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Fifteen new thiobarbituric acids have been prepared by the Knoevenagel condensation of appropriate aldehydes with 1-(2,3, 2,5, 2,6 dichlorophenyl)-3-morpholinophenyl thiobarbituric acids. Five compounds of this series were found to be biphasic in nature whereas some compounds showed appreciable hypotensive activity of varying magnitude and duration.
OBJECTIVES: In determining the plasma malondialdehyde MDA levels in some Taiwanese college students, we found rather different results by using different thiobarbituric acid TBA tests, even by the high-performance liquid chromatography HPLC-based methods. Here, we re-evaluated four commonly used TBA tests and improved the HPLC-based test. DESIGN AND METHODS: We used the blood plasma of 16 college volunteers to determine plasma MDA by using four methods: a spectrophotometric measurement of thiobarbituric acid-reactive substances (TBARS) in the TCA-supernatant of plasma (Method A); a fluorescence measurement of plasma lipid peroxides (Method B); and two different HPLC-based measurements of MDA with either 532-nm measurement (Method C, HPLC/532 nm) or fluorescence measurement (Method D, HPLC/fluor.). RESULTS: The levels of MDA or TBA reactive substances obtained from the four methods differed substantially (0.39 +/- 0.15; 2.14 +/- 0.73; 0.75 +/- 0.22; and 0.38 +/- 0.15 microM for Methods A, B, C, and D, respectively). The results were positively correlated between Methods A and B (r = 0.740, p < 0.02) and between Methods C and D (r = 0.516, p < 0.05). However, results were negatively correlated between Methods B and D (r = -0.548, p < 0.05). Because most plasma MDA is bound to proteins, we modified the HPLC-based methods (C and D) by adding an alkaline hydrolysis step, and the plasma TBA-MDA adduct detected by HPLC/532 nm was referred to as total MDA. RESULTS show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma because this treatment led to release of MDA from plasma proteins. We also adapted the potassium iodide (KI) treatment of plasma from Method D to reduce lipid hydroperoxides. Our modified method gave a total MDA level in the 16 volunteers of approximately 1.5 microM, which was equal to protein-bound MDA plus free MDA. This total MDA level was positively (p < 0.05) correlated with the level of TBA reactive substances obtained from Methods C (r = 0.63, p < 0.05) and D (r = 0.48, p < 0.07), but was not correlated with those from Methods A and B. The recovery (84 approximately 105%), precision (within-assay coefficient of variation: 2.4%, between-assay coefficient of variation: 4 approximately 8%) and sensitivity of the modified procedure were comparable to other HPLC-based methods. CONCLUSION: By using a validated modification of HPLC-based TBA method, the total plasma MDA in 16 Taiwanese college students was found to be 1.54 microM, which was relatively high compared to those obtained by other HPLC-based method, primarily due to the release of protein-bound MDA by alkaline hydrolysis. This level equaled the sum of protein-bound MDA and free MDA in plasma, confirming that this level represents total plasma MDA.
The circular dichroism (CD) and carbon-13 nuclear magnetic resonance (13C NMR) spectra of (S)-5-(2'-pentyl)-2-thiobarbituric acid and several (S)-5-alkyl-5-(2'-pentyl)-2-thiobarbituric acids were obtained. The results were compared to CD and 13C NMR spectral data obtained from (S)-5-(2'-pentyl)bartituric acid and (S)-5-alkyl-5-(2'-pentyl)barbituric acids.
