Transcript Antioxidant assays

Antioxidants – an overview
• Antioxidants are molecules capable of reducing the causes
or effects of oxidative stress
• Oxidative stress can be caused by environmental factors,
disease, infection, inflammation, aging (ROS production)
• ROS or “reactive oxygen species” include free radicals and
other oxygenated molecules resulting from these factors
• The body produces some endogenous antioxidants, but
dietary antioxidants may provide additional line of defense
• Flavonoids & other polyphenolics, Vitamins C & E, and
carotenoids are the most common dietary antioxidants
Resources:
Gordon, M. H., “Dietary Antioxidants in Disease Prevention,” Natural Product Reports (1996)
13: 265-273; Pietta, P.-G., “Flavonoids as Antioxidants”, Journal of Natural Products (2000)
63: 1035-1042; Scalbert, A., Johnson, I.T., Saltmarsh, M. “Polyphenols: antioxidants and
beyond,” American Journal of Clinical Nutrition (2005) 81: 215S-217S
Sources of antioxidants in the diet
Sources of antioxidants in the diet:
Polyphenols, carotenoids & vitamins
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Red wine (tannins, resveratrol, flavonoids)
Cranberries & blueberries (flavonoids & tannins)
Strawberries (ellagic acid, ellagitannins)
Tea (EGCG & other catechins, tannins)
Chocolate (catechins)
Onions (quercetin)
Spinach & leafy greens (lutein & zeaxanthin)
Eggs (lutein)
Citrus fruits (Vitamin C)
Plant oils (Vitamin E & omega-3)
Radicals
The enemy: “Reactive Oxygen Species” (ROS)
are highly reactive free radicals
 Superoxide (O2-.)
 Hydroxyl radical (.OH)
 Peroxyl radicals (.OOH)
They form as the result of stress, inflammation,
and the human body’s natural defenses
Many are formed in the mitochondria, by
phagocytes and peroxisomes, and by CYP450
activities.
They target tissue, proteins, lipids and DNA
Aging = cumulative damage over the years
What do antioxidants do?
 Prevent formation of ROS
 Inhibit xanthine oxidase, COX, LOX, GST
monooxygenases, chelate metals
 Scavenge/remove ROS before they can damage
important biomolecules
 Aid the human body’s natural defenses
 Upregulate SOD, catalase, glutathione peroxidase
 Repair oxidative damage
 Eliminate damaged molecules
 Prevent mutations
Structures of some “polyphenolic” antioxidants found
in fruits, vegetables & legumes
OH
cyanidin, an
"anthocyanin"
HO
OH
HO
O
OR
OH
OH
Found in blueberries,
blackberries and cranberries
OH
OH
HO
caffeic acid, a phenolic acid
OH
O
OR
catechins
OH
quercetin,
a "flavonol"
O
O
Found in herbs, coffee
and fruits
Found in berries, onions,
and citrus fruit
resveratrol, a
"stilbene"
OH
genistein, an
"isoflavone"
HO
HO
O
OR
OH
Found in chocolate
and tea
OH
Found in red wine,
peanuts
OH
O
OH
Found in soy products
and legumes
On a molecular level, all of these compounds react with harmful free radicals and can
chelate metal ions that act as pro-oxidants
Flavonoids are especially effective due to because of structural features including:
Conjugation, o-dihydroxysubstituted B ring, a,b-unsaturated ketone, 3-OH on C-ring
They also modulate cellular biochemical reactions and the expression of genes and
proteins associated with oxidative stress
Resveratrol…
the fountain of youth?
