthine as the

substrate, superoxide, hydrogen peroxide and nicotinamide adenine dinucleotide (NADH) are

produced, respectively. The generation of superoxide and peroxides during this enzymatic action

is known to contribute to oxidative stress and tissue injury. It is also known that xanthine

oxidoreductase (XOR) oxidizes a variety of pyrimidines, aldehydes and pterins. Xanthine

oxidoreductase is cytosolic and the optimal pH at which maximum enzyme activity is obtained

varies between 5.6 with pterin as the substrate and 8.4 with xanthine as substrate (Yokoyama, Y

et al., 1990).

The polypeptide chain of XOR is known to consist of 1331 amino acids in human, 1331

amino acids in rat, 1335 amino acids in mouse and 1358 in chicken liver enzyme. The purified

protein is a homodimer, and consists of identical subunits of size which is about 150 kDa,

estimated by SDS-PAGE (Wright, M.R. et al., 1997; Ravio, O.K. et al., 2005; Okamato, K. et

al., 2007). Each subunit is known to contain a molybdenum center (C-terminal; 85 KDa), an

FAD center (40 KDa with NAD+) and two iron sulphur centers (N-terminal; 20 KDa). Xanthine

oxidoreductase is coded by a single gene which is located on chromosome 2p22. This gene

consists of 36 exons and 35 introns (Xu, P. et al., 1996; Wright, M.R. et al., 1997). The exon -

intron structure is highly conserved and generates about 1330 1355 amino acid residues.

In humans, substantial xanthine oxidoreductase activity is found in the mucosal lining of

the liver and small intestine (Hare, M.J. and Berry, E.C. 2004). It has been reported that low

activity of xanthine oxidoreductase is found in the plasma, the endothelium, bronchial wall,









heart, lungs and kidneys (Moriwaki, Y. et al., 1993). Xanthine oxidoreductase reacts with

xanthine or hypoxanthine through a two electron mechanism. In its enzyme mechanism, a base

abstracts a proton from the Mo-OH group, initiating a nucleophile which attacks the purine

substrate. In the process oxygen is incorporated into the purine substrate. The hydroxyl group on

the molybdenum is then replaced by hydroxide from the surrounding aqueous medium.

Xanthine oxidoreductase has varying Michealis Menten constants (Km) values. This

enzyme is known to have Km value of 9 iM for hypoxanthine, 7 iM for xanthine and 5 iM for

adenine (Yokoyama, Y. et al., 1990). A purine analog, allopurinol is readily oxidized by XOR

with a Km of 2 riM.

Aldehyde Oxidase

Aldehyde oxidase is a dimeric molecule of approximately 300,000 molecular weight. The

two subunits are independent in function. Each subunit contains a flavin adenine dinuleotide

(FAD), two iron-sulphur clusters and molybdenum (Berry, C.C. and Hare, J.M. 2004). Aldehyde

oxidase has similar gene sequence as xanthine oxidase and each subunit is approximately 150

KDa. Aldehyde oxidase is located on chromosome 2q33 in humans and is found in the

homologous region of chromosome 1 in mouse (Mcanaman, L.J. et al., 2000). This cytosolic

enzyme is involved in the catalytic oxidation of N-oxides, nitrosamines, and hydroxamic acids. It

uses molecular oxygen as its physiological electron acceptor and is known to produce superoxide

radicals and hydrogen peroxides.









De novo purine
synthesis


IDH
I]1


Guanine


X


ATP


ADP


SASL AS ASL AMP
5' NT AK t5'NT
iosine Adenosine APRT
PNP

poxanthine d
Adenine
XO
anthine


XO

Uric acid


Fig 2-1: Schematic of metabolic pathway in purine nucleotide metabolism in man. This figure
was adapted from Biochemistry by Mathews, K.C., Van Holde, K.E. and Ahem,
G.K. 3rd Edition pages 804 806 (2001). HPRT: Hypoxanthine guanine
phosphoribosyl transferase, PNP: Purine nucleoside phosphorylase, IDH: IMP
dehydrogenase, ASL: Adenylsuccinate synthase, GA: Guanase, XO: Xanthine
oxidase, APRT: Adenine phosphoribosyl transferase; 5'NT: 5' Nucleotidase


GTP

It
GDP


SGMP XA









CHAPTER 3
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Chromatography

Chromatography was invented by a Russian botanist named Tswett. Chromatography is a

union of two Greek words chromos (color) and grafe (writing). It is a separation technique that

utilizes a stationary phase and a mobile phase. In chromatography, the separation process is

achieved by a distribution of analyte between the two phases. The stronger the forces of

interaction between the analyte and the mobile phase the greater the amount of solute that will be

held in the mobile phase.

Similarly, the stronger the interaction between the analyte and the stationary phase the

greater the amount of solute that will be held in the stationary phase. The distribution coefficient

(KD) of an analyte, which is the ratio of the concentration of analyte in the stationary phase to its

concentration in the mobile phase (Raymond, P.W.S. 1994), indicates the extent to which an

analyte interacts with the stationary phase during elution. Another important factor to consider is

the retention time. Solute retention and consequently resolution is determined by the magnitude

of the distribution coefficients of the solutes with respect to the stationary phase and relative to

each other.

Instrumentation

The kind of instrument that is used for a particular chromatography separation determines

the quality of the analytical method. Since the results obtained from high performance liquid

chromatography are reproducible, it is important that the instrumentation used for the HPLC

separation is emphasized. In addition, the components of an HPLC system must be within

acceptable and prescribed limits, without which the system is rendered unsuitable for a particular

chromatographic separation.









The main components of an HPLC system are the pump, injector, column and the detector.

The pump consists of one or more pistons/pump-head assemblies. The pump head is made of 316

stainless steel and has a cavity into which the pistons move in and out, which cause the mobile

phase solvent to be pushed into the column. The flow rate range of an HPLC pump is determined

from a combination of the cavity volume, piston diameter and speed of the piston stroke.

The two main types of elution methods used in chromatography are isocratic and gradient

elution. Isocratic elution requires one piston/pump-head assembly (shown in Figure 3 2)

whereas gradient elution involves multiple piston/pump head assemblies. Pump assemblies

typically utilizes one-way check-valves (inlet check valve and outlet check valve) to direct flow

of mobile phase.

A pulseless flow of mobile phase is achieved by dual-piston pump configurations. A

pressure transducer is placed between the outlet check valve and the column to monitor back

pressure changes. To withstand the high back pressures generated in the HPLC system, stainless

steel tubing of moderate inner diameter (0.01 in to 0.02 in) is utilized (Sadek, P.C. 2000).

The replaceable in-line filter prevents particulate matters generated by the mobile phase or

piston seal from reaching the HPLC column. The inline filter is usually made of stainless steel or

polyetheretherketones (PEEK) with 0.5 [m to 2.0 [m pore size.

Another important component of the HPLC system is the injection valve. The injector

provides a well defined volume in which the sample is contained prior to introduction into the

mobile phase. Injection of sample into the mobile phase is achieved by a manual or an automated

injection through a valve. The valve has a sample port and a sample loop (Shown in Figure 3 -

3). The sample is introduced into the injector through a syringe. When the injection loop is

switched to an on-flow position, the sample loop is connected to the mobile phase flow path.









However, if it is switched to an off-flow position the sample is loaded into the loop. Figure 3 3

shows an internal loop valve with four ports.

Another important component of the analytical HPLC system is the column connection.

Usually, a guard column is placed at the head of the column to prevent particulate matter in the

mobile phase and the pump from reaching the analytical HPLC column. A small internal

diameter of about 0.007 inches stainless tubing is used to connect the guard column to the HPLC

column. Similarly, it is important that a stainless tubing of equal dimension links the column to

the detector. The HPLC column contains the stationary phase and column dimensions vary with

application, and it dictates the volume of the mobile phase that is consumed in the separation

process.

The final component of the HPLC system is the detector. The common detectors usually

associated with HPLC separations are: (1) fluorescence; (2) ultraviolet-visible; (3)

electrochemical; (4) conductivity; (5) refractive index and (6) mass spectrometry. The detector is

chosen to generate the optimum response from each analyte in the sample mixture. Finally, the

eluent from the detector is collected into reservoir and classified as waste. It is important that

HPLC components mentioned above are properly calibrated in order to achieve accurate, precise,

and reproducible results to be obtained. A schematic assembly of a typical HPLC system is

shown in Figure 3 4.

Reverse Phase High Performance Liquid Chromatography (RPLC)

This is a kind of chromatography in which the mobile phase is polar, such as water (H20)

and the stationary phase is non polar alkyl chains (-CH2CH3). The main materials used as

stationary phases are alkyl modified silica (Si03(CH2)nCH3) and alkylated polystyrene-

divinylbenzene (PS DVB) polymers (Katz, E et al., 1998). This includes octadecyl (C-18);









octyl (C-8); cyano (CN) and phenyl (C6H5) etc. The silica surface is modified by alkyl chains to

prevent the silanol groups (-SiOH) reacting with water, which tend to wet the surface of the

stationary phase. A schematic of silica bonded stationary phase is shown in Figure 3 5.

Various organic modifiers such as methanol, acetonitrile, and tetrahydrfuran are used in the

mobile phase of a reverse phase separation process to tune retention, improve selectivity, and

enhance peak shape and resolution of analytes (Drumm P. et al., 2005). Reverse phase

chromatography is the first choice for most samples separations. This is because the reverse

phase column used is known to be efficient, stable, and reproducible and suitable for wide range

of sample separations. In addition, it is suitable for separation of analytes that show different

concentrations in aqueous solutions, different sizes because of their hydrophobic structures or of

different numbers of polar groups.

Principle of Operation

In reverse phase high performance liquid chromatography nonpolar compounds have

relatively higher retention time than ionic compounds. The driving forces for retention in RPLC

include: (1) london dispersion interactions that exist between nonpolar surface ligands and the

nonpolar components of the analyte and (2) the hydrophobic effect, that is, the tendency to

minimize the disturbance of the water structure (Katz, et al., 1998). This variation in retention

time for ionic and neutral analytes is supported by the solvophobic theory (that is a process

involving solute transfer into or onto the stationary phase which may involve partition or

adsorption or both) of retention. Based on the solvophobic theory, it is expected that nucleotides

will elute first, followed by nucleosides and bases (Zakaria, M. et al., 1983).

Solvophobic theory, in general, considers the stationary phase to have a passive role in the

retention process, and that retention is largely due to the thermodynamic interactions between the









solute and the mobile phase. Indeed, solvophobic theory doesn't provide a complete description

of the retention process. Various molecular interactions such as hydrophobicity (repulsive

interactions between non-polar compounds and a polar environment), electrostatic (ion ion. ion

- dipole and dipole dipole interactions dependent on the dielectric constant of the solvent) and

hydrogen bonding are known to govern significantly the retention process.

Further, retention is influenced by the pH and ionic strength of the mobile phase. When the

pH of the mobile phase is below the pKa of the analyte, the percent populations of the cationic

form can predominate over the neutral form. Retention time tends to be higher for neutral

molecules compared to their cationic forms. Thus, retention is dependent on the pKa of the

compound to be separated and the pH of the mobile phase. A plot of the retention time versus

varying pH for an ionizable compound (Figure 3 6) depicts a sigmoid shape with inflection

point which corresponds to the pKa of the compound.

It must be noted that all pH related changes in retention occur for pH values within + 1.5

units of the pKa value (Glajch, J.L. et al., 1997). At pH of at least two units higher than the

analyte pKa, there is retention of the anionic form of the acidic analyte. When the acid or basic

groups on a compound are similar, retention behavior is simpler; however mixed basic and acid

groups on a compound present a complex retention behavior. The relationship between the

cationic and the nonionic form of an analyte dependent on the pH of the mobile phase is

described by the Henderson- Hasselbach equation.


pH = pka+ Log[A (3- 1)
[HA ]

Where A represents the base form; and HA represents the acid form; pH represents -

logio[H+]









Another parameter which affects retention time is organic modifier added to the mobile

phase in chromatography separation. Organic modifiers such as methanol are added to aqueous

mobile phases to elute non-polar compounds that have high hydrophobic properties and interact

with the stationary phase, to decrease retention time.

Analytes that elute from the chromatographic column are identified by their retention time

(tr), selectivity factor (a) or by capacity factor (k'):

k' = t (3 2)
to

Where to (min) is void time (defined as the time taken by an unretained analyte to reach

the detector), and tr (min) is the retention time; k'capacity factor (5 < k' < 20)

The capacity factor may also be expressed in terms of retention volume.


k' r V (3-.3)
V

Where Vr (mL) is the retention volume and Vo (mL) is the void volume (mL) (defined as

the volume of mobile used by an unretrained analyte to reach the detector.

Column Selection and Efficiency

The HPLC column is the most critical part of the HPLC system. The dimensions of an

HPLC column dictate the kind of separation method that needs to be adapted. The standard

HPLC column used in reverse phase chromatography is 25 cm long x 0.46 cm inner diameter.

The silica or porous-polymer particle in HPLC columns provide adsorbable surfaces which serve

as support for organic surface layers. These particles are available in different diameters, pore

size, and surface area. The physical properties of these particles are considered during purchase

of an HPLC column. In the separation of smaller molecules, porous particles with 7 to 12 nm (70

- 120 angstrom) pores size and surface area of 150 to 400m2/g are used while bigger molecules

33









require pore diameters larger than 15 nm. An HPLC column with a 3 5 [im particle size is

considered suitable for analytical purposes. A column with small particle size (3 [tm) provides

faster separation because diffusion length of the particle is reduced, thus minimizing eddy

diffusion and enhancing mass transfer kinetics in the particles (Guiochon, G. and Gritty, F.

2007). Pore diameters of at least four times the hydrodynamic diameter of the solute ensure that

restricted diffusion (confinement of analyte to a small pore in the stationary phase) of the solute

does not degrade the column efficiency (Snyder, L.R. and Stadalius, M.A. 1986).

The surface area of wide pore particles usually range from 10 to 150 m2/g. Rigid high

strength particles such as silica, tend to produce low back pressures. Silica based columns are

easily dissolved at pH values higher than pH = 8 (Snyder, L.R. and Stadalius, M.A. 1986). Free

silanols can cause strong deleterious interaction with basic analytes which could lead to

increased retention and broad tailing peaks (Yacoub, M.H. et al., 1990). Basic analyte and silanol

interactions are prevented by endcaping the free silanol hydroxyl groups with short carbon

chains, such as -CH3 groups. Porous polymer particles have high utility at a wide pH range (1 to

13). Column stability is dependent on the pH of the mobile phase, buffer, and organic modifier.

Specifications of an HPLC column such as asymmetric factor (ratio of peak widths at 10% of

peak height), plate number, selectivity factor, back pressure, retention reproducibility, bonded

phase concentration, and column stability must be known before it is used for any

chromatographic separation, so that its condition can be evaluated with respect to specifications

after designated period of usage.

The efficiency of an HPLC column defines the maximum number of equilibrations

measured as theoretical plates (N) that an analyte will have with the stationary phase as it elutes

from the HPLC column. It defines the ability of the HPLC column to produce sharp and narrow









peaks. The theoretical plate (N) of an HPLC column is best determined at the optimum flow rate

of the mobile phase. Further, the theoretical number of plates is expressed as the ratio of column

length (L) to the plate height equivalent to a theoretical plate (H).


N=L (3-4)
H

Plate height equivalent to a theoretical plate ratio, which gives the reduced plate height (h),


h= (3-5)
dp


Where dp (pm) is the diameter of the silica particle.

For a particular Gaussian peak at half height the theoretical number of plates is given as


N=5.54x, trj (3-6)



Where W0.5 (cm) is the width length at 50 % of the full peak height.

The theoretical number of plates is related to resolution (Rs), separation factor (a) and

capacity factor. The relationship is given by the equation:

N a-i k
R =-x--x-- (3 -7)
S4 a k +1

Where selectivity factor (a)


a = k2- (3 -8)
k'


Where k' represents capacity factor; kl' represents capacity factor of first analyte; k2'

capacity factor of second analyte

t2 t
R = (3 9)









Wl and W2 are base width length of analyte 1 and analyte 2 respectively. tl and t2

represent retention time of analyte 1 and 2 respectively.

Mobile phase selection

Solvents that are used in the HPLC play critical role in the chromatography separation

process. The choice of a mobile phase is important because it could either enhance or reduce the

selectivity of the separation. In the separation process, mobile phase interacts (for example,

through hydrogen bonding, dipole-dipole, London dispersion and pi-pi interactions) with the

analyte to either extend or reduce its residence time in the stationary phase. Further, the mobile

phase interacts with the analyte to minimize or prevent strong interactions with the surface of the

stationary phase. The mobile phase also determines the elution order of the separating analyte as

it passes through the HPLC column.

Flow rate selection

Flow rate of the mobile phase defines the volume of solvent that passes through the HPLC

column per unit time. Flow rate provides the most convenient and predictable changes in

separation process. The selection of flow rate for a particular HPLC system is often determined

from a Van Deemter plot, which is a graph of the theoretical or reduced plate height (H) versus

mobile phase flow rate (Knox, H.J. 2002; Knox, J.H. 1999; Mulholland, M. 004). A theoretical

Van Deemter plot is shown in Figure 3 7.

The plate height (H) of an HPLC column has contributions from eddy diffusion (A),

longitudinal diffusion (B) and resistance to mass transport both in the stationary phase and the

mobile phase (C), involved in a chromatographic separation. The band broadening parameters

are explained by the Van Deemter equation (Mulholland, M et al., 2004; Knox, J.H. 1999;









Lifang, S. and Carr, P.W. 1998; Knox, H.J. 2002; Scott, P.W.R. and Cazes, J. 2002) is expressed

as:


H =A+B+Cu (3-9)
C

(Van Deemter equation as shown in figure 3 7).

The variables A and B and C are constants; H represents theoretical plate height and u

represents average linear velocity of mobile phase (cms-1).

In explicit form, the Van Deemter equation written above is expressed as:

27D, f(k')d2 f(k')d2
H = 2d + + + u (3-10)
u Dm D

Where X represents column packing factor (- 0.5 1.5) ; Dm represents solute diffusion in

mobile phase (cm2s-1); Ds represents solute diffusion in stationary phase (cm2s-1); dp represents

average size of filling particle (.im); y represents tortuosity factor dimensionlesss); and df

represents thickness of stationary phase (.im); and k' capacity factor; f(k') represents function of

capacity factor dimensionlesss).

First, assuming that solute diffusion in the mobile phase is equivalent to solute diffusion in

the stationary phase, then

D, =D (3 -11)

Second representing the uncommon variables in the resistance to mass transfer in both the

stationary and the mobile phase by variable b:

b= f(k)d2 +f2(k)d2 (3-12)

Then,


H = 2AdP + 2 + (3 13)
u D









At the minimum plate height:

OH 2yD b
= -+ (3-14)
du u D

Equating to zero and solving for optimum linear flow rate (Uopt):


U2 2yD (3- 15)




b


Substituting equation 15 into equation 13, the minimum plate

H_ =2yMAd + yb (3- 17)

Minimum plate height represents by Hmin

The constant A is independent of the linear velocity. Solute molecules take different paths

through the stationary phase. The path lengths of individual molecules differ from one another in

a random fashion leading to band broadening. The A term is dependent on the diameter of

particles packed into the HPLC column. The B term represent longitudinal diffusion and is

dependent on the flow rate, that is, analytes injected onto the HPLC column tend to diffuse as

they elutes from the column towards the detector.

The C term is the resistance to mass transfer from both the stationary phase and the mobile

phase and is also dependent on linear velocity. When the mobile phase velocity is high and the

analyte has a strong affinity to the stationary phase, the analyte in the mobile phase elutes faster

than in the stationary phase. Van Deemter plot in Figure 3 7 shows that the plate height

decreases to a minimum and increases linearly as the linear flow rate increases. At the minimum









plate height all the three parameters that contribute to plate height are equal and represent the

optimal condition suitable for analyte separation.

At a minimum plate height the flow rate is optimum and the total theoretical number of

theoretical plates is increased. When HPLC columns are operated at optimum flow rate, the

separating analyte experiences maximum equilibration between the stationary phase and the

mobile phase. The time a solute remains in the HPLC column is inversely proportional to the

flow rate of the mobile phase.

However, in most chromatographic separations, flow rates are often operated at higher than

the optimum flow rate in order to decrease analysis time. Another criteria that will require a flow

rate higher than optimum flow rate is when the linear portion of the Van Deemter plot is almost

flat (Glajch, J.L. et al., 1997;Snyder, L.R. and Stadalius, M.A. 1986; Heftmann, E. 1975). In

addition, the symmetrical nature of the analyte peak needs to be critically considered. Flow rate

selection is important because it determines the void volume (Vo) of the mobile phase that is

used to achieve a particular separation. Mobile phase flow rate (F) is expressed as:


F = (mLs-1) (3-12)
to

Where Vo (mL) is the mobile phase volume; to is void time (min)










Outer check valve


A
*--.--........ Column pressure (>>atmosphere)

Soot

Cylinder
........... Prssre slightly


Atmosphere pressure
B Column bhek pressure
""I Inlet check valve


.--............ Pressur from atmosphere > column
BSck pressure



Atmospere pressure
Fig 3-2 Piston and check valve of a reciprocating pump: The inlet check valve (A) is open during
the suction segment of the stroke, but closes during the exhaust segment (B), which
forces open the outer check valve. The liquid is thus displaced towards the column
during the exhaust stroke. Adapted from reference 30. (Katz, E., Eksteen, R.,
Schoenmakers, P. and Miller N. Handbook of High Performance Liquid
Chromatography (HPLC).Marcel Dekker Inc, New York, United State of America.
1998. Page 1 500).











Position A
Position B
Mobile Sample
P SSG *t Mobill
To Phe
To
Column To
Sample Colu Sample
InIn
To Semple To
waste Slot tTo
waste




Fig 3-3: An internal loop valve with four pots. Diagram adapted from Raymond P.W.S. Liquid
Chromatography for the Analyst. Marcel Dekker, New York. United States of
America 1994, Page 140.










Rheodynevalve In-linefllter


UV detector
I HPLC column I


Mobilephase \
reservoir


Stand
Chart recorder

HPLC pump

Fig 3-4: A schematic of the self integrated HPLC system used for the HPLC separation of
xanthine and uric acid.










cs ligand


H 3 C H IH, 2 HJC HH2
H CH2 H/CH2 H2 H H2H
H CH HCH2 H2H
H 2 HpC H CH2 >H 2 C /H2
H2C\ 2\ H2C\
End capping /CH2 /H2 CHH2 CH2 H2 CH2
H \ H H2 H o H2
o 0 \ 0\ /1 2
N I H2C
SC .H3 / CH3 H / CH3 3C H H

/ / H3C CH3



I 0 0 0 0 0\ 0 0 0 0
o o
Fig 3-5 A typical aklylated silica surface for reverse phase stationary phase
Fig 3-5: A typical aklylated silica surface for reverse phase stationary phase












10-


pKa + 1.5
I I



6t


E pKa





2



0 4-
0 1 2 3 4 5 6 7 8 9 10

pH of mobile phase
Fig 3-6: Typical retention time versus pH graph for a cationic species. Adapted from Glajch J.
L., Kirkland J.J, Snyder L.R. Practical High Performance Liquid Chromatography
(HPLC) Method Development.2nd. John Wiley and Sons, Inc, New York, United
States of America. 1997. Page 295 300.













0.350


S 0.300 -
E

c 0.250

-c
3 0.200
CL

0.150 *


0.100


0.050


0.000 -...
0 2 4 6 8 10 12 14

linear velocity (cm/s)

Fig 3-7: The figure above represents the theoretical Van Demeter plot for uric acid. Standard
column and mobile conditions were assumed in determine this curve.









CHAPTER 4
BIOLOGICAL FLUIDS WITH URIC ACID AND XANTHINE

Urine

Urine is a biological effluent that is produced from the kidney. The composition of urine

provides information on the metabolic rate in the human body. The volume and solute

composition of urine vary depending on the individuals' diet, physical activity and health. Urine

is filtered from blood through ultra-filtration process in the nephron of the kidney. For this

reason, urine could be used to monitor and evaluate progress of metabolic disease. Therefore, a

routine analysis of urine could provide information on the diagnosis of diseases, screen for

asymptomatic congenital and hereditary diseases (Bruzel, A.N. 1994)

Composition of Urine
The main solutes in urine include urea, chloride, sodium, potassium, phosphates, sulphates,

creatinine and uric acid. The protein concentration range in normal urine is from 8 to 10 mg/dL.

The main protein in urine is albumin. Proteins such as Tamm Horse Fall Mucoprotein and

Bilirubin are present as well, but are minimal in concentration. The concentration of these

proteins determines the efficiency of the Bowman's capsule. The normal specific gravity of urine

ranges from 1.002 to 1.035 g/ml. The established pH of urine varies from 4.5 to 8.0 (Bruzel,

A.N. 1994).

