Antioxidant Activity Measured by Fluorescence

97 Pages·2011·3.03 MB·English

by  Roland Meyer

Antioxidant Activity Measured by Fluorescence:
Investigation of Antioxidant and Probe Structure as well as
their Mobility and Position
by
Roland Meyer
A thesis submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
in Chemistry

Approved, Thesis Committee
Prof. Dr. Werner M. Nau, supervisor
Prof. Dr. Mathias Winterhalter, internal member

Prof. Dr. Detlef Gabel, external member

Date of defense: 13.05.2011
School of Engineering and Science
Jacobs University Bremen, Germany

To my parents

Optimisten sind Menschen,
die wissen wie schlecht die Welt ist;
Pessimisten sind Menschen,
die es täglich neu erleben müssen.
Sir Peter Ustinov

vii

Table of Contents

Acknowledgements......................................................................................................ix

List of Publications........................................................................................................x

List of Attended Conferences........................................................................................x

List of Acronyms..........................................................................................................xi

Abstract.....................................................................................................................xiii
Chapter 1 Introduction

1.1
 Scope
of
the
thesis...........................................................................................................................................3

1.1.1
 Summary
and
aims.................................................................................................................................3

1.2
 Free
radicals,
and
their
importance
in
biological
systems.............................................................5

1.3
 Antioxidant
defense
systems:
An
overview..........................................................................................6

1.4
 Low
molecular
weight
chain‐breaking
antioxidants
in
and
associated
with
lipids............7

1.4.1
 Vitamin
E....................................................................................................................................................8

1.4.2
 Vitamin
C..................................................................................................................................................12

1.5
 Experimental
approaches
to
elucidate
antioxidant
action..........................................................13

1.5.1
 DBO
as
a
fluorescent
probe
for
antioxidants............................................................................15

1.5.1.1
 Fluorazophore‐L:
A
lipid‐soluble
DBO
derivative..............................................................17

1.5.1.2
 Synthesis
of
Fluorazophore‐L.....................................................................................................18

1.6

 DBO
and
its
derivatives
as
fluorescent
probes
in
the
determination
of
antioxidant

action.....................................................................................................................................................................20

1.6.1
 Quenching
of
Fluorazophore‐L
by
vitamin
C............................................................................21

1.6.2
 Quenching
of
Fluorazophore‐L
by
vitamin
E............................................................................22

Chapter 2 Materials and Methods

2.1
 Materials............................................................................................................................................................29

2.2
 Liposome
preparation..................................................................................................................................29

2.3
 Fluorescence
spectroscopy........................................................................................................................30

2.4
 Liposome
characterization........................................................................................................................30

2.5
 Mathematical
analysis
for
determination
of
the
mutual
lateral
diffusion
coefficient......31
viii

Chapter 3 Antioxidant Activity Measured by Fluorescence: Investigation
of Antioxidant and Probe Structure as well as their Mobility and Position

3.1
 Analysis
of
a
synthetic
analogue
of
α‐tocopherol............................................................................35

3.2
 Effects
of
substitution
on
DBO
fluorescence......................................................................................39

3.3
 Phase
dependence
of
antioxidant
diffusion.......................................................................................43

3.4
 pH
dependence
of
L‐ascorbyl‐6‐palmitate
antioxidant
activity................................................47

3.5
 Conclusions......................................................................................................................................................51

3.6
 Perspectives.....................................................................................................................................................51

Chapter 4 Industry Project

4.1
 Samples..............................................................................................................................................................57

4.2
 Parameter
determination
techniques..................................................................................................57

4.3
 Vitamin
E...........................................................................................................................................................58

4.4
 Vitamin
C
and
Uric
Acid..............................................................................................................................58

4.5
 Total
antioxidant
power[92].......................................................................................................................59

4.6
 Isoprostane[93]
and
creatinine[94]............................................................................................................59

4.7
 GSH[101]...............................................................................................................................................................62

References...............................................................................................65

Chapter 5 Appendix

Appendix
I:
How
to
perform
a
global
fit
to
determine
D ..........................................................................73

L
Appendix II: Article, Journal of the American Chemical Society.....................................................79
Appendix III: Article, Photochemical & Photobiological Sciences................................................111
Appendix IV: Article, Langmuir......................................................................................................119
Appendix
V:
Lebenslauf..........................................................................................................................................127
ix

Acknowledgements
I would like to express my gratitude to my supervisor Prof. Dr. Werner M. Nau, who
took a wayward biologist under his wing and allowed him to delve into the
fascinating world of photochemistry. Without his ideas, capable guidance, constant
support and many fruitful discussions this thesis would not have been possible.
I would like to thank Prof. Dr. Mathias Winterhalter and Prof. Dr. Detlef Gabel for
their participation as co-referees.
I am grateful to the members of the Nau workgroup, both past and present, for a
dynamic, friendly, and enjoyable atmosphere. In particular I would like to thank Dr.
Harekrushna Sahoo for all the knowledge regarding instrumentation and
methodology he passed on to me and Thomas Schwarzlose for helping me out with
the synthetic procedures, and for supplying most of the raw materials used in my
experiments. I am grateful to Dr. Andreas Sonnen for introducing me to the global
fitting procedures and the lifetime measurements for lateral diffusion, and to Anja
Müller for working with me in the framework of the industry project. I also thank
Roy D’Souza for his tireless help with the more artistic endeavors in order to provide
good-looking figures and schemes.
I am indebted to Prof. Dr. Porter who chose to include our workgroup in his analysis
of the synthetic antioxidant N-tocopherol.
I would like to thank the workgroups of Prof. Dr. Schwaneberg for their support in
measuring multiplates and in handling biological waste, and of Prof. Dr. Winterhalter
for allowing me to perform light-scattering and differential scanning calorimetry
measurements with their instruments.
Finally, I am extremely grateful to my father, without whose constant support, both
morally and financially, I would not have been able to finish.