Thiobarbituric Acid Test Research Articles

Influence of pH on Egg Yolk Lipid Oxidation

ABSTRACTThe influence of pH on lipid oxidation of liquid egg yolk was studied. Liquid yolk samples were prepared to contain 60% yolk, 40% distilled water, 0.012% sodium azide, and pH was adjusted from the normal pH of 6.0 to 2.0, 3.0, 4.0, and 5.0 with 1N HCl. Samples were agitated at room temperature (ca 23°C) for 20 days and lipid oxidation measured using the distillation thiobarbituric acid test. No increase in thiobarbituric acid reactive substances occurred in the control (pH 6) or pH 5 yolk samples. The degree of oxidation significantly increased (P<0.01) in the more acidic samples in the following order: pH 3 > pH 2 > pH 4. Addition of 1.2% EDTA significantly lowered (P<0.01) the degree of oxidation but did not inhibit oxidation completely.

Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: nonspecificity of thiobarbituric acid-reactivity.

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (approximately 20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced approximately 40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA, TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result.

Polyunsaturated fatty acid-induced cytotoxicity against tumor cells and its relationship to lipid peroxidation.

The contribution of lipid peroxidation to the killing of human breast cancer cells by gamma-linolenate (GLA) was examined. Other fatty acids of different cytotoxic potential containing 2, 4, 5, and 6 double bonds were also tested for comparison. It was found that the cytotoxic potential varied with the ability of the fatty acids to stimulate the production of superoxide radicals. Neither hydrogen peroxide nor hydroxy radicals are significantly involved in cell killing. As nonspecific indicators of lipid peroxidation, measurements of the loss of unsaturated fatty acid in the phospholipids together with the generation of hydroperoxide breakdown products were done with the use of the thiobarbituric acid test. The results of these experiments showed that the effectiveness of a given fatty acid in killing cancer cells correlated with the intracellular thiobarbituric acid-reactive material (TBARM) content: GLA and arachidonate with 3 and 4 double bonds generated the most TBARM and were the most cytotoxic fatty acids, whereas docosahexaenoate with 6 double bonds was the least effective either in raising TBARM or in killing the malignant cells. Iron and copper accelerated the rate of cell death, whereas antioxidants such as vitamin E and butylated hydroxyanisole inhibited the effect of GLA dose dependently. Indomethacin, an inhibitor of endoperoxide formation, did not reduce either cell kill or TBARM amounts. In contrast, the addition of vitamin E acetate to the cancer cell cultures challenged with eicosapentaenoate reduced both cell killing and TBARM content. These results suggest that the effectiveness of a given fatty acid in killing cancer cells correlated with the extent of lipid peroxidation of the added fatty acid in the cells.

Free malondialdehyde determination in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for quantification of malondialdehyde (MDA) in human plasma. Deproteinized samples were injected onto a Waters carbohydrate analysis column which was eluted with 20% ( v v ) 0.03 m Tris buffer, pH 7.4, in acetonitrile. Peak absorbancy was measured at 267 nm. In contrast to data already published, we did not detect any free MDA in normal human plasma. This suggests that the classical thiobarbituric acid test is not suitable for the determination of MDA in human plasma.

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl)-methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non-enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10(-2)-10(-5) M CH 402 dose-dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.

Radurization of commercial freshwater fish species

The effect of radurization on the shelf life of fresh Whitefish obtained through ordinary commercial channels has been determined. Whitefish fillets irradiated at 1.2 kGy and stored at 3°C have a shelf life three times longer than the unirradiated fish. When the fish was irradiated at 0.82 kGy a two fold shelf-life extension was obtained. The shelf life was estimated by sensory, chemical and microbiological evaluations. Sensory evaluation involved organoleptic assessment of raw and cooked samples. Since freshwater fish do not contain trimethylamine oxide (TMAO), alternate tests for freshness were required. It was found the determination of hypoxanthine and total volatile acid number (VAN) are excellent tests for freshness and quality of freshwater fish; thus, these analyses were adopted. The degree of radiation-induced lipid oxidation was measured by the thiobarbituric acid test (TBA). It was found at doses of 0.82 and 1.2 kGy the TBA number remained within acceptable limits in all samples. Microbiological analyses consisted of the total microbial load assessment in the sample, as well as Pseudomonas and total psychrotrophic counts. The estimated shelf lives as determined by the three separate evaluations were in very good agreement.

