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
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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
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.
2.2 Materials and Methods
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
Are
a Und
er C
urve
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
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.
AIOR STD Curve at 1.00, 2.00, 4.00 mM
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
80000Mean Area Under Curve
R2 = 0.9589, n = 24
STD mM
Are
a Und
er C
urve
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.
AIOR STD Curve at 1.25, 2.50, 5.00 mM
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
Mean Area Under Curve
R2 = 0.9201, n = 36
STD mM
Are
a Und
er C
urve
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
AIOR STD Curve at 0.50, 1.00, 2.00 mM
0.0 0.5 1.0 1.5 2.010000
20000
30000
40000
50000
60000
70000
Mean Area Under Curve
R2 = 0.9919, n = 24
STD mM
Are
a Und
er C
urve
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
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
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
AIOR STD Curve at 0.01, 0.02, 0.04 mM
0.00 0.01 0.02 0.03 0.0425000
35000
45000
55000
65000
Mean Area Under Curve
R2 = 0.8419, n = 32
STD mM
Are
a Und
er C
urve
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
AIOR STD Curve at 0.50, 1.00, 2.00 mM
0.0 0.5 1.0 1.5 2.040000
50000
60000
70000
80000
90000
100000
110000
Mean Area Under Curve
R2 = 0.9779, n = 96
STD mM
Are
a Und
er C
urve
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
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
Are
a Und
er C
urve
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
n = 12 for each column
X Axis of 96-well plate
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
n = 12 for each column
X Axis of 96-well plate
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
n = 12 for each column
X Axis of 96-well plate
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
Y Axis of 96-well plate
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
Y Axis of 96-well plate
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
Y Axis of 96-well plate
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.
3.2 Materials and Methods
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) 15 min uroporphyrin I + AAPH 380 163
(4) 30 min uroporphyrin I + AAPH 319 133
(5) 60 min uroporphyrin I + AAPH 240 103
(6) 120 min uroporphyrin I + AAPH 106 46
(7) 180 min uroporphyrin I + AAPH 34 19
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.
Table 3.3. UV-visible scan from 200 - 450 nm in a cuvette of uroporphyrin I (urop. I) and AAPH
before and during the reaction
Time Abs. intensity 368 - 379 nm
Abs. intensity 397 nm
Abs. intensity 401 - 2 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).
Table 3.4. UV-visible scan from 200 - 700 nm in a cuvette of uroporphyrin I (urop. I) and AAPH
before and during the reaction
Time Abs. intensity 368 nm
Abs. intensity 398 nm
Abs. intensity 400 - 1 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
(4) 5 min (gallic acid + AAPH) 1.613 0.301
(5) 15 min (gallic acid + AAPH) 1.060 0.303
(6) 30 min (gallic acid + AAPH) 1.599 0.307
(7) 60 min (gallic acid + AAPH) 1.599 0.318
(8) 120 min (gallic acid + AAPH) 1.584 0.304
(9) 180 min (gallic acid + AAPH) 1.584 0.317
(10) 240 min (gallic acid + AAPH) 1.577 0.327
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
Abs. intensity 259 nm
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
(6) 5 min (urop. I + gallic acid + AAPH) 1.635 0.679
(7) 15 min (urop. I + gallic acid + AAPH) 1.635 0.680
(8) 30 min (urop. I + gallic acid + AAPH) 1.635 0.683
(9) 60 min (urop. I + gallic acid + AAPH) 1.635 0.691
(10) 120 min (urop. I + gallic acid + AAPH) 1.628 0.700
(11) 180 min (urop. I + gallic acid + AAPH) 1.620 0.713
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
Fig. 3.47. Fluorescence intensity versus time (min)
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
Fig. 3.48. Fluorescence intensity versus time (min)
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
Fig. 3.50. First-order kinetic curve between [uroporphyrin I] = 180 nM and [AAPH] = 292 mM measured at Ex = 405 nm and Em = 624 nm at room temperature with rate constant k = 0.0232 min-1
Ex = 405 nm, Em = 624 nmURO = 180 nM, 2.5 mL; AAPH = 292 mM, 0.5 mL
k = 0.0232 min-1 at room temp.
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
00 50 100 150 200
Time (minutes)
Ln([
R]t/[
R]o)
Fig. 3.51. First-order kinetic curve between [uroporphyrin I] = 180 nM and [AAPH] = 583 mM measured at Ex = 405 nm and Em = 624 nm at room temperature with rate constant k = 0.0276 min-1
Ex = 405 nm, Em = 624 nmURO = 180 nM, 2.5 mL; AAPH = 583 mM, 0.5 mL
k = 0.0276 min-1 at room temp.
-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
Fig. 4.17. HPLC monitored by PDA 397 nm at 120 min in the presence of gallic acid of the reaction between uroporphyrin I and AAPH
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
5.2 Materials and Methods
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