Uric Acid (2, 6, 8 trihydroxypurine)

Uric acid is a known diagnostic analyte in human urine. Uric acid (2, 6, 8-

trihydroxypurine) is the end product in the purine catabolic pathway. Purines (adenine and

guanine) involved in this pathway can be derived from dietary sources and the degradation of

nucleic acids in cells. Uric acid is a weak acid and undergoes two proton dissociations in basic









medium (Fig 4 8). The reported pKa values of uric acid are 5.74 and 10.3 (Scriver, B.V. et al.,

2001).

The established uric acid concentrations in adult male and female urine are respectively

250 800 mg/dL and 250 -750 mg/dL and plasma concentrations are 302 60 [tM and 234 52

ItM respectively. It is documented that an increase in uric acid concentration correlates with

hyperuricemia, hypertension, gout arthritis and cardiovascular diseases (Nakagawa, T. et al.,

2006). Uric acid is a known biomarker in cardiovascular disease and hypertension (Schechter, M.

2005).

Uric acid is a potent antioxidant. Its antioxidant properties involve the quenching of

reactive oxygen species such as hydroxyl radicals, singlet oxygen species, and oxo-heme

oxidants, which are major causes of cancer, heart disease and aging (Machoy, Z. and Safranow,

K. 2005). Purine analogue that is used to reduce uric acid concentration in vivo is allopurinol.

Allopurinol inhibits xanthine oxidase activity in the nucleotide catabolic pathway.

Xanthine (2, 6 dihydroxypurine)

Xanthine is a precursor of uric acid and metabolite in purine catabolic pathway. The

conversion of xanthine to uric acid generates reactive oxygen species. Reactive oxygen species

have been implicated in inflammation and cancer (Khandurina, J. 2000). Xanthine is obtained

from both the adenine and guanine catabolic pathways (See Figure 2 1).

It has been established that the main source of xanthine is derived through the guanine

pathway since hypoxanthine is continuously salvaged to inosine 5'-monophosphate (IMP). Cells

tend to lose more energy when high energy phosphates bonds are formed through the de-novo

pathway. To prevent this loss of energy, cells rely on hypoxanthine salvaged pathway. The

established pKa values (Figure 4 9) of xanthine are 7.7 and 11.3 (Scriver, B.V. et al., 2001).









Solubility of xanthine and uric acid

Uric acid and xanthine solubility in aqueous medium is primarily determined by the pH of

the medium, the pKa and temperature .As mentioned, uric acid has pKa's of 5.74 and 10.3, while

xanthine has pKa of 7.7 and 11.7 (Figure 4 8 and Figure 4 7). At a constant temperature the

solubility of uric acid and xanthine is solely dependent on the pH of the dissolving medium. The

first dissociated proton makes a major contribution to the solubility of xanthine and uric acid.

In solubility studies on uric acid as a function of pH at constant temperature of 38oC by

Smith, A. and Finlayson, B., 1974, the maximum uric acid concentration achievable for pH

values less than 5 was 0.257 + 0.006 mM. This, uric acid concentration was independent on pH

values less than 5.0. At 5 < pH < 7 the solubility of uric acid increased to 3 mM 19 mM. At pH

values below its pKa, uric acid exists as a cationic compound. Subsequently, at pH above its first

pKa value uric acid exists predominantly as a monoanion (Smith, A. and Finlayson, B. 1974;

Konigsberger, E. and Wang, Z. 1998; Konigsberger, E and Wang Z. 1999). Similarly, xanthine

has lower solubility at pH value below its first pKa whereas the anionic form is predominant at

pH above its pka. At pH > 7.5, the anionic form is the predominant species due to the increased

dissociation of xanthine (Konigsberger, E and Wang, Z. 2001).










0

HN 6 N pKal = 5.74
41 8 =
H


pKa2=10.3


O


Fig 4-8: Acid dissociation of uric acid and pKa values. This is an adaptation from The Metabolic
and Molecular Basis of Inherited Disease, Scriver B.V., Sly C, Kinsler W.K. and
Vogelstein, B. volume II, 8th, pp 2513 2530 (2001).


0 H



!C
I
H


pKa2=11.7


pKal = 7.7


0

N0


Fig 4-9: Acid dissociation of xanthine, pKa values adapted from The Metabolic and Molecular
Basis of Inherited Disease, Scriver, B.V., Sly C, Kinsler, W.K. and Vogelstein, B.
volume II, 8th, pp 2513 2530 (2001).









CHAPTER 5
EXPERIMENTAL

Materials and Chemicals

All chemicals were of the American Chemical Society (ACS) reagent grade. Xanthine

(2,6-dihydroxypurine, C5H402N4), uric acid (2,4,8 trioxypurine, C5H403N4), potassium

dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate Na2HPO4 and sodium

dihydrogen phosphate (NaH2PO4) were obtained from Sigma Aldrich (St. Louis, Missouri,

USA.). The HPLC grade methanol was obtained from Fischer Scientific (Pittsburgh,

Pennsylvania, U.S.A), Hanks Balance Salt Solution (HB SS) and RPMI 1640 were obtained from

Invitrogen Corporation (Carldbad, California, U.S.A). Magna Nylon membrane filter of 0.45 tm

pore size, and 47 mm diameter was obtained from GE Osmotics Labstore (Minnetonka,

Minnesota, U.S.A). Millex-HV 0.2 [tm syringe driven filter unit was obtained from Millipore

Corporation (Billerica, Massachusetts, U.S.A).

Instrumentation

The HPLC system (Figure 3 -4) consisted of an HPLC constant flow pump, HP 1050

series (Hewlett Packard Company, Austin, Texas, U.S.A) with a four port injection valve and

20.0 [iL injection loop. A Burdick and Jackson ODS C-18, 5 [tm particle size, 250 mm long x

4.6 mm internal diameter, (Burdick and Jackson Laboratories Inc, Muskegon, Michigan, U.S.A)

was connected between an in-line filter containing a 0.2 [im, 0.118 x 0.062 x 0.250 IN frit

(Upchurch Scientific, Oak Harbor, Washington, U.S.A), and MACS 700 UV-absorbance

spectrophotometer (EM Science, Gibbstown, New Jersey, U.S.A), which has a cell volume of 9

ItL. The stainless steel tubing utilized was a 1/16 in o.d x 0.01 in i.d chromatographic grade 316,

(Upchurch Scientific, Oak Harbor, Washington, U.S.A). The signal output was traced with a









strip chart recorder (Hewlett Packard Company, Houston, TA, U.S.A). The void volume was

2.57 mL (See section 5.7.7 for details on void volume determination).

Biological Sample Collection and Treatment

Biological samples that were used in this HPLC work include xanthinuria urine, normal

human urine, and extracellular fluid from the porcine endothelial cells of the pulmonary arteries

Xanthinuria Urine

Xanthinuric urine was received from Guy's Hospital, United Kingdom (courtesy of Dr.

Anne Simmonds). Xanthinuric urine was diluted 600 fold with 3 ImM NaH2PO4/Na2HPO4 at pH

7.4 and filtered through 0.2 [im Millex-HV Nylon membrane filter before injection into the

HPLC column.

Normal Urine

Normal urine was collected at random from healthy individuals without known purine

metabolic disorders. Normal urine was diluted 2000 fold with 31 mM NaH2PO4/Na2HPO4 at pH

7.4 and filtered through 0.2 [m Millex-HV Nylon membrane filter before injection into the

HPLC column.

Cell Culture

Endothelial cells were taken from the main pulmonary arteries of 6 to 7 month old pigs.

Third to sixth passage cells in the monolayer culture were maintained in RPMI 1640 medium

containing 4% fetal bovine serum and antibiotics (10 U/mL penicillin, 100 [g/mL streptomycin,

20 [g/ml gentamicin and 2 [g/ml fungizone).

Extracellular Fluid

The media from the confluent monolayer was removed and cells washed with Hanks

Balanced Salt Solution (HBSS). Approximately 5.0 mL ofHBSS was added to cells in lieu of the









media. The cells were placed in an incubation chamber under normoxia (5 % C02, 20 % 02 and

75 % N2) and hypoxia (5 % C02, 3% 02 and 92% N2) for 24 and 48 hours. The supernatant was

collected in 2 x 2 ml vials and analysed by RPLC.

HPLC Solvents
The solvents used in the HPLC work includes 20 mM KH2PO4 at pH 5.1 (See appendix B

for a description of mobile phase preparation), double distilled water, 50% (v/v) methanol (See

appendix B for methanol-water preparation) and water, and 100% absolute methanol.

Filtration of HPLC Solvents

Mobile phase of ionic strength 20 mM KH2PO4, at pH 5.1, doubly distilled water, and

50% (v/v) methanol/water solution were filtered through 0.45 [m pore size Magna Nylon

membrane filter obtained from GE Osmotics Labstore (Minnetonka, Minnesota, U.S.A).

Absolute methanol was not filtered.

Degassing of HPLC solvent

HPLC solvents were degassed immediately after filtration. Mobile phase of ionic strength

20 mM KH2PO4 at pH 5.1, 50 %( v/v), methanol water mixture, and doubly distilled water

were degassed with stirring under constant vacuum for thirty minutes. 100% absolute methanol

was not degassed.

Conditioning of HPLC Column

Before Analysis

The HPLC column was flushed first with a filtered and degassed 50 % (v/v) methanol -

water at a flow rate of 1.0 ml/min for thirty minutes. This was then followed with filtered and

degassed doubly distilled water for another thirty minutes. Finally, the HPLC column was

flushed for fourty five minutes with the mobile phase.









After Analysis

At the end of day's work, the HPLC column was flushed with filtered and degassed doubly

distilled water for an hour and with 50% (v/v) methanol-water for another hour. Finally, the

HPLC column was flushed with 100% absolute methanol for one hour. The HPLC column was

preserved in 100% absolute methanol.

Calibration Curve

Xanthine and uric calibration curves were determined under chromatographic conditions

delineated above. The details are explained below.

Xanthine Calibration Curve

Exactly 50.0 [tM xanthine stock solution was prepared in 31 mM Na2HPO4/NaH2PO4 at

pH 7.4. Subsequently, lower xanthine working concentrations were prepared from the original

xanthine stock solution (See Table 6 1 for details) and filtered through 0.2 .im sterile Millex-

HV syringe driven Nylon filter unit, (Millipore Corporation, Billerica, U.S.A), before final

injection onto the HPLC column. Xanthine detection was achieved at 270 nm; its signal was

recorded on a strip chart recorder (Fischer Recordall Series 5000, Houston Instruments, Texas,

U.S.A). Retention time and peak heights of xanthine signal were measured manually and a

calibration curve of peak height versus concentration of xanthine was determined.

Uric Acid Calibration Curve

Exactly, 20.0 [tM uric acid stock solutions were prepared in 31 mM Na2HPO4/NaH2PO4 at

pH 7.4. Similarly, uric acid working concentration was prepared from the original uric acid stock

solution and was filtered through 0.2 [tm sterile Millex-HV syringe driven Nylon filter unit

(Millipore Corporation, Billerica, U.S.A), before final injection onto the HPLC column. Uric

acid detection was at 293 nm and the signal was recorded on a strip chart recorder (Fischer









Recordall series 5000, Houston Instruments, Texas, U.S.A). Retention time and peak height of

uric acid signal was manually measured with a ruler and a calibration curve of peak height versus

uric acid concentration was obtained.

Chromatography Conditions

An optimised chromatographic condition for this separation was determined before

xanthine and uric acid separation. The details of the chromatographic conditions are as follows.

Selection of Mobile Phase

In the selection of the mobile phase for uric acid and xanthine separation on a reverse

phase column, structural and chemical properties of these analytes were considered. In addition,

the type of intermolecular forces of attraction between uric acid or xanthine and the mobile phase

was of primary importance. Uric acid is a polar organic compound, which has three hydrogen

bond acceptors and four hydrogen bond donors. Similarly, xanthine is also a polar organic

compound, which has three hydrogen bond donors and two hydrogen bond acceptors. Thus,

xanthine and uric acid form hydrogen bond with water.

Although both compounds are known to be polar, their dissolution in the aqueous phase is

limited and pH dependent (Smith, A. and Finlayson, B. 1974; Konigsberger, E. and Wang, Z.

1998; Konigsnerger, E. and Wang, Z. 1999; Konigsberger, E. and Wang, Z. 2000). To improve

the solubility of these compounds in the aqueous phase, potassium or sodium phosphate buffers

have been recommended (Brajter-Toth, A. and Childers-Peterson, T. 1987). Based on the above

mentioned solubility properties of uric acid and xanthine, 20 mM potassium dihydrogen

phosphate (KH2PO4) at pH 5.1 was selected. This mobile phase has been used to achieve

separation of purine metabolites from electro-oxidation products of uric acid and tubercidin by









Brajter Toth, A. and Childers Peterson, T., 1987 and is a modification of mobile phase

reported by Brown, P. R. and Mon, Z. 1983.

A low mobile phase ionic strength of 20 mM KH2PO4 at pH 5.1 was used to extend the

retention time of xanthine and uric acid to achieve a baseline resolution. A mobile phase pH of

5.1 was selected to maintain the neutral and cationic form of uric acid and xanthine as well as

their hydrophobic interaction with the C-18 alkyl chains of the stationary phase. At a pH of 5.1,

hydroxyl ionization in the stationary phase is likely to be suppressed. This mobile phase was

prepared from 20 mM potassium dihydrogen phosphate and its pH was adjusted with 1.0 M

potassium hydroxide.

UV-Absorbance Maximum for Xanthine.

Exactly 100.0 [iM xanthine concentration was prepared in mobile phase of ionic strength

20 mM KH2PO4,at pH 5.1 and 3 1mM Na2HPO4/NaH2PO4, pH 7.4(physiological buffer). The

ultraviolet absorption spectrum of xanthine was taken with optical fibre HP 8450A UV/Vis

spectrophotometer between 200 to 400 nm.

UV-Absorbance Maximum for Uric Acid.

Exactly 100.0 iM uric acid was prepared in the mobile phase of ionic strength 20 mM

KH2PO4, pH 5.1 and physiological buffer of 31 mM Na2HPO4/NaH2PO4, at pH 7.4. The

ultraviolet absorption spectrum of uric acid was taken with optical fibre, HP 8450A UV/Vis

spectrophotometer between 200 to 400 nm.

Selection of Optimum Flow Rate

Approximately 10.0 [iM uric acid working solution from 50 [iM uric acid stock solution

was prepared in sodium phosphate buffer at pH 7.4. Exactly 20.0 uL of the working uric acid

concentration was injected into the HPLC column and flow rate varied from 0.1 mL/min to 1.5









mL/min at 0.2 mL/min interval. At each flow rate, back pressure, retention time, uric acid peak

height, and width at 50 % peak height were recorded.

The theoretical number of plates (See equation 6) was calculated at each flow rate (See

equation 12). Subsequently, the plate height (H) was determined (See equation 4). Since uric

acid and xanthine have similar chemical and physical properties it was assumed that both

analytes would have similar Van Deemter curve. An optimum flow rate of 1.0 mL/min was

selected based on the symmetrical shape of uric acid peak. This flow rate was located in the flat

region of the Van Deemter curve relative to the 0.2 mL/min obtained at the minimum plate

height. In addition, at this flow rate, time of analysis was less (7.4 minutes) compared to the

optimum flow rate of 0.3 mL/min (18.0 minutes). Further, this flow rate has been used to achieve

suitable separation of xanthine and uric acid (Brajter-Toth A and Childers -Perterson T. 1987).

Selection of HPLC Column

Selection of HPLC column was determined from the physical and chemical properties of

uric acid and xanthine. First, based on hydrogen bonding and hydrophobic retention mechanism

expected for xanthine and uric acid in the aqueous mobile phase and nonpolar stationary phase, a

C-18 HPLC column was selected. In order to avoid a possible hydrophilic interaction between

the aqueous mobile phase and hydroxyl groups in the stationary phase, a trimethylsyl end-capped

C-18 column was considered. Subsequently, a standard ODS C-18, 5 [m particle size, 250 mm x

4.6 mm column was used. The stationary phase of the HPLC column had a pore size of 80

angstroms, surface area of 225 m2/g, 12.5% percent carbon, and is endcaped with trimethylsilyl.

General Chromatographic Conditions

The HPLC analysis was performed on an integrated HPLC system consisting of a pulseless

pump, ODS C-18 column, MACS 700 spectrophotometer (EM Sciences, Gibblestown, U.S.A)









and a strip chart recorder, Fischer Recordall Series 5000, (Houston Instruments, Texas, U.S.A).

A 20.0 iL injection volume was used throughout the HPLC analysis. The mobile phase

consisted of 20 mM potassium dihydrogen phosphate, (KH2PO4) at pH 5.1, adjusted with 1.0 M

potassium hydroxide (KOH). The flow rate was 1.0 mL/min. A Burdick and Jackson ODS C-18,

25 cm x 0.46 cm, 5 [m particle size column was connected between the pump and detector. The

back pressure was kept relatively constant within 115 119 bars. Peak height was measured as a

function of uric acid or xanthine concentration. All solvents were degassed by stirring under

constant vacuum for forty minutes. The HPLC column was conditioned with mobile phase for

forty minutes before HPLC analysis. The equilibration time between injections was ten minutes.

After analysis, the HPLC column was kept clean by flushing with 50 % methanol-water solution,

followed by water and 100 % absolute methanol for three hours at a flow rate of 1.0 mL/min.

Determination of Void Volume

Void volume, which is the total volume of mobile phase in the HPLC column, was

determined by an injection of a 0.1% of acetone into water mobile phase. The HPLC column was

equilibrated for fouty-five minutes with the water as mobile phase before acetone injection. The

flow rate was maintained at 1.0 mL/min in this analysis. Acetone signal was observed at 254 nm

and was recorded on the strip chart recorder. The retention time, and back pressure were

recorded. The volume of mobile phase in the column was calculated from the flow rate equation

(See equation 12).

HPLC Column Validation

The HPLC column was validated based on the manufacturer's recommendation for the

procedure for determining column efficiency. A 65/35 % C2H5OH/H20 mixture which had been

filtered and degassed was passed through ODS C-18, 5 im particle size, 250 mm long x 4.6 mm









internal diameter HPLC column at a flow rate of 1.0 mL/min. After 45 minutes of run, 50/50

(v/v %) methanol-water solvent flow, exactly 10.0 [iL of pre-made mixture of toluene, uracil,

anisole, and acetophenone was injected into the HPLC column. Analyte peak signal at 254 nm

was recorded on the strip chart recorder. Retention time, and back pressure were recorded. The

column efficiency was calculated for each analyte (See Table E 5) and compared with the

manufactures specifications (See Table E 6).

Standard Addition Method

A constant volume standard addition method was used to determine uric acid and xanthine

concentration in both xanthinuric urine and normal urine. This method was used to prevent the

matrix effect from the urine sample on xanthine and uric acid concentration in both xanthinuric

and normal urine. Below are the details of this method:

Xanthinuric Urine.

A 600 fold dilution of xanthinuric urine was made in 31 mM NaH2PO4/Na2HPO4 at pH

7.4. Approximately 15.0 [L of xanthinuria urine, which had been filtered through a Hellex-HV

0.2 [m syringe driven filter unit was pipette into four 10.0 mL volumetric flask. Three of the

volumetric flasks were spiked with 3.0 mL, 6.0 mL and 9.0 mL of 30 [M xanthine working

solution. The final volume in each volumetric flask was made-up with 31 mM

Na2HPO4/NaH2PO4 at pH 7.4. Xanthine signal which was observed at 270 nm was recorded on

the strip chart recorder. The retention time, back pressure and xanthine peak height were

recorded. A graph of xanthine peak height was plotted against xanthine concentration added to

xanthinuria urine. Xanthine concentration in xanthinuric urine was calculated (See appendix G

for details).









Normal Urine

A 2000 fold dilution of normal urine prepared in 31 mM NaH2PO4/Na2HPO4 at pH 7.4.

was used in this analysis. Approximately 5.0 [iL of 2000 fold normal urine was filtered through

0.2 [tm filter into four 10.0 mL volumetric flask. Three of the volumetric flasks were spiked with

5.0 mL, 6.5 mL and 8.0 mL of 10 [iM uric acid working solution. The final volume in each

volumetric flask was made-up with 31 mM Na2HPO4/NaH2PO4 at pH 7.4. A trace of uric acid

signal observed at 293 nm was recorded on the strip chart recorder. The retention time, back

pressure and uric acid peak height was recorded. A graph of uric acid peak height was plotted

against uric acid standard added to normal urine. Uric acid concentration in normal urine was

calculated (See appendix C for details).

Qualitative Analysis of Extracellular Fluid

A preliminary High Performance Liquid Chromatography analysis was used to determine

the oxypurine profile of normoxic (48 hours) extracellular fluid from porcine endothelial cells

from the pulmonary arteries. In this determination, 100 [tL of extracellular fluid was lyophilized

for 30 minutes and reconstituted to 10.0 [iL in 20 mM KH2PO4 at pH 5.1. Approximately 10.0

mL of reconstituted xanthinuric urine was injected into the HPLC column and an oxypurine

signal at 293 nm was recorded on the strip chart recorder. Oxypurine peak assignments were

achieved from a comparison of the chromatogram obtained from extracellular fluid to uric acid,

xanthine, and hypoxanthine standards achieved under similar chromatography conditions.

Oxypurine metabolite profile from the extracellular fluid of porcine endothelial cells is shown in

Figure 6 21.









CHAPTER 6
RESULTS AND DISCUSSION

Xanthine and Uric Acid Ultra-violet Absorption Spectra

In order to optimize the sensitivity and selectivity of xanthine and uric acid detection in the

extracellular fluid and xanthinuric urine, ultra-violet absorption spectra of uric acid and xanthine

were obtained. The UV spectra of xanthine and uric acid were determined in the in physiological

buffer of ionic strength 31 mM NaH2PO4/Na2HPO4 at pH 7.4 (Fig A 22 and A- 24) and in the

mobile phase of ionic strength 20 mM KH2PO4 at pH 5.1 (Figure A 23 and A 25). This was

done to determine the wavelength of maximum absorption in UV absorption spectrum and to

verify the effect of solvent pH on the UV- spectra of these analytes. Xanthine and uric acid were

prepared in 31 mM NaH2PO4/Na2HPO4 at pH 7.4 to mimic physiological conditions as it

pertains in live cells.

Although spectra of uric acid and xanthine obtained in mobile phase and physiological

buffer were superimposed (Figure 6 10 and Figure 6 11) to find a common wavelength that is

suitable for detection of these analytes, we used 270 nm and 293 nm to improve on the

sensitivity for xanthine and uric acid detection respectively. We found that uric acid had two UV

maxima in the physiological buffer, with molar absorptivities of s238 nm, Na2HPO4/NaH2PO4,

pH 7.4 = 9.78 x 103 cm-1M1 and s293 nm, Na2HPO4/NaH2PO4, pH 7.4 = 1.25 x 104 cm-M1.

Similarly, in the mobile phase, uric acid molar absorptivities were E235 nm,KH2PO4, pH 5.1 =

8.59 x 103 cmM-11 and s289 nm, KH2PO4 pH 5.1= 1.14 x 104 cm-M-1 On the other hand,

xanthine showed a single maximum wavelength in both the physiological buffer with molar

absorptivity of E272 nm, Na2HPO4/NaH2PO4, pH 7.4 = 8.99 x 103 cm-lM1 and in the mobile

phase with a molar absorptivity of E269 nm, KH2PO4 pH 5.1 = 9.48 x 103 cm-M1.









The molar absorptivity ofxanthine determined experimentally in this HPLC work was in

agreement with the literature values ( s268 nm (water, pH 6.58) = 9.23 x 103 M-cm-1; s277nm

(water, pH 9.02)= 8.87 x 103 M-cm-1; s240 nm (water, pH 9.02)= 8.09 x 103 M-cm-1) as

reported by Cavalier L.F. et al., 1948. Similarly, molar absorptivity of uric acid agreed with

literature values (uric acid:E290 nm( borate buffer, pH 8.5)= 1.22 x 104 M^cm-1; s292nm

(100mM phosphate buffer, pH 7.0)= 1.27 x 104 M-cm-1; s235 nm (100mM phosphate buffer, pH

7.0)= 1.01 x 104 M-cm-1 as determined by Cavalier, L.F. et al., 1998 and Nakaminami, T. et al.,

1999. Uric acid and xanthine UV absorption maxima were red shifted in the physiological buffer

relative to the mobile phase, possibly due to the dependence of xanthine and uric acid structure

on the pH of the solvent. The molar absorptivities of uric acid at the two maxima wavelengths

were higher in the physiological buffer than in the mobile phase. In addition, molar absorptivity

of xanthine was lower in the physiological buffer than in the mobile phase. Physiological buffer

was used to mimic in-vivo conditions expected in live cells.

