Lipids
Class Presentations Will be
Discussed at the End of Class
Exams back next Monday
No class this Wednesday !!!!!
Lipids
Main functions of lipids in foods
 Energy and maintain human health
 Influence on food flavor
 Fatty
acids impart flavor
 Lipids carry flavors/nutrients
 Influence on
 Solids
food texture
or liquids at room temperature
 Change with changing temperature
 Participation in emulsions
Lipids
 Lipids
are soluble in many organic solvents
 Ethers
(n-alkanes)
 Alcohols
 Benzene
 DMSO (dimethyl sulfoxide)
 They
are generally NOT soluble in water
 C, H, O and sometimes P, N, S
Lipids

Neutral Lipids


Waxes





Long-chain alcohols (20+ carbons in length)
Cholesterol esters
Vitamin A esters
Vitamin D esters
Conjugated Lipids


Triacylglycerols
Phospholipids, glycolipids, sulfolipids
“Derived” Lipids


Fatty acids, fatty alcohols/aldehydes, hydrocarbons
Fat-soluble vitamins
Lipids
Structure
 Triglycerides or triacylglycerols
 Glycerol + 3 fatty acids
 >20 different fatty acids
Lipids 101
 Fatty
acids- the building block of fats
 A fat with no double bonds in it’s structure is said to
be “saturated” (with hydrogen)
 Fats with double bonds are referred to as mono-, di-,
or tri- Unsaturated, referring to the number of
double bonds. Some fish oils may have 4 or 5
double bonds (polyunsat).
 Fats are named based on carbon number and number
of double bonds (16:0, 16:1, 18:2 etc)
Lipids
liquid triacylglycerides “Oleins”
 Fat- solid or semi-solid mixtures of crystalline
and liquid TAG’s “Stearins”
 Lipid content, physical properties, and
preservation are all highly important areas for
food research, analysis, and product
development.
 Many preservation and packaging schemes are
aimed at prevention of lipid oxidation.
 Oil-
Nomenclature
 The
first letter C represents Carbon
 The number after C and before the colon
indicates the Number of Carbons
 The letter after the colon shows the Number of
Double Bonds
 ·The letter n (or w) and the last number indicate
the Position of the Double Bonds
Saturated Fatty Acids
Saturated Fatty Acids
8
7
CH3 CH2
O
5
3
4
6
2
1
CH2 CH2 CH2 CH2 CH2 C
Octanoic Acid
OH
Mono-Unsaturated Fatty Acids
Poly-Unsaturated Fatty Acids
Fatty Acids
Melting Points and Solubility in Water
Melting Point
z
Solubility in H2O 2
Fatty acid chain length
Unsaturated Fatty Acids
8
CH3
7
CH2
5
6
CH2 CH2
4
CH2
O
3
2
1
CH2 CH2 C
OH
3 - Octenoic Acid
8
7
CH3 CH2
O
5
3
4
6
2
1
CH2 CH2 CH2 CH2 CH2 C
3, 6 - Octadienoic Acid
OH
Lipids
Properties depend on structure
 Length of fatty acids (# of carbons)
 Position of fatty acids (1st, 2nd, 3rd)
 Degree of unsaturation:

Double bonds tend to make them a liquid oil


Hydrogenation: tends to make a solid fat



Significantly lowers the melting point
Significantly increases the melting point
Unsaturated fats oxidize faster
Preventing lipid oxidation is a constant battle in the
food industry
Fatty Acids
Melting Points and Solubility in Water
Melting Point
z
Solubility in H2O 2
Fatty acid chain length
Characteristics of Fatty Acids
Fatty Acids
M.P.(C)
mg/100 ml
in H2O
C4
-8
C6
-4
970
C8
16
75
C10
31
6
C12
44
0.55
C14
54
0.18
C16
63
0.08
C18
70
0.04
Fatty Acids
O
R C OH
#1 Carbon
O
R C OH
Acid Group
Polar End - Hydrophilic End
Non-polar End - Hydrophobic End
(Fat-soluble tail)
Lipids 101
 Fatty
acid profile- quantitative determination of the
amount and type of fatty acids present following
hydrolysis.
 To help orient ourselves, we start counting the
number of carbons starting with “1” at the
carboxylic acid end.
O
C C C C C C C C C C C C C C C C C C
18
1
OH
Lipids 101
 For
the “18-series” (18:0, 18:1, 18:2, 18:3) the
double bonds are usually located between carbons
9=10 12=13 15=16.
O
C C C C C C C C C C C C C C C C C C
18
16 15 13 12 10 9
1
OH
Lipids 101
 The
biomedical field started using the OMEGA (w)
system (or “n” fatty acids).
 With this system, you count just the opposite.
 Begin counting with the methyl end
 Now the 15=16 double bond is a 3=4 double bond or
as the medical folks call it….an w-3 fatty acid
C
C C C C C C C C C C C C C C C C C C
1
6 7
3 4
18
9 10
OH
Tuning Fork Analogy-TAG’s
Envision a Triacylglyceride as a loosely-jointed E
 Now, pick up the compound by the middle chain,
allowing the bottom chain to hang downward in a
straight line.
 The top chain will then curve forward and form an

h
Thus the “tuning fork” shape
 Fats will tilt and twist to the lowest free energy
level

