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PubMed1985

Aminophenylboronic acid affinity chromatography and thiobarbituric acid colorimetry compared for measuring glycated albumin.

Two techniques originally developed for measurement of glycated ("glycosylated") hemoglobin but also applicable to determination of glycated albumin are the thiobarbituric acid colorimetric technique (I) and the aminophenylboronic acid affinity chromatographic procedure (II). The latter reliably distinguishes diabetics from nondiabetics, and concentrations of glycated hemoglobin and glycated albumin are linearly correlated. I is nonspecific; it neither correlates with diabetic status nor with values derived via the affinity technique. Most of the chromogenic material is present in the fraction of albumin that does not bind to aminophenylboronic acid. Glucose interferes significantly with I but only slightly with II. Prolonged incubation of plasma with glucose dramatically increases the II-determined glycated albumin. Reactivity with thiobarbituric acid increases much less, and mainly in the II-bound fraction. This fraction contains a high proportion of nonspecifically reactive material. The percentage of glycated albumin determined in crude plasma samples by II differs only slightly from the value determined by purifying the albumin from the plasma. This technique appears more promising than I for eventual clinical applications.

Blood Glucose↗
PubMed1982

Plasma lipoperoxides and aging. Critical assessment of the thiobarbituric acid method for the measurement of lipoperoxides and malondialdehyde.

Thiobarbituric acid reactive products were measured in plasma of 66 normal subjects ranging from 19 to 92 years of age. A significant correlation (r = 0.54, p less than 10(-6) was formed between the level of these metabolites and the age of the subjects. As a matter of fact this method analyzes two kinds of substances from two different origins, free malondialdehyde or lipid peroxides. These two fractions were identified by extracting lipids using Folch's method and leaving all free malondialdehyde in the plasma (95.6 +/- 11.8% recovery for a known amount added to the plasma). Plasma analysis prior to and after extraction showed that thiobarbituric acid reactive products were lipid peroxides for 74.8 +/- 4.3%, the remainder being free malondialdehyde. The origin of these two fractions is discussed.

Adult↗
PubMed1993

Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage.

A simple, reliable, and reproducible fluorometric method for measuring thiobarbituric acid-reactive substances (TBARS) in serum is proposed, based on the reaction between malondialdehyde (MDA) and thiobarbituric acid. Formation of TBARS was complete at pH 2.4-2.6, but extraction with n-butanol proved complete only at lower pH, i.e., 1.6-1.7. Analytical recoveries of MDA added to serum were 94%-101%; within- and between-run CVs were 2.4-3.6% and 4.6-5.5%; and the detection limit for TBARS in serum was 0.10 mumol/L. Optimized conditions included: (a) collection of either serum or heparinized plasma, (b) preservation from in vitro autoxidation by glutathione and EDTA, and (c) storage at -20 degrees C up to 35 days. The mean (+/- SD) TBARS concentration in 47 healthy adults was 1.01 (0.21) mumol/L; no sex-related difference was observed. Higher concentrations were measured in patients with renal insufficiency undergoing hemodialysis and in patients with insulin-dependent diabetes, chronic pancreatitis, or liver cirrhosis.

1-Butanol↗
PubMedDate not supplied

[Evaluation of lipid peroxidation by measuring thiobarbituric acid reactive substances].

Malondialdehyde assay is the most generally used test in the appreciation of the role of oxidative stress in disease. Malondialdehyde is one of several products formed during the radical induced decomposition of polyunsaturated fatty acids. Most often, malondialdehyde assay used its reactivity at high temperature and low pH, towards thiobarbituric acid. This reaction is very sensitive but its specificity, even with improvement of pre-analytical (sampling, preservatives), and analytical stages (fluorescence, HPLC) is still a matter of debate. At present, the concept of "thiobarbituric acid reactive substances" (TBARS) have merged and progressively replaced the initial malondialdehyde assay. In this review, we presented the main results concerning the assays of TBARS and malondialdehyde in blood and different biological medium. In the future, oxidative stress appreciation will need the precise analytical determination of different molecules triggered by free radicals. The TBARS assay should be considered as a global test, allowing a global approach of lipoperoxidation whereas specific determination of malondialdehyde can only appreciate one of the end-product formed during oxidative stress.

Animals↗
PubMed1991

Age-dependent alterations in the response of isolated rat aortas to thiobarbiturates.

