According to the method defined by Ohkawa et al. [19 (link)], MDA forms a pink complex with thiobarbi-turic acid (TBA) at 95°C, which can be measured using spectrophotometry at a wavelength of 532 nm. In the experiment, 0.1 mL of homogenate was added to a solution containing 0.1 mL of 8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid (Merck, Darmstadt, Germany), 1.5 mL of 0.9% TBA (Sigma-Aldrich, Schnelldorf, Germany), and 0.3 mL of DH2O. The mixture was incubated at 95°C for 1 hour. Upon cooling, 5 mL of n-butanol: pyridine (v/v, 15:1; Merck) was added. The mixture was vortexed for 1 minute and centrifuged for 30 minutes at 4,000 rpm. The absorbance of the 0.15 mL supernatant was measured at 532 nm by spectrophotometry. The standard curve was obtained using 1,1,3,3-tetramethoxypropane (Sigma-Aldrich).
N butanol pyridine
Manufactured by Merck Group
Sourced in Germany
N-butanol: pyridine is a laboratory reagent. It is a mixture of n-butanol and pyridine. The function of this mixture is to serve as a solvent and extraction agent for various chemical processes and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Acetic acid, by Merck Group (4 mentions)
1 1 3 3 tetramethoxypropane, by Merck Group (3 mentions)
Tetramethoxypropane, by Merck Group (2 mentions)
2 thiobarbiturate, by Merck Group (2 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Optizen pop, by Mecasys (1 mentions)
5 protocols using n butanol pyridine
1
Spectrophotometric Quantification of MDA
2
Spectrophotometric Determination of MDA Levels
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The MDA levels were determined spectrophotometrically at 532 nm according to the method described by Ohkawa et al. [14 (link)]. This method is based on spectrophotometric measurement of absorbance of the pink-colored complex which is formed by thiobarbituric acid and MDA at a high temperature (95°C). The 0.1 ml of supernatant separated for the measurement of GSH-Px and MDA was added to a solution containing 0.1 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (Merck), 1.5 ml of 0.9% thiobarbituric acid (Sigma-Aldrich) and 0.3 ml of dH2O. The mixture was incubated at 95°C for 1 h. Upon cooling, 5 ml of n-butanol:pyridine (v/v, 15:1; Merck) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4,000 rpm. The absorbances of the 0.15-ml final solutions were measured by spectrophotometry at 532 nm. The standard curve was obtained by using 1,1,3,3-tetramethoxypropane (Sigma-Aldrich).
3
Spectrophotometric Determination of MDA Levels
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According to the method defined by Ohkawa, et al.22 (link) (1979), MDA forms a pink complex with thiobarbituric acid (TBA) at 95°C, which can be measured using spectrophotometry at a wavelength of 532 nm22 (link). The volume of 0.1 mL homogenat was added to a solution containing 0.1 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL of 20% acetic acid (Merck, Darmstadt, Hessen, Germany) 1.5 mL of 0.9% TBA (Sigma-Aldrich, Steinheim, Nordrhein-Westfalen, Germany), and 0.3 mL dH2O. The mixture was incubated at 95°C for 1 h. Upon cooling, 5 mL of n-butanol: pyridine (v/v, 15:1, Merck, Darmstadt, Hessen, Germany) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4000 rpm. The absorbance of the 0.15 mL supernatant was measured at 532 nm by spectrophotometry. The Standard curve was obtained by using 1,1,3,3-tetramethoxypro pane (Sigma-Aldrich, Steinheim, Nordrhein-Westfalen, Germany).
4
Gastric Mucosal Lipid Peroxidation Analysis
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Thiobarbituric acid method was used to determine the concentration of gastric mucosal lipid peroxidation [31 (link)]. The corpus mucosa of stomach was scraped and homogenized in 10 mL of KCl solution (100 g L− 1). Five hundred microliter of the homogenate was added into a new tube containing thiobarbituric acid solution [1.5 mL of 8 g L− 1 2-thiobarbiturate (Sigma-Aldrich), 0.2 mL of 80 g L− 1 SDS, 1.5 mL of 200 g L− 1 acetic acid, and 0.3 mL of DW], heated at 98 °C for 1 h, cooled at room temperature and added 5 mL of n-butanol: pyridine (15:1; Sigma-Aldrich). The mixture was vortexed, centrifuged at 3000×g for 30 min, and measured the absorbance of the supernatant by UV/Vis spectrophotometer at 532 nm. The 1,1,3,3-tetramethoxypropane (Sigma-Aldrich) was used to draw the standard curve and the results were displayed as nM of MDA g− 1 of wet tissue (nM/g tissue).
5
Quantifying Lipid Peroxidation in Mouse Stomach
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Determination of malondialdehyde (MDA) formation by thiobarbituric acid was used as an index for the extent of lipid peroxidation using published method (Ohkawa et al., 1979) . Briefly, mouse stomach was promptly excised and rinsed with cold saline. To minimize the possibility of hemoglobin interfering with free radicals, any blood adhering to the mucosa was carefully removed. The corpus mucosa was scraped, weighed, and homogenized in 10 mL of 100 g/L KCl (Sigma-Aldrich). The homogenate (0.5 mL) was added to a solution containing 0.2 mL of 80 g/L SLS (Sigma-Aldrich), 1.5 mL of 200 g/L acetic acid (Merck, Darmstadt, Germany), 1.5 mL of 8 g/L 2-thiobarbiturate (Sigma-Aldrich), and 0.3 mL of distilled water. This mixture was then heated at 98°C for 1 h. After cooling to room temperature, 5 mL of n -butanol: pyridine (15:1: Sigma-Aldrich) was added. The reaction mixture was vortexed for 1 min and centrifuged at 2,000 × g for 30 min at 4°C. The supernatant was collected and its absorbance was measured at the wavelength of 532 nm using a UV/Vis spectrometer (Optizen Pop, Mecasys). A standard curve was obtained by using 1,1,3,3-tetramethoxypropane (Sigma-Aldrich). Results are expressed as nanomoles of MDA per gram of wet tissue.
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