Easily measure the anti-oxidative potential of your samples

Oxidative stress - kits to measure the anti-oxidative potential of samples
Figure 1: Oxidative stress and related diseases.

Oxidative stress can lead to diverse pathophysiological changes in the body. Neurodegenerative diseases such as Parkinson and Alzheimer are linked to oxidative stress, as well as several cancers and diseases such as chronic fatigue syndrome, fragile X syndrome, heart and blood vessel disorders, heart attack, heart failure, and atherosclerosis (for an overview see Fig. 1). In this post, let’s look at some convenient kits to measure the anti-oxidative potential of your samples!

Oxidative stress is based on the imbalance between the production of free radicals and the ability of the body to detoxify their hazardous effects through neutralization by antioxidants. Free radicals and reactive oxygen species (ROS) are highly reactive molecules that are generated by normal cellular processes, environmental stress, and UV irradiation. ROS react with cellular components, damaging DNA, carbohydrates, proteins, and lipids causing cellular and tissue injury. Organisms have developed complex antioxidant systems to protect themselves from oxidative stress, however, excess ROS can overwhelm the systems and cause severe damage.

14 versatile methods to measure the anti-oxidative potential of your samples

Fourteen convenient kits to measure the total antioxidant capacity of biological fluids, cells, and tissue (developed by ZenBio). They can also be used to assay the antioxidant activity of naturally occurring or synthetic compounds for use as dietary supplements, topical protection, and therapeutics. Let’s take a look at them.

5 fluorescence based assays

ORAC Antioxidant Assay Kit
HORAC Antioxidant Assay Kit
NORAC Antioxidant Assay Kit
CLORAC Antioxidant Assay Kit
CAA Cellular Antioxidant Assay Kit

9 colorimetry based assays

ABTS (TEAC) Antioxidant Assay Kit
DPPH Antioxidant Assay Kit
Cu-TAC Antioxidant Assay Kit
TAC-BCS Antioxidant Assay Kit
FRAP Assay Kit
Activated ABTS Assay Kit
Ferrous Iron Chelating (FIC) Assay
Cupric Ion Chelating (CIC) Assay
Total Phenolic Content Assay

ORAC assay

ORAC principle
Figure 2: Principle of the ORAC assay.

ORAC curve
Figure 3: Effects of antioxidants in ORAC assay: Trolox, Epogallocatechin gallate (EGCG), and Gallic acid were tested for their antioxidant activity in the ORAC assay.

The ORAC assay measures the loss of fluorescein fluorescence over time due to peroxyl-radical formation by the breakdown of AAPH (2,2′-azobis-2-methyl-propanimidamide, dihydrochloride) (Fig. 2). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting fluorescein decay in a dose dependent manner. The ORAC assay is a kinetic assay measuring fluroescein decay and antioxidant protection over time. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present (see Fig. 3).

ABTS assay

ABTS principle
Figure 4: Principle of the ABTS assay.

The assay measures ABTS + radical cation formation induced by metmyoglobin and hydrogen peroxide (Fig. 4). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting the formation of the radical cation in a dose dependent manner. As with the ORAC assay the antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present (see Fig. 5).

ABTS curve
Figure 5: Effects of antioxidants in ABTS assay Trolox, Sodium L-ascorbate (L-Asc), Epigallocatechin gallate (EGCG), and Gallic acid were tested for their antioxidant activity in the ABTS assay.

DPPH assay

Principle of DPPH
Figure 6: Principle of the DPPH assay.

ZenBio DPPH Antioxidant Assay Kit measures the reduction of the stable DPPH radical by electron transfer (Fig. 6). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control reducing the DPPH radical in a dose dependent manner. The antioxidant activity in the test samples can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present (Fig. 7).

DPPH curve
Figure 7: DPPH radical and reduced DPPH absorbance specta.

Cu-TAC assay

Cu-TAC principle
Figure 8 : Principle of the Cu-TAC assay.

The Cu-TAC Antioxidant Assay Kit measures the reduction of Copper(II) to Copper(I) in the presence of the aromatic chelator, neocuproine (Fig. 8). Uric acid or Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control for the reduction reaction in a dose dependent manner. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Uric acid or Trolox units to quantify the composite antioxidant activity present (Fig. 9).

Cu-TAC curve
Figure 9: Cu(I) neocuproine (Cu(I)Nc) complex absorbance spectrum.

TAC-BCS assay

TAC-BCS principle
Figure 10: Principle of the TAC-BCS assay.

The ZenBio TAC-BCS Antioxidant Assay Kit measures the reduction of copper(II) to copper(I) in the presence of the aromatic chelator, bathocuproinedisulfonate (BCS) (Fig. 10). Uric acid or Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control for the reduction reaction in a dose dependent manner. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Uric acid or Trolox units to quantify the composite antioxidant activity present (Fig. 11).

TAC-BCS principle
Figure 11: Cu(I) bathocuproine (Cu(I)BCS) complex absorbance spectrum.

HORAC assay

HORAC principle
Figure 12: Principle of the HORAC assay.

