Antioxidant Inhibition of Oxygen Radicals for Measurement ... · Antioxidant Inhibition of Oxygen Radicals for Measurement of Total Antioxidant Capacity in Chemical and Biological

Antioxidant Inhibition of Oxygen Radicals

for Measurement of Total Antioxidant Capacity

in Chemical and Biological Samples

by

Simon Y. L. Ching B.Sc. (Hons), M.Sc.

This thesis is presented for the Degree of Doctor of Philosophy of

The University of Western Australia

School of Biomedical, Biomolecular and Chemical Sciences

Discipline of Chemistry

2007

I

Abstract

A new method has been developed to measure the total antioxidant capacity of

chemicals, biological samples such as serum, and everyday substances such as vitamins,

tea, coffee, wine, spices, fruits, and vegetables to address important unresolved concerns

arising from intervention trials and clinical studies of the beneficial protective effect of

antioxidants versus the damaging effect of free radicals and reactive oxygen species in

oxidative stress in vivo. A review of previous attempts to resolve these concerns have

shown to be hindered by a lack of methods which take into account the two parameters

of measurement for assessing total antioxidant capacity: firstly, the degree of inhibition

and secondly, the duration of inhibition by antioxidants. In addition, existing

fluorescence methods with these fundamentals still require either above ambient

temperature incubation, reaction pre-heating and/or separate assays for testing

hydrophilic and hydrophobic antioxidant samples.

This high throughput "antioxidant inhibition of oxygen radicals" (AIOR) method

is performed at ambient temperature and is applicable to samples either in aqueous

solution or common organic solvents. The method has good linearity, within- and

between-run precision and recovery. The AIOR method uses peroxyl radicals to trigger

a decrease in fluorescence of the indicator molecule uroporphyrin I, which is delayed by

the presence of antioxidants. The area under the fluorescence curve is measured by a

fluorescence spectrophotometer in a 96-well microplate format with total antioxidant

capacity results expressed in mmol/L Trolox equivalents. Many of the concerns

associated with the measurement of total antioxidant capacity have been overcome and

AIOR has been applied to measure total antioxidant capacity of chemicals and

biological samples such as serum.

In addition, the kinetics and the reaction mechanism of the AIOR reaction have

been studied using UV-visible and fluorescence spectrophotometry, high performance

liquid chromatography (HPLC), liquid chromatography mass spectroscopy (LC-MS)

and electron spin resonance (ESR) analysis. The reaction between the indicator

II

molecule uroporphyrin I and the alkoxyl radicals generated from 2,2’-azobis(2-

amidinopropane) dihydrochloride (AAPH) was found to be first order kinetics with a

mean rate constant (k) of 0.0254. A mechanism for the reaction and the breakdown by-

products of the reaction is proposed based on the results from these experiments.

III

Acknowledgements

I would like to express my deepest gratitude to Associate Professor Emilio

Ghisalberti of the School of Biomedical and Chemical Sciences, The University of

Western Australia for his help and tireless guidance, and to Dr Jon Hall of Varian

Australia Pty Ltd for the expert technical advice given to me on this project.

Appreciation to PathWest Laboratory Medicine WA of Queen Elizabeth II

Medical Centre for a permission to undertake this work at the Department of Clinical

Biochemistry, and many thanks to Dr Keith Shilkin, the head of department Dr Chotoo

Bhagat and principal biochemist Dr John Beilby for their support.

Special thanks to Dr Alan Mckinley of the School of Biomedical and Chemical

Sciences, The University of Western Australia for assisting with the electron spin

resonance analysis; Associate Professor Kevin Croft of the School of Medicine and

Pharmacololgy, The University of Western Australia, for permission to use the LC-MS

instrument, and Dr Natalie Ward and Dr Trevor Mori of the School of Medicine and

Pharmacololgy, The University of Western Australia for providing the samples for the

hypertension study.

To my lovely wife Susan Yap and son David Ching for my source of renewable

motivation and inspiration.

To my sister Helen Ching and brother-in-law Dr Peter Kircher for their constant

encouragement, and to my brother Peter Ching and Jerry Villanueva for being good

friends.

To my mother Shuk Ming Cheng and my deceased father Ching Cham Ching for

the many fond memories and the opportunities given to me.

IV

Preface

Unless specifically stated, all work in this thesis was performed by the

candidate. This thesis describes the work carried out in the Department of Clinical

Biochemistry, PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre,

Western Australia, under the supervision of Associate Professor Emilio Ghisalberti of

the Department of Organic Chemistry, School of Biomedical, Biomolecular and

Chemical Sciences, The University of Western Australia, Dr Jon Hall of Varian

Australia Pty.,Ltd., Western Australia and Dr. John Beilby of the Department of

Clinical Biochemistry, PathWest Laboratory Medicine WA, Queen Elizabeth II Medical

Centre.

Publications, patent and presentations arising from the work for this thesis are as

follows:

Publication.

Ching SYL, Hall J, Croft K, Beilby J, Rossi E, Ghisalberti E. Antioxidant

inhibition of oxygen radicals for measurement of total antioxidant capacity in

biological samples. Analytical Biochemistry, 2006; 353/2:257-265.

Patent.

A method for measuring antioxidant status. International patent application

PCT/AU03/01324.

A method for measuring antioxidant status. Unites States patent and trademark

office publication number US-2005-0244983-A1.

V

Presentations.

Ching S, Hall J, Beilby J, Ghisalberti E. Measurement of total antioxidant

capacity by a novel antioxidant inhibition of oxygen radicals (AIOR) method.

The 43rd annual scientific conference of the Australasian Association of Clinical

Biochemists, 2005; October: Sydney.

Ching S, Croft K, Ward N, Mori T, Beilby J, Ghisalberti E, Hall J. Oxidative

stress ratio calculated from the antioxidant inhibition of oxygen radicals (AIOR)

assay in a hypertension cohort study. The 43rd annual scientific conference of the

Australasian Association of Clinical Biochemists, 2005; October: Sydney.

Ching S, Hall J, Beilby J, Ghisalberti E. A novel method (AIOR) to measure

total antioxidant capacity of serum, grapes, wine, tea, coffee and spices. The 44rd

annual scientific conference of the Australasian Association of Clinical

Biochemists, 2006; October: Hobart.

Publications from the use of the AIOR assay but the results have not been discussed and

did not form part of this thesis.

Dunstan JA, Breckler L, Hale J, Lehmann H, Franklin P, Lyons G, Ching SYL,

Mori TA, Barden A, Prescott SL. Association between antioxidant status,

markers of oxidative stress and immune responses in allergic adults. Clinical and

Experimental Allergy, 2006;36:993-1000.

Dunstan JA, Breckler L, Hale J, Lehmann H, Franklin P, Lyons G, Ching SYL,

Mori TA, Barden A, Prescott SL. Supplementation with vitamins C, E, β-

carotene and selenium has no effect on antioxidant status and immune responses

in allergic adults: a randomized controlled trial. Clinical and Experimental

Allergy, 2007;37:180-187.

VI

Table of Contents

Abstract

Acknowledgements

Preface

Table of Contents

List of Figures

List of Tables

Abbreviations

References

Chapter 1 Introduction

1.1 Oxidative Stress Assessment

1.2 Total Antioxidant Capacity Methods

Chapter 2 Method Development of the Antioxidant Inhibition

of Oxygen Radicals (AIOR) Assay

2.1 Introduction

2.2 Materials and Methods

VII

2.3 Results

2.3.1 Study of the Effect of Temperature on the ORAC Method with

Phycoerythrin versus the AIOR Method with Uroporphyrin I in Cuvette

2.3.2 Measurement of Antioxidant Capacity by the AIOR Method

2.3.3 Effect of 2,2’Azobis(2-methylpropionamidine) dihydrochloride (AAPH)

Concentration on the AIOR Method

2.3.4 Effect of Uroporphyrin I Concentration on the AIOR Method

2.3.5 Study of Standard Ranges for the AIOR Method

2.3.6 Types of 96-Well Microplate for the AIOR Method

2.3.7 Adaptation of the AIOR Method to Measure Both Hydrophilic and

Hydrophobic Compounds

2.3.8 Calibration of Fluorescence Spectrophotometer

2.3.9 Interference from Pipettes

2.4 Discussion

Chapter 3 Kinetic Study of the AIOR Reaction by Fluorescence

and UV-visible Spectroscopy

3.1 Introduction


3.3 Results

3.3.1 Emission and Excitation Fluorescence Scan in a

96-Well Microplate

3.3.2 Emission and Excitation Fluorescence Scan in a Cuvette

3.3.3 UV-visible Scan in a Cuvette of the Reaction between AAPH and

Uroporphyrin I

3.3.4 UV-visible Scan in a Cuvette from 200 - 700 nm of the Reaction

between AAPH and Gallic Acid

VIII

3.3.5 UV-visible Scan in a Cuvette from 200 - 700 nm of Gallic Acid,

Uroporphyrin I and AAPH

3.3.6 Kinetics Study of the AIOR Reaction by UV-visible Spectroscopy

3.3.7 Kinetics Study of the AIOR Reaction by Fluorescence

Spectrophotometry

3.4 Discussion

3.5 Appendix

3.5.1 Emission and Excitation Fluorescence Scans of the Reaction between

AAPH and Uroporphyrin

3.5.2 UV-visible Scans from 300 - 700 nm of the Reagents and the Reaction

between AAPH and Uroporphyrin I








between AAPH, Gallic Acid and Uroporphyrin I

Chapter 4 Study of the AIOR Reaction by Electron Spin

Resonance Analysis and HPLC

4.1 Introduction


4.3 Results

4.3.1 ESR Analysis of AAPH and Uroporphyrin I with DMPO as a Spin Trap

IX

4.3.2 HPLC Experiments

4.3.3 HPLC Analysis of the Reaction between AAPH and Uroporphyrin I

4.3.4 HPLC Analysis of the Reaction between AAPH and Uroporphyrin I in

the presence of Gallic Acid

4.4 Discussion

Chapter 5 A Study of the Reaction Mechanism of the AIOR

Assay by LC-MS

5.1 Introduction


5.3 Results

5.3.1 LC-MS of the Reaction between Uroporphyrin I and AAPH

5.3.2 LC-MS of the Reaction between Gallic Acid and AAPH

5.3.3 LC-MS in the Presence of Gallic Acid of the Reaction between AAPH

and Uroporphyrin I

5.3.4 MS/MS of Ions Detected in the Reaction between AAPH and

Uroporphyrin I

5.4 Discussion

Chapter 6 Antioxidant Capacity Measurement of Individual

Compounds by the AIOR Method

6.1 Introduction

X

6.1.1 Uric Acid

6.1.2 Vitamin C

6.1.3 Vitamin A

6.1.4 VitaminE

6.1.5 Thiols

6.1.6 Albumin

6.1.7 Total Protein

6.1.8 Polyphenols

6.1.9 Caffeine


6.3 Results

6.3.1 Uric Acid

6.3.2 Vitamin C

6.3.3 Vitamin A

6.3.4 Vitamin E

6.3.5 Thiols

6.3.6 Albumin

6.3.7 Total Protein

6.3.8 Polyphenols

6.3.9 Caffeine

6.4 Discussion

Chapter 7 Total Antioxidant Capacity Measurement of

Complex Mixtures by the AIOR Method

7.1 Introduction


XI

7.3 Results

7.3.1 Teas

7.3.2 Coffee, Chocolate Beverages and Green Powder Tea

7.3.3 Wines

7.3.4 Grape Juice

7.3.5 Spices

7.3.6 Rutin and Flavonoids Supplement Tablet

7.4 Discussion

Chapter 8 Validation of the AIOR Method

8.1 Introduction


8.2.1 Reagents and Equipment

8.2.2 Method Protocol

8.3 Results

8.3.1 Linearity

8.3.2 Recovery

8.3.3 Precision

8.3.4 Functional Sensitivity

8.3.5 Correlation with Individual Antioxidant

8.3.6 Sample Preservation and Storage Conditions

8.3.7 Stability of the Uroporphyrin I Reagent

8.4 Discussion

XII

Chapter 9 Oxidative Stress and Total Antioxidant Capacity in a

Hypertensive Study

9.1 Introduction


9.2.1 Subjects

9.2.2 Methods

9.2.3 Statistics

9.3 Results

9.4 Discussion

Chapter 10 Concluding Remarks

References

XIII

List of Figures

Figure Title Page

Chapter 1 1.1 Reactions of intracellular antioxidant enzymes 2

1.2 Examples of polyphenols and their molecular structures 3

1.3 Hydroxyl radical generation 4

1.4 Examples of free radicals and ROS propagation 4

1.5 Common features of inhibition assay 8

1.6 Molecular structure of Trolox 9

1.7 Molecular structure of uric acid 9

Chapter 2 2.1 Steps in the measurement of total antioxidant capacity 14

2.2 Molecular structure of AAPH and DMPO 15

2.3 Thermal decomposition of the azo-initiator AAPH 16

2.4 Molecular structure of uroporphyrin I 17

2.5 Molecular structure of

6-hydroxy-2,5,7,8-tetramethethylchroman-2-carboxylic acid

(Trolox) 17

2.6 Time versus fluorescence intensity of Trolox by the

ORAC method with R-phycoerythrin in cuvettes at 37oC 19


ORAC method with R-phycoerythrin in cuvettes at 25oC 20


AIOR method with uroporphyrin I in cuvettes at 37oC 20


AIOR method with uroporphyrin I in cuvettes at 25oC 20

XIV


AIOR method in 96-well microplate at ambient room

temperature 21

2.11 Concentrations versus AUC of the standard Trolox by

the AIOR method in mmol/L by the AIOR method in

96-well microplate at ambient room temperature 21

2.12 Time versus fluorescence intensity of the standard

Trolox in mmol/L (n = 64) by the AIOR method in

96-well microplate at ambient room temperature 22

2.13 Concentration versus AUC of the standard Trolox in

mmol/L (n = 64) by the AIOR method in 96-well microplate

at ambient room temperature 23

2.14 Time versus fluorescence intensity of the standard Trolox

in mmol/L with 583 mM AAPH (45 μL) by the

AIOR method 24

2.15 Time versus fluorescence intensity of the standard Trolox

in mmol/L with 640 mM AAPH (45 μL) by the

AIOR method 25

2.16 to 2.21 Concentration versus area under the curve of the

Trolox standard in different concentrations 27,28

2.22 Concentration versus area under the curve of the Trolox

standard. Comparing the effect of 20 μL and 100 μL

of Brij 35 per 15 mL of uroporphyrin I solution 31

2.23 to 2.26 Variation in fluorescence intensity of 96-well microplate

columns across the X axis pre-levelling, post-levelling,

photomulitplier calibration and new lamp 32

2.27 to 2.30 Variation in fluorescence intensity of 96-well microplate

rows across the Y axis pre-levelling, post-levelling,

photomultiplier calibration and new lamp 33

2.31 Ethanol delivered to 96-well microplate colmun A1 to A12,

B1 to B12 etc. The 96-well microplate read in the Eclipse

in the normal position 34

2.32 Ethanol delivered to 96-well microplate column A1 to A12,

B1 to B12 etc. The 96-well microplate read in the Eclipse

upside down position 34

XV

2.33 Ethanol delivered to 96-well microplate from row 1A to 1H,

row 2A to 2H etc. The 96-well microplate read in the

Eclipse in the normal position 35

2.34 Deionized H2O (2 μL) delivered to 96-well microplate

from column A1 to A12, B1 to B12 etc. The 96-well

microplate read in the Eclipse in the normal position 35

2.35 Ethanol delivered to 96-well microplate column A1 to A12,

B1 to B12 etc. with special precaution. The 96-well

microplate read in the Eclipse in the normal position 36

Chapter 3 3.1 Emission and excitation scans of uroporphyrin I with

Brij. before addition of AAPH in 96-well microplate 42

3.2 Emission and excitation scans of uroporphyrin I with

Brij. after addition of AAPH in 96-well microplate 42

3.3 Emission scan. Time = 0. Uroporphyrin I 43

3.4 Excitation scan. Time = 0. Uroporphyrin I 43

3.5 to 3.10 Emission scan. Time = 5 to 180 min after addition of

AAPH to uroporphyrin I (Appendix) 59 - 61

3.11 to 3.16 Excitation scan. Time = 5 to 180 min after addition of

AAPH to uroporphyrin I (Appendix) 61 - 62

3.17 to 3.22 UV-visible scans from 300 - 700 nm of the reagents

and the reaction between AAPH

and uroporphyrin I (Appendix) 63








and the reaction between AAPH and gallic acid (Appendix) 66


and the reaction between AAPH, gallic acid and

XVI

uroporphyrin I (Appendix) 67

3.45 First-order kinetic curve between uroporphyrin I and

AAPH measured at 300 to 650 nm by photodiode

array detector (PDA) 50

3.46 Fluorescence intensity versus time (min) of 292 mM

AAPH and 90 nM uroporphyrin 51

3.47 Fluorescence intensity versus time (min) of 292 mM


3.48 Fluorescence intensity versus time (min.) of 583 mM


3.49 First-order kinetic curves between 90 nM uroporphyrin I

and 292 mM AAPH measured at Ex = 405 nm and

Em = 624 nm at room temperature with rate constants 52







3.52 Fluorescence wavelength shifts after the addition of AAPH.

to uroporphyrin I 55

3.53 UV-visible wavelength shifts after addition of AAPH to

the uroporphyrin I 56

Chapter 4 4.1 AAPH with the spin trap (DMPO). Time = 2 min 71

4.2 AAPH with the spin trap (DMPO). Time = 15 min 72

4.3 AAPH (3 hours old) with the spin trap (DMPO) 72

4.4 AAPH, uroporphyrin I and the spin trap (DMPO).

Time = 2 min 73


Time = 15 min 73


Time = 30 min 74

XVII

4.7 HPLC monitored by PDA at 402 nm at 1 min of the

reaction between AAPH and uroporphyrin I 76















4.15 HPLC monitored by PDA 259 nm at 1 min in the presence

of gallic acid of the reaction between uroporphyrin I

and AAPH 79

4.16 HPLC monitored by PDA 397 nm at 1 min in the presence


and AAPH 79

4.17 HPLC monitored by PDA 397 at 120 min in the presence


and AAPH 79

4.18 HPLC monitoring of the absorbance at 402 nm versus

time (min) of the reaction between AAPH

and uroporphyrin I 81

4.19 HPLC monitoring of the absorbance at 397 nm versus

time (min) in the presence of gallic acid of the reaction

between uroporphyrin I and AAPH 81

Chapter 5 5.1 Chromatograms of uroporphyrin I at 402 nm at time = 0 85

XVIII

5.2 LC-MS of uroporphyrin I (45 μM) in PBS at time = 0 86

5.3 Positive mass ions (m/z) of uroporphyrin I (45 μM) in PBS

at time = 0 86

5.4 Chromatograms of the reation between uroporphyrin I and

AAPH at 402 nm at time = 5 min 86

5.5 LC-MS of the reaction between uroporphyrin I and

AAPH at time = 5 min 87

5.6 Positive mass ions (m/z) of the reaction between

uroporphyrin I and AAPH at time = 5 min 87

5.7 Chromatograms of the reaction between uroporphyrin I

and AAPH at 402 nm at time = 180 min 88

5.8 LC-MS of the reaction between uroporphyrin and AAPH

at time = 180 min 88

5.9 Positive mass ions (m/z) of the reaction between

uroporphyrin I and AAPH at time = 180 min 88

5.10 Chromatograms of gallic acid at 259 nm at time = 0 90

5.11 LC-MS of gallic acid at time = 0 90

5.12 Negative mass ions (m/z) of gallic acid at time = 0 90

5.13 Chromatograms of the reaction between gallic acid and

AAPH at time = 30 min 90

5.14 LC-MS of the reaction between gallic acid and AAPH at

time = 30 min 91

5.15 Negative mass ions (m/z) of the reaction between gallic acid

and AAPH at time = 30 min 91

5.16 Chromatograms of the reaction between uroporphyrin I,

gallic acid and AAPH at 259 and 397 nm at

time = 5 min 92

5.17 LC-MS of the reaction between uroporphyrin I, gallic acid

and AAPH at time = 5 min 92

5.18 Positive mass ions (m/z) of the reaction between uroporphyrin I,

gallic acid and AAPH at time = 5 min 92

5.19 MS/MS of the positive mass ion m/z 831 of the reaction between

uroporphyrin I and AAPH 94



XIX





5.23 Ion intensity (%) of extracted ion chromatogram (EIC) versus

time (min) of AAPH and uroporphyrin I 96

5.24 Ion intensity (%) of extracted ion chromatogram (EIC) versus

time (min) of AAPH, gallic acid and uroporphyrin I 97

5.25 Summary of MS/MS fragmentation of the mass ions

observed and identified 98

5.26 Porphyrin oxidation scheme 99

5.27 Proposed structure of the mass ions identified and those

putatively involved in MS/MS 100

Chapter 6 6.1 Uric acid 104

6.2 Vitamin C 104

6.3 Vitamin A 105

6.4 Vitamin E 105

6.5 Molecular structure of thiols 106

6.6 Caffeine 108

6.7 Trolox standard and uric acid by the AIOR assay 110

6.8 Trolox standard and vitamin C by the AIOR assay 111

6.9 Trolox standard and vitamin A by the AIOR assay 112

6.10 Trolox standard and vitamin E by the AIOR assay 113

6.11 Trolox standard (2.0 mM) with reduced form [R] and

oxidised form [O] thiols (5.0 mM) 114

6.12 Trolox standard and albumin by the AIOR assay 115

6.13 Trolox standard and total protein by the AIOR assay 116

6.14 Trolox standard (2.0 mM) and polyphenols (0.25 mM)

by the AIOR assay 117

6.15 Trolox standard (2.0 mM) and polyphenols (0.50 mM)


6.16 Trolox standard (2.0 mM) with mixture of four

XX

polyphenols and the four individual polyphenols at

respective concentration 118

6.17 Trolox standard and caffeine by the AIOR assay 120

6.18 Comparison of antioxidant capacity in mM TE of different

concentrations of albumin and total protein 122

Chapter 7 7.1 Examples of molecular structure of polyphenols 125,126

7.2 Comparison of antioxidant capacity of red teas,

green teas and a tea infusion by the AIOR assay 127

7.3 Comparison of coffee, chocolate beverages and

green tea powder by the AIOR assay 128

7.4 Comparison of two dilutions of red and white wine


7.5 Comparison of Trolox standard and grape juice by

the AIOR assay 132

7.6 Comparison of Trolox standard and two dilutions of

turmeric and curry powder by the AIOR assay 133

7.7 Comparison of Trolox standard and different dilutions

of the extract from a tablet containing rutin and

flavonoids by the AIOR assay 134

7.8 Comparison of antioxidant capacity in mmol/L TE

by the AIOR method of different teas 135


by the AIOR method of red and white wines 136


of grape juice by the AIOR assay 137


of spices by the AIOR assay 137

7.12 Comparison of antioxidant capacity in mmol/L of

different dilutions of a polyphenol supplement tablet


XXI

Chapter 8 8.1 Trolox standard curve of the AIOR assay in a

96-well microplate 141

8.2 Linearity of the Trolox standard curve of the AIOR assay

in a 96-well microplate 141

8.3 Linearity of serum sample dilutions with PBS of the

AIOR assay 142

8.4 Linearity of the dilutions of the serum protein precipitation

with ethanol of the AIOR assay 143

8.5 Structures of polyphenols used for validation 145

8.6 Functional sensitivity of serum sample dilution with

PBS of the AIOR assay in n = 4 runs (8 repeats per dilution) 147

8.7 Functional sensitivity of sample protein precipitation

with ethanol of the AIOR assay in n = 3 runs

(15 repeats per dilution) 148

Chapter 9 9.1 Molecular structure of arachidonic acid 153

9.2 Examples of the molecular structure of isoprostanes in

comparison to a prostaglandin 153,154

9.3 Comparison of oxidative stress ratio (OSR) between

untreated and treated hypertensive and normotensive

subjects 158

XXII

List of Tables

Table Title Page

Chapter 1 1.1 Half-lives of some free radicals and reactive

oxygen species (ROS) 5

1.2 Methods used to measure lipid oxidation or peroxidation 6

1.3 Selected examples of extracellular antioxidants in serum 7

1.4 Examples of methods used to measure total antioxidant

capacity 8

Chapter 2 2.1 Standard curve of AIOR assay with differing Trolox

concentrations versus mean area under the curve 22

2.2 Effect of AAPH concentration on area under the curve 24

2.3 Effect of uroporphyrin I concentration and PMT voltage

on fluorescence intensity 25

2.4 Comparison of fluorescence intensity of two

fluorescence spectrophotometers 26

2.5 Linearity comparison of standard curves with various

ratios of sample to reagent concentrations 27

2.6 Comparison of fluorescence intensity versus different

types of 96-well microplate 29

2.7 Antioxidant capacity of organic solvents 30

2.8 Effect of 20 μL of Brij 35 per 15 mL of uroporphyrin I

on the standard curve 30

2.9 Effect of 100 μL of Brij 35 per 15 mL of uroporphyrin I

on the standard curve 31

XXIII

Chapter 3 3.1 Fluorescence emission and excitation intensity with time

after addition of AAPH to uroporphyrin I monitored

in a cuvette 44

3.2 UV-visible scan from 300 - 700 nm in a cuvette of

uroporphyrin I (urop. I) and AAPH before and

during the reaction 45








gallic acid and AAPH before and during the reaction 48

3.6 UV-visible scan from 200 - 700 nm in a cuvette between

uroporphyrin I (urop. I) and AAPH in the presence

of gallic acid 49

3.7 Natural log conversion of the PDA absorbance maxima

of the reaction between AAPH and uroporphyrin I 50

3.8 First-order rate constant κ (min-1) at room temperature

from 3 experiments with different combinations of

uroporphyrin I and AAPH concentration 54

Chapter 4 4.1 HPLC first mobile phase tested with flow gradient 70

4.2 HPLC second mobile phase to be used for LC-MS

experiments with flow gradient 70

4.3 Effect of mobile phase composition on uroporphyrin I

precipitation 75

4.4 HPLC separation of products from the reaction of

uroporphyrin I and AAPH monitored at 402 nm 78

XXIV

4.5 HPLC separation of products from the reaction of

uroporphyrin I(Urop. I) in the presence of gallic acid (G. A.)

monitored at 397 nm 80

Chapter 5 5.1 PDA at 402 nm, EIC (m/z) and mass ion

intensity (%) of the HPLC peaks detected between the

AAPH and uroporphyrin I reaction 89

5.2 PDA at 259 and 397 nm, EIC (m/z) and mass ion

intensity (%) of the HPLC peaks detected in the presence

of gallic acid between the AAPH and uroporphyrin I

reaction 93

Chapter 6 6.1 Human serum standard protein from Behring (g/L) 109

6.2 Correlation of concentrations with antioxidant capacity

of uric acid in mmol/L Trolox equivalents 111


of vitamin C in mmol/L Trolox equivalents 112


of vitamin A in mmol/L Trolox equivalents 113


of vitamin E in mmol/L Trolox equivalents 114

6.6 Antioxidant capacity of thiols (5.0 mM) in

mmol/L Trolox equivalents 115


of albumin in mmol/L Trolox equivalents 116


of total protein in mmol/L Trolox equivalents 117

6.9 Antioxidant capacity of polyphenols (0.50 mM) in


6.10 Antioxidant capacity of polyphenols (0.25 mM) in


XXV

6.11 Additive effect of antioxidant capacity of 4 polyphenols

in mmol/L Trolox equivalents 119

Chapter 7 7.1 Antioxidant capacity in mmol/L TE of different

types of tea 128

7.2 Antioxidant capacity in mmol/L TE of coffee, chocolate

beverages and a green powder drink 129


of a red and white wine in 1/10 and 1/20 dilution 130


of different types of red and white wines 131


of grape juice 132


of spices 133

7.7 Antioxidant capacity in mmol/L TE of an antioxidant

supplement tablet in different concentrations 135

Chapter 8 8.1 Serum sample dilution linearity of the AIOR assay


8.2 Protein precipitation linearity of the AIOR assay


8.3 Range, mean, recovery and precision in n = 3 runs of

the AIOR assay (10 repeats per run) 144

8.4 Additive effect of total antioxidant capacity in mM Trolox

equivalents(TE) with polyphenols of the AIOR assay 146

8.5 Functional sensitivity of serum sample dilution with PBS

of the AIOR assay in n = 4 runs (8 repeats per run) 147

8.6 Functional sensitivity of sample protein precipitation

with ethanol of the AIOR assay in n = 3 runs

(15 repeats per run) 148

XXVI

8.7 Correlation of concentration and antioxidant capacity

(mM Trolox equivalents) of the AIOR assay 149

8.8 Comparison of serum, EDTA, heparin samples

antioxidant capacity (mM Trolox equivalents) of the AIOR

assay, n = 2 subjects (12 repeats per subject) 150

8.9 Comparison of serum antioxidant capacity

(mM Trolox equivalents) same day, stored at room

temperature overnight, 4oC for 4 days and –20oC for

28 months of the AIOR assay, n = 2 subjects

(6 repeats per subject) 150

Chapter 9 9.1 Biochemical analysis of untreated and treated hypertensive

and normotensive subjects, values are means ± SEM 157

XXVII

Abbreviation

AAPH 2,2'-azobis(2-amidinopropane) dihydrochloride

Abs Absorbance

ABTS 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonate)

AIOR Antioxidant inhibition of oxygen radicals

ARE Antioxidant response element

AUC Area under the curve

CV coefficient of variation

DMPO 5,5-dimethyl-4,5-dihydro-3H-pyrrole N-oxide

ECD electrochemical detection

EHT Extra high tension

EIC Extracted ion chromatogram

Em Emission

ESI Electrospray ionization

ESR Electron spin resonance spectroscopy

Ex Excitation

FRAP Ferric reducing/antioxidant power

GC-MS Gas liquid chromatography and mass spectrometry

GLC Gas liquid chromatography

HPLC High performance liquid chromatography

LC-MS Liquid chromatography and mass spectrometry

LDL Light density lipoprotein

MS Mass spectrometry

ORAC Oxygen radical absorbance capacity

OSR Oxidative stress ratio

RT Retention time

PDA Photodiode array detector

PMT Photomultiplier tube

ROS Reactive oxygen species

SD Standard of deviation

XXVIII

SOD superoxide dismutase

STD Standard

TAC Total antioxidant capacity

TEAC Trolox equivalent antioxidant capacity

TRAP Total radical trapping antioxidant parameter

TE Trolox equivalents

XXIX

Chapter 1

Introduction

1.1 Oxidative Stress Assessment

Reactive oxygen species (ROS) have been implicated in more than a hundred

diseases, from malaria and cancer to acquired immunodeficiency syndrome.1 ROS is a

collective term that describes free radicals and other non-radical reactive oxygen

species. Generated in vivo from processes such as metabolism, infection, inflammation,

poisoning, cell death and/or cell repair,2, 3 ROS react with key organic substrates such as

DNA, proteins and lipids, and the damage so caused can disturb normal function and

contribute to a range of diseases.5-8 When the presence or generation of ROS exceeds

the system’s ability to neutralize or eliminate them, then the system is said to be under

oxidative stress. A recent definition of oxidative stress9 states that it is a "condition

characterized by the accumulation of nonenzymatic oxidative damage to molecules that

threaten the normal function of the cell or the organism".