Lipids

Lipids are categorized into two broad classes.

The first, simple lipids, upon hydrolysis, yield up to two types
of primary products, i.e., a glycerol molecule and fatty acid(s).

The other, complex lipids, yields three or more primary
hydrolysis products.

Most complex lipids are either glycerophospholipids, or
simply phospholipids


contain a polar phosphorus moiety and a glycerol backbone
or glycolipids, which contain a polar carbohydrate moiety
instead of phosphorus.
Lipids
Other types of lipids
Phospholipids
 Structure similar to triacylglycerol
 High in vegetable oil
 Egg yolks
 Act as emulsifiers
Where Do We Get Fats and Oils?






“Crude” fats and oils are derived from plant and animal sources
Several commercial processes exist to extract food grade oils
Most can not be used without first “refining” before they reach
consumers
During oil refining, water, carbohydrates, proteins, pigments,
phospholipids, and free fatty acids are removed.
Crude fats and oils can therefore be converted into high quality
edible oils
In general, fat and oil undergo four processing steps:





Extraction
Neutralization
Bleaching
Deodorization
Oilseeds, nuts, olives, beef tallow, fish skins, etc.

Rendering, mechanical pressing, and solvent extraction.
Fats and Oils: Processing
Extraction
 Rendering
 Pressing oilseeds
 Solvent extraction
Soybean
Peanut
Rape Seed
Safflower
Sesame
Fats and Oils
Further Processing

Degumming


Refining


Remove free fatty acids (alkali +
water)
Bleaching


Remove phospholipids with water
Remove pigments (charcoal filters)
Deodorization

Remove off-odors (steam, vacuum)
Where Do We Get Fats and Oils?

Rendering
 Primarily for extracting oils from animal tissues.
 Oil-bearing tissues are chopped into small pieces and
boiled in water.
 The oil floats to the surface of the water and skimmed.
 Water, carbohydrates, proteins, and phospholipids
remain in the aqueous phase and are removed from the
oil.
 Degumming may be performed to remove excess
phospholipids.
 Remaining proteins are often used as animal feeds or
fertilizers.
Where Do We Get Fats and Oils?


Mechanical Pressing
Mechanical pressing is often used to extract oil from
seeds and nuts with oil >50%.
 Prior to pressing, seed kernels or meats are ground into
small sized to rupture cellular structures.
 The coarse meal is then heated (optional) and pressed in
hydraulic or screw presses to extract the oil.
 Olive oils is commonly cold pressed to get extra virgin
or virgin olive oil. It contains the least amount of
impurities and is often edible without further processing.
 Some oilseeds are first pressed or placed into a screwpress to remove a large proportion of the oil before
solvent extraction.
Where Do We Get Fats and Oils?
Solvent Extraction
 Organic solvents such as petroleum ether, hexane, and 2-propanol can be added
to ground or flaked oilseeds to recover oil.
 The solvent is separated from the meal, and evaporated from the oil.
Neutralization
 Free fatty acids, phospholipids, pigments, and waxes exist in the crude oil
 These promote lipid oxidation and off-flavors (in due time)
 Removed by heating fats and adding caustic soda (sodium hydroxide) or soda
ash (sodium carbonate).
 Impurities settle to the bottom and are drawn off.
 The refined oils are lighter in color, less viscous, and more susceptible to
oxidation (without protection).
Bleaching
 The removal of colored materials in the oil.
 Heated oil can be treated with diatomaceous earth, activated carbon, or
activated clays.
 Colored impurities include chlorophyll and carotenoids
 Bleaching can promote lipid oxidation since some natural antioxidants are
removed.
Where Do We Get Fats and Oils?
Deodorization
 The final step in the refining of oils.
 Steam distillation under reduced pressure (vacuum).
 Conducted at high temperatures of 235 - 250ºC.
 Volatile compounds with undesirable odors and tastes
can be removed.
 The resultant oil is referred to as "refined" and is ready
to be consumed.
 About 0.01% citric acid may be added to inactivate prooxidant metals.
Fats and Oils
Further Processing
Hydrogenation
 Add hydrogen to an oil to “saturate” the fatty acid
double bonds




