ORAC - Presentación COST 927 Training School-PL

COST-927 Action
THERMALLY PROCESSED FOODS: Possible health implications

Building Skills on the Determination of the Overall Antioxidant Capacity of Thermally Processed Foods
Olsztyn, 26 - 30 January 2009 Institute of Animal Reproduction and Food Research, Department of Food Technology in Olsztyn, Poland
Dr. Dolores del Castillo
Antioxidant activity by the Oxygen Radical Absorbance Capacity assay
ORAC assay
Dr. Dolores del Castillo delcastillo@ifi.csic.es
STARTING POINT
DID EXIST ANY PREVIOUS JOB?
Title: PHYCOERYTHRIN FLUORESCENCE-BASED ASSAY FOR PEROXY-RADICALS - A SCREEN FOR BIOLOGICALLY RELEVANT PROTECTIVE AGENTS Author(s): DELANGE RJ, GLAZER AN Source: ANALYTICAL BIOCHEMISTRY Volume: 177 Issue: 2 Pages: 300-306 Published: MAR 1989 Times Cited: 99
WHAT ARE THE MAIN DISADVANTAGES OF THAT METHOD ?
1. IT IS NOT CAPABLE OF QUANTITATING RESULTS
2. IT IS LIMITATED ONLY TO SCREENING THE RADICAL SCAVENING CAPACITY OF THE SAMPLE
Basic definitions for easy understanding of the PRINCIPLES of different analytical methods for assessing total antioxidant capacity
REDUCTION= A GAIN OF ELECTRONS OXIDATION= A LOSS OF ELECTRONS REDUCTANT OR REDUCING AGENT= A SUBSTANCE THAT DONATES ELECTRONS AND CAUSES TO ANOTHER REACTANT TO BE REDUCED OXIDANT OR OXIDATING AGENT= A SUBSTANCE THAT ACCEPTS ELECTRONS AND CAUSES ANOTHER REACTANT TO BE OXIDIZED ANTIOXIDANT= SUBSTANCE THAT WHEN PRESENT AT LOW CONCENTRATIONS COMPARED WITH THOSE OF AN OXIDIZABLE SUBSTRATE, SIGNIFICANTLY PREVENTS OR DELAYS A PRO-OXIDANT INITIATED OXIDATION OF THE SUBSTRATE PRO-OXIDANT= SUBSTANCE THAT CAN CAUSE OXIDATIVE DAMAGE TO LIPIDS, PROTEINS AND NUECLEIC ACIDS. IT SYNONYM FOR REACTIVE SPECIES. CHEMICALLY, IT IS AN OXIDANT. AN ANTIOXIDANT CAN EFFICIENTLY REDUCE A PRO-OXIDANT
REACTIVE SPECIES IN BIOLOGICAL SYSTMES: HYDROXYL RADICALS (OH) SUPEROXIDE ANIONS (O2-) SINGLET OXYGEN (1O2) HYDROGEN PEROXIDES (H2O2) ORGANIC PEROXIDES (R-OOH) NITRIC OXIDE PEROXYNITRITE
DEFINITION OF ANTIOXIDANT ACTIVITY
ABILITY OF A COMPOUND TO REDUCE PROOXIDANTS OR REACTIVE SPECIES OF PATHOLOGIC SIGNIFICANCE
AN ANTIOXIDANT IS A REDUCTANT, BUT A REDUCTANT IS NOT NECESSARILY AN ANTIOXIDANT
Na, GLUCOSE, ETHANOL AND MANY OTHER REDUCTANT ARE NOT ANTIOXIDANTS
DIETARY ANTIOXIDANTS
ESSENTIAL
VITAMINE E (TOCOPHEROL) VITAMINE C (ASCORBIC ACID) VITAMIN A (RETINOL AND CAROTENOIDS) NUMEROUS MINERALS (Cu, Mn, Zn, Se, Fe)
NON-ESSENTIAL
GLUTATHIONE, SMALL PEPTIDES PHYTOCHEMICALS (THOUNDS IN FOOD SUPPLY)
Basis of the ORAC assay

