David B' Min

1
Kinetics of Reactive Oxygen Species and
Antioxidant in Lipid Oxidation

• David B. Min
•  Department of Food Science and Technology
• The Ohio State University
• Columbus, Ohio
• U.S.A

2
Undesirable Effects of Lipid Oxidation
3
1772 Priestley Discovered Oxygen 1811
Avogadro Oxygen is a Diatomic Molecule 1848
Faraday Oxygen is a Paramagnetic Molecule 1934
Herzberg Observation of Singlet Oxygen 1964
Foote Wexler Rediscovered Singlet Oxygen  
History of Oxygen Molecule
4
Mechanism of Lipid Oxidation

• Triplet oxygen oxidation
• Singlet oxygen oxidation

5
Molecular Orbital of Triplet Oxygen
Molecular
?
Atomic
Atomic
?
?
?
?
2Px 2Py 2Pz
2Pz 2Py 2Px
?
Energy
?
2S
2S
?
?
1S
1S
?
6
Lipid Oxidation
14 13 12 11 10 9
C
H
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
2
2
3
3
Initiation
Reactive oxygen species
?
H
Preventive Antioxidants
13 12 11 10 9
(
C
H
)
C
H
C
H
R
C
H
C
H
C
H
C
H
C
H
3
2
2
4
?
E0 600mV
3O2
K109 M-1sec-1
7
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
2
3
4
2
O
O
K 1 M-1sec-1
E01000mV
?
3O
R. ROO
H from RH (Lipid)
2
Propagation
?
E01000mV
.
H
O
O
C
(
C
H
)
C
(
)
C
H
Radical Scavenging Antioxidant
.
3
3
3
3
H from
O
C
H
O
C
H
3
3
E0 400mV
K 107 M-1sec-1
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
4
3
O
O
H
8
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
4
3
O
O
OH
E02300 mV
?
H
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
O
E01600 mV
?
Termination
C
H
(
C
H
)
C
HO
3
2
4
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
O
O
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
3
2
2
4
9
Molecular Orbital of Singlet Oxygen
Molecular
?
Atomic
Atomic
?
?
?
?
2Px 2Py 2Pz
2Pz 2Py 2Px
?
Energy
?
2S
2S
?
?
1S
1S
?
10
Photosensitizers in Foods

• Chlorophyll
• Protoporphyrine
• Riboflavin

11
Chlorophyll
H
C
H
H
C
R
2
C
H
C
H
H
C
2
3
3
N
N
Mg
H
H
N
N
C
H
3
H
C
3
O
C
O
C
H
C
H
2
3
2
C
O
C
H

3
O
C
H
C
H
C
H
C
H
3
3
3
3
12
Singlet Oxygen Formation by Photosensitizer
Excited State
1Chlo
K1-20?108/sec
ISC
Fluorescence K 2?108/sec
3Chlo
hv
K1-3?109/sec
Phosphorescence
3O2
K10-104/sec
1Chlorphyll
Ground State
Singlet Oxygen
13
Singlet Oxygen Determination by Trapping
O
O
H3C
H3C
CH3
CH3
1O2
N
N
H3C
CH3
H3C
CH3
O
H

Tetramethyl-4-Piperidone-N-Oxyl
Tetramethyl-4-Piperidone
(TAN)
(TMPD)
14
Electron Spin Resonance Spectrum of
Tetramethyl-4-Piperidone-N-Oxyl
3370 G 3390 G
3410 G
15
Reaction of 3O2 and 1O2 with Linoleic Acid
OOH
.
(?)
O
3
3
O
2
2
R
R
R
R
R
R
OOH
1
O
O
2
R
R
R
R
H
O
OOH
gtgtgt
3
1
R
R
O
O
2
2
16
Reversion Beany Flavor of Soybean Oil
17
Soybean Oil Reversion Beany Flavor Compounds
C
H
C
H
C
H
C
H
C
H
3
2
2
O
2-(1-pentenyl)-furan
C
H
C
H
C
H
C
H
C
H
2
2
3
O
2-(2-pentenyl)-furan
18
Soybean Oil Purification by Chromatography
Soybean oil
Silicic acid
Sugar
Charcoal-Celite
Silicic acid
Vacuum
Purified soybean oil
19
Identified Compounds in the Column

• Phospholipids
• Tocopherols
• Monoglycerides
• Diglycerides
• Oxidized triglycerides
• Free fatty acids
• Chlorophylls

