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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
PAGE 19
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
PAGE 42
SEM Imaging Concentration Determination HPLC -UV Measurements
PAGE 43
Enzyme Assays
PAGE 44
Cells
PAGE 45
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
PAGE 52
R esults and Discussion Nanostructured Carbon Fiber Sensor E lectrode (N -CFS ) Background Current in Physiological B uffers
PAGE 55
Voltammetry of F erricyanide at the Carbon Fiber Sensor B efore and After Surface Fabrication
PAGE 56
Voltammetry o f Uric Acid (U A) i n Physiological Media
PAGE 57
N-CFS Fabrication for Use in Physiological Media
PAGE 61
Conclusions
PAGE 70
Introduction
PAGE 72
R esults and Discussion Background Current and Surface Structure of the Carbon Fiber Sensor
PAGE 75
Voltammetry of Ferricyanide Voltammetry of Xanthine
PAGE 76
Sensitivity in Fast Scan Voltammetry of Xanthine
PAGE 78
Xanthine and Uric Acid in Xanthinuric and Normal U rine
PAGE 79
Direct Determinations of Metabolite Concentrations in Urine
PAGE 80
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
PAGE 94
Metabolite Identification in Cell Supernatant by HPLC -UV
PAGE 96
Oxidative Stress
PAGE 97
Effect of Hyperoxia on Adaptation of PAECs
PAGE 98
in vivo H ypoxia at AECs
PAGE 99
Observations of the Effects of Oxidative Stress
PAGE 102
de novo in vivo in vivo
PAGE 104
de novo de novo
PAGE 105
- de novo
PAGE 106
PAGE 107
Conclusions
PAGE 117
I ntroduction
PAGE 118
In vivo in vivo
PAGE 119
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
Free Radic. Biol. Med 30 Am. J. Physiol .Cell Physiol 292 Plant Physiol., 141 Am. J. Physiol -Cell Physiol., 282 J. Clin. Invest., 82 Circ. Res., 86 Radiat. Res., 143 Circ. Res., 97 Anal. Biochem., 238 Am J. Physiol .Lung Cell Molec Physiol 270 J. Neurochem., 51 Am. J. Respir. Cell Mol. Biol., 6 Mol. Immunol., 38 Life Sci., 59 J. Biol. Chem., 256 Crit. Care Med., 32 Chest, 88 Ann. Neurol., 57
PAGE 139
Neuroimaging Clin. N. Am., 15 J. Biol. Chem., 279 Physiol. Genomics, 30 Physiol. Genomics, 30 Biochem. J., 390 J. Clin. Invest., 95 Am. J. Physiol., 258 J. Cell Biol., 109 Am. J. Physiol. -Cell Physiol., 276 J. Bacteriol., 82 J. Neurochem., 46 Brain and Nerve (Tokyo), 41 Neurosurgery, 25 Am. J. Physiol., 266 Am. J. Respir. Crit. Care Med., 158 Biochem. J., 266
PAGE 140
Pharmacol. Ther., 84 Hoppe -Seylers Zeitschrift Fur Physiologische Chemie, 355 Circulation, 107 Am. J. Physiol. -Cell Physiol., 289 Am. J. Physiol. -Lung Cell. Mol. Physiol., 270 Am. J. Physiol. -Lung Cell. Mol. Physiol., 269 J. Cardiovasc. Pharmacol. Ther., 13 J. Biol. Chem., 273 J. B iol. Chem., 273 J. Hypertens., 18 Antioxid. Redox Signal., 5 J. Chromatogr. B, 877 Clin. Chem., 45 Arch. Biochem. Biophys., 376 Proc Natl Acad Sci U. S A. 78 Physio l. Rev., 87 J. Neurochem., 69
PAGE 141
J. Biol. Chem., 279 Pflugers Arch., 452 Mol. Cell. Biochem., 321 Circ. Res., 47 Jpn. J. Cancer Res., 84 J. Nutr., 126 IUBMB Life, 58 Appl. Environ. Microbiol., 61 J. Bacteriol., 188 Analyst, 128 Analyst, 133 Chem. Anal., 44 Anal. Biochem., 129 Pediatr. Nephrol., 22 Aktuelle Urol., 35 Clin. Chim. Acta, 335 J. Clin. Invest., 79 Clin. Biochem., 40 Clin. Chim Acta 160
PAGE 142
J. Braz. Chem. Soc., 11 J. Pharm. Biomed. Anal., 19 Anal. Chem., 72 Anal. Chi m. Acta, 349 Analyst, 132 Brain Res., 381 J. Phys. Chem., 88 Analyst, 128 J. Neurosci. Methods, 95 Science, 240 Anal. Chem., 66 48 Anal. Chem., 66 Chem. Eng. J., 116 Carbon Fibers Carbon: Electrochemical and Physical Properties In Laboratory Techniques in Electroanalytical Chemistry In Electroanalytical Chemistry Trends Neurosci., 12