In the physiological buffer at pH 7.4, uric acid undergoes the first proton dissociation (pKa

=5.74) and exists in the enol form (Figure 4 8 and Figure 4 9) while xanthine remains neutral

(pKa = 7.7) and in keto form (Figure 4 9). In the mobile phase ionic strength of 20 mM

KH2PO4 pH of 5.1 both analytes remain neutral and in the keto and cationic forms. Xanthine has

four 7t bonds, three a bonds, and nine lone pairs of electrons while uric acid has four t bonds,

four c bonds, and ten lone pairs of electrons, The molar absorptivities, of xanthine and uric acid,

showed an absorption band which corresponds to 7t to 7* transition. The 7t bonds in both analytes

become polarized by the physiological buffer of ionic strength 31 mM Na2HPO4/NaH2PO4 at pH

7.4. As a result of this transition, the amount of energy (longer wavelengths) required to excite

an electron from the bonding 7t orbital into the anti-bonding 7t orbital is decreased (Hart, J.M.et









al.,1943; http://teaching. shu.ac.uk/hwb / chemistry.tutorials/molspec/uvvisable.html.2007). In

order to obtain the highest detection sensitivity, the wavelength of the detector was set at where

the absorbance for uric acid and xanthine were highest.

Xanthine Calibration Curve

In order to determine the minimum concentration of xanthine detectable by the HPLC-UV

method, a calibration curve was obtained for xanthine. Xanthine calibration curve was linear and

related peak height to xanthine concentration over the range of 5.0 iM 50 [M. The linear

dynamic range that was tested for xanthine was 5 tM to 50 aM. The limit of detection for

xanthine was 5.0 aM while the minimum xanthine concentration detected was 5 PM at a signal

to noise ratio of 2. Xanthine calibration curve is shown in Figure 6 12.

The retention time of xanthine in this HPLC analysis was 13.4 + 0.1 min. Xanthine

retention time in xanthinuric urine was confirmed with the retention time of xanthine standard.

Further, xanthine peak was identified when xanthinuric urine was spiked with 30 [M xanthine

standard. The relative standard deviation in retention time was 0.7 %. The percent recovery of

xanthine in xanthinuric urine was 98 %. Thus, only a minimal amount of xanthine was lost

during the separation process. A Solvophobic mechanism is primarily responsible for the

retention of xanthine on the reverse phase column (Westermeryer, F.A. and Maquire, H.A.

1986). Thus, xanthine retention in the HPLC column was achieved through hydrogen bonding

with the mobile phase and hydrophobic interaction with the stationary phase.

Since the pH of the mobile phase was kept below the pKa of xanthine and uric acid, the

hypothesis that the retention mechanism could involve binding to free silanol resulting in mixed-

mode separation cannot be corroborated in our HPLC separation (Nahum A. and Hovarth, C.

1981; Bij, K.E. et al., 1981; Zakaria, M. et al., 1983).The sensitivity of xanthine was 0.078









AU/giM. Nevertheless, in a preliminary experiment to verify the effect of physiological buffer

and Hanks Balance Salt Solution on sensitivity, it was concluded that these buffers did not alter

sensitivity for xanthine detection (data not provided). The intercept on the xanthine peak height

The asymmetric value of xanthine peak (Figure 6 -13) determined at 10 % of peak height was

0.7. The Linear correlation coefficient of xanthine obtained from xanthine calibration curve was

0.995. The signal to noise for xanthine detection as shown in Table 6 1 was determined from

the ratio of xanthine peak height to noise height (See Figure D 27 for details on signal to noise

calculations)axis obtained in the calibration curve seemly corresponds to the background

interference from the mobile phase.

Uric Acid Calibration Curve

The minimum uric acid concentration detectable with the HPLC-UV method was

determined from uric acid calibration curve. Uric acid calibration curve was linear for a plot of

uric acid peak versus concentration of analyte injected into the HPLC column. The limit of

detection for uric acid was 2 gtM at a signal to noise of 3. The calibration curve for uric acid is

shown in Figure 6 14.

The retention time of uric acid was 7.0 + 0.1 min. The relative standard deviation in

retention time was 2.5 %, while the standard deviation in uric acid peak height for a triplicate

injection of 10 [tM uric acid a was 0.1. Similarly, uric acid retention time in normal urine was

confirmed by comparison with the retention time of uric acid standard. Solvophobic mechanisms

are believed to be primarily responsible for the retention of uric acid on this reverse phase

column (Westermeryer, F.A. and Maquire, H.A. 1986). At the pH of the mobile phase the

hypothesis that uric acid, retention mechanism could involve binding to free silanol, which lead









to a mixed-mode separation cannot be corroborated in our HPLC separation (Nahum, A. and

Hovarth, C. 1981; Bij, K.E. etal., 1981; Zakaria, M. etal., 1983)

The sensitivity for uric acid was 0.211 AU/iM. Nevertheless, in a preliminary experiment

to verify the effect of physiological buffer and Hanks Balance Salt Solution on sensitivity, it was

found that these buffers had no effect on sensitivity for uric acid detection (data not provided).

Linear regression analysis of the dependence of uric acid peak height of the standards gave a

correlation coefficient of 0.995. The signal to noise ratio as shown in Table 6 2 was determined

from the ratio of uric acid peak height to noise height (See Figure D 27 for details on signal to

noise ratio calculation).

Xanthine Concentration in Xanthinuric Urine

A constant volume standard addition method was used to measure xanthine concentration

in xanthinuric and normal urine. The normal urine, which was obtained from a healthy person

was clinically unrelated to the xanthinuric patient. A constant volume standard addition method

was used to avoid urine matrix effect on uric acid concentration. Xanthine standard was prepared

in 31 mM sodium phosphate buffer at a pH of 7.4 to mimic physiological conditions.

Furthermore, the slopes of the xanthinuric standard addition curve (Figure 6 15), which

corresponds to the sensitivity of HPLC method for xanthine detection was similar to the

sensitivity of the xanthine calibration curve obtained in physiological buffer (Figure 6 12).

This suggests that matrix in the 600 fold diluted xanthinuria urine had no effect on xanthine

concentration as determined by the HPLC method. Xanthine concentration in the undiluted

xanthinuric urine was 2.8 x 103 aM, and is in agreement with the value provided by Dr.

Simmonds. The clinical reference range for xanthine in xanthinuric urine is 358 gM to 3400 aM

(Boulieu, R. et al., 1982; Mei, D.A. et al., 1996; Hassoun, P.M. et al., 1992). Xanthine standard









addition curve and its chromatogram is shown in Figure 6 -15, Figure 6 16 and Figure 6 17.

Clinical reference range for xanthine in normal urine is from 41 tM to 161 PM (Machoy, Z. and

Safranow, K. 2005). Interestingly, uric acid was not detected in the xanthinuric urine at 600 fold

dilution of xanthinuric urine. This is because xanthine oxidase is absent in purine catabolic

pathway of xanthinuric patient. The chromatogram of xanthine standard addition of xanthinuric

urine is shown in Figure 6 17.

Uric Acid Concentration in Normal Urine

A constant volume standard addition method was used to measure uric acid concentration

in normal urine. The normal urine was obtained from a healthy person who was clinically

unrelated to the xanthinuric patient. A 2000 fold dilution of urine was made to avoid matrix

effect on uric acid concentration. The linear calibration range for uric acid was 2 [iM to 20 riM.

Therefore, a constant volume standard addition method was used to determine uric acid

concentration in normal urine. Uric acid standard was prepared in 31 mM sodium phosphate

buffer at a pH of 7.4 to mimic physiological conditions.

Uric acid concentration in normal urine was 5.76 + 0.1 mM. The standard addition curve

and the chromatogram obtained for uric acid in normal urine is shown in Figure 6 -18, Figure

6 19, and Figure 6 20. Interestingly, uric acid was not detected in the xanthinuric urine.

However, a five-fold dilution of the normal urine at a decreased attenuation of 0.001AUFS,

showed xanthine peak which is about 150 iM in normal urine (See Figure A 26).

Extracellular Fluid from Endothelial Cells

A preliminary investigation on the oxypurine profile in the porcine extracellular fluid from

the endothelial cells showed five significant peaks (See Figure 6 21). Peak assignments based

on comparative studies with the retention time of their respective standards, identified these









peaks as uric acid, xanthine, and hypoxanthine. An attenuation of 0.001 absorbance unit full

scale was used for this qualitative determination of oxypurines in extracellular fluid form porcine

endothelial cells of the pulmonary arteries.

The oxypurine profile we obtained in this extracellular medium was similar to the

chromatographic profile reported by Hassoun, P.M. et al., 1992. In the research work of

Hassoun, P.M. et al., 1992 on the extracellular fluid from endothelial cells of the pulmonary

artery of bovine, xanthine, and hypoxanthine co-eluted. However, in our isocratic HPLC method

a baseline separation was achieved for xanthine and hypoxanthine, thus making our isocratic

HPLC method a better choice for oxypurine profiling in biological fluids.

The high uric acid peak detected in the extracellular fluid suggests that uric acid is actively

excreted from the cells into the extracellular medium as soon as it is formed or the conversion of

hypoxanthine to uric acid takes place at the extracellular surface of the endothelial cells. In

addition, the prominent uric acid peak suggests that hypoxanthine and xanthine are rapidly

converted to uric acid. Uric acid is a potent antioxidant, inhibits lipid peroxidation, and protects

erythrocytes against damage by singlet oxygen and free radical damage to deoxyribonucleic acid

(DNA). Uric acid peak height was compared to the peak height of uric acid standards, and uric

acid concentration in the extracellular fluid from endothelial cells of porcine pulmonary artery

was estimated to be 0.5 iM.











2.0


1.8

1.6 -

1.4 -"

1.2

C 1.0 -

0 0-8
S0.8

< 0-6 -

0-4

0.2 -

0.6
200 220 240 260 280 300 320 340 360 380 400

Wavelength (nm)
Fig 6-10: Ultra-violet absorption spectra of xanthine and uric acid in 20 mM KH2PO4 at pH 5.1.
Molar absorptivities of uric acid: s289 nm, KH2PO4, pH 5.1 = 1.14 x 104 cm-1M-1;
s235 nm, KH2PO4, pH 5.1 = 8.59 x 103 cm-1M-1. Molar absorptivity ofxanthine:
s269 nm, KH2PO4, pH 5.1 = 9.48 x 103 cm-1M-1. Legend: ----- uric acid;
xanthine.










2.0

1.8

1.6

1.4

S1.2 -r

1.0

o 0.8 "

S0.6

0.4

0.2

0.0
200 220 240 260 280 300 320 340 360 380 400

Wavelength (nm)
Fig 6-11: UV-absorption spectra of xanthine and uric acid in the 31 mM Na2HPO4/NaH2PO4 pH
7.4. Molar absorptivities of uric acid: s293 nm, Na2HPO4/NaH2PO4, pH 7.4 = 1.25 x
104 cm-1M1; s238 nm, Na2HPO4/NaH2PO4, pH 7.4 = 9.78 x 103 cmM-1MMolar
absorptivity of xanthine: F 272 nm,Na2HPO4/Na2HPO4, pH.7.4 = 8.99 x 103 cm-1M1.









Table 6-1: Peak height, retention time, and signal to noise ratio of xanthine in 31 mM
Na2HPO4/NaH2PO4 at pH 7.4. Column: B & J ODS C-18, 5 [im particle size, 25
cmx0.46 cm i.d. Mobile phase: 20 mM KH2PO4 at pH 5.1, flow rate: 1.0 ml/min;
wavelength of detection: 293 nm; attenuation: 0.01 absorbance unit full scale (AUFS)
Xanthine Xanthine Peak Retention Signal to noise
concentration height time ratio
(pM) (cm) (min) (S/N)
50.0 3.8 13.5 19
45.0 3.3 13.6 17
40.0 3.1 13.5 16
35.0 2.7 13.4 14
30.0 2.3 13.3 12
25.0 1.9 13.4 10
20.0 1.5 13.3 8
15.0 1.1 13.4 6
10.0 0.5 13.3 3
5.0 0.3 13.2 2










4.0

35

C.) 3.0

S 2.5

2.0

S5 y= 0.078x 0.12
SR'= 0.995
; 1.0

0.5



0.0 5.0 10.0 15.0 20.0 25.0 .30.0 35.0 40.0 -150 50.0

Xanthine Coiiceilltia loil (tiM)
Fig 6-12: Calibration curve ofxanthine. Column: B & J ODS C-18, 5 [am particle size, 25 cm x
4.6 cm i.d; flow rate: 1.0 mL/min; ultra-violet wavelength of detection: 270 nm;
attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; xanthine standards
were prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4









10.0



8.0
I-I


*t 6.0



S 4.0



2.0



0.0 -I

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

Retention time (mini
Fig 6-13: Peak shape of 50.0 pM xanthine prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4.
Column: B & J ODS C-18, 5 [tm, particle size, 25 cm x 4.6 cm i.d; flow rate: 1.0
mL/min; ultra-violet wavelength of detection: 270 nm; attenuation: 0.01 AUFS;
mobile phase: 20 mM KH2PO4 at pH 5.1; injection volume: 20.0 [L. asymmetric
value : 0.7.









Table 6-2: Peak height, retention time, and signal to noise ratio of uric acid prepared in 31 mM
Na2HPO4/NaH2PO4 at pH 7.4; column: B & J ODS C-18, 5 tm particle size, 25 cm x
0.46 cm; mobile phase: 20 mM KH2PO4 at pH 5.1; flow Rate: 1.0 ml/min;
wavelength of detection: 293 nm; attenuation: 0.01 AUFS. Injection volume: 20 pL.
Concentration of Uric acid peak Retention Signal to noise
uric acid height time ratio
(tiM) (cm) (min) (S/N)
20.0 4.3 7.7 7
15.0 3.2 7.7 5
10.0 2.1 7.8 3
8.0 1.7 8.2 3
6.0 1.4 7.7 2
4.0 0.9 7.5 1
2.0 0.7 7.6 1









6.0


I 5.0

76
t 4.0


0 3.0


2.0 -y = 0.211x + 0.0405
.0 ,- R2= 0.9981
1.0 -


0.0 -
0.0 5.0 10.0 15.0 20.0 25.0

Cocn of 11ic acid (uM)
Fig 6-14: Calibration curve of uric acid in 31mM Na2HPO4/NaH2PO4 pH 7.4. Column: B & J
ODS C-18, 5gLm particle size, 25 cm x 0.46 cm i.d; mobile phase: 20 mM KH2PO4 at
pH of 5.1; uv-absorbance wavelength: 293nm. Injection volume: 20.01g; attenuation:
0.01 AUF. 0.5 cm peak height is equivalent to 2.8 x 108 AUFS.









Table 6-3: Standard addition determination of xanthine in xanthinuric urine. Column: B & J
ODS C-18, 5 tm particle size, 25 cm x 0.46 cm i.d; mobile phase: 20 mM KH2PO4 at
pH 5.1; flow rate: 1.0 mL /min; wavelength of detection: 270 nm, attenuation: 0.01
AUFS; xanthinuric urine dilution factor: 600 fold.
Volume xanthine Xanthine peak Retention time of Calculated xanthine
standard height xanthine concentration added
(ml) (cm) (min) (iM)
0.0 0.3 13.2 0.0
3.0 0.8 13.2 9.0
6.0 1.4 13.1 18.0
9.0 2.0 13.2 27.0
















5*
1.5





Sy=0.063x+ 0.27
5 R- = 0.998





-10.0 -5.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0


Xantllne Conc. Added (iIM)
Fig 6-15: Constant volume standard addition curve of xanthinuric urine. Column: B & J ODS C-
18, 5 am particle size, 25 cm x 0.46 cm i.d; flow rate: 1.0 mL/min; ultra-violet
wavelength of detection: 270 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM
KH2PO4 at pH 5.1; xanthinuric urine dilution factor 600 fold.










7.0


6.0

5.0

4.0
-z
S.0 -

2.0

1.0

0.0
0.0 4.0 8.0 12.0 16.0

Retention time (mlill)
Fig 6-16: Unspiked xanthine in xanthinuric urine. Column: B & J ODS C-18, 5 [pm particle size,
25 cm x 0.46 cm i.d; flow rate: 1.0 mL/min; ultra-violet wavelength of detection: 270
nm; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; Urine sample:
15.0 pL; xanthinuric urine dilution : 600 fold.











7.0


6.0
Cs)
2 5.0

4.0

3.0

2.0

1.0-__

0.0 -
0.0 4.0 8.0 12.0 16.0

Retention time (min)

Fig 6-17: Spiked xanthinuric urine (9.0 mL of 31 [M xanthine standard prepared in 31 mM
Na2HPO4/NaH2PO4 at pH 7.4 plus 15.0 gL of xanthinuric urine). Column: B & J
ODS C-18, 5[am particle size, 25 cm x 0.46 cm i.d; flow rate: 1.0 mL/min; ultra-violet
wavelength of detection: 270 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM
KH2PO4 pH 5.1.









Table 6-4: Standard addition determination of uric acid in normal urine. Column: B & J ODS C-
18, 5 tm particle size, 25 cm x 0.46 cm; mobile phase: 20 mM KH2PO4 at pH 5.1;
flow rate: 1.0 mL/min; wavelength of detection: 293 nm; attenuation: normal urine
dilution: 2000.


Uric acid standard
added
(ml)
0.0
5.0
6.5
8.0


Uric acid peak
height
(cm)


Retention time of
uric acid
(min)


Uric acid
concentration added
(GM)
0.0
5.0
18.0
27.0















S 1.2


- 1.0


0.8
.,-C


y= 0.117x+ 0.339
t xr 'r ,


0.6 u.y-

0.4


/ .2



-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Uric Acid Cone. Added p IM)
Fig 6-18: Constant volume standard addition of normal urine. Column: B & J ODS C-18, 5 gm
particle size, 25 cm x 0.46 cm i.d; mobile phase: 20 mM KH2PO4 at pH 5.1; flow
rate: 1.0 mL/min; wavelength of detection: 293 nm; attenuation: 0.01 AUFS. Dilution
factor of normal urine: 2000 fold.











7.0


6.0

5.0 -

4.0

a 3.0

S 2.0

1.0

0.0
0.0 2.0 4.0 6.0 8.0 10.0

Retention Time (liin)
Fig 6-19: Unspiked uric acid in normal urine. Column: B & J ODS C-18 5 [pm particle size, 25
cm x 0.46 cm; flow rate: 1.0 mL/min; ultra-violet wavelength of detection: 293 nm;
attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; normal urine
volume: 5.0 pL; normal urine dilution: 2000 fold.











7.0


S 6.0

5.0

4.0

3.0

2.0 -

1.0 -

0.0 -
0.0 2.0 4.0 6.0 8.0 10.0

Retention Time (min)
Fig 6-20: Uric acid in normal urine, spiked with 8.0 mL of 10 [M uric acid standard prepared in
31 mM Na2HPO4/ NaH2PO4 at pH 7.4. Column: B & J ODS C-18, 5 pm, particle
size, 250 mm x 4.6 mm i.d; flow rate: 1.0 mL/min; ultra-violet wavelength of
detection: 293 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1;
normal urine volume: 5.0 pL









6E-4














v 3E-4
S1 2







3





0 5 10 15
Retention time (min)
Fig 6-21 The oxypurine profile of extracellular fluid from normoxic endothelial cells of porcine.
Mobile phase: 20mM KH2PO4 pH 5.1; column: ODS C-18 5 pm particle size, 250 cm
x 0.46 mm; flow rate: 1.0 ml/min; wavelength: 270 nm; Sample dilution 5 diluted
healthy human urine; physiological buffer: 31 mM Na2HPO4/NaH2PO4 pH 7.4;
attenuation: 0.001 AUFS. Peak 1: solvent front. Peak 2: uric acid, Peak 3: unknown,
Peak 4: Hypoxanthine, Peak 5: Xanthine









CHAPTER 7
SUMMARY AND CONCLUSION

The results indicate that xanthine and uric acid can be separated from xanthinuric urine and

extracellular fluid from porcine endothelial cells of the pulmonary arteries. In the xanthinuric

urine, a xanthine concentration of 2.8 + 0.1 mM was achieved. This value falls within the

accepted clinical reference range of 41 161 iM (Boulieu, R et al., 1983). Uric acid was not

detected in xanthinuric urine because the enzyme responsible for the conversion of xanthine to

uric acid is absent. We also demonstrated that the matrix effect in human urine analysis could be

drastically minimized at higher fold dilution of urine.

The oxypurine profile of the extracellular fluid from porcine endothelial cells of the

pulmonary arteries achieved a baseline separation. The identified peaks in extracellular fluid

were uric acid, xanthine, and hypoxanthine. Xanthine concentration in normoxic extracellular

fluid can be used as a baseline against the hypoxic exposed porcine endothelial cells from the

pulmonary arteries. The selectivity of the HPLC column was better than previously reported, In

addition, the sensitivity and the limit of detection of xanthine and uric acid were not significantly

improved over the previously reported values mentioned above..

The sample preparation technique we adapted was effective since there was a minimal

sample loss during the HPLC process (xanthine reproducibility = 98 %). Previous sample

preparation methods invoked protein precipitation with perchloric acid which was vigorous and

showed poor analyte recovery.

The UV wavelengths of detection used in this HPLC analysis corresponded to the

maximum molar absorptivities of each analyte and not the common wavelength obtained from

the superimposed UV spectrum of xanthine and uric acid in the mobile phase. This HPLC system









is adaptable and could be used for qualitative and quantitative analysis of biological fluid which

contains xanthine and uric acid.









CHAPTER 8
RECOMMENDATION

The oxypurine concentration in extracellular fluid from the endothelial cells of porcine

pulmonary arteries exposed to 48 hours of normoxic and hypoxic conditions could be measured

through standard addition method by utilising of the reverse phase high performance liquid

chromatography system assembled in the laboratory. The sensitivity of this method could be

improved by adopting a new detection technique such as mass spectrometry and/or amperometric

detection.

The high retention time obtained from the current chromatography system could be

reduced by the use of a column with reduced dimensions such as 15 cm x 4.6 cm. In the event

that the oxypurines co-elute during the separation process an organic modifier such as ionic

liquid solvent could be used to achieve a baseline separation. This will be the first time an ionic

liquid has been used to achieve separation of an extracellular fluid.









APPENDIX A
UV-ABSORPTION SPECTRUM OF XANTHINE AND URIC ACID


200 220 240 260 280 300 320 340 360 380 400
Wavelength (nm)

Fig A-22: UV-absorption spectrum of 100.0 !iM uric acid in 31 mM /NaH2PO4, pH 7.4. Molar
absorptivities: s293nm, Na2HPO4/NaH2PO4, Na2HPO4, at pH 7.4 = 1.25 x 104 cm-lM
1; s238 nm, Na2HPO4/NaH2PO4, pH 7.4 = 9.78 x 103 cm-M-1










2.0

1.8

1.6

1.4

1.2

1.0
-a

US 0.8

0.6

0.4 \

0.2

0.0 -
200 220 240 260 280 300 320 340 360 380 400
Wavelength (nm)

Fig A-23: UV-absorption spectrum of 100.0 pM uric acid in 20 mM KH2PO4, pH 5.1; molar
absorptivities: s289 nm, KH2PO4, pH 5.1= 1.14 x 104 cm-1M-1; s234.5 nm,
KH2PO4, pH 5.1 = 8.59 x 103 cm-1M-1











2.0

1.8

16

14

1.2
,)



0 ,

0.4


0.4

0.2

00
200 220 240 260 280 300 320 340 360 380 400

Wavelength (nm)
Fig A-24: UV-absorption spectrum of 100.0 gM xanthine in 31 mM Na2HPO4/NaH2PO4, at pH
7.4; molar absorptivity: s272nm, Na2HPO4/Na2HPO4, pH 7.4 = 8.99 x 103 cm-1M1




















14 -

<, 1 2

S1 0

0 0.8

< 06 \

04

02

0 0 i i i i i *
200 220 240 260 280 300 320 340 360 3

W avelength (nm )

Fig A-25: UV-absorption spectrum of 100.0 pM xanthine in KH2PO4, pH 5.1. Molar
absorptivity s269 nm, KH2PO4, pH 5.1 = 9.48 x 103 cm-1M1












APPENDIX B
NORMAL URINE CHROMATOGRAM


6E-4


3 5


0 10


5 10


Retention time (min)


Fig A-26. A 1 in 5 dilution of normal urine. Mobile phase: 20 mM KH2PO4, at pH 5.1; Column:
ODS C-18, 5 rm particle size, 250 mm x 4.6 mm; Flow rate: 1.0 ml/min;
Wavelength: 270 nm; Sample: Attenuation: 0.001 AUFS. Xanthine concentration:
150 + 0.1 riM. Peak 1- 9, 11, 12, 13: unknown, Peak 10: Uric acid Peak 14:
Hypoxanthine, Peak 15: Xanthine. Detector: 9 iL cell of 6 mm pathlength. Spectra
100 UV-Vis, Spectra-Physics Inc. (Autolab Division, San Jose, California, U.S.A)


3E-4


11--i











APPENDIX C:
LIMIT OF DETECTION OF XANTHINE AND URIC ACID


Table A-5: Limit of detection and minimum concentration of uric acid and xanthine prepared in
sodium phosphate buffer at pH 7.4. Column: B & J ODS C-18, 5[m particle size, 25
cm x 0.46 cm. Mobile phase: 20 mM KH2PO4 at pH 5.1; Diluting solvent: 31 mM
Na2HPO4/NaH2PO4 at pH 7.4; Flow rate: 1.0 ml/min; Wavelength of detection: 293
nm/270 nm. Attenuation: 0.01 AUFS. Limit of detection (LOD) = 3 x Sbk/M; where
M is sensitivity.
Physiological buffer Analyte UV-absorbance Minimum Detection limit


wavelength concentration at S/N = 3 or 2
detected
(nm) (GM)


31 mM
Na2HPO4/NaH2PO4
at pH 7.4


Xanthine

Uric acid


293


2.0


270










APPENDIX D
SIGNAL TO NOISE RATIO


7.0

6.0

5.0
-c
4.0
I
3.0
6 2.0cm
2.0 -

< 1.0 cm

S0.0
-1.0
Retention Time (min)
Fig D-27 Signal to noise ratio for 10 giM uric acid prepared in 31 mM Na2HPO4/NaH2PO4 at pH
7.4,. Retention time = 7.4 minutes. Column: ODS C-18, 5um particle size, 250 mm x
4.6 mm, Mobile phase: 20 mM KH2PO4 at pH = 5.1, flow rate = 1.0 mL/min.
Signal/Noise = 2.1 cm/0.6 cm = 3 (See Table 6 2).









