Antioxidants TBARS assay

Figure 8.2 Pomegranate juice polyphenols and antioxidant potency in comparison to other fruit juices. Total polyphenol concentration in the different juices was determined using quercetin as a standard. LDL (100 pg of protein/milliliter) was preincubated with increasing volume concentration (0-25 pL) of the juices. Then, 5 pmol/L of CuS04 was added, and the LDL was further incubated for 2 hours at 37°C. The extent of LDL oxidation was measured by the thiobar-bituric acid reactive substance (TBAR) assay, and the ICh, values (the concentration needed to get 50% inhibition) are given. Results are given as mean S.D. of three different experiments.

In the second experiment, mice were irradiated with the highest dose from the dose-response experiment. Concentrations of lipid hydroperoxides and lipophilic antioxidants were measured simultaneously on single skin samples from irradiated and non-irradiated sides of each mouse. The lipid hydroperoxide assay directly measured lipid peroxidation and thus was superior to the TBARS assay, which is indirect. Lipid-peroxyl radicals have been linked to chemically induced cutaneous carcinogenesis [31] as well as to UV-light-induced cutaneous carcinogenesis [32],... 

Figure 3 Alcohol augments grape skin polyphenol extraction Into white wine and Increases Its antioxidant capacity. Whole squeezed Muscat grapes were Incubated for 18 h with Increasing concentrations of alcohol up to 18%, after which the juice was separated from the grape skin and allowed to ferment Into wine. (A) Polyphenol concentration In wine samples was determined. (B) Wine samples at a final concentration of 2 pL/mL were added to LDL (100 mg of proteln/L) and Incubated with 5 pmol/L CUSO4 for 2 h at 37 °C. LDL oxidation was measured by LDL TBARS assay. LDL, low-density lipoprotein TBARS, thiobarbituric acld-reactlve substance. (C) Linear regression analysis of the total polyphenol concentration of wine and the wIne-Induced Inhibition of LDL oxidation.

The assays most widely employed are the measurement of thiobarbituric acid-reactive species (TBARS) and the formation of conjugated dienes, markers of lipid peroxidation [31-33] the determination of advanced oxidation protein products (AOPP), a marker of protein oxidation, and of advanced glycation end-products (AGE) [34-37] the measurement of erythrocyte antioxidant potential [38]. Of particular importance is the isoprostanes determination, since these compounds are formed by the free radical catalysed peroxidation of arachidonic acid, which is independent of the cyclooxygenase enzyme, giving rise to stable compounds, measurable in urine [39]. As recently assessed in a Workshop on markers of oxidative damage and antioxidant protection [40], currently available methods for the determination of antioxidant and redox status are not yet generally suitable for routine clinical applications, essentially for the lack of standardized tests. 

The EO of Crithmum maritimum (=Cachrys maritima, Apiaceae, rock samphire) comprises limonene and y-terpinene with an amount of 22.3% and 22.9%, respectively, as the major components. Two different test methods (TBA assay and a micellar model system where the antioxidative activity in different stages of the oxidative process of the lipid matrix was monitored) were used. Both assays explain the very high activity of this EO. In the TBA assay BHT and a-tocopherol were used as positive standards and the oil showed a better capacity than those substances. Comparable results were obtained by the micellar method system where the EO acts as a protector of the oxidation of linoleic acid and inhibits the formation of conjugated dienes (Ruberto et al., 2000). The modification of LDL by an oxidative process for instance can lead to atherosclerosis. Natural antioxidants such as P-carotene, ascorbic acid, a-tocopherol, EOs, and so on are able to protect LDL against this oxidative modification. y-Terpinene proved itself to be the strongest inhibitor of all used authentic compounds for the formation of TBARS in the Cu -induced lipid oxidation system (Grassmann et al, 2003). So, the addition of y-terpinene to food can possibly stop the oxidative modification of LDL and reduce the atherosclerosis risk. 

Two bioassays were used to evaluate the potency of Sal B and SM-EW-1 fraction to inhibit human LDL oxidation in vitro. The first assay was based on inhibition of malondialdehyde (MDA) formation, determined as TBARS by reacting with thiobaibituric acid (TBA), at fixed tin (2-6 hr). The ICso values indicated that Sal B (1.1 pM) was 4.0 times more potent than probucol (4.5 pM). The time course study of Sal B to inhibit conjugated diene formation in a standard LDL oxidation assay (LDL at 50 pg/mL was reacted with 5 pM Cu was also carried out in a microtiter plate (quartz) (44,45). Sal B prolonged the lag phase (ATiag) of LDL oxidation 7.5 times longer than probucol both at 1.0 pM (Table 1) (Figure 4). Sal B accounted for up to 75% of antioxidant activity in SM-EW-1 fraction. 

The relative antioxidant activities of carotenoids in multilamellar liposomes, as assayed by inhibition of the formation of thiobarbituric acid-reactive substances (TBARs), were lycopene>a-carotene>p-cryptoxanthin >zeaxanthin= P-carotene>lutein (Stahl et al, 1998). Mixtures of carotenoids were more effective than the single compounds, and this synergistic effect was most pronounced when lycopene or lutein was present. The superior antioxidant activity of mixtures of carotenoids may be related to the specific positioning of different carotenoids within cell membranes. 

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