Natural course of iron delocalization and lipid peroxidation during the first eight hours following a 15-minute cardiac arrest in dogs

Lipid peroxidation is thought to be a major contributing factor in neurological injury following cardiac arrest. Because iron availability is a prerequisite for lipid peroxidation, this experiment was designed to examine the natural time course of iron release, lipid peroxidation, and cerebral polyunsaturated fatty acid content following a 15-minute cardiac arrest in dogs. Large mongrel dogs were anesthetized with ketamine and halothane and divided into three groups of five each. In two groups, cardiac arrest was induced with KCl. After 15 minutes of cardiac arrest, the dogs were resuscitated by five minutes of internal cardiac massage, epinephrine, bicarbonate, and internal defibrillation. All ten dogs were resuscitated and supported by a standard intensive care protocol until tissue harvest. A 3-g portion of parietal cerebral cortex was obtained from the nonischemic dogs (n = 5), or at two hours (n = 5), or eight hours (n = 5) after resuscitation. Total tissue iron was measured by an atomic emission spectrometer; low molecular weight species (LMWS) iron by the o-phenanthroline test on an ultrafiltered sample; and lipid peroxidation by both the thiobarbituric acid test (TBARS) and determination of the tissue content of lipid double bonds, calculated by first fractionating the lipids by gas-liquid chromatography and then measuring the double bonds in each fraction by spectrometry and summing the results. Univariate ANOVA demonstrated all variables except total tissue iron to have significance at P less than .02. At two hours of reperfusion, LMWS iron and TBARS were significantly elevated above nonischemic control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative Stability and Flavour Acceptability of Nitrite-Free Meat-Curing Systems

The oxidative stability imparted to meat by combinations of antioxidants and/or chelators was assessed by the 2-thiobarbituric acid (TBA) test, and their effect on flavour acceptability was determined by sensory evaluation. The TBA results show that, at the 30ppm level, tert-butylhydroquinone (TBHQ) was the most effective antioxidant tested followed by butylated hydroxyanisole (BHA). Using surface response methodology, effective concentration combinations of sodium ascobate (SA) with either sodium tripolyphoshate (STPP) or ethylenediaminetetraacetic acid disodium salt (Na2EDTA) added to meat samples to give specified range of TBA values were predicted. Sensory evaluation indicated that the flavour of the untreated control meat sample was significantly (p≤0.05) less acceptable than that of the nitrite-cured reference. The flavour scores of samples treated with a mixture consisting of 3000ppm STPP, 550ppm SA, and 30ppm of TBHQ or BHA were not significantly different from that of nitrite-cured meat.

Heat Treatment Effects on Warmed‐Over Flavor in Chicken Breast Meat

ABSTRACTThe effects of various heat treatments on warmed‐over flavor (WOF) in ground chicken breast meat were examined. WOF was measured by thiobarbituric acid test and taste panel. Integral temperature‐time history functions (TTH) were used to quantify constant and variable temperature‐time heat treatments. The effect of processing on WOF was modeled as a modified first‐order kinetic absolute reaction. A reaction threshold temperature of 74°C and an activation energy of 15,000–20,000 Cal/g‐mole were calculated. The accumulated TTH value above 74°C was found to have a major effect on WOF development. WOF in precooked chicken breast meat can be controlled by choice of processing conditions.

Measurement of free and bound malondialdehyde in vitamin E-deficient and -supplemented rat liver tissues.

The quantity of free malondialdehyde (MDA) in liver tissues of rats fed vitamin E-deficient or -supplemented diets for 43 wk was measured by a newly developed high performance liquid chromatographic (HPLC) method. Bound MDA was quantified by the same HPLC method after alkaline hydrolysis of tissue homogenates. Tissues from vitamin E-deficient animals showed levels of free MDA about 15 times higher but levels of bound MDA less than 2 times higher than the vitamin E-supplemented animals. Free MDA is the major form in vitamin E-deficient tissues, but bound MDA is predominant in vitamin E-supplemented tissues. Conventional thiobarbituric acid (TBA) test results revealed that the content of TBA-reactive substances expressed in MDA equivalents was much higher than the actual free MDA levels in all groups. Results indicate that free MDA level measured by HPLC is a more sensitive index than the TBA value for lipid peroxidation. Some other TBA-reactive substances seem to exist in liver tissue regardless of the dietary treatment.

