8/8/2019 AN148 ORAC Trolox Antioxidants Fluorescence FLUOstar

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ORAC Assay on the FLUOstar OPTIMAto Determine Antioxidant CapacityFranka Ganske, BMG LABTECH, Oenburg, GermanyE.J. Dell, BMG LABTECH, Durham, USA

Application Note 148 Rev. 12/2006

Antioxidants are able to neutralize Reactive Oxygen Species

(ROS)

Antioxidant capacity successfully determined with the ORAC

assay on the FLUOstar OPTIMA

ORAC assay uses Trolox as reference substance

Introduction

In all oxygen consuming cells, metabolism and oxidative stress

generate several intermediates and byproducts that are collectively

known as reactive oxygen species (ROS). ROS are necessaryintermediates in the human body, but they are also involved in the

aging process and in the development o many degenerative diseases,

including cancer, heart disease, Alzheimers and Parkinsons. ROS are

dangerous to cellular structures and unctional molecules (i.e. DNA,

proteins, lipids) as they act as strong oxidizing agents or ree radicals.

Biological antioxidants are able to dispose o ROS; however, they are

not completely eective in eliminating all o the ree radicals, oxygen

ions and peroxides that can do damage to the body. Furthermore, ROS

can be generated rom exposure to other external sources such as

cigarette smoke, pollutants, chemicals and environmental toxins.

In recent years, clinical trials and epidemiology studies have shown an

inverse relationship between the consumption o ruits and vegetables

and degenerative diseases1. This data suggests a correlation between

antioxidant laden ood and good health; though an inverse relationship

with a specic antioxidant (i.e. carotenoid, vitamin C or vitamin E) and

a specic disease has not been completely successul. One major

drawback to these latter studies has been determining the antioxidant

capacity o these oods and their specic molecules, as well as the

antioxidant capacity o plasma or other biological samples.

One standardized method or determining the antioxidant capacity

o a substance is the ORAC (oxygen radical absorbance capacity)

assay2. The ORAC assay is based upon the inhibition o the peroxyl-

radical-induced oxidation initiated by thermal decomposition o azo-

compounds such as [2,2-azobis(2-amidino-propane) dihydrochloride

(AAPH)]3

. In this manner, the ORAC assay uses a biological relevantradical source and it combines both inhibition time and degree

o inhibition into one quantity. Recent modications to this assay

include the use o fuorescein as the probe4, the adaptation to a

high-throughput ormat, and the ability to measure the lipophilic,

hydrophilic, and total5 antioxidant capacity o a substance. These

modications, along with no washing steps, have greatly simplied

the ORAC assay; thereby making it ideally suited to measure the

antioxidant capacity o a substance.

Herein we describe the application o the ORAC-FL assay on a FLUOstar

OPTIMA using Trolox (a water-soluble analogue o vitamin E) as a

standard by which all other antioxidant compounds are compared.

Fig. 2: BMG LABTECHs FLUOstar OPTIMA

Fig. 1: ORAC Assay Principle

Assay Principle

Over time ROS, generated rom the thermal decomposition o AAPH,

will quench the signal rom the fuorescent probe fuorescein. The

subsequent addition o an antioxidant produces a more stable fuo-

rescence signal, with signal stability depending on the antioxidants

capacity (Fig. 1). The data points are summarized over the time by the

evaluation sotware. This is then compared to the standard, Trolox ,

and is expressed as micromoles o Trolox equivalents (TE) per gram

or per milliliter o sample (mole o TE/g or mole o TE/mL).

Materials and Methods

All materials were obtained through normal distribution channelsrom the manuacturer stated.

Costar 96 well black opaque plate, Corning Costar Corporation,

Cambridge, MA, cat. no. 3792

Fluorescein Sodium, 6-Hydroxy-2,5,7,8-tetra-methylchroman-2-

carboxylic acid (Trolox), L (+)-ascorbic acid, Epicatechin gallate,

[2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH)]

were obtained rom Sigma-Aldrich

Plate sealer, BMG LABTECH, Aylesbury, UK, Cat. No. 77400-05

Thermostar, BMG LABTECH, Oenburg, Germany

FLUOstar OPTIMA, BMG LABTECH, Oenburg, Germany

ROS (Reactive Oxygene Species)

Fluorecent Probe Fluorecent Probe Fluorescent Probe+ + +

Buffer Trolox Sample

Loss offluorescence Loss offluorescence Loss offluorescence

SumBlank SumStandard SumSample

Antioxidant Capacity relating to Trolox = (SumSample - SumBlank) / (SumStandard- SumBlank)

Although a FLUOstar OPTIMA was utilized or this application note,

BMG LABTECHs POLARstar also have been used or luorescence

measurements.