Reactive Oxygen Species (ROS) generators 1. Thermal control chemical generator a. Aqueous soluble b. Lipid soluble 2. Hydrogen peroxide, superoxide generator 3. Radiation related hydroxyl radical generator
-, H O , OH) ROS (R, ROO, O2 2 2
ANTIOXIDANTS Exc=540 nm OXIDATION Decreased 565 nm emission fluorescence light
PE INDICATOR PROTEIN Oxidation
Properties of Phycobiliproteins
Pigment Absorbance maximum1 (nm) 565 (495) 545 ~495 (545) 615 617 (555) 652 566 575 Fluorescence emission2 (nm) 575 575 ~563 647 637 660 617 625 220 100 100 55 100 Molecular weight3 (kda) 240 240

delcastillo@ifi.csic.es
Absorbtivity4 (L/g-cm)
Molar absorptivity4 (M-cm)-1(10-6) 1.97 2.40
Fluorescence: Absorbance5 (relative to RPE) 1.00 1.40 0.50
RPhycoerythrin BPhycoerythrin Y-Phycoerythrin C-Phycocyanin R-Phycocyanin Allophycocyanin Phycoerythrin 566 Phycoerythrocy anin
1 2
8.2 10.0
7.0 7.0 7.3 8.0 8.5
1.54 0.70 0.73 0.44 0.85
0.15 0.14 0.30 0.25 0.50
Values in parentheses indicate secondary absorbance maxima. Phycobiliproteins are aggregates of subunits, and various aggregates may occur in aqueous solution. Values given are for most common reported aggregates; aggregates of both larger and smaller size may occur. 3 Value for Phycoerythrin 566 is an estimate. 4 An approximate relative indicator of the quantum efficiency of the pigment (measured at absorbance and emission maxima).
Phycobiliproteins are water soluble fluorescent proteins derived from cyanobacteria and eukaryotic algae.
Nomenclature: Phycobiliproteins are classified on the basis of their color into two large groups, the phycoerythrins (red) and the phycocyanins (blue). Absorption maxima for phycoerythrins lie between 490 and 570 nm while absorption maxima for phycocyanins are found between 610 and 665 nm. These large groups have been subdivided to reflect variation among the proteins in the exact location of the absorbance maximum and the specific shape of the absorbance spectrum.
The B-phycoerythrin (B-PE) protein is the major light-harvesting pigment of red microalgae Porphyridium cruentum. B-PE finds application mainly in the pharmaceutical sector as fluorescent probe in biomedical analysis, and also in the food and cosmetics industries. In spite of its numerous uses, however, its large-scale purification remains problematic and expensive. B-PE is used in the pharmaceutical industry as a fluorescent probe and reagent. It is also utilised as a natural dye in the food and cosmetics industries due to its high fluorescence efficiency. The protein's purification from algal extracts involves a number of often-crude, highly complex steps, many of which are non-scaleable and result in unsatisfactory yields.
WHY DID THE AUTHORS SELECT B-PE AS FLUORESCENCE PORBE?
1. B-PES DISTINCT EXCITATION AND EMISSION WAVELENGTHS 2. HIGH FLUORESCENCE YIELD 3. SENSITIVITY TO ROS 4. WATER SOLUBILITY
How to classify ORAC assay ?

INHIBITION METHOD
Involve a pro-oxidant and oxidizable substrate

Antioxidant capacity of the antioxidant
The pro-oxidant induces oxidative damage to the substrate which is inhibited in the presence of an antioxidant
ORAC ASSAY MEASURES THE CAPICITY OF THE ANTIOXIDANT TO DIRECTLY QUENCH FREE RADICALS
Very high molar ratio of AAPH to antioxidants (mora than 2000) Dr. Dolores del Castillo delcastillo@ifi.csic.es
ADVANTAGES COMPARED TO PREVIOUS METHODS