20
Purified Soybean Oils
Colorless Tasteless Odorless
21
Light Box for Soybean Oil Flavor Study
22
Effect of 0 and 5 ppm Chlorophyll on
2-Pentenylfurans in Soybean Oil
0 ppm
No beany flavor ESR No singlet oxygen No singlet
oxygen, no
2-pentenylfuran
5 ppm
trans-2-heptenal
2-pentenylfuran

Strong beany flavor ESR Singlet oxygen
Singlet oxygen, 2-pentenylfuran
23
Mass Spectrum of 2-Pentenylfuran
Relative Abundance
m/z
24
2-Pentenylfuran from Linolenic Acid by 1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
(
C
H
)
C
O
O
H
2
2
2
6
3
2
2
1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
O
O
H
(
C
H
)
C
H
C
H
3
2
2
2
2
6
2
O
O
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
O
O
H
(
C
H
)
C
H
C
H
2
2
6
2
3
2
2
O
O


C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
O
1O2
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2
O
O
O
25
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2

O
O
O
H
- OH

C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
C
H
2
3
2
2

O
O

C
H
C
H
C
H
H
C
H
C
C
H
C
H
C
H
C
3
2
2
2
2
O
O
C
H
C
H
C
H
C
H
C
H
C
C
H
C
H
C
H
3
2
2
O
H
O
H
- H2O
2-(2-pentenyl)-furan
C
H
C
H
C
H
C
H
C
H
3
2
2
O
26
Formation of Reactive Oxygen Species
.
2
R
O
O
R
H2O2
.
HOO
Sensitizer
O

3
3
2
Irradiation
Sensitizer
1
1
O
Fe3
2
2

.
-
H2O2 Fe2
F
H
O
e
O
H

H
Irradiation
3

F
H2O2
e
.
-
.
Sensitize
O

O
H
2
2

H
Sensitizer
O
aqe
3
-
3

2
O
3


2

R
.
Xanthine Oxidase
O
3
H
O
O
2
H
O
2
.
R
O
O
.
H2O2
H
O

27
Reaction Rates (M-1sec-1 ) of
ReactiveOxygen Species with Lipids
28
Are You Ready to Fight Reactive Oxygen Species?
O-2, 1O2, .OH, H2O2, R, RO, ROO
Soybean Oil
Antioxidant
R, RO, ROO, 1O2, O-2, -OH, H2O2,
ROS Jail
29
Mechanism of Antioxidant
14 13 12 11 10 9
C
H
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
2
2
3
3
Initiation
Reactive oxygen species
?
H
Preventive Antioxidants
13 12 11 10 9
(
C
H
)
C
H
C
H
R
C
H
C
H
C
H
C
H
C
H
3
2
2
4
?
E0 600mV
3O2
K109 M-1sec-1
30
(
C
H
)
C
H
C
H
C
H
C
H
C
H
C
H
R
C
H
2
3
4
2
O
O
E01000mV
?
3O
R. ROO
H from RH (Lipid)
2
Propagation
?
E01000mV
K 1 M-1sec-1
.
H
O
O
C
(
C
H
)
C
(
)
C
H
Radical Scavenging Antioxidant
.
3
3
3
3
H from
O
C
H
O
C
H
3
3
E0 400mV
K 107 M-1sec-1
C
H
C
H
C
H
C
H
C
H
C
H
R
(
C
H
)
C
H
2
2
4
3
O
O
H
31
Types of Antioxidant

• Preventive Antioxidant
• Singlet oxygen quencher
• Antioxidant Enzymes
• Radical Scavenging Antioxidant
• Hydrogen donating antioxidants

32
Chlorophyll Effect on the Volatile Compounds of
Soybean Oil under Light at 10oC
Headspace Volatile Content
Storage in Hours
ESR
33
Carotenoid
C
H
O
b -
Apo - 8'- carotenal
Carotene
b -
O
Canthaxanthin
O
34
Effects of ?-Apo-8-Carotenal, ?-Carotene or
Canthaxanthin on Soybean Oil Oxidation
Peroxide Value (meq/kg oil)
Storage Time (hr)
35
Effects of ?-Apo-8-Carotenal, ?-Carotene or
Canthaxanthin on Soybean Oil Oxidation
Peroxide Value (meq/kg oil)
Storage Time (hr)
ESR
36
Diagram for Chlorophyll Photosensitized Singlet
Oxygen Soybean Oil Oxidation
3O2
A
hv
ISC
Sensitizer
3Sen
AO2
1Sen
1O2
ko
kr
Q
Q
Q
kQ
Q
kd
kQ
kox-Q
Sen
Sen
3O2
3O2
QO2
dAO2
ko3O2
k rA
K

ko3O2kQQ
k rA (kox-Q kQ)Q kd
dt

Plot of AO2 -1 vs. A -1 at various Q
37
Singlet Oxygen Quenching Plot
-1
K
Slope
Q4
?
1/AO2
?
?
Q3
Intercept K-1
?
?
Q2
?
?
?
1 K
?
Q1
?
?
?
?
1/A
38
Triplet Sensitizer Quenching Plot
Q3
?
Q2

?

