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Measurement of Stress and Disease Markers in Biological Fluids by Fast Scan Cyclic Voltammetry and HPLC-UV

Permanent Link: http://ufdc.ufl.edu/UFE0024706/00001

Material Information

Title: Measurement of Stress and Disease Markers in Biological Fluids by Fast Scan Cyclic Voltammetry and HPLC-UV
Physical Description: 1 online resource (151 p.)
Language: english
Creator: Kathiwala, Mehjabin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adenine, dihydroxyadenine, hplc, hypoxanthine, nanostructured, polyamines, purines, pyrimidines, sensor, uracil, uric, urine, voltammetry, xanthine, xanthinuria
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Changes in the concentrations of purine metabolites released from endothelial cells of the pulmonary artery (PAECs) and aorta (AECs) exposed to oxidative stress were studied to identify possible stress markers. Detection of purine metabolites was achieved using HPLC-UV and fast scan cyclic voltammetry (FSV) at nanostructured carbon fiber sensor (N-CFS). An N-CFS that was compatible in the measurements in biological media was obtained by potential cycling in phosphate buffer between +1.5 V and -1.0 V for 30 min. Initial compatibility of the N-CFS with FSV was performed in diluted urine sample. Xanthinuria is a rare disease, in which xanthine oxidase deficiency leads to the accumulation of xanthine (XA) in urine. Sensitivity of XA measurements at the N-CFS with FSV was optimized and XA was measured in 2000-fold diluted xanthinuric urine. This work demonstrated that the sensor could be used to measure XA as a disease marker. Results showed good agreement for measurement of metabolites in urine sample by HPLC-UV. However, for measurement of metabolites in cell supernatants, the fabrication of N-CFS and the FSV signal acquisition method required further optimization to obtain a low and stable background signal. The optimization procedure for the measurements in cell supernatants involved fabrication of the nanostructured sensor surface in simple physiological media and FSV signal acquisition in diluted cell supernatant to minimize matrix effects. The dilution was possible due to the high sensitivity of the sensor with FSV methods. The optimized FSV method with the N-CFS was used for the measurements of the metabolites in cell supernatants. The results showed a large sensor response in supernatants of PAECs and AECs exposed to oxidative stress, and indicated the presence of a mixture of metabolites, which were identified by a HPLC-UV method. Metabolites included uracil (Ur), 2,8-dihydroxyadenine (2,8-DHA), uric acid (UA), hypoxanthine (Hy) and XA, with 2,8-DHA present at highest concentrations. This was the first time 2,8-DHA was detected and identified in endothelial cell supernatant exposed to oxidative stress. The HPLC-UV results indicated that the analyte detected with the N-CFS and FSV in the cell supernatants was 2,8-DHA, since the levels of Ur, UA, Hy and XA were low compared to those of 2,8-DHA. A preliminary method using the N-CFS was developed for the measurement of 2,8-DHA in cell supernatants.
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 Mehjabin Kathiwala.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Brajter-Toth, Anna F.
Local: Co-adviser: Young, Vaneica Y.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024706:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024706/00001

Material Information

Title: Measurement of Stress and Disease Markers in Biological Fluids by Fast Scan Cyclic Voltammetry and HPLC-UV
Physical Description: 1 online resource (151 p.)
Language: english
Creator: Kathiwala, Mehjabin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adenine, dihydroxyadenine, hplc, hypoxanthine, nanostructured, polyamines, purines, pyrimidines, sensor, uracil, uric, urine, voltammetry, xanthine, xanthinuria
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Changes in the concentrations of purine metabolites released from endothelial cells of the pulmonary artery (PAECs) and aorta (AECs) exposed to oxidative stress were studied to identify possible stress markers. Detection of purine metabolites was achieved using HPLC-UV and fast scan cyclic voltammetry (FSV) at nanostructured carbon fiber sensor (N-CFS). An N-CFS that was compatible in the measurements in biological media was obtained by potential cycling in phosphate buffer between +1.5 V and -1.0 V for 30 min. Initial compatibility of the N-CFS with FSV was performed in diluted urine sample. Xanthinuria is a rare disease, in which xanthine oxidase deficiency leads to the accumulation of xanthine (XA) in urine. Sensitivity of XA measurements at the N-CFS with FSV was optimized and XA was measured in 2000-fold diluted xanthinuric urine. This work demonstrated that the sensor could be used to measure XA as a disease marker. Results showed good agreement for measurement of metabolites in urine sample by HPLC-UV. However, for measurement of metabolites in cell supernatants, the fabrication of N-CFS and the FSV signal acquisition method required further optimization to obtain a low and stable background signal. The optimization procedure for the measurements in cell supernatants involved fabrication of the nanostructured sensor surface in simple physiological media and FSV signal acquisition in diluted cell supernatant to minimize matrix effects. The dilution was possible due to the high sensitivity of the sensor with FSV methods. The optimized FSV method with the N-CFS was used for the measurements of the metabolites in cell supernatants. The results showed a large sensor response in supernatants of PAECs and AECs exposed to oxidative stress, and indicated the presence of a mixture of metabolites, which were identified by a HPLC-UV method. Metabolites included uracil (Ur), 2,8-dihydroxyadenine (2,8-DHA), uric acid (UA), hypoxanthine (Hy) and XA, with 2,8-DHA present at highest concentrations. This was the first time 2,8-DHA was detected and identified in endothelial cell supernatant exposed to oxidative stress. The HPLC-UV results indicated that the analyte detected with the N-CFS and FSV in the cell supernatants was 2,8-DHA, since the levels of Ur, UA, Hy and XA were low compared to those of 2,8-DHA. A preliminary method using the N-CFS was developed for the measurement of 2,8-DHA in cell supernatants.
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 Mehjabin Kathiwala.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Brajter-Toth, Anna F.
Local: Co-adviser: Young, Vaneica Y.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024706:00001