Cells have developed a defence mechanism to neutralize ROS or to prevent them

from being generated. Well known intracellular antioxidants include enzymes such as

superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)10-12 and

compounds like glutathione (GSH),13 oxidized glutathione (GSSG),14 ubiquinone,15, 16

and tocopherol.17 When intracellular antioxidants are low, free radicals and ROS may

activate oxidant-sensitive transcription factors such as nuclear factor-κβ.18-21 Binding of

transcription factor to the antioxidant response element (ARE) on the loci of the

oxidative responsive genes leads to the synthesis of cell survival proteins such as

1

antiapoptotic proteins Bfl-1 and Bcl-XL, and the intracellular antioxidant enzymes such

as manganese superoxide dismutase that protect the mitochondria. Reactions of the

protective antioxidant enzymes SOD (Equation 1), GPx (Equation 2) and CAT

(Equation 3) are shown in Fig. 1.1.

Fig.1.1. Reactions of intracellular antioxidant enzymes

2 O2•− + 2 H+ H2O2 + O2 (1)

SOD

2 GSH + H2O2 GSSG + 2 H2O (2) GPx

2 H2O2 2 H2O + O2(3) CAT

Extracellular antioxidants include compounds from endogenous and exogenous

sources. Endogenous compounds can be proteins, lipids, purines, pyrimidines, bilirubin,

urates, and thiols (glutathione, cysteine, homocysteine etc.); all contributing to the

extracellular antioxidant capacity. Exogenous compounds can be vitamins A, C, E and

complex mixtures of alkaloids, terpenoids, flavonoids, isoflavonoids, anthocyanins,

lignans, carotenoids, phenolic acids, sterols and amines from plant sources. These are

likely to function as protection and defense agents for plants, but analogously exert

similar antioxidant action in animals in vivo after ingestion and absorption. For

example, the polyphenols, found in plants, fruits and vegetables, are of particular

interest as antioxidants.22 Wine polyphenols such as resveratrol (Fig. 1.2) are proven

antioxidants23-25 and possess other biological properties26 that include reduction of

platelet aggregation,27 inhibition of LDL oxidation28-30 and inhibition of platelet-derived

growth factor (PDGF). Green tea polyphenols, especially (−)-epigallocatechin-3-gallate

(Fig. 1.2) possess anticarcinogenic activity as well as antioxidant properties.31-35

2

Fig. 1.2. Examples of polyphenols and their molecular structures.

OH

OH

OH

Trans-resveratrol

(−)-Epigallocatechin-3-gallate

OH

OH

OHOH

OH

CO

OH

OH

OH

O

O

Free radicals and ROS in vivo have complex origins. It has been suggested that

biological processes, glycolysis, the citric acid cycle and mitochondrial oxidative

phosphorylation can form reactive oxygen species and radical by-products such as

hydroperoxyl (HO2•), hydrogen peroxide (H2O2), hydroxyl (HO•) and superoxide anion

(O2•-). Monocytes, macrophages, eosinophils, and neutrophils may also generate

hydroxyl radicals (HO•) and hypochlorous acid (HOCl).2, 3, 36 Endogenous nitric oxide

(NO•)37 has an important role as mediator of vascular dilation, neural transmission, and

defense against microorganisms and inhibitors of platelet adhesion. Extracellular free

radicals may be formed when cells are exposed to radiation and pollutants, nitrogen

dioxide (NO2), toxic gases, herbicides, drugs and poisons. Oxidative stresses such as

vigorous exercise, heat shock, trauma, ultrasound, hyperoxia, radiation and disease

could induce the release of H2O2, HO• and O2•- from cells.1, 6

3

Many free radicals and ROS are unstable and have very short half-lives.38, 39 The

hydroxyl radical (HO•) is thought to be the most powerful oxidant formed in biological

systems. The generation of this radical (Fig. 1.3), as indicated by the Haber and Weiss

reaction (Equation 6), is maintained by the Fenton reaction (Equation 4) supported via

the recycling of iron from the ferric to the ferrous form by reducing agents, e.g.

superoxide (Equation 5). Further reaction of the hydroxyl radical and radical by-

products with organic substances may result in other free radicals and ROS (Fig. 1.4).

Fe2+ + H2O2 Fe3+ + HO• + OH−

•O2− + Fe3+ O2 + Fe2+

•O2− + H2O2 O2 + HO• + OH−

(4)

(5)

(6)

Fig.1.3. Hydroxyl radical generation

HO• + R •ROH

HO• + RH R• + H2O

R• + O2 ROO•

Fig. 1.4. Examples of free radical and ROS propagation

The detrimental effect of oxidative stress may occur when free radicals and ROS

levels are greater than the ability of the antioxidant store to quench them. How then can

oxidative stress be assessed? A brief outline of the common problems associated with

the measurement of oxidative stress is presented. The measurement choices have been

categorized into four groups for discussion: free radicals; oxidative markers; individual

antioxidants; and total antioxidant capacity.

Direct measurement of free radicals and ROS is extremely difficult because of

their short half-life (Table 1.1) and low concentrations.38 For instance, HO• has a half-

4

life of about 10-9 seconds and concentrations in the picomolar range. Another important

consideration is the location of the free radicals and ROS at the time of formation inside

the body, which is unpredictable. For these reasons, methods used to measure free

radicals and ROS directly are impractical.

Table 1.1. Half-lives of some free radicals and reactive oxygen species (ROS)38

Reactive oxygen species (ROS) Half-life at 37oC (seconds)

hydroxyl radical (HO•) 10-9

alkoxyl radical (RO•) 10-6

peroxyl radical (ROO•) 7

hydrogen peroxide (H2O2) environment dependent and variable

superoxide radical (O2•-) environment dependent and variable

singlet oxygen (O2) 10-6

nitric oxide (NO•) 1-10

peroxynitrite (ONOO-) 0.05 - 1

The measurement of oxidative by-products of proteins, DNA and lipids has been

employed to assess oxidative stress. However the increase of these oxidative markers

has often been found to be unrelated to oxidative stress. A detailed knowledge of the

confounding and unrelated causes leading to an increase in the level of these markers in

clinical situations has to be considered and evaluated for a meaningful interpretation of

the results obtained.5, 8, 40 Reactions of oxidatively damaged protein with 2,4-

dinitrophenylhydrazine, fluorescein thiosemicarbazide, and fluorescein

amine/cyanoborohydride41 have been reported. A method to measure protein carbonyls

of oxidized LDL in plasma by ELISA has been described.42 Other altered proteins such

as glutathionyl hemoglobin,43, 44 thioredoxin45 and ischemia-modified albumin46-48 have

also been measured.

Oxidative damage of deoxyribonucleic acid (DNA) produces altered DNA bases

such as 8-hydroxydeoxyguanosine (8-OhdG) in urine that is measured by liquid

chromatography with electrochemical detection (ECD) or gas chromatography and mass

5

spectrometry (GCMS). However isolation and hydrolysis of this compound gave

variable results and further method development is required for practical use. In other

words, the damaged DNA bases did not reflect oxidative stress due to excessive non-

specific production of these oxidative products.49-53

There are a number of lipid peroxidation by-products such as lipofuscin-like

compounds, oxidized-LDL,54-56 hydroxyalkenals,57, 58 malondialdehyde,59-61 conjugated

dienes, and F2-isoprostanes.62-66 Examples of lipid oxidative markers used to measure

lipid oxidation or lipid peroxidation are listed in Table 1.2.67, 68 These methods are

generally non-specific to a particular disease, require special expertise, are time

consuming and costly.

Table 1.2. Methods used to measure lipid oxidation or peroxidation67, 68

Analysis Method

loss of unsaturated fatty acids analysis of fatty acids by GLC and HPLC

uptake of oxygen by carbon-centered radicals and during peroxide decomposition reactions oxygen electrode

lipid peroxides iodine liberation

lipid peroxides heme degradation of peroxides (often first separated by HPLC)

lipid peroxides/aldehydes GC-MS

intermediate radicals spin trapping

thiobarbituric acid reacting substances (TBARS) such as malondialdehyde (MDA) thiobarbituric acid test

excited carbonyls, singlet oxygen light emission

aldehydes and their reaction products fluorescence; GCMS; HPLC

cytotoxic aldehydes HPLC/antibody techniques

diene-conjugated structures UV spectrophotometry

F2-isoprostanes solid phase extraction/EIA; GC-MS

free malondialdehyde high performance capillary electrophoresis

6

The measurement of individual antioxidants has been employed as a means of

assessing antioxidant capacity. In reality, it is not practical to measure every individual

potential antioxidant. The concentration of vitamins and antioxidants in vivo depends on

absorption, distribution, metabolism and transport. Many studies have failed to find the

connection between antioxidant supplements and the improvement of health and the

prevention of diseases. A number of large trials such as the Alpha-Tocopherol, Beta-

Carotene Cancer Prevention Study (ATBCCPS), the Beta-Carotene and Retinol

Efficacy Trial (CARET) and the Women’s Health Study (WHS) failed to reveal a

beneficial effect of any individual antioxidant against diseases.69-73 Vitamin

concentrations in vivo are very low and only constitute a small portion of the total

antioxidant capacity.

The measurement of a selection of antioxidants is also not the answer. There are

many extracellular antioxidants to choose from, including both endogenous and

exogenous compounds (Table 1.3). In particular, measurement of these compounds

requires special expertise and/or equipment, are time consuming procedures and are

costly.

Table 1.3. Selected examples of extracellular antioxidants in serum

Types Examples

vitamins C and E

thiols cysteine, homocysteine, cysteinyl glycine, glutathione

enzymes superoxide dismutase, glutathione peroxidase, catalase

endogenous compounds transferrin, caeruloplasmin, ferritin, lactoferrin, haemopexin, hatoglobin, uric acid, bilirubin, total protein, albumin

exogenous compounds carotenoids, flavonoids (quercetin, rutin, catechin), polyphenols

trace element zinc, selenium

Total antioxidant capacity measurements have the advantage of measuring all

antioxidants together. The idea of measuring total antioxidant capacity in vivo is not

new. The method, in principle, is based on the inhibition by antioxidants of the reaction

of an indicator molecule with free radicals. Examples of methods employed are shown

in Table 1.4.74

7

Table 1.4. Examples of methods used to measure total antioxidant capacity74

Free radical generation Target Measurement Time required

per sample

peroxyl radicals (ABAP) lipid peroxidation O2 uptake up to 2h

H2O2/peroxidase O-phenylene diamine A430nm 2h

peroxyl radicals (AAPH) phycoerythrin fluorescence inhibition 50 min

Cu2+/H2O2 thiobarbituric acid A530nm 2h

ferric metmyoglobin ABTS A734nm 6 min

HRP/H2O2 luminol chemiluminescence 10 min

ABAP = AAPH = 2,2’-azobis(2-amidinopropane) hydrochloride

AAPH = 2,2’-azobis(2-amidinopropane) dihydrochloride

ABTS = 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonate)

HRP = horseradish peroxidase

The common feature of these inhibition assays involves the production of a free

radical that is allowed to react with an indicator molecule, thereby generating an

endpoint that can be observed and quantified. Addition of antioxidants inhibits the

development of this endpoint as shown diagrammatically in Fig. 1.5.

Free radicals

Free radicals plus target indicator molecule

Observed difference in measurement

Free radicals plus target indicator molecule and antioxidants

Fig. 1.5. Common features of inhibition assay

The inhibition of free radicals by antioxidants in these assays depends on two

measurement factors: the degree of inhibition and the lag-phase.75 Lag-phase is defined

8

as the time where there is a total inhibition of free radical action by an antioxidant. For

example, Trolox (Fig. 1.6) and uric acid (Fig. 1.7) are compounds that show a good lag-

phase, but other compounds like albumin show no lag-phase unless at very high

concentration.76 The use of lag-phase measurements assumes a linear reaction rate

between the free radical generator and target indicator molecule. Body fluids with their

high protein content do not produce lag-phases at low concentrations.

OO

O

H

CH3

CH3

CH3

CH3

HO

Fig. 1.6. Molecular structure of Trolox

N

NN

NO

H

H

H

O

HO

Fig. 1.7. Molecular structure of uric acid

1.2 Total Antioxidant Capacity Methods

The measurement of total antioxidant capacity has followed two different

approaches. There are the methods that do not involve oxygen radicals, e.g. the ferric

reducing/antioxidant power (FRAP)77 and Trolox equivalent antioxidant capacity

(TEAC)78, 79 methods. Then there are the methods in which the reaction between

peroxyl radical and the indicator molecule is inhibited by antioxidants. Examples of this

approach are the total radical trapping antioxidant parameter (TRAP)80, 81 and the

oxygen radical absorbance capacity (ORAC)75 methods.

9

The FRAP method measures the reduction of a ferric tripyridyltriazine complex

to the ferrous form, which has a blue colour measurable at 593 nm at low pH. The

TEAC method is based on the quenching of the absorbance of the radical cation

(ABTS•+) formed by the reaction of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonate)

(ABTS) with metmyoglobin and H2O2. An example of this is the commercial Randox

total antioxidant method (Randox Laboratories Ltd., UK). A report by Schofield D et

al76 has described the shortcomings of the Randox total antioxidant method. The FRAP

methods are severely affected by interference from endogenous reducing agents such as

trace elements. In addition, non-involvement of oxygen radicals in the Randox total

antioxidant and the FRAP methods could compromise the validity of the measured

antioxidant capacity in vivo.82, 83

Importantly, FRAP, TEAC and TRAP methods are all single point measurement

methods which do not address, concomitantly, the degree of inhibition or lag-phase of

the antioxidants to be measured. Keeping in mind that many antioxidants have to be

measured simultaneously, these methods are unlikely to give a reliable total antioxidant

capacity result as they take only a single point in time to measure the entire reaction.

To address the concern of a single point in time measurement, the ORAC

method was developed by Cao GH et al. 1995.75 This method is a modification of that

developed by Glazer AN et al 1990,84 employing the indicator molecule B- or R-

phycoerythrin, but using an area under the curve calculation for quantification.

Although the ORAC method combines both the degree of inhibition and the lag-

phase for measurement, there are still several drawbacks. Firstly, it suffers from

chemical inconsistency and instability problems associated with the fluorescence probe

B- or R-phycoerythrin. This reagent is approximately 30% protein and the rest primarily

sucrose, dithioerythritol and sodium azide. As a result, the reagent varies from batch to

batch because it is formulated on the basis of protein content. Secondly, the ORAC

assay is conducted at 37oC. At above room temperature, there is a problem of stability

of B- or R-phycoerythrin and the free radical generating reagent, 2,2’-azobis(2-

amidinopropane) dihydrochloride (AAPH). In addition, B- or R-phycoerythrin is prone

to photo-degradation on exposure to excitation light. Thus in a 96-well microplate

reader, the fluorescence signal declined significantly even in the absence of AAPH.

Moreover, B-phycoerythrin interacts with polyphenols due to non-specific protein

10

binding and it is highly toxic.85 Thirdly, this method assumes linearity and uses a one-

point calibration rather than a multi-point standard curve.

There have been further modifications to the ORAC method, but the issue of

incubation and maintenance of the temperature of the reaction in the assay has not been

addressed. Other researchers have introduced different indicator molecules replacing B-

or R-phycoerythrin as the fluorescence probe. Fluorescein was introduced by Ou BX et

al85, 86 and 6-carboxyfluoroscein by Naguib YM et al.87 All these assays however still

require heating at 37oC.

Clearly, there is a need for a generally acceptable method to measure total

antioxidant capacity in the investigation of oxidative stress. The limited choice of

reliable methods however has greatly handicapped the progress in this area. This thesis

describes the development of a new method, named "Antioxidants Inhibition of Oxygen

Radicals" (AIOR) to measure total antioxidant capacity at ambient room temperature.88

The details of the AIOR method are discussed in the following chapters.

The use of a new fluorescence probe that produces a reaction curve using the

free radical generating reagent AAPH at ambient room temperature is presented in

chapter 2. On addition of antioxidants, there is inhibition in the decay of the indicator

molecule. Any need for preheating, or concerns for maintenance of the reaction

temperature during fluorescence measurement, are removed.

Other improvements of the assay are described in chapter 2. These include the

measurement of both hydrophobic and hydrophilic antioxidants; the measurement of the

lag-phase and degree of inhibition of antioxidants by calculating the area under the

curve; and the utilization of 96-well microplate to improve the throughput of the assay.

Also included are considerations for pipetting and mechanical factors influencing the

fluorescence intensity acquired by the 96-well microplate reader.

Chapters 3, 4 and 5 present investigations of the kinetics and the reaction

mechanism of the AIOR reaction by fluorescence and UV-visible spectrophotometry,

electron spin resonance (ESR) spectroscopy, high performance liquid chromatography

(HPLC) and mass spectrometry (MS). These experiments were conducted in an attempt

11

to gain insight into the AIOR reaction and thereby allow optimization of the assay

conditions.

Chapters 6, 7 and 8 describe the application of the AIOR assay to measure the

total antioxidant capacity in selected chemicals as well as tea, coffee and cocoa, wines,

spices and antioxidant supplements. The evaluation of the AIOR method for linearity,

recovery, imprecision and functional sensitivity for routine analysis is discussed.

Using the AIOR method, a study of the balance between the total antioxidant

capacity and the oxidative stress in treated and untreated hypertensive and normotensive

human subjects is discussed in chapter 9. An oxidative stress ratio (OSR) is calculated

from the ratio of the total antioxidant capacity obtained by the AIOR assay and the

oxidative stress marker F2-isoprostanes. The aim was to investigate the balance between

total antioxidant capacity and oxidative stress.

The final chapter presents concluding remarks and future investigations that

follow from this research.

12

Chapter 2

Method Development of the Antioxidant Inhibition

of Oxygen Radicals (AIOR) Assay

2.1 Introduction

The fluorescent compounds used to measure total antioxidant capacity are

referred to as the indicator compounds of the reaction in the assay. The presence of

antioxidants inhibits the destruction of the fluorescent indicator compound upon the

addition of free radicals.

A schematic diagram (Fig. 2.1) shows the reactions involved in the quantitative

measurement of total antioxidant capacity in samples with 2,2'-azobis(2-

amidinopropane) dihydrochloride (AAPH) as the free radical generating reagent.

The operational temperature of the reaction is an important consideration in the

measurement of total antioxidant capacity. The methods in the literature such as the

oxygen radical absorbance capacity (ORAC) method use B- or R-phycoerythrin at

37oC.75, 85, 87 Some methods use indicator compounds that require even higher

temperatures.

Whereas most of the other methods in the literature use a single point

measurement of the reaction of total antioxidant capacity, the ORAC based method

provides measurement by a process of continuous monitoring and calculating the area

under the curve (AUC) of the reaction from start to finish. Accordingly, variable factors

13

such as the lag time and the degree of inhibition of antioxidants are taken into account

in the calculation, resulting in a more reliable measurement of total antioxidant capacity.

Alkoxyl radicals generated by AAPH

Indicator + Blank (No antioxidant)

Indicator + Trolox Standard

(Trolox as antioxidant)

Indicator + Sample

(antioxidant)

Continuous monitoring of loss of fluorescence of Blank

Continuous monitoring of loss of fluorescence of Trolox Standard

Continuous monitoring of loss of fluorescence of Sample

Area Under the Curve (AUC) Blank

Area Under the Curve (AUC) Standard

Area Under the Curve (AUC) Sample

Reaction Curve Blank Reaction Curve Standard Reaction Curve Sample

Total Antioxidant Capacity Standard = AUC Standard − AUC Blank

Total Antioxidant Capacity Sample = AUC Sample − AUC Blank

Fig. 2.1. Steps in the measurement of total antioxidant capacity

14

It has long been accepted that the mechanism for the thermal decomposition of

AAPH (Fig. 2.2) is as shown in scheme Fig. 2.3. In the presence of oxygen a constant

flux of the alkyperoxyl radical (ROO•) is generated, but in organic solvents, the

tetroxide (ROOOOR) is formed. Further reaction of the tetroxide in the presence of

oxygen generates the alkoxyl radical (RO•). However recent evidence suggests that the

alkylperoxyl radicals are too unstable at room temperature and that the predominant

species is the alkoxyl radical.4 This evidence comes mainly from studies involving spin-

trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (Fig. 2.2) as the trap.89, 90 It

was found that only alkoxyl radicals added to the spin trap, and there was no evidence

for the presence of the alkylperoxyl radical-spin adducts.4

Fig. 2.2. Molecular structure of AAPH and DMPO

N

O

+

−

DMPO

AAPH

·2 HCl NN C C

CH3

NH

NH 2

CH3

CC

CH3

NH

NH2

CH3

15

Fig. 2.3. Thermal decomposition of the azo-initiator AAPH4-6

2R• 2ROO• 2 O2 Alkylperoxyl

radical

2ROOOOR

2 RO•

Alkoxyl radical

R − N = N − R N2 + 2R•

AAPH

R C C

CH3

NH

NH 2

CH3

=

In this thesis, the use of uroporphyrin I (Fig. 2.4) as the indicator compound for

the measurement of total antioxidant capacity is described. This fluorescent indicator is

conveniently used at ambient room temperature with no observable temperature

dependency. As for the ORAC method, the new method "Antioxidant Inhibition of

Oxygen Radicals" (AIOR) continuously monitors the reaction and calculates the area

under the curve.

16

Fig. 2.4. Molecular structure of uroporphyrin I

N

N

N

N

CH2COOH

CH2COOHCH2

A

R

A

A

A

A

R

R

R

R

H H

The compound Trolox, a water-soluble vitamin E analogue (Fig. 2.5), is

commonly used as a standard for the measurement of total antioxidant capacity because

of its stability and solubility in aqueous and organic solvents.

Fig. 2.5. Structure of 6-hydroxy-2,5,7,8-tetramethethylchroman-2-carboxylic acid (Trolox)

OO

O

H

CH3

CH3

CH3

CH3

HO

The concentration of AAPH is an important factor in the AIOR reaction

affecting the area under the curve. To find the appropriate concentration for the alkoxyl

radical generating reagent AAPH, different concentrations of AAPH have been

investigated. The effects of varying the uroporphyrin I concentrations, which control the

fluorescence intensity of the reaction and the shape of the curve, have been studied. The

17

linearity of the Trolox standard ranges of the AIOR assay and the ratio of sample to

reagents have also been examined.

The AIOR assay is capable of measuring the total antioxidant capacity of both

hydrophilic and hydrophobic antioxidants. Other factors which may cause imprecision,

such as the calibration of the fluorescence spectrophotometer and the pipetting of

samples dissolved in organic solvents, have been examined. The advantages of using

96-well microplate format are small sample size, high throughput and the potential for

automation. Different types of 96-well microplate have been evaluated. The use of 384-

well microplates is a possible alternative in the future. Considerations for pipetting

techniques and microplate reader technologies are also addressed. The measurement of

a large number of samples for total antioxidant capacity has been undertaken and

addressed. For example, testing wine, tea, coffee, fruits, vegetables and antioxidant

supplements could involve a large number of samples.


A Cary Eclipse fluorescence spectrophotometer (Varian Australia Pty. Ltd.,

Mulgrave, Victoria, Australia) equipped with either an exchangeable cuvette or a 96-

well microplate reader accessory was used. To make comparisons uniform, all the

curves acquired by the AIOR assay were normalised to 1000 as the starting point. A

program was written to perform normalisation of the curves. The area under the curve

(AUC) was calculated by the installed software of the Cary Eclipse fluorescence

spectrophotometer using the Visual Basic® Advanced Development Language (ADL)

incorporated within the software. The AUC measurement was achieved by normalising

the curve to the lowest intensity and scaling the highest intensity to a nominal value of

1000. There are 80 data points collected per curve for each well by the fluorescence

spectrophotometer for each of the 96 wells in the microplate. The area under the

resultant curve was then computed automatically using the ADL software. Linear

regression, correlation and statistics were performed by Prism 4.0 from GraphPad

Software Inc. San Diego, CA 92121 USA.

18

Uroporphyrin I dihydrochloride, Trolox, R-phycoerythrin, Brij 35 solution and

AAPH) were obtained from Sigma-Aldrich Pty. Ltd. (NSW, Australia). Stock

uroporphyrin I solution was prepared by dissolving 1.2 mg of uroporphyrin I

dihydrochloride in 5.9 mL of 0.075 mol/L phosphate buffer solution (PBS), pH = 7.0.

The concentration of the stock uroporphyrin I solution was 225 μmol/L. All dilutions

were made with PBS. The glass, polystyrene and polypropylene 96-well microplates

were obtained from Alltech Australia (NSW, Australia).

2.3 Results

2.3.1 Study of the Effect of Temperature on the ORAC Method with

Phycoerythrin versus the AIOR Method with Uroporphyrin I in Cuvette

The AIOR method was operational at ambient room temperature, whereas the

ORAC method had to be heated to 37oC. Both methods were performed in cuvettes at

37oC and at ambient room temperature (25oC). Fig. 2.6 and 2.7 show the effect of

temperature on the ORAC method with the standard curve at 37oC and 25oC

respectively. The ORAC method did not work at 25oC. In contrast, the AIOR method

showed no effect of temperature dependency with similar curves obtained at 37oC and

25oC (Fig. 2.8 and 2.9)

0 20 40 600

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.6. Time versus fluorescence intensity of the standard Trolox in μmol/L by the ORAC method with R-phycoerythrin in cuvettes at 37oC

Blank

STD 20 μM

STD 10 μM

STD 5 μM

19

0 20 40 600

500

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.7. Time versus fluorescence intensity of the standard Trolox in μmol/L by the ORAC method with R-phycoerythrin in cuvettes at 25oC

Blank

STD 5 μM STD 10 μM STD 20 μM

0 20 40 600

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.8. Time versus fluorescence intensity of the standard Trolox in μmol/L by the AIOR method with uroporphyrin I in cuvettes at 37oC

Blank

STD 5 μM

STD 10 μM

STD 20 μM

0 20 40 600

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.9. Time versus fluorescence intensity of the standard Trolox in μmol/L by the AIOR method with uroporphyrin I in cuvettes at 25oC

Blank STD 5 μM

STD 10 μM

STD 20 μM

20

2.3.2 Measurement of Antioxidant Capacity by the AIOR Method

The linearity of the Trolox standard values 0.5, 1.0 and 2.0 mmol/L of the AIOR

assay was checked. The curve of time versus fluorescence intensity produced by the

AIOR assay in a 96-well microplate at ambient room temperature is shown in Fig. 2.10.

Plotting the concentrations versus the area under the curve (AUC) of the Trolox

standard showed that the standard curve was linear (Fig. 2.11).

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.10. Time versus fluorescence intensity of the standard Trolox in mmol/L by the AIOR method in 96-well microplate at ambient room temperature

Blank

STD 0.5 mM

STD 1.0 mM

STD 2.0 mM

Fig. 2.11. Concentrations versus AUC of the standard Trolox in mmol/L by the AIOR method in 96-well microplate at ambient room temperature

AIOR STD Curve at 0.50, 1.00, 2.00 mM

0.0 0.5 1.0 1.5 2.020000

30000

40000

50000

60000Mean Area Under Curve

R2 = 0.9957, n = 24

STD mM

Are

a Und

er C

urve

21

To demonstrate the reproducibility of this assay, the Trolox standard curve was

repeated 16 times in the same run. The precision was shown to be excellent. The results

show that the intra-assay precision of the standard curves (n = 16 per Trolox standard)

had a coefficient of variation (CV) of < 5% for the blank and CV of < 3% for all three

Trolox standard concentrations as shown in Table 2.1.

