Role of prostaglandin endoperoxides in the serum thiobarbituric acid reaction

In the presence of heme and reduced glutathione, prostaglandin (PG) endoperoxides underwent rapid conversion to malondialdehyde and 12l-hydroxy-5,8,10-heptadecatrienoic acid. In addition, PG endoperoxides as well as lipid peroxides produced malondialdehyde to yield a red pigment during the thiobarbituric acid reaction with different efficiencies. The relative rates of the reaction were: 1,1,3,3-tetraethoxypropane, 100; PGG₂, 55; PGH₂, 32; and 15-hydroperoxyarachidonic acid, 6. The thiobarbituric acid reactive materials in rabbit serum decreased by 25–60%, after intravenous administration of aspirin (a cyclo-oxygenase inhibitor) and with a concomitant decline of serum PG levels. These results, taken together, suggested that serum thiobarbituric acid values, considered to be an indicator of lipid peroxide levels, were to a significant extent due to PG endoperoxides and their derivatives.     

Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid

Inhalation of toxic materials such as asbestos, silica, 100% oxygen, ozone, or nitrogen dioxide may lead to an increased production of reactive oxygen metabolites which may initiate lipid peroxidation. Measurement of lipid peroxidation in cells and fluid obtained by bronchoalveolar lavage (BAL), as well as in lung tissue, may aid in monitoring the development and extent of pulmonary damage after inhalation of a toxic substance. In this study, we employed a sensitive assay for detection of malondialdehyde (MDA), a breakdown product of lipid peroxidation. By separation of the adduct with thiobarbituric acid, using a reverse phase high pressure liquid chromatographic technique, we accurately and sensitively measured the content of MDA in BAL cells, lavage fluid, and lavaged lung tissue homogenates of rats. The amounts of sample required for detection of MDA were small enough possibly to be applied to use with human specimens; in addition, recovery of added MDA was acceptable with all types of samples. Inclusion of a metal chelator in the preparation of samples appeared necessary to prevent metal-catalyzed propagation of lipid peroxidation during the assay. Overall, the method described here using samples from rats may be applicable to detecting lipid peroxidation in BAL samples from humans.     

Assay of Malondialdehyde in Biological Fluids by Gas Chromatography-Mass Spectrometry

Malondialdehyde (MDA) is assayed in femtomole quantities in biological samples by gas chromatography-mass spectrometry (GC-MS). The MDA trapped in protein as a Schiff base is released by H₂SO₄, the protein precipitated using Na₂WO₄, and the MDA derivatized with pentafluorophenylhydrazine to form the stable adduct, N-pentafluorophenylpyrazole. Negative chemical ionization (NCI) capability allows the sensitive detection of this MDA adduct in biological samples at a level of 5 nM on-column. A stable-isotope-labeled MDA, [²H₂]MDA, was used as an internal standard for quantitation. MDA recovery from plasma was 76%. This assay provides two forms of confirmation of the analyte, retention time and mass ion, thus minimizing error due to interfering compounds. The commonly used thiobarbituric acid assay for MDA overestimates the MDA levels by over 10-fold, possibly resulting from cross-reactivity with other aldehydes and artifactual oxidation due to 100°C temperature conditions. In our assay, all steps were performed at room temperature thereby suppressing artifactual oxidation of the sample. We have successfully applied this assay to biological samples including plasma, tissue homogenates, and sperm.     

Method for the simultaneous determination of free/protein malondialdehyde and lipid/protein hydroperoxides

A simple and sensitive method is presented for the simultaneous quantification (spectrophotometric and spectrofluorimetric) of the main lipid and protein peroxidation products after their initial fractionation: free malondialdehyde (FrMDA), protein-bound malondialdehyde (PrMDA), total hydroperoxides (LOOH), and protein hydroperoxides (PrOOH). FrMDA and PrMDA (released from proteins by alkaline hydrolysis) are measured after the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions, by the specific fluorimetric quantification of the resulting MDA–(TBA)₂ adduct chromophore. The measurement of LOOH and PrOOH is based on the reaction of Fe³⁺ (resulting from the reaction of LOOH and PrOOH with Fe²⁺) with xylenol orange (XO) and the photometric quantification of the resulting XO–Fe complex. The sensitivity of the assays for FrMDA/PrMDA and LOOH/PrOOH is 20 and 100 pmol, respectively. The method was applied successfully on human plasma and can be used for the evaluation of oxidative stress in both basic and clinical research.     

