Measurement of fluorescent lipid peroxidation products in biological systems and tissues

A sensitive fluorometric assay for measurement of lipid peroxidation damage to biological preparations and tissues is described. The method consists of a chloroform-methanol extract of tissue followed by measurement of fluorescence characteristics of products derived from lipid peroxidation. Fluorescence analysis has been used successfully with rats and mice in vivo with stress induced during dietary and aging experiments and in peroxidation of subcellular organelles in vitro. Replicate extractions of fluorescent tissue showed a standard error of 2%. In a test of linearity with concentration, the amount of lipid-soluble fluorescent material in extracted samples was directly proportional to that added before extraction. Specific and general applications to in vivo and in vitro lipid peroxidation experiments are diseussed.     

Effect of gamma-radiation on contents of lipid peroxidation products in animal blood

The dynamics of malonic dialdehyde (MD) dependence in the blood serum of Vistar line male rats on the intensity of irradiation after a total single irradiation with 60Co gamma-quantums in 1.0 and 5.0 Gy doses (during 30 days); 9.0 Gy (during 15 days) were studied. The MD contents values both in the norm and in 0.5, 1, 2, 4, 6, 8, 10, 15, 20 and 30 days after irradiation are given. The experimental data demonstrated the different sensitivity of the final LPO parameters to the doses of 1.0, 5.0, 9.0 Gy radiation depending on the dose power, the changes of these parameters being kept for a long time after irradiation.     

Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

Chemistry and biochemistry of lipid peroxidation products

Abstract

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as ‘second messengers’ of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.     

Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins

Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and malonaldehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5-dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Schiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanals, alk-2-enals and alka-2,4-dienals can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.     

Effect of melanins on lipid peroxidation

Effects of melanins obtained from cultured Cladosporium cladosporidae fungi and Alpha grape on Fe(2+)-induced, Fe(2+)-ascorbate-induced, and NADPH-induced lipid peroxidation in rat liver, brain, and eye were studied. Melanins were shown to inhibit the accumulation of lipid peroxidation products in vitro. The inhibitory effects of melanins were not due to direct interactions of these pigments with superoxide anion (O2). However, melanins may interact with other free radicals. Melanins were demonstrated to have the ability to oxidize NADPH, which is probably one of the mechanisms of their antioxidant effects.     

Cellular response to bioactive lipid peroxidation products

Reactive aldehydes, such as 4-hydroxy-2-nonenal, have been implicated as inducers in generating intracellular reactive oxygen species and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli. Here, I briefly summarize a novel signaling pathway in cellular response, in which aldehyde-stimulated detoxification response is mediated by cyclooxygenase metabolites. These findings argue that lipid mediators could induce a cellular process that represents a cellular defense program against toxic compounds.     

Cellular response to bioactive lipid peroxidation products

Reactive aldehydes, such as 4-hydroxy-2-nonenal, have been implicated as inducers in generating intracellular reactive oxygen species and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli. Here, I briefly summarize a novel signaling pathway in cellular response, in which aldehyde-stimulated detoxification response is mediated by cyclooxygenase metabolites. These findings argue that lipid mediators could induce a cellular process that represents a cellular defense program against toxic compounds.     

DNA damage caused by lipid peroxidation products

Lipid peroxidation is a process involving the oxidation of polyunsaturated fatty acids (PUFAs), which are basic components of biological membranes. Reactive electrophilic compounds are formed during lipid peroxidation, mainly alpha, beta-unsaturated aldehydes. These compounds yield a number of adducts with DNA. Among them, propeno and substituted propano adducts of deoxyguanosine with malondialdehyde (MDA), acrolein, crotonaldehyde and etheno adducts, resulting from the reactions of DNA bases with epoxy aldehydes, are a very important group of adducts. The epoxy aldehydes are more reactive towards DNA than the parent unsaturated aldehydes. The compounds resulting from lipid peroxidation mostly react with DNA showing both genotoxic and mutagenic action; among them, 4-hydroxynonenal is the most genotoxic, while MDA is the most mutagenic. DNA damage caused by the adducts of lipid peroxidation products with DNA can be removed by the repairing action of glycosylases. The formed adducts have been hitherto analyzed using the IPPA (Imunopurification-(32)P-postlabelling assay) method and via gas chromatography/electron capture negtive chemical ionization/mass spectrometry (GC/EC NCI/MS). A combination of liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used in recent years.     