Table 2.1. Standard curve of AIOR assay with differing Trolox concentrations versus mean area

under the curve

STD Concentration Mean Area Under Curve SD CV (%) n

Blank 37362 1721 4.6 16

0.5 mM Trolox 47456 991 2.1 16

1.0 mM Trolox 57535 1368 2.4 16

2.0 mM Trolox 76499 1850 2.4 16

Fig. 2.12 shows the time versus fluorescence intensity of the Trolox standard

curve (n = 64) by the AIOR assay in a 96-well microplate at ambient room temperature

monitored by the fluorescence spectrophotometer.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.12. Time versus fluorescence intensity of the standard Trolox in mmol/L (n = 64) by the AIOR method in 96-well microplate at ambient room temperature

Blank

STD 0.5 mM

STD 1.0 mM

STD 2.0 mM

22

The results show that the blank and Trolox standard concentrations at 0.5, 1.0

and 2.0 mmol/L versus the area under the curves was linear with a coefficient of

determination R2 = 0.9895. The plot of the concentrations of the Trolox standard in

mmol/L versus area under the curve (AUC) by the AIOR assay in a 96-well microplate

at ambient room temperature is shown in Fig. 2.13.

Fig. 2.13. Concentration versus AUC of the standard Trolox in mmol/L (n = 64) by the AIOR method in a 96-well microplate at ambient room temperature

0.0 0.5 1.0 1.5 2.030000

40000

50000

60000

70000

80000

90000

Mean Area Under Curve

R2 = 0.9895, n = 64

AIOR STD Curve

STD mM

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2.3.3 Effect of 2,2’Azobis(2-methylpropionamidine) dihydrochloride (AAPH)

Concentration on the AIOR Method

The shape of the area under the curve for two AAPH concentrations at 583 and

640 mM were compared using uroporphyrin I (225 nmol/L) and Trolox standard of 1.0,

2.0, 4.0 mmol/L. The ratio of sample to AAPH to uroporphyrin I was 1 : 45 : 280 μL.

The two tailed P values showed that there was a significant difference between the two

AAPH concentrations in the mean area under the curve for both standard curves (Table

2.2). At the higher AAPH concentration, with more radicals generated to attack the

23

fluorescent indicator compound, the corresponding standard curve expectedly gave a

smaller mean AUC.

Table 2.2. Effect of AAPH concentration on area under the curve

Area Under Curve (mean ± SD) AAPH

Concentrations Blank STD 1.0 mM STD 2.0 mM STD 4.0 mM n

583 mM 16400 ± 760 41000 ± 4000 55000 ± 3200 77000 ± 3700 6

640 mM 21000 ± 1600 36000 ± 2300 44000 ± 2300 69000 ± 6600 6

two tailed P value < 0.0001 < 0.02 < 0.0001 < 0.03

Fig. 2.14 and 2.15 show the fluorescence intensity versus time of the Trolox

standard curves with AAPH concentrations at 583 and 640 mmol/L but otherwise under

the same conditions. The results indicate that the area under the curve was larger with

the less concentrated AAPH solution.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 2.14. Time versus fluorescence intensity of the standard Trolox in mmol/L with 583 mM AAPH (45μL) by the AIOR method

Blank

STD 1.0 mM

STD 2.0 mM

STD 4.0 mM

24

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Blank

STD 1.0 mM

STD 2.0 mM

STD 4.0 mM

Fig. 2.15. Time versus fluorescence intensity of the standard Trolox in mmol/L with 640 mM AAPH (45μL) by the AIOR method

2.3.4 Effect of Uroporphyrin I Concentration on the AIOR Method

Fluorescence intensity was directly proportional to the concentration of

uroporphyrin I. Decreasing the concentration of uroporphyrin I from 300 to 225 nmol/L

lowered the mean fluorescence intensity from 644 to 400. The fluorescence intensity

was also increased or decreased by changes to the photomulitplier tube (PMT) voltage

(V) of the fluorescence spectrophotometer. Increasing the PMT voltage from 600 to 650

volts compensated for the decrease in the concentration of uroporphyrin I from 450 to

300 nmol/L and increased the mean fluorescence intensity from 508 to 644 (Table 2.3).

Table 2.3. Effect of uroporphyrin I concentration and PMT voltage on fluorescence intensity

Uroporphyrin I

Concentration

Reagent Volume

(μL) PMT Voltage (V)

Mean

Fluorescence

Intensity

SD n

450 nmol/L 280 600 508 10 60

300 nmol/L 280 650 644 28 60

225 nmol/L 280 650 400 17 60

25

Using uroporphyrin I at a concentration of 300 nmol/L, a reduction of the

reagent volume from 280 to 136 μL, also decreased the mean fluorescence intensity

from 644 to 171 at the same PMT voltage of 650 V. Factors found to influence the

performance of the fluorescence spectrophotometer included the alignment of the 96-

well microplate, the voltage of the PMT and the age of the lamp in the instrument.

Comparison of two fluorescence spectrophotometers from the same manufacturer gave

a significantly different mean fluorescence intensity of 171 and 466 respectively as

shown in Table 2.4.

Table 2.4. Comparison of fluorescence intensity of two fluorescence spectrophotometers

Varian

Eclipse

Uroporphyrin I

Concentration

Reagent

Volume (μL)

PMT Voltage

(V)

Mean

Fluorescence

Intensity

SD n

I 300 nmol/L 136 650 171 7 60

II 300 nmol/L 136 650 466 25 60

2.3.5 Study of Standard Ranges for the AIOR Method

A range of Trolox standard curves from 0.01 - 0.04 mmol/L to 1.25 - 5.00

mmol/L were examined for linearity as shown in Table 2.5. All standard curves showed

a good linear relationship between the area under the curve and the standard

concentrations. At the lower concentrations the precision was not as good as the higher

standard concentrations. The ratio of sample to reagents did not affect the linearity of

the standard ranges. The linearity was maintained regardless of the standard

concentrations as shown in Fig. 2.16 to 2.21.

26

Table 2.5. Linearity comparison of standard curves with various ratios of sample to reagent

concentrations

Three Concentrations of Trolox

for each Standard Curve

(mmol/L)

Uroporphyrin I

Concentration

(R2) (nmol/L)

AAPH

Concentration

(R1) (mmol/L)

Ratio of Sample (S) to

Reagents (R1 : R2) (μL)

1.25, 2.50, 5.00 (Fig 2.16) 225 640 1 : 45 : 280

1.00, 2.00, 4.00 (Fig 2.17) 225 640 1 : 45 : 280

0.50, 1.00, 2.00 (Fig. 2.18) 300 583 2 : 40 : 270

0.50, 1.00, 2.00 (Fig. 2.19) 300 583 1 : 22 : 136

0.50, 1.00, 2.00 (Fig. 2.20) 300 583 2 : 28 : 170

0.01, 0.02, 0.04 (Fig. 2.21) 180 320 20 : 25 : 280

The standard concentrations of 0.5, 1.0, and 2.0 mmol/L were used in the initial

developmental experiments which allowed a sample dilution of 1/6 for serum and 1/15

for red and white wine samples. The polypropylene round bottom 96-well microplate

has a maximum capacity of 330 μL. A total reaction volume of 159 μL with a ratio of

sample (S) to AAPH (R1) to uroporphyrin I (R2) (1 : 22 : 136 μL) was found to be a

suitable volume for mixing without spillage by a plateshaker. For higher fluorescence

intensity, a ratio of sample to reagent S : R1 : R2 (2 : 30 : 186 μL) was used with a total

reaction volume of 218 μL.

Fig. 2.17. Concentration versus AUC of the Trolox standard at 0, 1.00, 2.00 and 4.00 mmol/L with sample to AAPH (640 mM) to Uro. I (225 nM) at (1 : 45 :280) μL.


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00

10000

20000

30000

40000

50000

60000

70000


R2 = 0.9589, n = 24

STD mM

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Fig. 2.16. Concentration versus AUC of the Trolox standard at 0, 1.25, 2.50 and 5.00 mmol/L with sample to AAPH (640 mM) to Uro. I (225 nM) at (1 : 45 :280) μL.


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.00

25000

50000

75000

100000

125000


R2 = 0.9201, n = 36

STD mM

Are

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27

Fig. 2.18. Concentration versus AUC of the Trolox standard at 0, 0.50, 1.00 and 2.00 mmol/L with sample to AAPH (583 mM) to Uro. I (300 nM) at (2 : 40 : 270) μL with Brij 35


0.0 0.5 1.0 1.5 2.010000

20000

30000

40000

50000

60000

70000


R2 = 0.9919, n = 24

STD mM

Are

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Fig. 2.19. Concetration versus AUC of the Trolox standard at 0, 0.50, 1.00 and 2.00 mmol/L with sample to AAPH (583 mM) to Uro. I (300 nM) at (1 : 22 : 136) μL with Bij 35


0.0 0.5 1.0 1.5 2.020000

30000

40000

50000


R2 = 0.9957, n = 24

STD mM

Are

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Fig. 2.21. Concentration versus AUC of the Trolox standard at 0, 0.01, 0.02 and 0.04 mmol/L with sample to AAPH (320 mM) to Uro. I (180 nM) at (20 : 25 :280) μL


0.00 0.01 0.02 0.03 0.0425000

35000

45000

55000

65000


R2 = 0.8419, n = 32

STD mM

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Fig. 2.20. Concentration versus AUC of the Trolox standard at 0, 0.50, 1.00 and 2.00 mmol/L with sample to AAPH (583 mM) to Uro. I (300 nM) at (2 : 28 : 170) μL with Brij 35


0.0 0.5 1.0 1.5 2.040000

50000

60000

70000

80000

90000

100000

110000


R2 = 0.9779, n = 96

STD mM

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2.3.6 Types of 96-Well Microplate for the AIOR Method

For the AIOR assay, the polypropylene 96-well microplate gave the best

fluorescence intensity with uroporphyrin I at 180 nmol/L (Table 2.6). The glass 96-well

28

microplate did not have good fluorescence intensity with low sensitivity, whereas the

white polystyrene 96-well microplate reflected too much light which gave a high

background. The round bottom polypropylene 96-well microplate provided the best

shape for mixing of the reaction mixture using a plateshaker.

Table 2.6. Comparison of fluorescence intensity versus different types of 96-well microplate

Types of 96-well microplate Uroporphyrin I

Concentration

PMT Voltage

(V)

Mean Fluorescence

Intensity n

Glass 180 nmo/L 660 270 ± 40 32

Polypropylene 180 nmol/L 700 430 ± 15 32

Polystyrene (white) 180 nmol/L 660 970 ± 20 32

2.3.7 Adaptation of the AIOR Method to Measure Both Hydrophilic and

Hydrophobic Compounds

The effect of organic solvents on the method is shown in Table 2.7. The aqueous

solvents used in these experiments were water and phosphate buffer. Common organic

solvents such as ethanol and isopropanol are often used to dissolve organic samples. In

these experiments, the area under the curve of the organic solvents was compared

against the blanks. The results showed that ethanol, isopropanol and acetone exhibited

no antioxidant activity. Dimethyl sulphoxide (DMSO) showed a mean antioxidant

capacity of 0.86 mM Trolox equivalents. The addition of surfactant improved the

precision of the AIOR assay. As shown in the standard curves, the introduction of a

nonionic surfactant Brij 35 into the uroporphyrin I reagent vastly improved the

precision of the assay as shown in the 0.5, 1.00 and 2.00 mmol/L standard curves at

differing sample to reagent ratios (Fig. 2.18 to 2.20). The standard curves (Fig. 2.16,

2.17 and 2.21) without the addition of Brij 35 showed less precision of the assay.

29

Table 2.7. Antioxidant capacity of organic solvents

Antioxidant capacity in

(mM Trolox equivalents) Ethanol Isopropanol DMSO Acetone

low value 0.0 0.0 0.6 0.0

high value 0.1 0.1 1.2 0.2

mean value 0.08 0.04 0.86 0.08

SD 0.04 0.05 0.17 0.07

n 9 9 9 9

The effect of adding different amounts of Brij 35 to uroporphyrin I (300 nmol/L)

on the standard curve was studied. Samples containing 20 μL and 100 μL of Brij 35 per

15 mL of uroporphyrin I (300 nmol/L) were compared. There was no significant

difference in precision and linearity of the standard curve (Fig. 2.22). The coefficient of

determination (R2) was 0.976 for the 20 μL and 0.936 for the 100 μL of Brij 35 per 15

mL of uroporphyrin I (300 nmol/L) respectively. The area under the curve was higher

with the 100 μL of Brij 35 solution. The results are summarised in Table 2.8 and 2.9.

Table 2.8. Effect of 20 μL of Brij 35 per 15 mL of uroporphyrin I on the standard curve

20 μL of Briij 35 per 15 mL of Uroporphyrin I (300 nmol/L)

Trolox Standard

Concentration Mean Area Under Curve SD CV (%) N

Blank 37028 1914 5.2 24

0.5 mM 45482 1432 3.1 24

1.0 mM 57154 2259 4.0 24

2.0 mM 72596 1635 2.3 24

30

Table 2.9. Effect of 100 μL of Brij 35 per 15 mL of uroporphyrin I on the standard curve

100 μL of Briij 35 per 15 mL of Uroporphyrin I (300 nmol/L)

Trolox Standard

Concentration Mean Area Under Curve SD CV (%) N

Blank 48861 1604 3.3 24

0.5 mM 52714 1818 3.4 24

1.0 mM 61482 2302 3.7 24

2.0 mM 71394 2261 3.2 24

Fig. 2.22. Concentration versus AUC of the Trolox standard at 0, 0.5, 1.0 and 2.0 mmol/L. Comparing the effect of 20 μL and 100 μL of Brij 35 per 15 mL of uroporphyrin I (300 nM).

AIOR STD Curves with Differing Amountof Brij 35

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.0020000

30000

40000

50000

60000

70000

80000

20μL of Brij, R2 = 0.9757, n = 96

100 μL of Brij, R2 = 0.9362, n = 96

STD mM

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2.3.8 Calibration of Fluorescence Spectrophotometer

The fluorescence spectrophotometer was equipped with a wellplate reader

accessory which utilized a platform of X and Y axis drive to align each well of the

microplate to the light beam. An assay, involving all the 96 wells of the microplate,

accounts for over 7000 discrete movements. As such, imprecisions were attributable to

the movements of the microplate. A slight tilt was found to cause an increase in

31

imprecision of the assay along the X axis (Fig. 2.23) and along the Y axis (Fig. 2.27).

The type of 96-well microplate and the elevation of the microplate to the light source

was also found to cause an increase in background reading. The 96-well microplate has

to be in a level position to provide maximum uniformity inside the fluorescence

spectrophotometer.

Fig. 2.24 to 2.26 show the improvement in precision (mean fluorescence

intensity ± SD) along the X axis after a levelling of the 96-well microplate platform, a

photomultiplier calibration and a change of lamp. It was important to calibrate the

wavelengths and the photomultiplier as part of the routine at the first use of a new lamp.

Fig. 2.23. Variation in fluorescence intensity of 96-well microplate columns across the X axis pre-levelling

Variation across X Axis of 96-well platePre-leveling

Col A

Col B

Col C

Col D

Col E

Col F

Col G

Col H

600

700

800

900

n = 12 for each column

X Axis of 96-well plate

A.U

. at 6

50 V

Fig. 2.24. Variation in fluorescence intensity of 96-well microplate across the X axis post-levelling

Variation across X Axis of 96-well platePost-leveling

Col A

Col B

Col C

Col D

Col E

Col F

Col G

Col H

400

500

600

700



A.U

. at 6

00 V

Fig. 2.25. Variation in fluorescence intensity of 96-well microplate columns across the X axis post-levelling and photomultiplier calibration

Variation across X Axis of 96-well platePost-leveling & EHT calibration

Col A

Col B

Col C

Col D

Col E

Col F

Col G

Col H

400

500

600

700



A.U

. at 7

00 V

Fig. 2.26. Variation in fluorescence intensity of 96-well microplate columns across the X axis post-levelling, photomulitplier calibration and new lamp

Variation across X Axis of 96-well plateNew lamp, post-alignment & EHT calibration

Col A

Col B

Col C

Col D

Col E

Col F

Col G

Col H

400

500

600

700



A.U

. at 6

00V

Fig. 2.28 to 2.30 show the improvement in precision (mean fluorescence

intensity ± SD) along the Y axis after a levelling of the 96-well microplate platform, a

photomultiplier calibration and a change of lamp.

32

Fig. 2.27. Variation in fluorescence intensity of 96-well microplate rows across the Y axis pre-levelling

Variation across Y Axis of 96-well platePre-Leveling

Row 1

Row 2

Row 3

Row 4

Row 5

Row 6

Row 7

Row 8

Row 9

Row 10

Row 11

Row 12

600

700

800

900

n = 8 for each row

Y Axis of 96-well plate

A.U

. at 6

50 V

Fig. 2.28. Variation in fluorescence intensity of 96-well microplate rows across the Y axis post-levelling

Variation across Y Axis of 96-well platePost-leveling

Row 1

Row 2

Row 3

Row4

Row 5

Row 6

Row 7

Row 8

Row 9

Row 10

Row 11

Row 12

400

500

600

700

n = 8 for each row


A.U

. at 6

00 V

Fig. 2.29. Variation in fluorescence intensity of 96-well microplate rows across the Y axis post-levelling and photomultiplier calibration

Variation across Y Axis of 96-well platePost-leveling & EHT calibration

Row 1

Row 2

Row 3

Row 4

Row 5

Row 6

Row 7

Row 8

Row 9

Row 10

Row 11

Row 12

400

500

600

700

n = 8 for each row


A.U

. at 7

00 V

Fig. 2.30. Variation in fluorescence intensity of 96-well microplate rows across the Y axis post-levelling, photomultiplier calibration and new lamp

Variation across Y Axis of 96-well plateNew lamp, post-alignment & EHT calibration

Row 1

Row 2

Row 3

Row 4

Row 5

Row 6

Row 7

Row 8

Row 9

Row 10

Row 11

Row 12

400

500

600

700

n = 8 for each row


A.U

. at 6

00 V

2.3.9 Interference from Pipettes

In an effort to investigate the considerations for pipetting samples in organic

solvents as well as automatic pipetting, several experiments were performed. A

Pipetman microlitre pipette and a pipette tip (2 μL) with filter (Molecular BioProducts,

ART 10 Pipet Tips) were used to deliver ethanol to the wells of the 96-well microplate.

In the first experiment, the samples were delivered from column A1 to A12, then

column B1 to B12 etc. for the whole length of the 96-well microplate. AIOR assay was

then performed on the plate (Fig. 2.31). In the second experiment, using exactly the

same conditions, the 96-well microplate was read upside down in the fluorescence

spectrophotometer (Fig. 2.32). This enabled the spectrofluorometer to read the samples

33

which were delivered last to be read first. In the third experiment, the samples were

delivered from row A1 to H1, then row A2 to H2 etc. for the length of the 96-well

microplate (Fig. 2.33). These experiments showed that the high background always

occurred in the wells which had samples delivered last. As shown in the Fig. 2.31 to

2.33, the imprecision of the assays under those pipetting conditions was unsatisfactory.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

These curves belonged to the samples from column H

Fig. 2.31. Ethanol (2 μL) delivered to 96-well microplate colmun A1 to A12, B1 to B12 etc. The 96-well microplate read in the Eclipse in the normal position

n = 96

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

These curves belonged to the samples from column G and H but indicated as from column A and B by Eclipse (read upside down)

Fig. 2.32. Ethanol (2 μL) delivered to 96-well microplate column A1 to A12, B1 to B12 etc. The 96-well microplate read in the Eclipse in upside down position

n = 96

34

50 100 150

200

400

600

800

1000

Time (m in)

Inte

nsity

(a.u

.)

These curves belonged to the samples from row 11 (A - H) and 12 (A - H)

Fig. 2.33. Ethanol (2 μL) delivered to 96-well microplate from row 1A to 1H, row 2A to 2H etc. The 96-well microplate read in the Eclipse in the normal position

n = 96

Aqueous samples did not present this problem. A 2 μL pipette tip with filter was

used to deliver water to the wells of the 96-well microplate as performed in the previous

experiments. There was no high background with aqueous samples (Fig. 2.34) and the

precision of the AIOR assay was good.

5 0 1 0 0 1 5 0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

T im e (m in)

Inte

nsity

(a.u

.)

Mean Area Under Curve = 49851 SD = 3379 CV = 6.8 % n = 96

Fig. 2.34. Deionized H2O (2 μL) delivered to 96-well microplate from column A1 to A12, B1 to B12 etc. The 96-well microplate read in the Eclipse in the normal position

Precision of AIOR assay with aqueous blank

The effect of high background towards the end of pipetting samples in ethanol

was found. The high background reading with ethanol was overcome by taking two

35

simple precautions. Firstly, use of a pipette tip without filter and secondly, priming the

pipette tip with air after disposal of the used pipette tip. The interference was due to the

high vapour pressure of ethanol. During continuous reuse of the pipette tip, the barrel of

the pipette became saturated with ethanol vapour which extracted substances from the

pipette. These substances found their way into the wells towards the end of multiple

sampling. As a result, a high background appeared in the wells. Fig. 2.35 shows that by

taking special precautions, the problem of high background with pipetting samples

dissolved in organic solvents was overcome.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Improved precision of AIOR assay with ethanol samples delivered with special precaution

Mean Area Under Curve = 48454 SD = 2420 CV = 5.0 % n = 96

Fig. 2.35. Ethanol (2 μL) delivered to 96-well microplate from column A1 to A12, B1 to B12 etc. with special precaution. The 96-well microplate read in the Eclipse in the normal position

2.4 Discussion

The advantage of the AIOR method is the elimination of temperature

dependence of the reaction in the measurement of total antioxidant capacity. Ultimately,

this means that the assay can be performed at ambient room temperature in any

laboratory. It is likely that the assay at ambient room temperature, instead of 37oC, is

better for the stability of the antioxidants in the samples being measured and the free

radicals generating reagent AAPH. In addition, the AIOR method does not require an

extra auxiliary temperature control for the 96-well microplate. In contrast, pre-heating at

37oC is required for the ORAC method, but the temperature of the reaction is not

maintained and, as a consequence, there would be a significant difference in

36

temperature at the start and end of the reaction. This will affect the precision of the

assay since it is well known that decreasing temperature has the effect of increasing

fluorescence intensity.

For the measurement of total antioxidant capacity, integration of the area under

the curve is more accurate than a single point measurement of the reaction and the

linearity and the reproducibility of the AIOR assay is reliable.

The optimum concentration of the radical generating reagent AAPH has been

investigated. Other water-soluble amidino-azo radical initiators such as 2,2’-azobis(2,4-

dimethylvaleronitrile), 2,2’-azobis, 2.17 and and 2,2’-azobis[2-(2-imidazolin-2-

yl)propane], which generate alkylperoxyl and/or alkoxyl radicals are likely to be

suitable for the AIOR assay, but their usage was not pursued since AAPH, as free

radical generator, adapts well to the assay.

As expected, the fluorescence intensity has been found to be directly

proportional to concentration as well as to the amount of uroporphyrin I used. In

addition, it has been shown that the lamp energy and the detector sensitivity of the

fluorescence spectrophotometer are important factors in determining the best

concentration and the amount of uroporphyrin I reagent for the AIOR assay.

The AIOR assay with the Trolox standard at various concentrations within a

range of 0.01 to 5.00 mmol/L have been shown to be linear. A standard range can be

chosen according to the quantity of the total antioxidant capacity of the samples to be

measured and/or multi-point calibration may be used because 96 wells are available for

standards and samples.

The 96-well microplate format was used to increase sample throughput and

because of its potential for automation. A change to the 384-well microplate may also

be possible enabling all manner of dedicated wellplate readers and a lowering of costs

for commercial scale-up operations.

The AIOR method can be used to measure a diversity of compounds in both

hydrophobic and hydrophilic conditions. To enable this particular environment protocol,

Brij 35 (30% w/v) was employed in the AIOR assay. Brij 35 is a polyoxyethylene

37

alcohol, a nonionic surfactant prepared by ethoxylation of fatty alcohols with ethylene

oxide. Furthermore, this compound improved the precision of the AIOR assay.

Uroporphyrin I, II, and III in 1 M HCl show absorption at λmax 406 nm (ε mM

505) and in 0.5 M HCl at λmax 406 nm (ε mM 541). Uroporphyrin I dihydrochloride has

good solubility in 75 mM PBS with Brij 35 at pH 7.0. It is also cheaper than

uroporphyrin II, III dihydrochloride and B- or R-phycoerythrin.

The alignment of the 96-well microplate in this particular instrument has been

found to be important in determining the precision of the assay. Optimisation of the

Eclipse fluorescence spectrophotometer was necessary before performing the assay. The

polypropylene 96-well microplate may be recycled and reused if washed properly. A

routine such as rinsing with distilled water, followed by rinsing with ethanol and

repeated rinsing with distilled water was found to be useful. The 96-well microplate

must be dried before use.

For the AIOR assay, the fluorescence spectrophotometer used was fitted with a

Xenon flash lamp which flashes only to acquire a data point, therefore photosensitive

samples are not exposed to continuous light. The sensitivity of this instrument and the

lamp energy was matched with the amount and concentration of uroporphyrin I for the

AIOR assay. The 96-well microplate must be aligned so that the source of light reaches

the centre of the well for fluorescence excitation and emission measurements. Similar

considerations are likely to apply to dedicated wellplate readers using filter based

technologies.

High readings were observed at the end of pipetting samples when organic

solvents such as ethanol were used. Ethanol did not have antioxidant activity as

measured in the AIOR assay. It was also found that the background interference was

related to the vapour of the organic solvents refluxing in the barrel of the pipette. A few

simple precautions described in section 2.3.9 eliminates this interference. Aqueous

samples did not show interference.

In conclusion, an efficient method has been developed for the measurement of

total antioxidant capacity.

38

Chapter 3

Kinetic Study of the AIOR Reaction by

Fluorescence and UV-visible Spectroscopy

3.1 Introduction

A study of the spectral characteristic of a compound by scanning before and

during a reaction can provide useful information about changes to the compound. To

study the reaction of the indicator molecule uroporphyrin I and the alkoxyl/alkylperoxyl

radical generating reagent AAPH, fluorescence and UV-visible spectroscopy were

employed to monitor the peak absorbance maxima and the formation of end-products.

Both fluorescence and UV-visible spectroscopy were used since AAPH and gallic acid

do not exhibit fluorescence. The inhibitory effect of antioxidants such as gallic acid on

the reaction between uroporphyrin I and AAPH was examined by UV-visible

spectroscopy. The kinetics of the reaction between uroporphyrin I and AAPH was

studied by UV-visible and fluorescence spectroscopy.

To ensure that the changes in spectral characteristic were detected, a UV-visible

scanning range from 200 - 700 nm was monitored. The 300 - 700 nm range was chosen

for the detection of the uroporphyrin I molecule and porphyrin-like end-products. A

shorter range from 200 - 450 nm was monitored for a closer observation of the

absorbance maximum of AAPH and uroporphyrin I at 368 and 397 nm respectively.

The effect of a 10 fold decrease in AAPH concentration on the uroporphyrin indicator

molecule was studied by monitoring the range from 200 - 700 nm.

39

Gallic acid was chosen as the antioxidant because of its property as a stable

antioxidant with a simple chemical structure. The reaction between gallic acid and

AAPH in the absence or presence of uroporphyrin I was studied in the 200 - 700 nm

UV-visible range.

The kinetics of the reaction between uroporphyrin I and AAPH was investigated

initially by UV-visible spectroscopy and then by a more detailed fluorescence

spectroscopy measurement at three different combinations of concentration of

uroporphyrin I and AAPH to obtain a statistically significant rate constant.


A Varian Cary IE/100 UV-visible spectrophotometer was used for UV-visible

scanning in the cuvettes. Cuvettes and 96-well microplates were used for fluorescence

scanning with a Varian Cary Eclipse fluorescence spectrophotometer.

For fluorescence scanning in the 96-well microplate, the emission scan was set

with excitation wavelength at 397 nm, and scanning emission wavelength from 500 -

700 nm. The excitation scan was set with emission wavelength at 615 nm, and scanning

excitation wavelength from 350 - 550 nm. The concentration of the uroporphyrin I

solution was 300 nmol/L and AAPH 583 mmol/L. In the 96-well microplate,

uroporphyrin I (170 μL) was reacted with AAPH (28 μL).

For fluorescence scanning in the cuvette, the emission scan was set with an

excitation wavelength at 397 nm, and scanning emission wavelengths from 450 - 650

nm. The excitation scan was set with an emission wavelength at 615 nm, and scanning

excitation wavelengths from 350 - 550 nm. The concentration of the uroporphyrin I

solution was 300 nmol/L and AAPH 583 mmol/L. In the reaction, AAPH (424 μL) was

reacted with uroporphyrin I (2576 μL).