Conducted with heated oil
Often under pressure
In the presence of a catalyst (usually nickel)
Converts liquid oils to solid fats
 Raises melting point
Hydrogenating Vegetable oils can
produce trans-fats
H H
C C
Cis-
H
C C
Trans-
H
The cis- and trans- forms of a fatty acid
Fats and Oils in Foods


SOLID FATS are made up of microscopic fat crystals. Many fats
are considered semi-solid, or “plastic”.
PLASTICITY is a term to describe a fat’s softness or the
temperature range over which it remains a solid.
Even a fat that appears liquid at room temperature contains a small number of
microscopic solid fat crystals suspended in the oil…..and vice versa

PLASTIC FATS are a 2 phase system:



Plasticity is a result of the ratio of solid to liquid components.



Solid phase (the fat crystals)
Liquid phase (the oil surrounding the crystals).
Plasticity ratio = volume of crystals / volume of oil
Measured by a ‘solid fat index’ or amount of solid fat or liquid oil in a lipid
As the temperature of a plastic fat increases the fat crystals melt
and the fat will soften and eventually turn to a liquid.
Fat and Oil: Further Processing
 Winterizing
(oil)
 Cooling
a lipid to precipitate solid fat crystals
 DIFFERENT from hydrogenation
 Plasticizing
 Modifying
(fat)
fats by melting (heating) and solidifying
(cooling)
 Tempering (fat)
 Holding
the fat at a low temperature for several
hours to several days to alter fat crystal properties
(Fat will hold more air, emulsify better, and have
a more consistent melting point)
Lipid Oxidation
Oleic acid
Radical Damage,
Hydrogen
Abstraction
Formation of a
Peroxyl Radical
Simplified scheme of lipoxidation
H H H H
H H H H
H H H H
R C C C C R
R C C C C R
R C C C C R
H
H
+ Catalyst
H
*
+ Oxygen
H
O
O
Primary Drivers
 Temperature-basic
 Water
rxn kinetics
Activity
 Both
high and low Aw
 At low Aw, peroxides decompose faster and metal
ions are better catalysts in a dry environment
 Metal
Ions-catalysts
 Light-energy source
 Singlet Oxygen- ROS, highly electrophilic
 Reacts
1,500 times faster at C=C than ground state O2
 Enzymes
ie. Lipoxygenase (LOX)
Initiation of Lipid Oxidation

There must be a catalytic event that causes the initiation of
the oxidative process


Enzyme catalyzed
“Auto-oxidation”

Excited oxygen states (i.e singlet oxygen): 1O2









Triplet oxygen (ground state) has 2 unpaired electrons in the same spin in
different orbitals.
Singlet oxygen (excited state) has 2 unpaired electrons of opposite spin in the
same orbital.
Metal ion induced (iron, copper, etc)
Light
Heat
Free radicals
Pro-oxidants
Chlorophyll
Water activity
Considerations for Lipid Oxidation
 Which
hydrogen will be lost from an unsaturated
fatty acid?
 The longer the chain and the more double
bonds….the lower the energy needed.
Bond Strength
85 kCal
103 kCal
65 kCal
65 kCal
Fatty acid
.OH
or other
Free radicals
..
.
Propagation Reactions
Initiation
Peroxyl radical
Ground state oxygen
Hydroperoxide
decomposition
Hydroperoxide
Alkoxyl radical
Start all over again…
New
Radical
Hydroxyl radical!!
Mechanism of Photooxidation
Chlorophyll
3O
1
or
2
Singlet Oxygen Oxidation
1
O2
HOOC
OOH
HOOC
OOH
HOOC
OOH
HOOC
OOH
HOOC
Autoxidation
9
HOOC
10
12
13
11
8
14
H
H
9
10
12
+
13
11
8
9
10
12
14
13
10
9
11
+
8
12
13
11
14
8
14
O2
OO
10
9
8
12
13
11
14
Singlet Oxygen Oxidation
1
O2
HOOC
HOOC
OOH
OH
O
Nonanal
Secondary Products: Aldehydes
Volatile Aldehydes from Oxidation
Lipid Oxidation

Peroxide decomposition can generate aldehydes, ketones, alcohols, various
hydrocarbons, and epoxides. Many are volatile, and many are unappealing.
Relative Oxidation Rates
 How
fast is lipid oxidation?
Fatty Acid
Relative Rate
 Steric acid (18:0)
1
 Oleic acid (18:1 n9)
100
 Linoleic acid (18:2 n6)
1,200
 Linolenic acid (18:3 n3)
2,500
Termination of Lipid Oxidation
Although radicals can “meet” and terminate propagation
by sharing electrons….
 The presence or addition of antioxidants is the best way in
a food system.
 Antioxidants can donate an electron without becoming a
free radical itself.