APPENDIX E:
HPLC COLUMN EFFICIENCY

Table E-6: Experimental HPLC column validation. Column: ODS C-18, 5 [m particle size, 250
mm x 4.6 mm i.d column; Flow rate: 1.0 mL/min; Mobile phase: 65/35% (v/v)
methanol/water; Back pressure: 144.
Test analyte Concentration Retention Capacity Theoretical
of analyte time factor number of plates
(mg/L) (min) (k') (N)
Uracil 5.0 2.60
Acetophenone 10.0 4.0 0.6 883
Anisole 250 5.2 1.0 1173
Toluene 700 7.2 1.8 788









Table E-7: Manufacture's HPLC column validation. Column: ODS C-18, 5 [tm particle size, 250
mm x 4.6 mm i.d column; Flow rate: 1.0 mL/min; Mobile phase: 65/35% (v/v)
methanol/water; Back pressure: 144.
Test analyte Concentration Retention Capacity Theoretical
of analyte time factor number of plates
(mg/L) (min) (k') (N)
Uracil 5.0 1.4
Acetophenone 10.0 3.0 1.1 10953
Anisole 250.0 5.2 2.6 13877
Toluene 700.0 9.5 5.6 15749









APPENDIX F
PREPARATION OF SOLUTIONS

Mobile Phase: 20.0 mM KH2PO4, pH 5.1

Approximately 2.72 g of KH2PO4 was weighed and dissolved in 5.0 L of double distilled

water. The resultant solution was stirred till KH2PO4 was completely dissolved in the water. The

pH of solution was adjusted to 5.1 with drops of 1.0 M potassium hydroxide. The final solution

was made up to 1.0 L with double distilled water in a 1.0 L volumetric flask.

Physiological Buffer: 31 mM Na2HPO4/NaH2PO4 pH 7.4

Approximately 1.65 g of NaH2PO4.H20 and 2.79 g of Na2HPO4 were weighed and

dissolved in 0.5 L of double distilled water. The resultant solution was stirred until

Na2HPO4/NaH2PO4 was completely dissolved. The pH of this solution was adjusted to 7.4 with

drops of 1.0 M NaOH and final solution was made up to 1L with double distilled water in a

volumetric flask.

50.0 IM Xanthine Stock Solution

Approximately 0.19 mg of xanthine was weighed and dissolved in 10.0 mL of double

distilled water. The solution was sonicated for thirty minutes and allowed to cool to room

temperature. The final solution was made up to 25.0 mL with double distilled water in a

volumetric flask. All subsequent dilutions less that 50.0 [tM xanthine solution was made by

following a simple dilution method (moles of xanthine before dilution = moles of xanthine after

dilution).

Preparation of 50.0 uiM UricAcid Stock Solutions

Approximately 0.210 mg of xanthine was weighed and dissolved in 10.0 mL of double

distilled water. The solution was sonicated for thirty minutes and allowed to cool to room

temperature. The final solution was made up to 25.0 mL with double distilled water in a









volumetric flask. All subsequent dilutions less that 50.0 iM xanthine solution was made by

following a simple dilution method (moles of xanthine before dilution = moles of xanthine after

dilution).

Preparation of 50% (v/v) Methanol Water Solution.

A 100.0 mL portion of methanol and 100.0 mL of filtered double distilled water were

measured separately into two 100 mL beakers. The two solutions were combined without any

adjustment to the final volume.

Degassing of Solvents

All HPLC solvents were degassed under constant vacuum with vigorous stirring for thirty

minutes before used in the HPLC system.









APPENDIX G
CALCULATION

Ms of K 4 20.0mM x 1000.0mL x136.09g / mol
I. Mass of KH2PO4 =
1000. OmL

= 2.72mg


II. Mass of xanthine


III. Mass of uric acid


50.0/iM x 25, OmL x152. lg / ml
1000.0mL


0. 19mg


50.0 /M x 25.0mL x 152. Ig / mol
1000.OmL


0.21mg


IV. Sodium Phosphate Buffer


[HPO4
pH = pKa + log [HP
[H2PO


[Buffer] total


[HPO ]+[HPO2]


[HPO2-]
7.4 7.2= log --H



[H2POO]
1.58= HPO-
[ H2PO]


31mM = 1.58[H2PO4] + [HPO2 ]









31m ImM
2.6 =[H2 ]


1.192 x102 x138g /mol= 1.645g


[HPO2 = 1.192x10-2 xl.58x141.96g /mol


=2.79g




Uric Acid Concentration in Normal Healthy Urine

Concentration of uric acid Cn = C td
nk nk
Vulnk


Where Cunk = concentration of unknown; Vtot = total reaction volume; Vo = volume of uric acid at

zero uric acid


1; Vo = 0.75 ml; Vunk = 5.0 jil; Cstd: 10 iM;


concentration; Vunk = volume of unknown.


Vtot = 10.0 mCuk


Cnk =1.5mM


750/L xl10.O/pM
5.0/uL










Xanthine Concentration in Xanthinuric Urine


Concentration of uric acid C ,k Ctd
Vunk
Vulnk


Where Cunk = concentration of unknown; Vtot = total reaction volume; Vo = volume of uric acid at

zero uric acid concentration; Vunk = volume of unknown.


Vtot = 10.0 ml; Vo = 1.42 ml Vo = 1420 il; Cstd = 30.0 riM; Vunk= 5.0 il


1420xL x 30.0 p
uk 5.0/pL



Cunk = 2.8mM









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BIOGRAPHICAL SKETCH

Mr. Andrews Obeng Affum was born at the Effia Nkwanta Hospital in Sekondi/Takoradi

of the Western Region of Ghana to a family of Mr. Francis Obeng Affum and Mrs. Mary Afful.

Mr. Affum had his kindergarten and primary education at the Aggrey Memorial Primary School.

He was studious and eager to excel in his academics. He sat for the common Entrance

Examination when he was 15 years of age. He had his Secondary School Education at the

Bompeh Secondary Technical School. Although issues of life delayed him for a while, he never

relented in his drive to achieve success in academics.

Mr Affum had his A-level Education at Ghana Secondary Technical School and excelled

again. He enrolled as a biochemistry student at the Biochemistry Department of University of

Ghana. He graduated with BSc (HONS) in biochemistry and was pronounced as one of the best

student in his graduating class. Immediately after his graduation he was invited to Gottingen

University in Germany. Presently He is pursuing a master's program in chemistry at the

Chemistry Department of University of Florida in the United States of America.





PAGE 1

1 DETERMINATION OF XANTHINE AND URIC ACID IN XANTHINURIC URINE AND EXTRACELLULAR FLUID OF PORCINE E NDOTHELIAL CELLS OF THE PULMONARY ARTERY BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY By ANDREWS OBENG AFFUM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Andrews Obeng Affum

PAGE 3

3 To my parents Mr. Francis Obeng Affum and Mary Afful, my siblings, and Kakra Biritwum who through their encouragement have made it possible for me to complete an MSc degree in chemistry, University of Florida.

PAGE 4

4 ACKNOWLEDGMENTS I expres s my sincere gratitude to my heaven ly father for making it possible for me to complete my MSc degree successfully. I extend my heart-felt appreciation to Dr. Br ajter-Anna Toth for her supervision and encouragement, without which, th is MSc wouldnt have been po ssible. I thank Dr. Ben Smith, Mrs Lori Clark, Dr. Thomas Lyons, laboratory colleagues (Mehjabin Kathiwala and Alpheus Mautjana), supporting staff, machine shop sta ff, electronic shop sta ff (Mr. Harry and Mr. Stephen), and Computer Desk staff in the Chemistry Department of the University of Florida for their assistance. I also wish to express my sincere gratitude to Dr. John Toth for donating an HPLC pump for my research work. I thank my committee me mbers for their time and support in my research work at the Chemistry Departme nt, University of Florida.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................8LIST OF FIGURES .........................................................................................................................9LIST OF ABBREVIATIONS ........................................................................................................ 11ABSTRACT ...................................................................................................................... .............13 CHAP TER 1 INTRODUCTION .................................................................................................................. 15General Introduction .......................................................................................................... .....15Analytical Methods for Measurement of Purines ................................................................... 16Application of HPLC in th e Analysis of Bio-fluids for Purine Metabolites ................... 17Clinical Method for Uric Acid ........................................................................................212 PURINE METABOLITES AND RE LATED ENZYMES IN MAN ..................................... 22Endothelial Cells of Pulmonary Arteries ................................................................................22Xanthinuria ................................................................................................................... ..........22Types of Xanthinuria .......................................................................................................23Enzymes Involved in Xanthinuria ...................................................................................24Xanthine Oxidoreductase ................................................................................................24Aldehyde Oxidase ...........................................................................................................263 HIGH PERFORMANCE LIQUI D CHROMATOGRAPHY .................................................28Chromatography ................................................................................................................ .....28Instrumentation ............................................................................................................... ........28Reverse Phase High Performance Liquid Chromatography (RPLC) .............................. 30Principle of Operation .....................................................................................................31Column Selection and Efficiency .................................................................................... 33Mobile phase selection .............................................................................................36Flow rate selection ...................................................................................................364 BIOLOGICAL FLUIDS WITH UR IC ACID AND XANTHI NE ......................................... 46Urine ......................................................................................................................... ..............46Composition of Urine .......................................................................................................... ...46Uric Acid (2, 6, 8 trihydroxypurine) ............................................................................ 46

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6 Xanthine (2, 6 dihydroxypurine) ..................................................................................47Solubility of xanthine and uric acid ................................................................................. 485 EXPERIMENTAL .................................................................................................................. 50Materials and Chemicals .........................................................................................................50Instrumentation ............................................................................................................... ........50Biological Sample Coll ection and Treatment ......................................................................... 51Xanthinuria Urine ............................................................................................................ 51Normal Urine ...................................................................................................................51Cell Culture .....................................................................................................................51Extracellular Fluid ...........................................................................................................51HPLC Solvents ................................................................................................................ 52Filtration of HPLC Solvents ............................................................................................52Degassing of HPLC solvent ............................................................................................ 52Conditioning of HPLC Column ....................................................................................... 52Before Analysis ...............................................................................................................52After Analysis ..................................................................................................................53Calibration Curve ............................................................................................................ 53Xanthine Calibration Curve ............................................................................................. 53Uric Acid Calibration Curve ...........................................................................................53Chromatography Conditions ...........................................................................................54Selection of Mobile Phase ...............................................................................................54UV-Absorbance Maximum for Xanthine. .......................................................................55UV-Absorbance Maximum for Uric Acid. ...................................................................... 55Selection of Optimum Flow Rate .................................................................................... 55Selection of HPLC Column ............................................................................................. 56General Chromatographic Conditions ............................................................................. 56Determination of Void Volume ....................................................................................... 57HPLC Column Validation ............................................................................................... 57Standard Addition Method ..............................................................................................58Xanthinuric Urine. ........................................................................................................... 58Normal Urine ...................................................................................................................59Qualitative Analysis of Extracellular Fluid ..................................................................... 596 RESULTS AND DISCUSSION ............................................................................................. 60Xanthine and Uric Acid Ultr a-violet Absorption Spectra ...................................................... 60Xanthine Calibration Curve .................................................................................................... 62Uric Acid Calibration Curve ...................................................................................................63Xanthine Concentration in Xanthinuric Urine ........................................................................ 64Uric Acid Concentration in Normal Urine .............................................................................65Extracellular Fluid from Endothelial Cells ............................................................................. 657 SUMMARY AND CONCLUSION .......................................................................................83

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7 APPENDIXA UV-ABSORPTION SPECTRUM OF XANT HINE AND URIC ACID ............................... 86B NORMAL URINE CHROMATOGRAM .............................................................................. 90C LIMIT OF DETECTION OF XAN THINE AND URIC ACID ............................................. 91D SIGNAL TO NOISE RATIO ................................................................................................. 92E HPLC COLUMN EFFICIENCY ............................................................................................ 93F PREPARATION OF SOLUTIONS ....................................................................................... 95G CALCULATION ....................................................................................................................97REFERENCES .................................................................................................................... ........100BIOGRAPHICAL SKETCH .......................................................................................................107

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8 LIST OF TABLES Table page 6-1 Peak height and signal to noise ratio of xanthine in physiological buffer ......................... 69 6-2 Peak height, and signal to noise ratio of uric acid in physiological Buffer ...................... 72 6-3 Standard addition determination of xanthine in xanthinuric urine. ................................... 74 6-4 Standard addition determination of uric acid in norm al urine. .......................................... 78 A-5 Limit of detection of xanthine and uric acid ......................................................................91 E-6 HPLC column validation. ..................................................................................................93 E-7 Manufactures HPLC column validation. .......................................................................... 94

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9 LIST OF FIGURES Figure page 2-1 Schematic of metabolic pathway in purine nucleotide m etabolism in man ....................... 273-2 Piston and check valve of a reciprocating pump ............................................................... 403-3 An internal loop valve with four pots ................................................................................413-4 A schematic of the self integrated HPLC system .............................................................. 423-5 A typical aklylated silica surface fo r reverse phase stationary phase ................................ 433-6 Typical retention time versus pH graph for a cationic species ..........................................443-7 The figure above represents the theore tical Van Demeter plot for uric acid ..................... 454-8 Acid dissociation of uric acid and pKa values ................................................................... 494-9 Acid dissociation of xanthine, pKa values .........................................................................496-10 Ultra-violet absorption spectra of xa nthine and uric acid in mobile phase 676-11 UV-absorption spectra of xanthine and uric acid in physiological buffer ......................... 686-12 Calibration curve of xanthine. ........................................................................................... .706-13 Peak shape of 50.0 M xanthine prepared in physiological buffer ................................... 716-14 Calibration curve of uric acid ........................................................................................... .736-15 Constant volume standard addi tion curve of xanthinuric urine .........................................756-16 Unspiked xanthine in xanthinuric urine ............................................................................. 766-17 Spiked xanthine in xanthinuric urine .................................................................................776-18 Constant volume standard addition of normal urine .......................................................... 796-19 Unspiked uric acid in normal urine ....................................................................................806-20 Spiked uric acid in normal urine ........................................................................................ 816-21 The oxypurine profile of extracellular fluid from normoxic endothelial cells .................. 82

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10 A-22 UV-absorption spectrum of uric acid in physiological buffer ........................................... 86A-23 UV-absorption spectrum uric acid in mobile phase ........................................................... 87A-24 UV-absorption spectrum of xant hine in physiological buffer ........................................... 88A-25 UV-absorption spectrum of xanthine in mobile phase ...................................................... 89A-26 A 1 in 5 dilution of normal urine .......................................................................................90D-27 Signal to noise ratio ...........................................................................................................92

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11 LIST OF ABBREVIATIONS S/N Signal to noise ratio Nm Nanometers M Micromolar XOR Xanthine oxidoreductase XDH Xanthine dehydrogenase NADH Nicotinamide adenine dinucleotide FAD Flavin adenine dinucleotide PEEK Polyetheretherkitone k Capacity factor tr Retention time (minutes) to Void time (minutes) V Retention volume (milliliters) Vo Void volume (milliliter) N Number of theoretical plates L Column length (cm) H Plate height (cm) dp Diameter of packing material Rs Resolution h Reduced plate height (cm) AUF Absorbance unit full scale Selectivity factor W1 Base width length of peak 1 t1 Retention time of peak 1 Dm Diffusion coefficient in stationary phase

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12 Ds Diffusion coefficient in mobile phase Column parking factor Tourtosity factor f (k) F unction of capacity factor

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13 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DETERMINATION OF XANTHINE AND URIC ACID IN XANTHINURIC URINE AND EXTRACELLULAR FLUID OF PORCINE E NDOTHELIAL CELLS OF THE PULMONARY ARTERY BY HIGH PERFORMANCE LIQUID CHROMATOGRAPY By Andrews Obeng Affum December 2007 Chair: Brajter-Anna Toth Major: Chemistry A reverse phase high performance liquid chro matography method with ultraviolet light detection was developed to determine the concen trations of xanthine and uric acid in the extracellular fluid of endotheli al cells of porcine pulmonary arteries and in normal and xanthinuric urine. Normal urine samples were collected randomly and filtered through a 0.45 m nylon filter before analysis. Xanthine and uric acid concentrations were determined by a constant volume standard addition method. The mobile phase was 20 mM KH2PO4 at pH 5.1 and the detection wavelength 270 nm (xanthine) and 293 nm (uri c acid). The injection volume was 20.0 L and attenuation was 0.01 absorbance unit full scale (AUFS). Retenti on times for xanthine a nd uric acid standards were respectively 13 0.1 minutes and 7.0 0.1 minutes. The linear correlation coefficient for xanthine and uric acid working cu rves were 0.995 and 0.998 respectively The linear dynamic range, at the low concentra tion limits of xanthine and uric acid, was 5 M to 40 M, and 2 M to 20 M, respectively. The sensitivity of xanthine in 31 mM Na2HPO4/NaH2PO4 at pH 7.4 was 0.08 AU/ M, whilst that of uric acid in the same physiological buffer was 0.21 AU/M. The limit of de tection (LOD) for xant hine and uric acid

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14 were respectively 5.1 M, (S/N = 2) and 1.6 M (S/N = 3). Xanthine in xanthinuric urine was 2.8 0.1 mM; while uric acid in normal urine was 5.7 0.1 mM. In the extracellular fluid, the oxypurine peaks were identified as uric acid, hypoxanthine and xanthine.

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15 CHAPTER 1 INTRODUCTION General Introduction The need to develop an analytical technique to s electively detect and quantitate purine metabolites such as xanthine and uric acid biomarkers in human urine and extracellular fluids is of interest to the analytical chemist and the cl inician in the diagnosis of diseases. A suitable analytical method for detection a nd quantitation of xanthine and ur ic acid must be fast, robust, less expensive, and reproducible. In addition, it is important that the developed method must be selective, for xanthine and uric acid detection and quantitation. Developing a suitable analytical method for these analyte is important because this method could be adapted and used to separate other analytes that have similar physical and chemical properties. Xanthine and uric acid are know n to be involved in many clini cally important diseases and metabolic disorders that are known to be geneti cally related, such as xanthinuria and Lesch Nyhan syndrome. In addition, endothelial cells of the pulmonary arteries under oxidative stress conditions generate xanthine and uric acid metabolites. Analytical method which can separate and detect uric acid and xanthine in biological fluids is relevant to biomedical research and in the prognosis and diagnosis of the diseases known to cause imbalance in xanthine and uric acid concen trations. In addition, xanthine and uric could serve as biomarkers for diseases. The common analytical methods, which have been used to detect purines in biological fl uids, include enzymatic assays, colorimetry, and chromatography. In these established methods, analyte detection used is important in biological analysis. The general objective of this project was to develop a comp rehensive HPLC method with ultraviolet detection to determine xanthine and ur ic acid concentration in biological fluids. The specific objectives are: (1) to set-up an HPLC in strument; (2) to determine the sensitivity of the

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16 HPLC method; (3) to determine the limit of det ection (LOD) for xanthine and uric acid. and (3) measure the concentration of xanthine and uric acid in (a) xanthinuric urine and (b) in the extracellular fluid of porcine endothe lial cells of the pulmonary arteries. Although popular, isocratic elution methods ar e less common for xanthine and uric acid separation in biological fl uids. Nevertheless, an isocratic el ution method was used to achieve a separation of uric acid and xanthine in xanthinuric and normal urine, as well as in hypoxic and normoxic extracellular fluid from porcine endoth elial cells of the pulmonary arteries, which, have been oxidatively stressed for forty-eight hours. Analytical Methods for Measurement of Purines A number of analytical methods have been utilized to separa te purines (adenine, guanine, xanthine, hypoxanthine and uric acid) from biological fluids. Fo r example, purines (oxypurines) were separated and detected from urine (Boulieu, R. et al ., 1982; Boulieu, R. et al ., 1983; Gonnet, C. et al; 1983), plasma (Boulieu, R. et al ., 1982; Boulieu, R. et al ., 1983; Gonnet, C. et al ; 1983), serum (Castilo, J.R et al ., 2001) cerebro spinal fluids (Michal, K. et al ., 2005) and saliva (Nakazawa, H. et al ., 2003). In addition, purines have al so been extracted and separated from biological tissues such as animal heart (Mei, D.A. et al ., 1996; Yacoub, M.H. et al ., 1990) and placenta (Westermeryer, F.A. et al ., 1986). To achieve a suitable separation and quantitation of purine metabolites in biological fluids, sample preparation a nd detection, which are dependent on the physical and chemical properties of uric acid and xanthine must be considered. Analytical methods that have been used to achieve oxypurine separation in biological fluids include: (a) chromatography methods such as ion-exchange chromatography (Katz, S. et al ., 1983; Iwase, H. et al., 1975); gas-liquid chromatography coupled to mass spectrometry (Iwase, H. et al .,Chadard, J.L. et al ., 1980) and reverse phase high performance liquid

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17 chromatography coupled to an ultra-violet light electrochemical or mass spectrometry detection (Baltassat, P. et al ., 1984; Baltassat, P. et al .,1982; Gonnet, C. et al ., 1983; Hassoun, P.M. et al ., 1992; Boulieu, R. et al ., 1983; Boulieu, R. et al ., 1983; Katz S. et al ., 1983; Machoy, Z. and Safranow, K. 2005; Mei, D.A. et al ., 1996; Nakaminami, T. et al ., 1999;Yacoub, M.H et al 1990. (b) enzymatic methods which involve measur ements of hydrogen peroxide levels from xanthine oxidase reaction with xant hine or uric acid (Davis, J. et al ., 2005; Hart, J.M. et al ., 1943). Application of HPLC in the Analysis of Bio-fluids for Purine Metabolites In a reverse phase chrom atography separation of purines from urinary calculi, Zygmunt, M and Safranow, K. 2005 used a gradie nt elution method and varied methanol concentration as well as pH to separate 16 oxypurines, which included uric acid and xanthine. In this method the average retention times of uric acid and xant hine were 3 and 5.5 minutes respectively. Although, the retention time was much shorter than that repor ted in other HPLC methods, the ternary mobile phase com position (solvent A: 50 mM KH2PO4 pH 4.6; solvent B: 50 mM KH2PO4/K2HPO4 at pH 6.4; solvent C: methanol) wa s highly complex to use in routine laboratory purine analysis. Moreover, the high ionic strength of the phosphate buffer (100 mM K2HPO4/KH2PO4) was not best because it could easily prec ipitate in the mobile phase containing methanol used in the separati on as an organic modifier. In addition, the extended use of highly aqueous mobile phase could cause a stationary phase collapse (poor wettability of stationary pha se) and can adversely affect analyte peak shape, retention time, and column life span. In anot her gradient method developed by Mei, D.A. et al ., 1998, a microbore column (ODS-2, C18, 5 m, 25 0 mm x 1.0 mm) was used to achieve a

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18 separation for xanthine, hypoxanthine and uric acid. The retention times for this separation were 10.3 and 6.9 minutes for xanthine and uric acid respectively. The problem associated with Mei, D.A. et al ., 1998 method is the high back pressure that is known to be associated with columns of redu ced internal diameter. The retention times (uric acid 7.5 and xanthine 10 minutes) we re relatively similar compared to previously established chromatography methods. Microbore columns have an advantage of improved sensitivity and less mobile phase consumption because of the reduced internal diameter of the column. Further, microbore columns are expensive if compared to the standard HPLC columns. The gradient elution method as mentioned above requires a careful equilibration of the column between samples to ensure repr oducible retention time. Purines have been separated by reversephase HPLC with organic modifier in the aqueous mobile phase, but, this method is time consuming because of longer equilibration time. In an isocratic HPLC method pr eviously used by Boulieu, R. et a.,l 1982, and modified by Hassoun, P.M. et al ., 1992, a mobile phase composition of 20 mM KH2PO4 at pH 3.60 was utilized to achieve xanthine, uric acid, and hypoxanthine separati on on a C-18 column. The retention times of uric acid and xanthine were 9 and 14 minutes respec tively. These retention times were longer than those achieved by gr adient methods reported by Mei, D. A. et al ., 1998. However, the limit of detection for xanthine ob tained in this isocratic method was lower (0.05 M) compared with the gradient method (8 M) of Mei, D.A. et al ., 1998. In a reverse phase method reported by Nakasawa, H et al ., 2003, with an amperometric detection (+0.6V) of uric acid in saliva, the limit of detection of uric acid wa s 3 nM at a flow rate of 0.2 mL/min with 74 mM potassium phosphate buffer at pH 3.0 as mobile phase. In this

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19 method, the low flow rate and high ionic strength of the mobile phase co uld cause phosphate to precipitate in the head of the HP LC pump and stainless tubing. To dissolve the phosphate deposit in the HPLC system, it is necessary to flush with water for a long time. The method of Nakasawa, H. et al 2003 is therefore time consuming because of extensive column flushing time. In addition, analyt es that have similar oxidation potentials tend to suppress uric acid detection. In an isocratic elution method by Gonnet, C. et al ., 1983, a shortest run time was obtained for uric acid whic h eluted at 4.1 minutes and xanthine at 5.3 minutes. Although retention time was inproved, Gonnet, C. et al ., 2003 did not achieve a baseline separation of oxypurines. In addition, uric acid and xanthi ne peaks appeared to nearly co-elute at 1.5 ml/min flow rate. Furthermore, the low pH of the mobile phase (0.02M KH2PO4 at pH 3.65) is not suitable for the stainless tubing in the HPLC system (Gonnet, C. et al., 1983; Chadard, J.L. et al ., 1980). Ion pair chromatography method for determina tion of uric acid in human brain dialysis fluid was reported by Marklund, N. et al ., 2000. At a flow rate of 0.8 mL/min, and with a 5.0 mM H3PO4 at pH 2.4 as mobile phase, the lim it of detection for uric acid was 0.25 M. This method requires control of column temperature (28oC) for biological fluid HPLC purine separation. A regular HPLC instrument without temperature controlling equipment is therefore not sufficient. In addition, the ion pairing agents (tetrabutylammonium hydrogensulphate) present in the mobile phase makes regeneration of HPLC column difficult, and may compromise the performance and shorten the shelf life of the HPLC column. Also changing HPLC column regularly could affect the reprodu cibility of retention time. In addition to HPLC chromatography techniques utilized in the separation of uric acid and xanthine from biological fluids, other analytical methods such as enzyme assays (Telefoncu, A.