40

UV-visible scanning ranges in the cuvettes were at 300 - 700, 200 - 450 and 200

- 700 nm. The concentration of the reagents used for this study was 4.5 μmol/L for the

uroporphyrin I solution, 583 mmol/L for the AAPH reagent and 225 μmol/L for gallic

acid.

The scans for the 300 - 700 and 200 - 450 nm range were monitored at time = 0,

1, 5, 15, 30 60 120, 180, 240, 300 and 360 min and overnight with the reaction of

uroporphyrin I (3000 μL) with AAPH (50 μL). The scans for the 200 - 700 nm range

were monitored at time = 0, 1, 5, 15, 30, 60, 120, 180, 240 min and overnight with the

reaction of uroporphyrin I (3000 μL) with AAPH (5 μL). The stability of uroporphyrin I

was checked at 240 min and overnight.

The reaction between gallic acid (3000 μL) and AAPH (50 μL) was monitored

at 200 - 700 nm at time = 0, 1, 5, 15, 30, 60, 120, 180 and 180 min. The stability of

gallic acid was checked after 90 min.

The reaction between uroporphyrin I (1500 μL), gallic acid (1500 μL) and

AAPH (50 μL) was monitored at 200 - 450 and 200 - 700 nm range at time = 0, 1, 5, 15,

30, 60, 120, and 180 min. Uroporphyrin I and gallic acid were mixed and observed in

the 200 - 700 nm range at time = 0, 15 and 90 min.

For the kinetics experiment, uroporphyrin I at concentrations of 90, 180 and 180

nmol/L were reacted with AAPH at 292, 292 and 583 mmol/L respectively.

Uroporphyrin I (2.5 mL) was reacted with AAPH (0.5 mL) in all three experiments. The

fluorescence intensity of the reaction between uroporphyrin I and AAPH was monitored

at excitation wavelength of 405 nm and emission wavelength of 624 nm.

For the HPLC monitoring by photodiode array detector (PDA), the absorbance

maxima was recorded at a spectral range of 300 - 650 nm at time = 0, 60, 120 and 180

min.

41

3.3 Results

3.3.1 Emission and Excitation Fluorescence Scan in a 96-Well Microplate

The emission and excitation fluorescence scans in a 96-well microplate before

and after the addition of AAPH to uroporphyrin I are shown in Fig. 3.1 and 3.2.

400 500 600 7000

100

200

300

400

500

600

700

W avelength (nm )

Inte

nsity

(a.u

.)

Em is s io n M axim a: 61 5 nm

Ex c itatio n M ax im a: 3 97 n m

Fig. 3.1. Emission and excitation scans of uroporphyrin I with Brij. Before the addition of AAPH in a 96-well microplate

400 500 600 700

50

100

150

200

250

W avelength (nm )

Inte

nsity

(a.u

.) Excita tio n M axima: 402 n m

Em is s ion M axim a: 622nm

Fig. 3.2. Emission and excitation scans of uroporphyrin I with Brij. After the addition of AAPH in a 96-well microplate

Before the addition of AAPH to uroporphyrin I, the emission and the excitation

maxima were 615 nm and 397 nm respectively (Fig. 3.1). With the addition of AAPH to

42

uroporphyrin I, the fluorescence emission intensity dropped by 60% and excitation

intensity dropped by 75% in 5 min. There was also an emission wavelength shift to 622

from 615 nm and an excitation wavelength shift to 402 from 397 nm (Fig. 3.2).

3.3.2 Emission and Excitation Fluorescence Scan in a Cuvette

Uroporphyrin I, AAPH and gallic acid in a cuvette were scanned by a

fluorescence spectrophotometer to establish their spectral characteristics. For

uroporphyrin I, the emission intensity maximum was at 615 nm (Fig. 3.3) and the

excitation intensity maximum at 397 nm (Fig 3.4). Gallic acid and AAPH did not

exhibit fluorescence.

450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)

Emission intensity at 615 nm = 753

Fig. 3.3. Emission scan. Time = 0. Uroporphyrin I

350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)

Fig. 3.4. Excitation scan. Time = 0. Uroporphyrin I

Excitation intensity at 397 nm = 749

43

The effect of adding AAPH to uroporphyrin I in a cuvette was monitored over

time. The results for the fluorescence emission intensity are shown in Fig 3.5 - 3.10

(appendix) and for the excitation intensity in Fig 3.11 - 3.16 (appendix). Interestingly, 5

min after addition, the emission wavelength maxima had shifted to 624 from 615 nm

and the excitation wavelength from 397 to 404/405 nm. At the same time, the emission

intensity dropped by 45% and the excitation intensity by 78%. These results are

summarized in Table 3.1.

Table 3.1. Fluorescence emission and excitation intensity with time after addition of AAPH to

uroporphyrin I monitored in a cuvette

Time Emission intensity 615 nm

Emission intensity 624 nm

Excitation intensity 397 nm

Excitation intensity

404 - 5 nm (1) 0 uroporphyrin I 753 749

(2) 5 min uroporphyrin I + AAPH 414 167






3.3.3 UV-visible Scan in a Cuvette of the Reaction between AAPH and

Uroporphyrin I

(a) Range monitored 300 - 700 nm:

The UV-visible spectra from 300 - 700 nm of AAPH and uroporphyrin I at time

= 0 are shown in Fig. 3.17 and Fig. 3.18 (appendix). To check the stability of

uroporphyrin I, it was left in the dark at ambient room temperature for 240 min (Fig.

44

3.19, appendix). AAPH and uroporphyrin I gave an UV-visible absorbance maxima at

368 and 397 nm respectively.

A summary of the UV-visible scans obtained for the reaction between

uroporphyrin I and AAPH 1 to 360 min (see Figs. 3.20 - 3.22 for examples, appendix) is

shown in Table 3.2. A shift of the absorption maximum from 397 to 402 nm was

observed in the first minute. This was accompanied by a 15% decrease in intensity.

Table 3.2. UV-visible scan from 300 - 700 nm in a cuvette of uroporphyrin I (urop. I) and AAPH

before and during the reaction

Time Abs. intensity 368 nm

Abs. intensity 397 nm

Abs. intensity 401 - 2 nm

(1) 0 (uroporphyrin I) 0.909

(2) 240 min (uroporphyrin I) 0.909

(3) 1 min (H2O + AAPH) 0.244

(4) 1 min (urop. I + AAPH) 0.774

(5) 5 min (urop. I + AAPH) 0.770

(6) 15 min (urop. I + AAPH) 0.765

(7) 30 min (urop. I + AAPH) 0.758

(8) 60 min (urop. I + AAPH) 0.737

(9) 120 min (urop. I + AAPH) 0.686

(10) 180 min (urop. I + AAPH) 0.607

(11) 240 min (urop. I + AAPH) 0.527

(12) 300 min (urop. I + AAPH) 0.383

(13) 360 min (urop. I + AAPH) 0.272

45

(b) Range monitored 200 - 450 nm:

Similar data were obtained by scanning the range between 200 - 450 nm (see

Figs. 3.23 - 3.27 for examples, appendix). These are listed in Table 3.3. No new

absorbance peaks were obtained. The shift of the absorption maximum from 397 to 402

nm and a 13% decrease in intensity were noted.



Time Abs. intensity 368 - 379 nm



(1) 0 (PBS + AAPH) 0.252 - -

(2) 0 (uroporphyrin I + PBS) - 0.768 -

(3) 1 min (urop. I + AAPH) 0.500 0.677

(4) 5 min (urop. I + AAPH) 0.493 0.671

(5) 15 min (urop. I + AAPH) 0.482 0.658

(6) 30 min (urop. I + AAPH) 0.460 0.634

(7) 60 min (urop. I + AAPH) 0.427 0.578

(8) 120 min (urop. I + AAPH) 0.372 0.462

(9) 180 min (urop. I + AAPH) 0.336 0.352

(10) 240 min (urop. I + AAPH) 0.315 0.277

(11) 300 min (urop. I + AAPH) 0.302 0.220

(12) Overnight (urop. I + AAPH) 0.265 -

46

(c) Range monitored 200 - 700 nm:

The effect of reducing the amount of AAPH by 10 fold was investigated by

monitoring the region from 200 - 700 nm (see Figs. 3.28 - 3.33 for examples, appendix).

Reducing AAPH by 10 fold showed no significant difference in the decrease in

absorbance intensity of about 13% in 1 min (Table 3.4).



Time Abs. intensity 368 nm



(1) 0 (PBS + AAPH) 0.083 - -

(2) 0 (uroporphyrin I) 0.770 -

(3) overnight (uroporphyrin I) 0.782 -

(4) 1 min (urop. I + AAPH) 0.672

(5) 5 min (urop. I + AAPH) 0.668

(6) 15 min (urop. I + AAPH) 0.667

(7) 30 min (urop. I + AAPH) 0.666

(8) 60 min (urop. I + AAPH) 0.661

(9) 120 min (urop. I + AAPH) 0.646

(10) 180 min (urop. I + AAPH) 0.631

(11) 240.min (urop. I + AAPH) 0.622

(12) overnight (urop. I + AAPH) 0.475

47

3.3.4 UV-visible Scan in a Cuvette from 200 - 700 nm of the Reaction between

AAPH and Gallic Acid

The reaction of AAPH with gallic acid (λmax 259 nm) was monitored by

scanning the 200 - 700 nm range (Figs. 3.34 - 3.38 for examples, appendix). The results

summarized in Table 3.5 show essentially no differences in absorbance wavelength or

intensity over 240 min.

Table 3.5. UV-visible scan from 200 - 700 nm in a cuvette of gallic acid and AAPH before and

during the reaction

Time

Gallic acid

Abs. intensity at 259 nm

AAPH

Abs. intensity at 368 nm (AU)

(1) 0 (PBS + AAPH) - 0.249

(2) 0 (gallic acid + PBS) 1.562 -

(3) 1 min (gallic acid + AAPH) 1.613 0.301








48

3.3.5 UV-visible Scan in a Cuvette from 200 - 700 nm of Gallic Acid,

Uroporphyrin I and AAPH

In the first min after the addition of AAPH, a shift in wavelength for

uroporphyrin I to 400 from 397 nm was observed (Figs 3.39 - 3.41 for examples,

appendix). Following a small decrease (7%) in absorbance intensity (Fig. 3.42,

appendix) after 1 min, no change occurred up to 180 min (Fig. 3.43 - 3.44). The results

are summarized in Table 3.6.

Table 3.6. UV-visible scan from 200 - 700 nm in a cuvette between uroporphyrin I (urop. I) and

AAPH in the presence of gallic acid

Time Gallic acid


Uroporphyrin I Abs. intensity

397 nm

Uroporphyrin I Abs. intensity

400 nm

(1) 0 (urop. I + PBS) 0.756

(2) 0 (urop. I + gallic acid) 1.590 0.729

(5) 1 min (urop. I + gallic acid + AAPH) 1.635 0.679







49

3.3.6 Kinetics Study of the AIOR Reaction by UV-visible Spectroscopy

Table 3.7 shows the UV-visible maxima absorbance and natural log conversion

Ln([R]t/[R]o) of absorbance intensity of uroporphyrin I and AAPH from HPLC

monitored by photodiode array detector (PDA) in the range of 300 - 650 nm at

injections time = 0, 60, 120 and 180 min (Fig. 3.45).

Table 3.7. Natural log conversion of the PDA absorbance maxima of the reaction between AAPH

and uroporphyrin I

Time (min) PDA Absorbance

intensity [R]t/[R]o Ln([R]t/[R]o)

0 0.04161 1 0

60 0.02403 0.5775 -0.5490

120 0.01412 0.3393 -1.0807

180 0.00854 0.2052 -1.5836

Decomposition curve from HPLC injections of uroporphyrin I working reagent with AAPH reaction monitored by photodiode array

detector at 300 - 650 nm.

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

00 50 100 150 200

Time (minutes)

Ln([

R]t/[

R]o)

Fig. 3.45. First-order kinetic curve between uroporphyrin I and AAPH measured at 300 - 650 nm by photodiode array detector

50

3.3.7 Kinetics Study of the AIOR Reaction by Fluorescence Spectrophotometry

To investigate the reaction kinetics of the uroporphyrin I reaction with AAPH,

the reaction was monitored continuously by fluorescence at an excitation wavelength

405 nm and emission wavelength at 624 nm for 150 min. Uroporphyrin I at two

concentrations of 90 nM, 180 nM and 180 nM was reacted with AAPH with

concentrations of 292 mM, 292 mM and 583 mM respectively, as shown in Fig. 3.46,

3.47 and 3.48.

0 50 100 1500

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.) [Uroporphyrin I] = 90 nM, 2.5 mL [AAPH] = 292 mM, 0.5 mL Ex = 405 nm Em = 624 nm

Fig. 3.46. Fluorescence intensity versus time (min)

0 50 100 1500

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

[Uroporphyrin I] = 180 nM, 2.5 mL [AAPH] = 292 mM, 0.5 mL Ex = 405 nm Em = 624 nm


51

0 50 100 1500

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

[Uroporphyrin I] = 180 nM, 2.5 mL [AAPH] = 583 mM, 0.5 mL Ex = 405 nm Em = 624 nm


Natural log (ln) conversion of fluorescence intensity ln([R]t/[R]o) versus time in

minutes of the fluorescence scanning experiments at the three concentrations of

uroporphyrin I of 90 nM, 180 nM and 180 nM and AAPH of 292 nM, 292 mM and 583

mM were plotted as shown in Fig. 3.49, 3.50 and 3.51.

Fig. 3.49. First-order kinetic curve between [uroporphyrin I] = 90 nM and [AAPH] = 292 mM measured at Ex = 405 nm and Em = 624 nm at room temperature with rate constant k = 0.0253 min-1

Ex = 405 nm, Em = 624 nmURO = 90 nM, 2.5 mL; AAPH = 292 mM, 0.5 mL

k = 0.0253 min-1 at room temp.

-4.5

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

00 50 100 150 200

Time (minutes)

Ln([

R]t/

[R]o

)

52




-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

00 50 100 150 200

Time (minutes)

Ln([

R]t/[

R]o)




-5

-4.5

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

00 50 100 150 200

Time (minutes)

Ln([

R]t/[

R]o)

53

Table 3.8 shows a summary of the reaction rate experiments. All three

experiments gave a kinetic curve with a straight line and a negative slope, which

confirms the reaction to be first-order. The average rate constant k from three

experiments was calculated to be 0.0254 min-1.

Table 3.8. First-order rate constant κ (min-1) at room temperature from 3 experiments with

different combinations of uroporphyrin I and AAPH concentration

Uroporphyrin I

concentration (nM)

AAPH

concentration (mM)

First-order rate constant k

(min-1) at room temp.

90 292 0.0253

180 292 0.0232

180 583 0.0276

n = 3 Average k = 0.0254

3.4 Discussion

For the fluorescence and UV-visible spectroscopy experiments reported, three

general conclusions can be made. Firstly, there was a rapid decrease in the fluorescence

and UV-visible absorbances at the beginning of the reaction after AAPH was added to

uroporphyrin I. Secondly, a shift in the absorbance maximum of the uroporphyrin I with

the addition of AAPH was observed. Thirdly, in the presence of gallic acid,

uroporphyrin I was protected, showing no further decrease in the UV-visible

absorbance. In the absence of gallic acid, the results showed that there was a progressive

destruction of uroporphyrin I as indicated by the decrease in UV-visible absorbance.

54

It has been shown that porphyrins can protect lipids against peroxidation by

scavenging radicals generated from AAPH.91 This results in the degradation of

porphyrins.

After the addition of AAPH to uroporphyrin I, the excitation wavelength shifted

from 397 to 405 nm and the emission wavelength from 615 to 624 nm (Fig. 3.52).

Moreover, the emission intensity decreased by 45% and the excitation intensity by 78%

(Table 3.1).

397 nm

405 nm

615 nm

624 nm

Δ = 8 nm

Δ = 9 nm

Time = 0, before addition of AAPH

Time = 1 - 5 min., after addition of AAPH

Excitation λ

Emission λ

Fig. 3.52. Fluorescence wavelength shifts after the addition of AAPH. to uroporphyrin I

The observed shifts in UV-visible absorption spectra were also significant (Fig

3.53). The wavelength accuracy and reproducibility of the spectrophotometer was stated

to be (0.02 - 0.008) nm at 656 nm and (0.04 - 0.008) nm at 486 nm. In this case also the

absorption intensity decreased by 13 - 15% (Table 3.2 – 3.4).

55

UV-visible absorbance of

Uroporphyrin I : H2O (3000 μL : 50 μL)

UV-visible absorbance of Uroporphyrin I : AAPH

(3000 μL : 50 μL)

397 nm

502 nm

538 nm

560 nm

612 nm

400-2 nm

500 nm

535 nm

567 nm

622 nm

Fig.3.53. UV-visible wavelength shifts after addition of AAPH to the uroporphyrin I

There was a significant drop in fluorescence intensity and UV-visible

absorbance immediately after the addition of AAPH to uroporphyrin I. For example, the

fluorescence emission intensity dropped by 45% and the excitation intensity by 78%

after AAPH was added to uroporphyrin I. The UV-visible absorbances all decreased

immediately after the addition of AAPH to uroporphyrin I.

This behaviour has been observed before in a study of the changes in the

absorption and emission spectra of porphyrins in various molecular complexes in

aqueous solution.92 For example, a solution of uroporphyrin in 0.1 M NH3, pH 9.5

showed absorption bands (nm) at 397.5 (Soret), 502 (IV), 538.5 (III), 560 (II) and 611.5

(I). Addition of 1-(2-hydroxyethyl)-3-carbamylpyridinium chloride gave a spectrum

with bands (nm) at 403.7, 503.5, 537.5, 566.5 and 619.5 that match well with the values

shown in Fig. 3.53. Similarly the fluorescence emission bands at 615 and 680 nm are

shifted to 622 and 690 nm in the complex. For the case of AAPH, these appear at 622

and 690 nm. The total intensity of the emission spectra, on the average, decreases 2 - 3

times (Fig. 3.1 - 3.2) as found by Mauzerall.92 These observations are not restricted to

the interactions of uroporphyrin and cationic addends; complexes are also formed with

planar neutral heterocyclic molecules. It has been suggested that these complexes are

induced micelles in which the addends cluster about the porphyrin plane and thus

transfer the chromophore from an aqueous to a polar organic environment.92

56

As expected, the largest changes in the absorption and emission spectra occur on

forming the uroporphyrin : addend 1:1 complex. Although there is evidence that a

sequential series of complexes are formed, results strongly indicate that the porphyrin is

monomeric in the complexes. In the case of AAPH, reduction of the amount of addend

by ten fold still resulted in a shift of the Soret band to higher wavelength with a

decrease of the absorbance intensity (Table 3.4).

No interaction was observed between uroporphyrin I and gallic acid. Addition of

AAPH resulted in a minimal decrease (7%) in the absorbance intensity after 1 minute,

but this remained constant up to 180 min. These results show that gallic acid protects

uroporphyrin I from the alkoxyl radicals generated from AAPH.

The kinetics of the reaction between uroporphyrin I and AAPH was investigated

initially with an experiment of UV-visible spectroscopy and then with a more detailed

study by a continuous monitoring of the reaction with fluorescence spectroscopy.

A first-order reaction is indicated if a straight line is produced by plotting natural

log (ln) of [Uroporphyrin I]t/[Uroporphyrin I]o versus time. The slope of the line gives

the rate constant k.93 The experiments to determine the rate constant k for this reaction

were all performed at room temperature. Only the fluorescence experiment was used to

calculate the rate constant, with continuous monitoring for 150 min of the reaction.

For a given reaction,

Reactant (R) Product (P)

The rate of the reaction may be expressed as:

Rate =

Δ R

Δ t= k [R] -

ln [R]t

[R]o

= - k t

57

[R] can be expressed in any convenient quantity unit. The ratio [R]t/[R]o is

dimensionless and < 1 because [R]t is always less than [R]o. Plotting ln [R]t/[R]o versus

time produces a straight line with a negative slope, k, the rate constant for a first-order

reaction. For the following reaction,

Uroporphyrin I + AAPH Product (P)

ln

[Uroporphyrin I]t

[Uroporphyrin I]o

= - k t

Both UV-visible and fluorescence experiments in the kinetic study showed that

the reaction between uroporphyrin I and AAPH is of first order kinetics.

The kinetic curve determined from HPLC monitored by photodiode array

detector at times of 0, 60, 120, and 180 min for the reaction between uroporphyrin I and

AAPH showed first order kinetics by plotting the natural log (ln) of [R]t/[R]o versus

time in min (Fig. 3.45).

Plotting ln [Uroporphyrin I]t/[Uroporphyrin I]o versus all three fluorescence

experiments gave first-order kinetics as shown in Fig. 3.49, 3.50 and 3.51. Table 3.8

summarizes the rate constants k calculated from the three curves at different

combinations of uroporphyrin I and AAPH concentration. They were 0.0253, 0.0232

and 0.0276 with a mean rate constant k = 0.0254.

The average time-half (t1/2) of the reaction was calculated to be 27.3 min at room

temperature.

t 1/2 = 0.693

k

t 1/2 = 0.693

0.0254 = 27.3 min

58

In conclusion, the observed shifts in wavelengths in both the fluorescence and

UV-visible scans after the addition of AAPH to the uroporphyrin I indicate that a

change of the uroporphyrin I molecule has taken place as a result of the reaction

between the uroporphyrin I molecule and AAPH. Further studies by HPLC, electron

spin resonance (ESR) analysis and LC-MS to look at the reaction mechanism are

described in chapters 4 and 5. The fluorescence and UV-visible studies have shown that

the reaction between uroporphyrin I and AAPH displays first order kinetics.

3.5 Appendix

3.5.1 Emission and Excitation Fluorescence Scans of the Reaction between AAPH

and Uroporphyrin

450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.) Emission intensity at 624 nm = 414

Fig. 3.5. Emission scan. Time = 5 min after addition of AAPH to uroporphyrin I

450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



59

450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)


Emission Maxima at 624 nm = 319

450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



60

450 500 550 600 6500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)

Fig. 3.11. Excitation scan. Time = 5 min after addition of AAPH to uroporphyrin I


350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



61

350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



350 400 450 500 5500

200

400

600

800

1000

Wavelength (nm)

Inte

nsity

(a.u

.)



62



Fig. 3.18. At time = 0. Uroporphyrin I

397 nm

Fig. 3.17. At time = 0. H2O + AAPH

Fig. 3.20. At time = 1 min. Uroporphyrin I + AAPH

400 nm

Fig. 3.19. At time = 240 min. Uroporphyrin I

397 nm


401 nm


402 nm

63



Fig. 3.23. At time = 0. AAPH + PBS Fig. 3.24. At time = 0.

Uroporphyrin I + PBS




64



Fig. 3.29. At time = 0. Uroporphyrin I

Fig. 3.28. At time = 0. AAPH + PBS

Fig. 3.31. At time = 1 min. Uroporphyrin I + AAPH Fig. 3.30. At time = overnight.

Uroporphyrin I


Fig. 3.33. At time = Overnight. Uroporphyrin I + AAPH

65



Fig. 3.35. At time = 90 min. Gallic acid + PBS

Fig. 3.34. At time = 0. Gallic acid + PBS

Fig. 3.36. At time = 1 min. AAPH + gallic acid



66


between AAPH, Gallic Acid and Uroporphyrin I

Fig. 3.39. At time = 0. Gallic acid + uroporphyrin I

Fig. 3.40. At time = 15 min. Gallic acid + uroporphyrin I

Fig. 3.41. At time = 90 min. Gallic acid + uroporphyrin I

Fig. 3.42. At time = 1 min. AAPH + Gallic acid + uroporphyrin I

Fig. 3.43. At time = 5 min. AAPH + gallic acid + uroporphyrin I

Fig. 3.44. At time = 180 min. AAPH + gallic acid + uroporphyrin I

67

Chapter 4

Study of the AIOR Reaction by

Electron Spin Resonance Analysis and HPLC

4.1 Introduction

As indicated previously in chapter 2, the decomposition of the azo-initiator

AAPH can give rise to alkylperoxyl radicals (ROO•) which can also form a tetraoxide

intermediate that subsequently collapses to the alkoxyl radical (RO•). Tetraoxides have

not been observed in aqueous solution.4 In the case of the reaction of uroporphyrin I

with AAPH-derived radicals, the formation of micelles of uroporphyrin I and AAPH

complexes may provide an organic environment that could affect the type of radicals

formed.

Short-lived free radicals are normally detected using spin traps that react with

radicals to form relatively stable adduct that can be studied by electron spin resonance

(ESR). It was considered worthwhile to monitor the reaction of uroporphyrin I and

AAPH by ESR to investigate the types of radical species produced. In a preliminary

attempt, the formation of the spin adducts between the radicals from AAPH and the spin

trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was studied by ESR.

In the expectation that distinct end-products would be generated from the

reaction of uroporphyrin I with radicals derived from AAPH, high performance liquid

chromatography was applied to the separation of such products.

68

The reaction between uroporphyrin I and AAPH, in the absence and presence of

the antioxidant gallic acid, was studied by monitoring with a fluorescence detector and a

photodiode array (PDA) detector.


ESR spectra were obtained with a Bruker ESR spectrometer for a mixture of

AAPH (583 mmol/L), deionized H2O and DMPO (3 mg/mL), each 100μL, at 2 min and

15 min. For the stability study, the AAPH solution was kept at 0oC and used 3 hours

after preparation. To test for the presence of free radical formation in the reaction of

uroporphyrin I and AAPH, the reaction mixture was examined by ESR analysis. The

spectra were collected for a mixture of AAPH (583 mmol/L), uroporphyrin I (1.7

mmol/L) and DMPO (3 mg/mL), each 100 μL, at times 2, 15 and 30 min.

A Waters Symmetry C18, 3.9 x 150 mm, 5 μm, column was used for the HPLC

experiments. A Waters 600E system controller, 510 pump, 717 autosampler, 996

photodiode array detector (PDA) and a Shimadzu RF551 spectrofluorometric detector

were used for the HPLC experiments. Wavelengths of 397, 402 and 259 nm were

selected for monitoring the absorbance. The spectrofluorometric detector was set up in

series behind the PDA detector for fluorescence monitoring at an excitation λ 397 nm

and emission λ 615 nm.

The mobile phase was degassed with helium at a sparge rate of 100 mL/min for

15 min before reducing to 20 mL/min, which was then maintained for the entire run.

Two mobile phases were tested. The first was composed of two solvents A and B.

Solvent A consisted of 0.005% formic acid (pH 3.45) and acetonitrile (9 : 1 v/v) and B

methanol and acetonitrile (9 : 1 v/v). The HPLC gradient conditions are summarised in

Table 4.1.

69

Table 4.1. HPLC first mobile phase tested with flow gradient

Time (min) Flow (mL/min) %A %B

1.0 50 50

10 1.0 20 80

25 1.0 0 100

30 1.0 0 100

45 1.0 50 50

The first mobile phase was subsequently found to be unsuitable due to gradual

precipitation of uroporphyrin I at low pH. A second mobile phase was developed for the

LC-MS experiments. Solvent A consisted of 1 mol/L ammonium acetate (pH 5.16) and

acetonitrile (9 : 1 v/v) and solvent B of methanol in acetonitrile (9 : 1 v/v). The HPLC

gradient conditions are summarised in Table 4.2.

Table 4.2. HPLC second mobile phase to be used for LC-MS experiments with flow gradient

Time (min) Flow (mL/min) %A %B

0 0.2 100 0

10 0.2 70 30

12 0.2 50 50

13 0.2 50 50

15 0.2 100 0

20 0.2 100 0

For the reaction between AAPH and uroporphyrin I, 583 mM AAPH (19.5 μL)

was reacted with 45 μM uroporphyrin I (125 μL). The reaction mixture (60 μL) was

monitored at time = 1, 30, 60, 90, 120, 180, 240 and 450 min. The reaction sample was

also left overnight at room temperature and injected the next day.

For the reaction between uroporphyrin I and AAPH in the presence of gallic

acid, the same concentration of uroporphyrin I was prepared. The mixture had a

70

concentration of 45 μM uroporphyrin I and gallic acid 2.25 mM. All dilutions were

made up with 0.075 M phosphate buffer solution (PBS), pH = 7.0. The reaction of gallic

acid, uroporphyrin I, and AAPH was performed with 583 mM AAPH (19.5 μL). A

sample of the reaction mixture (60 μL) was injected at time = 1, 30, 60, 90 and 120 min.

4.3 Results

4.3.1 ESR Analysis of AAPH and Uroporphyrin I with DMPO as a Spin Trap

The formation and stability of the radicals generated from AAPH was

investigated by ESR using the spin trap DMPO. Typical ESR spectra are displayed in

Fig. 4.1 - 4.3. These are essentially identical to those reported in the literature.4, 89 The

hyperfine coupling constants (G) observed, aN 14.47 and βaN 14.84 were determined by

simulation of the spectrum and were taken to be indicative of a DMPO-OR adduct.

Fig. 4.1. AAPH with the spin trap (DMPO). Time = 2 min

71

Fig. 4.2. AAPH with the spin trap (DMPO). Time = 15 min

Fig. 4.3. AAPH (3 hours old) with the spin trap (DMPO)

Significantly, alkoxyl radicals generated by AAPH 3 hours earlier were found to

be stable (Fig. 4.3). This reaffirmed the suitability of AAPH as a free radical generating

reagent for the AIOR method, which may take from 1 to 3 hours for the assay to be

completed when using the 96-well microplate.

