Chapter 3.

Total Antioxidant Capacity Measurement

by Colorimetric Assay
Sajal Gupta, Meaghanne Caraballo and Ashok Agarwal
American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States

INTRODUCTION
Reactive oxygen species (ROS) have been implicated as causative factors for many diseases including male infertility
[1e3]. Elevated levels of ROS in semen samples are generated from immature spermatozoa and leukocytes present in the
semen [2]. On the other hand, seminal fluid is rich in antioxidants that are able to neutralize these ROS. There are several
published literature reports that have observed a correlation between low antioxidant capacity and male infertility [3,4].
Assessment of total antioxidant capacity (TAC) is a diagnostic tool in male infertility evaluation. However, the antioxidant
capacity has to be assessed in combination with the amount of ROS produced in the semen and the sperm DNA damage to
provide a complete picture of the impact of oxidative stress on semen quality and sperm function. Therefore, TAC
determination is a crucial component of the assessment of redox status in semen samples of infertile male patients [5].

METHODS FOR MEASURING ANTIOXIDANTS

Assessment of antioxidants in biological fluid can be performed for individual antioxidants or as aggregate of all
antioxidants, which is also referred to as TAC. Antioxidants present in the semen offer formidable protection against
excessive oxidative stress. The assays are based on principles of enhanced chemiluminescence (ECL), spectrophotometry-
based ferric reducing of antioxidant power (FRAP) assay, principle of formation of 2,20 ,-azinobis-3-ethylbenzothiazoline-
6-sulphonate (ABTSþ) radical and electrochemical methodologyebased voltametry.

TYPES OF ASSAYS FOR TOTAL ANTIOXIDANT MEASUREMENT

Chemiluminescence Assays (ECL Assays)
The enhanced chemiluminecsence assay detects nonenzymatic antioxidants in seminal plasma using very stringent
conditions. Therefore, the assay is very cumbersome with limitations of preparing fresh reagents each time. Luminol plus
para-iodophenol are the chemical inducers of luminescence utilized in the assay. The chemical inductors are then mixed
with immunoglobulin linked to horseradish to produce the ROS. In the next step hydrogen peroxide is added to the
mixture. Trolox is a water-soluble tocopherol analog and is used as the reference standard in this assay. To evaluate the
results, a comparison is made between the ability of the seminal plasma versus Trolox to inhibit the induced
chemiluminescence [6].

Spectrophotometric Assay: Ferric Reducing of Antioxidant Power Assay

TAC is also measured by utilizing the FRAP assay, and the measurement is conducted with the help of a spectropho-
tometer. In this assay, the colorless oxidized Fe3þ form of iron is converted to a blue-colored Fe2þ tri-pyridyl triazine

Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction. https://doi.org/10.1016/B978-0-12-812501-4.00019-5 207
Copyright © 2019 Elsevier Inc. All rights reserved.
208 PART j III Clinical Methods to Determine and Treat Oxidative Stress

(TPTZ)-reduced form, which is due to the action of the electron donation from antioxidants [7]. The assay measures a
change in the absorbance at a wavelength of 593 nm. The reagent for the FRAP assay is constituted with mixing of TPTZ,
HCl, and FeCl3 [7].

Assay-based on Formation of ABTS Radical: ELISA Assay

The TAC assessment technique based on colorimetry was described by Miller et al. in 1993 [8]. The compound 2,20 -
azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid) (ABTS) is incubated with met-myoglobin, which is a peroxidase, and
with hydrogen peroxide [9]. This coincubation results in the production of a stable product, ABTSþ, which produces a
stable green color and is measured at a wavelength of 600 nm. The antioxidants in the seminal plasma will suppress the
green color in an intensity proportional to its concentration [10].

MEASUREMENT OF ANTIOXIDANT CAPACITY BY COLORIMETRIC ASSAY

ROS are natural products of cellular metabolism in physiological amounts and are essential to trigger capacitation and
induce acrosome reaction leading to successful fertilization. Free radicals are unstable due to the presence of one unpaired
electron in their outer orbit causing high chemical reactivity. Therefore, such molecules react very easily with cellular
components and damage lipids, proteins, and DNA. The sperm plasma membrane contains an extraordinarily high amount
of polyunsaturated fatty acids, and in addition to that, have only very limited antioxidant defense mechanisms, including
low concentrations of ROS-scavenging enzymes due to absence of cytoplasm [11,12]. Living organisms have developed
complex antioxidant systems to counteract the effects of ROS and reduce damage. Antioxidants can help protect the
spermatozoa and cells by using three mechanisms: prevention, interception, and repair [13]. The antioxidant system
includes enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; macromolecules such as albumin,
ceruloplasmin, and ferritin; and an array of small molecules, including ascorbic acid, a-tocopherol, b-carotene, reduced
glutathione, uric acid, and bilirubin [14]. A step-by-step protocol for the TAC assessment by ELISA assay is provided next.
This is an in-house protocol developed in the Andrology Lab [15].