Method development and validation for monitoring in vivo oxidative stress: evaluation of lipid peroxidation and fat-soluble vitamin status by HPLC in rat plasma

Monitoring in vivo oxidative stress implicates the evaluation of damage and defence parameters by well-established, validated methods. We report two optimized and validated HPLC methods for measurement of malondialdehyde (MDA) and fat-soluble vitamins in rat plasma. For the MDA method, optimization experiments of the thiobarbituric acid test resulted in the addition of 1% butylhydroxytoluene to the reaction mixture and in a heating time reduction to 40 min, ensuring inhibition of further lipid peroxidation during the test. Validation experiments showed good linearity, precision and recovery. The use of HPLC with coulometric array detection technology permits simultaneous and sensitive analysis of different fat-soluble vitamins and related compounds (tocopherols, retinoids, carotenoids and coenzyme Q10), which are identified by both retention time and electrochemical characteristics. Furthermore, this method is extended to the analysis of coenzyme Q9, the predominant homologue in rats. Validation experiments with rat plasma gave good results.     

Methods for estimating lipid peroxidation: an analysis of merits and demerits

Among the cellular molecules, lipids that contain unsaturated fatty acids with more than one double bond are particularly susceptible to action of free radicals. The resulting reaction, known as lipid peroxidation, disrupts biological membranes and is thereby highly deleterious to their structure and function. Lipid peroxidation is being studied extensively in relation to disease, modulation by antioxidants and other contexts. A large number of by-products are formed during this process. These can be measured by different assays. The most common method used is the estimation of aldehydic products by their ability to react with thiobarbituric acid (TBA) that yield 'thiobarbituric acid reactive substances' (TBARS), which can be easily measured by spectrophotometry. Though this assay is sensitive and widely used, it is not specific and TBA reacts with a number of components present in biological samples. Hence caution should be used while employing this method. Wherever possible this assay should be combined with other assays for lipid peroxidation. Such methods are measurement of conjugated dienes, lipid hydroperoxides, individual aldehydes, exhaled gases like pentane, isoprostanes, etc. The modern methods also involve newer techniques involving HPLC, spectrofluorimetry, mass spectrometry, chemiluminescence etc. These and other modern methods are more specific and can be applied to measure lipid peroxidation. There are certain restraints, in terms of high cost and certain artifacts, and these should be considered while selecting the method for estimation. This review analyses the merits and demerits of various assays to measure lipid peroxidation.     

Glucosamine prevents <Emphasis Type="Italic">in vitro</Emphasis> collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.     

Determination of a guanosine—malonaldehyde adduct in urine by high-performance liquid chromatography with a thiobarbituric acid reaction detector

A new method for the determination of a guanosine—malonaldehyde adduct, β- -ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine—malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.     

Analysis of lipid peroxidation mechanisms in human spermatozoa

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal* and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe²⁺-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe²⁺-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation. © 1993 Wiley-Liss, Inc.     

Rapid monitoring of diabetes-induced lipid peroxidation by Fourier transform infrared spectroscopy: evidence from rat liver microsomal membranes

Increased oxidative stress is the consequence of either enhanced reactive oxygen species (ROS) production or attenuated ROS scavenging capacity, resulting in tissue damage that in most instances is assessed by the measurement of lipid peroxides. In the current study, diabetes-induced lipid peroxidation in rat liver microsomal membranes was investigated by Fourier transform infrared (FT-IR) spectroscopy at different temperatures. The olefinic (CH) band at 3012 cm-1 was used to probe diabetes-induced lipid peroxidation. The intensity and area values of this band of diabetic samples were found to be increased significantly (P<0.05) compared with nondiabetic samples. The increase in olefinic band intensity is attributed mainly to the lipid peroxidation end products. The results of the FT-IR study were found to be in agreement with biochemical studies that revealed a significant increase in malondialdehyde levels of diabetic samples compared with control samples (P<0.05) using the thiobarbituric acid test.     

Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein: antioxidant implications

Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2(+)-mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.     

Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose. Quantitative determination of malondialdehyde.

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.     

The measurement of malondialdehyde in peroxidised ox-brain phospholipid liposomes

The preparation of ox-brain phospholipid liposomes and their peroxidation using different catalysts have been described in detail. The degree of peroxidation is related to the formation of thiobarbituric acid (TBA)-reacting compounds and expressed as malondialdehyde (MDA). As confirmatory data, fluorescent MDA-phospholipid complexes were measured in parallel. Close agreement between the polar TBA-reactive compounds and the nonpolar fluorescent compounds confirmed the usefulness of the simple TBA test as a measure of peroxidative activity in pure lipid liposomes. Relative differences in the catalytic activity of ascorbic acid and cupric ions when assayed by the two methods are discussed.     

The role of nitric oxide and lipid peroxidation in patients with Plasmodium vivax malaria

In this study, we investigated the role of nitric oxide metabolism and lipid peroxidation in patients with P. vivax malaria. The levels of nitrite and nitrate were analyzed using a procedure based on the Griess reaction and malondialdehyde levels which index of lipid peroxidation was determined by thiobarbituric acid reaction. The levels of nitrite/nitrate and malondialdehyde in patients were higher than controls and found to be statistically significant (p < 0.001). We performed this study to determine whether nitric oxide and lipid peroxidation is produced during blood-stage P. vivax malaria. This present study shows that lipid peroxidation occurs in P. vivax malaria. The levels of nitric oxide are associated with lipid peroxidation in this disease.     

Is there evidence for excessive free radical production in vivo during ultrasound-assisted liposuction?

The surgical technique of ultrasound-assisted liposuction has become a standard procedure for the treatment of lipodystrophy. However, little is known about the impact of this therapy on fatty tissue on the molecular level. There are concerns about possible adverse effects related to the high-intensity ultrasound energy, because in vitro studies have shown a substantial generation of free radicals. In this study, the authors investigated whether ultrasound waves can create an excessive free radical production in vivo by measuring lipid peroxidation products in the form of malondialdehyde equivalents. For this purpose, the thiobarbituric acid-reactive substances (TBARS) assay was chosen. In this test, malondialdehyde, a major product of lipid peroxidation, reacts with thiobarbituric acid to produce a pink adduct that can be measured spectrophotometrically. The authors determined oxidation products in 28 aspirates of 17 treated patients before ultrasound-assisted liposuction (0 minutes) to establish a baseline concentration and at 2, 5, and 10 minutes after the treatment was begun. Median malondialdehyde concentration of the control group (conventional liposuction, 0 minutes) was 3.40 nmol of malondialdehyde per gram of adipose tissue. Median concentrations after 2, 5, and 10 minutes of ultrasound-assisted liposuction were 7.45 (n = 28), 8.84 (n = 21), and 4.07 (n = 8) nmol malondialdehyde per gram adipose tissue, respectively. The differences were not statistically significant. The data suggest that there is no excessive formation of lipid oxidation products in response to free radicals. The antioxidative capacity of adipose tissue does not seem to be overwhelmed by the standard application regimen of ultrasound-assisted liposuction.     

Tannic acid interferes with the commonly used laccase-detection assay based on ABTS as the substrate

Laccase enzymatic activity in biological samples is usually detected spectrophotometrically through its capacity to oxidize several specific aromatic compounds. One of the most commonly used substrates is the compound 2-2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which becomes green-blue coloured when it is oxidized by laccase. In this work we study the interference of tannic acid with the spectrophotometric assay to detect laccase by using ABTS as the substrate. Our data show that under the normal reaction conditions of this assay, but in the absence of any catalyst, tannic acid is able to carry out the chemical reduction of the oxidized specie of ABTS, thus decreasing the overall detectable laccase-activity values observed when this enzyme is present in the reaction mixture. Therefore, our results represent an important warning concerning a commonly used method for measuring, detecting or screening laccases in biological samples that may content tannic acid or structural-related molecules.     

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.     