1. The use of peroxyl and hydroxyl radicals as pro-oxidants in the ORAC assay makes its different and unique from the assays that involve oxidants that are necessarily prooxidants. 2. An improve in quantitation is achieved in ORAC assay by: a) Taking the reaction between substrate and free radicals to completation b) Using an area-under-curve technique for quantitation compared to the assay that measure the lag phase.
ANTIOXIDANT ASSAYS
Involving oxidants that are not necessarily pro-oxidants
Involving oxidants that are prooxidants
TRAP assay FRAP assay TEAC assay Cyclic voltammetry assay Luminol-based assays Dichlorofluorescin-diacetate (DCFH-DA) based assay Crocin based assay Phycoerythrin (PE) based assay ORAC assay
Prior and Cao. Free Radical Biology & Medicine 27 (11/12), 1173-1181, 1999
Title: OXYGEN-RADICAL ABSORBENCY CAPACITY ASSAY FOR ANTIOXIDANTS Author(s): CAO GH, ALESSIO HM, CUTLER RG Source: FREE RADICAL BIOLOGY AND MEDICINE Volume: 14 Issue: 3 Pages: 303311 Published: MAR 1993 Times Cited: 441
THE AMOUNT OF SAMPLE CAN BE SUSTANTIALLY REDUCED AND THE SIMULTANEOUS KINETIC ANALYSIS OF MANY SAMPLES IS MADE POSSIBLE BY USSING FLUORIMETRY MICROPLATE READER USING A 96-WELL PLATE
Cao, G, Verdon, CP, Wu AH, Wang H and Prior RL. Automated assay of oxigen radical absorbance capacity with COBAS FARA II. Clinical Chemistry 41, 1738-1744, 1995.
Title: AUTOMATED-ASSAY OF OXYGEN RADICAL ABSORBENCY CAPACITY WITH THE COBAS FARA-II Author(s): CAO G, VERDON CP, WU AHB, et al. Source: CLINICAL CHEMISTRY Volume: 41 Issue: 12 Pages: 17381744 Part: Part 1 Published: DEC 1995 Times Cited: 218
Title: Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe Author(s): Ou BX, Hampsch-Woodill M, Prior RL Source: JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY Volume: 49 Issue: 10 Pages: 4619-4626 Published: OCT 2001 Times Cited: 216
WHAT DID THEY PROPOSE THIS TIME ?
QUANTITATION
SCHEMATIC ILUSTRATION OF THE PRINCIPLE OF THE ORACFL ASSAY
HOW DOES THE CALIBRATION CURVE LOOK?
Molecular formula Molar mass
C20H12O5 332.306 g/mol
Chemical and physical properties 1. Very high fluorescence 2. Excitation occurs at 494 nm and emission at 521. 3. pKa of 6.4 4. Ionization equilibrium leads to pHdependent absorption and emission over the range of 5 to 9 5. Fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns
07367 Fluka B-Phycoerythrin concentration~10.0 mg/mL (UV)fluorescenceex 542 nm; em 576 nm in 0.1 M phosphate pH7.2 storage temp.2-8C Prices: 1mg 82.80 euros; 5mg 318.00 euros P128 Sigma B-Phycoerythrin from Porphyridium cruentum (lyophilized powder) 1mg 356 euros 46960 Fluka Fluorescein sodium salt Fluorescence ex 490 nm; em 514 nm in 0.1 M Tris pH 8.0 Prices: 25 g 12.90 euros, 100 g 37.10 euros, 500 g 142.50 euros Dr. Dolores del Castillo delcastillo@ifi.csic.es
ARE THERE OTHER REASONS FOR CHANGING THE FLUORESCENCE PROBE?

1. DIFFERENT PEs, SUCH AS B-PE AND R-PE, POSSESS DIFERENT FLUORESCENCE INTENSITY AND REACTIVITY TO PEROXY RADICAL
2. DIFFERENCES EXIST IN THE SAME PE WITH DIFFERENT LOTS
3. A SINGLE PE LOT NUMBER SHOULD BE USED FOR A PLANNED PROJECT
HOW TO EXPLAIN THE INCONSISTENCY OF PE FROM LOT TO LOT?
PURIFICATION PROCESS B-PE 30% PURE Dr. Dolores del Castillo delcastillo@ifi.csic.es
MORE?
B-PE INTERACT WITH POLYPHENOLS
COMPARISON OF FL WITH B-PE

ANALYSIS IN ABSENCE OF AAPH B-PE
FL
STRONG PROTEIN BINDING
B-PE DECLINED MORE RAPIDLY THAN DID FL FLUORESCENCE Dr. Dolores del Castillo delcastillo@ifi.csic.es
THE FL YIELDS A CONSISTENTLY HIGHER ORAC VALUE AS COMPARED TO B-PE (~1.6-3.5 FOLD)
HOW DO THEY INTERACT?
HYDROPHOBIC INTERACTION
HYDROGEN BONDING
ORACFL MECHANISM
HAT= HYDROGEN ATOM TRANSFER
ORACFL MECHANISM
ORACFL MECHANISM
THE FOLLOWINGS EQUATIONS ILUSTRATES THE STEPWISE PROCESS OF HAT

RADICAL INICIATOR ABSTRACTS A HYDROGEN ATOM FROM THE ANTIOXIDANT
REACTION BEWTEEN ROO AND THE TARGET MOLECULE IS INHIBITED
SUMMARY OF IMPROVEMENTS
1. ROGGEDNESS 2. FL DOES NOT INTERACT WITH ANTIOXIDANT SAMPLES 3. FL SHOWS EXCELLENTE PHOTOSTABILITY SO THAT THE ORACFL ASSAY CAN BE TRANSFERRED TO A 96WELL PLATE READER. 4. THE USE OF FL REDUCES THE COST OF THE EXPERIMENT 5. ORACFL ASSAY DIRECTLY ESTIMATES THE CHAINBREAKING ANTIOXIDANT ACTIVITY
WHAT DOES ORACFL ASSAY MEASURE?