Antioxidants and Lipid Oxidation
BHT – butylated hydroxytoluene
 BHA – butylated hydroxyanisole
 TBHQ – tertiary butylhydroquinone
 Propyl gallate
 Tocopherol – vitamin E
 NDGA – nordihydroguaiaretic acid
 Carotenoids

Polar Antioxidants

Most effective in
nonpolar or less polar
environment


Bulk oils
Located at the oil-air
interface or in reverse
micelles

High amount of
oxidants present here
Yellow Oil
Blue water
Phospholipids
Non-polar Antioxidants

Most effective in polar
environment


Oil-in-water emulsions
Located at the water-oil
interface


Dissolved in oil droplets
of the emulsion
Allows access to
oxidizing agents located
in the water phase


Peroxides
Oxidizing metals
Chemical Tests for
Lipid Characterizations
Iodine Value
 Measure
of the degree of unsaturation in an oil
or the number of double bonds in relation to
the amount of lipid present
 Defined as the grams of iodine absorbed per
100-g of sample.
 The
higher the amount of unsaturation, the
more iodine is absorbed.
 Therefore the higher the iodine value, the
greater the degree of unsaturation.
Iodine Value
A
known solution of KI is used to reduce excess ICl
(or IBr) to free iodine
R-C-C = C-C-R + ICl  R-C-CI - CCl-C-R + ICl
[Excess]
(remaining)
scheme: ICl + 2KI  KCl + KI + I2
 The liberated iodine is then titrated with a
standardized solution of sodium thiosulfate using a
starch indicator
 I2 + Starch + thiosulfate = colorless endpoint
 Reaction
(Blue colored)
Iodine Value
Used to characterize oils:

Following hydrogenation
 During oil refining (edible oils)
 Degree of oxidation (unsaturation decreases
during oxidation)
 Comparison of oils
 Quality control
Iodine value: g absorbed I2/ 100 g fat
Iodine Value of some oils (Table 14-2)
Source
Beef Tallow
Olive, Palm,
Peanut
Corn, Cottonseed
I2 Value
<50
< 100
100 - 130
Highly saturated
High in 18:1
High in 18:1 and 18:2)
Linseed, Soybean,
safflower, conola
> 130
18:1, 18:2, 18:3
Fish
>150
18:1, 18:2, 18:3
(longer chains)
What can we conclude about the COMPOSITION
or STRUCTURE of each of these oil types?
Automated
Iodine Value
Determination
Standard Iodine Value
A = 23
B = 44
C = 67
D = 89
E = 111
Measures IBr or ICl
Consumption (neg. peak)
Consumption over
time
Chemical Tests
Saponification Value
Saponification Value
Saponification is the process of breaking down or
degrading a neutral fat into glycerol and fatty acids
by treating the sample with alkali.
Heat
Triacylglyceride ---> Fatty acids + Glycerol
KOH
Definition: mg KOH required to titrate 1g fat
(amount of alkali needed to saponify a given amount of fat)
Typical values: Peanut = 190, Butterfat = 220
Saponification Value




The mg KOH required to saponify
triacylglycerides into glycerol plus fatty acids
is related to:
average fatty acid chain length or
average fatty acid molecular weight
Divide molecular weight by 3 to get average of
the fatty acids present
Chemical Tests for Oxidation
Lipid Oxidation
Hydrolysis
Peroxide Value
Oxidation Tests
LIPID OXIDATION
Reactants and Products
35
Lipid System Under
Oxidizing Conditions
30
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10
5
0
1
2
3
4
5
Time
6
7
8
9
Free Fatty Acids (FFA’s)


