Formation of Reactive Oxygen
Species
Ground-state oxygen (3O2)
Oxygen is the most important factor on the development of lipid
peroxidation.
Ground state oxygen is itself a radical, with two unpaired electrons
each located in a π* antibonding orbital
Ground state oxygen has its outermost pair of electrons parallel spins:
does not allow them to react with most molecules
Ground-state or triplet oxygen is not very reactive
Can be activated by the addition of energy, and transformed into
reactive oxygen species
Singlet oxygen (1O2)
Formed from triplet oxygen
Conversion of oxygen to singlet state can be accomplished
by photosensitization in the presence of suitable
sensitizers, such as chlorophyl, or heme pigments
myoglobin or hemoglobin or by their derivatives
Has a pair of electrons with opposite spins
Singlet oxygen (1O2)
1ΣgO
has no unpaired electrons and thus does not qualify
as a radical. 37.5 kCla above the ground state O2
1ΣgO usually decay to 1ΔgO 22.4 kCla above the ground
2
2.
state O2
1ΔgO is not a true radical but is reported to be an
2
important ROS in reactions related to ultraviolet
exposition (UVA, 320-400 nm)
Excess singlet oxygen formation can lead to certain
diseases: porphyrias
2
Superoxide anion (O2-)
Monovalent reduction of triplet oxygen produces
superoxide
Formed in almost all aerobic cells: mitochondria
Outside of mitochondria: ER through oxidation process of
cytochrome P-450 and NADPH-cytochrome c reductase
Cu+2 + ascorbate + O2 ---Æ O2Fenton reaction also produces superoxide
Superoxide anion (O2-)
Not reactive enough to abstract hydrogen from lipids
Cannot enter the hydrophobic interior of membrane
because of its charged nature
Can produce hydrogen peroxide (Dismutation)
Involved in hydroxyl radical formation
Can also react with nitric oxide (NO.) to produce
peroxynitrate (OONO-)
Hydrogen peroxide (H2O2)
Not a radical
Important in biological systems because it can pass readily
through cell membranes
Superoxide-generating systems produces H2O2 by nonenzymatic or SOD-catalyzed dismutation
2 O2- + 2 H+ -----> H2O2 + O2 (SOD)
Hydroperoxyl radical (HO2.)
Protonation of O2- yields the HO2.
Hydroperoxyl radical (HO2.) is more reactive than superoxide
and can enter membrane fairly easily.
The pKa of HO2. is 4.7-4.8, and so only 0.25% of O2generated in physiological conditions is hydroperoxyl
radical. Localized pH drop can exist
Hydroxyl radical (OH·)
The most reactive oxygen species known: site specific
reaction
Can be produced by high-energy ionizing radiation
H2O -----> ·OH + ·H + H2 + H2O2 + H3O+ + e-aq
(ionizing radiation)
In vivo production comes from metal-dependent (Fe, Cu)
breakdown of hydrogen peroxide
Fe2+-dependent decomposition of H2O2 (Fenton reaction)
Fe2+ + H2O2 -----> Fe3+ + ·OH + OHFe2+ + H2O2 -----> ferryl? -----> Fe3+ + ·OH + OH-
Hydroxyl radical (OH·)
Fe3+-dependent multi-stage decomposition of H2O2
Fe3+ + H2O2 -----> ferryl + H2O2 -----> perferryl + H2O2 ----> .OH
Fe2+-EDTA dependent decomposition of H2O2
Fe2+-EDTA + H2O2 -----> intermediate species (ferryl) ----> Fe3+-EDTA + ·OH + OH-
Ozone (O3)
Ozone is not a free radical
Ionizing radiation of oxygen produces ozone
As singlet oxygen, it stimulates lipid peroxidation
Can induce damages at the lipid and proteins
Lipid (R.), peroxyl (ROO.) and alkoxyl radical (RO.)
Very strong reactivity and can abstract hydrogen atom
from lipids
Can be formed from the lipid radicals by iron complexes
ROOH + Fe3+-complex -----> ROO. + H+ + Fe2+complex
ROOH + Fe2+-complex -----> RO. + OH- + Fe3+-complex
Iron-Oxygen Complexes
Ferryl (Fe4+) and perferryl (Fe5+) radicals
Powerful oxidants as a component of enzyme or simple iron
complex
Ferryl species are generated by the interaction of H2O2 with
metmyoglobin
Fe(II)-complex + ROO• + H+ Æ Fe(III)-complex + ROOH
Fe(II)-complex + RO• + H+ Æ Fe(III)-complex + ROH
Nitric oxide (.NO)
Produced in various types of cells
Is not too reactive (poorly oxidizing function), even antioxidant under
physiological concentrations (up to 100 nM)
Reacts rapidly with oxygen to yield nitrogen dioxide (.NO2) which in
turn may react with .NO to yield nitrogen trioxide (N2O3)
Rapidly react with O2- and produce extremely reactive peroxinitrite
(ONOO-) which mediates oxidation, nitrosation, and nitration
reactions
ONOO- also decomposes to produce OH. radical
Thiyl radicals (RS.)
