2--7--dichlorodihydrofluorescein and 2-2--azobis(2-amidinopropane)

This study aimed to develop a fluorometric method to determine total antioxidant activity of plant foods. The antioxidant activities in plant foods were determined after extracting (1) hydrophilic components with acidified methanol (methanol:glacial acetate acid:water=50:3.7:46.3), (2) lipophilic components with methanol followed by tetrahydrofuran (THF), or (3) both hydrophilic and lipophilic components using sequential extraction of acidified methanol and THF together. Both the hydrophilic assay [using the hydrophilic radical initiator 2,2'-azobis-(2-amidinopropane)dihydrochloride (10 mmol/L) and hydrophilic probe 2,7-dichlorodihydrofluorescein (DCFH)] and the lipophilic assay [using the lipophilic radical initiator [2,2'-azobis (4-methoxiy-2,4-dimethylvaleronitrile), 2 mmol/L], and the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY 581/591) (BODIPY: 2 micromol/L)] were used to measure antioxidant activity. The inhibition of BODIPY oxidation was significantly increased (P<.01) when both the hydrophilic and lipophilic components were extracted using acidified methanol and organic solvent as compared to those extracted by organic solvent alone. In addition, the rate of DCFH oxidation was significantly delayed (P<.05) when both components coexisted compared to DCFH oxidation of the hydrophilic component alone. The combination of lipophilic and hydrophilic components in these plant foods showed significantly greater antioxidant activity than that of either hydrophilic or lipophilic component alone. Thus, both hydrophilic and lipophilic components in plant foods and their interactions should be considered when determining their antioxidant activity.

Topics: Amidines; Angelica; Antioxidants; Azo Compounds; Boron Compounds; Chemical Fractionation; Fluoresceins; Fluorometry; Nitriles; Oxidation-Reduction; Perilla; Plants; Solubility; Vegetables

The oxidation of endogenous antioxidant nutrients in human plasma was determined to examine their activities against free radicals generated in the aqueous and lipid compartments of plasma. Free radicals were induced at a constant rate in the aqueous compartment by the hydrophilic radical generator, 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH; 10-20 mmol/L) and in the lipid compartment by the lipophilic radical generator, 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN; 1-2 mmol/L). The depletion of endogenous plasma antioxidant nutrients (lutein, cryptoxanthin, beta-carotene, lycopene, alpha-tocopherol, ascorbic acid, uric acid) was determined after incubation with either AAPH or MeO-AMVN at 37 degrees C using HPLC. The oxidation of the aqueous and lipid compartments of plasma was selectively monitored by a fluorimetric method using either the hydrophilic probe, 2',7'-dichlorodihydrofluorescein (DCFH) or the lipophilic probe, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591). When plasma was incubated with AAPH, the rates of consumption of the antioxidant nutrients were as follows: ascorbic acid > alpha-tocopherol > uric acid > lycopene > lutein > cryptoxanthin > beta-carotene. When plasma was incubated with MeO-AMVN, alpha-tocopherol and carotenoids were depleted at similar rates and ahead of the major water-soluble antioxidants. Our study indicates that the antioxidant nutrients present in both the lipid and aqueous compartments can remove free radicals generated in plasma, and their activity depends on the localization of the attacking radical species.

Topics: Amidines; Antioxidants; Aza Compounds; Azo Compounds; Body Water; Chromatography, High Pressure Liquid; Fatty Acids; Fluoresceins; Fluorometry; Humans; Lipids; Nitriles; Nutritional Physiological Phenomena; Oxidants; Oxidation-Reduction; Reactive Oxygen Species; Tissue Distribution

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BODIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizability of human plasma. Aqueous plasma oxidizability was initiated by the aqueous peroxyl radical generator, AAPH, and 2',7'-dichlorodihydrofluorescein (DCFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determined by measuring the rate of consumption of the plasma hydrophilic and lipophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > alpha-tocopherol > uric acid > beta-carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), beta-carotene was consumed earlier than uric acid and almost at the same time as alpha-tocopherol, reflecting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly increased after the depletion of endogenous alpha-tocopherol and beta-carotene, whereas it was delayed for 180 min when AAPH was used instead of MeO-AMVN. The measurement of lipid oxidation in plasma was validated by adding to plasma the two lipophilic antioxidants, alpha-tocopherol and beta-carotene, whose inhibitory effects on BODIPY oxidation were dependent on the duration of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aqueous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma.

Topics: Adult; alpha-Tocopherol; Amidines; Antioxidants; Azo Compounds; beta Carotene; Biomarkers; Boron Compounds; Fluoresceins; Fluorometry; Humans; Lipids; Nitriles; Oxidation-Reduction; Plasma; Water