72

A mixture consisting of AAPH, uroporphyrin I and the spin trap DMPO was

examined by ESR at 2 min (Fig. 4.4), 15 min (Fig. 4.5) and 30 min (Fig. 4.6). The

results showed that the ESR spin signals are positive and there is a noticeable increase

in radicals trapped by DMPO at 15 and 30 min as compared to 2 min. This is consistent

with the presence of uroporphyrin I aggregated with a number of AAPH molecules.

However no distinct signals attributable to uroporphyrin I radicals could be

distinguished.

Fig. 4.4. AAPH, uroporphyrin I and the spin trap (DMPO). Time = 2 min


73


When other indicator compounds such as fluorescein were mixed with AAPH

under the same condition, no ESR signal was observed. Antioxidants such as Trolox

and uric acid also did not produce any observable ESR signals when reacted with

AAPH.

4.3.2 HPLC Experiments

Beckman Ultrasphere C18 and the Waters Symmetry C18 columns were tested

and found to give good resolution of the Type I porphyrins. The Waters Symmetry C18

was chosen after comparison as it provided a slightly better resolution of the LC peaks.

To investigate the solubility of uroporphyrin I in the mobile phase, the reagent

was monitored by PDA at time = 0, 30, 60, 120, 210 and 310 min for changes in the

absorbance caused by precipitation under acidic condition (Table 4.3). The results

showed that the mobile phase containing formic acid had a diminishing absorbance

because uroporphyrin I was precipitating from the mobile phase. A new mobile phase

was developed without formic acid for the subsequent HPLC and LC-MS experiments.

74

A suitable mobile phase was made up with 1 mol/L ammonium acetate (pH 5.16) and

acetonitrile (9 : 1 v/v) as solvent A, and with methanol and acetonitrile (9 : 1 v/v) as

solvent B.

Table 4.3. Effect of mobile phase composition on uroporphyrin I precipitation

Absorbance at time (min)

Mobile Phases 0 30 60 120 210 310

1. PBS, 0.075 M, pH = 7 0.968 0.967 0.968 0.969 0.970 0.974

2. aqueous formic acid 0.015% with acetonitrile (9 : 1 v/v) 0.306 ppt. ppt. ppt. ppt. ppt.

3. aqueous formic acid 0.005% with acetonitrile (9 : 1 v/v) 0.623 0.607 0.578 0.439 0.266 0.262

4. methanol with acetonitrile (9 : 1 v/v) 0.873 0.888 0.897 0.914 0.945 0.973

5. ammonium acetate, 1 M pH 5.16 with acetonitrile (9 : 1 v/v) 0.887 0.887 0.892 0.886 0.882 0.878

4.3.3 HPLC Analysis of the Reaction between AAPH and Uroporphyrin I

The reaction between AAPH and uroporphyrin I was monitored at 397 nm, 402

nm and by fluorescence spectroscopy. Similar results were obtained from these

measurements, with the exception of the AAPH peak which was not observed with

fluorescence. Therefore only those obtained at 402 nm are reported.

The reaction between uroporphyrin I and AAPH at 1 min showed a major HPLC

peak at a retention time of 5.1 min and minor peaks at 8.2 and 10.2 min. An example of

the chromatogram is shown in Fig. 4.7. With fluorescence monitoring, the peaks were

seen at retention times of 5.4 and 8.4 min. Fig. 4.8 - 4.14 show the progression of the

reaction at 30 min intervals up to 180 min and a final measurement at 450 min.

AU

0 1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

75

Uro

porp

hyrin

I - 5

.056

- 10

.255

- 8.

159

ss

AU

0 .00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minu tes0 .00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16 .00 18.00 20.00

AA

PH

- 1.

700

Uro

porp

hyrin

I - 5

.233

Pea

k at

8 m

inut

es -

8.23

0

Pea

k at

10

min

utes

- 10

.137

Fig. 4.8. HPLC monitored by PDA 402 nm at 30 min of the reaction between AAPH and uroporphyrin I

AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

Min u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AA

PH

- 1.

700

Uro

porp

hyrin

I - 5

.263

Pea

k at

8 m

inut

es -

8.21

9

Pea

k at

10

min

utes

- 10

.088

11.8

62


AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

Min u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AA

PH

- 1.

698

Uro

porp

hyrin

I - 5

.289

Pea

k at

8 m

inut

es -

8.23

4

Pea

k at

10

min

utes

- 10

.096

11.8

63


AU 0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

76

- 5.3

55

nute

s - 8

.254

nute

s - 1

0.11

2

AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

Min u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AA

PH

- 1.

704

Uro

porp

hyrin

I - 5

.608

Pea

k at

8 m

inut

es -

8.42

9

Pea

k at

10

min

utes

- 10

.284

12.0

02

13.5

63


AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

M in u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AA

PH

- 1.

707

Uro

porp

hyrin

I - 5

.655

Pea

k at

8 m

inut

es -

8.46

2

Pea

k at

10

min

utes

- 10

.334

12.0

34

13.6

12


AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

Min u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AA

PH

- 1.

679

Uro

porp

hyrin

I - 5

.981

Pea

k at

8 m

inut

es -

8.09

0

Pea

k at

10

min

utes

- 10

.065

11.7

93


77

From 1 min onwards, the major HPLC peak decreased to zero overnight while

the two smaller peaks (8.2 and 10.2 min) increased up to 120 min before decreasing to

baseline. After 60 min, a fourth peak at 11.9 min was apparent (Fig. 4.9) and at 90 min a

fifth peak was observed at 13.5 min (Fig. 4.10). These results are summarized in Table

4.4.

Table 4.4. HPLC separation of products from the reaction of uroporphyrin I (urop. I) and AAPH

monitored at 402 nm

Time (min)

AAPH R.T. 1.7 min

Abs. 402 nm

Peak R.T. 5.0 – 6.0 min

Abs. 402 nm

Peak R.T. 8.0 – 8.5 min

Abs. 402 nm

Peak R.T. 10.0 – 10.4 min

Abs. 402 nm

(1) 1 urop. I + AAPH 0.0182 0.2256 0.0073 0.0026

(2) 30 urop.I + AAPH 0.0200 0.1495 0.0340 0.0251

(3) 60 urop. I + AAPH 0.0218 0.0944 0.0440 0.0353

(4) 90 urop. I + AAPH 0.0247 0.0593 0.0449 0.0384

(5) 120 urop. I + AAPH 0.0254 0.0388 0.0410 0.0370

(6) 180 urop. I + AAPH 0.0275 0.0179 0.0331 0.0328

(7) 240 urop. I + AAPH 0.0284 0.0080 0.0242 0.0263

(8) = 450 urop. I + AAPH 0.0305 0.0011 0.0033 0.0064

(9) Overnight urop. I + AAPH 0.0324 0 0 0

4.3.4 HPLC Analysis of the Reaction between AAPH and Uroporphyrin I in the

Presence of Gallic Acid

The reaction was monitored at 397 nm and by fluorescence spectroscopy. The

results were essentially identical and only the 397 nm traces are presented below.

78

Monitoring at 259 nm showed the gallic acid peak only (Fig. 4.15). After 1 min a major

peak at 5.0 min and a minor peak at 8.1 min were observed. AAPH gave a peak at 1.7

min. No significant changes in peak intensities could be detected from 1 - 120 min of

the reaction (Fig. 4.16 - 4.17). These results are summarized in Table 4.5.

AU

0 .0 0

0 .1 0

0 .2 0

0 .3 0

0 .4 0

0 .5 0

0 .6 0

0 .7 0

0 .8 0

0 .9 0

1 .0 0

M in u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

Gal

lic a

cid

- 1.5

63

Fig. 4.15. HPLC monitored by PDA 259 nm at 1 min in the presence of gallic acid of the reaction between uroporphyrin I and AAPH

AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

M in u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AAP

H -

1.68

6

Uro

porp

hyrin

I - 4

.976

Pea

k at

8 m

inut

es -

8.08

2


AU

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

0 .1 2

0 .1 4

0 .1 6

0 .1 8

0 .2 0

0 .2 2

0 .2 4

Min u te s0 .0 0 2 .0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .00 1 8 .00 2 0 .0 0

AA

PH

- 1.

706

Uro

porp

hyrin

I - 5

.281

Pea

k at

8 m

inut

es -

8.24

0


79

Table 4.5. HPLC separation of products from the reaction of uroporphyrin I (urop. I) and AAPH

in the presence of gallic acid (G. A.) monitored at 397 nm

Time (min) AAPH Abs.

397 nm

Peak R.T. 5.0 – 5.3 min

Abs. 397 nm

Peak R.T. 8.0 – 8.3 min

Abs. 397 nm

(1) 1 urop. I + G. A. + AAPH 0.0418 0.2231 0.0059

(2) 30 urop. I + G. A. + AAPH 0.0424 0.2245 0.0061

(3) 60 urop. I + G. A. + AAPH 0.0428 0.2242 0.0062

(4) 90 urop. I + G. A. + AAPH 0.0435 0.2251 0.0062

(5) 120 urop. I + G. A. + AAPH 0.0443 0.2264 0.0064

4.4 Discussion

The HPLC peak absorbances detected in the reaction between uroporphyrin I

and AAPH in the absence (Fig. 4.18; monitored at 402 nm) and presence of gallic acid

(Fig. 4.19; monitored at 397 nm) are shown.

For comparison, it is interesting to note that the one-electron oxidation product

of metal-free porphyrin is a porphyrin π-cation radical as described by Morehouse K et

al.94 The absorption spectra of the porphyrin during oxidation changes significantly.

Using coproporphyrin III, oxidation by lactoperoxidase and horseradish peroxidase

systems, results in a decrease (by half) of the Soret band and an increase in intensity of

band I accompanied by a shift to longer wavelength (630 nm from 610 nm). Overall

these changes are qualitatively similar, but quantitatively different, to those arising from

aggregation.92

HPLC of reaction between uroporphyrin I and AAPH monitored at 402 nm

80

0.2

0.225

0.25

U)

0 175A

Fig. 4.19. HPLC monitoring of the absorbance at 397 nm versus time (min) in the presence of gallic acid of the reaction between uroporphyrin I and AAPH

HPLC of reaction between uroporphyrin I, gallic acid and AAPH monitored at 397 nm

0

0.05

0.1

0.15

0.2

0.25

0 30 60 90 120 150

Time (minutes)

Abs

orba

nce

at 3

97 n

m (A

U)

AAPH at retention time 1.7 min.Peak at retention time of 5.0 - 5.3 min.Peak at retention time of 8.0 - 8.3 min.

81

The steady-state concentrations varied considerably from coproporphyrin III

(relative peak height: 1000) to uroporphyrin I (18).94 For the case involving the reaction

between uroporphyrin I and AAPH, the concentration of the porphyrin (4.5 μmol/L)

was less than half that (10 μmol/L) mentioned94 for coproporphyrin III. Effectively, this

is equivalent to a dilution of 100 fold. Thus it is unlikely that the UV-visible spectral

changes induced by formation of the π-cation radical of uroporphyrin I would be

observed. However, the suggested fate of this radical is of some interest. It has been

shown that this radical decays via a disproportionation to give the corresponding di-

cation which is a strong electrophile and reacts with water to form a dihydroxy-

derivative.

2H2P 2H2P•+ H2P + H2P2+ H2P(OH)2 H2O

For the reaction of coproporphyrin III, the intermediate that gives rise to an

absorption maximum at 630 nm, was tentatively suggested to be a 5,6-

dihydroxyporphyrin.94

In conclusion, the HPLC study has shown the presence of a major peak and

other smaller peaks in the reaction between uroporphyrin I and AAPH. In the presence

of gallic acid, the formation of end-products was inhibited while uroporphyrin I was

being protected by the antioxidant. The spin trap DMPO has shown to form spin

adducts with AAPH. The ESR experiments have provided evidence to suggest that it is

probable that a free radical reaction has occurred when AAPH reacts with uroporphyrin

I.

82

Chapter 5

A Study of the Reaction Mechanism of the AIOR

Assay by LC-MS

5.1 Introduction

Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique

that combines the separation ability of LC with the sensitive and information rich

detection capability of MS. A recent refinement of the technique incorporates tandem

mass spectrometry (MS/MS) that involves two stages of MS. In the first, ions of a

desired m/z are selected (precursor ions) from the ions produced in the ion source. The

isolated ions are induced to fragment by collision with a neutral gas (collision-induced

dissociation) and the product ions are analyzed by a second MS. Tandem MS/MS is

particularly useful in the analysis of complex mixtures and, since the first stage MS acts

as a filter, it has the advantage that only product ions associated with a particular

precursor ion are observed. This can facilitate the structural determination of the

precursor ion.

It was thought worthwhile to apply this technique to obtain a better

understanding of the reaction mechanism of the AIOR assay. Thus LC-MS/MS was

used to study the reaction between uroporphyrin I and the radical (alkoxyl or/and

alkylperoxyl) from AAPH.

83


Uroporphyrin I (45 μM, 125 μL) was allowed to react with AAPH (583 mM,

19.5 μL). For the LC-MS, aliquots at time = 0, 5, 30, 60, 90, 120 and 180 min were

taken for analysis. A solution (2.25 mM) of gallic acid (C7H6O5, MW = 170.1) was

prepared in 0.075 M pH 7.0 PBS. For LCMS and UV monitoring at 259 nm, gallic acid

solution (6 μL) was injected.

For the reaction between gallic acid and AAPH, 2.25 mM gallic acid (250 μL)

was reacted with 583 mM AAPH (39 μL). For the LC-MS, the reaction mixture (6 μL)

was injected at time = 5, 30, 60 min to monitor the reaction at 259 nm.

For the reaction between gallic acid, uroporphyrin I and AAPH, 583 mM AAPH

(39 μL) was added to a mixture of gallic acid and uroporphyrin I (250 μL) with final

concentrations of 2.25 mM and 45 μM respectively. The reaction mixture (6 μL) was

injected at time = 0, 5, 30, 60, 90, 120 and 180 min.

For the LC-MS, a Waters Xterra MS microbore column, C18, 2.5 μm, 2.1 x 50

mm column, with a flow rate of 0.2 mL/min was used. The LC-MS experiments were

performed with an Agilent 1100 LC/MSD Trap instrument. The LC consisted of

degasser, capillary pump, photodiode array detector (PDA), column compartment,

microarray loading system, and thermo regulator. The PDA was set at 397 and 402 nm

for detection. A mobile phase composed of solvent A with ammonium acetate, 1 mol/L

(pH 5.16) in acetonitrile (9 : 1 v/v) and solvent B with methanol in acetonitrile (9 : 1

v/v) was used with the following gradient program, starting from 0 to 10 min, with a

linear increase in solvent B to 30% followed by a linear increase in solvent B to 50%

from 10 to 12 min. From 12 to 13 min solvent B was held at 50% before a linear

decrease in solvent B to 0% from 13 to 15 min. Solvent B was held at 0% from 15 to 20

min. The total run time was 20 min. The column was maintained at 20oC.

For mass spectrometry (MS), the ion trap mass spectrometer was interfaced via

an atmospheric pressure electrospray ionization (ESI) source in the positive mode.

Nitrogen was used as the desolvation gas at a flow rate of 8 L/min and also as the

84

nebulizing gas at 20 psi. Target mass was set at 831 m/z. The trap parameter for scan

delay was 0 μs and the accumulation time of 300000 μs was used. The multiplier

voltage was 1750 volt and the dynode voltage was 7.0 kV. MS/MS was set on auto

MS(2). Precursor ions included were 831, 803, 819 and 801 m/z. MS/MS fragmentation

amplitude was 1.0 volt. The scan range was 500 to 1000 m/z.

For the mass spectrometry (MS) of gallic acid and the reaction of gallic acid

with AAPH, all conditions were the same as for the uroporphyrin I experiment except

for the interface via atmospheric pressure ESI source operating in the negative mode.

The target mass was set at 170 m/z. For MS/MS fragmentation, auto MS(2) was selected

as was the precursor ion 169 m/z with fragmentation amplitude 1.0 volt.

5.3 Results

5.3.1 LC-MS of the Reaction between Uroporphyrin I and AAPH

LC-MS analysis of uroporphyrin I at time = 0 showed a major peak at retention

time = 7.5 min and a smaller peak at 9.6 min detected at 402 nm (Fig. 5.1). The

extracted ion chromatogram (EIC) showed that the major peak had a positive mass ion

at m/z 831 that corresponded to uroporphyrin I whereas the smaller peak showed an ion

at m/z 803 (Fig. 5.2 and Fig. 5.3).

Fig. 5.1. Chromatograms of uroporphyrin I at 402 nm at time = 0

PDA at 402 nm

85

S IM 0 0 0 9 7 .D : E IC 8 3 1 ±A l l

S IM 0 0 0 9 7 .D : E IC 8 0 3 ±A l l0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 56x 1 0

In te n s.

0 .0

0 .5

1 .0

1 .5

2 .0

4x 1 0

2 4 6 8 1 0 1 2 1 4 1 6 1 8 T i m e [m i n ]

Fig. 5.2. LC-MS of uroporphyrin I (45 μM) in PBS at time = 0

EIC m/z = 831

EIC m/z = 803

611 .6 629 .0 677 .2 711 .3 725 .6 743 .2 759 .8 781 .2

803 .1

825 .1866 .0 888 .8 956 .0 973 .1

+M S , 9 .6m in (#371 )

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

4x10In tens.

600 650 700 750 800 850 900 950 m /z

8 3 1 .3

+ M S , 7 .6 m i n (# 2 8 2 )

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

6x1 0In te n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m /z

Fig. 5.3. Positive mass ions (m/z) of uroporphyrin I (45 μM) in PBS at time = 0. Retention time (RT)

RT = 7.5 min

RT = 9.6 min

After 5 min, two more minor peaks were observed at 9.9 and 11.2 min (Fig. 5.4 -

5.6). LC-MS scans taken from 30 - 180 min all showed four peaks with retention time

of ∼7.5, 9.6, 9.9 and 11.2 min (Fig 5.4). From the EIC, these peaks could be correlated

with positive mass ions at m/z 831, 803, 819 and 801 respectively (Fig. 5.5, 5.6). At

time = 180 min the same peaks were observed, but with significant changes in relative

amounts (Fig. 5.7 - 5.9).

Fig. 5.4. Chromatograms of the reaction between uroporphyrin I and AAPH at 402 nm at time = 5 min

PDA at 402 nm

86

SIM00098.D: EIC 831 ±All




0.0

0.5

1.0

6x10Intens.

0

2

4

4x10

0.0

0.5

1.0

1.54x10

0

1

2

34x10

2 4 6 8 10 12 14 16 18 Time [min]

Fig. 5.5. LC-MS of the reaction between uroporphyrin I and AAPH at time = 5 min

EIC m/z = 831

EIC m/z = 803

EIC m/z = 819

EIC m/z = 801

8 3 1 . 3

8 5 3 . 0

+ M S , 7 . 5 m i n (# 3 1 6 )

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

6x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

6 4 2 . 7 6 6 2 . 4 6 9 6 . 7 7 3 0 . 4 7 4 6 .2 7 8 5 .0

8 0 3 . 1

8 2 4 . 9 8 4 6 . 9 8 6 5 . 0 9 4 6 . 1 9 8 9 .0

+ M S , 9 . 6 m i n (# 4 0 2 )

0

1

2

3

4x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

6 0 5 . 0 6 2 2 . 4 6 4 0 . 8 6 8 7 . 2 7 1 0 . 1 7 2 5 . 3 7 6 2 . 0 7 8 7 . 2

8 0 3 . 0

8 1 9 . 0

8 5 4 . 0 8 9 4 . 2 9 1 6 . 2 9 4 5 . 8 9 8 0 . 7

+ M S , 9 . 9 m i n (# 4 1 6 )

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

6 0 7 . 7 6 3 0 . 2 6 4 6 . 2 6 7 4 . 3 7 0 7 . 0 7 3 5 . 1 7 5 4 . 3

8 0 1 . 1

8 2 2 .98 4 5 . 0 8 6 6 . 9 8 8 3 . 7 9 2 4 .4 9 4 2 . 8 9 6 0 . 0

+ M S , 1 1 . 2 m i n (# 4 6 1 )

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

4x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

Fig. 5.6. Positive mass ions (m/z) of the reaction between uroporphyrin I and AAPH at time = 5 min. Retention time (RT)

RT = 7.5 min

RT = 9.6 min

RT = 9.9 min

RT = 11.2 min

87

Fig. 5.7. Chromatograms of the reaction between uroporphyrin I and AAPH at 402 nm at time = 180 min

PDA at 402 nm





0

2

4

64x10

Intens.

0

2

4

4x10

0

2

4

6

84x10

0

2

4

6

4x10

2 4 6 8 10 12 14 16 18 Time [min]

Fig. 5.8. LC-MS of the reaction between uroporphyrin I and AAPH at time = 180 min

EIC m/z = 831

EIC m/z = 803

EIC m/z = 819

EIC m/z = 801

8 3 1 . 3

8 5 2 . 9

+ M S , 7 . 5 m i n (# 2 6 2 )

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

5x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

8 0 3 . 2

8 2 5 . 18 4 1 . 0

+ M S , 9 . 5 m i n (# 3 2 8 )

0

1

2

3

4

5

4x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

8 0 3 . 1

8 1 9 . 2

+ M S , 9 . 7 m i n (# 3 3 9 )

0 . 0

0 . 2

0 . 4

0 . 6

0 . 8

1 . 0

5x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

7 7 5 . 0

8 0 1 . 2

8 2 3 . 0 8 4 5 . 0

+ M S , 1 1 . 0 m i n (# 3 8 5 )

0

2

4

6

4x 1 0I n t e n s.

6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 m / z

Fig. 5.9. Positive mass ions (m/z) of the reaction between uroporphyrin I and AAPH at time = 180 min. Retention time (RT)

RT = 7.5 min

RT = 9.5 min

RT = 9.7 min

RT = 11.0 min

88

Table 5.1 shows a summary of all the results at time = 0, 5 30, 60, 90, 120 and

180 min. Mass ions (m/z) and the intensity for the mass ions expressed in (%) are shown

for the peaks detected according to their retention time (RT).

Table 5.1. PDA at 402 nm, EIC (m/z) and mass ion intensity (%) of the HPLC peaks detected

between the AAPH and uroporphyrin I reaction. Retention time (RT). Extracted ion

chromatograms (EIC)

RT ∼7.5 min

RT ∼9.6 min

RT ∼9.9 min

RT ∼11.2 min

Event

abs 402 m/z (%)

0.332 831 99.0

0.010 803 1.0

- - -

- - -

uroporphyrin I

abs 402 m/z (%)

0.268 831 94.4

0.010 803 2.5

0.007 819 0.9

0.003 801 2.2

uroporphyrin I + AAPH at time = 5 min

abs 402 m/z (%)

0.182 831 69.7

0.022 803 8.7

0.051 819 10.3

0.009 801 11.3


abs 402 m/z (%)

0.120 831 51.2

0.028 803 12.7

0.071 819 17.2

0.012 801 18.9


abs 402 m/z (%)

0.084 831 39.8

0.028 803 15.7

0.074 819 20.1

0.014 801 24.4


abs 402 m/z (%)

0.062 831 32.4

0.027 803 17.7

0.070 819 22.2

0.015 801 27.6


abs 402 m/z (%)

0.037 831 24.6

0.023 803 19.4

0.059 819 24.8

0.014 801 31.3


5.3.2 LC-MS of the Reaction between Gallic Acid and AAPH

The LC-MS of gallic acid showed a peak with retention time at 0.8 min and an

absorbance maximum at 259 nm (Fig. 5.10). The extracted ion chromatogram (EIC)

89

showed negative mass ions at m/z 169 and 125 (Fig. 5.11), representing the loss of CO2.

Another ion at m/z 141 probably arises by loss of 28 amu (CO) from the molecular ion

(Fig. 5.12). LC-MS of the reaction mixture of gallic acid with AAPH after 30 min

showed little change (Fig. 5.13 - 5.15).

Fig. 5.10. Chromatograms of gallic acid at 259 nm at time = 0

PDA at 259

Fig. 5.11. LC-MS of gallic acid at time = 0


SIM00114.D: EIC 169 ±All0.0

0.5

1.0

1.5

2.0

4x10Intens.

0.00

0.25

0.50

0.75

1.00

1.25

1.505x10

2 4 6 8 10 12 14 16 18 Time [min]

EIC m/z = 125

EIC m/z = 169

Fig. 5.12. Negative mass ions (m/z) of gallic acid at time = 0. Retention time (RT)

125.0 141.0 156.9

168.9

178.8 194.8216.8

232.7

-MS, 0.8min (#29)

0.00

0.25

0.50

0.75

1.00

1.25

5x10Intens.

100 120 140 160 180 200 220 240 m/z

RT = 0.8 min

Fig. 5.13. Chromatograms of the reaction between gallic acid and AAPH at time = 30 min

PDA at 259 nm

90

Fig. 5.14. LC-MS of the reaction between gallic acid and AAPH at time = 30 min

EIC m/z = 125

EIC m/z = 169

124.8

168.7

-MS, 0.8min (#30)

0

2

4

6

5x10Intens.

100 120 140 160 180 200 220 240 m/z

Fig. 5.15. Negative mass ions (m/z) of the reaction between gallic acid and AAPH at time = 30 min. Retention time (RT)

RT = 0.8 min

5.3.3 LC-MS in the Presence of Gallic Acid of the Reaction between AAPH and

Uroporphyrin I

The progress of this reaction was followed by LC-MS monitoring at 259 nm

(gallic acid) and 397 nm (uroporphyrin I). The chromatogram obtained at 259 nm

showed a peak for gallic acid at 0.8 min. This was correlated with positive mass ions of

gallic acid at m/z 213, 196 and 175 (see Discussion 5.4 for an explanation). The

chromatogram obtained at 397 nm showed uroporphyrin I at 9.0 min accompanied by

two other compounds at 10.7 and 12.2 min (Fig. 5.16). The major peak at 9.0 min was

due to uroporphyrin I (m/z 831), whereas the compounds at 10.7 and 12.2 min

correlated with m/z 803 and 801 respectively (Fig. 5.17 - 5.18).

91

min0 2 4 6 8 10 12 14 16 18

mAU

0

500

1000

1500

2000

DAD1 A, Sig=259,4 Ref=360,100 (SIMON\SIM00124.D)

0.6

48 0

.768

1.0

29 1

.214

min0 2 4 6 8 10 12 14 16 18

mAU

0

100

200

300

400

DAD1 B, Sig=397,4 Ref=360,100 (SIMON\SIM00124.D)

0.7

26 0

.885

9.0

06

9.6

76

10.

695

12.

169

PDA at 259 nm

PDA at 397 nm

Fig. 5.16. Chromatograms of the reaction between uroporphyrin I, gallic acid and AAPH at 259 and 397 nm at time = 5 min

1 SIM 00124.D: EIC 175 ±Al l

SIM 00124.D: EIC 831 ±Al l

SIM 00124.D: EIC 803 ±Al l

SIM 00124.D: EIC 801 ±Al l

0 .0

0.5

1.0

1.5

2.0

5x10Intens.

0.00

0.25

0.50

0.75

1.00

1.25

6x10

0.0

0.5

1.0

1.5

2.0

2.5

3.04x10

0.0

0.5

1.0

1.5

4x10

2 4 6 8 10 12 14 16 18 T im e [m in]

EIC m/z = 175

EIC m/z = 831

EIC m/z = 803

EIC m/z = 801

Fig. 5.17. LC-MS of the reaction between uroporphyrin I, gallic acid and AAPH at time = 5 min

1 7 4 .6

2 1 2 .6

2 8 2 .63 1 6 .6 3 4 6 .4

4 2 0 .14 5 2 .5 4 8 9 .8

5 8 8 .3 6 2 8 .3 6 7 4 .0 7 0 8 .3 7 4 6 .28 0 4 .38 3 0 .3

+ M S , 0 .7 m i n (# 3 8 )

0

1

2

3

5x 1 0In te n s.

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 m /z

8 3 1 .2

+ M S , 9 .0 m i n (# 6 1 4 )

0 .0

0 .5

1 .0

1 .5

6x 1 0In te n s.

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 m /z

1 9 8 .9

2 3 3 .8 2 6 0 .8 2 8 7 .8 3 4 1 .84 4 0 .7 4 7 6 .2 5 2 4 .6 5 8 4 .6

6 4 3 .2 7 0 2 .0 7 5 0 .5

8 0 3 .0

+ M S , 1 0 .7 m i n (# 7 3 5 )

0

1

2

3

4x 1 0In te n s.

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 m /z

1 9 8 .8

2 3 3 . 82 6 0 .8 3 1 0 .8

3 3 9 . 9

3 9 3 . 14 3 5 . 0

4 6 4 . 65 5 4 .9 5 8 6 . 0 6 1 0 .9 6 6 2 .4 6 9 0 . 9 7 5 6 . 0

8 0 0 . 9

8 2 3 .0 8 8 8 .3

+ M S , 1 2 . 2 m i n (# 8 3 6 )

0 .0

0 .5

1 .0

1 .5

2 .0

4x 1 0In t e n s.

