Thiobarbituric Acid Test Research Articles

The Effect of 2-Chloroadenosine on Lipid Peroxide Level During Experimental Cerebral Ischemia–Reperfusion in Gerbils

Oxygen free radicals may be implicated in the pathogenesis of ischemia–reperfusion damage. It is known that 2-chloroadenosine (2-CADO) has neuromodulatory effects and prevents the neuronal damage seen in the period of postischemia reperfusion. However, direct effects of 2-CADO on lipid peroxidation have not been investigated previously. The attack on the cell membrane by free radicals leads to lipid peroxidation, which can be assayed by the malondialdehyde (MDA) level. The aim of this study was to determine the effect of 2-CADO therapy on lipid peroxidation in experimental forebrain ischemia and postischemia reperfusion in Mongolian gerbils. Cerebral ischemia was induced by a bilateral 30-mm occlusion of the common carotid arteries. 2-Chloroadenosine (0.6 mg/kg, IV) was administered 5 min subsequent to ischemia. Ischemia was followed by reperfusion for 30 min. The MDA level was measured by the thiobarbituric acid (TBA) test. Bilateral carotid artery occlusion for 30 min in gerbils resulted in no significant change in MDA level in the brain. The MDA level was higher in postischemia reperfusion than in the ischemic group. 2-Chloroadenosine treatment did not change the MDA level in the ischemic period. However, the MDA level recovered significantly upon 2-CADO therapy during reperfusion following ischemia. These results suggest that 2-CADO may offer some degree of protection against oxidative stress in cerebral ischemia–reperfusion damage. Copyright © 1996 Elsevier Science Inc.

Selection of induced mutants with improved linolenic acid content in camelina

AbstractCamelina (Camelina sativa (L.) Crtz.) is regarded as a potential crop producing a seed oil rich in linolenic acid (C18:3), which could be utilized for different oleochemical applications. Seeds of a camelina breeding line have been irradiated with gamma‐rays in order to induce genetic variation in fatty acid composition. In the M2‐generation, 8017 plants were subjected to a thiobarbituric acid test to identify mutants with increased linolenic acid content. Subsequently, M3‐lines were isolated, which showed significantly higher concentrations of linolenic acid (up to 40.8%) than the control (34–36%). Moreover, genotypes with an erucic acid content of less than 2% were also found in the mutant population. Different mutant lines can thus be combined in order to obtain transgressive segregants, which could give a further increase in linolenic acid content.

A critical examination of some experimental variables in the 2-thiobarbituric acid (TBA) test for lipid oxidation in meat products

By comparison of various procedures for the determination of TBARS (2-thiobarbituric-acid-reactive substances) in non-cured meat samples, it was found that a direct extraction procedure with trichloroacetic acid gave significantly higher reaction yields than a rapid Kjeltec steam distillation procedure (50 ml distillate in 2.5 min). When optimizing the distillation procedure with regard to distillation principle (heating by thermal conductivity or steam injection) and rate, the reaction yield was more affected by changes in distillation principle than by changes in distillation rate. Simple distillation (conduction heating) yielded significantly higher TBA values than steam distillation at the same distillation rate. The reproducibility of the distillation procedure was found to be about 5–10 times poorer than of the extraction procedure and, because of the high reaction yields and robustness of the extraction procedure, the use of the extraction procedure is recommended with food matrices such as non-cured meat samples with less than 14–18% fat and no artificial colorants.

Suppression of Iron Catalyzed Free Radical Generation by Iron Tyrosinate Protein Models

Novel antioxidants, N-(2-Hydroxy-1-naphthal)amino acids which mimic iron proteins, have been intensively studied for suppressive effect on iron-catalyzed free radical generation. These compounds exhibit inhibition of the Fenton reaction in electron spin resonance assessment. In addition, it is shown that the compounds inhibit iron-induced peroxidation by thiobarbituric acid test. Since these antioxidants form stable complexes with Fe3+, this antioxidative activity is expected to be derived from sequestration of catalytic iron.

TBA test by an extractive method applied to ‘paté’

The aim of this work was to study the influence of different conditions on the thiobarbituric acid (TBA) test, determined by the extractive method, as a measure of lipid oxidation in ‘paté’. Different extracting agents (trichloroacetic acid and trichloroacetic acid in 2 M phosphoric acid), different concentrations of these acids (10, 15 and 20%), different reaction times and temperatures (35 min at 100 °C and 900 min at room temperature) and the effect of sulfanilamide additions were evaluated. All the samples were measured by HP 8451A Diode ARRAY spectrophotometer at 532 nm. Significant differences between 35 and 900 min of MA-TBA reaction time were found. Highest TBA numbers were found in samples with a reaction time of 35 min in a thermostatically controlled waterbath at 100 °C, rather than 900 min at room temperature. Lowest TBA numbers were found in samples with the addition of sulfanilamide rather than without it. The 10% TCA solution gave the best recovery percentages for ‘paté’, making it the preferred extractant.