ORACFL ASSAY MEASURES HYDROPHYLIC ANTIOXIDANT ACTIVITY AGAINST PEROXYL RADICAL ORACFL ASSAY CANNOT BE CONSIDERED A TOTAL ANTIOXIDANT ACTIVITY ASSAY
IT IS IMPOSSIBLE TO MEASURE TOTAL ANTIOXIDANT ACTIVITY USING A SINGLE ASSAY

FOOD APPLICATION
FOOD APPLICATION
WHAT DID THE AUTHORS CONCLUDE?
FURTHER IMPROVEMENTS
Times Cited: 103 Dr. Dolores del Castillo delcastillo@ifi.csic.es
ADVANTAGES
1. THE EFFICIENCY IS IMPROVED WITH AT LEAST A 10 FOLD INCREASE IN SAMPLE PREPARATION AND IN INSTRUMENT UTILIZATION (130 SAMPLES/DAY) 2. THE USE OF THE ROBOTIC LIQUID HANDLING AND PLATE READER TECHNOLOGIES HAS NOT COMPROMISED THE QUALITY OF DATA OBTAINED
NEXT STEP
Volume: 50 Issue: 7 Pages: 1815-1821 Published: MAR 27 2002 Times Cited: 90
WHAT DID THEY DO?
ORACFL-LIPO ASSAY
MECHANISMS FOR OXIDATION
MECHANISMS FOR OXIDATION
FOOD APPLICATION
MAIN IMPROVEMENT
FURTHER DATA ON FOOD
DATA ON LIPOPHILIC AND HYDROPHILIC ORACFLVALUES FOR OVER 100 COMMON FOODS IN U.S. MARKETS Dr. Dolores del Castillo delcastillo@ifi.csic.es
FURTHER DATA ON FOOD
WHAT ABOUT CONCLUSIONS?
Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods - 2007
http://www.ars.usda.gov/Services/docs.htm?docid=15866
This database contains ORAC data for 277 food items and represents a collaboration between the Nutrient Data Laboratory, Beltsville Human Nutrition Research Center and the Arkansas Children's Nutrition Center in Little Rock, Arkansas.
The database will be used by scientists to help guide ongoing research into how antioxidants may correlate to health benefits. For example, many fruits and vegetables are known to be good sources of antioxidant vitamins, such as E, C, and beta carotene, a form of vitamin A. But these natural foods also contain other compounds, collectively known as phytonutrients, that may contribute to health.
Dr. Dolores del Castillo http://www.ars.usda.gov/is/pr/2007/071106.htm delcastillo@ifi.csic.es
How to access to the databse?. Go to:

http://www.ars.usda.gov/nutrientdata/ORAC
CAN FOOD PROCESSING GIVE RISE NEOANTIOXIDANTS?
SEVERAL CHEMICAL EVENTS CONTRIBUTE TO THE OVERALL ANTIOXIDANT PROPERTIES OF PROCESSED FOODS
CAN BE MEASURED THE NEOANTIOXIDANTS BY ORAC ASSAY?
KEY CHEMICAL EVENTS INDUCING FORMATION OF NEW ANTIOXIDANTS DURING THERMAL FOOD PROCESSING
PAY ATENTION TO SAMPLES PREPARATION
DO YOU HAVE ANY FURTHER QUESTION?

HMW MRPs contain reductone and amino reductone structures, which provide a potential for reducing and metal chelation activities, both of which are relevant to the antioxidant mechanisms noted forMRPs. In the present study, all three sugar-lysine HMW MRPs displayed a similar, significant capacity to scavenge DDPH radicals, which was also found for earlier products of the MR generated from heated histidine-xylose reactants. Measuring total antioxidant activity of MRPs by the ORAC method provided an excellent basis for assessing MRP scavenging capacity for oxygen radicals between different sugar reactant sources. It is of interest that the ORAC activity of all three HMW MRPs was not only similar when expressed in Trolox equivalents, but also agree well with the DPPH measurements. Moreover, the Trolox equivalent for all three MRP HMW mixtures far exceeds the total antioxidant activity determined previously for ORAC measures of different soft fruits, such as strawberries (approx. 150 mol Trolox/g dry matter [dm]), blueberries (approx. 200 mol Trolox/g dm), and raspberries (approx.100 mol Trolox/g dm). The potential for a large contribution from HMW MRPs to total antioxidant intake, in terms of ORAC values, suggests that the estimated 1.21.7 mmol Trolox/day value proposed for US consumers may be underestimated if MRPs derived from processed foods are considered in the ORAC range for total antioxidant intake.
WHAT OTHER REACTIONS MAY CONTRIBUTE TO THE OVERALL ANTIOXIDANT ACTIVITY OF PROCESSED FOODS?
WHAT DID WE DO?
DOES THE METHOD NEED FURTHER IMPROVEMENTS?
WHAT DO THEY THINK ABOUT THAT?