PAGE 143
Anal. Chem., 68 Analyst, 128 Electroanalysis, 12 Analyst, 123 J. Neurosci. Methods, 10 Anal. Chem., 56 J Electroanal Chem, 272 Anal. Chim. Acta, 321 Zeitschrift Fur Elektrochemie, 57 Anal. Chem., 67 Anal. Chim. Acta, 202 Clin. Biochem., 38 Chem. Lett., J. Clin. Invest., 56 Am. J. Physiol., 257 J. Am. Chem. Soc., 104 Anal. Chem., 56 Anal. Chem., 67 J. Phys. Chem., 90 Anal. Chem., 57
PAGE 144
Ann. Anat. -Anat. Anz., 191 Placenta, 26 Am. J. Obstet. Gynecol 185 Free Radic. Biol. Med., 14 J. Leukoc. Biol., 61 Anal. Chem., 67 Anal. Chem., 71 Anal. Chem., 71 Anal. Chem., 57 Anal. Chem., 55 Electroanalysis, 19 Anal. Chem., 79 Electrochem. Commun., 6 J. Mass Spectrom 41 Circulation, 115 Nephron. Cli n. Pract., 105 Clin. J. Am. Soc. Nephrol., 2 Hypertension, 49
PAGE 145
J. Chromatogr. B, 781 Clin. Chem., 46 J. Chromatogr. B, 837 Arch. Med. Res., 38 Electrophoresis, 28 J. Liq. Chromatogr. Rel. Technol., 25 Chromatographia, 65 Anal. Biochem., 108 Anal. Chim. Acta, 89 Nanotechnol. 17 Methods in Molecular Biology Anal. Chem., 59 Anal. Chem., 65 Anal. Chem., 70 J. Electrochem. Soc., 147 Carbon, 39 Polym. Bull., 59
PAGE 146
J. Raman Spectrosc., 35 Electrochemical Methods, Fundamentals and Applications J. Chim. Phys 94 J. Chim Phys 93 Radiat. Res., 97 J. Electrochem. Soc., 119 Chem. Res. Toxicol., 16 Anal. Chem., 65 Acta Neurochir., 142 Arch. Biochem. Biophys., 348 Proc. Natl. Acad. Sci. U. S. A., 99 Mutat. Res 402 Circ. Res., 87 Am. J. Physiol. -Heart Circul. Physiol., 292 Proc. Natl. Acad. Sci. U. S. A., 103 Am J. Physiol 289 Am. J. Physiol. -Heart Circul. Physiol., 293 Pharmacol. Rev., 50
PAGE 147
Cancer Res., 54 Pathobiology, 58 J. Chromatogr. B, 707 J. Chromatogr. B, 704 Gen. Pharmacol., 27 Basic Res. Cardiol., 92 Clin. Chem., 29 QJM Mon. J. Assoc. Physicians, 93 Cancer Res., 16 Chromatographia, 47 Cardiovasc. Res., 28 Am. J. Physiol., 263 In Vitro Mol. Toxicol. -J. Basic Appl. Res., 14 Clinica Chimica Acta, 245 FASEB J., 23 J. Exp. Med., 198 Trends Pharmacol. Sci., 10
PAGE 148
Am. J. Physiol., 262 Am. J. Physiol. -Heart Circul. Physiol., 277 Am. J. Pathol., 145 J. Appl. Physiol., 59 Am. J. Respir. Cell Mol. Biol., 28 Biochem. -Moscow, 69 Biochem. Biophys. Res. Commun., 113 Am. J. Respir. Cell Mol. Biol., 7 Brain Res., 1008 Proc. Natl. Acad. Sci. U. S. A., 95 Proc. Natl. Acad. Sci. U. S. A., 89 Biochem. J., 249 Biochem. Biophys. Res. Commun., 122 Annu. Rev. Biochem., 30 Proc. Natl. Acad. Sci. U. S. A., 87 Free Radic. Biol. Med., 11
PAGE 149
Phytochemistry, 25 Topics and perspectives in adenosine research. Proc. Natl. Acad. Sci. U. S. A., 93 Int. J. Cardiol., 100 J. Cell. Mol. Med., 12 News. Physiol Sci 16 Am. J. Physiol. Cell Physiol., 42 Arch. Biochem. Biophys., 273 J. Appl. Physiol., 65 J. Cell. Physiol., 162 J. Biol. Chem. 283 Biochem. Biophys. Res. Commun., 251 Am. J. Physiol. -Cell Physiol., 295 Biochem. J., 255 Metab. -Clin. Exp., 40 Arch. Biochem. Biophys., 70 J. Chem. Soc
PAGE 150
Bioelectrochem 70 Indian J. Chem. Sect A -Inorg. Phys. Theor. Anal. Chem., 32 Aust. N. Z. J. Med., 5 Journal of Chromatography A, 894 J. Chromatogr. B -Biomed. Appl., 672 Clin. Chim. Acta, 181 J Electroanal Chem, 557 J Electroanal Chem, 521 Free Radic. Biol. Med., 40
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