The imbalance between oxidants and antioxidants is known to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoking is the most frequent factor responsible for development of COPD by leading to oxidant overload in the lower airways, due to presence of its own oxidants and to recruitment and activation of pulmonary phagocytes. We aimed to determine whether (1) patients with stable COPD have higher thiobarbituric acid-reactive substances (TBARs, an end-product of lipid peroxidation) and H2O2 levels in expired breath condensate than healthy subjects who have never smoked; (2) COPD subjects who are current smokers exhale more TBARs and H2O2 than COPD ex-smokers and those who have never smoked; and (3) concentration of TBARs correlates with H2O2 levels in the breath condensate of COPD patients. The TBAR and H2O2 content in expired breath condensate of 17 healthy nonsmoking subjects and 44 patients (11 current smokers, 20 ex-smokers and 13 who had never smoked) with stable COPD [forced expiratory volume in 1 s (FEV1) 63.3 +/- 16.3% and FEV1 reversibility 5.2 +/- 4.3% predicted value] was measured spectrofluorimetrically by the thiobarbituric acid and homovanillic acid methods, respectively. The mean concentrations of TBARs and H2O2 in the expired breath condensate of COPD subjects were 12 (0.48-0.86 microM vs. 0.04 +/- 0.14 microM; P < 0.05) and 10 times (0.48 +/- 0.67 microM vs. 0.05 +/- 0.07 microM; P < 0.005) higher than in healthy controls. Current smokers with COPD did not exhale more H2O2 than COPD ex-smokers and those who had never smoked. TBARs levels shared only a tendency to be higher in the breath condensate of smoking COPD subjects than in that of ex-smokers (0.92 +/- 1.49 microM vs. 0.35 +/- 0.44 microM) and of COPD subjects who had never smoked (0.92 +/- 1.49 microM vs. 0.30 +/- 0.53 microM). No correlation was found between TBAR and H2O2 levels in the whole COPD group. These variables did not correlate with cigarette smoking status and the time from smoking cessation. Subjects with stable COPD exhibit increased lipid peroxidation and H2O2 generation in the airways. Current cigarette smoking does not distinguish COPD subjects with respect to TBARs and H2O2 exhalation.
OBJECTIVE: To evaluate the effect of arthrocentesis and sodium hyaluronate (SH) injections on nitrite, nitrate, and thiobarbituric acid-reactive substance (TBA-RS) levels in temporomandibular joint internal derangements. STUDY DESIGN: Arthrocentesis was performed on 10 patients, and 15 patients received a supplemental injection of SH after arthrocentesis. All these patients received an SH injection 15 days after the first intervention. The synovial fluid samples were obtained before arthrocentesis on the first appointment and before the SH injection 15 days later. Nitrite and nitrate levels were measured with a highly sensitive and specific chemiluminescence detection method, and the concentration of lipid peroxidation products was assessed by means of the thiobarbituric acid reaction. RESULTS: Symptomatic improvement was seen in both groups. Nitrite, nitrate, and TBA-RS levels only decreased significantly (P <.05) with a supplemental SH injection after arthrocentesis. CONCLUSIONS: Intra-articular injections of SH may reduce nitrite, nitrate, and TBA-RS levels that play a role in the pathogenesis of various temporomandibular disorders.
BACKGROUND: Patients with end-stage renal disease are thought to be in a highly peroxidative state, based on studies showing decreased serum antioxidant activity and increased peroxidative products. In this study we confirm these findings by examining lipid peroxidation in subcutaneous fat tissue in uraemia. SUBJECTS AND METHODS: Twenty-seven subcutaneous fat samples were taken from patients with end-stage renal disease when they underwent intervention for arteriovenous fistula for haemodialyis or for catheter insertion for continuous ambulatory peritoneal dialysis. The control samples were taken from 11 patients with normal renal function and without any history of renal disease who had surgical interventions. Lipid peroxides were measured as thiobarbituric acid reactive substance. RESULTS: The concentration of thiobarbituric acid reactive substances in subcutaneous fat tissue in the patients with end-stage renal disease is 1.223 +/- 0.636 nmol/mg fat tissue (mean +/- SD) whereas the level in the control group is 0.097 +/- 0.054 nmol/mg fat tissue. A comparison of the two groups by Student's t test revealed a highly significant difference (P < 0.001). CONCLUSIONS: This study supports the finding of a severe peroxidative state in patients with end-stage renal disease.