• Produced by plants in response
to stress
• Found in red wine, grape and
cranberry juice, legumes
• Thought to contribute to the “French
Paradox”
• Decreases lipoprotein oxidation leading to
cardiovascular disease (early 90’s)
• Anticancer & antiinflammatory activity
(1997)
• Extends lifespan through sirtuin activation,
enhancing mitochondrial function (2006)
Extending lifespan: the sirtuins
• Sir2 family of proteins (silent information
regulator) regulate aging &
longevity in lower organisms
• NAD+-dependent protein acetylases that
regulate gene silencing, DNA repair &
recombination
• Sirtuins mediate life-extending effect of caloric
restriction
• Analogous SIRT1 gene found in mammals
• May regulate apoptosis (downregulation of p53
tumor suppressor) and differentiation
• Modulates adipogenesis by deactivating PPARg,
triggering loss of fat, similar to caloric restriction
de la Lastra & Villegas (2005) Mol. Nutr. Food Res. 49: 405-430
Despite its mythical powers, flavonoids identified in
Acai are similar to those found in other fruits
OH
cyanidin, an
"anthocyanin"
HO
Orientin = luteolin-8C-glucoside (above)
Homoorientin = luteolin-6C-glucoside (below)
Luteolin is a flavone.
These compounds are unusual because the sugar
is attached to a C instead of O, making it more
difficult to hydrolyze the glycosidic linkage
OH
O
OR
OH
R = glucose or rutin
Evaluation of antioxidant efficacy: Antioxidant assays
1)
Free – radical scavenging (DPPH or TEAC assay)
2)
Lipid oxidation / peroxidation assay (TBARS)
3)
LDL oxidation assay
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ORAC assay
5)
Cellular antioxidant assay (CAA)
6)
Assays measuring redox reactions of iron
Resources: excerpts from:
Yan, X., Murphy, B.T., Hammond, G.B., Vinson, J. A., Neto, C.C. “Antioxidant activities and antitumor
screening of extracts from cranberry fruit” J. Agric. Food Chem. (2002) 50: 5844-5849.
Seeram, N. and Nair, M. “Inhibition of lipid peroxidation and structure-activity related studies of the
dietary constituents anthocyanins, anthocyanidins and catechins” J. Agric. Food Chem (2002) 50:
5308-5312.
Vinson, J. et al, “Vitamins and especially flavonoids in common beverages are powerful in vitro
antioxidants which enrich LDL and increase their oxidative resistance after ex vivo spiking in
human plasma” (1999) J. Agric. Food Chem. 47: 2502-2504.
Wolfe, K. and Liu, R.H. “Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and
Dietary Supplements” J. Agric. Food Chem. (2007) 55, 8896–8907.
General free radical-scavenging ability:
the DPPH Assay
Antioxidant activity of extracts and compounds can be
evaluated by a general radical-scavenging assay that
predicts ability to quench OH., ROO. and other ROS.
DPPH: 2,2-diphenyl-1picrylhydrazyl radical
lmax = 517nm
O2N
.
(Ph)2-N-N
H
NO 2
O2N
antioxidant
Violet ------------------> Yellow
• Radical-scavenging activity is determined by measuring degree
of absorbance quenching for varying sample concentrations
• Activity expressed as EC50 = concentration required to quench
50% of DPPH radical
Lipid peroxidation as the target
TBARS assay for LDL oxidation: Joe Vinson, Univ. of Scranton
• Used to test flavonoids and other dietary antioxidants
for ability to prevent lipoprotein oxidation
• LDL / VLDL are reacted with varying concentrations of antioxidant in the
presence of cupric ion (Cu2+) to induce formation of oxidation
products from unsaturated FA
• After 6 hrs @ 37oC, thiobarbituric acid (TBA) added
• Formation of conjugated diene oxidation products measured by fluorescence
% inhibition = control - native LDL – sample fluorescence x 100/control fluorescence
Oxidation products of
lipids or LDL react
with TBA to form
colored adducts that
can be detected by
absorbance or fluorescence
How do these
popular
antioxidants
and beverages
stack up in
protecting
plasma lipids?
Protecting against iron-induced lipid oxidation
ORAC assay
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ORAC (oxygen radical absorbance
capacity) assay is used extensively to
compare antioxidant activities of foods,
beverages, and antioxidant capacity of
human blood samples in a clinical setting.
ORAC is based on the inhibition of
peroxyl-radical-induced oxidation initiated
by thermal decomposition of azocompounds such as 2,2’-azobis(2amidino-propane) dihydrochloride (AAPH)
ORAC measures free radical damage to a
fluorescent probe through the change in
its fluorescence intensity, an index of the
degree of free radical damage.