The effect of treatment with coenzyme Q 10 on the mitochondrial function and superoxide radical formation in cardiac muscle hypertrophied by mild aortic stenosis

A 40% reduction of the diameter of the ascending aorta maintained for 60 days induced the formation of a compensate cardiac hypertrophy in rabbits without changing the value of the azide insensitive Ca2+-ATPase activity in comparison to control hearts. The cardiac mitochondria isolated from constricted animals assayed in presence of glutamate and succinate did not show a change in the R.C.I. and ADP/O values in comparison to the controls, whilst the QO2 value enhanced or decreased respectively when determined with glutamate or succinate. The intramuscular injections of CoQ10 (12 mg/kg body weight/48 h) enhanced the mitochondrial CoQ10 concentrations both in the control and in the constricted animals and further increased the QO2 value determined in both groups of animals when glutamate was used as the substrate. The production of O2.- radicals by the level of the complexes I and III of the respiratory chain, did not change in the constricted animals, nor in the animals administered with CoQ10 in comparison to the control. CoQ10 augmented the rate of oxygen consumption by the submitochondrial particles only in the constricted animals. Moreover, the treatment with the coenzyme or the constriction of the aorta, did not modify the cardiac superoxide dismutase activity, but increased the glutathione peroxidase activity only in the banded animals. In addition, in the CoQ10 treated animals there was a reduction of NADH-diaphorase activity both in the control and constricted animals, while the malondialdehyde, generated during the thiobarbituric acid test, and the cardiac content of lipofuscin were decreased.

Control of lipid oxidation in cooked meats by combinations of antioxidants and chelators

The degree of oxidation in cooked meats treated with phosphates or polyphosphates and sodium ascorbate (SA) or one of its related compounds was determined using the 2-thiobarbituric acid (TBA) test. Combinations of ascorbates with a polyphosphate effectively retarded lipid oxidation in cooked pork during a 5-week storage period at refrigerator temperature. Addition of a phenolic antioxidant—butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), tert-butylhydroquinone (TBHQ) or trihydroxybutyrophenone (THBP)—to the above mixtures did not affect the TBA numbers substantially (≤0·1 TBA unit). Ethylenediaminetetraacetic acid disodium salt (Na 2 EDTA) with sodium ascorbate, with or without a phenolic antioxidant, was effective in protecting cooked pork from oxidation (TBA numbers of ≤1). Commercial antioxidant systems, Tenox A and Tenox II, were also effective in retarding meat rancidity, but Ronoxan A and Ronoxan D20 were only slightly effective.

Effect of Inorganic Polyphosphates on Ground Beef Characteristics: Some Chemical, Physical, and Sensory Effects on Frozen Beef Patties

ABSTRACTChemical, physical, and sensory effects of 0.4% sodium tripolyphosphate (STPP), tetrasodium pyrophosphate (TSPP) and three commercial phosphate blends were studied in frozen beef patties over a 90‐day storage period at −20°C. Addition of phosphates significantly (P<0.05) increased pH, soluble orthophosphates, Hunter color a values, cook yields and overall acceptability scores. Phosphate addition did not affect (p>0.05) proximate analysis, texture, and flavor scores. Hunter L (lightness) and b (yellowness) values were also unaffected by phosphates. Overall quality of patties, as measured by thiobarbituric acid (TBA) numbers and cooking yields, was improved by all phosphates. There were indications that phosphates interfered with the distillation TBA test.

Warmed‐Over Flavor Development in Beef Patties Prepared by Three Dry Heat Methods

ABSTRACTTHE THIOBARBITURIC ACID (TBA) test and sensory evaluations were used to assess the effectiveness of three dry heat preparations—microwave, microwave/convection combination and oven broiling—in retarding warmed‐over flavor (WOF) development in ground beef patties. Animals finished on pasture, 60 and 120‐day grain were used. The oven broiling method was found to delay WOF development in refrigerated patties. Taste panelists failed to detect differences in flavor as a result of cooking or animal diet. There was a significant (P < 0.001) negative correlation (r=−0.66) between aroma and flavor scores and TBA values over time. Panelists were able to detect deteriorating changes in flavor and aroma after refrigerated storage.