Instrument and Consumables

1. Antioxidant Assay Kit (Cat # 709001; Cayman Chemical, Ann Arbor, Michigan) (Fig. 1)
2. Epoch Biotek Gen 5 Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, Vermont) (Fig. 2)
3. 96-well plate (seen in Fig. 2)
4. Horizontal plate shaker (Eppendorf MixMate) (Fig. 3)
5. Pipettes (20, 200 and 100 mL)
6. Pipette tips (20, 200 and 100 mL)
7. Multichannel pipettes (8 channel, 30e300 mL) (Fig. 9)
8. Ultrapure water (Cat # 400000; Cayman Chemicals, Ann Arbor, Michigan) (Fig. 5)
9. Polystyrene centrifuge tubes (50 and 15 mL) (Fig. 7)
10. Eppendorf Safe Lock tubes (12 x 75 mm) (Figs. 4e8)
11. Plastic boats for reagents (Cat # P5078-23, Vistalab Technologies) (Fig. 9)
12. Microfuge (Fig. 6)

FIGURE 1 Antioxidant assay kit.

 Measurement of Total Antioxidant Capacity Chapter | 3.3 209

FIGURE 2 Epoch Biotek Gen 5 absorbance microplate reader.

FIGURE 3 Horizontal plate shaker.

FIGURE 4 Eppendorf tubes.

210 PART j III Clinical Methods to Determine and Treat Oxidative Stress

FIGURE 5 Ultrapure water.

FIGURE 6 Microfuge, for centrifugation of thawed seminal plasma.

Assay Reagents
1. Antioxidant assay buffer (10)
2. Lyophilized met-myoglobin
3. Trolox standard (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid)
4. Hydrogen peroxide (441 mM)
5. Chromogen (containing ABTS)
 Measurement of Total Antioxidant Capacity Chapter | 3.3 211

FIGURE 7 Seminal plasma samples ordered and numbered for identification.

FIGURE 8 Samples AeG: Trolox standard controls. Samples 1e25: diluted patients samples (10 mL patient seminal plasma þ90 mL assay buffer).

FIGURE 9 Reagent boat containing reconstituted chromogen and a multichannel pipette.

212 PART j III Clinical Methods to Determine and Treat Oxidative Stress

Methodology
Specimen Collection
A semen sample should be collected by masturbation and ejaculation into a clean wide-mouthed plastic specimen cup
between 48 and 72 h of sexual abstinence. The sample should be collected in the privacy of a room near the laboratory. If
not, it should be delivered to the laboratory within 1 h of collection. Lubrication should not be used to facilitate the semen
collection, except for those provided by the laboratory. The sample should be protected from extreme temperatures (not
less than 20 C and not more than 40 C) when transporting to the laboratory if collected outside the facility grounds. After
complete liquefaction (37 C for 20 min), the sample has to be centrifuged at 300g for 10 min at room temperature. Finally,
the clear seminal plasma has to be aliquoted into cryogenic vials and frozen at 80 C until the time of the TAC assay.

Instrument Setup
1. Open the Gen 5 software, 2.09 in One Micro Manipulator Reader software.
2. Power on the Epoch Biotek Gen 5 absorbance microplate reader.
3. Choose the Experiments icon from the Task Manager tab.
4. Choose the proper Protocol per laboratory requirements.
5. Select the green Read New tab, enter the Assay Kit Lot Number, and select OK.
6. In the data box for the plate reader, select 750.
7. The Load Plate icon will appear; select OK to run a test on the software and the reader connection.
8. Test Run Setup: Select the green Read New Button, enter the Assay Kit lot number, and select OK. Verify that the data
box for the plate reader is selected at 750 nm.

Principle of the Assay

Seminal plasma TAC measurement is done using the Antioxidant Assay Kit supplied by Cayman Chemical, Ann Arbor,
Michigan Cat # 709001 (Fig. 1). TAC assay is dependent on the capability of the antioxidants in the seminal plasma to
inhibit the oxidation of ABTS (2,20 -azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS$þ by met-myoglobin [1] [14].
As a result of the induced reaction the antioxidants suppress the absorbance at 750 nm to a level that is equivalent to their
concentration. The capacity of the antioxidants present in the sample to prevent ABTS oxidation was compared to standard
Trolox (6-hydroxy-2,5,7,8 etramethylchroman-2-carboxylic acid); Trolox is a water-soluble tocopherol analog. Results are
reported as micromoles of Trolox equivalent.
Assay steps are given next.

