Lipids TBARS assay

Hassoun et al. (1993) examined the effects of various pesticides on lipid peroxidation and DNA single strand breakage in the hepatic cells of female Sprague-Dawley rats. Animals were dosed orally once with endrin at 4.5 mg/kg, lindane at 30 mg/kg, chlordane at 120 mg/kg, or DDT (dichlorodiphenyl trichloro-ethane) at 40 mg/kg, or vehicle only (com oil, control). At 6, 12, and 24 hours post-dosing, 4 animals from each group were sacrificed, their livers removed, and prepared for lipid peroxidation assay. Lipid peroxidation was measured calorimetrically by determining the amount of thiobarbituric acid reactive substances (TBARS) formed. Exposure to endrin resulted in a 14.5% increase in hepatic mitochondrial... 

In the UVB study we also measured malondialdehyde, a marker of lipid peroxidation, but were unable to detect any increase in response to UV light, in contrast to other reports. There are a number of possible explanations. In our experiment, a-tocopherol levels remained high after irradiation, and it is known that lipid peroxidation does not begin in in vitro membrane systems until a-tocopherol is completely depleted [29], Several studies have shown a time lag of one to several hours after irradiation to occur before a measurable increase in cutaneous lipid peroxidation can be detected [26-28] since skin was processed immediately in our experiments, lipid peroxidation may not have reached detectable limits. Finally, the TBARS assay for malondialdehyde is notoriously fraught with artifact and has a relatively high background [30], and noise levels may simply have been too high to detect peroxidation. Results... 

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],... 

The immediate appearance of lipid hydroperoxides after irradiation in this study contrasts with several other studies, which have either reported no increase in lipid-peroxidation products in skin after UV irradiation [21] or an increase only several hours after irradiation [27-29], In these studies the background level of lipid-peroxidation products has been high, even in control, non-irradiated skin. In contrast, in our experiments lipid hydroperoxides were undetectable before irradiation, but appeared at very high concentrations in skin immediately after irradiation. The discrepancies between various studies may be due to differences in technique. The HPLC-chemiluminescence assay used in the present study is specific for lipid hydroperoxides, and has less potential for artifact than the TBARS assay, which is known to suffer from many possibilities for artifact [30] -... 

Following treatment of the rats with chlorpyrifos and fenthion. 4,3- and 4.8-foId increases, respectively, were observed in hepatic lipid peroxidation, whereas at the same doses, incrca.ses of 4.6- and 5.3-fold, respectively, were observed in brain homogenates. The effects of the pesticides on lipid peroxidation in hepatic and brain homogenates based on the TBARS assay are summarized in Table I (Bagchi et al., 1995). 

This test is used for both in vitro and in vivo determinations. It involves reacting thiobarbituric acid (TBA) with malondialdehyde (MDA), produced by lipid hydroperoxide decomposition, to form a red chromophore with peak absorbance at 532 nm (Fig. 10.1). The TBARS reaction is not specific. Many other substances, including other alkanals, proteins, sucrose, or urea, may react with TBA to form colored species that can interfere with this assay. 

To confirm that the effects of NO on O2 consumption are due to inhibition of hpid peroxidation, we also examined the effect of NO on TBARS, a product of lipid peroxidation. Cells were oxidized by Fe ", and 0.9 (jM N0 was added 1 min later. Cells were collected after 5 min for assay. Figure 5 shows that Fe " increased lipid peroxidation, and NO inhibited it by 63% (after subtracting basal levels). It is noteworthy that the percentage inhibition of O2 uptake by NO as measured by the change in O2 concentration under similar conditions was similar (78%) lending verification to these complementary methods. These results confirm the relationship of O2 consumption and lipid peroxidation in these experiments... 

Transesterification, fatty acid analysis of lipids, 437, 439 Triacetin, lipase assays, 378 Triacylglycerol acylhydrolase, 371, 375, 378. See also Lipases Triacylglycerols, 432 Tributyrin, lipase assays, 378 Trichloroacetic acid (TCA) solubility index for protein hydrolysis, 152 in TBARS determination, 548-550 Trienes, conjugated, determination of, 515-517, 523-524, 526, 528 Trifluoroacetic acid (TFA), for determination of neutral sugars, 721-722, 724-725, 729-730... 

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. 

Because of its convenience, the TB A method has become a common assay to determine the degree of peroxidation and oxidative susceptibility of a wide range of biological materials, including LDL. However, the validity of the TBA determination as an index of lipid peroxidation in biological samples has been a matter of considerable debate in the literature. The determination of TBARS inherently lacks specificity, and is subject to interference by many compounds including materials that are not due to lipid peroxidation (see Chapter 5.E). This method is also flawed by analytical artifacts, and is affected by the same factors as lipid peroxidation. 

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. 

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