In vivo and in vitro studies on heavy doses and fluorescent-labelled vitamin C using ESR and fluorescence spectroscopy [Elektronische Ressource] / vorgelegt von Fabian Rozario

Rozario

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: technische_universitat_kaiserslautern
• Publié le: 01 janvier 2005
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

In vivo and in vitro studies on heavy doses and fluorescent-
labelled vitamin C using ESR and fluorescence
spectroscopy



















Fabian Rozario TU Kaiserslautern









II





Vom Fachbereich Chemie der Technischen Universität Kaiserslautern





zur Verleihung des akademischen Grades

“Doktor der Naturwissenschaften“
genehmigte



DISSERTATION
(D 386)
vorgelegt von




Dipl.-Chem. Fabian Rozario


Betreuer: Prof. Dr. Wolfgang Trommer


Tag der wissenschaftlichen Aussprache: 22. Februar 2005


III




















For Penny, Joshua and Jasmine

























IV








































Lack of ascorbic acid caused scurvy, so if there was no scurvy there was no lack of ascorbic
acid. Nothing could be clearer than this. The only trouble was that scurvy is not a first
symptom of lack but a final collapse, a premortal syndrome, and there is a very wide gap
between scurvy and full health. But nobody knows what full health is!
Albert Szent-Gyorgyi, M.D., Ph.D.
Nobel Laureate
V


Die vorliegende Arbeit entstand zwischen 2000 and 2004 im Fachbereich Chemie / Abteilung
Biochemie der TU Kaiserslautern






























Ich danke Herrn Prof. Dr. W. E. Trommer für die Überlassung des Themas und seine
Betreuung bei der Durchführung dieser Arbeit. Weiterhin möchte ich ihm für seine stets
wohlwollende Unterstützung, Hilfe und Ratschläge bei Problemen danken, sowohl im
Rahmen dieser Arbeit als auch außerhalb davon.

Herrn Prof. Dr. H. K. Biesalski möchte ich für die Überlassung und Betreuung eines Teils
des Themas danken, das maßgeblich zum Gelingen dieser Arbeit beigetragen hat.






VI
































Tag der wissenschaftlichen Aussprache: 22. Februar 2005


Promotionskommission


Vorsitzender : Prof. Dr.-Ing. S. Ernst

1. Berichterstatter : Prof. Dr. W. E. Trommer
2. : Prof. Dr. H. K. Biesalski






Contents

1 Introduction 1

1.1 Reactive oxygen species (ROS) and cellular damage ................................................ 1
1.2 Ascorbic acid and oxidative stress .............................................................................. 3
1.2.1 Oxidative stress and antioxidant protection........................................................ 3
1.2.2 Ascorbate as an antioxidant................................................................................ 3
1.2.3 Ischaemia-reperfusion.........................................................................................5
1.2.4 Consequences of hypoxia ................................................................................... 6
1.2.5 Rexoygenation injury.........................................................................................7
1.2.6 Biochemical measures of oxidative stress .......................................................... 8
1.2.7 Systemic free radical activation is a major event involved in
myocardial oxidative stress related to cardiopulmonary bypass (CPB) ........... 10
1.3 ESR spectroscopy ....................................................................................................... 11
1.3.1 Electron spin resonance and ascorbyl radicals ................................................. 11
1.4 Fluorescence spectroscopy 15

2 Tasks and objectives 17

2.1 Biochemical studies of the efficacy of heavy-dose vitamin C
supplementation on healthy subjects........................................................................ 17
2.2 Biochemical correlations of oxidative stress parameters from patients
undergoing an aorta-coronary bypass operation after heavy doses
vitamin C therapy....................................................................................................... 17
2.3 Chemistry and detection of ascorbyl free radicals compared to spin
trapping techniques are studied in vitro as markers of oxidative stress................ 18
2.4 Fluorescent labelling of ascorbic acid with N-methylisatoic anhydride................ 18
2.5 Validation of routine ESR spectrometers with respect to the
determination of the ascorbyl radical concentration in plasma
samples in comparism with the Bruker ELEXSYS E500 Series
spectrometer (E580) ................................................................................................... 19




Contents

3 Results and discussion 20

3.1 Biochemical studies of the efficacy of heavy-dose vitamin C
supplementation on healthy subjects........................................................................ 20
3.1.1 Protocol of healthy subjects with intravenous infusion of 7.5 g and/or
750 mg vitamin C ............................................................................................. 21
3.1.2 Classification of the clinical parameters........................................................... 21
3.1.3 Primary Parameter: Determination of ascorbyl radicals in blood
plasma of healthy subjects using ESR spectroscopy ........................................ 21
3.1.4 Stability of ascorbyl radicals in plasma under laboratory conditions at
physiological pH in the presence of anticoagulant and ferrous irons............... 23
3.1.5 Primary Parameter: ascorbic acid concentrations in blood plasma .................. 31
3.1.6 Biochemical correlations between secondary parameters in blood
plasma and urine from healthy subjects with ascorbyl radical
concentrations................................................................................................... 35
3.2 Biochemical correlations of oxidative stress parameters from patients
undergoing an aorta-coronary bypass operation after heavy doses
vitamin C therapy....................................................................................................... 39
3.2.1 Classification of patients in treatment groups .................................................. 39
3.2.2 Determination of ascorbyl radical concentrations using ESR
spectroscopy and its relation to ascorbic acid concentrations .......................... 40
3.2.3 The relationship between ascorbyl free radical concentrations and
secondary parameters of oxidative stress; TBARS, sICAM and
sVCAM and total antioxidant capacity assays determined in
Hohenheim........................................................................................................ 47
3.2.4 Discussion on the effects of heavy doses of vitamin C on healthy
subjects and patients undergoing bypass surgery ............................................. 51
3.3 Chemistry and detection of ascorbyl free radicals and spin trapping
techniques in vitro as markers of oxidative stress.................................................... 54
3.3.1 Redox reactions of ascorbate as a function of pH ............................................ 54
3.3.2 Ascorbate oxidation with hypoxanthine/xanthine-xanthine oxidase
(XOD) in vitro as source of ROS ..................................................................... 56
3.3.3 Spin trapping methods in hypoxanthine/xanthine-xanthine oxidase
(XOD) systems ................................................................................................. 61 Contents

3.3.4 Vitamin C and vitamin E interactions in biphasic systems .............................. 63
3.3.5 Ascorbate and spin trapping studies on oxidative stress observed
under ischemia/anoxia and reoxygenation conditions...................................... 65
3.3.6 Nitroxide activity of fluorescent-labelled spin probes in the presence
of ROS ......................................................................................................