The inhibition of free radical damage by
an antioxidant is reflected in its protection
against the change of probe fluorescence
in the ORAC assay.
Grandfathers of ORAC: method was
developed by Dr. Guohua Cao in 1992. In
1995, Dr. Cao joined Dr. Ronald L. Prior's
group at Jean Mayer USDA Human
Nutrition Research Center on Aging to
develop a semi-automated ORAC assay.
Use of ORAC to compare antioxidant power of foods or change in
plasma antioxidant capacity over time in response to a treatment
ORAC values are expressed as mmoles of Trolox equivalents per unit mass or volume
Trolox = water-soluble Vitamin E analog
Source: Brunswick labs (http://brunswicklabs.com/app_orac.shtml)
Cellular Antioxidant Activity (CAA) assay
Can antioxidant activity be measured directly inside cells?
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Dye precursor
DCFH diffuses into
the cell
Cells treated with
ABAP, azo
compound that
forms peroxyl
radicals
Peroxyl radicals
oxidize dye to
fluorescent form
Cells are treated
with antioxidants
If AO makes it into
cell and scavenges
the radicals,
fluorescence
decreases
Fe induced formation of
hydroxyl radical
Fenton Reaction:
Fe2+
+ H2O2
H. J. H. Fenton, J. Chem. Soc., Trans. 1894: 899
Fe3+
_
+ OH + OH
Ascorbate(AscH-) + Fe3+ → •Asc- + Fe2+
Hydroxyl radical OH, very reactive, t1/2 ca 10-9 s
Consequences: Oxidative DNA damage, protein
modification, lipid peroxidation, etc. Even small amounts of
ferrous iron in the body can lead to the production of a
large number of hydroxyl radicals.
Fluorescent sensing of iron-induced
oxidation in cells (Guo, 2010)
Fig. 3. The RS-BE sensor can detect iron/H2O2-induced oxidative stress in live cells.
Confocal fluorescence images of live human SH-SY5Y cells with the treatment of RSBE/Fe/ H2O2 (scale bar 10 µm). (a) DIC; (b) the cells incubated with 10 µM RS-BE for 30
min; (c) the cells were then incubated with 10 µM Fe(8-HQ) for 30 min; (d) and (e) the cells
were further treated with 100 µM H2O2 for 10 and 25 min, respectively, (f) Integrated
emission (547-703 nm) intensity of (a), (b), (c), (d) and (e) images.
FRAP and similar assays measure
ability to reduce Fe3+  Fe2+
• Benzie & Strain (1999) Methods in
Enzymology
• FRAP reagent contains ferric (Fe3+ )
tripyridyl triazine complex
• Reduction to ferrous (Fe2+) tripyridyl
triazine forms a blue complex
• Reducing capacity of foods or plasma are
measured based on change in absorbance
at 593 nm
Antioxidants and more…quinones and isoprenoids
Coenzyme Q10:
Redox carrier for
electrons in human
mitochondrial ETS
Vitamin K1
Sources: plants, primarily green veggies
Role: blood clotting (carboxylation of glu in prothrombin
Inhibited by warfarins (coumadin)
Carotenoids
OH
Vitamin E
Sources: cereals, seed oils
eggs, soybean, corn oil, barley
--Free radical scavenger
--Protects lipids in LDL and cell
membranes from oxidation
--decreases coronary artery
lesions, but effect on CVD
mortality still not proven
HO
Derived from 40 carbon isoprenoid chain (phytoene) through the
mevalonate pathway; conjugation makes good antioxidant.
Biosynthesis of Vitamin E:
Shikimate pathway-derived
4-hydroxyphenyl pyruvic acid
is alkylated with isoprenoid chain
made through mevalonate pathway
Rings are then methylated by SAM
Cyclization of phenol with chain
gives tocopherol chroman ring
Tocopherols differ in pattern of
methylation on the ring
Oxidation to quinone occurs in
plastoquinones, ubiquinone
Coenzyme Q10 comes from
4-hydroxybenzoic acid which is
decarboxylated, oxidized to quinone.