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20 et al ., 2004; Korf, J. et al ., 1995), colorimetry (Miller, P. and Oberhozer, V. 1990; Carrol, J.J. et al ., 1971) and nuclear magnetic resonance have been used to detect and quantitate either uric acid or xanthine in biological fluids. In most of these methods only uric acid was analyzed; however it is a generally believed that this method is applicable to xanthine. Unfortunately, these methods besides chromat ography have major deficiencies such as poor sensitivity and selectivity, which makes th em a poor choice for xanthine and uric acid detection in the urine of man a nd extracellular fluid of porcine e ndothelial cells in the pulmonary arteries. For instance, in the uricase assay used to determine uric acid in serum, the redox conversion of phosphotungsten to tungsten, whic h is used as a measure of uric acid concentration, is affected by matrix components of the reaction medium. Visible light absorption by the phosphotungsten complex or the amount of hydrogen peroxide produced is used as a measure of the concentration of uric acid in the serum. Despite the suitability of the uricase method, phosphotungsten or hydrogen peroxide measurements have not been su ccessful for uric acid detectio n and quantitation in biological samples because of turbidity of the reacti on medium from proteins and other possible interferences that accompany the analyte of interest. A recent sh ift from ultraviolet detection mode for enzyme assay to fluorescence detecti on has had a limited success, because of possible interference from other fluorescent substa nces in the sample (Castillo, J.R. et al ., 2001). One popular enzyme assay that has been used to determine uric ac id concentration in biological fluids is the xanthine oxidase assay. In this method, the alkalinity (pH ~ 9) of the reaction medium showed a deleterious effect on th e structure of xanthine oxidase (Castilo, J.R. et al ., 2001). In another enzyme assay, xanthine ox idase modified glassy carbon paste electrode developed by Telefoncu, A. et al ., 2004, was used to determine xa nthine concentrations in blood

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21 plasma. This method may not show the true uric acid concentration because it does not include any prior sample treatment, thus fluorescent interferences may mask the uric acid concentration through signal cross talk. Furthermore, this method has low reproducibility and is time consuming. Clinical Method for Uric Acid Clinical m ethods that have been used to detect uric acid and xanthine includes spectrophotometry, enzymatic assays, and electrochemical assa ys. In the spectrophotometry methods, ultra-violet light at 293 nm is used to determine the concentration of uric acid in biofluids. However, this method is not spec ific because of interferences from endogenous components which seem to absorb ultra-violet lig ht at same wavelength as uric acid. With enzymatic assay, uricase method is used to determin e the concentration of ur ic acid in biofluids. Uricase converts uric acid to allantoin and the de crease in absorbance of uric acid at 293 nm is measured as a function of uric acid concentration (Skoug, J.W. et al ., 1986; Roland, E. et al ., 1979). Electrochemical sensors have been used to de tect and measure uric acid in biofluids. For example, carbon fibre sensors were used by Davis, J. et al ., 2006 to detect uric acid directly from serum (Davis, J et al ., 2006). In a xanthine oxidase modified glassy carbon paste electrode to detect and quantitate uric acid, an oxidation poten tial of 0.6 Volts was applie d to this electrode to produce a current which was proportional to uric acid concentration pr esent in biofluid (Telefoncu, A. et al ., 2004). The only problem associated with this technique is the cross signal talk from other analytes such as ascorbic acid which have similar oxidation potential as uric acid.

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22 CHAPTER 2 PURINE METABOLITES AND RELATED ENZYMES IN MAN Endothelial Cells of Pulmonary Arteries The pulm onary artery is lined with smooth m onolayer of cells called endothelial cells. Endothelial cells form a barrier between the blood and vascular ti ssue of the pulmonary artery. In addition, endothelial cells facilitate a two way transport of biomolecules between the blood and the vascular tissue. Pulmonary ar tery is known to be affected by diseases such as pulmonary edema: a condition in which fluid accumulates in the lungs usually because the heart left ventricle does not pump adequately (Seki, T. et al ., 2007), and pulmonary thromboembolism: a blockage of the pulmonary artery by a bl ood clot (Steiner, I. 2007; Scalea, M.T. et al ., 2007). These diseases present low oxygen concentration (< 3% O2) below the physiological oxygen concentration which is exposed to endothelial cells. At such low oxygen tensions, purine catabolism in endothelial cells can be enhanced, which result in the generation of uric acid and xanthine from the purine catabolic pathway (Hassoun, P.M. et al ., 1992). These biomolecules are excreted into the extracellular sp ace of the endothelial cells. The ability to detect uric acid and xanthine in normoxic endotheli al cells will serve as a gold standard for detecting these biomolecules in a diseased pulmonary artery. Xanthinuria In the m etabolic pathways of purine, many clinical defects can occur which may lead to diseases. Changes in purine concentrations in biol ogical fluids such as urine could be used as a diagnostic measure of these defects and an indi cator of the effectiven ess of the associated enzymes in the purine catabolic pathway. One well known and a rare clinical defect is xanthinuria. Xanthinuria is a pur ine metabolic disorder, which is usually clinically mild and

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23 asymptomatic. As an inherited autosomal recessive disorder, it is known to be associated with low uric acid and high xa nthine concentrations in the plasma and urine. The physiological plasma xanthine level in a healthy person is less than 5 M while in xanthinuric person is over 10 M (Scriver, B.V. et al ., 2001). Boulieu, R. et al ., 1983 found by HPLC method that the mean normal xanthine con centration in plasma is 1.4 0.7M; clinical range: < 0.5 2.5 M while the ur inary xanthine is 68 42 M; clinical range: 41 161 M. Xanthinuric individuals have hi gh xanthine and moderate hypoxant hine concentrations in urine and in blood, because hypoxanthine is salv aged to inosine monophosphate by hypoxanthine guanine phosphoribosyl phosphate (HGPT) in the purine salvage pathway. High xanthine concentrations can cause xanthine deposits in the urinary tract, and is known to result in hematuria or renal colic, and acute renal failure or chronic complications related to urolithiasis ( http://teaching.shu.ac.uk/hwb/chemistry/tut otrials/ m o lspec/uvvisabl.html). In addition, xanthine could also be deposited in the muscles, which is normally associated with severe pain. The main source of xanthi ne is through the guanine nucleotide catabolic pathway. The schematic of nucleotide catabolic pathway is clearly shown in Figure 2 1. Types of Xanthinuria The two m ajor sub-types of xanthinuria, which have been reported, are xanthinuria type I and xanthinuria type II. These s ubtypes are a result of the effect of mutations in the molybdenum cofactor sulfurase on th e active sites of xanthine dehydroge nase/xanthine oxidase and aldehyde oxidase. Mutations that have been determined in the molybdenun cofactor sulfurase are a G to C in nucleotide 466,GCC (Ala) to CCC (Pro) in exon 2 and C to T in nucleotide 1255, CGA (Arg) to TGA (Ter),(Hada et al., 2003). A deficiency in molybdenum cofactor sulfurase causes xanthine dehydrogenase/xanthine oxidase and aldehyde oxidase to remain desulfonated and

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24 deficient. Xanthinuria type I therefore results from a gene tic deficiency of xanthine dehydrogenase, and aldehyde oxidase, while xanthinuria type I is known to be involved with a deficiency in xanthine ox idoreductase. A mutational an alysis by Topalogue, R. et al ., 2003, on the XDH gene at exon 20 identified an A to T base change in nucleotide 1264, AAG (Lys) to TAG (Tyr). This mutation distinguishes xanthinuri a type I from xanthinuria type II. The main physiological substrates of xant hine oxidoreductase are hypoxant hine and xanthine, which are derived from the breakdown of the major purines in the cell: adenine and guanine. It has been established that xanthine oxidore ductase is the last enzyme in the purine catabolic pathway in humans and higher apes. In other animals such as birds and reptiles, uric acid is converted to allantoin by uricase. Enzymes Involved in Xanthinuria Biological enzym es that have been known to be associated with xanthinuria include xanthine oxidoreductase, aldehyde oxidase and sulfite oxidase. These enzymes belong to molybdenum iron-sulfur flavin hydroxylases (Hada et al ., 2003). Molybdenum is essential in their catalytic activities. They wo rk in synergy to effect a part icular sub-type of xanthinuria. Xanthine Oxidoreductase Under physiological con ditions, xanthine oxidore ductase (XOR) exists in two convertible forms as xanthine oxidase (XO) and xanthine dehydrogenase (XDH). Xant hine oxidoreductase (XOR) is found both in the cytoplasm and on ce ll membranes (Hare, M. J. and Berry, C.E. 2004). It has been established that proteolysis (pro tein treated with protea ses, such as trypsin, chymotrypsin, and pancreatin) a nd cysteine oxidation (Cyst 535 and Cyst 992) of XOR results in an irreversible conversion of XDH to XO. In xanthine dehydrogenase th e electron acceptor is nicotinamide adenine dinucleotide (NAD) while in xanthine oxidase it is molecular oxygen (O2).

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25 However, it has been established that under so me physiological conditions such as hypoxia, molecular oxygen could also serve as electron acceptor for XDH (Hare, M.J. and Berry, E.C. 2004). In the catalysis of xanthine oxidase and xanthine dehydroge nase with xanthine as the substrate, superoxide, hydrogen peroxide and nicotinamide adenine dinucleotide (NADH) are produced, respectively. The generati on of superoxide and peroxide s during this enzymatic action is known to contribute to oxidati ve stress and tissue injury. It is also known that xanthine oxidoreductase (XOR) oxidizes a variety of pyr imidines, aldehydes and pterins. Xanthine oxidoreductase is cytosolic and the optimal pH at which maximu m enzyme activity is obtained varies between 5.6 with pterin as the substrate and 8.4 with xanthine as substrate (Yokoyama, Y et al ., 1990). The polypeptide chain of XOR is known to consist of 1331 amino acids in human, 1331 amino acids in rat, 1335 amino acids in mouse and 1358 in chicken liver enzyme. The purified protein is a homodimer, and consists of iden tical subunits of size wh ich is about 150 kDa, estimated by SDS-PAGE (Wright, M.R. et al ., 1997; Ravio, O.K. et al ., 2005; Okamato, K. et al ., 2007). Each subunit is known to contain a mo lybdenum center (C-terminal; 85 KDa), an FAD center (40 KDa with NAD+) and two iron sulphur centers (N-terminal; 20 KDa). Xanthine oxidoreductase is coded by a single gene whic h is located on chromosome 2p22. This gene consists of 36 exons and 35 introns (Xu, P. et al ., 1996; Wright, M.R. et al ., 1997). The exon intron structure is highly c onserved and generates about 1330 1355 amino acid residues. In humans, substantial xanthine oxidoreductase activity is found in the mucosal lining of the liver and small intestine (Hare, M.J. and Berry, E.C. 2004). It has been reported that low activity of xanthine oxidoreduc tase is found in the plasma, the endothelium, bronchial wall,

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26 heart, lungs and kidneys (Moriwaki, Y. et al ., 1993). Xanthine oxidored uctase reacts with xanthine or hypoxanthine through a two electron mechanism. In its enzyme mechanism, a base abstracts a proton from the Mo-OH group, initia ting a nucleophile whic h attacks the purine substrate. In the process oxygen is incorporated into the purine substrate. The hydroxyl group on the molybdenum is then replaced by hydr oxide from the surrounding aqueous medium. Xanthine oxidoreductase has varying Michea lis Menten constants (Km) values. This enzyme is known to have Km value of 9 M fo r hypoxanthine, 7 M for xanthine and 5 M for adenine (Yokoyama, Y. et al ., 1990). A purine analog, allopurinol is readily oxidized by XOR with a Km of 2 M. Aldehyde Oxidase Aldehyde oxidase is a dim eric molecule of approximately 300,000 molecular weight. The two subunits are independent in function. Each subunit contains a flavin adenine dinuleotide (FAD), two iron-sulphur clusters and molybdenum (Berry, C.C. and Hare, J.M. 2004). Aldehyde oxidase has similar gene sequence as xanthine oxidase and each subun it is approximately 150 KDa. Aldehyde oxidase is located on chro mosome 2q33 in humans and is found in the homologous region of chromosome 1 in mouse (Mcanaman, L.J. et al ., 2000). This cytosolic enzyme is involved in the catalytic oxidation of N-oxides, nitrosamines, and hydroxamic acids. It uses molecular oxygen as its physiological elec tron acceptor and is known to produce superoxide radicals and hydrogen peroxides.

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27 GTP GDP GMP Guanosine Guanine XA IMP AS AMP ATP ADP Adenosine Adenine Inosine Hypoxanthine Xanthine Uricacid HPRT HPRT APRT XO XOG APNP 5'NT AK 5'NT IDHASLASL 5'NT PNP Denovopurine synthesis Fig 2-1: Schematic of metabolic pathway in purine nucleotide metabolis m in man. This figure was adapted from Biochemistry by Mathews, K.C., Van Holde, K.E. and Ahern, G.K. 3rd Edition pages 804 806 (2001). HPRT: Hypoxanthine guanine phosphoribosyl transferase, PNP: Puri ne nucleoside phosphorylase, IDH: IMP dehydrogenase, ASL: Adenylsuccinate sy nthase, GA: Guanase, XO: Xanthine oxidase, APRT: Adenine phosphoribosyl tr ansferase; 5NT: 5 Nucleotidase

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28 CHAPTER 3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Chromatography Chrom atography was invented by a Russian bot anist named Tswett. Chromatography is a union of two Greek words chromos (color) and graf e (writing). It is a separation technique that utilizes a stationary phase a nd a mobile phase. In chromatogr aphy, the separation process is achieved by a distributio n of analyte between the two phase s. The stronger the forces of interaction between the analyte and the mobile phase the greater the amount of solute that will be held in the mobile phase. Similarly, the stronger the inte raction between the analyte and the stationary phase the greater the amount of solute that will be held in the stationary phase. Th e distribution coefficient (KD) of an analyte, which is the ratio of the conc entration of analyte in th e stationary phase to its concentration in the mobile phase (Raymond, P.W.S. 1994), indicates the extent to which an analyte interacts with the stationary phase during elution. Another important factor to consider is the retention time. Solute retention and consequently resolution is determined by the magnitude of the distribution coefficients of the solutes with respect to the stationary phase and relative to each other. Instrumentation The kind of instrum ent that is used for a pa rticular chromatography separation determines the quality of the analytical method. Since th e results obtained from high performance liquid chromatography are reproducible, it is important that the instrumentation used for the HPLC separation is emphasized. In addi tion, the components of an HPLC system must be within acceptable and prescribed limits, without which the system is rendered unsuitable for a particular chromatographic separation.

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29 The main components of an HPLC system are the pump, injector, column and the detector. The pump consists of one or more pistons/pump-head assemblies. The pump head is made of 316 stainless steel and has a cavity into which the pi stons move in and out, which cause the mobile phase solvent to be pushed into the column. The fl ow rate range of an HPLC pump is determined from a combination of the cavity volume, pist on diameter and speed of the piston stroke. The two main types of elution methods used in chromatography are isocratic and gradient elution. Isocratic elution requi res one piston/pump-head assembly (shown in Figure 3 2) whereas gradient elution involves multiple pi ston/pump head assemblies. Pump assemblies typically utilizes one-way check-valves (inlet check valve and outlet check va lve) to direct flow of mobile phase. A pulseless flow of mobile phase is achieved by dualpist on pump configurations. A pressure transducer is placed between the outle t check valve and the column to monitor back pressure changes. To withstand the high back pr essures generated in the HPLC system, stainless steel tubing of moderate inner diameter (0.01 in to 0.02 in) is utilized (Sadek, P.C. 2000). The replaceable in-line filter prevents partic ulate matters generated by the mobile phase or piston seal from reaching the HPLC column. The inlin e filter is usually made of stainless steel or polyetheretherketones (PEEK) with 0.5 m to 2.0 m pore size. Another important component of the HPLC syst em is the injection valve. The injector provides a well defined volume in which the samp le is contained prior to introduction into the mobile phase. Injection of sample into the mobile phase is achieved by a manual or an automated injection through a valve. The valve has a samp le port and a sample loop (Shown in Figure 3 3). The sample is introduced in to the injector through a syring e. When the injection loop is switched to an on-flow position, the sample loop is connected to the mobile phase flow path.

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30 However, if it is switched to an off-flow position the sample is loaded into the loop. Figure 3 3 shows an internal loop valve with four ports. Another important component of the analytic al HPLC system is the column connection. Usually, a guard column is placed at the head of the column to prevent particulate matter in the mobile phase and the pump from reaching the analytical HPLC column. A small internal diameter of about 0.007 inches stainless tubing is used to connect the guard column to the HPLC column. Similarly, it is important that a stainless tubi ng of equal dimension links the column to the detector. The HPLC column contains the stat ionary phase and column dimensions vary with application, and it dictates the vo lume of the mobile phase that is consumed in the separation process. The final component of the HPLC system is the detector. The common detectors usually associated with HPLC separations are: (1) fluorescence; (2) ultraviolet-visible; (3) electrochemical; (4) conductivity; (5) refractive i ndex and (6) mass spectrome try. The detector is chosen to generate the optimum response from each analyte in the sample mixture. Finally, the eluent from the detector is collec ted into reservoir and classified as waste. It is important that HPLC components mentioned above ar e properly calibrated in order to achieve accurate, precise, and reproducible results to be obtained. A schematic assembly of a typical HPLC system is shown in Figure 3 4. Reverse Phase High Performance Liquid Chromatography (RPLC) This is a kind of chrom atography in which the mobile phase is pol ar, such as water (H2O) and the stationary phase is non polar alkyl chains (-CH2CH3). The main materials used as stationary phases are alkyl modified silica (SiO3(CH2)nCH3) and alkylated polystyrenedivinylbenzene (PS DVB) polymers (Katz, E et al ., 1998). This includes octadecyl (C-18);

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31 octyl (C-8); cyano (CN) and phenyl (C6H5) etc. The silica surface is modified by alkyl chains to prevent the silanol groups (-SiOH) reacting with water, which tend to wet the surface of the stationary phase. A schematic of silica bonded stationary phase is shown in Figure 3 5. Various organic modifiers such as methanol, acetonitrile, and tetrahydrfuran are used in the mobile phase of a reverse phase separation proc ess to tune retention, improve selectivity, and enhance peak shape and resolution of analytes (Drumm P et al ., 2005). Reverse phase chromatography is the first choice for most samp les separations. This is because the reverse phase column used is known to be efficient, st able, and reproducible a nd suitable for wide range of sample separations. In addition, it is suitable for separation of analytes that show different concentrations in aqueous solutions, different si zes because of their hydrophobic structures or of different numbers of polar groups. Principle of Operation In reverse phase high perform ance liquid chromatography nonpolar compounds have relatively higher retentio n time than ionic compounds. The driv ing forces for retention in RPLC include: (1) london dispersi on interactions that exist between nonpolar surface ligands and the nonpolar components of the analyt e and (2) the hydrophobi c effect, that is, the tendency to minimize the disturbance of the water structure (Katz, et al ., 1998). This variation in retention time for ionic and neutral analytes is supported by the solvophobic theory (that is a process involving solute transfer into or onto the stationary phase which may involve partition or adsorption or both) of retention. Based on the sol vophobic theory, it is expected that nucleotides will elute first, followed by nucleosides and bases (Zakaria, M. et al ., 1983). Solvophobic theory, in general, c onsiders the stationary phase to have a passive role in the retention process, and that retention is largely due to the thermodynamic interactions between the

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32 solute and the mobile phase. Indeed, solvophobic th eory doesnt provide a complete description of the retention process. Various molecular in teractions such as hydr ophobicity (repulsive interactions between non-polar compounds and a polar environment) electrostatic (ion ion. ion dipole and dipole dipole interactions dependent on the dielectric constant of the solvent) and hydrogen bonding are known to govern significantly the retent ion process. Further, retention is influenced by the pH and ionic strength of the mobile phase. When the pH of the mobile phase is belo w the pKa of the analyte, the pe rcent populations of the cationic form can predominate over the neutral form. Re tention time tends to be higher for neutral molecules compared to their cationic forms. T hus, retention is dependent on the pKa of the compound to be separated and the pH of the mobile phase. A plot of the retention time versus varying pH for an ionizable compound (Figure 3 6) depicts a sigmoid shape with inflection point which corresponds to the pKa of the compound. It must be noted that all pH related changes in retention occur for pH values within 1.5 units of the pKa value (Glajch, J.L. et al ., 1997). At pH of at leas t two units higher than the analyte pKa, there is retention of the anionic form of the acidic an alyte. When the acid or basic groups on a compound are similar, retention behavior is simpler; however mixed basic and acid groups on a compound present a complex retentio n behavior. The relationship between the cationic and the nonionic form of an analyte dependent on the pH of the mobile phase is described by the HendersonHasselbach equation. A pHpkaLog HA (3 1) Where A represents the base form; and HA represents the acid form; pH represents log10[H+]