Accumulation of lipid peroxidation products in cataractous lenses

A study was made of the content of lipid peroxidation products (LPP) in the lenses extracted during operations for cataract as well as in transparent human lenses. In a mature cataract, the elevated content of primary, secondary and end products of lipid peroxidation (diene and triene conjugates, Schiff bases) was revealed. The content of LPP was identical in different clinical patterns of a mature cataract (senile, traumatic, complicated), which points to the universal role of lipid peroxidation in lenticular opacity.     

Susceptibility to lipid peroxidation and accumulation of fluorescent products with age is greater in T-cells than B-cells

The age-related loss of immune function, which is primarily due to loss of T-lymphocyte function, is also associated with accumulation in spleen lymphocytes of autofluorescent products indicative of peroxidation damage. In this study, we examined T-cell membranes for age-related changes which could be related to lipid peroxidation. Using fluorescence spectroscopy of CHCl3:CH3OH membrane extracts, we observed that old T-cells have a 2-fold greater accumulation of fluorescent products than old B-cells and that young T-cells, when exposed to free radicals in an in vitro system, accumulate significantly more fluorescent products over time than young B-cells. We used fluorescence polarization to show that young T-cell membranes are more fluid than young B-cell membranes. However, T-cell membrane fluidity decreases with age, whereas B-cell membrane fluidity does not change; in old mice, T-cell membranes are significantly less fluid than old B-cell membranes. Using two-dimensional electrophoresis, we showed that membrane extracts of old T-cells contain many more proteins than extracts of young T-cells. Our results indicate that age-related changes occur in T-cell membranes which could be due to their increased susceptibility to lipid peroxidation and these changes may contribute to the age-related decline in immune function.     

Seizure-induced formation of isofurans: novel products of lipid peroxidation whose formation is positively modulated by oxygen tension

We have previously shown that seizures induce the formation of F₂-isoprostanes (F₂-IsoPs), one of the most reliable indices of oxidative stress in vivo . Isofurans (IsoFs) are novel products of lipid peroxidation whose formation is favored by high oxygen tensions. In contrast, high oxygen tensions suppress the formation of F₂-IsoPs. The present study determined seizure-induced formation of IsoFs and its relationship with cellular oxygen levels (pO₂). Status epilepticus (SE) resulted in F₂-IsoP and IsoF formation, with overlapping but distinct time courses in hippocampal subregions. IsoF, but not F₂-IsoP formation coincided with mitochondrial oxidative stress. SE resulted in a transient decrease in hippocampal pO₂ measured by in vivo electron paramagnetic resonance oximetry suggesting an early phase of seizure-induced hypoxia. Seizure-induced F₂-IsoP formation coincided with the peak hypoxia phase, whereas IsoF formation coincided with the 'reoxygenation' phase. These results demonstrate seizure-induced increase in IsoF formation and its correlation with changes in hippocampal pO₂ and mitochondrial dysfunction.     

Methods for determination of aldehydic lipid peroxidation products

A complex pattern of aldehydes (alkanals, 2-alkenals, 2,4-alkadienals, 4-hydroxyalkenals) is generated by peroxidizing biological samples. Several methods based on HPLC or GC-MS have been developed to qualitatively and quantitatively measure the aldehydes in tissues, cells and cell fractions exposed to various pro-oxidative stimuli. 4-Hydroxynonenal, hexanal and propanal are, besides malonaldehyde, the most abundant aldehydes formed. The high sensitivity of the methods also allows the measurement of physiological aldehyde levels in plasma or low density lipoproteins and this could be of great importance for in vivo studies.     

Interaction of lipid peroxidation products with nuclear macromolecules

The interaction of lipid peroxidation products with nuclear macromolecules was investigated in rat liver nuclei labelled with [3H]arachidonic acid. Lipid peroxidation reactions were driven both non-enzymatically and enzymatically by the addition of ascorbate-Fe2+ or NADPH-ADP-Fe3+, respectively, to the incubation mixtures. The extent of peroxidation was evaluated by the formation of thiobarbituric acid chromophore and of radioactive hydrophilic peroxidation products. The results obtained show that: (1) nuclear membrane lipid peroxidation products formed during incubation interact with DNA and total nuclear proteins; (2) non-enzymatic lipid peroxidation processes induced a 40% larger association of peroxidation products to DNA compared to processes driven enzymatically, whereas the corresponding interaction with total nuclear proteins was similar in both peroxidation systems; (3) the radioactivity associated with histones decreased during incubation in the presence of ascorbate-Fe2+ or NADPH-ADP-Fe3+, and increased in control samples (no additions); (4) inhibition of lipid peroxidation by the iron chelator Desferrioxamine B prevented the association of peroxidation products to nuclear macromolecules; (5) the levels of radioactivity found in DNA after 180 min of incubation would represent the formation of 0.6-1.0 adducts per 10(6) DNA bases. The results obtained provide evidence for an interaction between lipid peroxidation products and chromatin in the interior of the cell nucleus.     

