Fluorescence characteristics of peroxidation products in porcine intestinal brush-border membranes

Treatment of the porcine intestinal brush-border membranes with 100 microM ascorbic acid and 10 microM Fe2+ in the presence of various concentrations of tert-butyl hydroperoxide (t-BuOOH) resulted in a marked fluorescence development at 430 nm, depending on the hydroperoxide concentration. This fluorescence formation was closely related to lipid peroxidation of the membranes as assessed by formation of conjugated diene. However there is no linear relation between thiobarbituric acid-reactive substances (TBARS) and fluorescence formation. On the other hand, fluorescence formation in the membranes by treatment with ascorbic acid/Fe2+ or t-BuOOH alone was negligible. The results with antioxidants and radical scavengers suggest that ascorbic acid/Fe2+/t-BuOOH-induced lipid peroxidation of the membranes is mainly due to t-butoxyl and/or t-butyl peroxy radicals. Most TBARS produced during the peroxidation reaction were released from the membranes, but fluorescent products remained in the membrane components. The fluorescence properties of products formed by lipid peroxidation of the membranes were compared with those of products derived from the interaction of malondialdehyde (MDA) or acetaldehyde with the membranes. The fluorescence products in the acetaldehyde-modified membranes also exhibited the emission maximum at 430 nm, while the emission maximum of MDA-modified membranes was 470 nm. The fluorescence intensity of MDA-modified membranes was markedly decreased by treatment with 10 mM NaBH4 but that of the peroxidized or acetaldehyde-modified membranes was enhanced by about two-fold with the treatment. In addition, a pH dependence profile revealed that the fluorescence intensity of the peroxidized or acetaldehyde-modified membranes decreases with increasing pH of the medium, whereas that of MDA-modified ones did not change over the pH range from 5.4 to 8.0. On the basis of these results, the fluorescence properties of products formed in the intestinal brush-border membranes by lipid peroxidation are discussed.     

Lipid fluorophores of the crystalline lens in cataracts

Microcolumn liquid and column chromatography technique is conjunction with UV-spectrophotometry and spectrofluorescent analysis were used to study lipid peroxidation products accumulated in human lenses during cataract formation by means of chromatographic separation in regard to the molecular weight and polarity properties. Cataract is characterized by the appearance of certain substances changing UV-absorption lipid spectra in the region of 230 and 274 nm and having special fluorescence (excitation--320-370 nm), (emission--405-460 nm). The same changes were observed by ultrasoundinduced lipid peroxidation of model lipid samples. The accumulated lipid peroxidation products are concentrated in the same chromatographic fractions that are responsible for the change of UV-absorption and fluorescent spectra of lipids of cataractous lenses. It is the evidence of free radical lipid peroxidation products accumulation in human lenses at cataract formation. Along with the formation of diene and triene conjugates in the lens lipids, cataract is characterized by the formation of cetodienes and of low molecular weight lipid fluorescent products of fatty acids oxidation with low polarity due to the appearance of tetraene derivatives of polyunsaturated fatty acids. The particular features of mature cataract are an increased intensity of long-wave lipid fluorescence in the blue-green region (430-460 nm) of the spectrum, formation of high molecular weight fluorescent lipid peroxidation products with high polarity, and smooth decrease in absorbance in the region of 220-330 nm. During cataract formation products of deep lipid peroxidation resulting from radical phospholipids and fatty acids polymerisation are accumulated. It is supposed that lipid peroxidation is an initial phase of membrane desintegration and formation of HMW-proteins in cataract.     

The origin of red cell fluorescence caused by hydrogen peroxide treatment

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.     

Olive phenols efficiently inhibit the oxidation of serum albumin-bound linoleic acid and butyrylcholine esterase

Background

Olive phenols are widely consumed in the Mediterranean diet and can be detected in human plasma. Here, the capacity of olive phenols and plasma metabolites to inhibit lipid and protein oxidations is investigated in two plasma models.

Methods

The accumulation of lipid oxidation products issued from the oxidation of linoleic acid bound to human serum albumin (HSA) by AAPH-derived peroxyl radicals is evaluated in the presence and absence of phenolic antioxidants. Phenol binding to HSA is addressed by quenching of the Trp214 fluorescence and displacement of probes (quercetin, dansylsarcosine and dansylamide). Next, the esterase activity of HSA-bound butyrylcholine esterase (BChE) is used as a marker of protein oxidative degradation.

Results

Hydroxytyrosol, oleuropein, caffeic and chlorogenic acids inhibit lipid peroxidation as well as HSA-bound BChE as efficiently as the potent flavonol quercetin. Hydroxycinnamic derivatives bind noncompetitively HSA subdomain IIA whereas no clear site could be identified for hydroxytyrosol derivatives.

General significance

In both models, olive phenols and their metabolites are much more efficient inhibitors of lipid and protein oxidations compared to vitamins C and E. Low postprandial concentrations of olive phenols may help to preserve the integrity of functional proteins and delay the appearance of toxic lipid oxidation products.     