The ORAC (Hydroxyl (HO•) Radical Absorbance Capacity) Antioxidant Assay Kit measures the loss of fluorescein fluorescence over time due to hydroxyl-radical formation by the mixture of hydrogen peroxide and oxidizable metal ions (Co(II)) (Fig. 12). Gallic Acid [3,4,5-Trihydroxybenzoic acid], a simple phenolic compound, serves as a positive control inhibiting fluorescein decay in a dose dependent manner. The HORAC assay is a kinetic assay measuring fluorescein decay and antioxidant protection over time. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Gallic Acid units to quantify the composite antioxidant activity present (Fig. 13).

HORAC graphic
Figure 13: Effects of antioxidants in HORAC assay. Caffeic Acid, Resveratrol, Pterostilbene and Quercetin were tested for their antioxidant activity in the HORAC antioxidant assay.

 

NORAC assay

NORAC principle
Figure 14: Principle of the NORAC assay

The NORAC (Peroxynitrite [ONOO-] Radical Absorbance Capacity) Antioxidant Assay Kit measures the increase in rhodamine 123 fluorescence over time due to the oxidation of dihydrorhodamine 123 by peroxynitrite radicals formed through SIN-1 (3-morpholinosydnonimine) degradation (Fig. 14). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting DHR123 oxidation in a dose dependent manner. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present in each sample. While the NORAC assay can be performed as a kinetic assay, typically it is performed as an endpoint assay due to the linear nature of the oxidation reaction over time (Fig. 15).

NORAC graphic
Figure 15: Effects of antioxidants in the NORAC assay. Qurecetin, Catechin, Epigallocatechin gallate (EGCG), Gallic acid, Caffeic acid, Resveratrol and Pterostilbene were tested for their antioxidant activity in the NORAC antioxidant assay.

CLORAC assay

CLORAC principle
Figure 16: Principle of the CLORAC assay.

The CLORAC (hypoChLORite [ClO-] Absorbance Capacity) Antioxidant Assay Kit measures the decrease in fluorescein fluorescence due to the oxidation of fluorescein by hypochlorite ions (Fig. 16). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting fluorescein oxidation in a dose dependent manner.

The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present in each sample. The CLORAC assay is performed as an endpoint assay due to the rapidity and linear nature of the oxidation reaction over time (Fig. 17).

CLORAC graphic
Figure 17: Effects of antioxidants in the CLORAC assay. EGCG. Catechin, Quercetin, Resveratrol and Caffeic acid were tested for their antioxidant activity in the CLORAC antioxidant assay.

CAA assay

CAA principle
Figure 18: Principle of the CAA assay.

The CAA (Cellular Antioxidant Activity) Assay Kit measures the capacity of antioxidants to inhibit the oxidation of the nonfluorescent probe, DCFH to the fluorescent species, DCF, by intracellular reactive oxygen species (ROS). Cellular ROS is induced by peroxyl radical formation from the Radical Initiator and leads to a gradual increase in fluorescence due to oxidation of DCFH within the cell. Cell permeant antioxidants inhibit this reaction by interfering with ROS activity, leading to a reduction in cellular fluorescence over time (Fig. 18). The fluorescence signal is measured over 60 minutes by excitation at 485 nm, emission at 538 nm. The CAA assay is a kinetic assay measuring increasing fluorescence and antioxidant protection over time. The antioxidant activity in the test samples can be normalized to equivalent quercetin units to quantify the composite antioxidant activity present (Fig. 19).

CAA graphic
Figure 19: Effects of antioxidants in CAA assay. Caffeic Acid, Catechin, EGCG, Gallic Acid, L-ascorbic acid, and Resveretrol were tested for their antioxidant activity in the CAA assay at 125 µM.

FRAP assay

FRAP assay Principle
Figure 20: Principle of th FRAP assay

The Zen-Bio FRAP (Ferric Reducing Antioxidant Power) Assay Kit measures the increase in absorbance due to the reduction of Fe(III) to Fe(II) at low pH in the presence of a chelating probe, tripyridyltriazine (TPTZ) (Fig. 20). An Fe(II) solution serves as the positive control comparator to determine the sample’s reducing capacity. The FRAP assay is an endpoint assay measuring the increase in blue absorbance at 540-600nm. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Fe(II) units to quantify the composite antioxidant activity present (Fig. 21).

FRAP graphic
Figure 21: Effects of antioxidants in FRAP assay. EGCG, Catechin, Caffeic Acid, Resveratrol, Pterostilbene and Quercetin were tested for their antioxidant activity in the FRAP antioxidant assay at 100 µM.

SOAC assay

The SOAC (Singlet Oxygen (1O2) Absorbance Capacity) Antioxidant Assay measures the increase in fluorescence caused by singlet oxygen oxidation of dihydroethidium (DHE) over time. Singlet oxygen is generated by a reaction between sodium molybdate and hydrogen peroxide in N,N-dimethylacetamide. Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting DHE oxidation in a dose dependent manner. The antioxidant activity in biological fluids, cells, tissues, synthetic compounds and natural extracts can be normalized to equivalent Trolox units to quantify the antioxidant activity present in each sample. The SOAC assay can be performed as a kinetic assay or endpoint assay due to the linear nature of the oxidation reaction over time.