Degree of hydrolysis (hydrolytic rancidity)
High level of FFA means a poorly refined fat
or fat breakdown after storage or use.
Measures of Oxidation
 Oxidation is a very complex reaction - no one test
will measure all of the reactants or products.
 Some assays measure intermediates while others
measure end products.
Peroxide Value
 Measures peroxides and hydroperoxides in an
oil which are the primary oxidation products
(usually the first things formed).
 The peroxide value measures the “present
status of the oil”. Since peroxides are
destroyed by heat and other oxidative
reactions, a seriously degraded oil could have
a low PV.
Peroxide Value
KI + peroxyl radical yields free Iodine (I2)
 The iodine released from the reaction is measured in the
same way as an iodine value.
 I2 in the presence of amylose is blue.
 I2 is reduced to KI and the endpoint determined by loss of
blue color.
4I + O2 + 4H
2I2 + 2H2O
Thiobarbituric acid (reactive substances) TBA OR TBARS
Tests for end products of oxidation – aldehydes, Malonaldehyde
(primary compound), alkenals, and 2,4-dienals
A pink pigment is formed and measured at ~530 nm.
TBARS is firmly entrenched in meat oxidation research and is a
method of choice.
TBARS measure compounds that are volatile and may react further
with proteins or related compounds.
High TBA = High Oxidative Rancidity
HEXANAL Determination
 Good indictor of the end products of oxidation
(if there are any).
 Standard method in many industries.
 Aldehyde formation from lipid oxidation.
 Nonenal is also a common end-product
Conjugated Fatty Acids
During oxidation, double bond migration
occurs and conjugated fatty acids are
formed.
They absorb light efficiently and can be
monitored in a spectrophotometer.
R C C C C C C C C R
TECHNIQUES OF MEASURING OXIDATIVE
STABILITY
Induction Period: is defined as the length of time
before detectable rancidity or time before rapid
acceleration of lipid oxidation
MEASURING OXIDATIVE STABILITY
Active
Oxygen Method - Air is bubbled through oil or fat at
97.8°C. Time required to reach peroxide value of 100 meq/kg
fat determined. (method replaced by OSI)
Stability Index – automated Rancimat (instrumental
method). Air bubbled through sample (110°C). Oil degrades
to many acidic volatiles (e.g. formic acid) which are carried by
the air into a water trap. Conductivity of the water can then be
assessed.
Oil
Free Radicals
 What
are free radicals?
 Where are free radicals from?
 How damaging are free radicals?
 How do we control free radicals?
What are free radicals?

Any molecular species capable of independent
existence, which contains one or more unpaired
valence electrons not contributing to intramolecular
bonding….is a free radical.
The most frequent radicals are oxygen-derived free radicals, also known
as reactive oxygen species (ROS):
Superoxide (O2·-)
Peroxyl (ROO˙)
Alkoxyl (RO˙)
Hydroxyl (HO˙)
Nitric oxide (NO˙)
Other ROS are non-radicals such as singlet oxygen (O2), hydrogen
peroxide (H2O2), and hypochlorous acid (HClO).
Where do they come from?

Free radicals are produced by oxidation/reduction reactions
in which there is a transfer of only one electron at a time, or
when a covalent bond is broken and one electron from each
pair remains with each atom.
1) Normal ongoing metabolism, especially from the
electron transport system in the mitochondria and
from a number of normally functioning enzymes
2) Environmental factors such as pollution, radiation,
cigarette smoke and toxins can also spawn
biologically-derived free radicals.
How damaging are free radicals?

ROS may be very damaging, since they can
attack:





Lipids in cell membranes
Proteins in tissues or enzymes
Carbohydrates
DNA
These cause cell membrane damage, protein
modification, and DNA damage.
 Thought to play a role in aging and several
degenerative diseases (heart disease, cataracts,
cognitive dysfunction, and cancer).
 Oxidative damage can accumulate with age.
Our Body vs. Our Food
 Biological radicals
 Food-based radicals
 Where
do these 2 areas cross?
Functional Foods Concept
 Certain
food ingredients have health benefits
beyond basic nutrition
 Recent development only: since ~1975
 The concept that ‘non-nutrients’ were beneficial
has taken off since then
 First idea in scientific community: antioxidant
compounds may protect against chronic diseases
Free Radicals
 Early
1950’s: cell damage is due to reactive
oxygen species called “free radicals”
 Unstable, ‘damaged’ molecule that is missing an
electron
 Highly reactive; reacting to some measurable
extent with any molecule they come in contact
with
 In living systems, cell injury or disease
 In foods, quality-degrading impact
Reactive Oxygen Species (ROS)
 Primary
target list: protein, lipid, DNA, and
carbohydrates
 End results: cancer, CHD, stroke, arterial
disease, rheumatoid arthritis,
Parkinson’s/Alzheimer disease, cataracts,
macular degeneration….many more
 Aging by slow oxidation?
The Defense
 Minimize
contact between free radicals and
important systems (like cellular components)
 Cell membranes are one of our best barriers
 Metal chelation system in-place
 Protease enzymes are in place to remove
damaged proteins for replacement by new
 “Repair enzymes” help to restore DNA
 “Antioxidant enzymes”-superoxide dismutase,
catalase, glutathione peroxidase
Best Defense…A Good Offense
 “Nutrients”
that can’t be synthesized in
vivo: vitamin C, vitamin E, (pro)vitamin A
 “Non-nutrients”: polyphenolics/carotenoids
 Diet
 What
is only source….are they “essential”?
about conditions of “oxidative stress”?
 This
is a condition when pro-oxidants
outnumber antioxidants (I.e. decreased immune
response, environmental factors, hypertension,
poor diet).
Foods and the Antioxidant Link