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PAGE 15

Oxidative Stress Hypoxia in vivo in vitro in vivo

PAGE 16

.6, 7 .11

PAGE 17

Hyperoxia

PAGE 18

Effect of Oxidative Stress on Endothelial Cell Metabolism

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de novo de novo

PAGE 22

Purine and Pyrimid ine Cycle in Oxidative Stress de novo de novo

PAGE 24

Carbon Fiber Ultrami croelectrodes in vitro in vivo

PAGE 27

Fast Scan Cyclic Volt ammetry (FSV) in vivo

PAGE 28

High P erformance L iquid C hromatography (HPLC)

PAGE 29

Study Overview

PAGE 31

.

PAGE 35

600400200 0 -200 0 2 4 6 current/ nApotential/ mV

PAGE 36

injector

PAGE 37

Reagents and Solutions

PAGE 38

Cyclic Voltammetry

PAGE 39

Fast Scan Cyclic Voltammetry (FSV) Instrumental Setup

PAGE 41

Electrodes

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SEM Imaging Concentration Determination HPLC -UV Measurements

PAGE 43

Enzyme Assays

PAGE 44

Cells

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Statistical Analysis

PAGE 48

600400200 0 -200 0 1 2 3 4 5

PAGE 49

1.51.00.50.0-0.5-1.0 -8 -6 -4 -2 0 2 4 6

PAGE 50

Introduction

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R esults and Discussion Nanostructured Carbon Fiber Sensor E lectrode (N -CFS ) Background Current in Physiological B uffers

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Voltammetry of F erricyanide at the Carbon Fiber Sensor B efore and After Surface Fabrication

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Voltammetry o f Uric Acid (U A) i n Physiological Media

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N-CFS Fabrication for Use in Physiological Media

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Conclusions

PAGE 70

Introduction

PAGE 72

R esults and Discussion Background Current and Surface Structure of the Carbon Fiber Sensor

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Voltammetry of Ferricyanide Voltammetry of Xanthine

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Sensitivity in Fast Scan Voltammetry of Xanthine

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Xanthine and Uric Acid in Xanthinuric and Normal U rine

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Direct Determinations of Metabolite Concentrations in Urine

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Conclusions

PAGE 88

Introduction de novo

PAGE 89

de novo .2931, 153

PAGE 92

inal metabolites of the purine and pyrimidine cycle released in the cell supernatant were expected to change after exposure to stress.12, 27 Isocrati c elution with HPLC -UV was used, since it and allows good separation of purines, pyrimidines and their oxidation products .100 Results and Discussion Measurements of Metabolites in Cell Supernatant

PAGE 93

In this work with a low ionic strength 20 mM buffer, pH 5.1. This allowed separation of neutral forms of structurally similar purine and pyrimidine metabolites Hy (pKa 8.8),169 XA (7.44),151 UA (5.8),170 Ur (9.5)171 and Ade (3.6)172 from their oxidation products. Longer retention times and higher capacity factors in isocratic elution favor separation of structurally similar metabolites such as 2,8 -D HA and UA, and Hy and XA. Some variations in retention times were observed, which may be due to changes in the pH of cell supernatants. Consequently, standards and enzyme assays were used to confirm metabolite identity. Ado and Ino were n ot detected in cell supernatants B reakdown of Ado to Hy is very rapid in endothelial cells due in part to the high activity of purine nucleoside phosphorylase.173 This suggests that ATP breakdown products are re leased from cells while some may be formed by the degradation of exogenous ATP Ado may be transported back into the cell to form Hy, XA and UA, which are then released .167 In vivo

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Metabolite Identification in Cell Supernatant by HPLC -UV

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Oxidative Stress

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Effect of Hyperoxia on Adaptation of PAECs

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in vivo H ypoxia at AECs

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Observations of the Effects of Oxidative Stress

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de novo in vivo in vivo

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de novo de novo

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- de novo

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PAGE 107

Conclusions

PAGE 117

I ntroduction

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In vivo in vivo

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R esults and Discussion FSV of 2,8-DHA in pH 7.4 P hosphate Buffer at N -CFS

PAGE 121

FSV of 2,8-DHA at N-CFS in Physiological Buffers

PAGE 123

Measurement of 2,8 -DHA in PAECs and AECs Supernatant in vivo

PAGE 126

Conclusions

PAGE 138

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PAGE 142

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PAGE 143

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