Analysis of DNA-protein crosslinking activity of malondialdehyde in vitro

In an attempt to identify endogenous chemicals producing DNA-protein crosslinks, we have studied in vitro crosslinking potential of malondialdehyde, a bifunctional chemical that is ubiquitously formed as a product of lipid peroxidation of polyunsaturated fatty acids. We have found that malondialdehyde readily forms crosslinks between DNA and histones under physiological ionic and pH conditions. Formation of DNA-protein crosslinks was limited to proteins that were able to bind to DNA. Malondialdehyde failed to form DNA-protein crosslinks when histone binding to DNA was prevented by elevated ionic strength or when bovine serum albumin was used in the reaction mixture. Malondialdehyde-produced DNA-histone crosslinks were relatively stable at 37 degrees C with t1/2=13.4 days. Crosslinking of histones to DNA proceeds through the initial formation of protein adduct followed by reaction with DNA. Modification of DNA by malondialdehyde does not lead to a subsequent crosslinking of proteins. Significant formation of DNA-protein crosslinks was also registered in isolated kidney and liver nuclei treated with malondialdehyde. Based on its reactivity and stability of the resulting crosslinks, it is suggested that malondialdehyde could be one of the significant sources of endogenous DNA-protein crosslinks.     

Factors relevant in the reaction of pyrroloquinoline quinone with amino acids. Analytical and mechanistic implications

In order to reveal the stability of pyrroloquinoline quinone (PQQ) in complex samples, its reaction on incubation with amino acids was followed spectrophotometrically by monitoring oxygen consumption, and with a biological assay. For several alpha-amino acids, the formation of a yellow coloured compound (lambda max = 420 nm) was accompanied by oxygen uptake and disappearance of biological activity from the reaction mixture. The yellow product appeared to be an oxazole of PQQ, the exact structure depending on the amino acid used. Oxazole formation also occurred under anaerobic conditions with concomitant formation of PQQH2, suggesting that PQQ is able to oxidize the presumed oxazoline to the oxazole. Besides the condensation reaction, there is also a catalytic cycle in which an aldimine adduct of PQQ and the amino acid is converted into the aminophenol form of the cofactor and an aldehyde resulting from oxidative decarboxylation of the amino acid. Addition of NH4+ salts, as well as that of certain divalent cations, greatly stimulated both the cyclic and the linear reaction. With basic amino acids, oxazole formation scarcely occurred. However, as oxygen consumption was observed (provided that certain divalent cations were present), conversion of these compounds took place. A reaction scheme is proposed accounting for the products formed and the effects observed. Since NH4+ ions activate several quinoproteins (PQQ-containing enzymes) and divalent cations (Ca2+, Fe2+, and Cu2+) are additional (co)factors in certain metallo quinoproteins, the effects of metal ions observed here could be related to the mechanistic features of these enzymes. Although all oxazoles were converted to PQQ by acid hydrolysis, PQQ was not detected when hydrolysis was carried out in the presence of tryptophan, a compound which appeared to have a deleterious effect on the cofactor under this condition. The results here described explain why analysis methods for free PQQ in complex samples fail in certain cases, or are not quantitative.     

The reaction stoichiometry between ozone and unsaturated fatty acids in an aqueous environment

The light absorption of ozone in an air stream allowed the monitoring of reactions of ozone with unsaturated fatty acids in solution. The kinetics for the reaction of ozone with linolenic acid was found to be of a pseudo-first-order after the first few minutes and did not vary with the concentration of ozone introduced into the solution. The reaction of ozone with linolenic acid in solution was found to be exceedingly rapid.When various combinations of polyunsaturated fatty acids were injected simultaneously, they reacted independently. The stoichiometry of ozone reacted to number of double bonds present in the fatty acid was one for mono- and diunsaturated; however, for triunsaturated fatty acid the stoichiometry was about 0.70.Malondialdehyde was produced upon the reaction of ozone with di- and triunsaturated fatty acids, as shown by both the thiobarbituric acid test and the characteristic UV absorption of malondialdehyde in solution. The true yield of malondialdehyde for the reaction of polyunsaturated fatty acids with ozone was found to be about 2%. In addition, other species, absorbing at 290–300 nm, were formed in solution during ozonolysis.