Soy- isoflavones, polyphenolics
 Tea- polyphenolics, flavans
 Coffee- polyphenolics
 Wine- polyphenolics
 Rosemary- carnosic acid, rosmaric acid
 Citrus- flavonoids
 Onions- sulfur cpds, flavonoids
 Berries- flavonoids, polyphenolics
 Vegetables- carotenoids, polyphenolics
Antioxidants in Food Systems
Oxidative Stress-the food remedy
 Diet:
 Inflammation- tocopherol
 Smoke-
ascorbic acid
 Physical stress- carotenoids
 Pollution- carotenoids
 Environment:
 Radiation- glutathione
 Carcinogens- antioxidant enzymes, diet
modification
Oxidative Stress and Foods
 Tocopherol- vegetable
oil, whole grains,
vegetables, fish/poultry
 Ascorbic acid- citrus, berries, tomato, leafy
veggies, brassicas (broccoli, cauliflower)
 Carotenoids- yellow/orange fruits and
veggies, tomatoes, green leafy veggies.
 Polyphenolics- coffee/tea, grains, all fruits
and vegetables
Magic Bullets…for our body?
Most likely not…
 Will increasing the intake of antioxidants
modulate disease prevention? Will we live longer
with no health problems?
 Lung cancer and ß-carotene: Whoa…
 Antioxidant compounds have demonstrated
benefits (both acute and long-term) of preventing
or postponing the onset of many degenerative
diseases, but clinical trials are full of holes, and
conclusive evidence of “the bullet” is still not with
us.

Magic Bullets…for our foods?

Will increasing the use of antioxidants in foods
modulate all oxidative damage?
 Will food products “live” longer with no quality
problems?
 Pro-oxidant nature of ascorbic acid: Whoa…
 Ascorbic acid does not always act linearly in food
systems
 In the presence of metal ions (ie. Fe/Cu) it can
generate reactive oxygen species (peroxides) or free
radicals (hydroxyl radicals)
Causes and Effects

ß-carotene and lung cancer: small, but significant
increase with smokers
 Tocopherols and CHD: protect lipoproteins or inhibit
blood clotting (which initiates heart attacks)
 Tocopherols and Alzheimer’s: reducing oxidative
stress by supplementation
 Cataracts and vitamins (A,C,E): inverse association
 Macular degeneration and carotenoids: inverse
 Vitamin C and the Common Cold: shorter, milder
colds
Structure-Based AOX
Polyphenolics
Structure of flavonoids
OH
3’
HO
7
6
8
O
A
5
C
4
B
2
3
X
OH O
Flavonols: X=OH
Flavones: X=H
Flavanones: No 2-3 db
4’
5’
OH
B-Ring Substitutions
HO
O
OH
B
Kaempferol
A C
OH
OH O
Quercetin
OH
OH
HO
O
Myricetin
OH
OH
OH O
OH
HO
O
OH
OH
OH O
Quercetin
4 –OH groups
OH
OH
HO
O
2-3 db
OH
OH O
3-OH
4-oxo function
Catechin
4 –OH groups
OH
OH
HO
O
OH
OH
3-OH
Cyanidin
4 –OH groups
OH
OH
+
HO
O
OH
OH
Structurally Similar Compounds
OH
OH
OH
OH
HO
HO
O
O
OH
OH
OH
OH O
Quercetin
AOX = 4.7
OH
OH
+
O
HO
Cyanidin
OH AOX = 4.4
OH
Catechin
AOX = 2.4
Importance of the
3-OH group
OH
OH
HO
O
O
O
OH
OH
HO
O
Quercetin-3-glucoside
AOX ~ 2.5
O
OH
O
O
OH
OH O
Quercetin
AOX = 4.7
O
OH O
OH
HO
O
OH O
Luteolin
AOX = 2.1
Importance of the 4-Oxo Function
 Works
with the 2-3 double bond in the C-ring and
is responsible for electron delocalization from the
B-ring.
 3-OH and 5-OH substitutions with the 4-oxo
function are best for maximum AOX properties
OH
OH
HO
Structurally, quercetin has
all the right components to
make for the “perfect” antioxidant.
O
OH
OH O
Importance of the 2-3 db
OH
OH
OH
OH
HO
HO
O
O
OH
OH
OH O
Quercetin
AOX = 4.7
OH O
Taxifolin
AOX = 1.9
More on the Phenolic Acids
COOH
COOH
COOH
1
2 Ortho
3
4
Para
Meta
Hydroxybenzoic
Acid
(HBA)
1
1
2
2
3
3
4
4
Hydroxyphenylacetic
Acid
(HPA)
Cinnamic
Acid
(CA)
COOH
HO
0.08
COOH
HO
1.19
p-OH-benzoic
p-coumaric
Protocatechuic
Caffeic
HO
COOH
2.22
HO
COOH
1.26
HO
HO
MeO
MeO
HO
COOH
1.43
Vanillic
Ferulic
HO
1.90
COOH
Antioxidants in Food Systems
What Makes a Good Antioxidant?
Polyphenolics- Radical scavengers
 Number
of hydroxy groups (-OH)
 Location of hydroxy groups (on benzene ring)
 Presence of a 2-3 double bond (flavylium ring)
 4-oxo function (flavylium ring)
 Synergistic/antagonistic reactions
with other
antioxidant compounds
OH
OH
HO
HO
O
COOH
COOH
OH
HO
OH O
What Makes a Good Antioxidant?
Carotenoids
The number of conjugated double bonds (9+ is best)
 Substitutions on ß-ionone group (on the end)