Responses to thiamylal and thiopental were compared in helical strips of rat thoracic aortas of different ages (5-6, 10-12 and 20-22 weeks old), precontracted partially with phenylephrine or prostaglandin F(2alpha) (PGF(2alpha)). Thiamylal and thiopental, in concentrations of 3 x 10(-5) to 10(-4) M, produced a dose-dependent relaxation in aortas at 5-6 weeks of age, no significant change of tension in those at 10-12 weeks of age, and a marked constriction in those at 20-22 weeks of age. These thiobarbiturates, in a high concentration of 10(-3) M, produced a profound relaxation in aortas at any age studied. It is concluded that the responses to thiobarbiturates of thoracic aorta precontracted with phenylephrine or PGF(2alpha) alter with age.

Journal Article↗
PubMed1975

Synthesis and absolute stereochemistry of 5-alkyl-5-(3'-hydroxy-1'-methylbutyl)barbituric acid and 5-alkyl-5-(3'-hydroxy-1'-methylbutyl)-2-thiobarbituric acids.

5-Alkyl-5-(3-hydroxy-1-methylbutyl) barbituric acid (2) and 5-alkyl-5-(3-hydroxy-1-methylbutyl)-2-thiobarbituric acids (3) are matabolites of 5-alkyl-5-(2-pentyl) barbituric acid and 5-alkyl-5-(2-pantyl)-2-thiobarbituric acid, respectively. We have synthesized the four possible optical isomers of 2 and 3 by a procedure which established the absolute stereochemistry of each isomer. The two racemic pairs in each case were also prepared. The properties of these synthetic samples of 2 and 3 of known stereochemistry are compared to the properties of 2 and 3 which have been isolated from metabolism studies.

Barbiturates↗
PubMed1998

Selenium levels, thiobarbituric acid-reactive substance concentrations and glutathione peroxidase activity in the blood of women with gestosis and imminent premature labour.

The aim of the study was to investigate antioxidant status, monitored by selenium and thiobarbituric acid-reactive substance concentrations in blood plasma, and glutathione peroxidase activity in erythrocytes and blood plasma in women with gestosis (n = 26), imminent premature labour (n = 48) and normal pregnancy (n = 23) during 19-38 weeks of pregnancy. Selenium concentrations in blood plasma were significantly higher in women with pathological pregnancies than in normal (45.5 +/- 10.5 micrograms l-1, p < 0.01 and 44.1 +/- 11.6 micrograms l-1, p < 0.05 vs. 38.6 +/- 8.3 micrograms l-1, respectively). In all groups of pregnant women Se concentrations were extremely low as compared with non-pregnant females. Glutathione peroxidase (GSH-Px) activity in blood plasma was significantly higher in complicated pregnancies than in healthy ones. There were no significant differences in thiobarbituric acid-reactive substance concentrations between all groups of pregnant women. Statistically significant correlations were found between blood plasma Se concentrations and GSH-Px activity in healthy pregnant (r = 0.53, p < 0.01), imminent premature labour (r = 0.39, p < 0.01), and non-pregnant females (r = 0.56, p < 0.001).

Adult↗
PubMed1993

Characteristics of the thiobarbituric acid reactivity of human urine as a possible consequence of lipid peroxidation.

A 532 nm red pigment formed in the thiobarbituric acid (TBA) assay of human urine was characterized after separation of the pigment by high-performance liquid chromatography. The yield of the red pigment was somewhat higher at pH 2 than at pH 5; its development was not inhibited by ethylenediaminetetraacetic acid. The characteristics of the pigment were similar to those of the pigment derived from standard malonaldehyde. The amount of the pigment formed was roughly equal to the content of malonaldehyde derivatives estimated as 1-(2,4-dinitrophenyl)pyrazole. Pigment formation was significantly enhanced by t-butyl hydroperoxide (t-BuOOH) and ferric ions, which may be due to pigment formed from aldehydes other than malonaldehyde; the presence of these aldehydes was confirmed by the formation of the corresponding 2,4-dinitrophenylhydrazones. The amount of pigment produced from 24-h urine samples of 12 healthy subjects was estimated to be 26-95 nmol/kg, and 65-182 nmol/kg in the presence of t-BuOOH. These values are lower than those for urine of rabbit or rat. The TBA reactivity in the absence and presence of t-BuOOH of human urine was not related to age or sex. The TBA reactivity of human urine collected in the afternoon and in the evening was higher than that of urine collected in the morning.

Animals↗
PubMed1993

A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials.