Reagent Preparation for Total Antioxidant Capacity

1. Antioxidant assay buffer is used diluted using a ratio 1:9, 1 mL of assay buffer concentrate to 9 mL of ultrapure water
in a 15 mL conical tube. The reconstituted vial is stable for 6 months when stored at 4 C.
2. Lyophilized met-myoglobin is reconstituted with 600 mL of assay buffer (prepared in the previous step). Once recon-
stituted, it is sufficient for 60 wells. The reconstituted reagent is stable for 1 month when stored at 20 C.
3. The Trolox standard (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) is used to prepare the standard curve.
One milliliter of ultrapure water is added to the lyophilized Trolox; the reconstituted vial is stable for 24 h at 4 C.
4. Hydrogen peroxide 441 mM working solution is prepared from 8.82 M solution of hydrogen peroxide. Dilute 10 mL of
hydrogen peroxide reagent with 990 mL of ultrapure water. Further dilute by removing 20 mL and diluting with
3.98 mL of ultrapure water to give a 441 mM working solution. The working solution is stable for 4 h at room
temperature.
5. Chromogen (containing ABTS) is reconstituted with 6 mL of ultrapure water. It is sufficient for 40 wells. The recon-
stituted vial is stable for 24 h at 4 C. Chromogen is light sensitive and must be prepared in indirect light.

Sample Preparation for Total Antioxidant Capacity

1. Bring all reagents and samples to room temperature for 30 min prior to starting the assay. All reagents are prepared per
the manufacturer’s instructions.
2. The frozen seminal plasma is brought to room temperature and centrifuged in a microfuge at 300g for 7 min. Remove
the clear seminal plasma and dilute each sample 1:9 (10 mL sample þ 90 mL assay buffer) in a microfuge tube.
 Measurement of Total Antioxidant Capacity Chapter | 3.3 213

3. Prepare Eppendorf Safe Lock tubes for the Trolox standards (seven tubes), internal controls (previously ran patients),
and current patient samples. Label the Trolox standard tubes AeG and the patient tubes numerically (e.g., 1, 2, 3, etc.).
a. Trolox standard (tubes AeG): Add the required amount of reconstituted Trolox and assay buffer to each tube as
shown in Table 1.
b. Patient sample (numerical tubes): Remove the clear seminal plasma and dilute each sample of seminal plasma, 1:9
(10 mL sample þ 90 mL assay buffer) in a microfuge tube.

TABLE 1 Preparation of Trolox standards for TAC assay

Final Concentration
Tube Reconstituted Trolox (mL) Assay Buffer (mL) (mM Trolox)
A 0 1000 0
B 30 970 0.044
C 60 940 0.088
D 90 910 0.135
E 120 880 0.18
F 150 850 0.225
G 220 780 0.330

4. Trolox standards and patient samples are added in duplicate to each well in the 96-well plate. Each sample is recorded
in a plate template form (Appendix A).
5. Add 10 mL of Trolox standard and samples in duplicate and 10 mL of met-myoglobin per well.
6. In the dark add 150 mL of chromogen per well. A multichannel pipette should be used to pipette the chromogen. Chro-
mogen can be pipetted from a flat container or reagent boat.
7. With the lights still turned off, initiate the reaction by adding 40 mL of hydrogen peroxide working solution using a
multichannel pipette from a reagent boat. Complete this step as quickly as possible (w45 s). Set the timer for 5 min, 5 s.
8. Cover the plate with a plate cover and incubate on a shaker at room temperature. Remove the plate from the shaker with
w30 s left on the timer and remove the plate cover.
9. Place the plate onto the plate holder on the Epoch Biotek Gen 5 Absorbance Microplate Reader. At the end of the in-
cubation time select OK and read the absorbance at 750 nm.

Calculating Assay Results

Calculating the average absorbance of each Trolox standard and sample determines the reaction rate. The standard curve
records the average absorbance of the standards as a function of the final Trolox concentration. The total antioxidant
concentration of each sample is calculated using the equation obtained from the linear regression of the standard curve by
substituting the average absorbance values for each sample into the equation:

Antioxidant (mM) ¼ [(Unknown average absorbance  Y intercept)/slope]  dilution  1000(14).

