Inhibitory Effect of Lutein and Pycnogenol on Lipid Peroxidation in Porcine Retinal Homogenate
Takako Nakanishi‐UedaMaki KamegawaSeiichiro IshigakiMasahiko TsukaharaSatoshi YanoKatsuya WadaHajime Yasuhara
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Abstract:
This study aimed to investigate the antioxidant effect of lutein, human macula pigment, and pycnogenol, which contains several flavonoids, on lipid peroxidation induced in 10% porcine retinal homogenate by the addition of 1 mM Ferric chloride (FeCl3), 50 mM 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH), or 25 mM 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN). Lipid hydroperoxide concentration was determined from the amount of thiobarbituric acid reactive substances (TBARS) in the sample following treatment. After 60 min of oxidation with FeCl3, AAPH and AMVN, the TBARS content in the retinal homogenates increased from 28.6 ± 1.6 to 85.4 ± 0.9, from 27.9 ± 1.2 to 57.2 ± 1.1, and from 26.0 ± 1.0 to 77.5 ± 2.0 nmol MDA/mg protein, respectively. Lutein did not show remarkable antioxidant activity in this experimental system. However, IC50 of pycnogenol for TBARS formation was decreased by combining 10 μM lutein in each initiator; from 12 to 5 μg/mL in FeCl3, from 2.8 to 0.5 μg/mL in AAPH, from 465 to 110 μg/mL in AMVN. These results suggested that a combination treatment of lutein and pycnogenol is more effective for inhibiting lipid peroxidation in porcine retinal homogenate. This synergy might be due to efficient functional antioxidants acting in both hydrophilic and lipophilic cellular environments.Keywords:
TBARS
Thiobarbituric acid
A simple and highly sensitive spectrophotometric method was developed for the determination of thiobarbituric acid reactive substances (TBARS) as a marker for lipid peroxidation in fried fast foods. The method uses the reaction of malondialdehyde (MDA) and TBA in the glacial acetic acid medium. The method was precise, sensitive, and highly reproducible for quantitative determination of TBARS. The precision of extractions and analytical procedure was very high as compared to the reported methods. The method was used to determine the TBARS contents in the fried fast foods such as Shami kebab, samosa, fried bread, and potato chips. Shami kebab, samosa, and potato chips have higher amount of TBARS in glacial acetic acid-water extraction system than their corresponding pure glacial acetic acid and vice versa in fried bread samples. The method can successfully be used for the determination of TBARS in other food matrices, especially in quality control of food industries.
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Abstract Background Oxidative stress has been proved to participate in a plethora of human and canine diseases. Among oxidative stress biomarkers, Thiobarbituric Acid Reactive Substances (TBARS) and Total Antioxidant Status (TAS) are two of the most widely used. Pre-analytical factors are highly relevant to obtain accurate results in these assays. Hemolysis, icterus and lipemia (HIL) are among the most frequent sources of pre-analytical errors in the laboratory, but limited information is available on the considerations for canine specimens. Hence, the objective of this study was to assess the potential interferences due to HIL on the determination of TBARS and TAS in canine serum. Methods Dilutions of canine pooled serum samples were prepared with increasing concentrations of hemolysate, bilirubin and a synthetic lipid emulsion. TBARS and TAS were determined, and biases from the control value due to the interferents were calculated. Results Hemolysis, icterus and lipemia induced significant interferences on TBARS and TAS, to a variable extent depending on the biomarker and interferent. TBARS seemed the most vulnerable method to interferences in this study. Slight hemolysis, moderate icterus and slight lipemia induced significant deviations of TBARS value, exceeding the acceptable interference threshold. TAS assay was also affected by HIL, but to a lesser extent compared to TBARS. Significant biases from TAS control value were observed when icterus was moderate, and hemolysis and lipemia were marked. Conclusions TBARS and TAS assays are widely used for oxidative stress evaluation. However, the literature on the interference of HIL on these biomarkers in canine serum is scarce. In light of our results, we conclude that hemolyzed, icteric and lipemic specimens are not suitable for TBARS and TAS determination in canine serum. Our findings seem of considerable practical utility, as a simple visual inspection would be sufficient for discarding these specimens.
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Summary: Plasma concentrations of thiobarbituric acid reactive substances (TBARS) are an index of lipid peroxidation and oxidative stress. The protocol describes how the DiaComp quantitates TBARS in the animal models. Diabetic Complication:
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Thiobarbituric acid reactive substances (TBARS), carbonyls and reactive oxygen species (ROS) are oxidant compounds that can provide useful information on the oxidative status. Pigs can be affected by oxidative stress in different situations including physiological conditions such as lactation and also in different diseases, and the measurement of these three analytes in saliva could be potentially useful as biomarkers of the redox status in this species. Assays for the measurement of TBARS and carbonyls by spectrophotometry and ROS by luminol-based chemiluminescence in pigs' saliva were analytically validated and were applied in saliva of pigs after an in vitro incubation with different doses of ascorbic acid (AA). All the assays showed a satisfactory analytical precision and accuracy. The 240 h-incubation of saliva samples with 60 mM of AA induced to an increased TBARS and carbonyls production. TBARS, carbonyls and ROS can be estimated in saliva of pigs by the assays validated in this report. In addition, these assays can detect changes in the concentration of these analytes associated to incubation of saliva samples with AA.
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Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 °C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 μM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.
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The ability of the spectrophotometric thiobarbituric acid reactive substances (TBARS) test to determine malondialdehyde (MDA) in various food matrices was evaluated. MDA was extracted from the foods; the extract reacted with thiobarbituric acid (TBA); and the formed TBA-MDA adduct was measured spectrophotometricaly at 532 nm. In parallel, the TBA-MDA adduct was analyzed with high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Oils and unprocessed and uncooked meat and fish products did not exhibit any significant difference in the amount of MDA measured by the two methods, indicating that the major substance reacting with TBA and forming an adduct that absorbs at 532 nm was MDA. However, in products such as dry nuts, pork sausages, cooked fish, and gouda cheese, an overestimation of MDA was observed, indicating that TBARS test was unsuitable for accurate determination of MDA. Furthermore, the results in the present work suggest that the overestimation of MDA by the TBARS test as it was applied is related to the interference of other than secondary lipid oxidation products.
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Malondialdehyde
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Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 °C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 μM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.
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Malondialdehyde (MDA) level measured with thiobarbituric acid (TBA) method has been widely used as diagnostic indices of peroxidative tissue injury and lipid peroxidation. However, the specificity of the TBAtest toward MDA is disputed. MDA is merely one of the main unsaturated carbonyl products detected by TBA method. Since thiobarbituric acid reactive substances (TBARS) including most, if not all, of the unsaturated aldehydes/ketones resulted from oxidative stress, the using of TBARS may be useful as an index of carbonyl stress.
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