A comparative evaluation was made of the conventional spectrophotometric procedure and three published high performance liquid chromatographic (HPLC) procedures for the determination of malondialdehyde (MDA) as the thiobarbituric acid (TBA) derivative when applied to liver, fish meal, serum, and urine. Except for urine, spectrophotometric analysis overestimated MDA content. Purification of the TBA-MDA complex obtained from liver and fish meal on reverse phase cartridges was found to entail a loss of complex bound to residual peptides in the trichloracetic acid (TCA) extract. Mincing as opposed to homogenizing liver samples led to a doubling of values for MDA content. Hexanal was a major TBA reactant, in addition to MDA, in all the samples. Acid hydrolysis and heat were necessary for the release of MDA bound to the amino groups of proteins and other amino compounds. Methods for free MDA have limited application to biological materials except short term in vitro preparations such as peroxidizing microsomes, in which free MDA accumulates. On the basis of these and other observations, a modified HPLC procedure for the determination of MDA as the TBA-MDA complex is proposed.

Animals↗
PubMed1999

Beta-carotene generates thiobarbituric acid-reactive substances by interaction with nitrogen dioxide in air.

Generation of thiobarbituric acid-reactive substances (TBARS) from methyl linoleate in the exposure of nitrogen dioxide/air was inhibited by beta-carotene in a dose-dependent manner. However, introduction of nitrogen dioxide/air or oxygen into a solution of beta-carotene generated a significant amount of TBARS accompanying loss of its characteristic yellow color. Storing beta-carotene in a solid state at ambient temperatures in air generated a large amount of TBARS accompanying loss of its yellow color. TBARS from beta-carotene may interfere the measurement of TBARS from polyunsaturated fatty acids, and may give undesirable effects on biomaterials.

Air↗
PubMed1988

Liver and biliary levels of glutathione and thiobarbituric acid reactants after acute lindane intoxication.

The i.p. administration of 60 mg kg-1 body weight of lindane, the gamma-isomer of hexachlorocyclohexane, to fed rats led to an enhancement of hepatic lipid peroxidation after 24 h of treatment. This was evidenced by significant increases in the hepatic production and biliary release of thiobarbituric acid reactive substances, and in the biliary release of glutathione disulphide. Under these conditions, the content of cytochrome P450 was enhanced concomitantly with increases in the total microsomal oxygen uptake, superoxide radical generation and (+)-catechin (cyanid-3-ol) sensitive respiration. The glutathione status of hepatocytes was altered by lindane as the content and biliary release of glutathione disulphide was drastically augmented, leading to a decrease in the cellular and biliary GSH/GSSG ratios. It is suggested that lindane treatment leads to an induced oxidative capacity, which, in turn, alters the glutathione status of the liver tissue.

Animals↗
PubMed1985

Modified determination of 2-thiobarbituric acid value in fats and oils.

Several methods for the determination of TBA-value were compared with a new direct modification (omitting the previous distillation), using 1-butanol as the sole solvent. The absorbance is measured at 530 nm after heating to 95 degrees C for 120 min, and cooling. The effect of variables was tested by the Youdens ruggedness test (reaction time, temperature, cooling, concentration of sample, thiobarbituric acid, water, heavy metals, antioxidants, chelating agents, bubbling with oxygen or nitrogen). The Lambert-Beers law is valid in the absorbance range between 0.05 and 0.8. The repeatability is better than that of the Dzikowskis procedure and comparable with those of more complicated distillation methods. The standard deviation is in a linear relation with the absorbance (range 0.1-1.0). The method was found satisfactory for evaluating lard, cooking fat, soybean, sunflower, and rapeseed oils in the stage of beginning rancidity.

Antioxidants↗
PubMed1986

Butylated hydroxytoluene addition improves the thiobarbituric acid assay for malonaldehyde from chicken plasma fat.

Samples of plasma lipid were tested for autoxidation during the heating step of the thiobarbituric acid (TBA) assay for malonaldehyde. Butylated hydroxytoluene (BHT) was used to quench sample autoxidation during the assay, and 25 ppm BHT was found effective in eliminating autoxidation in fresh plasma fat. At this concentration, BHT does not interfere with the reaction of malonaldehyde with TBA. Depending on fat concentration, absorbance at 532 nm was 2-5 times higher when samples were assayed without BHT compared to identical samples with 25 ppm BHT addition. It is concluded that BHT addition to the TBA assay before the heating step improves the reliability of the assay by eliminating autoxidation which may generate the majority of oxidative products measured in TBA assays lacking antioxidant protection.

Animals↗
PubMed1986

Detection of in vivo lipid peroxidation using the thiobarbituric acid assay for lipid hydroperoxides.

Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides.

Animals↗
PubMed1976

IR spectroscopic characterization of 2-thiohydantoins and 2-thiobarbiturates.

A characterization of 2-thiohydantoins and 2-thiobarbiturates by IR spectra is proposed, using three characteristic group frequencies: the "thioureide band" around 1500 cm-1 and the antisymmetric-symmetric stretching modes of NCS bonds around 1400 and 1200 cm-1. The general characteristic absorption areas are found by comparison with N-phenylthioureas and thioanilides.

Chemical Phenomena↗
PubMed1997

Assay of aldehydes from lipid peroxidation: gas chromatography-mass spectrometry compared to thiobarbituric acid.

The oxidation of lipids, lipid peroxidation, is usually assayed with thiobarbituric acid (TBA). We compare the TBA assay measuring TBA-reactive substances (TBARS), and a new gas chromatography-mass spectrometric (GC-MS) assay measuring malondialdehyde (MDA) with unsaturated fatty acids and biological samples. The extent of oxidation to different unsaturated fatty acids is related to the total number of bis-allylic positions, the position of the first double bond from the methyl terminus, and the lipid chain length. The extent of oxidation of different biological samples or organs is related to the component polyunsaturated fatty acids. Both the GC-MS and TBA assays give parallel results for oxidation of unsaturated fatty acids and biological samples. The GC-MS assay is about two- to sixfold more sensitive than the TBA assay for oxidation of unsaturated fatty acids. In contrast, the TBA assay gives about two- to sixfold higher TBARS than MDA by GC-MS assay in biological samples, possibly due to the nonspecificity and artifactual formation of derivatives in the acid-heating step of the TBA assay. The GC-MS assay is shown to be useful in oxidation-related cell culture studies with as few as 250,000 neural cells. These results suggest that the GC-MS assay is a useful, sensitive, and specific assay for lipid peroxidation. The TBA assay is also quite useful because of its sensitivity and simplicity, if one clearly understands its nonspecificity.

Animals↗
PubMed1997

The peroxisome proliferator-activated receptor alpha (PPAR alpha) regulates the plasma thiobarbituric acid-reactive substance (TBARS) level.

We investigated whether liver expression of the peroxisome proliferator-activated receptor alpha (PPAR alpha) gene is related to the plasma thiobarbituric acid-reactive substance (TBARS) level, as well as to plasma cholesterol (TC) level and plasma triglyceride (TG) level in rats fed a high fat chow containing a variety of fatty acids. Only the plasma TBARS level showed a significant negative correlation with the liver PPAR alpha mRNA level (TC, R = 0.001, p = 0.9967; TG, R = 0.248, p = 0.1276; TBARS, R = 0.439, p = 0.0046). Although further studies are needed to clarify whether the increase of the liver PPAR alpha mRNA level confers a reduction in plasma TBARS levels, it is likely that PPAR alpha activity plays a regulatory role in the pathogenesis of hyperlipidemia and atherosclerosis.

Animals↗
PubMed2000

Interactions of nitric oxide with lipid peroxidation products under aerobic conditions: inhibitory effects on the formation of malondialdehyde and related thiobarbituric acid-reactive substances.

Under aerobic conditions, exposure of peroxidized lipids to nitric oxide (NO) was found to result in a rapid decrease in the levels of thiobarbituric acid-reactive substances (TBARS). Addition of 10-100 microM NO to rat brain homogenates preincubated for 2 h at 37 degrees C caused up to a 20% decrease in the levels of TBARS compared to controls. A similar inhibitory effect was observed on TBARS produced by Fe(2+)-induced decomposition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), due apparently to NO-induced decomposition of the hydroperoxide (ferrous oxidation/xylenol orange assay). Prostaglandin G(2) (PGG(2), 35 microM), as a model bicyclic endoperoxide, and malondialdehyde (MDA, 20 microM), the main component of TBARS, proved also susceptible to degradation by NO or NO donors (diethylamine NONOate, DEA/NO) at concentrations of 100 microM or higher in 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C, as indicated by the reduced response to the TBA assay. No significant effect on TBARS determination was caused by nitrite ions. These and other data indicate that NO can inhibit TBARS formation by decomposing primary lipid peroxidation products, chiefly 15-HPETE and related hydroperoxides, and, to a lesser extent, later stage TBARS precursors, including bicyclic endoperoxides and MDA, via nitrosation and other oxidative routes, without however affecting chromogenic reactions during the assay.

Animals↗