Fluorescence formation and heme degradation at different stages of lipid peroxidation

Secondary oxidative products of autoxidized methyl linoleate were divided into three groups (SP-I, SP-II and SP-III), which were then compared as to their abilities to form fluorescent substances and to degrade heme. SP-III showed a marked ability to produce two fluorescent substances exhibiting an excitation maximum at 350-360 nm and an emission maximum at 410-430 nm, while SP-I showed a more strongly degradative effect on heme than SP-III. The heme degradation was observed in parallel with the changes of TBA value in an early stage of lipid peroxidation and the fluorescence formation markedly increased according to the decrease of TBA value in a later stage. The results suggested that there are different reactive substances which bring about fluorescence formation and heme degradation and that they are produced at different stages of lipid peroxidation.     

Antioxidant Activity of Vitamin E and Trolox: Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro

Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the processing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO^•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information to that obtained currently from assays performed in aqueous buffer media.     

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.     

Peroxidative Changes in Brain Cortical Fatty Acids and Phospholipids, as Characterized During Fe2+- and Ascorbic Acid-Stimulated Lipid Peroxidation In Vitro

Abstract: The occurrence of peroxidative damage, as distinguished from anaerobic damage, to brain fatty acids and phospholipids was characterized in vitro. Fe²⁺ and ascorbic acid were used to stimulate peroxidation in cortical homogenates from rat brain incubated with or without oxygen. Lipid peroxidation was established in samples incubated with oxygen by increased diene conjugation, accumulation of thiobarbituric acid-reactive material (TBAR) and of lipid-soluble fluorescent products. No peroxidation occurred in samples incubated in the absence of oxygen (100% N₂). Lipid peroxidation was characterized by a selective loss of arachidonic acid and docosahexaenoic acid and by degradation of ethanolamine phosphoglyceride, while choline phosphoglyceride did not change. During the course of peroxidation there were parallel increases in products of lipid peroxidation concomitant with the decrease in polyenoic fatty acids. The maximal changes in diene conjugation and TBAR occurred earlier than the maximal changes in fluorescent material and fatty acids. It is concluded that measurements of changes in brain fatty acid and phospholipid composition may be a useful tool to establishment of whether peroxidative damage is important in vivo in situations with a critically reduced oxygen supply. Estimation of lipid-soluble fluorescence in vivo may also be useful, since it is considered to reflect the accumulation of stable end products of peroxidation.     

Inhibition of copper-induced lipid peroxidation by sinapic acid and its derivatives in correlation to their effect on the membrane structural properties

The efficiencies of sinapic acid and its derivatives syringic acid, syringaldehyde, three sinapoyl esters (ethyl, propyl, butyl sinapates), 4-vinylsyringol and sinapine were investigated for prevention of lipid peroxidation in correlation with their interactions with model lipid membrane systems. Significant antioxidant activities of propyl and butyl sinapates were seen by fluorimetric assay in phosphatidylcholine liposomes as model membrane using C11-BODIPY^581/591 lipophilic fluorescent probe. The sinapic acid esters also had the highest impact on membrane structural properties, as observed by differential scanning calorimetry and fluorescence polarisation measurements. The greatest protection of phospholipids from peroxidation by these esters correlated well with their polarity and insertion into the lipid bilayer.     

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.     

The chemical nature of the fluorescing products accumulating in the lipids of the crystalline lenses of mice with hereditary cataract

The content of diene conjugates (lipid hydroperoxides) was shown to be significantly higher in lipids extracted from the lenses of mice with hereditary cataract than in the controls. The same holds true for characteristics of fluorescence of the end-product of lipid peroxidation. Two (low- and high-molecular weight) peaks were detected in chromatographic lipid profile of cataract lenses measured by fluorescence on Sephadex LH-20 column, whereas only one (high-molecular weight) peak was found in lipids from normal lenses. It was established that high-molecular weight fluorescent fractions corresponded to lipid components of lipofuscin-like pigments. NMR and mass spectrometry of low-molecular weight fractions suggested that they contained predominantly products of free radical oxidation of long chain polyunsaturated fatty acids (C22:6).     

Quinolinic acid is a potent lipid peroxidant in rat brain homogenates

In this study, we describe the lipoperoxidative effect of quinolinic acid (QUIN) in vitro. The formation of thiobarbituric acid reactive products (TBA-RP), an index of lipid peroxidation, was measured in rat brain homogenates after incubation at 37°C for 30 min in the presence of QUIN and some structurally and metabolically related compounds such as Kynurenine, Kynurenic acid, Glutamate, Aspartate and Kainate. Concentrations of QUIN in the range of 20 to 80 M increased lipid peroxidation in a concentration-dependent manner from about 15% to about 50%. Kynurenic acid, a compound metabollically related to QUIN that can block its neurotoxic actions in vivo, also inhibited completely the QUIN-induced TBA-RP formation in our system. Lipid fluorescent material, another index of lipid peroxidation was also found increased by 49% after incubation with 40 M QUIN. It is concluded that lipid peroxidation may be a damaging process involved in the neurotoxicity of QUIN.     

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.     