The effect of irradiation on thiobarbituric acid reactive substances (TBARS) and volatile compounds in raw and cooked nonirradiated and irradiated chicken breast meat infused with green tea and grape seed extracts was investigated. Chicken breast meat was vacuum infused with green tea extract (3,000 ppm), grape seed extract (3,000 ppm), or their combination (at a total of 6,000 ppm), irradiated with an electron beam, and stored at 5 degrees C for 12 d. The targeted irradiation dosage was 3.0 kGy and the average absorbed dosage was 3.12 kGy. Values of TBARS and volatile compound contents of raw and cooked chicken meat were determined during the 12-d storage period. Thiobarbituric acid reactive substances values ranged from 15.5 to 71.4 mg of malondialdehyde/kg for nonirradiated raw chicken and 17.3 to 80.1 mg of malondialdehyde/kg for irradiated raw chicken. Values for cooked chicken ranged from 31.4 to 386.2 and 38.4 to 504.1 mg of malondialdehyde/kg for nonirradiated and irradiated chicken, respectively. Irradiation increased TBARS and hexanal values of controls and meat infused with plant extracts. Hexanal had the highest intensity of volatiles followed by pentanal and other volatiles. Cooking the samples significantly (P < 0.05) increased the amounts of TBARS and volatiles. Addition of plant extracts decreased the amount of TBARS as well as hexanal and pentanal values. Although irradiation increases lipid oxidation, infusion of chicken meat with plant extracts could reduce lipid oxidation caused by irradiation.
Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating metabolism of oxygen free radicals, and some of the related enzymes were studied in 10 areas of adult and aged rat brains. Thiobarbituric acid-reactive products were lower in cerebral cortex, septal area, hippocampus, caudate-putamen, and substantia nigra compared with other areas studied in adult rats; however, they increased significantly in the former areas with aging. A slight but significant reduction in superoxide dismutase activity was noted in frontal cortex, septal area, caudate-putamen, and substantia nigra with aging. Glutathione peroxidase and reductase activities were highest in caudate-putamen and in substantia nigra. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were lowest in cortical areas. Phosphofructokinase activity was lowest in septal area and hippocampus in aged rats. Glyceraldehyde-3-phosphate dehydrogenase activity showed only small regional and evolutional changes. Lactate dehydrogenase activity declined with age in most of the areas studied. sn-Glycerol-3-phosphate dehydrogenase activity showed small changes with aging except in hippocampus, where 40% reduction was noted. Generally, cerebral cortical areas, hippocampus, and septal areas were not particularly enriched in enzymes regulating the metabolism of oxygen free radicals. The results were discussed in relation to the role of free radicals in aging.
Four nonsteroidal antiinflammatory drugs--namely, acetylsalicylic acid, naproxen, nimesulide, and dipyrone--were assayed to search their capability in preventing the hepatic increase of triacylglycerols and thiobarbituric acid reactive substances, as an indication of lipid peroxidation, resulting from the acute intoxication of rats with ethanol. The four antiinflammatory compounds inhibited the ethanol-mediated increase in triacylglycerols and thiobarbituric acid reactive substances. Furthermore, each one of these antiinflammatory drugs produced distinctive actions on blood ethanol levels: a temporal diminution with aspirin and naproxen, no changes with nimesulide, and an increase with dipyrone. It is concluded that the nonsteroidal antiinflammatory drugs contributed to controlling hepatic lipid peroxidation, and hence the oxidative stress promoted by ethanol intoxication.