 Radical
scavengers
Beta-Carotene
R* + CAR => R- + CAR*+
 Chain
breakers
ROO*+ CAR => ROO-CAR*
ROO-CAR* + ROO* => ROO-CAR-ROO
 Singlet
1
oxygen quenchers
O2* + 1CAR =>
3O
2
+ 3CAR*
Tocopherol

Alpha-tocopherol = Vitamin E

beta and gamma forms also

Synergist with carotenoids and selenium and is
regenerated by vitamin C
 Efficiency determined by the bond dissociation energy
of the phenolic -OH bond
 The heterocyclic chromanol ring is optimized for
resonance stabilization of an unpaired electron.
HO
O
Antagonism-Synergism-Metals

Many antioxidant work for and against each other
 An antioxidant in a biological system my be
regenerated
 In mixed ROS…inefficiency of one antioxidant to
quench all the different radicals.
 No way of knowing if the “better” antioxidant for a
particular radical is doing all the work or not.
 Will a better antioxidant for a given food system “beat
out” a lesser antioxidant (antagonistic response) in
order to quench the radicals.
Example: Factors Affecting
AOX of Bell Peppers
Chemical interactions
 In vitro models
 Find synergistic/antagonistic effects
Free metal ions
 Diluted isolates
 Add metal chelator
Flavonoid Ascorbic
AOX ?
AOX with Quercetin Interactions
(ß-Carotene Bleaching)
Rate of Inhibition
60
Quercetin Only
50
Quercetin + 5 mM Ascorbic acid
40
Quercetin + 2.5 mM Caffeic acid
30
20
10
0
-10
10
20
30
450 ppm Caffeic = 47 %
880 ppm Ascorbic = 15%
40
50
60
70
Quercetin (ppm)
80
90 100
HO
OH
OH
O
OH
OH O
AOX with Luteolin Interactions
(ß-Carotene Bleaching)
Rate of Inhibition
90
80
Luteolin only
70
Luteolin + 2.5 mMCaffeic acid
60
Luteolin + 5 mM Ascorbic acid
50
40
30
20
10
0
OH
OH
-10
10
20
30
450 ppm Caffeic = 47 %
880 ppm Ascorbic = 15%
40
50
60
70
80
HO
90
O
Luteolin (ppm)
OH O
100
Theoretical Quercetin
“Regeneration” Scheme
OH
O
B
HO
OH O
Quercetin
Quercetin
HO
O
A C
OH
OH
OH
OH
HO
OH
OH
O
OH
O
H.
O
OH
O
O
Reduced resonation in A and B-ring
Minor regeneration by ascorbic acid
Reduced resonation
in A and
B rings
Minor regeneration
by caffeic
acid
Delocalization of C-ring
Delocalization
of C-ring
Minor regeneration by ascorbic acid
Minor regeneration by caffeic acid
Theoretical Luteolin
“Regeneration” Scheme
OH
OH
OH
HO
O
B
OH
OH
HO
O
OH
HO
O
A C
OH O
Luteolin
Luteolin
H.
O
O
O
O
Excellent resonance stability in A-ring
Excellent resonance
stability
in A-ring
Highly regenerated
by ascorbic
acid
No regeneration
by caffeic acid
acid
Highly regenerated
by ascorbic
Electron donation by C-ring
Electron donation
by C-ring
No regeneration by caffeic acid
Antioxidant Activity after Dilution
% Inhibition of Carotene Bleaching
50
Bell Pepper Juice (Boiled)
40
30
20
10
0
-10
-20
-30
1X
2X
4X
8X
Dilutions
16X
32X
Antioxidant Activity with Chelator
% Inhibition of Bleaching
100
Bell Pepper Juice (Boiled)
Bell Pepper Juice + 500 ppm EDTA
80
60
40
20
0
-20
100 %
Juice
75%
Juice
50%
Juice
25%
Juice
Antioxidant Methods
HAT and SET Reactions
 Hydrogen Atom
Transfer (HAT) vs. Single
Electron Transfer (SET)
 Antioxidants can work in one of two ways (HAT
or SET).
 End result is the same for both, differing in
kinetics and side rxns.
 HAT and SET rxns may occur in parallel
 Determined
by antioxidant structure and properties
 Solubility and partition coefficient
 System solvent, system pH
HAT
 HAT-based methods
measure the classical
ability of an antioxidant to quench free radicals
by hydrogen donation (AH = any H donor)
SET
 SET-based
methods detect the ability of a
potential antioxidant to transfer one electron to
reduce any compound, including metals,
carbonyls, and radicals.
 Also based on deprotonation, so pH dependent
HAT vs SET
HAT
 Selectivity in HAT rxs are determined by the bond dissociation
energy of the H-donating group in the antioxidant
 Antioxidant reactivity or capacity measurements are therefore
based on competition kinetics.
 Reactions are solvent and pH independent and are very fast
 Common reducing agents (Vitamin C) are an interference
SET
 Usually slow and can require long times to reach completion
 Antioxidant reactivity is based on a percent decrease, rather than
kinetics
 Very sensitive to ascorbic acid and other reducing agents.
 Trace amounts of metal ions will interfere, and cause overestimation and inconsistent results.
Antioxidants and Radicals