Lipoxygenase Activity of Pityrosporum In Vitro and In Vivo

Lipid peroxidation has been investigated both in cultures of Pityrosporum supplemented with different lipid classes and in skin surface lipids from patients affected with pityriasis versicolor. Thin-layer chromatography (TLC) and 2 spectrophotometric methods were used: the indirect thiobarbituric acid test and the direct N,N-diethyl-1,4-phenylene-diammonium sulfate (DEPD) test. The coupling of the DEPD test with the TLC technique performed by different eluent systems allowed the detection of the specific lipoperoxides deriving from the oxidation of the different lipid classes. In the cultures, Pityrosporum was capable of peroxidating not only unsaturated free fatty acids, but also unsaturated triglycerides, cholesterol, and squalene. A similar lipid peroxidation was observed in patients with pityriasis versicolor in skin lipids from areas positive for fungal hyphae and spores and fluorescent under the UV lamp (366 nm). The lipoperoxide values were significantly higher (p less than 0.05) than in skin lipids from normal controls. Hyphae and spore-negative areas of patients with pityriasis versicolor, whether apparently normal or achromic, showed no evidence of a significant lipid peroxidation and neither did skin areas of patients with pityriasis alba. Though further investigations are necessary, it seems reasonable to suggest, in analogy with other biologic systems, that the presence in skin lipids of a significant amount of highly reactive and cytotoxic lipoperoxides may play a role in the pathogenesis of skin alterations in pityriasis versicolor, including damage to melanocytes and resulting achromia.

Increased in vitro lipid peroxidation of gerbil cerebral cortex as compared with rat

The in vitro thiobarbituric acid test was used as a measure of lipid peroxidation in the gerbil and rat. Synaptosomal preparations were isolated from the cerebral cortex of each species and incubated with a free radical generating system. Varying concentrations of ADP-Fe 3+, with ascorbate and oxygenated incubation medium were used to generate hydroxy-radicals. Peroxidation of the synaptosomal membrane lipids was determined using malondialdehyde (MDA) accumulation. Both the gerbil and rat demonstrated significant increases in MDA in the presence of the generating system, while the gerbil P 2 fraction consistently showed an increased level of MDA accumulation as compared with rat at each of the concentrations of ADP-Fe 3+. Across a range of concentrations, there was a 2–2.6-fold greater increase in MDA accumulation in gerbil as compared with rat. Free radical generation is currently thought to be involved in the associated damage following cerebral ischemia. An in vitro model capable of producing biochemically similar damage to membrane systems by means of a controlled free-radical generating system may prove useful in studying possible mechanisms of ischemic damage.

New data on kinetics of lipid peroxidation in experimental hepatomas and preneoplastic nodules.

Lipid peroxidation has been found decreased in several hepatomas. The decline has been shown already at the level of preneoplastic nodules obtained after DEN treatment of rats. A substantial exception is represented by the hepatoma cell line MH1C1, deriving from a slightly deviated Morris tumor. Most of the described experiments estimated lipid peroxidation levels in terms of malonaldehyde production by the thiobarbituric acid test. It is now clear that this test does not account for several other aldehydes produced during lipid peroxidation. We now investigated by high performance liquid chromatography (HPLC) the whole range of non-polar aldehydes produced by tumor homogenates and by preneoplastic nodules both in basal conditions and after stimulation with ADP-iron or ascorbate. It was reduced in the preneoplastic nodules as well as in the DEN-induced hepatoma. The susceptibility to the prooxidant effect of ADP-iron or ascorbate was strongly decreased in all hepatomas as well as in preneoplastic nodules. It has been recently published that hepatoma cells are more susceptible than normal liver to the toxic action of aldehydes. This was attributed at least in part to the decreased activity of aldehyde dehydrogenases, as well as to their different distribution in tumor cells. A deeper study on aldehyde metabolism in hepatomas has shown that alcohol dehydrogenase and NADPH-aldehyde reductase also are markedly decreased in Yoshida hepatoma cells and the MH1C1 cell line. However, glutathione transferase, that can use hydroxynonenal as a substrate, is strongly decreased in Yoshida hepatoma cells but not in MH1C1 cells.

