Xylenol orange disodium salt

The hydrogen peroxide (H₂O₂) scavenging activity, based on transition metal chelation, was determined by using a method modified from Hazra et al. [49 (link)] and Jiang et al. [50 (link)] with a microplate reader in 96-well format with four technical replicates on each plate. An aliquot of 2 mmol/L H₂O₂ (Merck KGaA, Darmstadt, Germany) was added to the reaction mixture with the sample, 2.56 mmol/L ammonium iron (II) sulphate·6H₂O (BDH Prolabo) and 111 µM xylenol orange disodium salt (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). After 30 min incubation, the absorbance of the ferric-xylenol orange complex at 560 nm was measured. The assay measures the ability of the sample to scavenge H₂O₂ and prevent the oxidation of Fe(II) to Fe(III), which is indicated by the formation of the ferric-xylenol orange complex. The H₂O₂ scavenging ability is expressed as inhibition percentage (%) of Fe(II) oxidation to Fe(III). Sodium pyruvate (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used as a reference compound.

AgNO₃ (>99.8%), HCl (32%), NaH₂PO₄ (99.5%), Na₂HPO₄ (99%) and NaOH (99%) were acquired from Merck, and HNO₃ (65%) and H₂SO₄ (95%) from Panreac. AgNO₃ aqueous solution (0.1 M) was obtained from Riedel-de Haën. Ascorbic acid (≥99.0%), D-Sorbitol (>8%), H₂O₂ (35%), H₂PtCl₆ (99.9%), (NH₄)₂Fe(SO₄)₂.6H₂O (≥9%), Ru(NH₃)₆Cl₃ (98%) and xylenol orange disodium salt were purchased from Sigma-Aldrich. KNO₃ (>99.5%) was obtained from Fluka and K₄Fe(CN)₆·3H₂O (98%) from Probus S.A. All the reagents were used as received without further purification. Ultrapure water (Milli-Q purification system, 18.2 MΩ cm, Millipore Corp, Bedford, MA, USA) was used for the preparation of all solutions. Two laundry detergent boosters and an antiseptic product were used as real samples for the determination of H₂O₂. The first ones are a bleaching agent (stated composition: 5–15% oxygenated whitening agents, <5% anionic and non-ionic surfactants, perfume and optical whiteners) and a brightener (stated composition: 5–15% oxygenated whiteners, <5% anionic and non-ionic surfactants, perfume, methylchloroisothiazolinone and methylisothiazolinone), while the antiseptic compound contains 3% hydrogen peroxide. All of them were purchased from a local supermarket.

Methanol (MeOH, >99%), (GVL, 99%), 2H-methylimidazole (MIM, 97%), Xylenol Orange disodium salt (XO), Bromophenol Blue sodium salt (BB), Congo red (CR) and zinc nitrate hexahydrate (Zn(NO₃)₂∙6H₂O, 99%), and deuterium chloride (DCl (35%)/D₂O) were purchased from Sigma Aldrich (Darmstadt, Germany); deuterated dimetyhlsulphoxide (DMSO-d6) was purchased from Eurisotop (Saint-Aubin, France) and was used without further purification.

The TOS analysis is based on the oxidation of ferrous ions-o-dianisidine complexes to ferric ions. This process is regulated by the oxidants present in the sample. The whole analysis is based on the two reaction solutions: TOS R1 and TOS R2. The TOS R1 is composed of 150 μmol xylenol orange disodium salt (Sigma-Aldrich, St. Louis, MO, USA), 140 mmol sodium chloride (Sigma-Aldrich, St. Louis, MO, USA), and 1.35 mol glycerol (Centralchem, Bratislava, SR) in 25 mmol H₂SO₄ (Sigma-Aldrich, St. Louis, MO, USA). The TOS R2 contains 5 mmol ferrous ammonium sulfate hexahydrate (Centralchem, Bratislava, Slovakia), and 10 mmol o-dianisidine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in 25 mmol sulfuric acid (Sigma-Aldrich, St. Louis, MO, USA). Standards (H₂O₂) and samples of SP (35 μL) were pipetted in doubles to 96-well plate. TOS R1 (225 μL) was added, and the reference reading at 560 nm was performed using a Glomax Multi+ Detection System plate reader (Promega, Madison, Wisconsin, USA). After 10-min incubation, 11 μL TOS R2 was added to each well. Following a 3-min incubation period, the absorbance was spectrophotometrically assessed at the same wavelength. The assay was calibrated using hydrogen peroxide, and the results are expressed as μmol H₂O₂ Eq/g of protein [6 (link),37 (link)].

