Document ID: IM­KPA071026E

For Research Use Only

ＰＡＯ Test kit for Total Antioxidant Capacity
Instruction manual
Catalog #KPA­050

Oxidative stress plays on important role in various diseases and aging. The control of oxidative stress is expected to be useful to prevent diseases
and aging.Oxidative stress is caused by the imbalance between reactive oxygen species (ROS) and antioxidant defense system. For accurate
assessment of oxidative stress, measurement of ROS, oxidative damage and antioxidant activity may be essential. PAO can detect not onlyhydrophilic antioxidants such as Vitamin C, glutathione, but also can detect hydrophobic antioxidants such as Vitamin E. Applicable for assessmentof total antioxidants of serum, foods and beverage samples.

1. Principles and components
Samples are mixed with Cu++ Solution. Cu++ are reduced by antioxidants to form Cu+. Reduced Cu+++
Ｃｕreact with Chromatic Solution (Bathocuproine) , and can be detected by absorbance at wavelength 480 toReduction by antioxidants

490 nm. Antioxidant capacity can be calculated from the Cu+ formed.

+Ｃｕ

Chrolatic reagent(Bathocuproine)Color 480～490nm(Bathocuproine-Cu+)

2. Specifications

３．Required but not provided

４．Assay procedure①Standard （Uric acid powder）②Sample diluent③Cu++ solution④Stop solution⑤Micro titer plate①Assay range:②Storage:③Expiry date:1 vial (for 2mM)
60 mL x 1 bottle
5 mL x 1 bottle
5 mL x 1 bottle
1 plateDissolve with distilled water.
Ready to use.
Ready to use.
Ready to use.

21.9～4378μmol/L（Cupric ion reducing power）
Room temperature
3 years （Indicated on the outer box）

①A micro plate reader (measuring wavelength 490 nm)②Pipettes and pipette chips③Plastic test tubes④Distilled water⑤NaOH, HCl solution and pH meter (Not required if standards are prepared with distilled water only)

1) Reconstitute of Standard (2mM Uric acid solution). There are two ways for preparation. Please select one.
Case 1：Add distilled water to ①Standard vial, and stand for 3 or 4 hours at room temperature. The volume of distilled water is indicatedon the label of the vial.
Case 2: If you wish to prepare standard solution immediately, please pour 1mL of 10% (w/v) NaOH to ①Standard vial, and dissolve
completely, followed by pH adjustment (pH7.4) by HCl solution. Add distilled water to make the total volume as indicated on the label.
2mM uric acid solution can be stored at below ­70℃ for 1 year.
2) Preparation of standards.
Dilute 2mM uric acid solution with distilled water for 2, 4, 8, 16 and 32 times, result in 5 levels of diluted standards (1 mM, 0.5 mM, 0.25 mM,0.125 mM and 0.063 mM respectively).
3) Preparation of samples.
If you measure serum samples, fresh frozen samples are recommended. Because some antioxidants such as vitamin C, uric acid andcoenzyme Q10 are unstable. For other samples such as beverages, see section 6) Assay examples, and dilute with distilled water.
4) Assay procedure.
A) Please prepare plastic test tubes for 6 levels of standards and each sample. Pour 390 μL of ② Sample Diluent, and add 10 μL ofstandards or diluted samples.
B) Pour 200μL of mixture to ⑤Micro titer plate. Use 200μL of ②Sample Diluent for blank well.
C) Read absorbance at 490 nm (as READ1).
D) Add 50μL of ③Cu++ solution to each well, mix gently, and incubate at room temperature for 3 minutes.
E) Add 50μL of ④Stop solution, mix gently, and read absorbance at 490 nm (as READ2). (Continue to the next page)(Page 1/2)

5) Determination of antioxidant power of samples.
Please draw standard curves by plotting the difference of absorbance readings (READ2 ­ READ1) as vertical axis, and concentration ofuric acid standards (mM) as horizontal axis. Calculate the corresponding uric acid concentration of samples. Multiply corresponding
uric acid concentration (mM) of samples by 2189, to estimate antioxidant power (μmol/L).
1mM of uric acid = 2189 μmol/L (copper reducing power)

5. Typical standard curves
PAO検量線curve
PAO Standard

吸光度変化（ΔA490nm）
Absorbance(READ2­ READ1)

0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.0

0.5

1.0

1.5

2.001094

2188

3282

4376

0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

2.5
Uric acid（mM）

Antioxidant
5470 power（μM）

6. Assay examples

D.W. : distilled water.

SamplePre-dilutionAntioxidant power (μmol/L)

Human serum

Not required

Human urine

Mix with 3 volumes of D.W.

5508

Red wine

Mix with 7 volumes of D.W.

45479

Japanese SAKE (rice wine) Not required
Black tea
Mix with 7 volumes of D.W.

18～211

CoffeeGreen tea

Mix with 27 volumes of D.W.
Mix with 7 volumes of D.W.

1069±145

8728～46687

Fresh frozen serum.

Green tea products.

A more dilution is recommended if the antioxidant power is over 2000μmol/L antioxidant power. For example, some green tea products
which contain high concentration of catechin should be diluted by 40 times (mix 1 volume of sample and 39 volume of distilled water).
Some samples which contain chelating agents such as EDTA can’t be applied.

7. References
Oxidative stress and its association with coronary artery disease and different atherogenic risk factors
C. VASSALLE, L. PETROZZI , N. BOTTO, M. G. ANDREASSI & G. C. ZUCCHELLI
Journal of Internal Medicine 256, p308–315 (2004)
Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: A pilot study
E Strafacea, P Matarresea, L Gambardella, R Vona, A Sgadari,MC Silveri, W Malorni
FEBS Letters 579, p2759­766 (2005)
Restored Antioxidant Capacity Parallels the Immunologic and Virologic Improvement in Children with Perinatal Human
Immunodeficiency Virus Infection Receiving Highly Active Antiretroviral Therapy.
M Martino, F Chiarelli, M Moriondo, M Torello, C Azzari, and L Galli
Clinical Immunology, Vol.100(1),p82­6 (2001)
Antioxidant capacity as a reliable marker of stress in dairy calves transported by road.
P Preget, E Bollo, FT Cannizzo, B Biolatti, E Contato, PG Biolatti: Veterinary Record Vol. 156, p53­54 (2005)

For more information and technical support, please visit JaICA web site http://www.jaica.com/biotech/e/Japan Institute for the Control of Aging (JaICA), Nikken SEIL Co., Ltd.
hｔｔｐ：//www.jaica.com/biotech/e/ e­mail: biotech@jaica.com
TEL：+81­538 49 0125
FAX： +81­538 49 1267
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