Symptoms of bronchial asthma are a manifestation of airway inflammation. Circulatory leucocytes (predominantly eosinophils, mast cells and neutrophils), release inflammatory mediators, including reactive oxygen species, i.e. superoxide anion which is dismutated to hydrogen peroxide (H2O2). Neutrophils from asthmatics generate greater amounts of these species than those of healthy subjects. Some of the H2O2 and thiobarbituric acid-reactive products (TBARs) can evaporate from alveolar lining fluid, and could be expired from the airways of asthmatics. In this study, therefore, we determined whether asthmatic patients exhale more H2O2 and TBARs than healthy subjects. We examined 10 healthy subjects as a control group and 21 asthmatic subjects. In asthmatic subjects, forced expiratory volume in one second (FEV1), was 68+/-9% of predicted value, peak expiratory flow rate (PEFR) was 65+/-8% pred, and bronchial reversibility was 34+/-5% of prebronchodilated FEV1. The mean H2O2 level measured spectrofluorimetrically in the expired breath condensate of asthmatic subjects was 26 fold higher than that in healthy controls (0.26+/-0.29 vs 0.01+/-0.03 nM; p<0.05). The concentration of TBARs in breath condensate was also higher in asthmatic patients compared with nonasthmatics (0.073+/-0.071 vs 0.004+/-0.009 nM; p<0.05). There was a significant correlation between H2O2 level and concentration of TBARs in asthmatic patients (r=0.74; p<0.01). There was also a strong inverse correlation between H2O2 content of all asthmatics and FEV1% pred (r=-0.63; p<0.005) and PEFR% pred (r=-0.52; p<0.05). We conclude that there are elevated levels of hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients, and that measurement of these substances in the expired breath condensate could be a simple, noninvasive method that could be used as a biochemical marker of airway inflammation.
A simple and sensitive spectrophotometric method for the determination of a thiobarbituric acid-reactive substance (TBARS) in human hair has been developed. The proposed method is based on the formation of a red-colored product by the reaction of products of lipid peroxidation with thiobarbituric acid in an acidic medium. The absorbance of the resulting red product was measured at 534 nm. The linear dynamic range was between 1.0 and 20 micromol/L. The recoveries were 98.3-105.0%, and the relative standard deviations (RSD) were 0.32-1.24, respectively. TBARS in digested hair sample was stable for 3 days at room temperature. It was found that, using this method, the hair TBARS concentration in smokers (0.116 +/- 0.030 micromol/g, n = 30) was significantly higher than that in non-smokers (0.096 +/- 0.015 micromol/g, n = 30) (p < 0.05).
Colorimetric techniques such as the thiobarbituric acid assay are widely used for the determination of nonenzymatic glycosylation of proteins. One of the major problems associated with this technique is the high nonspecific background absorbance which, due to its variability, results in loss of sensitivity. This report describes a method of removing the nonspecific absorbance by extracting the final chromogen into cyclohexanone. Using this method for the determination of nonenzymatic glycosylation of hair and epidermal keratin, at least 90% of the background absorbance is removed, increasing the sensitivity of the technique. This allows more effective discrimination of the level of glycolsylation of proteins from control and diabetic patients, by reducing the degree of overlap. The requirement for individual borohydride reduced samples is also avoided, thus simplifying the technique. Cyclohexanone extraction provides a simple addition to the standard thiobarbituric acid technique with significant improvement in results.
Peroxidation of human erythrocyte membranes was followed in vitro with head space analysis of ethane and pentane and a thiobarbituric acid assay in a standardized system liberating free oxygen radicals. Simultaneously, the decrease of the membrane palmitic, linoleic, arachidonic and docosahexaenoic acid was monitored. The recoveries of the peroxidation products of the red cell ghost preparations were compared with those obtained by peroxidation of pure fatty acids. Experiments using purified fatty acids revealed that ethane was preferentially produced from docosahexaenoic and linolenic, and pentane from linoleic and arachidonic acids. Thiobarbituric acid-reactive material (TBAR) was produced from each unsaturated fatty acid tested, but the amount was dependent on the number of carbon chain double bonds. During peroxidation of the erythrocyte ghosts, 72% of ethane and 51% pentane were produced during the first 12 h of incubation, whereas TBAR was produced at a constant rate throughout the 36-h test period. Hydrocarbon and TBAR production were similarly inhibited by desferoxamine (at p less than 0.005 and p less than 0.0001, respectively). The total recoveries of ethane, pentane and TBAR exceeded the amount expected by 7.8-, 1.4- and 5.5-fold, respectively. It was concluded that measurement of pentane is a reliable method to monitor lipid peroxidation during oxidative damage of the erythrocyte membrane.