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33 Another parameter which affects retention time is organic modifier added to the mobile phase in chromatography separation. Organic modi fiers such as methanol are added to aqueous mobile phases to elute non-pol ar compounds that have high hydrophobic properties and interact with the stationary phase, to decrease retention time. Analytes that elute from the chromatographi c column are identified by their retention time (tr), selectivity factor ( ) or by capacity factor (k): 0 0 rtt k t (3 2) Where to (min) is void time (defined as the time taken by an unretain ed analyte to reach the detector), and tr (min) is the retention time; kcapacity factor (5 k 20) The capacity factor may also be expr essed in terms of retention volume. 0 0 rVV k V (3 -.3) Where Vr (mL) is the retention volume and Vo (mL) is the void volume (mL) (defined as the volume of mobile used by an unretra ined analyte to reach the detector. Column Selection and Efficiency The HPLC colum n is the most critical part of the HPLC system. The dimensions of an HPLC column dictate the kind of separation method that needs to be adapted. The standard HPLC column used in reverse phase chromatography is 25 cm long x 0.46 cm inner diameter. The silica or porous-polymer par ticle in HPLC columns provide adsorbable surfaces which serve as support for organic surface layers. These partic les are available in different diameters, pore size, and surface area. The physical properties of these particles are considered during purchase of an HPLC column. In the separa tion of smaller molecules, porous particles with 7 to 12 nm (70 120 angstrom) pores size and surface area of 150 to 400m2/g are used while bigger molecules

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34 require pore diameters larger th an 15 nm. An HPLC column with a 3 5 m particle size is considered suitable for analytical purposes A column with small particle size (3 m) provides faster separation because diffusion length of th e particle is reduced, thus minimizing eddy diffusion and enhancing mass transfer kinetics in the particles (Gui ochon, G. and Gritty, F. 2007). Pore diameters of at least four times the hydrodynamic diameter of the solute ensure that restricted diffusion (confinement of analyte to a small pore in the stationary phase) of the solute does not degrade the column efficiency (Snyder, L.R. and Stadalius, M.A. 1986). The surface area of wide pore particles usually range from 10 to 150 m2/g. Rigid high strength particles such as silica, tend to produ ce low back pressures. Silica based columns are easily dissolved at pH values higher than pH = 8 (Snyder, L.R. and Stadalius, M.A. 1986). Free silanols can cause strong dele terious interaction with basic analytes which could lead to increased retention and broad tailing peaks (Yacoub, M.H. et al., 1990). Basic analyte and silanol interactions are prevented by endcaping the free silanol hydro xyl groups with short carbon chains, such as CH3 groups. Porous polymer particles have high utility at a wide pH range (1 to 13). Column stability is dependent on the pH of the mobile phase, buffer, and organic modifier. Specifications of an HPLC column such as asymme tric factor (ratio of peak widths at 10% of peak height), plate number, selectivity factor back pressure, retention reproducibility, bonded phase concentration, and column stability mu st be known before it is used for any chromatographic separation, so that its condition can be evaluated with respect to specifications after designated period of usage. The efficiency of an HPLC column defines the maximum number of equilibrations measured as theoretical plates (N) that an analyt e will have with the stationary phase as it elutes from the HPLC column. It defines the ability of the HPLC column to produce sharp and narrow

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35 peaks. The theoretical plate (N) of an HPLC column is best determined at the optimum flow rate of the mobile phase. Further, the theoretical number of plates is expressed as the ratio of column length (L) to the plate height equiva lent to a theoretical plate (H). L N H (3 4) Plate height equivalent to a theo retical plate ratio, which gives the reduced plate height (h), H h dp (3 5) Where dp (m) is the diameter of the silica particle. For a particular Gaussian peak at half height the theoretical number of plates is given as 2 0.55.54rt Nx W (3 6) Where W0.5 (cm) is the width length at 50 % of the full peak height. The theoretical number of plates is relate d to resolution (Rs), separation factor ( ) and capacity factor. The relationshi p is given by the equation: '1 41sNk Rxx k (3 7) Where selectivity factor ( ) 2 1k k (3 8) Where k represents capacity factor; k1 repr esents capacity factor of first analyte; k2 capacity factor of second analyte 21 21 stt R WW (3 9)

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36 W1 and W2 are base width le ngth of analyte 1 and analyte 2 respectively. t1 and t2 represent retention time of an alyte 1 and 2 respectively. Mobile phase selection Solvents that are used in the HPLC play cr itical role in the chromatography separation process. The choice of a mobile phase is important because it could either enhance or reduce the selectivity of the separation. In the separation process, mobile phase in teracts (for example, through hydrogen bonding, dipole-dipole, London di spersion and pi-pi inte ractions) with the analyte to either extend or reduce its residence tim e in the stationary phase. Further, the mobile phase interacts with the analyte to minimize or prevent strong interactions with the surface of the stationary phase. The mobile phase also determines the elution or der of the separating analyte as it passes through the HPLC column. Flow rate selection Flow rate of the mobile phase defines the volume of solvent that passes through the HPLC column per unit time. Flow rate provides th e most convenient and predictable changes in separation process. The selection of flow rate for a particular HP LC system is often determined from a Van Deemter plot, which is a graph of the theore tical or reduced plate height (H) versus mobile phase flow rate (Knox, H.J. 2002; Knox, J.H. 1999; Mulholland, M. 004). A theoretical Van Deemter plot is shown in Figure 3 7. The plate height (H) of an HPLC column has contributions from eddy diffusion (A), longitudinal diffusion (B) and resistance to mass tr ansport both in the stationary phase and the mobile phase (C), involved in a chromatographic separation. The band broadening parameters are explained by the Van Deem ter equation (Mulholland, M et al ., 2004; Knox, J.H. 1999;

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37 Lifang, S. and Carr, P.W. 1998; Knox, H.J. 2002; Scott, P.W.R. and Cazes, J. 2002) is expressed as: B HACu C (3 9) (Van Deemter equation as shown in figure 3 7). The variables A and B and C are constants; H represents theoretical plate height and u represents average linear velocity of mobile phase (cms-1). In explicit form, the Van Deemter e quation written above is expressed as: '2'2 11()() 2 2pf m p ms f kdfkd D Hd u uDD (3 10) Where represents column packing factor (~ 0.5 1.5) ; Dm represents solute diffusion in mobile phase (cm2s-1); Ds represents solute diffusion in stationary phase (cm2s-1); dp represents average size of filling particle (m); represents tortuosity factor (dimensionless) ; and df represents thickness of st ationary phase (m); and k capacity factor; f(k) represents function of capacity factor (dimensionless). First, assuming that solute diffusion in the mobile phase is equivalent to solute diffusion in the stationary phase, then s mDD (3 -11) Second representing the uncommon variables in the resistance to mass transfer in both the stationary and the mobile phase by variable b: 22 12()()pfbfkdfkd (3 12) Then, 2 2m p mD bu Hd uD (3 13)

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38 At the minimum plate height: 2m mD Hb uuD (3 14) Equating to zero and solving fo r optimum linear flow rate (Uopt): 2 22m optD U b (3 15) 2m optD Uu b (3 16) Substituting equation 15 into equation 13, the minimum plate 2 min22pHdb (3 17) Minimum plate height represents by Hmin The constant A is independent of the linear velocity. Solute molecules take different paths through the stationary phase. The path lengths of indivi dual molecules differ from one another in a random fashion leading to band broadening. The A term is dependent on the diameter of particles packed into the HPLC column. The B term represent longitudinal diffusion and is dependent on the flow rate, that is, analytes in jected onto the HPLC column tend to diffuse as they elutes from the column towards the detector. The C term is the resistance to mass transfer from both the stationary phase and the mobile phase and is also dependent on linear velocity. When the mobile phase velocity is high and the analyte has a strong affinity to th e stationary phase, the analyte in the mobile phase elutes faster than in the stationary phase. Van Deemter plot in Figure 3 7 shows that the plate height decreases to a minimum and increas es linearly as the linear flow rate increases. At the minimum

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39 plate height all the three parameters that contri bute to plate height are equal and represent the optimal condition suitable for analyte separation. At a minimum plate height the flow rate is optimum and the total theoretical number of theoretical plates is increased. When HPLC columns are operated at optimum flow rate, the separating analyte experiences maximum equilibration between the stationary phase and the mobile phase. The time a solute remains in the HPLC column is inversely proportional to the flow rate of the mobile phase. However, in most chromatographic separations, flow rates are often ope rated at higher than the optimum flow rate in order to decrease analysis time. Another criteria that will require a flow rate higher than optimum flow rate is when the li near portion of the Van Deemter plot is almost flat (Glajch, J.L. et al., 1997; Snyder, L.R. and Stadalius, M. A. 1986; Heftmann, E. 1975). In addition, the symmetrical nature of the analyte peak needs to be critically considered. Flow rate selection is important because it determines the void volume (Vo) of the mobile phase that is used to achieve a particular separation. Mobile phase flow rate (F) is expressed as: 0oV F t (mLs-1) (3 12) Where Vo (mL) is the mobile phase volume; to is void time (min)

PAGE 40

40 Fig 3-2 Piston and check valve of a reciprocatin g pump: The inlet check valve (A) is open during the suction segment of the stroke, but closes during the exhaust segment (B), which forces open the outer check valve. The liqui d is thus displaced towards the column during the exhaust stroke. Adapted from reference 30. (Katz, E., Eksteen, R., Schoenmakers, P. and Miller N. Handbook of High Performance Liquid Chromatography (HPLC).Marcel Dekker Inc, New York, United State of America. 1998. Page 1 500). A

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41 Fig 3-3: An internal loop va lve with four pots. Diagram adapted from Raymond P.W.S. Liquid Chromatography for the Analyst. Marcel Dekker, New York. United States of America 1994, Page 140.

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42 Fig 3-4: A schematic of the self integrated HPLC system used for the HPLC separation of xanthine and uric acid.

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43 Fig 3-5: A typical aklylated silica surf ace for reverse phase stationary phase S 0 o S o o S o o o S o o S o S o o o S o o o S o o S o o o S o o S o o o S iCH3 H3C CH2 H2C CH2 H2C CH2 H2C C H2 H3C o oH2CH2C CH2 H2C CH2 H2C CH2 H3C oH2CH2C CH2 H2C CH2 H2C CH2 H3C o S iCH2 H2C CH2 H2C CH2 H2C CH2 H3C oS iCH3 H3C CH3 S iCH3 H3C CH3 S iCH3 H3C CH3 Si CH3 H3C Si H3C CH3 H2CH2C CH2 H2C CH2 H2C CH2 H3C Si H3C CH3 H2CH2C CH2 H2C CH2 H2C CH2 H3C Si H3C CH3 H H S i l a n o l E n d c a p p i n gC 8 l i g a n d

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44 Fig 3-6: Typical retention time ve rsus pH graph for a cationic sp ecies. Adapted from Glajch J. L., Kirkland J.J, Snyder L.R. Practical High Performance Liquid Chromatography (HPLC) Method Development.2nd. John Wiley and Sons, Inc, New York, United States of America. 1997. Page 295 300.

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45 Fig 3-7: The figure above represents the theoretical Van Demeter plot for uric acid. Standard column and mobile conditions were assumed in determing this curve.

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46 CHAPTER 4 BIOLOGICAL FLUIDS WITH UR IC ACID AND XANTHI NE Urine Urine is a biological effluent that is produced from the ki dney. The composition of urine provides information on the metabolic rate in the human body. The volume and solute composition of urine vary depending on the indivi duals diet, physical activity and health. Urine is filtered from blood through ultra-filtration pr ocess in the nephron of the kidney. For this reason, urine could be used to monitor and evalua te progress of metabolic disease. Therefore, a routine analysis of urine could provide inform ation on the diagnosis of diseases, screen for asymptomatic congenital and heredi tary diseases (Bruzel, A.N. 1994) Composition of Urine The main solutes in urine include urea, chloride, sodium, potassium, phosphates, sulphates, creatinine and uric acid. The protein concentrati on range in normal urine is from 8 to 10 mg/dL. The main protein in urine is albumin. Protei ns such as Tamm Horse Fall Mucoprotein and Bilirubin are present as well, but are minimal in concentration. The concentration of these proteins determines the efficiency of the Bowm ans capsule. The normal specific gravity of urine ranges from 1.002 to 1.035 g/ml. The established pH of urine varies from 4.5 to 8.0 (Bruzel, A.N. 1994). Uric Acid (2, 6, 8 trihydroxypurine) Uric acid is a known diagnostic analyte in human urine. Uric acid (2, 6, 8trihydroxypurine) is the end pr oduct in the purine cat abolic pathway. Purines (adenine and guanine) involved in this pathway can be derive d from dietary sources and the degradation of nucleic acids in cells. Uric acid is a weak acid and undergoes two proton dissociations in basic

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47 medium (Fig 4 8). The reported pKa values of uric acid are 5.74 and 10.3 (Scriver, B.V. et al., 2001). The established uric acid concentrations in adult male and female urine are respectively 250 800 mg/dL and 250 -750 mg/dL and plasma concentrations are 302 60 M and 234 52 M respectively. It is documented that an increa se in uric acid concentration correlates with hyperuricemia, hypertension, gout arthritis and cardiovascular diseases (Nakagawa, T. et al., 2006). Uric acid is a known biomarker in cardiova scular disease and hyper tension (Schechter, M. 2005). Uric acid is a potent antioxidant. Its antio xidant properties invol ve the quenching of reactive oxygen species such as hydroxyl radi cals, singlet oxygen species, and oxo-heme oxidants, which are major causes of cancer, hear t disease and aging (Mac hoy, Z. and Safranow, K. 2005). Purine analogue that is used to reduce uric acid concentration in vivo is allopurinol. Allopurinol inhibits xanthine oxidase activity in the nuc leotide catabolic pathway. Xanthine (2, 6 dihydroxypurine) Xanthine is a precursor of uric acid and metabolite in purine catabolic pathway. The conversion of xanthine to uric acid generate s reactive oxygen species. Reactive oxygen species have been implicated in inflammation and can cer (Khandurina, J. 2000). Xanthine is obtained from both the adenine and guanine cata bolic pathways (See Figure 2 1). It has been established that the main source of xanthine is derived through the guanine pathway since hypoxanthine is continuously salv aged to inosine 5-monophosphate (IMP). Cells tend to lose more energy when high ener gy phosphates bonds are form ed through the de-novo pathway. To prevent this loss of energy, cel ls rely on hypoxanthine salvaged pathway. The established pKa values (Figure 4 9) of xanthine are 7.7 and 11.3 (Scriver, B.V. et al., 2001).

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48 Solubility of xanthine and uric acid Uric acid and xanthine solubil ity in aqueous medium is primarily determined by the pH of the medium, the pKa and temperature .As mentione d, uric acid has pKas of 5.74 and 10.3, while xanthine has pKa of 7.7 and 11.7 (Figure 4 8 an d Figure 4 7). At a constant temperature the solubility of uric acid and xant hine is solely dependent on the pH of the dissolving medium. The first dissociated proton makes a major contribution to the solubility of xanthine and uric acid. In solubility studies on uric acid as a function of pH at constant temperature of 38oC by Smith, A. and Finlayson, B., 1974, the maximum uric acid concentration achievable for pH values less than 5 was 0.257 0.006 mM. This, ur ic acid concentration was independent on pH values less than 5.0. At 5 < pH 7 the solubility of uric acid increased to 3 mM 19 mM. At pH values below its pKa, uric acid exists as a cationic compound. Subsequently, at pH above its first pKa value uric acid exists predominantly as a monoanion (Smith, A. and Finlayson, B. 1974; Konigsberger, E. and Wang, Z. 1998; Konigsberg er, E and Wang Z. 1999). Similarly, xanthine has lower solubility at pH value below its first pKa whereas the anionic form is predominant at pH above its pka. At pH > 7.5, the anionic form is the predominant specie s due to the increased dissociation of xanthine (Konigs berger, E and Wang, Z. 2001).

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49 Fig 4-8: Acid dissociation of uric acid and pKa va lues. This is an adaptation from The Metabolic and Molecular Basis of Inherited Disease, Scriver B.V., Sly C, Kinsler W.K. and Vogelstein, B. volume II 8th, pp 2513 2530 (2001). Fig 4-9: Acid dissociation of xanthine, pKa va lues adapted from The Metabolic and Molecular Basis of Inherited Disease, Scriver, B.V., Sly C, Kinsler, W.K. and Vogelstein, B. volume II, 8th, pp 2513 2530 (2001). HN N H N H H N O O O 2 3 4 6 1 7 8 95 HN N H N H N O O O HN N N H N O O O + 2H+pKa1 = 5.74 p Ka2=10.3N N N N O O H H H N N N N O O H H N N N N O O H pKa1 = 7.7 p Ka2=11.7

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50 CHAPTER 5 EXPERIMENTAL Materials and Chemicals All chemicals were of the American Chemi cal Society (ACS) reagent grade. Xanthine (2,6dihydroxypurine, C5H4O2N4), uric acid (2,4,8 trioxypurine, C5H4O3N4), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate Na2HPO4 and sodium dihydrogen phosphate (NaH2PO4) were obtained from Sigma Al drich (St. Louis, Missouri, USA.). The HPLC grade methanol was obtai ned from Fischer Scientific (Pittsburgh, Pennsylvania, U.S.A), Hanks Balance Salt Solu tion (HBSS) and RPMI 1640 were obtained from Invitrogen Corporation (Carldbad, California, U.S.A). Magna Nylon membrane filter of 0.45 m pore size, and 47 mm diameter was obtained from GE Osmotics Labstore (Minnetonka, Minnesota, U.S.A). Millex-HV 0.2 m syringe driven filter unit was obtained from Millipore Corporation (Billerica, Massachusetts, U.S.A). Instrumentation The HPLC system (Figure 3 4) consisted of an HPLC constant flow pump, HP 1050 series (Hewlett Packard Company, Austin, Texas, U.S.A) with a four port injection valve and 20.0 L injection loop. A Burdick and Jacks on ODS C-18, 5 m particle size, 250 mm long x 4.6 mm internal diameter, (Burdick and Jacks on Laboratories Inc, Muskegon, Michigan, U.S.A) was connected between an in-line filter co ntaining a 0.2 m, 0.118 x 0.062 x 0.250 IN frit (Upchurch Scientific, Oak Harbor, Washi ngton, U.S.A), and MACS 700 UV-absorbance spectrophotometer (EM Science, Gibbstown, New Jersey, U.S.A), which has a cell volume of 9 L. The stainless steel tubing uti lized was a 1/16 in o.d x 0.01 in i.d chromatographic grade 316, (Upchurch Scientific, Oak Harbor, Washington, U.S.A). The signal output was traced with a

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51 strip chart recorder (Hewlett Packard Company, Houston, TA, U.S.A). The void volume was 2.57 mL (See section 5.7.7 for details on void volume determination). Biological Sample Collection and Treatment Biological samples that were used in this HP LC work include xanthinuria urine, normal human urine, and extracellular fluid from the porci ne endothelial cells of the pulmonary arteries Xanthinuria Urine Xanthinuric urine was receive d from Guys Hospital, Unit ed Kingdom (courtesy of Dr. Anne Simmonds). Xanthinuric urine was diluted 600 fold with 31mM NaH2PO4/Na2HPO4 at pH 7.4 and filtered through 0.2 m Millex-HV Nylon membrane filter before injection into the HPLC column. Normal Urine Normal urine was collected at random from healthy indi viduals without known purine metabolic disorders. Normal urine was diluted 2000 fold with 31 mM NaH2PO4/Na2HPO4 at pH 7.4 and filtered through 0.2 m Millex-HV Nylon membrane filter before injection into the HPLC column. Cell Culture Endothelial cells were taken from the main pul monary arteries of 6 to 7 month old pigs. Third to sixth passage cells in the monolayer culture were maintained in RPMI 1640 medium containing 4% fetal bovine serum and antibiotics (10 U/mL penicillin, 100 g/mL streptomycin, 20 g/ml gentamicin and 2 g/ml fungizone). Extracellular Fluid The media from the confluent monolayer was removed and cells washed with Hanks Balanced Salt Solution (HBSS). Approximately 5.0 mL of HBSS was added to cells in lieu of the

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52 media. The cells were placed in an incubation chamber under normoxia (5 % CO2, 20 % O2 and 75 % N2) and hypoxia (5 % CO2, 3% O2 and 92% N2) for 24 and 48 hours. The supernatant was collected in 2 x 2 ml vi als and analysed by RPLC. HPLC Solvents The solvents used in the HPLC work includes 20 mM KH2PO4 at pH 5.1 (See appendix B for a description of mobile phase preparation), double distilled water, 50% (v/v) methanol (See appendix B for methanol-water preparation) and water, a nd 100% absolute methanol. Filtration of HPLC Solvents Mobile phase of ionic strength 20 mM KH2PO4, at pH 5.1, doubly distilled water, and 50% (v/v) methanol/water solution were filt ered through 0.45 m pore size Magna Nylon membrane filter obtained from GE Osmotics Labstore (Minnetonka, Minnesota, U.S.A). Absolute methanol was not filtered. Degassing of HPLC solvent HPLC solvents were degassed immediately after filtration. Mobile phase of ionic strength 20 mM KH2PO4 at pH 5.1, 50 %( v/v), methanol wa ter mixture, and doubly distilled water were degassed with stirring under constant vacuum for thirty minutes. 100% absolute methanol was not degassed. Conditioning of HPLC Column Before Analysis The HPLC column was flushed first with a f iltered and degassed 50 % (v/v) methanol water at a flow rate of 1.0 ml/min for thirty minutes. This was then followed with filtered and degassed doubly distilled water for another thirty minutes. Fi nally, the HPLC column was flushed for fourty five minutes with the mobile phase.