Isoprostanes: novel bioactive products of lipid peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F₂-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F₂-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F₂-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

Effect of alimentary factors on the production of primary and final products of lipid peroxidation

In blood plasma, liver, and brain of pubertal female Wistar line rats the level of diene conjugates, fluorescent lipopigments and vitamin E were studied as a function of different diets: control, fish and fish with addition of vitamin E. The obtained results show that in the brain tissue the role of vitamin E as an antioxidant is more pronounced, but dynamical equilibrium between the oxidation substrates and antioxidants after alimentary deficiency of vitamin E is restored in the nervous tissue more slowly than in the liver and blood plasma. In the organism of young animals fluorescent pigments are not accumulated in form of inert products, but behave as normal metabolites.     

Site-specific DNA damage caused by lipid peroxidation products

DNA damage induced by autoxidized lipids was investigated using covalently closed circular (supercoiled) DNA and DNA fragments of defined sequence. DNA-strand-breaking substances accumulated during autoxidation of methyl linolenate, and strand breakage was measured with samples taken at different times. The DNA-strand-breaking activity reached its maximum a little after the peak value of peroxide and decreased upon further autoxidation. The peak of the DNA-strand-breaking activity did not always coincide with the peak of thiobarbituric acid reactants or of conjugated diene, either. The DNA-strand-breaking reaction was dependent on metal ions and was inhibited by potassium iodide and tiron and partially by catalase, suggesting the involvement of radical species and/or oxygen radicals. No direct cleavage of singly end-labeled 100-200 basepair DNA fragments by autoxidized methyl linolenate and cupric ion was detected under the conditions used. Cleavage occurred during subsequent heating in piperidine after the reaction. The alkali-labile damage was preferentially induced at pyrimidine residues, especially in dinucleotide sequences of pyrimidine-guanine (5'----3'), which was determined by sequencing.     

Aldehydic lipid peroxidation products derived from linoleic acid

Lipid peroxidation (LPO) processes observed in diseases connected with inflammation involve mainly linoleic acid. Its primary LPO products, 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) and 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), decompose in multistep degradation reactions. These reactions were investigated in model studies: decomposition of either 9-HPODE or 13-HPODE by Fe(2+) catalyzed air oxidation generates (with the exception of corresponding hydroxy and oxo derivatives) identical products in often nearly equal amounts, pointing to a common intermediate. Pairs of carbonyl compounds were recognized by reacting the oxidation mixtures with pentafluorobenzylhydroxylamine. Even if a pure lipid hydroperoxide is subjected to decomposition a great variety of products is generated, since primary products suffer further transformations. Therefore pure primarily decomposition products of HPODEs were exposed to stirring in air with or without addition of iron ions. Thus we observed that primary products containing the structural element R-CH=CH-CH=CH-CH=O add water and then they are cleaved by retroaldol reactions. 2,4-Decadienal is degraded in the absence of iron ions to 2-butenal, hexanal and 5-oxodecanal. Small amounts of buten-1,4-dial were also detected. Addition of m-chloroperbenzoic acid transforms 2,4-decadienal to 4-hydroxy-2-nonenal. 4,5-Epoxy-2-decenal, synthetically available by treatment of 2,4-decadienal with dimethyldioxirane, is hydrolyzed to 4,5-dihydroxy-2-decenal.     

Damage to photosystem II by lipid peroxidation products

BackgroundPhotosystem II proteins of higher plant chloroplasts are prone to oxidative stress, and most prominently the reaction center-binding D1 protein is damaged under abiotic stress. The reactive oxygen species produced under these stress conditions have been suggested to be responsible for the protein injury.Scope of reviewRecently, it has been shown that the primary and secondary products of non-enzymatic and enzymatic lipid peroxidation have a capability to modify photosystem II proteins. Here, we give an overview showing how lipid peroxidation products formed under light stress and heat stress in the thylakoid membranes cause oxidative modification of proteins in higher plant photosystem II.Major conclusionsDamage to photosystem II proteins by lipid peroxidation products represents a new mechanism underlying photoinhibition and heat inactivation.General significanceComplete characterization of photosystem II protein damage is of crucial importance because avoidance of the damage makes plants to survive under various abiotic stresses. Further physiological significance of photosystem II protein oxidation by lipid peroxidation product should have a potential relevance to plant acclimation because the oxidized proteins might serve as signaling molecules.