2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 m / z

RT = 0.8 min

RT = 9.0 min

RT = 10.7 min

RT = 12.2 min

Fig. 5.18. Positive mass ions (m/z) of the reaction between uroporphyrin I, gallic acid and AAPH at time = 5 min. Retention time (RT)

92

Table 5.2 shows a summary of all the results at time = 0, 5, 30, 60, 90, 120 and

180 min. Mass ions (m/z) and the intensity for the mass ions expressed in (%) are shown

for the peaks detected according to their retention time (RT). The retention time of the

peaks have shifted comparing to Table 5.1. The addition of gallic acid to the reaction

mixture and a batch to batch variation of the mobile phase may be the cause of the shift

in the retention time of the peaks.

Table 5.2. PDA at 259 and 397 nm, EIC (m/z) and mass ion intensity (%) of the HPLC peaks

detected in the presence of gallic acid between the AAPH and uroporphyrin I reaction. Retention

time (RT). Extracted ion chromatogram (EIC)

RT ∼0.8 min

RT ∼9.0 min

RT ∼10.7 min

RT ∼12.2 min

Event

abs 259 abs 397 m/z (%)

2.490 -

175, 213 16.8

0.559 831 81.0

0.016 803 1.3

0.002 801 0.8

uroporphyrin I and gallic acid

abs 259 abs 397 m/z (%)

2.350 -

175, 213 9.5

0.490 831 88.0

0.014 803 1.4

0.002 801 1.1

uroporphyrin I and gallic acid + AAPH at time = 5 min

abs 259 abs 397 m/z (%)

2.346 -

175, 213 6.8

0.482 831 90.8

0.013 803 1.4

0.002 801 1.0


abs 259 abs 397 m/z (%)

2.334 -

175, 213 3.9

0.480 831 93.2

0.013 803 1.7

0.002 801 1.1


abs 259 abs 397 m/z (%)

2.333 -

175, 213 2.7

0.479 831 93.9

0.014 803 2.0

0.002 801 1.4


abs 259 abs 397 m/z (%)

2.319 -

175, 213 2.0

0.479 831 93.0

0.013 803 3.0

0.002 801 2.0


abs 259 abs 397 m/z (%)

2.307 -

175, 213 0.8

0.485 831 92.8

0.014 803 3.6

0.002 801 2.8


93

5.3.4 MS/MS of Ions Detected in the Reaction between AAPH and Uroporphyrin

I

In an attempt to identify the products generated in this reaction, MS/MS spectra

were obtained. MS/MS fragmentation of the mass ion at m/z 831 (Fig. 5.19), 803 (Fig.

5.20), 819 (Fig. 5.21) and 801 (Fig. 5.22) are presented below.

Fig. 5.19. MS/MS of the positive mass ion m/z 831 of the reaction between uroporphyrin I and AAPH.

831.3

853.0

+MS, 7.4min (#216)

0

2

4

6

4x10Intens.

500 550 600 650 700 750 800 850 900 950 m/z

518.1 603.9 694.0

727.1

767.1

785.1 813.0831.0

+MS2(831.2), 7.3min (#215)

0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.

500 550 600 650 700 750 800 850 900 950 m/z

781.1

803.2

825.0

+MS, 9.4min (#272)

0

1

2

3

4x10Intens.

500 550 600 650 700 750 800 850 900 950 m/z

520.8 591.1 628.7 667.2 698.9

726.0

739.1

773.0

785.1

802.9

+MS2(803.2), 9.4min (#273)

0

2000

4000

6000

Intens.

500 550 600 650 700 750 800 850 900 950 m/z


94

803.1

819.2

840.9

+MS, 9.7min (#281)

0

1

2

3

4

5

4x10Intens.

500 550 600 650 700 750 800 850 900 950 m/z

654.8 685.0

729.0

743.1

757.1

773.0 802.0

+MS2(819.2), 9.7min (#280)

0.00

0.25

0.50

0.75

1.00

1.254x10

Intens.

500 550 600 650 700 750 800 850 900 950 m/z


775.1

801.2

823.0845.0

+MS, 11.0min (#316)

0

1

2

3

4

54x10

Intens.

500 550 600 650 700 750 800 850 900 950 m/z

617.9 669.0 691.9

709.0 737.1

755.0

773.1

+MS2(801.2), 11.0min (#317)

0.0

0.5

1.0

1.5

4x10Intens.

500 550 600 650 700 750 800 850 900 950 m/z


95

5.4 Discussion

As mentioned previously, uroporphyrin I forms molecular complexes through

interaction of the porphyrin π-electron core with other π-electron systems. These

complexes are manifested by small shifts in the visible spectra, a drop in the intensity of

the Soret band and a decrease in the total intensity of the emission spectra by a factor of

between two and five. Mauzerall92 observed the formation of 1:1 and 1:2 complexes

between uroporphyrin I and addend. This is of some significance in the case of the

porphyrin : AAPH complex since it allows formation of the radicals in essentially an

organic environment and in close juxtaposition to the porphyrin. This reaction slowly

leads to the formation of three end-products of uroporphyrin I.

A plot of the ion intensity (%) of the mass ions (m/z) versus time at 0, 5, 30, 60,

90 120 and 180 min of the reaction between uroporphyrin I and AAPH is shown in Fig.

5.23. The result indicates that the ion intensity (%) of the mass ion m/z 831 diminished

while the mass ions m/z 803, 801 and 819 increased with time during the reaction.

Uro I with AAPH

0 30 60 90 120 150 180 2100

25

50

75

100

125m/z 831m/z 803m/z 801m/z 819

Time

EIC

%

Fig. 5.23. Ion intensity (%) of extracted ion chromatogram (EIC) versus time (min) of AAPH and uroporphyrin I

96

A similar plot for the reaction with the addition of gallic acid is shown in Fig.

5.24. The ion intensity (%) of the mass ion m/z 831 remained high while the other mass

ions m/z 803 and 801 remained constant. The gallic acid mass ion m/z 175 diminished

with time. This clearly shows that as expected gallic acid protects uroporphyrin I from

the radical species generated from AAPH.

Uro I, Gallic acid with AAPH

0 30 60 90 120 150 180 2100

25

50

75

100

125m/z 831m/z 803m/z 801m/z 175

Time

EIC

%

Fig. 5.24. Ion intensity (%) of extracted ion chromatogram (EIC) versus time (min) of AAPH, gallic acid and uroporphyrin I

The positive ion mass of gallic acid (MW = 170) obtained in the electrospray

ionization (ESI) mode contained some unusual peaks at m/z 213, 196 and 175. It is well

known that some analytes are cationized by sodium under these conditions.95 This

results in replacement of protons with Na+ and then proton addition or addition of some

combination of protons and sodium ions. Thus the ion m/z 213 arises from an ion

containing two Na+. The loss of OH leads to m/z 196 and this ion can further lose Na+ to

generate an ion at m/z 175.

In the presence of AAPH, uroporphyrin I m/z 831 was converted into three

products that showed the highest mass peaks at m/z 819, 803 and 801. It is noteworthy

that the compound showing m/z 803 was a minor contaminant of uroporphyrin I and

probably an oxidation product of the porphyrin. Application of the MS/MS technique

97

allowed the suite of fragment ions from each of these compounds to be observed. This

provided a starting point from which tentative structures for the three degradation

compounds could be derived (Fig. 5.25).

N N

N N

H

H

P

A

A

P

A

PA

P

m/z 831

m/z 801

-H2O m/z 813

m/z 785 m/z 767

-HCO2H

m/z 819

m/z 802 m/z 757

m/z 773

-CO2H

m/z 743

-CH2CO2H

m/z 729

-CO

m/z 803

-H2O

-CH2O

m/z 785

m/z 773m/z 726

-CH2CO2H -CO m/z 773

m/z 755-HCO2H

m/z 709m/z 737

-H2O

m/z 787-CO2

m/z 727

-CH2CO2H2

UROPORPHYRIN I

-HCO2H

-HCO2H

-HCO2H

-HOA = CH2CO2HP = CH2CH2CO2H

Fig. 5.25. Summary of the proposed MS/MS fragmentation of the mass ions observed and identified

Examples of free radical reactions that proceed by attack on metal-free

porphyrins are known.96, 97 These reactions lead to the introduction of a meso-oxygen

function. Thus, homolysis of benzoyl peroxide generates the benzoyloxy radical that

reacts with a porphyrin to afford mainly the monobenzoyloxy derivative together with a

small amount of the disubstituted derivative (Fig. 5.26).

98

N N

N N

H

H

P

A

A

P

A

PA

P

PORPHYRIN

56

N NH

P

A

A

P

OHOH

5,6-DIHYDROXYPORPHYRIN

N NH

P

A

A

P

O

OPh

(PhCO.O)2(2OH)

N NH

P

A

A

P

O

RO

BENZOYLOXYPORPHYRIN

AAPH

N NH

P

A

A

P

O

OXOPHLORIN PEROXYPORPHYRIN Fig. 5.26. Porphyrin oxidation scheme

A similar reaction with the alkoxyl or/and alkylperoxyl radical from AAPH and

uroporphyrin I could generate the peroxyl species that could decompose to give the

corresponding oxophlorin. However the mass spectra of these oxoporphyrins generally

show the molecular ion as the base peak.98 Considering the case of uroporphyrin I with

a molecular weight of 831 amu, the ion due to the keto derivative should be seen at m/z

847 (Fig. 5.27). No such peak was observed. It has been suggested by Morehouse et al94

that one-electron oxidation of porphyrins can lead to radical cations that

disproportionate to provide two-electron oxidation intermediates.94 These are highly

99

electrophilic species that can react with water. In fact, one of these compounds has been

assigned the tentative structure of a 5,6-dihydroxyporphyrin derivative (Fig. 5.26). Such

a compound would have a molecular weight of 865 and it would be expected to be

unstable under mass spectral conditions. The presence of a dihydroxy moiety could

allow lactone formation (m/z 847) with subsequent loss of CO2 to generate an ion at m/z

803. A loss of the equivalent of formic acid from the dihydroxyporphyrin, a radical

reaction that has precedents in porphyrin chemistry, would give a compound with an ion

at m/z 819. Loss of water would result in an ion at m/z 801.99

N N

N N

H

H

A P

A

PA

P

HO2CHO2C

N NHA P

HO2CHO2C

HOHO

UROPORPHYRIN I m/z 831

M. W. 865

N NHA P

HO2C

M. W. 847

HO2C

CO2

N NHA P

HOO

O

HCO2H

m/z 801

N NHA P

HO2CHO

HO

m/z 819H2O

A = CH2CO2HP = CH2CH2CO2H

OH

N NHA P

HO2COH

m/z 803

(Putative)

(Putative)

Fig. 5.27. Proposed structure of the mass ions identified and those putatively involved in MS/MS

100

The proposed reaction mechanism (Fig. 5.27) is hypothetical at this stage and

the evidence for the structures of the end-products is incomplete. Further experiments

would be required to isolate and characterize the compounds generated in the reaction

between uroporphyrin I and AAPH.

101

Chapter 6

Antioxidant Capacity Measurement of Individual

Compounds by the AIOR Method

6.1 Introduction

There have been many attempts to determine if antioxidant supplements, for

example vitamins and flavonoids, are beneficial in reducing oxidative stress. Generally

the results have been ambiguous and discouraging, despite antioxidants having been

found to be effective in vitro.69-73, 100 Two important points appear to have been

overlooked in the in vivo studies. The quality and the quantity of the antioxidants used

in these studies have not been adequately evaluated. In the next two chapters, the two

ingredients necessary for a successful investigation of oxidative stress are explored.

In terms of quality, in most cases, only a few selected vitamins or antioxidants

were measured to correlate with an outcome. The overall total antioxidant capacity was

not always measured. In those cases, where the total antioxidant capacity was measured,

the methods are now known to have been misleading. In the cases of epidemiological

studies, the amount of antioxidant supplements taken was presumed from the dietary

history.

In terms of quantity, the supplementation of vitamins or antioxidants in many

instances did not reach a level high enough to raise the total antioxidant capacity. For

example, the concentration of vitamin E at physiological level is 12 - 42 µmol/L and

even after supplementation may increase by 1.5 to 2 fold. However, to raise the total

102

antioxidant capacity to a significant level, the vitamin E level would have to be

increased by about 30 to 50 fold if only one antioxidant was taken. The slight increase

in concentration of antioxidant compounds at physiological concentration after

supplementation, although measured, does not necessarily mean an effective total

antioxidant capacity in vivo.101, 102 In many studies, the combined supplement

concentration is still too low to significantly contribute to an increase in the overall total


The aim of the study described in this chapter was firstly, to study at what

concentration antioxidants, for example uric acid, vitamin C, A and E, thiols, albumin,

total protein, polyphenols and caffeine, show an effective antioxidant capacity;

secondly, to investigate if antioxidants display a linear relationship between

concentration and antioxidant capacity; and thirdly, to determine if there is an additive

effect of antioxidant capacity of antioxidants that can give a meaningful result to the

total antioxidant capacity measurement.

The AIOR method was used to measure and examine the total antioxidant

capacity in mmol/L Trolox equivalents (mM TE). A brief discussion of the selected

antioxidants with attention to their physiological concentration in vivo is presented.

6.1.1 Uric Acid

Uric acid is an important antioxidant in the extracellular compartment. It is a

metabolic end-product produced via both de novo synthesis and salvage pathway of

purine metabolism of adenine, guanine, hypoxanthine and xanthine.103 The overall

production rate of urate is 5 - 6 mmol/day on a normal diet with 3 - 4 mmol derived

from the de novo route and 1 - 2 mmol from the diet. About one third of urate is

secreted into the gastrointestinal tract where it is destroyed by bacterial uricases. The

kidney excretes the remaining two thirds. The human adult uric acid reference ranges

are 200 - 500 μmol/L for males and 130 - 460 μmol/L for females.104

103

N

NN

NO

H

H

H

O

HO

Fig. 6.1. Uric acid

6.1.2 Vitamin C

The adult human vitamin C (Fig. 6.2) reference range is 23 - 85 μmol/L.104 The

concentrations of vitamin C in fruits, vegetables, drinks and supplements in diets cover

a wide range and the effective antioxidant capacity at any time is variable. Consumption

of vitamin C via supplement and diet is not a good indication of antioxidant capacity.

O

C

CH2OH

OHH

O

OHOH

Fig. 6.2. Vitamin C

6.1.3 Vitamin A

Vitamin A, also known as retinol (Fig. 6.3), and its biologically active

derivatives such as retinoic acid are collectively referred to as retinoids. The reference

range of vitamin A for the human adult is around 1 - 3 μmol/L.104 Vitamin A has been

investigated as one of the antioxidants at low concentrations in vivo. However, in high

concentration, vitamin A was found to be toxic. The main food sources of vitamin A are

liver, fish, whole milk, cheese, yoghurt, butter, and margarine supplemented with

104

vitamin A. A major precursor of vitamin A is β-carotene, the most abundant carotenoid.

It is estimated that it takes 12 μg of β-carotene to produce 1 μg of vitamin A in vivo.103

OH

C HCH

CH

33

3

Fig. 6.3. Vitamin A

6.1.4 Vitamin E

The reference range of vitamin E (Fig. 6.4) in an adult human is 12 - 42

μmol/L.104 Vitamin E is generally used to describe four tocopherols (α, β, γ and δ) and

four tocotrienols. In vivo, levels of α-tocopherol are about 15 - 40 μmol/L and γ-

tocopherol 3 - 5 μmol/L. The main food sources of vitamin E are nuts, wheat germ,

vegetable oils, green-leafy vegetables, milk, egg yolk and fish-liver oils.103 The U.S.

recommended daily allowance for vitamin E is 30 IU and higher intakes have not been

found to have toxic effects.

R

H

C H

O

O

RH

CH H CH HC H

C HC

1

2

33

3 33

3

Fig. 6.4. Vitamin E

6.1.5 Thiols

Thiols are found mainly in red blood cells. There are protein bound thiols,

oxidised thiols, mixed disulphide thiols and reduced thiols.105-110 Homocysteine and

glutathione (Fig. 6.5) are two of the best known thiols in blood. Less well known thiols

105

include cysteine, cysteinylglycine (Fig. 6.5), cystathione, methionine, N-acetylcysteine,

γ-glutamylcysteine and the oxidised form of all these compounds. Thiols in the reduced

form are not very stable and are easily oxidised to the more stable disulphide (Fig. 6.5).

Reduced glutathione disappears within minutes in cell-free plasma because of oxidation.

In other words, thiols exist in different forms in whole blood and plasma. For example,

glutathione concentration in whole blood or red blood cell lysate is in the mmol range,

whereas in plasma it is in the μmol range.

R1⎯SH = Glutathione [R] Reduced form R1⎯SS⎯R1 = Glutathione [O] Oxidized form

C H

NH2

CO2H

C H2C H2

C

O

NH

C H

C

O

NH C H2CO

2H

C H2

SH

R1

R2⎯SH = Homocysteine [R] Reduced form R2⎯SS⎯R2 = Homocystine [O] Oxidized form

C H

NH2

CO2H

C H2C H2

SH

R2

R4⎯SH = Cysteine [R] Reduced form R4⎯SS⎯R4 = Cystine [O] Oxidized form

C H

NH 2

CO2H

C H2SH

R4

R3⎯SH = Cysteinylgylcine [R] Reduced form R3⎯SS⎯R3 = Cystinylglycine [O] Oxidized form

C H

NH2

C H2C H2

SH

C

O

NH C H2CO

2H

R3

Fig. 6.5. Molecular structure of thiols

106

6.1.6 Albumin

The adult human albumin reference range is around 30 - 50 g/L.104 Albumin is

the predominant contributor to the osmotic pressure in plasma and hence plays an

important role in controlling the distribution of water between the intra- and

extracellular compartments. It is also an important carrier protein for calcium,

magnesium, thyroid hormones, unconjugated bilirubin, fatty acids and some drugs.103

Less well known is its contribution as an antioxidant.

6.1.7 Total Protein

There are more than a hundred different proteins in plasma, the majority of

which are enzymes and polypeptide hormones.103 The structural heterogeneity of

plasma proteins is matched by their functional diversity. In the experiment to be

described, human serum standard protein from Behring with known concentration (g/L)

of all the proteins in solution was used to measure the mean antioxidant capacity in mM

Trolox equivalents. The effect of total protein concentration on total antioxidant

capacity is still being discussed. To investigate the antioxidant capacity of the non-

protein fraction of serum, deproteinized serum samples can be used to assess total


6.1.8 Polyphenols

A major class of natural products from plants are the polyphenols. The

concentration of these compounds in vivo is determined by intake, absorption,

distribution and metabolism.111 Much research has focused on the antimutagenic,

anticarcinogenic and antioxidant capacity of this class of compounds.26, 112 Chemically

polyphenols may be subdivided into two categories; the flavonoid polyphenols such as

rutin, catechin, quercetin, myricetin and apigenin and the non-flavonoid polyphenols

107

such as hydroquinone, gallic acid and chlorogenic acid. The chemical structures of

polyphenols play an important part in determining the potency of their antioxidant

capacity.

6.1.9 Caffeine

The antioxidant capacity effect of caffeine (Fig. 6.6) has been the subject of

some discussion. In this study, caffeine has been tested for antioxidant capacity.

HC 3

HC 3

H C3

N

NN

NO

O

Fig. 6.6. Caffeine


All chemicals, except total protein, were obtained from Sigma-Aldrich Pty. Ltd.

(NSW, Australia). Human serum standard protein was obtained from Behring.

Uric acid (0.0285 g) was dissolved in 100 mL of phosphate buffer and

concentrations at 424, 848 and 1696 μmol/L were prepared. Caffeine (0.0468 g) was

dissolved in 25 mL of phosphate buffer.

Vitamin C (0.1417 g) was dissolved in 500 mL of deionized water and

concentrations at 2.65, 5.30 and 10.60 mmol/L were prepared.

108

Vitamin A (0.02224 g) and vitamin E (0.01643 g) were dissolved in 10 mL of

isopropanol and concentrations at 1940, 3880, and 5820 μmol/L for vitamin A and

concentrations of 950, 1900 and 3800 μmol/L for vitamin E were obtained.

The reduced and oxidized form of homocysteine, glutathione, cysteinylglycine

and cysteine at concentration of 5.0 mM were prepared.

Rutin, quercetin, (−)-epicatechin, (±)-catechin and gallic acid were dissolved in

1 mL of ethanol. Two dilutions were made with ethanol to give concentration of 0.50

and 0.25 mM for each of the compounds.

Albumin was dissolved in phosphate buffer to give concentrations of 20 and 40

g/L. The total protein concentration of human serum standard protein was 64.8 g/L

(Table 6.1). Albumin was the major contributor with 41.5 g/L followed by IgG with

11.0 g/L.

Table 6.1. Human serum standard protein from Behring (g/L)

Human Serum Standard Protein (Behring) g / L

IgG 11.0

IgA 2.2

IgM 1.1

C3 0.65

C4 0.17

transferrin 3.0

albumin 41.5

α1-antitrypsin 2.0

α2-macroglobulin 1.6

haptoglobin 1.7

acid α1-glycoprotein 0.8

pre-albumin 0.3

α1-antichymotrypsin 0.4

haemopexin 0.4

caeruloplasmin 0.18

retinol-binding protein 0.04

Ig κ light chain 2.7

Ig λ light chain 1.5

total 64.8

109

6.3 Results

6.3.1 Uric Acid

Fig. 6.7 shows a diagram of the AIOR assay fluorescence intensity versus time

of uric acid at different concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Blank

STD 0.5 mM

STD 1.0 mM

STD 2.0 mM

Uric Acid 424 μM Uric Acid 1696 μM

Uric Acid 848 μM

Fig. 6.7. Trolox standard and uric acid by the AIOR assay

The mean antioxidant capacities for uric acid at concentrations of 424, 848, 1696

μM were 0.6, 1.2, and 2.4 mmol/L Trolox equivalents respectively as shown in Table

6.2. The urate concentration was directly proportional to the antioxidant capacity in

mmol/L Trolox equivalents. The correlation between antioxidant capacity and

concentration was good and followed a linear relationship. The coefficient of

determination R2 = 1.00, p < 0.004. At the upper physiological range for male and

female of 420 and 340 μmol/L, the mean antioxidant capacity for uric acid was

estimated to be at 0.59 and 0.48 mmol/L Trolox equivalents respectively.

110

Table 6.2. Correlation of concentrations with antioxidant capacity of uric acid in mmol/L Trolox

equivalents

Uric acid Concentration Antioxidant capacity (mM TE ± SD) CV (%) n

424 μM 0.6 ± 0.1 12.3 12

848 μM 1.2 ± 0.1 7.1 12

1696 μM 2.4 ± 0.1 4.7 12

6.3.2 Vitamin C


of vitamin C at different concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Vit C 10.6 mM Blank

STD 0.5 mM

Vit C 2.65 mM

STD 1.0 mM

Vit C 5.30 mM

STD 2.0 mM

Fig. 6.8. Trolox standard and vitamin C by the AIOR assay

The mean antioxidant capacities for vitamin C at concentrations of 2.65, 5.30

and 10.6 mM were 0.9, 1.8 and 4.0 mM Trolox equivalents respectively as shown in

Table 6.3. The antioxidant capacity for vitamin C showed a fairly linear relationship

with concentration. The vitamin C concentration at 10.60 mmol/L used for this

experiment is abnormally high when compared with a physiological range of 23 - 80

μmol/L for adults. The purpose was to examine the linearity at high antioxidant

concentration with antioxidant capacity. The measured antioxidant capacity of 4.0 mM

Trolox equivalents was outside the range of the highest standard but only showed a

slight deviation from linearity. Overall, the vitamin C concentration was directly

111

proportional to the antioxidant capacity in mM Trolox equivalents. The correlation

between concentration and antioxidant capacity was good with the coefficient of

determination R2 = 0.99, p < 0.022.

Table 6.3. Correlation of concentrations with antioxidant capacity of vitamin C in mmol/L Trolox

equivalents

Vitamin C concentration Antioxidant Capacity (mM TE ± SD) CV (%) n

2.65 mM 0.9 ± 0.1 12.2 12

5.30 mM 1.8 ± 0.1 8.1 12

10.6 mM 4.0 ± 0.2 4.6 12

6.3.3 Vitamin A


of vitamin A at different concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Blank

STD 0.5 mM

STD 1.0 mM

STD 2.0 mM

Vit A 5820 μM

Vit A 3880 μM

Vit A 1940 μM

Fig. 6.9. Trolox standard and vitamin A by the AIOR assay

The mean antioxidant capacities for vitamin A at the concentrations of 1940,

3880 and 5820 μM were 0.56, 1.11 and 1.41 mM in Trolox equivalents respectively.

The correlation between antioxidant capacity and concentration was acceptable with the

coefficient of determination R2 = 0.97, p < 0.104 (Table 6.4). There was a slight

deviation at the highest concentration, which may be due to matrix effects since the

112

vitamin A samples were dissolved in isopropanol. Overall, the vitamin A concentration

was directly proportional to antioxidant capacity in mM Trolox equivalents.

Table 6.4. Correlation of concentrations with antioxidant capacity of vitamin A in mmol/L Trolox

equivalents

Vitamin A Concentration Antioxidant Capacity (mM TE ± SD) CV (%) n

1940 μM 0.6 ± 0.1 15.6 10

3880 μM 1.1 ± 0.1 6.5 10

5820 μM 1.4 ± 0.1 4.7 10

6.3.4 Vitamin E

Fig. 6.10 shows a diagram of the AIOR assay fluorescence intensity verses time

of vitamin E at different concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Blank

STD 0.5 mM Vit E 952 μM

STD 1.0 mM

Vit E 1905 μM

STD 2.0 mM

Vit E 3810 μM

Fig. 6.10. Trolox standard and vitamin E by the AIOR assay

The mean antioxidant capacities for vitamin E at the concentrations of 952, 1905

and 3810 μM were 0.4, 1.1 and 2.7 mM Trolox equivalents respectively. It was

interesting to compare the antioxidant capacity of vitamin E with the water-soluble

vitamin E derivative Trolox which is commonly used as the standard for total

antioxidant capacity measurement. The correlation between vitamin E concentration and

antioxidant capacity was good with the coefficient of determination R2 = 0.99, p < 0.021

(Table 6.5). There was a slight deviation at the highest concentration, which may be due

113

to matrix effects since the vitamin E samples were also dissolved in isopropanol.

Generally, the vitamin E concentration was directly proportional to antioxidant capacity

in mM Trolox equivalents.

Table 6.5. Correlation of concentrations with antioxidant capacity of vitamin E in mmol/L Trolox

equivalents

Vitamin E Concentration Antioxidant Capacity (mM TE ± SD) CV (%) n

952 μM 0.4 ± 0.1 25.4 10

1905 μM 1.1 ± 0.1 5.8 10

3810 μM 2.7 ± 0.1 3.0 10

6.3.5 Thiols


of the thiols at 5.0 mM in the AIOR assay.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 6.11. Trolox standard (2.0 mM) with thiols in reduced form [R] and oxidised form [O] (5.0 mM)

Blank

Glutathione [O]

STD 2.0 mM

Glutathione [R]

Homocysteine [R]

Cysteinylglycine [R]

Homocystine [O] Cysteine [R]

Cystinylglycine [O] Cystine [O]

The mean antioxidant capacities for glutathione at the concentrations of 0.37,

0.73, 1.46 and 2.93 mM were 0.3, 0.4, 0.6 and 1.3 mM Trolox equivalents respectively.

The correlation between glutathione concentration and antioxidant capacity was good

114

with the coefficient of determination R2 = 0.98, p < 0.009. The antioxidant capacity of

thiols at concentration of 5.0 mM was compared with glutathione and the results in mM

Trolox equivalents are shown in Table 6.6.

Table 6.6. Antioxidant capacity of thiols ( 5.0 mM) in mmol/L Trolox equivalents

Thiols Concentration Antioxidant Capacity (mM TE ± SD) CV (%) n

homocysteine (reduced form) 5.0 mM 1.91 ± 0.12 6.1 9

glutathione (reduced form) 5.0 mM 1.84 ± 0.08 4.4 9

cysteinylglycine (reduced form) 5.0 mM 1.12± 0.25 21.8 9

cysteine (reduced form) 5.0 mM 0.81 ± 0.12 15.1 9

homocystine (oxidized form) 5.0 mM 0.99 ± 0.15 15.5 9

cystinylglycine (oxidized form) 5.0 mM 0.74 ± 0.13 17.2 9

cystine (oxidized form) 5.0 mM 0.35± 0.11 31.7 9

glutathione (oxidized form) 5.0 mM 0.18 ± 0.03 16.6 9

6.3.6 Albumin


of albumin at the different concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

STD 2.0 mM

Blank

STD 0.5 mM

STD 1.0 mM Albumin 40 g/L

Albumin 20 g/L

Fig. 6.12. Trolox standard and albumin by the AIOR assay

115

The mean antioxidant capacities at concentrations of 20 and 40 g/L were 1.2 and

2.4 mM Trolox equivalents respectively (Table 6.7). The antioxidant capacity showed

good correlation with concentration and showed a linear relationship with the

coefficient of determination R2 = 1.00, p < 0.001. The albumin concentration was

directly proportional to antioxidant capacity in mM Trolox equivalents.