The 2-thiobarbituric acid reaction with malondialdehyde has been used to assess lipid peroxidation in a variety of biologic systems. However, in an attempt to measure plasma thiobarbituric acid-reactive substances (TBARS) during extracorporeal membrane oxygenation, a form of sustained cardiopulmonary bypass, it became apparent that the absorbance signal at the 532-nm wavelength was composed not only of the peak absorbance of TBARS, but also of interfering substances from heme pigments and bilirubin. A method of subtracting interfering substances was developed and applied to normal human plasma. The method was tested by adding varying amounts of red blood cell hemolysate, bilirubin, and 1,1,3,3-tetramethoxypropane (TMP) standard to plasma and determining TBARS in the resulting mixture. In addition, varying the amount of added desferoxamine was investigated to determine the effects of iron chelation on the assay. This was important because the different samples would have varying amounts of free iron from hemoglobin to catalyze the reaction. It was found that the following equation could be used in this system to determine that amount of 532-nm absorption due to TBARS: MDA532 = 1.22[(A532) - (0.56)(A510) + (0.44)(A560)]. Regression analysis revealed an 86.6% recovery of the TMP spike. Analysis of variance showed that the variability in the model could be explained mainly by the additive increments of TMP spike (94.6%).
The linoleic acid hydroperoxide obtained by enzymatic peroxidation of linoleic acid was found to react with thiobarbituric acid to yield a red pigment. The optimum pH for the reaction was found to be 4.0. In the early stages of peroxidation of linoleic acid, thiobarbituric acid value, the amount of conjugated diene, oxygen consumption, and peroxide value were in parallel with one another. The data were compared with those on peroxidation of linolenic acid and arachidonic acid.
A simple, inexpensive spectrofluorometric ultramicroassay for determining the plasma lipid peroxidation index is described. Plasma is incubated for 60 minutes at 95 degrees C in a thiobarbituric acetate buffer medium (pH 3,5) with butylhydroxytoluene and Fe . The chromogen is then extracted in butanol 1-pyridine and quantified by spectrofluorometry (exc.: 532 nm; em: 553 nm). The mean value of the plasma thiobarbituric lipid peroxidation index determined by this method in 101 healthy non-smokers (age 39.1 +/- 8.7 years) was found to be 1.61 +/- 0.72.
Male Wistar-H-rats were exposed monthly to a 60cobalt-source low dose whole body irradiation (0.25 Gy, total dose: 4.5 Gy). The glutathione disulphide:total glutathione ratio, the concentration of thiobarbituric acid-reactive substances and the activities of glutathione peroxidase and glutathione transferase in eight different organs and blood were analysed. The low dose irradiation is accompanied by distinct peroxidative changes in organs. These oxidative loadings occur in the small intestine, the spleen and the kidneys. The measurements of glutathione status and of thiobarbituric acid-reactive substances are proposed as sensitive parameters for low dose radiation induced changes.
We assessed the distribution of malondialdehyde (MDA) in lipoproteins and proteins in serum after using two procedures to separate the lipoproteins: sequential ultracentrifugation or selective precipitation with a sodium phosphotungstate and magnesium chloride reagent followed by ultracentrifugation of the supernate. MDA concentrations were determined by the thiobarbituric acid reaction and quantified by fluorometry. We found that 43% of the thiobarbituric acid-reactive substances (TBARS) was bound to the lipoproteins--27% to very-low- and low-density lipoproteins (VLDL-LDL) and 16% to high-density lipoproteins (HDL)--and from 11.5% to 15.8% to proteins, depending on the separation procedure. Residual unbound TBARS were located in the ultracentrifugation layers that contained no lipoproteins or proteins. The TBARS concentration in serum lipoproteins containing apolipoprotein B (i.e., VLDL-LDL) was the same after ultracentrifugation or selective precipitation. We therefore consider the precipitation method more suitable for routine TBARS determination in these lipoproteins, because it is easier to handle and faster. However, for determination of TBARS in HDL, selective precipitation requires subsequent ultracentrifugation at a density of 1.21 kg/L.