Effect of sequestering agents on lipid oxidation in cooked meats

To retard lipid oxidation, which affects the sensory and nutritional quality of cooked meats, a number of sequestering agents (ortho- and polyphosphates, citric acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), salicylic acid, the sodium salts of oxalic, phytic and lactic acids, glycine, cysteine, 8-hydroxy-quinoline and catechol) were added to ground pork prior to cooking. The TBA numbers of meat were evaluated after cooking (day 1) and after 7, 14, 21, 28 and 35 days of storage at 4°C by the 2-thiobarbituric acid (TBA) test. It was found that catechol, EDTA, DTPA, sodium pyrophosphate and, to a lesser extent, sodium tripolyphosphate, substantially lowered the TBA numbers while the other sequestering agents had only a marginal effect. Mixed-chelating systems composed of salicylic acid and either EDTA or DTPA did not show any synergistic effects.

Cardiac arrest and resuscitation: Brain iron delocalization during reperfusion

We hypothesize that brain injury from cardiac arrest occurs during reperfusion and is in part mediated by iron-dependent lipid peroxidation. We conducted a study to examine the time course of brain iron delocalization and lipid peroxidation in an animal model of cardiac arrest and resuscitation. Assays for brain tissue iron in low-molecular-weight species (LMWS iron) used the o-phenanthroline test on an ultrafiltered (molecular weight less than 30,000) tissue sample; malondialdehyde (MDA), a product of lipid peroxidation, in brain tissue was assayed by the thiobarbituric acid test (TBA). Samples of the parietal cortex from 11 nonischemic control dogs (Group 1) had LMWS iron levels of 9.6 +/- 4.9 nmol/100 mg tissue and MDA levels of 7.7 +/- 2.0 nmol/100 mg tissue. Samples from the parietal cortex taken from five dogs after 15 minutes of cardiac arrest (Group 2) had LMWS iron levels of 9.3 +/- 3.1 nmol/100 mg tissue and MDA levels of 6.1 +/- 1.0 nmol/100 mg tissue. Samples from the parietal cortex taken from five dogs after 45 minutes of cardiac arrest (Group 3) had LMWS iron levels of 6.7 +/- 3.3 nmol/100 mg tissue and MDA levels of 5.6 +/- 0.4 nmol/100 mg tissue. There was no significant difference among the three groups for either LMWS iron or MDA. Five dogs were subjected to 15 minutes of cardiac arrest and definitive resuscitation by internal cardiac massage and defibrillation (Group 4). Following resuscitation the chest was closed and the dogs were given intensive care for two hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of prostanoids and lipid peroxides as mediators of the 12-O-tetradecanoylphorbol-13-acetate effect on cell growth.

Confluent cultures of guinea pig smooth muscle cells (SMC) or human fibroblasts (HNF) were treated with 12-tetradecanoylphorbol-13-acetate (TPA). Prostanoid levels were measured by the radioimmunoassay of 6-keto-PGF1 alpha and PGE2, and lipid peroxides were measured by the thiobarbituric acid test for malondialdehyde (MDA). Cells were seeded at low densities, and growth was calculated both from the cell count (Coulter Counter) and the colony number (image analysis). When confluent SMC and HNF were incubated in media alone, 6-keto-PGF1 alpha levels were a function of the TPA concentration, increasing to a maximum at 10(-8) M TPA and then decreasing at higher TPC concentrations. When confluent SMC and HNF were incubated in media containing exogenous arachidonic acid, 6-keto-PGF1 alpha levels again increased to a maximum at 10(-8) M TPA but decreased at higher TPA concentrations only with SMC. The increase in 6-keto-PGF1 alpha levels was much greater in HNF (1310%) than SMC (680%). SMC synthesized similar amounts of 6-keto-PGF1 alpha and PGE2, and the stimulatory effect of TPA was similar with 6-keto-PGF1 alpha and PGE2. Indomethacin (IM) blocked prostanoid synthesis at all TPA concentrations. TPA did not have a significant effect on MDA levels in either cell line. The lipid antioxidants alpha-tocopherol and alpha-tocopherylquinone blocked lipid peroxidation without affecting the stimulation of prostanoid synthesis with TPA. Cell number decreased to a minimum at 10(-8) M TPA in both cell lines. The decrease in cell number was much greater in HNF (72%) than SMC (30%). SMC colony number decreased at 10(-8) TPA and then increased at 10(-6) M TPA. IM did not block the TPA effect on cell number in either cell line.(ABSTRACT TRUNCATED AT 250 WORDS)