Thiol compounds (RSH) are frequently oxidized in the
presence of iron or copper ions:
RSH + Cu2+ ----> RS. + Cu+ + H+
These thiyl radicals have strong reactivity in combining with
O2
RS. + O2 ---> RSO2.
They are able to oxidize NADH into NAD., ascorbic acid and
to generate various free radicals (.OH and O2-).
Thiyl radicals can also be formed by homolytic fission of
disulfide bonds in proteins
Irradiation
Radiation: Energy moving through space in invisible waves
Light, infrared heat, microwaves, TVs all use radiant energy
Ionizing radiation: Shorter wavelengths in radiation. Capable of converting
atoms and molecules to ions via the removal of electrons. Destroy DNA
bonds in bacteria, pathogens and insects
Types of Irradiation
Radio Active Nuclides
X-ray
Gamma Rays
Cobalt-60
Cesium-137
Electron Beam
Linear Accelerator
E-beam Irradiation
Better Consumer Reception for Food Irradiation
X-ray can be generated
Other Industrial Uses
Physical and Chemical Modification and Cross-linking
Effects
Provides increased tensile, impact, and abrasion strength for
wire, cable, and tubing
Improves product performance
Gamma Irradiation
The method of choice for
Sterilization of single-use medical supplies such as syringes,
catheters, IV sets, gloves, face masks and more
Elimination of organisms from pharmaceuticals such as ointments
and solutions
Advantages
Offers superior material penetration
Economical for high and/or low volume operation
Is highly time-efficient
Offers versatile irradiator designs Imposes minimal restrictions on
product design and packaging
Irradiated Foods Marketed in the U.S.
Irradiated ground beef and poultry
18-20 M Lb sold in 2004
Fruits & vegetables
2 M lb sold annually
Mango, papaya, guava are currently sold by US retailers
Growing interest in blueberries, cherries, raspberries
The amount of irradiated tropical fruits will increase
rapidly in the future
Spices and Botanics
Commercially irradiated since 1986
175 M Lb of (1/3 of commercial spices consumed in the
US) are irradiated annually
Effect of Irradiation
Maximum Irradiation Dose
year
Food
Dose
Purpose
1963
Wheat flour
0.2-0.5
Control molds
1986
Fresh fruit & vegetables
1.0
Inhibit sprouting
Delay ripening
Disinfestation
1990
Poultry meat
3.0
1999
Refrigerated meat
Frozen meat
4.5
7.0
Dehydrated enzymes
10
Dehydrated spices & herbs
30
1986
(kGy)
Control pathogens
Shelf-lives of Meat Products after Irradiation
Meat Products
Dose
Untreated shelf
Irradiated shelf-
(kGy )
life (days)
life (days)
Beef top round
2.0
8-11
28
Beef burgers
1.54
8-10
26-28
Beef cuts under vacuum
2.0
NA
70
Corned beef
4.0
14-21
35
Whole and minced Lamb 2.5
Andrew et al. (1998)
7
28-35
Is Irradiated Food Safe?
Exhaustive chemical, biological, and feeding studies have shown
irradiated foods to be both safe and wholesome
In 1980, FDA and a Joint FAO/IAEA/WHO Expert Committee had
concluded on the wholesomeness of irradiated food that
irradiation of any food at < 10 kGy causes no toxicological
hazard and nutritional or microbiological problems
In 1983, a worldwide standard covering irradiated foods was adopted
by the Codex Alimentarius Commission
2-ACBs, benzene and methyl benzene (toluene)
Lipid Oxidation in Precooked Turkey Breast
TBARS (mg MDA/kg meat)
5
Frozen storage for 3 months
4
3
2
1
0
0 kGy 2.5 kGy 5 kGy
Aerobic
0 kGy
2.5 kGy 5 kGy
Vacuum
Cholesterol Oxidation in Turkey Thigh
Day 7
Vacuum pkg
Aerobic pkg
0 kGy
4.5 kGy
0 kGy
4.5 kGy
7α & 7β-hydroxychol.
36.0c
30.0c
51.9b
86.7a
β-epoxide
0b
0b
0b
7.2a
α-epoxide
0c
0c
6.4b
11.5a
20α-hydroxycholesterol
0b
0b
0b
1.4a
cholestanetriol
0
0
0
1.0
7-ketocholesterol
2.7c
1.5c
19.0b
27.1a
Total*
38.7c
31.6c
77.3b
134.7a
* μg COPs/g lipid
Vacuum-Packaged Raw Turkey Breast
Color of Irradiated Ground Beef
Nonirradiated
4.5 kGy irradiated
Production of Sulfur Volatiles
COOI
H3N-CH
I
CH2
I
CH2
I
S
I
CH3
CH3-S-CH3
(dimethyl sulfide)
H3C-S-S-CH3
(dimethyl disulfide)
S=C=S
(carbon disulfide)
H3C-S-S-S-CH3
(dimethyl trisulfide)
Methionine
…
COOI
H3N-CH
I
CH2
I
SH
Cysteine