'Free' iron, as detected by electron paramagnetic resonance spectroscopy, increases unequally in different tissues during dietary iron overload in the rat.

'Free' iron concentration, as determined by electron paramagnetic resonance (EPR) spectroscopy, and lipid peroxidation (LPO), as determined by thiobarbituric acid test, were assessed in the lung, heart, liver, spleen, brain and kidney of rats subjected to experimental iron overload. Two tests, Desferal- and NO-available iron, were used to measure 'free' iron and gave comparable results. The most pronounced accumulation of 'free' iron was observed in liver, kidney and spleen. Differences between control and iron loaded animals increased during the initial 90 days of treatment. Between 90 and 180 days 'free' iron concentration reached a steady state level, or even decreased, as in the case of liver. Lipid peroxidation level, measured in the organs of both treated and matched controls, did not give any significant difference during the initial 90 days of treatment. A significant augmentation was observed in liver, kidney, spleen and heart at 180 days. The results of the present research show that, under conditions of moderate siderosis, the occurrence of LPO is partially related to the level of 'free' iron.

No direct evidence of increased lipid peroxidation in hemodialysis patients.

Lipid peroxidation, as measured by the thiobarbituric acid test, has been reported to have increased in hemodialysis (HD) patients, even though the test has low specificity in vivo. Conjugated diene fatty acid (CDFA) hydroperoxides are formed during lipid peroxidation, but not all conjugated dienes (CD) detected in humans originate from lipid peroxidation: octadeca-9,11-dienoic acid, a nonhydroperoxide CD derivative of linoleic acid (CDLA), has a dietary origin. We evaluated CDFA hydroperoxides, CDLA and linoleic acid, using high-performance liquid chromatography, in lipids extracted from plasma, adipose tissue and RBC membranes obtained from 25 patients treated with HD, 16 patients treated with hemodiafiltration (HDF) and 29 controls. No differences in the levels of CDFA hydroperoxides and linoleic acid were seen in any of the groups. Concentrations of CDLA were found to be significantly high in the adipose tissue and low in the RBC membranes of HD patients. HDF-treated patients showed the same results as HD patients. No direct evidence of increased lipid peroxidation was found in HD patients. This does not exclude the possibility that lipid peroxidation is increased and escapes direct detection due to the body's homeostatic control eliminating the increased production of hydroperoxides. Both HD- and HDF-treated patients showed a significant change in CDLA concentrations, either in the adipose tissue, or in the RBC membranes. These dietary CD may be mistaken for markers of lipid peroxidation by conventional methodologies.

Effect of aging on lipid peroxide levels induced by L-glutamic acid and estimated by means of a thiobarbituric acid test in rat brain tissue

Studies of age-related changes based on the thiobarbituric acid (TBA) test appear to be inconsistent and contradictory. In our work, real basal (hypothetical, corrected to the zero concentration of atmospheric oxygen), basal (atmospheric oxygen-stimulated) and stimulated (L-glutamic and hydrochloric acids) levels of TBA-reactive products (TBARP) were estimated in the brain (hippocampus--HPC, cortex--COR, cerebellum--CRB) from young (3 months) and old (28 months) male and female Wistar rats. The values of basal levels of TBARP were different in young (HPC > COR > CRB) as well as in old animals (COR > CRB > HPC). Thus, the process of aging caused a significant decrease in the HPC (to 72%) and no change in the COR and CRB. Levels stimulated by L-glutamic acid were significantly decreased (COR, CRB) and unchanged (HPC) in old compared to young animals. The real basal levels estimated by regression analysis seem to be higher in the brain tissue damaged by aging. We discuss the usefulness of the TBA test applied to aged tissue, the effect of acidosis, the effectivity of L-glutamic acid to generate free radicals and the differences between individual brain areas.

Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain

We used several biochemical assays to evaluate age-related changes in antioxidant enzyme levels vs. free-radical damage in the murine brain. We found levels of several free-radical scavenging enzymes in the brains of 24-month-old C57B1 male mice vs. 12-month-old animals were decreased, including superoxide dismutase (SOD), catalase, and glutathione reductase (GSSG-Rd). In addition, we found concomitant increases in the levels of several forms of free-radical damage including sensitivity to lipid peroxidation as measured by the thiobarbituric acid test, protein oxidation as measured by glutamine synthetase (Gln Syn) activity, as well as increases in oxidized glutathione (GSSG) levels, a measure of oxidative stress. These data suggest that decreases in levels of enzymes which ordinarily protect neuronal cells against oxidative stress with age may be responsible for increased levels of free-radical damage in the murine brain, or that these enzymes themselves are susceptible to inactivation by free radical molecules which increase with age in the brain.

Antioxidant and cytotoxic tocopheryl quinones in normal and cancer cells

We found previously that [ d]-α-tocopherol (α-T) and [ d]-α-tocopherol (α-T) are lipid antioxidants (thiobarbituric acid test) in model systems containing arachidonic acid (AA), cumene hydroperoxide, and Fe 3+ and in smooth muscle cell (SMC) cultures challenged with AA. We now show that [d]-α-tocopherylquinone (α-TQ), [ d]-δ-tocopherylquinone (δ-TQ), and [ d]-γ-tocopherylquinone (γ-TQ) are antioxidants at low concentrations and prooxidants at high concentrations in the model system. Prooxidant activity is greater with γ-TQ than either α-TQ or δ-TQ. Low concentrations of α-TQ, γ-TQ, and δ-TQ are also antioxidants in SMC cultures challenged with AA. Unlike α-TQ, partially substituted γ-TQ and glutathione (GSH) form a Michael adduct which has been purified and characterized. We found previously that α-T, γ-T, and α-TQ are mitogenic in SMC. We now report that both δ-TQ and δ-TQ but not α-TQ show concentration-dependent cytotoxicity (changes in morphology, propidium iodide stain) in SMC cultures. Cytotoxicity is greater with γ-TQ than δ-TQ. An acute lymphoblastic leukemia (ALL) cell line shows greater chemosensitivity (MTF and Neutral Red assays) to γ-TQ than to either doxombicin (DOX) or vinblastine (VLB). An ALL cell line resistant to both DOX and VLB retains the same chemosensitivity to γ-TQ as the drug-sensitive ALL cell line. ALL cell lines are unaffected by either α-TQ or the GSH Michael adduct of γ-TQ. These data show that partially substituted tocopheryl quinones capable of forming Michael adducts are potential chemotherapeutic agents for multidrug-resistant cancer cells.

Production and characteristics of protein hydrolysates from capelin ( Mallotus villosus)

Protein hydrolysates were prepared from male and spent capelin ( Mallotus villosus) using commercially available Alcalase, Neutrase and papain. Short-time autolysis of proteins by endogenous enzymes in fish viscera was also investigated and compared to procedures of accelerated enzymatic hydrolysis. While protein recovery varied from 51.6 to 70.6% for commercial enzymes, a yield of 22.9% was obtained for autolyzed products. All methods of preparation afforded products containing about 71–78% proteins after dehydration. Alcalase served best for preparation of capelin protein hydrolysates (CPH). Thus, products of Alcalase-assisted hydrolysis of capelin proteins were further assessed for their nutritional and functional characteristics. The amino acid composition of CPH was similar to that of the starting capelin, except for methionine and tryptophan which were present in smaller amounts. The products had excellent solubility (≥84%) over a pH range of 2–11. Incorporation of CPH (up to 3%) in meat model systems resulted in an increase of 4% in cooking yield and inhibition of oxidation (determined by the 2-thiobarbituric acid test) by 17.7–60.4%.

Aldehydes in Food and its Relation with the TBA Test for Rancidity

AbstractAldehydes have been found in fruits and essential oils. But their presence in the off‐flavor compounds (rancidity of foods) is well known. Aldehydes are characteristic compounds of secondary oxidation in the autoxidative process of fats, oils, lipidic foods and biological membranes. Malonaldehyde and other aldehydes responsible for rancidity can be evaluated through the thiobarbituric acid (TBA) assay. Alkanals, alkenals and dienals give characteristic pigments (yellow, orange, red pigments) with TBA reagent, which could be spectrophotometrically evaluated.