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53 After Analysis At the end of days work, the HPLC column was flushed with filtered and degassed doubly distilled water for an hour and with 50% (v/v ) methanol-water for another hour. Finally, the HPLC column was flushed with 100% absolute methanol for one hour. The HPLC column was preserved in 100% absolute methanol. Calibration Curve Xanthine and uric calibration curves were determined unde r chromatographic conditions delineated above. The details are explained below. Xanthine Calibration Curve Exactly 50.0 M xanthine stock solution was prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4. Subsequently, lower xanthine working c oncentrations were prepared from the original xanthine stock solution (See Tabl e 6 1 for details) and filtered through 0.2 m sterile MillexHV syringe driven Nylon filter unit, (Millipore Corporation, Billerica, U.S.A), before final injection onto the HPLC column Xanthine detection was achieve d at 270 nm; its signal was recorded on a strip chart recorder (Fischer Recordall Series 5000, Houston Instruments, Texas, U.S.A). Retention time and peak heights of xa nthine signal were measured manually and a calibration curve of peak height versus concentration of xanthine was determined. Uric Acid Calibration Curve Exactly, 20.0 M uric acid stock so lutions were prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4. Similarly, uric acid working concentratio n was prepared from the original uric acid stock solution and was filtered through 0.2 m steril e Millex-HV syringe driven Nylon filter unit (Millipore Corporation, Billerica, U.S.A), before final injection onto the HPLC column. Uric acid detection was at 293 nm and the signal was recorded on a strip chart recorder (Fischer

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54 Recordall series 5000, Houston Instruments, Texas, U.S.A). Retention time and peak height of uric acid signal was manually measured with a ru ler and a calibration curve of peak height versus uric acid concentration was obtained. Chromatography Conditions An optimised chromatographic condition for this separation was determined before xanthine and uric acid separation. The details of the chromatographic conditions are as follows. Selection of Mobile Phase In the selection of the mobile phase for ur ic acid and xanthine se paration on a reverse phase column, structural and chem ical properties of these analytes were considered. In addition, the type of intermolecular forces of attraction betw een uric acid or xanthine and the mobile phase was of primary importance. Uric acid is a polar organic compound, wh ich has three hydrogen bond acceptors and four hydrogen bond donors. Similarly, xanthine is also a polar organic compound, which has three hydrogen bond donors and two hydrogen bond acceptors. Thus, xanthine and uric acid form hydrogen bond with water. Although both compounds are known to be polar, their dissolution in the aqueous phase is limited and pH dependent (Smith, A. and Finlay son, B. 1974; Konigsberger, E. and Wang, Z. 1998; Konigsnerger, E. and Wang, Z. 1999; Koni gsberger, E. and Wang, Z. 2000). To improve the solubility of these compounds in the aqueous phase, potassium or sodium phosphate buffers have been recommended (Brajter-Toth, A. and Childers-Peterson, T. 1987). Based on the above mentioned solubility properties of uric acid and xanthine, 20 mM potassium dihydrogen phosphate (KH2PO4) at pH 5.1 was selected. This mobile phase has been used to achieve separation of purine metabolites from electro-oxidation products of uric acid and tubercidin by

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55 Brajter Toth, A. and Childers Peterson, T., 1987 and is a modification of mobile phase reported by Brown, P. R. and Mon, Z. 1983. A low mobile phase ionic strength of 20 mM KH2PO4 at pH 5.1 was used to extend the retention time of xanthine and uric acid to achieve a baseline re solution. A mobile phase pH of 5.1 was selected to maintain the neutral and catio nic form of uric acid and xanthine as well as their hydrophobic interaction with the C-18 alkyl chains of the st ationary phase. At a pH of 5.1, hydroxyl ionization in the stationary phase is likely to be suppr essed. This mobile phase was prepared from 20 mM potassium dihydrogen phos phate and its pH was adjusted with 1.0 M potassium hydroxide. UV-Absorbance Maximum for Xanthine. Exactly 100.0 M xanthine concentration was pr epared in mobile phase of ionic strength 20 mM KH2PO4,at pH 5.1 and 31mM Na2HPO4/NaH2PO4, pH 7.4(physiological buffer). The ultraviolet absorption spectrum of xanthine was taken with optical fibre HP 8450A UV/Vis spectrophotometer between 200 to 400 nm. UV-Absorbance Maximum for Uric Acid. Exactly 100.0 M uric acid was prepared in the mobile phase of ionic strength 20 mM KH2PO4, pH 5.1 and physiological buffer of 31 mM Na2HPO4/NaH2PO4, at pH 7.4. The ultraviolet absorption spectrum of uric acid was taken with optical fibre, HP 8450A UV/Vis spectrophotometer between 200 to 400 nm. Selection of Optimum Flow Rate Approximately 10.0 M uric acid working so lution from 50 M uric acid stock solution was prepared in sodium phosphate buffer at pH 7. 4. Exactly 20.0 uL of the working uric acid concentration was injected into the HPLC column and flow rate varied from 0.1 mL/min to 1.5

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56 mL/min at 0.2 mL/min interval. At each flow ra te, back pressure, retention time, uric acid peak height, and width at 50 % peak height were recorded. The theoretical number of plates (See equation 6) was calculated at each flow rate (See equation 12). Subsequently, the plate height (H) was determined (See equation 4). Since uric acid and xanthine have similar chemical and physical properties it was assumed that both analytes would have similar Van Deemter curve. An optimum flow rate of 1.0 mL/min was selected based on the symmetrical shape of uric aci d peak. This flow rate was located in the flat region of the Van Deemter curve relative to th e 0.2 mL/min obtained at the minimum plate height. In addition, at this flow rate, time of analysis was less (7.4 minutes) compared to the optimum flow rate of 0.3 mL/min (18.0 minutes). Fu rther, this flow rate has been used to achieve suitable separation of xanthine and uric acid (Brajter-Toth A and Childers Perterson T. 1987). Selection of HPLC Column Selection of HPLC column was determined from the physical and chemical properties of uric acid and xanthine. First, based on hydrogen bonding and hydrophobic retention mechanism expected for xanthine and uric acid in the aque ous mobile phase and nonpolar stationary phase, a C-18 HPLC column was selected. In order to avoid a possible hydrophili c interaction between the aqueous mobile phase and hydroxyl groups in the stationary phase, a trimethylsyl end-capped C-18 column was considered. Subsequently, a sta ndard ODS C-18, 5 m particle size, 250 mm x 4.6 mm column was used. The stationary phase of the HPLC column had a pore size of 80 angstroms, surface area of 225 m2/g, 12.5% percent carbon, and is endcaped with trimethylsilyl. General Chromatographic Conditions The HPLC analysis was performed on an inte grated HPLC system consisting of a pulseless pump, ODS C-18 column, MACS 700 spectrophotometer (EM Sc iences, Gibblestown, U.S.A)

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57 and a strip chart recorder, Fisc her Recordall Series 5000, (Houston Instruments, Texas, U.S.A). A 20.0 L injection volume was used throughout the HPLC analysis. The mobile phase consisted of 20 mM potassium dihydrogen phosphate, (KH2PO4) at pH 5.1, adjusted with 1.0 M potassium hydroxide (KOH). The flow rate was 1.0 mL/min. A Burdick and Jackson ODS C-18, 25 cm x 0.46 cm, 5 m particle size column wa s connected between the pump and detector. The back pressure was kept relatively constant within 115 119 bars. Peak height was measured as a function of uric acid or xanthi ne concentration. All solvents were degassed by stirring under constant vacuum for forty minutes. The HPLC column was conditioned with mobile phase for forty minutes before HPLC analysis. The equili bration time between inje ctions was ten minutes. After analysis, the HPLC column was kept clea n by flushing with 50 % me thanol-water solution, followed by water and 100 % absolute methanol fo r three hours at a flow rate of 1.0 mL/min. Determination of Void Volume Void volume, which is the total volume of mobile phase in the HPLC column, was determined by an injection of a 0.1% of acetone into water mobile phase. The HPLC column was equilibrated for fouty-fi ve minutes with the water as mobile phase before acetone injection. The flow rate was maintained at 1.0 mL/min in this analysis. Acetone signal was observed at 254 nm and was recorded on the strip chart recorder. The retention time, and back pressure were recorded. The volume of mobile phase in the colu mn was calculated from the flow rate equation (See equation 12). HPLC Column Validation The HPLC column was validated based on the manufacturers recommendation for the procedure for determining column efficiency. A 65/35 % C2H5OH/H2O mixture which had been filtered and degassed was passed through ODS C-18, 5 m particle size, 250 mm long x 4.6 mm

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58 internal diameter HPLC column at a flow ra te of 1.0 mL/min. After 45 minutes of run, 50/50 (v/v %) methanol-water solvent flow, exactly 10. 0 L of pre-made mixtur e of toluene, uracil, anisole, and acetophenone was inje cted into the HPLC column. Analyte peak signal at 254 nm was recorded on the strip chart recorder. Retenti on time, and back pressure were recorded. The column efficiency was calculated for each anal yte (See Table E 5) and compared with the manufactures specifications (See Table E 6). Standard Addition Method A constant volume standard addition method was used to determine uric acid and xanthine concentration in both xanthinuric urine and norma l urine. This method was used to prevent the matrix effect from the urine sample on xanthine and uric acid concentrat ion in both xanthinuric and normal urine. Below are the details of this method: Xanthinuric Urine. A 600 fold dilution of xanthinuric urine was made in 31 mM NaH2PO4/Na2HPO4 at pH 7.4. Approximately 15.0 L of xanthinuria ur ine, which had been filtered through a Hellex-HV 0.2 m syringe driven filter unit was pipette in to four 10.0 mL volumetric flask. Three of the volumetric flasks were spiked with 3.0 mL, 6. 0 mL and 9.0 mL of 30 M xanthine working solution. The final volume in each volumetric flask was made-up with 31 mM Na2HPO4/NaH2PO4 at pH 7.4. Xanthine signal which was observed at 270 nm was recorded on the strip chart recorder. The retention time, back pressure and xanthine peak height were recorded. A graph of xanthine peak height was plotted against xanthine concentration added to xanthinuria urine. Xanthine c oncentration in xanthinuric urin e was calculated (See appendix G for details).

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59 Normal Urine A 2000 fold dilution of normal urine prepared in 31 mM NaH2PO4/Na2HPO4 at pH 7.4. was used in this analysis. Approximately 5.0 L of 2000 fold normal urine was filtered through 0.2 m filter into four 10.0 mL volumetric flask. Three of the volumetric flasks were spiked with 5.0 mL, 6.5 mL and 8.0 mL of 10 M uric acid working solution. The final volume in each volumetric flask was made-up with 31 mM Na2HPO4/NaH2PO4 at pH 7.4. A trace of uric acid signal observed at 293 nm was recorded on the st rip chart recorder. The retention time, back pressure and uric acid peak height was recorded A graph of uric acid peak height was plotted against uric acid standard added to normal urin e. Uric acid concentraton in normal urine was calculated (See appendix C for details). Qualitative Analysis of Extracellular Fluid A preliminary High Performance Liquid Chromatography analysis was used to determine the oxypurine profile of normoxic (48 hours) extracel lular fluid from porci ne endothelial cells from the pulmonary arteries. In this determination, 100 L of ex tracellular fluid was lyophilized for 30 minutes and reconstituted to 10.0 L in 20 mM KH2PO4 at pH 5.1. Approximately 10.0 mL of reconstituted xanthinuric urine was inje cted into the HPLC column and an oxypurine signal at 293 nm was recorded on the strip ch art recorder. Oxypurine peak assignments were achieved from a comparison of the chromatogram obtained from extracellular fluid to uric acid, xanthine, and hypoxanthine standards achieved under similar chromatography conditions. Oxypurine metabolite profile from the extracellular fl uid of porcine endothelial cells is shown in Figure 6 21.

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60 CHAPTER 6 RESULTS AND DISCUSSION Xanthine and Uric Acid Ultra-violet Absorption Spectra In order to optimize the sensitivity and selectiv ity of xanthine and uric acid detection in the extracellular fluid and xanthinuric urine, ultra-violet absorption sp ectra of uric acid and xanthine were obtained. The UV spectra of xa nthine and uric acid were determined in the in physiological buffer of ionic strength 31 mM NaH2PO4/Na2HPO4 at pH 7.4 (Fig A 22 and A24) and in the mobile phase of ionic strength 20 mM KH2PO4 at pH 5.1 (Figure A 23 and A 25). This was done to determine the wavelength of maximum absorption in UV absorption spectrum and to verify the effect of solvent pH on the UVspectra of these analytes. Xant hine and uric acid were prepared in 31 mM NaH2PO4/Na2HPO4 at pH 7.4 to mimic phys iological conditions as it pertains in live cells. Although spectra of uric acid a nd xanthine obtained in mob ile phase and physiological buffer were superimposed (Figure 6 10 and Figur e 6 11) to find a common wavelength that is suitable for detection of thes e analytes, we used 270 nm and 293 nm to improve on the sensitivity for xanthine and uric acid detection respectively. We found that uric acid had two UV maxima in the physiological buffer, with molar absorptivities of 238 nm, Na2HPO4/NaH2PO4, pH 7.4 = 9.78 x 103 cm-1M-1 and 293 nm, Na2HPO4/NaH2PO4, pH 7.4 = 1.25 x 104 cm-1M-1. Similarly, in the mobile phase, uric acid molar absorptivities were 235 nm,KH2PO4, pH 5.1 = 8.59 x 103 cm-1M-1 and 289 nm, KH2PO4 pH 5.1= 1.14 x 104 cm-1M-1 On the other hand, xanthine showed a single maxi mum wavelength in both the phys iological buffer with molar absorptivity of 272 nm, Na2HPO4/NaH2PO4, pH 7.4 = 8.99 x 103 cm-1M-1 and in the mobile phase with a molar absorptivity of 269 nm, KH2PO4 pH 5.1 = 9.48 x 103 cm-1M-1.

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61 The molar absorptivity of xanthine determined experimentally in this HPLC work was in agreement with the literature values ( 268 nm (water, pH 6.58) = 9.23 x 103 M-1cm-1; 277nm (water, pH 9.02) = 8.87 x 103 M-1cm-1; 240 nm (water, pH 9.02) = 8.09 x 103 M-1cm-1) as reported by Cavalier L.F. et al., 1948. Similarly, molar absorptivity of uric acid agreed with literature values (uric acid: 290 nm( borate buffer, pH 8.5) = 1.22 x 104 M-1cm-1; 292nm (100mM phosphate buffer, pH 7.0) = 1.27 x 104 M-1cm-1; 235 nm (100mM phosphate buffer, pH 7.0) = 1.01 x 104 M-1cm-1 as determined by Cavalier, L.F. et al., 1998 and Nakaminami, T. et al., 1999. Uric acid and xanthine UV absorption maxima were red shifted in the physiological buffer relative to the mobile phase, possibly due to the dependence of xanthine a nd uric acid structure on the pH of the solvent. The molar absorptivitie s of uric acid at the two maxima wavelengths were higher in the physiological bu ffer than in the mobile phase. In addition, molar absorptivity of xanthine was lower in the phys iological buffer than in the m obile phase. Physiological buffer was used to mimic in-vivo condi tions expected in live cells. In the physiological buffer at pH 7.4, uric ac id undergoes the first proton dissociation (pKa =5.74) and exists in the enol form (Figure 4 8 and Figure 4 9) while xanthine remains neutral (pKa = 7.7) and in keto form (Figure 4 9). In the mobile phase i onic strength of 20 mM KH2PO4 pH of 5.1 both analytes remain neutral and in the keto and cationic forms. Xanthine has four bonds, three bonds, and nine lone pairs of el ectrons while uric acid has four bonds, four bonds, and ten lone pairs of electrons, The mola r absorptivities, of xa nthine and uric acid, showed an absorption band which corresponds to to transition. The bonds in both analytes become polarized by the physiologica l buffer of ionic strength 31 mM Na2HPO4/NaH2PO4 at pH 7.4. As a result of this transition, the amount of energy (longer wavelengths) required to excite an electron from the bonding orbital into the anti-bonding orbital is decreased (Hart, J.M.et

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62 al.,1943; http://teaching. shu.ac.uk/hwb / chemistry.tutorials/molspec/uvvisable.html.2007). In order to obtain the highest detec tion sensitivity, the wavelength of the detector was set at where the absorb ance for uric acid and xanthine were highest. Xanthine Calibration Curve In order to determine the minimum concentra tion of xanthine detect able by the HPLC-UV method, a calibration curve was obtained for xant hine. Xanthine calibratio n curve was linear and related peak height to xanthine concentration over the range of 5.0 M 50 M. The linear dynamic range that was tested for xanthine was 5 M to 50 M. The limit of detection for xanthine was 5.0 M while the minimum xanthine concentration detected was 5 M at a signal to noise ratio of 2. Xanthine calibra tion curve is shown in Figure 6 12. The retention time of xanthine in this HP LC analysis was 13.4 0.1 min. Xanthine retention time in xanthinuric ur ine was confirmed with the retent ion time of xanthine standard. Further, xanthine peak was identified when xant hinuric urine was spiked with 30 M xanthine standard. The relative standard de viation in retention time was 0.7 %. The percent recovery of xanthine in xanthinuric urine was 98 %. Thus, only a minimal amount of xanthine was lost during the separation process. A Solvophobic m echanism is primarily responsible for the retention of xanthine on the reverse phase co lumn (Westermeryer, F.A. and Maquire, H.A. 1986). Thus, xanthine retenti on in the HPLC column was achieved through hydrogen bonding with the mobile phase and hydrophobic inte raction with the st ationary phase. Since the pH of the mobile phase was kept below the pKa of xanthine and uric acid, the hypothesis that the retention mechanism could invol ve binding to free sila nol resulting in mixedmode separation cannot be corrobo rated in our HPLC separation (Nahum A. and Hovarth, C. 1981; Bij, K.E. et al., 1981; Zakaria, M. et al., 1983).The sensitivity of xanthine was 0.078

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63 AU/M. Nevertheless, in a preliminary experiment to verify the effect of physiological buffer and Hanks Balance Salt Solution on sensitivity, it was concluded that thes e buffers did not alter sensitivity for xanthine detection (data not provided). The intercept on the xanthine peak height The asymmetric value of xanthine peak (Figure 6 -13) determined at 10 % of peak height was 0.7. The Linear correlation coefficient of xanthi ne obtained from xanthine calibration curve was 0.995. The signal to noise for xanthine detection as shown in Table 6 1 was determined from the ratio of xanthine peak height to noise height (See Figure D 27 for details on signal to noise calculations)axis obtained in the calibration curve seemly corresponds to the background interferences from the mobile phase. Uric Acid Calibration Curve The minimum uric acid concentration de tectable with the HPLC-UV method was determined from uric acid calibration curve. Ur ic acid calibration curve was linear for a plot of uric acid peak versus concentration of analyte injected into the HPLC column. The limit of detection for uric acid was 2 M at a signal to noise of 3. The calibration curve for uric acid is shown in Figure 6 14. The retention time of uric acid was 7.0 0. 1 min. The relative standard deviation in retention time was 2.5 %, while th e standard deviation in uric ac id peak height for a triplicate injection of 10 M uric acid a was 0.1. Similarl y, uric acid retention time in normal urine was confirmed by comparison with the retention time of uric acid standard. Solvophobic mechanisms are believed to be primarily responsible for the retention of uric acid on this reverse phase column (Westermeryer, F.A. and Maquire, H.A. 1986). At the pH of the mobile phase the hypothesis that uric acid, reten tion mechanism could involve bindi ng to free silanol, which lead

PAGE 64

64 to a mixed-mode separation ca nnot be corroborated in our HPLC separation (Nahum, A. and Hovarth, C. 1981; Bij, K.E. et al., 1981; Zakaria, M. et al., 1983) The sensitivity for uric acid was 0.211 AU/M Nevertheless, in a preliminary experiment to verify the effect of physiological buffer and Hanks Balance Sa lt Solution on sensitivity, it was found that these buffers had no effect on sensitivity for uric acid detecti on (data not provided). Linear regression analysis of the dependence of ur ic acid peak height of the standards gave a correlation coefficient of 0.995. The signal to noise ratio as shown in Table 6 2 was determined from the ratio of uric acid peak height to noise height (See Figure D 27 for details on signal to noise ratio calculation). Xanthine Concentration in Xanthinuric Urine A constant volume standard addition method wa s used to measure xanthine concentration in xanthinuric and normal urine. The normal ur ine, which was obtained from a healthy person was clinically unrelated to the xanthinuric pa tient. A constant volume standard addition method was used to avoid urine matrix effect on uric aci d concentration. Xanthine standard was prepared in 31 mM sodium phosphate buffer at a pH of 7.4 to mimic physiological conditions. Furthermore, the slopes of the xanthinuric standard addition curve (Figure 6 15), which corresponds to the sensitivity of HPLC method for xanthine detection was similar to the sensitivity of the xanthine calibration curve obtained in physiological buffer (Figure 6 12). This suggests that matrix in the 600 fold dilu ted xanthinuria urine ha d no effect on xanthine concentration as determined by the HPLC method. Xanthine c oncentration in the undiluted xanthinuric urine was 2.8 x 103 M, and is in agreement w ith the value provided by Dr. Simmonds. The clinical reference range for xanthine in xanthinuric urine is 358 M to 3400 M (Boulieu, R. et al., 1982; Mei, D.A. et al., 1996; Hassoun, P.M. et al., 1992). Xanthine standard

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65 addition curve and its chromatogram is shown in Figure 6 -15, Figure 6 16 and Figure 6 17. Clinical reference range for xant hine in normal urine is from 41 M to 161 M (Machoy, Z. and Safranow, K. 2005). Interestingly, uric acid was no t detected in the xanthinuric urine at 600 fold dilution of xanthinuric urine. This is because xanthine oxidase is absent in purine catabolic pathway of xanthinuric patient. Th e chromatogram of xanthine st andard addition of xanthinuric urine is shown in Figure 6 17. Uric Acid Concentration in Normal Urine A constant volume standard addition method wa s used to measure uric acid concentration in normal urine. The normal urine was obtained from a healthy person who was clinically unrelated to the xanthinuric pa tient. A 2000 fold dilution of ur ine was made to avoid matrix effect on uric acid concentration. The linear cali bration range for uric acid was 2 M to 20 M. Therefore, a constant volume standard additi on method was used to determine uric acid concentration in normal urine. Uric acid standard was prepared in 31 mM sodium phosphate buffer at a pH of 7.4 to mimic physiological conditions. Uric acid concentration in normal urine wa s 5.76 0.1 mM. The standard addition curve and the chromatogram obtained for uric acid in normal urine is shown in Figure 6 -18, Figure 6 19, and Figure 6 20. Interestingly, uric ac id was not detected in the xanthinuric urine. However, a five-fold dilution of the normal ur ine at a decreased attenuation of 0.001AUFS, showed xanthine peak which is about 150 M in normal urine (See Figure A 26). Extracellular Fluid from Endothelial Cells A preliminary investigation on the oxypurine prof ile in the porcine extr acellular fluid from the endothelial cells showed five significant peaks (See Figure 6 21). Peak assignments based on comparative studies with the retention time of their respective standards, identified these

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66 peaks as uric acid, xanthine, and hypoxanthine. An attenuation of 0.001 absorbance unit full scale was used for this qualitative determination of oxypurines in extracellular fluid form porcine endothelial cells of th e pulmonary arteries. The oxypurine profile we obtained in this extracellular medium was similar to the chromatographic profile reported by Hassoun, P.M. et al., 1992. In the research work of Hassoun, P.M. et al., 1992 on the extracellular fluid from endothelial cells of the pulmonary artery of bovine, xanthine, and hypoxanthine co-eluted. However, in our isocratic HPLC method a baseline separation was achieved for xanthi ne and hypoxanthine, thus making our isocratic HPLC method a better choice for oxypur ine profiling in biological fluids. The high uric acid peak detected in the extracellu lar fluid suggests that uric acid is actively excreted from the cells into the extracellular medi um as soon as it is form ed or the conversion of hypoxanthine to uric acid takes place at the ex tracellular surface of the endothelial cells. In addition, the prominent uric acid peak suggests that hypoxanthine and xanthine are rapidly converted to uric acid. Uric ac id is a potent antioxidant, inhibits lipid peroxidation, and protects erythrocytes against damage by singlet oxygen an d free radical damage to deoxyribonucleic acid (DNA). Uric acid peak height was compared to the peak height of uric ac id standards, and uric acid concentration in the extracellular fluid from endothelial cells of porcine pulmonary artery was estimated to be 0.5 M.

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67 Fig 6-10: Ultra-violet absorption spectra of xanthine and uric aci d in 20 mM KH2PO4 at pH 5.1. Molar absorptivities of uric acid: 289 nm, KH2PO4, pH 5.1 = 1.14 x 104 cm-1M-1; 235 nm, KH2PO4, pH 5.1 = 8.59 x 103 cm-1M-1. Molar abso rptivity of xanthine: 269 nm, KH2PO4, pH 5.1 = 9.48 x 103 cm-1M-1. Legend: ----uric acid; xanthine.

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68 Fig 6-11: UV-absorption spectra of xant hine and uric acid in the 31 mM Na2HPO4/NaH2PO4 pH 7.4. Molar absorptivities of uric acid: 293 nm, Na2HPO4/NaH2PO4, pH 7.4 = 1.25 x 104 cm-1M-1; 238 nm, Na2HPO4/NaH2PO4, pH 7.4 = 9.78 x 103 cm-1M-1.Molar absorptivity of xanthine: 272 nm,Na2HPO4/Na2HPO4, pH.7.4 = 8.99 x 103 cm-1M-1.

PAGE 69

69 Table 6-1: Peak height, retention time, and signal to noise ratio of xanthine in 31 mM Na2HPO4/NaH2PO4 at pH 7.4. Column: B & J ODS C-18, 5 m particle size, 25 cmx0.46 cm i.d. Mobile phase: 20 mM KH2PO4 at pH 5.1, flow rate: 1.0 ml/min; wavelength of detection: 293 nm; attenuation: 0.01 absorbance unit full scale (AUFS) Xanthine concentration (M) Xanthine Peak height (cm) Retention time (min) Signal to noise ratio (S/N) 50.0 3.8 13.5 19 45.0 3.3 13.6 17 40.0 3.1 13.5 16 35.0 2.7 13.4 14 30.0 2.3 13.3 12 25.0 1.9 13.4 10 20.0 1.5 13.3 8 15.0 1.1 13.4 6 10.0 0.5 13.3 3 5.0 0.3 13.2 2

PAGE 70

70 Fig 6-12: Calibration curve of xanthine. Column: B & J ODS C-18, 5 m particle size, 25 cm x 4.6 cm i.d; flow rate: 1.0 mL/min; ultra-violet wavelength of detection: 270 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; xanthine standards were prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4

PAGE 71

71 Fig 6-13: Peak shape of 50.0 M xanthine prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4. Column: B & J ODS C-18, 5 m, particle size, 25 cm x 4.6 cm i.d; flow rate: 1.0 mL/min; ultra-violet wavele ngth of detection: 270 nm ; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 at pH 5.1; injection volume: 20.0 L. asymmetric value : 0.7.

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72 Table 6-2: Peak height, retenti on time, and signal to noise ratio of uric acid prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4; column: B & J ODS C-18, 5 m particle size, 25 cm x 0.46 cm; mobile phase: 20 mM KH2PO4 at pH 5.1; flow Rate: 1.0 ml/min; wavelength of detection: 293 nm; attenuatio n: 0.01 AUFS. Injection volume: 20 L. Concentration of uric acid (M) Uric acid peak height (cm) Retention time (min) Signal to noise ratio (S/N) 20.0 4.3 7.7 7 15.0 3.2 7.7 5 10.0 2.1 7.8 3 8.0 1.7 8.2 3 6.0 1.4 7.7 2 4.0 0.9 7.5 1 2.0 0.7 7.6 1

PAGE 73

73 Fig 6-14: Calibration curve of uric acid in 31mM Na2HPO4/NaH2PO4 pH 7.4. Column: B & J ODS C-18, 5m particle size, 25 cm x 0.46 cm i.d; mobile phase: 20 mM KH2PO4 at pH of 5.1; uv-absorbance wavelength: 293nm. Injection volume: 20.0l; attenuation: 0.01 AUF. 0.5 cm peak height is equivalent to 2.8 x 10-8 AUFS.