Table 6.7. Correlation of concentrations with antioxidant capacity of albumin in mmol/L Trolox

equivalents

Albumin Concentration Antioxidant Capacity (mM TE ± SD) CV (%) n

20 g/L 1.2 ± 0.1 9.4 12

40 g/L 2.4 ± 0.1 2.9 12

6.3.7 Total Protein


of total protein at the different concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

BlankSTD 0.5 mM

STD 1.0 mM

STD 2.0 mM

TP 43.2 g/L

TP 64.8 g/L

Fig. 6.13. Trolox standard and total protein (TP) by the AIOR assay

116

The mean antioxidant capacities for human serum total protein at 32.4, 43.2 and

64.8 g/l were 1.1, 2.0 and 3.3 mM Trolox equivalents respectively (Table 6.8). At the

adult human total protein reference range of 60 - 80 g/L, the antioxidant capacity of

total protein is substantial. The correlation between concentration and antioxidant

capacity was good with the coefficient of determination R2 = 0.99, p < 0.054. The total

protein concentration was directly proportional to the antioxidant capacity in mM

Trolox equivalents.

Table 6.8. Correlation of concentrations with antioxidant capacity of total protein in mmol/L

Trolox equivalents

Total Protein

Concentration Antioxidant Capacity (mM TE ± SD) CV (%) n

32.4 g/L 1.1 ± 0.1 12.4 20

43.2 g/L 2.0 ± 0.2 12.0 20

64.8 g/L 3.3 ± 0.2 4.9 20

6.3.8 Polyphenols

Fig. 6.14 and 6.15 show diagrams of the AIOR assay fluorescence intensity

versus time of five polyphenols quercetin, (−)-epicatechin, (±)-catechin, rutin and gallic

acid at 0.25 and 0.50 mM respectively.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 6.14. Trolox standard (2.0 mM) and polyphenols (0.25 mM) by the AIOR assay

Blank

Gallic acid 0.25 mM

Quercetin 0.25 mM

Rutin 0.25 mM

STD 2.0 mM

(±) Catechin 0.25 mM

(−) Epicatechin 0.25 mM

117

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Blank Quercetin 0.5 mM

(−) Epicatechin 0.5 mM

(±) Catechin 0.5 mM

Rutin 0.5 mM

Gallic acid 0.5 mM

STD 2.0 mM

Fig. 6.15. Trolox standard (2.0 mM) and polyphenols (0.5 mM) by the AIOR assay

Fig. 6.16 shows the fluorescence intensity versus time of different

concentrations of rutin (0.44 mM), quercetin (0.38 mM), (−)-epicatechin (0.44 mM) and

gallic acid (0.57 mM) and a mixture of these polyphenols with the same concentrations.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Blank

STD 2.0 mM

Gallic acid 0.57 mM

Quercetin 0.38 mM

Epicatechin 0.45 mM

Rutin 0.45 mM

Mixture of Rutin (0.45 mM), Quercetin (0.38 mM), Epicatechin (0.45 mM), Gallic acid (0.57 mM)

Fig. 6.16. Trolox standard (2.0 mM) with mixture of four polyphenols and the four individual polyphenols at respective concentration

The antioxidant capacity of five polyphenols, quercetin, (−)-epicatechin, (±)-

catechin, rutin and gallic acid at 0.50 and 0.25 mM are summarised in Tables 6.9 and

6.10 respectively.

118

Table 6.9. Antioxidant capacity of polyphenols (0.50 mM) in mmol/L Trolox equivalents

Polyphenols Concentration Antioxidant Capacity

(mM TE± SD) CV (%) n

quercetin 0.5 mM 4.58 ± 0.24 5.3 12

(−)-epicatechin 0.5 mM 3.69 ± 0.23 6.2 12

(±)-catechin 0.5 mM 3.47 ± 0.19 5.4 12

rutin 0.5 mM 3.39 ± 0.40 11.7 12

gallic acid 0.5 mM 1.22 ± 0.17 14.3 12

Table 6.10. Antioxidant capacity of polyphenols (0.25 mM) in mmol/L Trolox equivalents

Polyphenols Concentration Antioxidant Capacity

(mM TE ± SD) CV (%) n

quercetin 0.25 mM 1.82 ± 0.13 7.3 12

(−)-epicatechin 0.25 mM 1.62 ± 0.13 8.0 12

(±)-catechin 0.25 mM 1.60 ± 0.07 4.6 12

rutin 0.25 mM 1.58 ± 0.09 6.0 12

gallic acid 0.25 mM 0.46 ± 0.07 14.5 9

Adding the antioxidant capacity of the individual polyphenol gave a similar

result to that of the total antioxidant capacity of the mixture (Table 6.11).

Table 6.11. Additive effect of antioxidant capacity of 4 polyphenols in mmol/L Trolox equivalents.

The concentrations are shown in Fig 6.16

Polyphenols Observed

AOC in mM TE

Calculated

AOC in mM TE

rutin 3.79 ± 0.25

quercetin 4.41 ± 0.44

(−)-epicatechin 4.03 ± 0.28

gallic acid 1.75 ± 0.11

calculated antioxidant capacity (AOC) of the 4

polyphenols together - 3.50

observed antioxidant capacity (AOC) of the mixture of

4 polyphenols (12 repeats) 3.93 ± 0.21

119

6.3.9 Caffeine


of caffeine

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Caffeine 9.6 mM

STD 2.0 mM

STD 1.0 mM

Blank

STD 0.5 mM

Fig. 6.17. Trolox standard and caffeine by the AIOR assay

The result of this experiment showed that the mean antioxidant capacity for

caffeine at a high concentration of 9.6 mmol/L was only 0.2 mM Trolox equivalents.

6.4 Discussion

The total antioxidant capacity for a concentration within the adult reference

ranges for males and females of 424 μmol/L of uric acid is measured and reflected

significantly at 0.6 mmol/L Trolox equivalents. Considering that the adult human uric

acid reference ranges are 200 - 500 for males and 130 - 460 μmol/L for females, uric

acid provides a substantial extracellular antioxidant capacity in vivo.

120

Vitamin C at very high concentration showed antioxidant capacity. The

concentration at 2650 μmol/L was shown to have an antioxidant capacity of 0.9 mmol/L

Trolox equivalents. Although it is common to find vitamin C supplemented drinks at

this level, the physiological vitamin C adult reference range is only 23 - 85 μmol/L. At

physiological concentration, the antioxidant capacity of vitamin C is hardly

noticeable.102

Vitamin A at a concentration of 1940 μmo/L is required to provide an

antioxidant capacity of 0.6 mmol/L Trolox equivalents. At physiological concentrations

of 1 - 3 μmol/L, changes in vitamin A concentration will not significantly alter the

extracellular total antioxidant capacity.

Vitamin E at a concentration of 952 μmol/L has an antioxidant capacity of 0.4

mmol/L Trolox equivalents. The adult reference range of vitamin E is 12 - 42 μmol/L.

Vitamin E, even after supplementation, will not improve the total antioxidant capacity

in vivo to a large degree.102 The result also showed that the antioxidant capacity of

vitamin E is about half that of the Trolox standard, a vitamin E derivative. This result

showed that a compound with a similar chemical structure can have a very different


Thiols in the reduced or oxidised form have been shown to possess antioxidant

activity in this study. In comparison, the reduced form of homocysteine was found to

have the highest antioxidant capacity in mM Trolox equivalents. The oxidised form of

homocysteine also showed antioxidant capacity. Homocysteine has been implicated in

oxidative stress disease and is used as a biochemical marker in cardiovascular disease.

Further studies are required to clarify the antioxidant activity and oxidative stress effects

of thiols.

Albumin concentration at 20 and 40 g/L showed antioxidant capacity of 1.2 and

2.4 mmol/L Trolox equivalents. The adult human albumin reference range is around 30

- 50 g/L. The results showed that albumin contributes significantly to total antioxidant

capacity in vivo. The measurement of extracellular antioxidants other than albumin and

proteins should be performed with deproteinized samples.

121

In the total protein experiment, the concentrations of albumin were 20.8, 27.7,

and 41.5 g/L in a human serum with total protein concentrations of 32.4, 43.2 and 64.8

g/L respectively. The measured mean antioxidant capacity was 1.1 mM in Trolox

equivalents at a total protein concentration of 32.4 g/L which had 20.8 g/L of albumin.

The result compared well with the measured mean antioxidant capacity of 1.2 mM in

Trolox equivalents of albumin at a concentration of 20 g/L. The AIOR is able to

compare the contribution of antioxidant capacity from the proteins in the mixture (Fig.

6.18).

Comparison of Antioxidant Capacity ofAlbumin and Total Protein

ALB 20 g/

L

ALB 40 g/

L

TP 32.4

g/L

TP 43.2

g/L

TP 64.8

g/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Albumin (ALB) and Total Protein (TP) concentrations

AO

C in

mM

Tro

lox

Equi

vale

nt

Fig. 6.18. Comparison of antioxidant capacity in mM TE of different concentrations of albumin and total protein

For the five polyphenols examined, quercetin has the highest antioxidant

capacity and gallic acid the lowest. At the same concentration, (−)-epicatechin and (±)-

catechin and rutin all showed different antioxidant capacity. The antioxidant capacity

for (−)-epicatechin was relatively higher than for (±)-catechin. Adding the antioxidant

capacity of the individual polyphenol gave a similar result to that of the total antioxidant

capacity of the mixture (Table 6.11). The results of this experiment showed that the

antioxidant capacity of polyphenols is additive.

The antioxidant capacity of caffeine at a very high concentration of 9600

μmol/L is only 0.2 mM Trolox equivalents. In humans, the mean plasma caffeine

concentrations are 15.5 μmol/L (range 10.3 - 20.6) and 40.7 μmol/L (range 30.9 - 46.4)

after oral ingestion of 120 and 300 mg of caffeine respectively.113

122

In conclusion, these experiments have shown a linear correlation between

concentration and total antioxidant capacity. The contribution to the total antioxidant

capacity in vivo of uric acid, albumin and total protein is substantial. However

individual antioxidants, like polyphenols, thiols, vitamin E and C, have been shown to

be present in a very low concentration at physiological levels. Individually these

antioxidants cannot make a significant contribution to the overall total antioxidant

capacity in vivo. To study antioxidants in oxidative stress, a total measurement of all the

antioxidants in vivo has to be performed. The AIOR method has been shown to be

effective in measuring different types of antioxidants as well as mixtures of

antioxidants. The results have shown that the total antioxidant capacity is additive. The

AIOR has been shown to be capable of measuring total antioxidant capacity.

123

Chapter 7

Total Antioxidant Capacity Measurement of

Complex Mixtures by the AIOR Method

7.1 Introduction

Fruits, vegetables, spices and beverages such as wine, tea and coffee contain

compounds which are potential antioxidants, for example polyphenols and vitamins.114

World-wide attention has now been directed towards the consumption of these

substances to boost antioxidant levels for good health.115-121 The assumption is that a

high intake of these substances will provide a significant change in antioxidant level to

combat oxidative stress. However, there is little evidence to indicate that an effective

antioxidant level has been reached after consumption of these substances in respect to

quality and quantity.122 Total antioxidant capacity has not been measured and the

beneficial effect reported was based on food consumption surveys.123-126 A few studies

have measured total antioxidant capacity but with the less reliable single point assay

such as the Randox total antioxidant status.127

Polyphenols are present in many fruits and vegetables. Plants polyphenols in

complex polymeric and glycosidic forms may not be readily absorbed by the digestive

system after ingestion.26 It is also possible that through the process of fermentation in

wine production, polyphenols are made more readily available for absorption into the

extracellular compartment. Polyphenols in wine such as resveratrol have been

mentioned as the main contributor for good health.23, 27, 28, 128 Previous studies have

shown that the concentration of flavonoids in red wine is about 20 fold more than in

124

white wine, and resveratrol was found mainly in red wine.129 In vitro experiments have

shown resveratrol to be effective in preventing LDL oxidation. An acceptable method

for the measurement of antioxidant capacity of these complex substances is still in

discussion. As a result, a common standard to compare total antioxidant capacity is not

yet available.

Green, black and oolong teas are common beverages in many cultures

worldwide. Green tea is produced by brief exposure to high temperature, just long

enough to deactivate enzymes such as polyphenol oxidase that cause oxidation. This has

the end result of preventing the oxidation of green leaf polyphenols and yielding more

of the polyphenol (−)-epigallocatechin-3-gallate (Fig. 7.1). Black tea is processed by

initial withering of the tea leaves at warm temperature. It is then rolled and allowed to

ferment before final roasting at high temperatures. Polyphenol oxidase catalyzes the

aerobic oxidation of the catechins when the leaf structure is disrupted. The various

quinones produced by the enzymatic oxidation undergo condensation reactions to form

compounds such as theaflavins, epitheaflavic acids, and thearubigens. Oolong tea is

semi-fermented, which means it contains partially oxidized products of polyphenols.

Green, black and oolong teas have been studied by liquid chromatgraphy mass

spectrometry (LC-MS)31 and found to contain polyphenols such as (−)-epicatechin, (−)-

epigallocatechin, (−)-epicatechin gallate, (−)-epigallocatechin-3-gallate, and gallic acid

(Fig. 7.1). Polyphenols are also found in spices such as turmeric.

OH

OH

OHOH

H

O

O

(−)-Epicatechin

OH

OH

OHOH

OH

HO

O

(−)-Epigallocatechin

OH

OHOHOH

C O

Gallic acid

125

OH

OH

OHOH

CO

OH

OH

OH

O

O

(−)-Epicatechin gallate

OH

OH

OHOH

OH

CO

OH

OH

OH

O

O

(−)-Epigallocatechin-3-gallate

Fig. 7.1. Examples of molecular structure of polyphenols

The aim of the experiments in this chapter was to determine the usefulness of

the AIOR method in the quantification of total antioxidant capacity in complex mixtures

such as teas, coffee, grapes, wines, spices and a flavonoid supplement tablet.

Ultimately, this method may be applied to food and beverage samples as well as

biological samples such as serum.


Wine samples were prepared by 1/10 and 1/20 dilutions with PBS. Tea was

weighed and placed in 100 mL of boiling water in glass beakers for 30 minutes. The

final concentration of tea was adjusted by dilution with buffer to a relative weight per

volume of 1.87 mg/mL. Coffee, cocoa and Milo were weighed and dissolved in hot

water (1.87 mg/mL). Three types of grapes, Shiraz, Cabernet, and Merlot were crushed

and the resulting grape juice were diluted 1/20 dilution with PBS to assess the total

antioxidant capacity. Turmeric and curry powder were weighed and extracted with 1

mL of a solution of ethanol in PBS (3:1) at room temperature. The final concentration

of the spices was adjusted with PBS to a relative weight per volume of 1.87 mg/mL of

126

the spice powder. Natural Nutrition bioflavonoid dietary supplement tablets were

obtained from Bullivant’s Natural Health Products Pty. Ltd. Each tablet contains rutin

(500 mg) plus mixed solubilised bioflavonoids (500 mg). A tablet (1.40 g) was crushed

and ground to a fine powder form. A portion of this powder was weighed (10.4 mg) and

extracted with isopropanol : water (1 : 1 : 1 mL). The mixture was centrifuged and the

supernatant separated. The final concentration of the extract was adjusted with buffer to

a relative weight per volume of 1.87 mg/mL of the original weight of the powder.

Further dilution of 1/2, 1/3, 1/5, and 1/7 of this solution was made with PBS to check

the linearity of the AIOR method.

7.3 Results

7.3.1 Teas


of the different types of tea measured.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Green Tea (Twinings)

Red Tea (Twinings)

Red Tea (Gibsons)

Green Tea (China)

Fruit Tea (Twinings)

Green Tea (Tetley)

Fig. 7.2. Comparison of antioxidant capacity of red teas, green teas and a tea infusion by the AIOR assay

127

The total antioxidant capacity of green and black teas was compared by the

AIOR method. The results showed that teas display a wide range of total antioxidant

capacity. Tea infusion was shown to have the least antioxidant capacity when compared

to green and black teas as shown in Table 7.1.

Table 7.1. Antioxidant capacity in mmol/L TE of different types of tea

Tea Brand Antioxidant Capacity in Trolox Equivalents (mM TE ± SD) Concentration CV

(%) n

Butterfly brand Fujian China 3.7 ± 0.3 1.87 mg/mL 6.5 24

Twinings traditional afternoon tea 3.5 ± 0.1 1.87 mg/mL 3.8 24

Gibsons traditional full strength 3.0 ± 0.2 1.87 mg/mL 5.0 24

Twinings green tea and mint 2.8 ± 0.1 1.87 mg/mL 4.8 24

Tetley emperor’s garden green tea and peppermint 2.7 ± 0.1 1.87 mg/mL 5.2 24

Twinings blackcurrant and apple 1.2 ± 0.1 1.87 mg/mL 10.1 24

7.3.2 Coffee, Chocolate Beverages and Green Powder Tea


of coffee, cocoa and other beverages.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

NesCafe Blend 43

Nestle Cocoa

NesCafe Decaffinated

NesCafe Gold

Milo

Japan Green Tea Powder

Fig. 7.3. Comparison of coffee, beverages and green tea powder by the AIOR assay

128

The mean total antioxidant capacity for Nescafe decaffinated, Nescafe Blend 43

and Nescafe Gold were 3.6, 3.5 and 3.3 mmol/L Trolox equivalents respectively. The

mean total antioxidant capacity of Nestle Cocoa and Milo were 1.3 and 0.4 mM Trolox

equivalents respectively (Table 7.2). The green powder tea drink had very little

antioxidant capacity. It seems likely that it was just a green coloured drink rather than

real tea!

Table 7.2. Antioxidant capacity in mmol/L TE of coffee, chocolate beverages and a green powder

drink

Beverage Brand Antioxidant Capacity in Trolox Equivalents (mM TE ± SD) Concentration CV (%) n

Nescafe Decaffinated 3.6 ± 0.2 1.87 mg/mL 4.6 24

Nescafe Blend 43 3.5 ± 0.2 1.87 mg/mL 4.9 24

Nescafe Gold 3.3 ± 0.1 1.87 mg/mL 4.3 24

Nestle Cocoa powder 1.3 ± 0.1 1.87 mg/mL 6.2 24

Milo 0.4 ± 0.1 1.87 mg/mL 13.6 24

green powder tea (Japan) 0.2 ± 0.1 1.87 mg/mL 27.0 24

7.3.3 Wines

Fig. 7.4 shows a diagram of the AIOR assay fluorescence versus time of a red

and white wine at two dilutions (1/10 and 1/20).

129

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Chardonnay 1/20 dilution

Chardonnay 1/10 dilution

Cabernet Sauvignon 1/20 dilution

Cabernet Sauvignon 1/10 dilution

Fig. 7.4. Comparison of two dilutions of red and white wine by the AIOR assay

A red wine was examined at two dilutions (1/10 and 1/20) and the mean

antioxidant capacities measured were 3.51 and 1.83 mM Trolox equivalents

respectively. A white wine was also examined at two dilutions (1/10 and 1/20) and the

mean antioxidant capacities measured were 1.06 and 0.54 mmol/L Trolox equivalents

respectively (Table 7.3).

Table 7.3. Comparison of antioxidant capacity in mmol/L TE of a red and white wine in 1/10 and

1/20 dilution

Dilution factor Antioxidant Capacity in Trolox

Equivalents (mM TE ± SD) CV (%) n

red wine 1/10 3.51 ± 0.14 4.1 12

red wine 1/20 1.83 ± 0.09 4.8 12

white wine 1/10 1.06 ± 0.05 4.9 12

white wine 1/20 0.54 ± 0.07 12.3 12

130

Table 7.4 shows a comparison of a selection of red and white wines measured by

the AIOR assay. The result further indicated that red and white wines have a wide range

of total antioxidant capacity. The red wines studied have consistently higher antioxidant

capacity than the white wines. The wines are ranked according to the antioxidant

capacity in mmol/L Trolox equivalents and the results are summarised in Table 7.4.

Table 7.4. Comparison of antioxidant capacity in mmol/L TE of different types of red and white

wines

Wine Type Name Antioxidant Capacity in Trolox

Equivalents (mM TE ± SD) Rank CV (%) n

red Cabernet / Merlot (Pemberton) 50.1 ± 1.3 1 2.6 6

red Pinot 1 (Pemberton) 47.0 ± 1.2 2 2.5 6

red Cabernet (Margaret River) 46.2 ± 1.2 3 2.6 6

red Cabernet Sauvignon (Wyndham) 45.4 ± 1.1 4 2.4 6

red Shiraz (Margaret River) 38.6 ± 0.7 5 1.9 6

red Pinot 2 (Pemberton) 34.7 ± 0.6 6 1.9 6

white Chardonnay (Jacob’s Creek) 24.3 ± 4.0 7 16.6 6

white Chardonnay (Dunsborough) 21.9 ± 2.3 8 10.6 6

white Chardonnay (Margaret River) 16.5 ± 1.3 9 8.1 6

white Chardonnay (Pemberton) 15.8 ± 1.4 10 8.7 6

white Mix White (Margaret River) 15.0± 1.7 11 11.1 6

7.3.4 Grape Juice

Three types of grapes, Shiraz, Cabernet, and Merlot, were crushed and the total

antioxidant capacity of the resulting grape juice measured by the AIOR assay. Fig. 7.5

131

shows a diagram of the AIOR assay fluorescence intensity versus time of the grape

juice (1/20 dilution).

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 7.5. Comparison of Trolox standard and grape juice by the AIOR assay

Blank

STD 4.0 mM

Shiraz 1/20 dilution

Cabernet 1/20 dilution

Merlot 1/20 dilution

The results are summarised in Table 7.5. The Merlot studied showed the highest

total antioxidant capacity followed by Cabernet and Shiraz.

Table 7.5. Comparison of antioxidant capacity in mmol/L TE of grape juice

Dilution Result times dilution

factor

Antioxidant Capacity in Trolox


Shiraz 1/20 X 20 18.5 ± 2.8 15.0 10

Cabernet 1/20 X 20 25.6 ± 4.7 18.5 10

Merlot 1/20 X 20 30.3 ± 3.5 11.7 10

132

7.3.5 Spices

Turmeric and curry powder extracts in neat and 1/2 dilution were measured. Fig.

7.6 shows a diagram of the AIOR assay fluorescence intensity versus time of the

antioxidant capacity measurement experiment.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

BlankSTD 0.5 mM

STD 1.0 mM

Turmeric 1/2 dilution

Curry 1/2 dilution

STD 2.0 mM

Curry neat

Turmeric neat

Fig. 7.6. Comparison of Trolox standard and two dilutions of turmeric and curry powder by the AIOR assay

Both the turmeric and curry powder neat extracts (1.87 mg/mL) showed a mean

total antioxidant capacity of 2.0 mmol/L Trolox equivalents (Table 7.6). The results of

the neat extracts and 1/2 dilution showed good agreement.

Table 7.6. Comparison of antioxidant capacity in mmol/L TE of spices

Dilution Concentration Antioxidant Capacity in Trolox

Equivalents (mM TE ± SD)

CV

(%) n

turmeric powder neat 1.87 mg/mL 2.0 ± 0.2 7.3 12

turmeric powder (1/2)

dilution 0.94 mg/mL 1.0 ± 0.1 7.5 12

curry powder neat 1.87 mg/mL 2.0 ± 0.3 13.9 12

curry powder (1/2) dilution 0.94 mg/mL 1.1 ± 0.2 15.7 12

133

7.3.6 Rutin and Flavonoids Supplement Tablet

Fig. 7.7 shows a diagram of the AIOR assay of fluorescence versus time of

different dilutions of a rutin and flavonoids supplement tablet.

The dilutions of 1/2, 1/3, 1/5 and 1/7 showed good linearity with the antioxidant

capacity in mM Trolox equivalents. A coefficient of determination R2 = 0.99 was

observed.

50 100 150

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Fig. 7.7. Comparison of Trolox standard and different dilutions of the extract from a tablet containing rutin and flavonoids by the AIOR assay

Blank

STD 0.5 mM

STD 2.0 mM

STD 1.0 mM

Dilution 1:7

Dilution 1:5

Dilution 1:3

Dilution 1:2

The antioxidant capacity of a rutin and flavonoids supplement tablet at a

concentration of 1.87 mg/mL showed very high antioxidant capacity and out of the

range of the standard curve. An estimated antioxidant capacity of 6.8 mmo/L Trolox

equivalents was calculated from the dilution curve. Table 7.7 shows the results of the

different dilutions of the tablet.

134

Table 7.7. Antioxidant capacity in mmol/L TE of an antioxidant supplement tablet in different

concentrations

Tablet extract dilution Extract

Concentration

Antioxidant Capacity in Trolox


1:7 dilution 0.24 mg/mL 1.2 ± 0.2 12.8 12

1:5 dilution 0.31 mg/mL 1.5 ± 0.2 10.1 12

1:3 dilution 0.47 mg/mL 2.2 ± 0.2 9.8 12

1:2 dilution 0.62 mg mL 2.6 ± 0.2 6.3 12

7.4 Discussion

The green and black teas were found to have different total antioxidant capacity.

Fig. 7.8 shows a diagram of the antioxidant capacity of the teas examined in

comparison. Coffee was also found to have different antioxidant capacity. Cocoa and

Milo have much less antioxidant capacity than teas and coffee. When compared to teas

in the same amount (mg/mL), coffee was found to have a similar level of antioxidant

capacity as shown in Table 7.1 and 7.2.

Fig. 7.8. Comparison of antioxidant capacity in mmol/L TE by the AIOR method of different teas

Comparison of Antioxidant Capacity of Tea

Gibson

s Trad

itiona

l

Twining

s Trad

itiona

l

Butterf

ly Fuji

an

Twining

s Gree

n

Tetley

Gree

n

Twining

s Frui

t0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Tea at 1.87 mg/mL

AO

C in

mM

Tro

lox

Equi

vale

nts

135

The AIOR method may be applied to measure the total antioxidant capacity in

wines. The total antioxidant capacity of the red and white wines in mM Trolox

equivalents has been compared (Fig. 7.9). The wine industry could use the measurement

of total antioxidant capacity in wine.

Fig. 7.9. Comparison of antioxidant capacity in mmol/L TE by the AIOR method of red and white wines

Comparison of Antioxidant Capacity of Red and White Wine

Cabern

et Merl

ot (P)

Pinot 1

(P)

Cabern

et (M

R)

Cabern

et Sau

vigno

r (W

)

Shiraz

MR)

Pinot 2

(P)

Chardo

nnay

(D)

Chardo

nnay

(JC)

Chardo

nnay

(MR)

Chardo

nnay

(P)

Mix W

hite (

MR)0

5

10

15

20

25

30

35

40

45

50

55

Red and White Wine

AO

C in

mM

Tro

lox

Equi

vale

nts

The antioxidant capacity of the different types of grape juice studied is different

(Fig. 7.10). A possible application is to measure the total antioxidant capacity of grapes

before harvest and during fermentation as a quality control measure.

136

Fig. 7.10. Comparison of antioxidant capacity in mmol/L TE of grape juice by the AIOR assay

Comparison of Antioxidant Capacity of Grape Juice

Shiraz

Cabern

et

Merlot

0

10

20

30

40

50

60

Juice from Grapes

AO

C in

mM

Tro

lox

Equi

vale

nts

It is also possible to compare the total antioxidant capacity of spices such as

turmeric and curry powder by the AIOR as shown in Fig. 7.11.

Fig. 7.11. Comparison of antioxidant capacity in mmol/L TE of spices by the AIOR assay

Comparison of Antioxidant Capacity of Spices

Turmeri

c 1/2

Dilutio

n

Turmeri

c

Curry 1

/2 Dilu

tion

Curry

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Spices at 1.87 mg/mL

AO

C in

mM

Tro

lox

Equi

vale

nts

The AIOR assay result indicated that it is capable of measuring the total

antioxidant capacity in a polyphenol supplement. The AIOR method may be useful

137

particularly in quality assurance and product labelling of products such as vitamin and

polyphenol supplements (Fig. 7.12).

Fig. 7.12. Comparison of antioxidant capacity in mmol/L of different dilutions of a polyphenol supplement tablet by the AIOR assay

Comparison of Antioxidant Capacity of Dilutions ofPolyphenol Supplement Tablet

Tablet

Extract

(1:2)

Tablet

Extract

(1:3)

Tablet

Extract

(1:5)

Tablet

Extract

(1:7)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Dilutions of Tablet Extract from 1.87 mg/mL

AO

C in

mM

Tro

lox

Equi

vale

nts

The AIOR assay was used to measure the total antioxidant capacity in complex

substances such as teas, coffee, chocolate beverages, wines, grapes, spices and a

polyphenol supplement tablet. The results showed that the AIOR assay can quantify the

antioxidants in different types of substances in different matrices.

In conclusion, the AIOR method can measure the total antioxidant capacity of

different substances and has many potential applications in industry such as quality

assurance, quality control and product labelling for antioxidants.

138

Chapter 8

Validation of the AIOR Method

8.1 Introduction

Analytical methods employed routinely for quantification are required to have a

validation of the methodology. Depending on the application of the analytical method,

different schemes have been used for validation, which may involve some or all of the

performance characteristics such as accuracy, precision, linearity, recovery, specificity

and limit of detection. A validation of the performance of the AIOR method has been

undertaken for the routine measurement of total antioxidant capacity in our laboratory.