Oxygen radical absorbance capacity (ORAC) values have been obtained for a series of teas and herbal infusions employing 2,20-azobis (2-amidinopropane) as free radical source, and fluorescein and pyrogallol red as target molecules. The amounts of phenols in the extracts were evaluated by Folins methodology. ORAC values are extremely dependent upon the employed target molecule. Even more, relative ORAC values measured for different infusions depend upon the employed methodology. For example, ORAC-fluorescein value of Aloysia citriodora is larger than that of green tea, while if pyrogallol red is employed as target molecule green tea appears as nearly nine times more efficient. Similarly, for extracts with comparable amounts of phenols, herbal infusions are more efficient than teas by ORAC fluorescein, while opposite conclusions are obtained if ORAC-pyrogallol red values are considered. Extreme care must then be taken for conclusions obtained from ORAC values estimated by employing a single target molecule.
IS THERE MORE WORK LEFT TO BE DONE?
WHAT ELSE CAN WE DO? FEEL FREE YOURSELF TO MAKE PROPOSALS. THE DISCUSSION SECTION IS OFICIALLY OPENED

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COST-927 Action

THERMALLY PROCESSED FOODS: Possible health implications

Dr. Dolores del Castillo

Antioxidant activity by the Oxygen Radical Absorbance Capacity assay

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

DID EXIST ANY PREVIOUS JOB?

Dr. Dolores del Castillo delcastillo@ifi.csic.es

WHAT ARE THE MAIN DISADVANTAGES OF THAT METHOD ?

1. IT IS NOT CAPABLE OF QUANTITATING RESULTS

2. IT IS LIMITATED ONLY TO SCREENING THE RADICAL SCAVENING CAPACITY OF THE SAMPLE

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

DEFINITION OF ANTIOXIDANT ACTIVITY

ABILITY OF A COMPOUND TO REDUCE PROOXIDANTS OR REACTIVE SPECIES OF PATHOLOGIC SIGNIFICANCE

Dr. Dolores del Castillo delcastillo@ifi.csic.es

AN ANTIOXIDANT IS A REDUCTANT, BUT A REDUCTANT IS NOT NECESSARILY AN ANTIOXIDANT

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Basis of the ORAC assay

ANTIOXIDANTS Exc=540 nm OXIDATION Decreased 565 nm emission fluorescence light

PE INDICATOR PROTEIN Oxidation

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo

Molar absorptivity4 (M-cm)-1(10-6) 1.97 2.40

Fluorescence: Absorbance5 (relative to RPE) 1.00 1.40 0.50

7.0 7.0 7.3 8.0 8.5

1.54 0.70 0.73 0.44 0.85

0.15 0.14 0.30 0.25 0.50

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

WHY DID THE AUTHORS SELECT B-PE AS FLUORESCENCE PORBE?

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

How to classify ORAC assay ?

Involve a pro-oxidant and oxidizable substrate

ADVANTAGES COMPARED TO PREVIOUS METHODS

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo

Involving oxidants that are not necessarily pro-oxidants

Involving oxidants that are prooxidants

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

WHAT DID THEY PROPOSE THIS TIME ?

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo

SCHEMATIC ILUSTRATION OF THE PRINCIPLE OF THE ORACFL ASSAY

Dr. Dolores del Castillo delcastillo@ifi.csic.es

HOW DOES THE CALIBRATION CURVE LOOK?

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Molecular formula Molar mass

C20H12O5 332.306 g/mol

Dr. Dolores del Castillo delcastillo@ifi.csic.es

ARE THERE OTHER REASONS FOR CHANGING THE FLUORESCENCE PROBE?

2. DIFFERENCES EXIST IN THE SAME PE WITH DIFFERENT LOTS

3. A SINGLE PE LOT NUMBER SHOULD BE USED FOR A PLANNED PROJECT

Dr. Dolores del Castillo delcastillo@ifi.csic.es