Four sources of antioxidants:

Enzymes


Large molecules


estrogen, angiotensin, melatonin
Multiple free radical and oxidant sources


ascorbic acid, glutathione, uric acid, tocopherol, carotenoids, phenols
Hormones


albumin, ferritin, other proteins
Small molecules


Superoxide dismutase, glutathione peroxidase, and catalase
O2, O2·-, HO˙, NO˙, ONOO-, HOCl, RO(O)˙, LO(O)
Oxidants and antioxidants have different chemical and
physical characteristics.
Complex Systems: Singlet Oxygen

Carotenoids are not good peroxyl radical quenchers compared to
polyphenolics
 Carotenoids are exceptional singlet oxygen quenchers compared
to polyphenolics
 However, singlet oxygen is not a radical and does not react via
radical mechanisms
 Singlet oxygen reacts by its addition to fatty acid double bonds,
forming endoperoxides, that can be reduced to alkoxyl radicals,
that initiate radical chain reactions.
 Now we have multiple reaction characteristics and multiple
mechanisms
 No single assay will accurately reflect all of the radical sources
or test all the antioxidants in such a complex system.
Method Selections for Antioxidants





Controversy exists over standard methods for antioxidant
determination
Historical use and peer-review acceptance is critical
Use my multiple labs to highlight strength, weakness, and
effectivness
New methods take time to adopt and accept
An “ideal” method:










Measures chemistry actually occurring in potential application
Utilizes a biologically relevant radical source
Simple to run
Uses a defined endpoint and chemical mechanism
Instrumentation is readily available
Good within-run and between-day reproducibility
Adaptable for both hydrophilic and lipophilic antioxidants
Adaptable for multiple radical sources
Adaptable for high-through-put analysis
Understanding of the range of use and recognition of interfering agents
HAT assays
 ORAC
 Oxygen
Radical Absorbance Capacity
 Measures inhibition of peroxyl radical induced
oxidations in chain breaking activity by H atom
transfer
 TRAP
 Total
Radical-Trapping Antioxidant Parameter
 Measures the ability to interfere with peroxyl
radicals or stable free radicals
SET assays
 FRAP
 Ferric
Reducing Antioxidant Power
 The reaction measures the reduction capacity of a
ferric compound to a color end-product
 CUPRAC
 Copper
Reduction Assay
 Variant of FRAP assay using Cu instead of Fe
 Folin-Ciocalteu assay
 Reduction
of oxidized iron and molybdenum