All solvents used for n-3 LC-PUFA, carotenoid and primary oxidation determination, and for lipid extraction were HPLC or GC-grade and were purchased from Roth (Karlsruhe, Germany) (chloroform, methanol, n-hexane ≥95%, acetonitrile) or VWR (Fontenay-sous-Bois, France) (toluene and ethyl acetate). Reagents were the following: xylenol orange disodium salt, iron(II) sulfate heptahydrate ≥99% (Sigma-Aldrich, Steinheim, Germany), barium chloride dihydrate p.a. (Chem-Lab NV, Zedelgem, Belgium), triphenylphosphine 99% (Sigma-Aldrich, Buchs, Switzerland), iron(III)chloride (Merck Schuchardt OHG, Hohenbrunn, Germany) and ammonium acetate (Merck KGaA, Darmstadt, Germany). Standards of fucoxanthin, diatoxanthin, and β-carotene for carotenoid analyses were purchased from DHI (Horsholm, Denmark) and standards for secondary oxidation analysis were acquired from Sigma-Aldrich (Steinheim, Germany) (2,4-heptadienal and (E)-2-pentenal) and Roth (Karlsruhe, Germany) (2-butenal).

The hydrogen peroxide (H₂O₂) scavenging activity was determined using a method modified from Hazra et al. [39 (link)] and Jiang et al. [40 (link)]. The assay was carried out according to Vaario et al. [36 (link)]. An aliquot of 2 mM H₂O₂ (Merck KGaA, Darmstadt, Germany) was added to the reaction mixture with the sample and a mixture containing 2.56 mM ammonium ferrous (II) sulfate (BDH Prolabo) in 0.25 mM H₂SO₄ (Merck KGaA) and 27.8 µM xylenol orange disodium salt (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 4.4 mM sorbitol (D(-)-sorbitol, AppliChem GmbH). After 30 min incubation, the absorbance of violet-colored ferric-xylenol orange complexes at 560 nm was measured. The assay measures the ability of the sample to scavenge H₂O₂ and prevent the oxidation of Fe(II) to Fe(III), which is indicated by the formation of a ferric–xylenol orange complex. The inhibition of the oxidation is expressed as an inhibition % of the reaction and the samples with 100% inhibition activity will remain yellowish. Sodium pyruvate (Sigma-Aldrich) was used as a reference compound.

Activity was determined using a method modified from Hazra et al. (2008) (link) and Jiang et al. (1990) (link). The experimental setup has been described in detail by Tienaho et al. (2020) (link). In brief, an aliquot of 2 mM H₂O₂ (Merck KGaA, Darmstadt, Germany) was added to the reaction mixture with the sample and a mixture containing 2.56 mM ammonium ferrous (II) sulfate (BDH Prolabo) in 0.25 mM H₂SO₄ (Merck KGaA) and 27.8 µM xylenol orange disodium salt (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 4.4 mM sorbitol (D(-)-sorbitol, AppliChem GmbH). After 30 min of incubation, the absorbance of violet-colored ferric-xylenol orange complexes at 560 nm was measured. The assay measures the ability of the sample to scavenge H₂O₂ and prevent the oxidation of Fe(II) to Fe(III), which is indicated by the formation of the ferric-xylenol orange complex. The inhibition of oxidation is expressed as the inhibition (%) of the reaction, and the samples with 100% inhibition activity will remain yellowish. Sodium pyruvate (Sigma-Aldrich) was used as a reference compound.