Peroxidation levels inEschericia coli as a potential toxicity marker: Effect of nickel and methyl-parathion

The effects of nickel and methyl-parathion were studied in subinhibiting concentrations (40 and 62.5 μg/mL, respectively) on the levels of cellular peroxidation in Escherichia coli. These effects were evaluated using the thiobarbituric acid (TBA) test and the determination of lipofuscin-like substances. Both toxicants caused an increase in cellular peroxidation levels, although at the concentrations used, nickel was shown to be a better inducer than methyl-parathion. The TBA test gave more significant differences than the determination of lipofuscin-like substances when the control cultures were compared to those containing the toxicant. This finding reveals that the TBA test could be used as a toxicity marker in E. coli for the development of ecotoxicity biotests. © 1994 by John Wiley & Sons, Inc..

A decrease of free radical production near critical targets as a cause of maximum longevity in animals

A comprehensive study was performed on the brains of various vertebrate species showing different life energy potentials in order to find out if free radicals are important determinants of species-specific maximum life span. Brain superoxide dismutase, catalase, Se-dependent and independent GSH-peroxidases, GSH-reductase, and ascorbic acid showed significant inverse correlations with maximum longevity, whereas GSH, uric acid, GSSG/GSH, in vitro peroxidation (thiobarbituric acid test), and malondialdehyde (measured by HPLC), did not correlate with maximum life span. Superoxide dismutase, catalase, GSH-peroxidase, GSH and ascorbate results agree with those previously reported in various independent works using different animal species. GSSG/GSH, and true malondialdehyde (HPLC) results are reported for the first time in relation to maximum longevity. The results suggest that longevous species simultaneously show low antioxidant concentrations and low levels of in vivo free radical production (a low free radical turnover) in their tissues. The “free radical production hypothesis of aging” is proposed: a decrease in oxygen radical production per unit of O 2 consumption near critical DNA targets (mitochondria or nucleus) increases the maximum life span of extraordinarily long-lived species like birds, primates, and man. Free radical production near these DNA sites would be a main factor responsible for aging in all the species, in those following Pearl's (Rubner's) metabolic rule as well as in those not following it.

Lipoperoxidation, trace elements and vitamin E in patients with liver cirrhosis

Objective: To evaluate serum levels of malonaldehyde (as a marker of lipoperoxidation), copper, zinc, manganese (trace elements) and vitamin E in cirrhotic patients with respect to the severity of cirrhosis. Methods: Twenty patients with compensated cirrhosis, 22 with decompensated cirrhosis and 21 healthy subjects were studied. Malonaldehyde was measured by the thiobarbituric acid test; copper, zinc, and manganese were determined by atomic absorption, while vitamin E was measured by high-performance liquid chromatography. Results: Malonaldehyde levels were significantly higher in both groups of cirrhotic patients when compared with healthy subjects. Zinc and vitamin E levels were significantly lower in patients with decompensated cirrhosis

Effects of EUK-8, a synthetic catalytic superoxide scavenger, on hypoxia- and acidosis-induced damage in hippocampal slices

Anoxia produces deleterious effects on synaptic transmission in the hippocampal slice preparation. A proposed source of damage is the Superoxide radical (O − 2) produced during the earlier period of reoxygenation. The present study tested the effects of a synthetic, catalytic Superoxide radical scavenger (EUK-8) on CA1 pyramidal cell responses elicited by electrical stimulation of the Schaffer-commissural pathway after severe anoxic episodes. Following reoxygenation, slices incubated with EUK-8 (50 μM) exhibited significantly better recovery of excitatory postsynaptic potentials (EPSPs) than control slices. In addition, repeated episodes of anoxia produced irreversible loss of synaptic transmission in the majority of control slices (93 ± 7%, n = 15), compared to a small fraction in EUK-8-incubated slices (27 ± 12%, n = 15). A thiobarbituric acid (TBA) test was used to assess the effect of EUK-8 on lipid peroxidation elicited in hippocampal slices by acidosis and lactic acid (pH 5.0 and 30 mM lactic acid). Incubation in the presence of EUK-8 totally prevented the increase in lipid peroxidation produced by acidosis and lactic acid in both the incubation medium and the slice homogenates. These results indicate that a Superoxide scavenger like EUK-8 prevents damage produced by acidosis and anoxia in hippocampal slices and suggest the possibility of using this type of molecule under various pathological conditions.