SORAC assay

The SORAC (Superoxide Radical (O-) Absorbance Capacity) Antioxidant Assay measures the increase in fluorescence caused by superoxide radical oxidation of dihydroethidium (DHE) over time. Superoxide radicals are formed by the enzymatic reaction of xanthine oxidase and xanthine substrate. Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting DHE oxidation in a dose dependent manner. The antioxidant activity in biological fluids, cells, tissues, synthetic compounds and natural extracts can be normalized to equivalent Trolox units to quantify the antioxidant activity present in each sample. The SORAC assay is performed as a kinetic assay taking fluorescence measurements over the course of 10 minutes.

Activated ABTS assay

Activated ABTS principle
Figure 22: Principle of the Activated ABTS assay.

The Activated ABTS Antioxidant Assay Kit improves upon the AOX-1 kit by generating the highly colored ABTS + radical cation prior to testing samples for their antioxidant activity. This isolates a sample’s ability to reduce the pre-formed ABTS+ radical cation from also interacting with ferryl metmyoglobin or HO• to overestimate its antioxidant activity (Fig. 22). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control reducing the pre-formed radical cation in a dose dependent manner. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present (Fig. 23).

Activated ABTS graphic
Figure 23: Effects of antioxidants in the Activated ABTS assay. Resveratrol, Quercetin, Caffeic Acid, Catechin, and Gallic acid were tested for their antioxidant activity in the Activated ABTS antioxidant assay as compared to Trolox.

FIC assay

FIC principle
Figure 24: Principle of the FIC assay.

Excess transition metal ions, such as Fe(II), can generate hydroxyl radicals [OH•] in biological systems through Fenton-like reactions. Some antioxidants are able to chelate Fe(II) thereby inhibiting the formation of hydroxyl radicals and oxidative damage. The ZenBio Ferrous Ion Chelating (FIC) Assay measures the capacity of test samples to chelate free ferrous ions in solution thereby inhibiting Fe(II) binding to ferrozine which generates a highly colored complex (Fig. 24). EDTA serves as a positive control capable of chelating ferrous ions in a dose dependent manner. The FIC assay is an endpoint assay measuring absorbance of the ferrous-ferrozine complex at λ = 562nm. FIC activity is determined as the percent of total ferrozine / Fe(II) binding (Fig. 25).

FIC graphic
Figure 25: Fe(II) Chelating Activity of Compounds in the FIC Assay. EDTA, Deferoxamine, Deferiprone, Deferasirox, and Gallic acid were tested at 50µM for their Fe(II) chelation activity in the FIC assay.

CIC assay

CIC principle
Figure 26: Principle of the CIC assay.

Excess transition metal ions, such as Cu(II), can generate hydroxyl radicals [OH•] in biological systems through Fenton-like reactions. Some antioxidants are able to chelate Cu(II) thereby inhibiting the formation of hydroxyl radicals and oxidative damage. The ZenBio Cupric Ion Chelating (CIC) Assay measures the capacity of test samples to chelate free cupric ions in solution thereby inhibiting Cu(II) binding to pyrocatechol violet (PV) which generates a highly colored complex (Fig. 26). EDTA serves as a positive control capable of chelating cupric ions in a dose dependent manner. The CIC assay is an endpoint assay measuring absorbance of the Cu(II)-PV complex at λ = 632nm. CIC activity is determined as the percent of total PV / Cu(II) binding (Fig. 27).

CIC curve
Figure 27: Cu(II) Chelating Activity of PV. Pyrocatechol Violet (2mM) chelates Cu(II) to produce a highly colored complex. Absorbance at 632nm increases with increasing Cu(II) concentration in solution.

Total Phenolic Content assay

Total phenolic content principle
Figure 28: Principle of the Total Phenolic Content assay.

Phenolic compounds are present in fruits, vegetables and medicinal plants and can impart antioxidant capacity to reduce the effects of reactive oxygen species. These compounds also can provide UV protection, are anti-inflammatory and anti-microbial. The ZenBio Total Phenolic Content Assay uses the Folin-Ciocalteu Reagent to react with phenolic compounds in a test sample producing a highly colored molybdenum species (Fig. 28). The phenolic content of a sample can be normalized to equivalent Gallic Acid units. The total phenolic content assay is an endpoint assay measuring absorbance at λ = 765nm (Fig. 29).

Total phenolic content graphic
Figure 29: Gallic Acid Equivalence of Common Antioxidants. Caffeic acid, resveratrol, Epigallocatechin gallate (EGCG), quercetin and catechin were tested for their total phenolic content relative to gallic acid.

So, how will you measure the anti-oxidative potential of your samples?

Besides the kits mentioned in this post, you can also choose to outsource your oxidative activity measurement, to labs such as tebu-bio who perform this service (saving time and resources for other activities!).

Interested in finding the best way to measure the anti-oxidative potential of your samples? Leave your questions and comments below, I’ll be pleased to get in touch with you!