The ferric reducing/antioxidant power (FRAP) test was used to measure total antioxidant capacity. In this test, the antioxidant agents in the sample decreased the ferric tripyridyltriazine (TPTZ‐Fe3+) complex to the ferrous (TPTZ‐Fe2+) complex, which is blue in the acidic medium. To prepare the FRAP solution, 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6‐tripyridyl‐s‐triazine (TPTZ) solution (Sigma–Aldrich) in 40 mM HC1, and 20 mM FeCl3·6H2O in a ratio of 10:1:1 were mixed together. Then, the FRAP solution was heated to 37°C. The 300 μl of FRAP solution and the 10 μl of each standard solution (FeSO4·7H2O) or plasma sample were mixed. The optical absorption of the samples was measured by spectrophotometer at 593 nm against the blanks (distilled water), and the FRAP content of the unknown sample was calculated as compared to the standard curve in Fe2+ (μM per liter).22
Tptz solution
Manufactured by Merck Group
Sourced in United States
TPTZ solution is a reagent that is used in various analytical and diagnostic applications. It is a colorimetric iron-chelating agent, which forms a blue-colored complex upon interaction with ferrous ions. The solution is commonly used in analytical techniques, such as spectrophotometry, to quantify iron content in samples.
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Lab products found in correlation
4 protocols using tptz solution
1
Measurement of Total Antioxidant Capacity
2
Serum Antioxidant Capacity via FRAP
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Serum antioxidant capacity measured by the FRAP method. This method is prepared by mixing 300 mM acetate buffer, 10 mM TPTZ solution (Sigma–Aldrich, St. Louis, MO, USA), in 40 mM HC1, and ferric chloride solution. FRAP reagents were mixed with samples and incubated for 10 min at 37 ° C. Then, the light absorption of the samples at 593 nm was read by ELISA Reader, and the concentration of the samples was determined using a standard curve.
3
Ferric Reducing Antioxidant Power Assay
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This method evaluates the capacity of the antioxidants in the samples by reducing the ferric ion (Fe3+) in an acidic medium in the presence of TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine) to form the ferrous form (Fe2+). This reaction leads to a reduced TPTZ–Fe (III) complex with blue color, measured at 593 nm. The working solution contained 300 mM acetate buffer (pH 3.6), a 40 mM TPTZ solution (Sigma-Aldrich Chemical Co., Louis, MO, USA), and a 20 mM FeCl3·6H2O solution (Merck Chemicals, Darmstadt, Germany) in water at a 10:1:1 ratio. Suspensions and the working FRAP solution were mixed at a 1:25 ratio for 10 min at 37 °C in a dark place. The absorbance was taken at 593 nm using a UV/Vis spectrophotometer (BioTek Instruments, Winooski, VT, USA) [10 (link)]. A calibration curve with Trolox (Sigma-Aldrich Chemical Co., Louis, MO, USA) was used. The results are expressed in µmol Trolox equivalents per gram of suspension and relative antioxidant capacity compared to the nonprocessed bulk material.
4
Quantifying Antioxidant Capacity via FRAP Assay
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FRAP reagent was freshly prepared by mixing 25 mL of acetate buffer (300 mmol L–1, pH 3.6) (Sigma, St Louis, MO, USA), 2.5 mL of TPTZ solution (10 mL of TPTZ in 40 mmol L–1 HCl) (Sigma, St Louis, MO, USA) and 2.5 mL of 20 mmol L–1 FeCl3 (Sigma, St Louis, MO, USA) in water. A 10 μL aliquot of each extract dissolved in the appropriate solvent was added to 300 μL of FRAP reagent in each well of a 96‐well plate and incubated for 30 min at 37 °C. Absorbance was measured at 593 nm using a microplate reader. All experiments were performed in triplicate, and all results are presented as the Trolox equivalent antioxidant capacity. Trolox (Sigma, St Louis, MO, USA) standard solution at concentrations of 0, 5, 10, 20, 40 and 80 mmol L–1 was prepared and the absorbance was measured at 593 nm to construct a standard calibration curve, where absorbance was plotted on the y‐axis and the concentration on the x‐axis. The gross dose rate was y = 0.0503x + 0.0387, and the correlation coefficient (R2) was 0.9998, indicating good linearity.
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