8.2.1 Reagents and Equipment

Trolox standard was prepared by dissolving Trolox (50 mg) in 100 mL of PBS

(75 mmol/L, pH = 7.0) to give a concentration of 2.000 mmol/L. Standards 0.250,

0.500, 0.625, 0.750, 0.925, 1.035, 1.500 mmol/L were prepared from the 2.000 mmol/L

standard by dilution with PBS. AAPH (0.474 g) completely dissolved in 6.0 mL of PBS

to give a concentration of 292 mmol/L. The AAPH reagent was prepared immediately

before use. The indicator reagent is the 300 nmol/L uroporphyrin I dihydrochloride. The

139

fluorescence spectrophotometer Eclipse was from Varian. The instrument was set with

an excitation at λ 402 nm and emission at λ 622 nm. The excitation slit was 20 nm with

the emission slit at 10 nm. The run time was 180 min but flexible depending on the

substances being measured.

8.2.2 Method Protocol

Uroporphyrin I solution (200 μL) was placed into a 96-well microplate followed

by the diluted samples to be measured (1 μL). This was mixed by plateshaker for 2 min.

Fresh AAPH solution was prepared, making sure that all the AAPH was completely

dissolved. A multi-channel pipettor was used to dispense the AAPH solution (36 μL).

The final mixture in the 96-well microplate was mixed for exactly 2 min before data

acquisition by fluorescence monitoring with the fluorescence spectrophotometer at 0.2

seconds per reading for 180 min.

The AUC was calculated automatically by the installed software using the

Advanced Development Language incorporated within the software. The curve was first

normalised to the lowest intensity and scaled to the highest intensity to a nominal value

of 1000, thus achieving the AUC measurement. The AUC was then computed

automatically using the software.

8.3 Results

8.3.1 Linearity

The linearity of the Trolox standard curve was determined by using standard

concentrations between 0.250 to 1.500 mmol/L. The fluorescence intensity of a Trolox

standard curve with uroporphyrin I in the presence of AAPH versus time is shown in

140

Fig. 8.1. The linearity of the Trolox standard curve was maintained between 0.250 to

1.500 mmol/L (6 repeats per standard). The equation describing the line with the slope,

intercept and coefficient of determination (R2) of the standard curves is shown in Fig.

8.2. Linear regression analysis of AUC with the seven Trolox standard (TS) between

0.25 to 1.50 mmol/L plus blank gave the equation: AUC (Y) = 41080 x TS + 27980 (R2

= 0.9901). The Sy|x was 1842 and the SE of the slope and intercept were 605 and 499

respectively.

0 50 100 1500

200

400

600

800

1000

Time (min)

Inte

nsity

(a.u

.)

Trolox 1.500 mM

Trolox 1.035 mM

Trolox 0.925 mM

Trolox 0.750 mM

Trolox 0.625 mM

Trolox 0.500 mM

Trolox 0.250 mM

Blank

Fig. 8.1. Trolox standard curve of the AIOR assay in a 96-well microplate

0.0 0.5 1.0 1.5 2.00

25000

50000

75000

100000 Mean Area Under Curve

STD mM

Are

a U

nder

Cur

ve

Fig. 8.2. Linearity of the Trolox standard (TS) curve of the AIOR assay in a 96-well microplate

Y (AUC) = 41080 ± 605 x TS + 27980 ± 499 R2 = 0.9901, Sy|x = 1842, n = 48.

141

Linear regression analysis of serum sample dilutions with PBS from 1 in 10 to 1

in 70 with concentrations in mmol/L Trolox equivalents gave the equation: mM TE (Y)

= 13.84 x dilution + 0.0013 (R2 = 0.9933). The Sy|x was 0.032 and the SE of the slope

and intercept were 0.14 and 0.0071 (Fig. 8.3) respectively. The linearity of the serum

sample dilutions was accomplished with eight dilutions (Table 8.1, 8 repeats per

dilution).

0.000 0.025 0.050 0.075 0.100 0.1250.00

0.25

0.50

0.75

1.00

1.25

1.50Mean mM Trolox equivalents

Dilution

mM

Tro

lox

equi

vale

nts

Fig. 8.3. Linearity of serum sample dilutions with PBS of the AIOR assay

Y (mM TE) = (13.84 ± 0.14) x Dilution + 0.0013 ± 0.0071; R2 = 0.9933, Sy|x = 0.032, n = 64.

Table 8.1. Serum sample dilution linearity of the AIOR assay (8 repeats per dilution)

Dilution Mean mM Trolox equivalents SD CV, %

1 in 10 1.345 0.030 2.3

1 in 15 0.970 0.026 2.7

1 in 20 0.720 0.023 3.3

1 in 30 0.479 0.021 4.3

1 in 40 0.349 0.016 4.6

1 in 50 0.265 0.011 4.3

1 in 60 0.221 0.008 3.6

1 in 70 0.173 0.010 5.8

142

Linear regression analysis of the dilutions of the serum protein precipitation with

ethanol was from 1 in 4 to 1 in 8 with concentrations in mmol/L Trolox equivalents

gave the equation: mM TE (Y) = 3.35 x dilution – 0.2408 (R2 = 0.9917). The Sy|x was

0.0198 and the SE of the slope and intercept were 0.22 and 0.0413 respectively (Fig.

8.4). The linearity of the dilutions of the serum protein precipitation was accomplished

with four dilutions (Table 8.2, 15 repeats per dilution).

Fig. 8.4. Linearity of the dilutions of the serum protein precipitation with ethanol of the AIOR assay

0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.2750.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Mean mM Trolox equivalents

Dilution

mM

Tro

lox

equi

vale

nts

Y (mM TE) = (3.35 ± 0.22) x dilution - 0.2408 ± 0.0413; R2 = 0.9917, Sy|x = 0.0198, n = 45.

Table 8.2. Protein precipitation linearity of the AIOR assay (15 repeats per dilution)

Dilution Mean mM Trolox equivalents SD CV, %

1 in 4 0.610 0.046 7.6

1 in 5 0.420 0.046 10.9

1 in 6 0.302 0.026 8.7

1 in 8 0.194 0.024 12.4

143

8.3.2 Recovery

Trolox was used in a recovery study (Table 8.3). The % recovery between the

actual and the measured concentration of Trolox between 0.25 to 1.50 mM in three runs

had a mean of 100 %.

Table 8.3. Range, mean, recovery and precision in n = 3 runs of the AIOR assay (10 repeats per

run)

Trolox Run 1 Run 2 Run 3

actual concentration 0.250 mM measured range mM 0.234 - 0.300 0.235 - 0.291 0.232 - 0.320 CV, % 9.3 8.5 12.6 mean mM 0.258 0.258 0.263 recovery % 103 103 105 actual concentration 0.500 mM measured range mM 0.450 - 0.590 0.486 - 0.572 0.486 - 0.596 CV, % 2.9 6.1 7.2 mean mM 0.528 0.520 0.526 recovery % 106 104 105 actual concentration 0.625 mM measured range mM 0.621 - 0.705 0.615 - 0.698 0.607 - 0.707 CV, % 4.0 4.5 5.5 mean mM 0.655 0.644 0.641 recovery % 105 103 103 actual concentration 0.750 mM measured range mM 0.720 - 0.827 0.719 - 0.806 0.704 - 0.814 CV, % 4.7 4.2 5.0 mean mM 0.759 0.750 0.746 recovery % 101 100 99 actual concentration 0.925 mM measured range mM 0.878 - 0.984 0.877 - 0.985 0.878 - 0.990 CV, % 3.8 4.2 4.3 mean mM 0.916 0.917 0.916 recovery % 99 99 99 actual concentration 1.035 mM measured range mM 0.976 - 1.063 0.974 - 1.074 0.971 - 1.105 CV, % 2.8 3.3 4.3 mean mM 1.004 1.011 1.015 recovery % 97 98 98 actual concentration 1.500 mM measured range mM 1.347 - 1.451 1.365 - 1.463 1.355 - 1.488 CV, % 2.4 2.6 3.2 mean mM 1.392 1.403 1.396 recovery % 93 94 93

144

There is no definitive or reference total antioxidant capacity method available

for accuracy comparison with the AIOR method. In this study, polyphenols were used

to determine the accuracy of the AIOR assay. The common polyphenols rutin,

quercetin, (−)-epicatechin and gallic acid (Fig. 8.5) at 0.89, 0.77, 0.89, and 1.13 mmol/L

respectively were found to have antioxidant capacities of 3.79, 4.41, 4.03, and 1.75

mmol/L Trolox equivalents respectively.

Fig. 8.5. Structures of polyphenols used for validation

O

OH

OH

OHOH

O

O rutinose

Rutin

O

OH

OH

OHOH

O

OH

Quercetin

OH

OHOHOH

C O

Gallic acid

OH

OH

OHOH

H

O

O

(−)-Epicatechin

A mixture of these four polyphenols with the same individual concentration was

made. The calculated total antioxidant capacity of the mixture of the four polyphenols

was 3.49 mmol/L Trolox equivalents (Table 8.4). The measured AIOR assay

antioxidant capacity of the mixture was 3.93 ± 0.21 mmol/L Trolox equivalents.

Comparing the calculated value with the measured total antioxidant capacity, the result

showed a recovery of 113%.

145

Table 8.4. Additive effect of total antioxidant capacity in mM Trolox equivalents(TE) with

polyphenols of the AIOR assay

Concn. (mM) Mean mM TE (SD) mM TE (x 1/4) n

rutin 0.89 3.79 (0.25) 0.95 9

quercetin 0.77 4.41 (0.44) 1.10 9

(−)-epicatechin 0.89 4.03 (0.28) 1.00 9

gallic acid 1.13 1.75 (0.11) 0.44 9

total of (x 1/4) 3.49

mixture of the 4 polyphenols (1 : 1 : 1 : 1) 3.93 (0.21) 12

recovery = 113 %

8.3.3 Precision

In the precision study using serum sample, the within-run CV was 5.7%, (mean,

15.30; SD, 0.88; n = 72) and the between-run CV was 7.3% (mean, 15.10; SD, 1.05; n =

25). Using serum protein precipitation with ethanol sample, the within-run CV was

7.7% (mean, 2.67; SD, 0.21; n = 40) and the between-run CV was 7.9% (mean, 2.44;

SD, 0.19; n = 25).

8.3.4 Functional Sensitivity

A functional sensitivity measured as a coefficient of variation (CV) of < 20% is

generally defined as an acceptable analytical performance. Fig. 8.6 shows the functional

sensitivity of a serum sample dilutions with PBS of 0.16 mmol/L Trolox equivalents at

1/70 dilution which gave a CV of 15.4% (Table 8.5). Fig. 8.7 shows the functional

146

sensitivity of a deproteinised serum sample with ethanol of 0.20 mmol/L Trolox

equivalents at (1/8) dilution which gave a CV of 14.4% (Table 8.6).

Fig. 8.6. Functional sensitivity of serum sample dilution with PBS of the AIOR assay in n = 4 runs (8 repeats per dilution)

1.26-1

.47

0.88-1

.11

0.63-0

.86

0.28-0

.40

0.16-0

.29

0.12-0

.20

0.04-0

.20

0

5

10

15

20

25

30

35

Measured concentration range of serumsample dilution with PBS (mM TE)

CV

(%)

Table 8.5. Functional sensitivity of serum sample dilution with PBS of the AIOR assay in n = 4 runs

(8 repeats per run)

Dilution Concn. range (mM TE) Mean (mM TE) SD CV, %

1/10 1.26 – 1.47 1.36 0.05 3.9

1/15 0.88 – 1.11 0.96 0.05 5.6

1/20 0.63 – 0.86 0.71 0.05 7.3

1/40 0.28 – 0.40 0.34 0.03 8.6

1/50 0.16 – 0.29 0.25 0.03 14.0

1/70 0.12 – 0.20 0.16 0.03 15.4

1/100 0.04 – 0.20 0.10 0.03 34.2

147

Fig. 8.7. Functional sensitivity of sample protein precipitation with ethanol of the AIOR assay in n = 3 runs (15 repeats per dilution)

0.50-0.70 0.22-0.37 0.13-0.250

5

10

15

20

25

30

35

Measured concentration range of sample proteinprecipitation with ethanol (mM Trolox equivalents)

CV

(%)

Table 8.6. Functional sensitivity of sample protein precipitation with ethanol of the AIOR assay in

n = 3 runs (15 repeats per run)

Dilution Concn. range (mM TE) Mean (mM TE) SD CV, %

1/4 0.52 – 0.76 0.62 0.05 8.5

1/6 0.23 – 0.41 0.31 0.04 13.2

1/8 0.15 – 0.28 0.20 0.03 14.4

8.3.5 Correlation with an Individual Antioxidant

Linear regression analysis of antioxidant capacity versus concentration of rutin,

quercetin, (−)-epicatechin, gallic acid, gluthathione, uric acid, vitamin C, vitamin E,

vitamin A, albumin and total protein samples provides slope, intercept and a coefficient

of determination (R2) ≥ 0.97. The equation of the line with concentration gives the

antioxidant capacity of the individual antioxidant (Table 8.7).

148

Table 8.7. Correlation of concentration and antioxidant capacity (mM Trolox equivalents) of the

AIOR assay.

Compound Concn. range in (mM) or (g/L) Slope Intercept R2

rutin 0.30 – 0.89 3.254 0.870 0.9920

quercetin 0.26 – 0.77 4.171 1.186 0.9972

(−)-epicatechin 0.30 – 0.89 2.793 1.526 0.9899

gallic acid 0.57 – 4.52 1.159 0.452 0.9804

glutathione 0.37 – 2.93 0.395 0.109 0.9828

uric acid 0.42 – 1.70 1.471 -0.043 1.0000

vitamin C 2.65 – 10.6 0.394 -0.195 0.9988

vitamin E 1.00 – 4.00 0.775 -0.405 0.9989

vitamin A 1.94 – 5.82 0.217 0.190 0.9735

albumin 20.0 – 40.0 g/L 0.060 0.000 1.0000

total protein 32.4 – 64.8 g/L 0.068 -1.003 0.9929

∗ Human serum albumin in PBS and total protein standard solution from Behring.

8.3.6 Sample Preservation and Storage Conditions

The total antioxidant capacity (TAC) of serum, heparin and EDTA preserved

samples were measured in two subjects. The Kruskal-Wallis test indicated significant

difference amongst samples collected in different preservatives (Table 8.8). Compared

with the heparin and EDTA samples, the serum sample had the lowest TAC mean.

Serum is recommended for routine assay as it gives the least possibility of interference

from preservative. To test the stability of samples, serum samples were assayed on the

day of collection, stored in the dark at room temperature for overnight, 4oC for 4 days

and –20oC for 28 months. The results showed no decline in TAC when the serum was

stored in the dark at –20oC for over two years (Table 8.9).

149

Table 8.8. Comparison of serum, EDTA, heparin samples total antioxidant capacity (mM Trolox

equivalents) of the AIOR assay, n = 2 subjects (12 repeats per subject)

Subject 1 Subject 2

preservative mean mM TE SD CV,

% mean

mM TE SD CV, %

serum 11.17 0.67 6.0 12.44 0.44 3.6

EDTA 12.25 0.53 4.4 13.37 0.38 2.9

heparin 13.19 0.52 4.0 12.98 0.60 4.6

Kruskal-Wallis test P < 0.0001 P = 0.0009

Table 8.9. Comparison of serum total antioxidant capacity (mM Trolox equivalents) same day,

stored at room temperature overnight, 4oC for 4 days and –20oC for 28 months of the AIOR assay,

n = 2 subjects (6 repeats per subject)

Subject 1 Subject 2

storage condition mean mM TE SD CV,

% mean

mM TE SD CV, %

room temperature same day 11.17 0.67 6.0 12.44 0.44 3.6

room temperature overnight 10.89 0.47 4.3 12.22 0.39 3.2

4oC for 4 days 11.65 0.83 7.2 13.03 0.61 4.7

-20oC for 28 months 11.77 0.37 3.1 13.48 0.29 2.1

8.3.7 Stability of the Uroporphyrin I Reagent

To test the stability of the working uroporphyrin I reagent, 200 μL of the reagent

was exposed to the excitation wavelength at 402 nm under the normal assay conditions

in the fluorescence spectrophotometer for 180 min with no AAPH added. The reagent

was found to be stable over this time period without photodecomposition.

150

8.4 Discussion

The linearity of the AIOR method has been demonstrated. The accuracy of the

measurement was checked using the recovery of a mixture of polyphenols. The

recovery of 113% may be due to an additive effect of the polyphenols.

The linearity in serum and deproteinised sample dilutions has been established

for dilutions of serum and ethanolic deproteinization supernatant. An effective

functional sensitivity defined as the concentration range in mmol/L Trolox equivalents

at which the analytical coefficient of variation (CV) increased to 20% has been noted in

the AIOR method.

The AIOR assay showed good precision and correlation of antioxidant capacity

with concentration with a coefficient of determination (R2) ≥ 0.97 in all cases. The

linear regression analysis with individual antioxidant rutin, quercetin, (−)-epicatechin,

gallic acid, glutathione, uric acid, vitamin C, E and A, albumin and total protein

provides conversion of concentration to antioxidant capacity. The results show that the

indicator molecule uroporphyrin I is stable over the reaction period. The effect of

sample preservative and storage conditions has been investigated. In conclusion, it has

been demonstrated that the AIOR assay is suitable for the measurement of total

antioxidant capacity in biological samples such as serum.

151

Chapter 9

Oxidative Stress and Total Antioxidant Capacity

in a Hypertensive Study

9.1 Introduction

Measurement of total antioxidant capacity in biological samples such as serum is

complex. Firstly, it presents a different matrix compared to food and beverage samples;

secondly, serum contains a wide range of hydrophilic and hydrophobic antioxidants;

and thirdly, high concentrations of albumin and proteins may mask the antioxidant

capacity effect of other antioxidants at lower concentrations.

Ideally, the oxidative stress status of the subjects studied should be considered

concomitantly with the total antioxidant capacity. To investigate this proposal, an index

designed to bring these two parameters together as a ratio, the oxidative stress ratio

(OSR), has been proposed. The OSR was calculated from the total antioxidant capacity

of the serum measured and divided by the value of the measured oxidative stress marker

plasma F2-isoprostanes.

Under normal physiological conditions, arachidonic acid (Fig. 9.1) is

metabolised by cyclooxygenase enzymes to prostaglandins. However, in the presence of

reactive oxygen species (ROS), prostaglandin (PG)-like compounds are formed as a

result of free radical reaction with arachidonic acid. These PG-like compounds are

commonly known as isoprostanes (IsoP)66 some of which are shown in Fig. 9.2. The

detection and quantitation of F2-isoprostanes62, 64 are determined either by GC-MS, LC-

152

MS or immunoassay. One of the F2-isoprostane, 15-F2t-IsoP also known as 8-iso-PGF2α

is regularly being used as an oxidative stress marker. In the present study, the

concentration of F2-isoprostanes in plasma was measured by GC-MS.64

COOH

Fig. 9.1. Molecular structure of arachidonic acid

OO

O

COOH

H

HH

15-F2t-IsoP

O

OCOOH

H

H

HO

a8-F2-IsoP

O

OCOOH

H

H

HO

a5-F2-IsoP

153

O

OO

COOH

HH

H

PGF2α

Fig. 9.2. Examples of the molecular structure of isoprostanes in comparison to a prostaglandin

Treated and untreated hypertensive and normotensive subjects all have different

oxidative stress levels which can be assessed by the plasma level of the oxidative

marker F2-isoprostanes in these subjects.130 The aims of this study were: firstly to

measure the total antioxidant capacity of these subjects with and without

deproteinization by the AIOR method; secondly, to measure oxidative stress from the

level of F2-isoprostanes; and thirdly, to calculate the OSR. In this study, all subjects had

ceased taking any type of antioxidant supplement.


9.2.1 Subjects

The hypertensive study and collection of samples were performed by Trevor

Mori and Natalie Ward130 of the School of Medicine and Pharmacology, University of

Western Australia. A population of 155 hypertensive subjects and 40 normotensive

subjects were recruited from the general population in Western Australia. Of the

hypertensive subjects, 85 were treated and 70 untreated. All volunteers had ceased

taking any vitamin, antioxidant, or fish oil supplement for a minimum of 4 weeks prior

to entry into the study. Where possible, all prescribed medications were taken as normal

154

on the morning of each visit. Exclusion criteria included: previous coronary or

cerebrovascular event < 6 months, heart failure, premenopausal women, use of oral

contraception, and body mass index (BMI) > 35 kg/m2. The study was approved by the

Royal Perth Hospital Human Ethics Committee.130 Written informed consent was

obtained before inclusion in the study. Blood pressure (BP) was defined using

ambulatory BP monitoring. Normotension was defined according to the World Health

Organization – International Society of Hypertension Guildelines as a mean 24 hour BP

< 125/80 mm Hg or day BP < 130/85 mm Hg. Untreated hypertensive subjects were

included if their mean 24 hour systolic BP (SBP) was ≥ 135 mm Hg or the mean awake

SBP was ≥ 140 mm Hg and they had never been treated. Fifty-seven of the subjects

recruited to the untreated hypertensive group had a mean 24 hour diastolic BP (DBP) ≥

80 mm Hg. Treated hypertensive subjects were included on the basis of a prior

physician’s diagnosis of essential hypertension and currently taking one or more

antihypertensive medications for ≥ 3 months.

9.2.2 Methods

The hypertensive study, collection of samples and chemical analysis, with the

exception of total antioxidant capacity measurement and oxidative stress ratio, were

performed by Trevor Mori and Natalie Ward130 of the School of Medicine and

Pharmacology, University of Western Australia. Plasma F2-isoprostanes were analyzed

by GCMS as previously described.64 Plasma vitamins C and E were analyzed by HPLC

using electrochemical detection and ultraviolet detection130 respectively. Total protein

and uric acid were analyzed using standard methods with the Hitachi 917 autoanalyzer

in the Department of Clinical Biochemistry at Royal Perth Hospital.130

The serum total antioxidant levels and the deproteinized antioxidant levels were

measured by the new AIOR method as described. The oxidative stress ratio (OSR) was

calculated by dividing the serum total antioxidant levels and plasma F2-isoprostanes

concentrations.

155

9.2.3 Statistics

Statistical analysis was performed using the GraphPad Prism Version 4.0.

Results are presented as means ± SEM or geometric means (SD). Pearson correlation

coefficient was calculated from the linear regression analysis. Variance analysis was

performed with Kruskal-Wallis and Dunn’s post test or one-way ANOVA and

Bonferroni post test for multiple comparisons of nonparametric or parametric data

between the groups.

9.3 Results

Table 9.1 outlines the biochemical analyses of the untreated and treated

hypertensive subjects, and normotensive subjects. The treated hypertensive subjects

were significantly older than the untreated hypertensive subjects and normotensive

controls. As expected, both the systolic and diastolic blood pressure were significantly

different in these groups. F2-isoprostanes concentrations were 2.98 ± 0.22, 3.41 ± 0.18

and 3.23 ± 0.27 ρmol/L in treated, untreated and control subjects respectively. The total

antioxidant capacities were 9.94 ± 0.11, 9.79 ± 0.13, and 9.62 ± 0.12 mM Trolox

equivalents and the deproteinized total antioxidant capacities were 1.48 ± 0.22, 1.45 ±

0.22 and 1.43 ± 0.22 mM Trolox equivalents in treated, untreated and control subjects

respectively.

Statistically, there were no significant differences in vitamin C, uric acid, serum

total antioxidant capacity and deproteinized total antioxidant capacity amongst these

groups. However F2-isoprostanes and vitamin E concentrations were lower in the

treated than untreated subjects. F2-isoprostanes and vitamin E showed significant

differences, (p < 0.034) and (p < 0.0001) respectively, between treated and untreated

subjects.130

156

The oxidative stress ratios (OSR) were 0.35 ± 0.02, 0.30 ± 0.02 and 0.34 ± 0.03

in untreated, treated and control subjects. There was a statistically significant difference

in the OSR between the treated and untreated subjects (p < 0.015).

Table 9.1. Biochemical anlysis of untreated and treated hypertensive and normotensive subjects, values are means ± SEM

Untreated

hypertensive subjects

Treated hypertensive

subjects

Normotensive Controls subjects

ANOVA P value

n 70 85 40

serum vitamin E (μmol/L) 38.1 ± 1.4 29.3 ± 1.2 33.7 ± 1.2 < 0.0001

serum vitamin C (μmol/L) 53.0 ± 2.5 53.6 ± 2.7 52.7 ± 3.7 0.982

plasma total protein (g/L) 69.8 ± 0.6 70.0 ± 0.5 67.5 ± 0.6 0.032

plasma uric acid (mmol/L) 0.34 ± 0.01 0.34 ± 0.01 0.32 ± 0.01 0.214

(A) plasma F2-isoprostanes (ρmol/L) 3.41 ± 0.18 2.98 ± 0.22 3.23 ± 0.27 0.034

(B) serum total antioxidant capacity (mmol/L TE) 9.79 ± 0.13 9.94 ± 0.11 9.62 ± 0.12 0.274

deproteinized total antioxidant capacity (mmol/L TE) 1.45 ± 0.22 1.48 ± 0.22 1.43 ± 0.22 0.495

oxidative stress ratio (OSR) = A/B 0.35 ± 0.02 0.30 ± 0.02 0.34 ± 0.03 0.015

9.4 Discussion

In this study, all subjects had ceased taking any vitamin, antioxidant, or fish oil

supplements for a minimum of 4 weeks prior to entry into the study. The energy and

nutrient intake did not show significant difference amongst the untreated and treated

hypertensive and normotensive groups. These results were supported by the serum total

antioxidant level, uric acid and vitamin C, which did not show significant differences in

these groups. The total antioxidant capacities measured by the AIOR assay in the three

groups were similar.

157

The oxidative stress marker F2-isoprostanes showed significant difference (p <

0.034) between the treated and untreated subjects.130 By comparison, the OSR indicates

that the treated hypertensive subjects had a significantly lower OSR than the untreated

hypertensive subjects (p < 0.015) (Fig. 9.3). From these results, it can be inferred that

the oxidative stress is higher in the untreated hypertensive subjects using either the OSR

or oxidative stress marker. Both serum total antioxidant capacity and the deproteinized

total antioxidant capacity were lower in the untreated hypertensive subjects, albeit not

significantly different. This study shows that an oxidative stress state existed within a

similar total antioxidant capacity in the treated and untreated subjects. However by

taking the total antioxidant capacity into consideration, the OSR showed more

convincingly the evidence of oxidative stress in untreated subjects than F2-isoprostanes

alone.

Oxidative Stress Ratio (OSR) in Hypertension Study

Untreat

ed

Treated

Normal

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Groups in the Hypertension Study

OSR

= O

S/A

IOR

Fig. 9.3. Comparison of oxidative stress ratio (OSR) between untreated and treated hypertensive and normotensive subjects

158

Chapter 10

Concluding Remarks

For many years, high levels of antioxidants have been hypothesized to lower

oxidative stress. However, recent research has conclusively shown that individual

antioxidant supplements are ineffective in preventing oxidative stress diseases. Multi-

coloured fruits and vegetables are now being promoted as the likely supplier of

antioxidants in vivo and implicated in lowering oxidative stress.

Although there are methods available to measure total antioxidant capacity, they

are not universally acceptable because of the various problems and pitfalls discussed in

chapter 1.

How then should we measure total antioxidant capacity associated with the

intake of fruits, vegetables, tea, coffee, red wine, chocolate, and spices in diets?

The most effective way is to measure the total antioxidant capacity before and

after the consumption of these substances.

The AIOR assay developed in this research has been evaluated and applied to

measure total antioxidant capacity in chemicals, foodstuffs and biological samples such

as serum.

The mechanism of reaction of the AIOR assay was examined by fluorescence

and UV spectrophotometry, electron spin resonance spectroscopy, HPLC and LC-MS.

The AIOR reaction has been shown to display first order kinetics and a reaction

mechanism for the AIOR reaction has been tentatively proposed.

159

The AIOR assay is temperature independent and free of the problems associated

with previous methods. Hence the AIOR assay is more likely to be acceptable to the

wider research community. The linearity, precision, recovery and functional sensitivity

of the method has been shown to be robust and reliable. The stability of the reagents and

sample preservation requirements and storage have been checked. The correlation

between high antioxidant status samples and the resulting antioxidant capacity has been

shown to have a coefficient of determination (R2) ≥ 0.97. The method uses multi-point

calibration and is capable of large scale study by the use of 96-well microplates.

The limitation of total antioxidant capacity measurement in serum is the high

concentration of albumin and proteins which mask the antioxidant capacity effect of the

other antioxidants. This limitation can be overcome by serum deproteinization by

organic solvent such as ethanol before application of the assay.

The total antioxidant capacity in the serum of hypertensive subjects has been

studied. The relationship between oxidative stress and antioxidant capacity can

potentially be assessed by bringing both parameters together as the oxidative stress

ratio. The results obtained from the ratio presents a better picture of the outcome of the

subjects after treatment than the oxidative stress marker alone.

The plan for the future direction of the AIOR method in this laboratory is to

apply the assay in clinical situations and in the food and beverage industries. The

beneficial effect of fruits, vegetables and healthy diets to provide antioxidants in

situations of oxidative stress such as cancer and cardiovascular disease could be further

studied by this method in conjunction with other indicators of oxidative stress markers

such as isoprostanes.

160

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