Factors affecting the thiobarbituric acid test as index of red blood cell susceptibility to lipid peroxidation: a multivariate analysis

The relationship between formation of thiobarbituric acid reactive substances (TBARS) in red blood cells (RBC) after exposure to H 2O 2 and factors potentially able to modulate it was investigated by a multivariate analysis in 92 healthy volunteers. The independent covariates considered were: RBC membrane fatty acids and cholesterol, RBC antioxidant enzymes and zinc, plasma vitamins A and E and serum selenium, zinc and copper. The stepwise multiple-linear-regression analysis revealed RBC membrane fatty acids and cholesterol as predictors of a consistent proportion of the RBC-TBARS variability whereas none of the antioxidants entered the equation. The unsaturation index was the most important individual predictor; RBC-TBARS increased with increasing concentrations of total ω-3 polyunsaturated fatty acids, C 20:5ω-3 and cholesterol, whereas they decreased with increasing concentrations of total monounsaturated fatty acids, saturated fatty acids, C 16:0 and C 18:0. It is suggested that formation of TBARS, at least in currently used conditions, reflects mainly the lipid composition of the tissue under investigation, without giving sufficient information about the status of the antioxidant defences.

Ascorbate protects guinea pig tissues against lipid peroxidation

In recent years we and others have shown that ascorbic acid (AH 2) is a potential scavenger of superoxide (O 2 ·−) and peroxyl (LOO ·) radicals, the species involved in lipid peroxiddation (LPO) in animals tissues. In this paper we have demonstrated that AH 2 protects guinea pig tissues from LPO both in vivo and in vitro. The extent of LPO has been determined by estimating malonaldehyde using the thiobarbituric acid test and HPLC and also by measuring the accumulation of fluorescent pigment and occurrence of protein changes in the microsomal membranes. In AH 2-deficiency, LPO occurs progressively in guinea pig tissues, despite the presence of adequate levels of antioxidants like α-tocopherol, GSH, protein thiols, and scavenging enzymes, namely, superoxide dismutase, catalase, and glutathione peroxidase. In a model in vitro system, microsomal LPO initiated by O 2 ·− is completely prevented by AH 2 but not by α-tocopherol, GSH, uric acid, and catalase. AH 2 is also the most effective antioxidant in preventing microsomal LPO mediated by tert-butylhydroperoxide or the chain propagating species LOO ·, generated from 2,2′-azobis (2-amidinopropane) hydrochloride. The results obtained with guinea pigs may be applicable to humans, because humans are also dependent on dietary AH 2. Our data suggest that an adequate vitamin C nutrition may prevent common cellular degenerative diseases associated with LPO.

Malondialdehyde measurement in urine

Malondialdehyde (MDA) is an end product of lipid peroxidation and is a frequently measured index of these processes. The thiobarbituric acid (TBA) test is commonly used to measured MDA, but its specificity is questionable due to the presence of interfering chromogens. Wade and van Rij described in 1988 a method which removes these chromogens by HPLC. However, the sensitivity and the resolution of this method was not adequate for measurements of MDA in urine. We have improved this method by replacing TBA with diethylthiobarbituric acid (DETBA). The less polar MDA—DETBA complexes were isolated on Bakerbond cartridges and quantified by HPLC without interference. MDA was detectable using a fluorescence or ultraviolet detector at picomole levels. This technique was applied to urine samples obtained from ten burns patients on different days following their hospitalization. Urinary MDA in burns patients was very high and reached 18.6 μmol/mmol creatinine in one patient compared with a mean value of 0.23 μmol/mmol creatinine in healthy controls. Maximum MDA levels were attained on the third day for the majority of patients and remained, on average, much higher than normal even after 20 days. Using this method, picomole quantities of MDA can be easily and specifically detected in urine samples. This method is useful for assessing an oxidative stress.

Susceptibility of myelin glycerophospholipids and sphingolipids to oxidative attack by hydroxyl free radicals as measured by the thiobarbituric acid test

Glycerophospholipids and sphingolipids typical of the myelin sheath have been examined for the formation of thiobarbituric acid reactive materials after attack by hydroxyl free radicals. Thiobarbituric acid (TBA) reactive materials are formed following exposure of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) to hydroxyl radical attack in the order PS > PC > PE while the sphingolipids, sphingomyelin and cerebroside and the sterol cholesterol, did not form TBA reactive materials. Inclusion of cholesterol into PC vesicles reduced the formation of TBA reactive materials on hydroxyl radical attack while with PS there was an increase. In mixed glycerophospholipid systems inclusion of cerebroside reduced formation of TBA reactive materials following hydroxyl radical attack on phosphatidylcholine and phosphatidylserine, but not phosphatidylethanolamine. The findings are discussed in relation to the oxidative damage we have observed in the central nervous system at different stages of chronic relapsing experimental allergic encephalomyelitis [Brett R. and Rumsby M.G. Neurochem. Int. 23, 35–44 (1993)].