Reference Values and Ranges

The normal value for TAC is greater than or equal to () 1950 mM Trolox [14].

Assay Quality Control

The standard reading of the Trolox standards (vials AeG) should be within the expected range. Standard A ranges are equal
to 0.35 to 0.45 and standard G ranges are equal to 0.100 to 0.150. If the readings are significantly different (higher or lower),
the samples must be rerun. Internal controls or previously run samples should be within 500  mM of the previous results.
214 PART j III Clinical Methods to Determine and Treat Oxidative Stress

Tips for Troubleshooting

Tips are provided for resolving common issues with the assay, including in sample preparation, reagent preparation,
sample incubation, and reagent addition for the assay [14].
1. Samples should be completely thawed for 20 min and centrifuged at 300g for 7 min to pellet the debris and
spermatozoa.
2. All reagents should be at room temperature for 30 min prior to starting the assay.
3. The water used to prepare the reagents should be ultrapure water and used before the expiration date (6 months).
4. To avoid great fluctuation in the readings, both chromogen and met-myoglobin should be from the same lot.
5. It is important that the incubation time after the addition of hydrogen peroxide is exactly 5 min and 5 s. The reaction
starts as soon as hydrogen peroxide is added to each well, and there is a reaction termination step. The absorbance will
keep changing with time.
6. Thirty seconds before the completion of the 5 min and 5 s, place the plate on the plate reader.
7. If you make any errors when adding the reagent(s) or sample to each well, note this fact in the template and final results.
8. The Trolox standard reading (A and G) well should be within the expected range (i.e., 0.35 to 0.45 for well A and 0.100
to 0.150 for well G). If the readings are significantly different (higher or lower), the samples must be rerun.

CONCLUSIONS
The colorimetric assay is a relatively inexpensive, simple, and reliable assay for assessment of total antioxidant capacity,
which is less time consuming. It was also reported to have a strong correlation with other established assays such as the
enhanced chemiluminescence method. The TAC estimation is an important marker associated with male infertility and
impaired sperm function.

REFERENCES
[1] (a) Gangel EK. AUA and ASRM produce recommendations for male infertility. American Urological Association, Inc and American Society for
Reproductive Medicine. Am Fam Physician 2002;65:2589e90.
(b) Tremellen K. Oxidative stress and male infertilityea clinical perspective. Hum Reprod Update 2008;14:243e58.
[2] Aitken RJ. A free radical theory of male infertility. Reprod Fertil Dev 1994;6:19e23 [discussion 23e24].
[3] Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma is different in fertile and infertile men.
Fertil Steril 1995;64:868e70.
[4] Lewis SE, Sterling ES, Young IS, Thompson W. Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men.
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[5] Smith R, Vantman D, Ponce J, Escobar J, Lissi E. Total antioxidant capacity of human seminal plasma. Hum Reprod 1996;11:1655e60.
[6] Said TM, Kattal N, Sharma RK, Sikka SC, Thomas Jr AJ, Mascha E, et al. Enhanced chemiluminescence assay vs colorimetric assay for mea-
surement of the total antioxidant capacity of human seminal plasma. J Androl 2003;24:676e80.
[7] Pahune PP, Choudhari AR, Muley PA. The total antioxidant power of semen and its correlation with the fertility potential of human male subjects.
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[8] Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring
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[9] Sharma RK, Pasqualotto FF, Nelson DR, Thomas Jr AJ, Agarwal A. The reactive oxygen species-total antioxidant capacity score is a new measure
of oxidative stress to predict male infertility. Hum Reprod 1999;14:2801e7.
[10] Roychoudhury S, Sharma R, Sikka S, Agarwal A. Diagnostic application of total antioxidant capacity in seminal plasma to assess oxidative stress in
male factor infertility. J Assist Reprod Genet 2016;33:627e35.
[11] Agarwal A, Durairajanayagam D, du Plessis SS. Utility of antioxidants during assisted reproductive techniques: an evidence based review. Reprod
Biol Endocrinol 2014;12:112.
[12] Parks JE, Lynch DV. Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology
1992;29:255e66.
[13] Sies H. Strategies of antioxidant defense. Eur J Biochem 1993;215:213e9.
[14] Makker K, Agarwal A, Sharma R. Oxidative stress & male infertility. Indian J Med Res 2009;129:357e67.
[15] Agarwal A, Gupta S, Sharma R. Andrological evaluation of male infertility. 2016.
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APPENDIX A
PLATE TEMPLATE

1 2 3 4 5 6 7 8 9 10 11 12