8/8/2019 AN148 ORAC Trolox Antioxidants Fluorescence FLUOstar

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Germany: BMG LABTECH GmbH Tel: +49 781 96968-0

Australia: BMG LABTECH Pty. Ltd. Tel: +61 3 59734744France: BMG LABTECH SARL Tel: +33 1 48 86 20 20Japan: BMG LABTECH JAPAN Ltd. Tel: +81 48 647 7217UK: BMG LABTECH Ltd. Tel: +44 1296 336650USA: BMG LABTECH Inc. Tel: +1 919 806 1735

Internet: www.bmglabtech.com info@bmglabtech.com

Results

Figure 3 shows Trolox signal curves (relative fuorescent units versus

time) at dierent concentrations. Ater 3 cycles AAPH was added,

which lead to a loss in fuorescence signal that depended upon the

concentration o Trolox.

1 World Cancer Research Fund, American Institute or Cancer Re-

search. Food, Nutrition and the PreVention o Cancer: A Global Per-

spectiVe; American Institute or Cancer Research: Washington, DC,

1997.

2 Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance

capacity assay or antioxidants. Free Radical Biol. Med. 1993, 14,

303-311.

3 Glazer, A. N. Phycoerythrin Flurorescence-Based Assay or Reactive

Oxygen Species. Methods Enzymol. 1990, 186, 161-168.

4 Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and valida-

tion o an improved oxygen radical absorbance capacity assay us-

ing fuorescein as the fuorescent probe. J. Agric. Food Chem. 2001,

49, 4619-4926.

5 Prior, R. L.; Hoang, H.; Gu, L.; Wu, X.; Bacchiocca, M.; Howard, L.;

Hampsch-Woodill, M.; Huang, D.; Ou, B.; Jacob, R. Assays or hy-

drophilic and lipophilic antioxidant capacity (oxygen radical ab-

sorbance capacity (ORACFL)) o plasma and other biological and

ood samples. J. Agric. Food Chem. 2003, 51, 3273-3279.

References

Fig. 3: Signal curves o dierent concentrations o Trolox compared to theBlank (blue line).

Fig. 4: Blank-corrected linear regression curves o Trolox, ascorbic acid andepicatechin gallate. The data points were summed over time and wereplotted on the y-axis vs. concentration.

Test Protocol

Dierent dilutions o Trolox (200 M 12.5 M) and sample com-

pounds (ascorbic acid and epicatechin gallate, two known antioxi-

dants) were prepared in phosphate buer (10 mM, pH 7.4). All solu-

tions were and should be prepared resh daily.

In every working well the ollowing was pipetted in triplicate:

Fluorescein, 150 l o a 10 nM solution

For standard, 25 l o Trolox

dilutionFor sample, 25 l o sample dilution

For blank, 25 l o phosphate buer

The microplates were sealed ollowed by an incubation or 30 min at 37C

in a Thermostar microplate incubator without shaking. Alternatively, the

FLUOstar OPTIMA itsel can perorm the incubation step.

Ater incubation, fuorescence measurements (Ex. 485 nm, Em. 520 nm)

were taken every 90 sec to determine the background signal. Ater 3

cycles, 25 l (240 mM) o AAPH was injected with the help o onboard

injectors. Alternatively AAPH can also be added manually with a

multi-channel-pipette. This has to be done as quickly as possible

since the ROS-generator displays immediate activity ater addition.

The test was resumed and fuorescent measurements were taken upto 90 minutes.

Instrument Settings Overview

Mode: Fluorescence intensity, plate mode

Optic : Top optic, combination head

Filter: Exc. 485 nm Em. 520 nm

No. o cycles: 60

Measurement start time: 0.0 sec

No. o fashes: 10

Cycle time: 90 sec

Gain: Adjusted or each plate

Since the sample concentrations are known, the sotware has a ea-

ture in layout (ound in test protocols setup) that allows the user to

simultaneously look at calibration curves rom up to 12 compounds.

Figure 4 depicts the blank-corrected linear regression curves o

Trolox, ascorbic acid and epicatechin gallate. Graphically one can

see that ascorbic acid is a weaker antioxidant than Trolox, whereas

epicatechin gallate is a much stronger one.

Conclusion

The ORAC assay is a common and popular tool used to determine the

antioxidant capacity o any substance. With the help o the FLUOstar

OPTIMA and its easy-to-use sotware, the antioxidant capacity o a

substance can be directly estimated by comparison to the standard

curve o Trolox. The progress o each reaction can be ollowed in real-

time using the current state option. Furthermore, the use o onboard

injectors allow or consistent and reproducible data.

UNITS

70000

60000

50000

40000

30000

20000

10000

00 1000 2000 3000 4000 5000

TIME[sec]

B25 M12.5 M6.25 M3.13 M1.56 M

Sum

2000000

1500000

1000000

500000

0 0 5 10 15 20 25 30

Concentration [M]

TroloxAscorbicacidEpicatechin gallate

To obtain the values or Trolox equivalents (TE) o antioxidants with

known concentration over the desired concentration range one can

divide the slopes o the regression curves:

In the case o compounds with unknown concentrations, the sotware

calculates the Trolox

equivalents o a special dilution using theTrolox calibration curve.

TE over considered concentration range =slope regression curve (sample)

slope regression curve (Trolox)