1/ AO2
?
Q1
?

?
?
?

Slope K-1kd (ko 3O2 kQQ) /
ko3O2kr Intercept K-1(ko 3O2 kQQ) /
ko3O2
?
1/A
39
Quenching Mechanism of b-Carotene
1/ Peroxide Value ( 1/ M )
1/ Soybean Oil (1/ M)
40
Singlet Oxygen Quenching Rate (kqkox-Q) of
Carotenoids
Carotenoids Number of Conjugated
Quenching Rate Double Bonds
(M-1 sec-1) ?- apo - 8- Carotenal
10 2.86 ? 109 ?-
Carotene 11
4.60 ? 109 Canthaxanthin
13
1.12 ? 1010

41
Singlet Oxygen Quenching Mechanism of Carotenoid
1O2 1?-Carotene 3O2 3?-Carotene
3 ?-Carotene
1?-Carotene
Energy Transfer
Radiationless Transfer
42
Singlet Oxygen Quenchers
Quencher Quenching Rate (M-1sec-1) ?-
Carotene 4.60 ? 109 Ascorbic acid 1.08 ?
108 ?-Tocopherol 2.70 ? 107
43
Initiation Preventing Antioxidant Enzymes
Catalase
Superoxide dismutase
2O2- H2O2 O2
H2O
2H
Glutathione Peroxidase
2GSH
GSSG 2H2O
Glutathione Reductase
NADP
NADPH H
NADP Reductase
44
Propagation Preventing Hydrogen Donating
Antioxidant

• Antioxidant should donate a hydrogen to
• peroxy radical, which is mainly responsible
  for
• free radical chain reaction in lipid.
• Antioxidant radical should be stable not to
• initiate free radical chain reaction in lipid.

45
Mechanism and Radical Stabilization of Antioxidant
H
O
C
(
C
H
)
3
3

ROO
(Lipid Radical)
ROOH
O
C
H
E01000mV
3
.
O
O
.
C
(
C
H
)
C
(
C
H
)
3
3
3
3
O
C
H
O
C
H
3
3
E0 400mV
O
O
C
(
C
H
)
C
(
C
H
)
.
3
3
3
3
.
O
C
H
O
C
H
3
3
46
Standard Electron Reduction Potentials of Peroxy
Radical and Antioxidants
47
Bond Dissociation Enthalpy of Antioxidant
Hydroxyl Group
H
O
H
CH3
H
OCH3
H
48
Effect of Type and Position of Substituents on
Bond Dissociation Enthalpy of Antioxidant
H
O
87 (kcal/mol)
49
Reaction Rates of Peroxy Radical with Oleic Acid
and Antioxidant
K (M-1sec-1)
Reaction

1
Oleic Acid
ROO?

107
Antioxidant
ROO?
50
Reaction Rates of a-Tochopherol with Peroxy
Radical in Food Systems

• Medium K (M-1sec-1)
• Hydrophobic System 7 106
• Aqueous System 8 105
• Phospholipid bilayer 3 103

Trolox
51
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52
Singlet Oxygen Quenching Mechanism
-1
dAO2
k rA (kox-Q kq)Q kd
-1
ko3O2kQQ
K
ko3O2
kr A
dt
If there is only singlet oxygen quenching, the
equation is

-1
dAO2
-1
1
K
dt

Plot of AO2 -1 vs. A -1 at various Q
53
Triplet Sensitizer Quenching Mechanism
If carotenoids quenched triplet chlorophyll, the
steady state equation is
dAO2
ko3O2
k rA
K

ko3O2kQQ
k rA (kox-Q kQ)Q kd
dt

(kox-Q kQ)Q 0
dAO2
ko3O2
k rA
K

ko3O2kQQ
k rA kd
dt
Plot of AO2 -1 vs. A -1 at various Q
54
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