PAGE 74

74 Table 6-3: Standard addition determination of xanthine in xanthinuric urine. Column: B & J ODS C-18, 5 m particle size, 25 cm x 0.46 cm i.d; mobile phase: 20 mM KH2PO4 at pH 5.1; flow rate: 1.0 mL /min; wavelengt h of detection: 270 nm, attenuation: 0.01 AUFS; xanthinuric urine dilution factor: 600 fold. Volume xanthine standard (ml) Xanthine peak height (cm) Retention time of xanthine (min) Calculated xanthine concentration added (M) 0.0 0.3 13.2 0.0 3.0 0.8 13.2 9.0 6.0 1.4 13.1 18.0 9.0 2.0 13.2 27.0

PAGE 75

75 Fig 6-15: Constant volume standard addition cu rve of xanthinuric urine. Column: B & J ODS C18, 5 m particle size, 25 cm x 0.46 cm i.d; flow rate: 1.0 mL/min; ultra-violet wavelength of detection: 270 nm; attenua tion: 0.01 AUFS; m obile phase: 20 mM KH2PO4 at pH 5.1; xanthinuric ur ine dilution factor 600 fold.

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76 Fig 6-16: Unspiked xanthine in xanthinuric urine. Column: B & J ODS C-18, 5 m particle size, 25 cm x 0.46 cm i.d; flow rate: 1.0 mL/min ; ultra-violet wavelengt h of detection: 270 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; Urine sample: 15.0 L; xanthinuric urine dilution : 600 fold.

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77 Fig 6-17: Spiked xanthinuric urin e (9.0 mL of 31 M xanthine standard prepared in 31 mM Na2HPO4/ NaH2PO4 at pH 7.4 plus 15.0 L of xanthinuric urine). Column: B & J ODS C-18, 5 m particle size, 25 cm x 0.46 cm i.d; flow rate: 1.0 mL/min; ultra-violet wavelength of detection: 270 nm; attenua tion: 0.01 AUFS; m obile phase: 20 mM KH2PO4 pH 5.1.

PAGE 78

78 Table 6-4: Standard addition determination of uric acid in normal urine. Column: B & J ODS C18, 5 m particle size, 25 cm x 0.46 cm; mobile phase: 20 mM KH2PO4 at pH 5.1; flow rate: 1.0 mL/min; wavelength of det ection: 293 nm; attenuation: normal urine dilution: 2000. Uric acid standard added (ml) Uric acid peak height (cm) Retention time of uric acid (min) Uric acid concentration added (M) 0.0 0.1 7.0 0.0 5.0 0.7 7.0 5.0 6.5 0.9 6.9 18.0 8.0 1.1 6.9 27.0

PAGE 79

79 Fig 6-18: Constant volume standard addition of normal urine. Column: B & J ODS C-18, 5 m particle size, 25 cm x 0.46 cm i.d; mobile phase: 20 mM KH2PO4 at pH 5.1; flow rate: 1.0 mL/min; wavelength of detectio n: 293 nm; attenuation: 0.01 AUFS. Dilution factor of normal urine: 2000 fold.

PAGE 80

80 Fig 6-19: Unspiked uric acid in norm al urine. Column: B & J ODS C-18 5 m particle size, 25 cm x 0.46 cm; flow rate: 1.0 mL/min; ultraviolet wavelength of detection: 293 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; normal urine volume: 5.0 L; normal urine dilution: 2000 fold.

PAGE 81

81 Fig 6-20: Uric acid in normal urine, spiked with 8.0 mL of 10 M uric acid standard prepared in 31 mM Na2HPO4/ NaH2PO4 at pH 7.4. Column: B & J ODS C-18, 5 m, particle size, 250 mm x 4.6 mm i.d; flow rate: 1. 0 mL/min; ultra-violet wavelength of detection: 293 nm; attenuation: 0.01 AUFS; mobile phase: 20 mM KH2PO4 pH 5.1; normal urine volume: 5.0 L

PAGE 82

82 Fig 6-21 The oxypurine profile of ex tracellular fluid from normoxi c endothelial cells of porcine. Mobile phase: 20mM KH2PO4 pH 5.1; column: ODS C-18 5 m particle size, 250 cm x 0.46 mm; flow rate: 1.0 ml/min; wavele ngth: 270 nm; Sample dilution 5 diluted healthy human urine; phys iological buffer: 31 mM Na2HPO4/NaH2PO4 pH 7.4; attenuation: 0.001 AUFS. Peak 1: solvent fr ont. Peak 2: uric acid, Peak 3: unknown, Peak 4: Hypoxanthine, Peak 5: Xanthine 5 10 15 Retention time (min) 2 6E 4 3E 4 1 0 3 4 5

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83 CHAPTER 7 SUMMARY AND CONCLUSION The results indicate that xanthine and uric acid can be separa ted from xanthinuric urine and extracellular fluid from porcine endothelial cells of the pulmonary arteries. In the xanthinuric urine, a xanthine concentration of 2.8 0.1 mM was achieved. This value falls within the accepted clinical reference range of 41 161 M (Boulieu, R et al., 1983). Uric acid was not detected in xanthinuric urine because the enzyme responsible for the conv ersion of xanthine to uric acid is absent. We also demonstrated that th e matrix effect in human urine analysis could be drastically minimized at highe r fold dilution of urine. The oxypurine profile of the extracellular fl uid from porcine endothe lial cells of the pulmonary arteries achieved a baseline separatio n. The identified peaks in extracellular fluid were uric acid, xanthine, and hypoxanthine. Xanthine concentrat ion in normoxic extracellular fluid can be used as a baseline against the hypox ic exposed porcine endothelial cells from the pulmonary arteries. The selectivity of the HPLC column was better than previously reported, In addition, the sensitivity and the limit of detecti on of xanthine and uric acid were not significantly improved over the previously reported values mentioned above.. The sample preparation technique we adapte d was effective since there was a minimal sample loss during the HPLC process (xanthin e reproducibility = 98 %). Previous sample preparation methods invoked protei n precipitation with perchloric acid which was vigorous and showed poor analyte recovery. The UV wavelengths of detection used in this HPLC analysis corresponded to the maximum molar absorptivities of each analyte and not the common wave length obtained from the superimposed UV spectrum of xa nthine and uric acid in the m obile phase. This HPLC system

PAGE 84

84 is adaptable and could be used for qualitative an d quantitative analysis of biological fluid which contains xanthine and uric acid.

PAGE 85

85 CHAPTER 8 RECOMMENDATION The oxypurine concentration in extracellular fluid from the e ndothelial cells of porcine pulmonary arteries exposed to 48 hours of nor moxic and hypoxic conditions could be measured through standard addition method by utilising of the reverse phase high performance liquid chromatography system assembled in the laboratory. The sensitivity of this method could be improved by adopting a new detectio n technique such as mass spect rometry and/or amperometric detection. The high retention time obtained from the current chromatography system could be reduced by the use of a column with reduced dime nsions such as 15 cm x 4.6 cm. In the event that the oxypurines co-elute du ring the separation process an or ganic modifier such as ionic liquid solvent could be used to achieve a baseline separation. This will be the first time an ionic liquid has been used to achieve sepa ration of an extracellular fluid.

PAGE 86

86 APPENDIX A UV-ABSORPTION SPECTRUM OF XANT HINE AND URIC ACID Fig A-22: UV-absorption spectrum of 100.0 M uric acid in 31 mM /NaH2PO4, pH 7.4. Molar absorptivities: 293nm, Na2HPO4/NaH2PO4, Na2HPO4, at pH 7.4 = 1.25 x 104 cm-1M1; 238 nm, Na2HPO4/NaH2PO4, pH 7.4 = 9.78 x 103 cm-1M-1

PAGE 87

87 Fig A-23: UV-absorption spectrum of 100.0 M ur ic acid in 20 mM KH2PO4, pH 5.1; molar absorptivities: 289 nm, KH2PO4, pH 5.1= 1.14 x 104 cm-1M-1; 234.5 nm, KH2PO4, pH 5.1 = 8.59 x 103 cm-1M-1

PAGE 88

88 Fig A-24: UV-absorption spectrum of 100.0 M xanthine in 31 mM Na2HPO4/NaH2PO4, at pH 7.4; molar absorptivity: 272nm, Na2HPO4/Na2HPO4, pH 7.4 = 8.99 x 103 cm-1M-1

PAGE 89

89 Fig A-25: UV-absorption spectru m of 100.0 M xanthine in KH2PO4, pH 5.1. Molar absorptivity 269 nm, KH2PO4, pH 5.1 = 9.48 x 103 cm-1M-1

PAGE 90

90 APPENDIX B NORMAL URINE CHROMATOGRAM Fig A-26. A 1 in 5 dilution of norma l urine. Mobile phase: 20 mM KH2PO4, at pH 5.1; Column: ODS C-18, 5 m particle size, 250 mm x 4.6 mm; Flow rate: 1.0 ml/min; Wavelength: 270 nm; Sample: Attenuation: 0.001 AUFS. Xanthine concentration: 150 0.1 M. Peak 19, 11, 12, 13: unknown, Peak 10: Uric acid Peak 14: Hypoxanthine, Peak 15: Xanthi ne. Detector: 9 L cell of 6 mm pathlength. Spectra 100 UV-Vis, Spectra-Physics Inc. (Autolab Division, San Jose, California, U.S.A) 6E 4 3E 4 Retention time (min) 0 5 10 15 1 15 10 3 7 9 5 14

PAGE 91

91 APPENDIX C: LIMIT OF DETECTION OF XANTHINE AND URIC ACID Table A-5: Lim it of detection a nd minimum concentration of uric acid and xanthine prepared in sodium phosphate buffer at pH 7.4. Column : B & J ODS C-18, 5m particle size, 25 cm x 0.46 cm. Mobile phase: 20 mM KH2PO4 at pH 5.1; Diluting solvent: 31 mM Na2HPO4/NaH2PO4 at pH 7.4; Flow rate: 1.0 m l/min; Wavelength of detection: 293 nm/270 nm. Attenuation: 0.01 AUFS. Limit of detection (LOD) = 3 x Sbk/M; where M is sensitivity. Physiological buffer Analyte UV-absorbance wavelength (nm) Minimum concentration detected (M) Detection limit at S/N = 3 or 2 31 mM Na2HPO4/NaH2PO4 at pH 7.4 Xanthine 293 2.0 1.6 Uric acid 270 5.0 5.1

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92 APPENDIX D SIGNAL TO NOISE RATIO Fig D-27 Signal to noise ratio for 10 M uric acid prepared in 31 mM Na2HPO4/NaH2PO4 at pH 7.4,. Retention time = 7.4 minutes. Column : ODS C-18, 5um particle size, 250 mm x 4.6 mm, Mobile phase: 20 mM KH2PO4 at pH = 5.1, flow rate = 1.0 mL/min. Signal/Noise = 2.1 cm/0.6 cm = 3 (See Table 6 2). 0.6 cm 2.0 cm

PAGE 93

93 APPENDIX E: HPLC COLUMN EFFICIENCY Table E-6: E xperimental HPLC column validation. Column: ODS C-18, 5 m particle size, 250 mm x 4.6 mm i.d column; Flow rate: 1.0 mL/min; Mobile phase: 65/35% (v/v) methanol/water; Back pressure: 144. Test analyte Concentration of analyte (mg/L) Retention time (min) Capacity factor (k) Theoretical number of plates (N) Uracil 5.0 2.60 Acetophenone 10.0 4.0 0.6 883 Anisole 250 5.2 1.0 1173 Toluene 700 7.2 1.8 788

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94 Table E-7: Manufactures HPLC column validation. Column: ODS C-18, 5 m particle size, 250 mm x 4.6 mm i.d column; Flow rate: 1.0 mL/min; Mobile phase: 65/35% (v/v) methanol/water; Back pressure: 144. Test analyte Concentration of analyte (mg/L) Retention time (min) Capacity factor (k) Theoretical number of plates (N) Uracil 5.0 1.4 Acetophenone 10.0 3.0 1.1 10953 Anisole 250.0 5.2 2.6 13877 Toluene 700.0 9.5 5.6 15749

PAGE 95

95 APPENDIX F PREPARATION OF SOLUTIONS Mobile Phase: 20.0 mM KH2PO4, pH 5.1 Approximately 2.72 g of KH2PO4 was weighed and dissolved in 5.0 L of double distilled water. The resultant solution was stirred till KH2PO4 was completely dissolved in the water. The pH of solution was adjusted to 5.1 with drops of 1.0 M potassium hydroxide. The final solution was made up to 1.0 L with double distilled water in a 1.0 L volumetric flask. Physiological Buffer: 31 mM Na2HPO4/NaH2PO4 pH 7.4 Approximately 1.65 g of NaH2PO4.H2O and 2.79 g of Na2HPO4 were weighed and dissolved in 0.5 L of double distilled water. The result ant solution was stirred until Na2HPO4/NaH2PO4 was completely dissolved. The pH of this solution was adjusted to 7.4 with drops of 1.0 M NaOH and final solution was made up to 1L with double distilled water in a volumetric flask. 50.0 M Xanthine Stock Solution Approximately 0.19 mg of xanthine was we ighed and dissolved in 10.0 mL of double distilled water. The solution was sonicated for thirty minutes and allo wed to cool to room temperature. The final solution was made up to 25.0 mL with double distilled water in a volumetric flask. All subsequent dilutions le ss that 50.0 M xanthine solution was made by following a simple dilution method (moles of xanthi ne before dilution = moles of xanthine after dilution). Preparation of 50.0 M UricAcid Stock Solutions Approximately 0.210 mg of xanthine was we ighed and dissolved in 10.0 mL of double distilled water. The solution was sonicated for thirty minutes and allo wed to cool to room temperature. The final solution was made up to 25.0 mL with double distilled water in a

PAGE 96

96 volumetric flask. All subsequent dilutions le ss that 50.0 M xanthine solution was made by following a simple dilution method (moles of xanthi ne before dilution = moles of xanthine after dilution). Preparation of 50% (v/v) Methanol Water Solution. A 100.0 mL portion of methanol and 100.0 mL of filtered dou ble distilled water were measured separately into two 100 mL beakers. The two solutions were combined without any adjustment to the final volume. Degassing of Solvents All HPLC solvents were degassed under consta nt vacuum with vigorous stirring for thirty minutes before used in the HPLC system.

PAGE 97

97 APPENDIX G CALCULATION I. Mass of 2420.01000.0136.09/ 1000.0mMmLgmol KHPO mL 2.72 mg II. Mass of xanthine 50.025,0152.1/ 1000.0 M mLgml mL 0.19 mg III. Mass of uric acid 50.025.0152.1/ 1000.0 M mLgmol mL 0.21 mg IV. Sodium Phosphate Buffer 2 4 24log HPO pHpKa HPO [Buffer] total 2 244HPOHPO 2 4 247.47.2logHPO HPO 2 4 241.58HPO HPO 2 244311.58 mMHPOHPO

PAGE 98

98 2431 2.6 mM HPO 21.19210138/1.645 gmolg 22 41.192101.58141.96/HPO gmol 2.79 g Uric Acid Concentration in Normal Healthy Urine Concentration of uric acid ostd unk unkVC C V Where Cunk = concentration of unknown; Vtot = total reaction volume; Vo = volume of uric acid at zero uric acid l; Vo = 0.75 ml; Vunk = 5.0 l; Cstd : 10 M; concentration; Vunk = volume of unknown. Vtot = 10.0 m 75010.0 5.0unkLM C L 1.5unkCmM

PAGE 99

99 Xanthine Concentration in Xanthinuric Urine Concentration of uric acid ostd unk unkVC C V Where Cunk = concentration of unknown; Vtot = total reaction volume; Vo = volume of uric acid at zero uric acid concentration; Vunk = volume of unknown. Vtot = 10.0 ml; Vo = 1.42 ml Vo = 1420 l; Cstd = 30.0 M; Vunk = 5.0 l 142030.0 5.0unkLM C L 2.8unkCmM

PAGE 100

100 REFERENCES 1. Alpheus, N.M., Jorn, D.S.M., Susan, A.B., Gei A., and Klaus, R.K.(2003) Tailoring Hydrophilic N,N-Dialkyl-N-Acylthioureas suita ble for Pt (II), Pd (II) and Rh (II) Chloride Pre-concentration from Acid Aqueous So lutions and Their Complex Separation by Reversed-Phase HPLC. Dalton Transaction 2003, 1952 1960. 2. Babson, L.A., Douglas, R., Coburn, H., Carrol, J.J. (1971) Simplified Alkaline Phosphotungstate Assay for Uric Acid in Serum. Clinical Chemistry 17, 158 163. 3. Baltassat, P., Bory, C., and Boulieu, R. (1982) High-Performance Liquid Chromatographic Determination of Hypoxanthine and Xanthine in Biological Fluids. Journal of Chromatography 233, 131 140. 4. Baltassat, P., Bory, C., and Boulieu, R. (1 984) Simultaneous Determination of Allopurinol, Oxipurinol, Hypoxanthine and Xanthine in Biological Fluids by High-Performance Liquid Chromatography. Journal of Chromatography 307, 469 474. 5. Berry, C.E., and Hare, J.M. (2004) Xanthine Oxidoreductase and Ca rdiovascular Disease: Molecular Mechanisms and Pathophysiologcal Implications. Journal of Physiology. 555.3, 589 606. 6. Bij, K.E., Horvath, C., Melander, W.R., and Nahum, A.(1981) Surf ace Silanols in SilicaBonded Hydrocarbonaceous stationary Phases: II Irregular Retention Behavior and Effect of Silanol Masking. Journal of Chromatography 203, 65 70 (1981). 7. Boulieu, R., Bory, C., Baltassat, P., and Gonnet, C.(1983) Hypoxa nthine and Xanthine Levels Determined by High Performance Liqu id Chromatography in Plasma, Erythrocyte, and Urine Samples from Healthy Subjects: Th e Problem of Hypoxanthine Level Evolution as a Function of Time. Analytical Biochemistry 129, 398 404. 8. Boulieu, R., Bory, C., and Baltassat, P. (1982) High Performance Liquid Chromatography Determination of Hypoxanthine and Xanthine in Biological Fluids. Journal of Chromatography 233, 131 140. 9. Brajter-Toth, A., and Childers Peters on, T. A. (1987) High-Performance Liquid Chromatography Assay of the Electro Oxidation of Purines Uric Acid and the Nucleotide Drug Tuberc idin-5-Monophosphate. Analytica Chemica Acta 2003, 167 174. 10. Brajter-Toth, A., and Mckenna, K. (1987) Tetrathiafulvalene Tetracyanoquinodimethane Xanthine Oxidase Amperometric Electrode for the Determination of Biological Purines. Analytical Chemistry 51, 958 962 (1987).

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104 45. Mcanaman, L.J., Costantino, D.A., Ginger, L.A., Weigel, L.K., Riley, M.G., and Wright, R.M.(2000) Activation of human Aldehy de Oxidase (hAOX) promoter by tandem cooperative Sp1/Sp3 binding sites. Identific ation of Complex Architecture in the hAOX Upstream DNA that includes a Proximal Promoter, Distal Activation and Silencer Elements. DNA and Cell Biology 19, 459 479. 46. Mei, D.A., Gross, G.J., and Nithipatikom, K.(1996) Simultaneous Determination of Adenosine, Inosine, Hypoxanthine, Xanthine, and Uric Acid in Microdialysis Samples using Microbore Column High-Performance Liquid Chromatography with Diode Array Detector. Analytical Biochemistry 238, 34 39. 47. Michal, K., Cieslak, M., and Czarnecka, J. (2005) Application of Solid Phase Extraction and High Performance Liquid Chromatography to Qualitative and Quantitative Analysis of Nucleosides and Nucleotides in the Human Cerebr ospinal Fluid. Journal of Chromatography B, 833, 85 90. 48. Moriwaki, Y., Yamamoto, M., Suda, M., Nasako, Y., Takahasi, O.E., and Agbedana, O.E.(1993) Purification and Im munohistochemical Tissue Local ization of Human Xanthine Oxidase. Biochimica T. Biophysica. Acta 1164, 327330 (1993). 49. Mulholland, M., Foot, M., and Kirkup, L. (2004) Comparison of Equations Describing Band Broadening in High-Perfo rmance Liquid Chromatography. Journal of Chromatography A, 1030, 25 31. 50. Nahum, A., and Horvath, C. (1981) Surf ace Silanols in Silica-B onded Hydrocarbonaceous Stationary Phases: I. Dual Retention M echanism in Reversed-Phase Chromatography Journal of Chromatography 203, 5357. 51. Nakagawa, T., Kang D-H., Feig, D., Sanch ez-Lozada, L.G., Srinivas, T.R., Sautin, Y., Ejaz, A.A., Segal, M. and Johnson, R.J. Unearthing Uric Acid: An Ancient Factor with Recently found Significance in Renal and Cardiovascular Disease. Kidney International 69, 1722 1725 (2006) 52. Nakaminami, T., Ito, S-H., Yomegame, H ., and Kuwaba, J. (1999) Uricase Catalysed Oxidation of Uric Acid Using Artificia l Electron Acceptor and Fabrication of Amperometric Uric Acid Sensor with use of Redox Ladder Polymer. Analytical Chemistry 71, 1928 1934. 53. Nakazawa, H., Yoshimura, Y., Iwasaki, Y., Namiki, T., and Inoue, T. (2003) Determination of Uric Acid in Human Saliva by High Performance Liquid Chromatography with Amperometric Electrochemical Detection. Journal of Chromatography B 785, 57 63.

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106 67. Steiner, I. (2007) Pulmonary Embolism Temporal Changes. Cardiovascular pathology 16: 248 251. 68. Tadahiko, S., Takashi, N., Kazuo O., and Matsuyama T. (2007) Neurogenic pulmonary Edema caused by a Medulla Oblongata Lesion after Head Injury. The Journal of Trauma Injury, Infection and Critical Care 63, 700 702. 69. Telefoncu, A., Wang, J., Timur, S., and Kirgoz A. U. (2004) Xant hine Oxidase Modified Glassy Carbon Paste Electrode. Electrochemistry Communications 6, 913 916. 70. Topalogue, R., Ichida, K., and Gok, F. (2003) Mutational Analys is of the Xanthine Dehydrogenase Gene in a Turkish Family with Autosomal Recessive Classical Xanthinuria. Nephrol Dial Transplant 18, 2278 2283. 71. Westermeryer, F.A., and Maquire, H.A. (1986) Measurement of Ad enosine, Inosine and Hypoxanthine in Human Term Placenta by Reverse-Phase High-Performance Liquid Chromatography. Journal of Chromatography 380, 55 66. 72. Wright, M.R., McManaman, L.J., Shibao, G.N., Piermattei, D., and Terada L.S.(1997) Hypoxia Regulates Xanthine Dehydrogenate Activ ity at Preand Post Translational Levels. Archives of Bioche mistry and Biophysics 348, 163 168. 73. Xu, P., Huecksteadt, T., and Hoidal, J.R. (1996) Molecular Cloning an d Characterization of the Human Xanthenes De hydrogenase Gene (XDH). Genomics 34, 173 180. 74. Yacoub, M.H., Ledingham, S.J.M., Lachno, D.R., and Smolensk R.T.(1990) Determination of Sixteen Nucleotides, and Bases Using High-Performance Liquid Chromatography and its Application to the Study of Purine Metabolism in Hearts for Transplantation. Journal of Chromatography 527, 414 420. 75. Yokoyama, Y., Beckman, J.S., Beckman, T.K., Wheat, J.K., Cash T.G., Freeman, B.A., and Parks D.A. (1990) Circulating Xanthenes Oxidase: Potential Mediator of Ischemic Injury. American. Journa l of Physiology 258, G564. 76. Zakaria, M., Brown, P.R., and Grushka, E. (1983) Mechanism of Retention in Reverse Phase Liquid Chromatography Separation of Ribonucleotides and Ribonucleosides and their Bases. Analytical Chemistry 55, 457 459.

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107 BIOGRAPHICAL SKETCH Mr. Andrews Obeng Affum was born at the Effia Nkwanta Hospital in Sekondi/Takoradi of the Western Region of Ghana to a family of Mr. Francis Obeng Affum and Mrs. Mary Afful. Mr. Affum had his kinder garten and primary education at th e Aggrey Memorial Primary School. He was studious and eager to excel in his academics. He sat for the common Entrance Examination when he was 15 years of age. He had his Secondary School Education at the Bompeh Secondary Technical School. Although issu es of life delayed him for a while, he never relented in his drive to achieve success in academics. Mr Affum had his A-level Education at Ghan a Secondary Technical School and excelled again. He enrolled as a biochemistry student at the Biochemistry Department of University of Ghana. He graduated with BSc (HONS) in bioc hemistry and was pronounced as one of the best student in his graduating class. Immediately after his graduati on he was invited to Gottingen University in Germany. Presently He is pursuing a masters program in chemistry at the Chemistry Department of University of Fl orida in the United States of America.


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