Tryptophan fluorescence of erythrocyte ghosts following irradiation and Fe2+ initiation of lipid peroxidation

It was shown that under the effect of Fe2+-initiated lipid peroxidation and ionizing radiation tryptophan fluorescence parameters (i.e. intensity and polarization) were subjected to similar changes. Shortly (15 min) after irradiation no changes were observed in the level of products reacting with thiobarbituric acid. It is concluded that the process and products of lipid peroxidation do not markedly contribute to the postirradiation alteration of tryptophan fluorescence. At the same time additional postirradiation damages to proteins can be attributed to activation of lipid peroxidation.     

The reaction of DNA with lipid oxidation products, metals and reducing agents

The interaction of lipid hydroperoxides and secondary oxidation products with DNA was investigated by evaluating the fluorescence formed in the presence of metals and reducing agents. We also investigated the effect of malonaldehyde, because it has been generally considered responsible for the formation of fluorescence with DNA. However, malonaldehyde usually has been estimated by the notoriously unspecific thiobarbituric acid test. At low concentration of oxidation products (1 mM), fluorescence formation required the presence of metals and ascorbic acid. In contrast, a positive thiobarbituric acid reaction was obtained with many lipid oxidation products without metals or ascorbic acid. Monohydroperoxides from autoxidized methyl linoleate and linolenate produced the highest level of fluorescence. Hydroperoxy epidioxides of linolenate and dihydroperoxides of linoleate and linolenate were among the most active secondary products in forming fluorescence with DNA. In contrast, malonaldehyde produced very little fluorescence under our conditions. The thiobarbituric acid values did not correlate with fluorescence formation. This study showed that, in our model reaction system, DNA forms fluorescent products by the breakdown of lipid oxidation products in the presence of metals and ascorbic acid into reactive materials other than malonaldehyde. Therefore, the importance of malonaldehyde in its crosslinking properties with DNA may have been exaggerated in the literature.     

Kinetics of lipid peroxidation in isolated surviving rat livers

The kinetics of accumulation of lipid peroxidation products (hydroperoxides as primary products and malonic dialdehyde and "fluorescent pigments" as secondary ones) was investigated in an isolated non-perfused and preliminarily perfused liver during aerobic incubation. In the course of surviving there takes place an intensive accumulation of primary, secondary and final products of lipid peroxidation whose kinetics is of an extreme character. The rate of this process in a non-perfused liver is considerably higher than in a preliminarily perfused liver.     

Iron-induced lipid peroxidation and protein modification in endoplasmic reticulum membranes. Protection by stobadine

Treatment with FeSO(4)/EDTA (0.2 micromol Fe(II) per mg of protein) was used to study the effect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence in a time-dependent manner. In contrast, fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO(4)/EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in fluorescence excitation (350-360 nm) and emission (440-450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic fluorescence of aromatic amino acids, suggesting modification or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS fluorescence and production of peroxidized lipid-protein conjugates whereas tryptophan fluorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent-modification of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modification.     

Activation of lipid peroxidation processes in chronic ischemic heart disease

The content of lipid peroxidation products--hydroperoxides with conjugated double bonds and fluorescent compounds, which are formed on interaction of primary lipid peroxidation products and proteins, considerably increases in blood plasma of patients suffering from coronary heart disease. Treatment with combined vitamins E and C enables the blood plasma lipid peroxidation products to be decreased to a far greater extent as compared with conventional therapy.     

Deliberately added and "cryptic" antioxidants in three artificial diets for insects

Characteristics of both deliberately added and "cryptic" antioxidants were assayed from hydrophilic and lipophilic extracts from artificial diets for plant bugs, lepidopteran larvae, and green lacewings. Cryptic antioxidants are defined as substances naturally existing in diet ingredients but not deliberately added because of their antioxidant potential. Diets were tested after 1) being freshly produced, 2) stored for 48 h at 4 degrees C, or 3) held for 48 h under rearing room conditions at 27 degrees C. Tests included 1) a general assay of antioxidant capacity known as the ferric-reducing antioxidant power (FRAP) assay. 2) a cation radical-scavenging assay, 3) an ascorbic acid assay, and 4) an assay of inhibition of lipid peroxidation. In all assays, the lepidopteran diet had the highest values for protection against reactive oxygen species (ROS). The lepidopteran diet (with 0.17-0.23-mg equivalents of gallic acid equals total phenolic compounds per gram of diet) had three- to four-fold higher concentrations of phenolic compounds than did either the plant bug diet or the lacewing diet. Unexpectedly, the plant bug and the lacewing diets caused more lipid peroxidation than did the positive controls. This was attributed to the high concentrations of iron in these diets (mainly from chicken eggs), causing an ascorbate-ferric ion-induced lipid peroxidation. Diet storage, measured after 2 d at 27 or 4-6 degrees C, caused no significant declines in overall antioxidant potential. However, storage did lead to decline in ascorbic acid. The FRAP assay offered the best potential as a general, routine test of the potential of various insect diets to resist the destructive effects of ROS. The importance of addressing issues of protection against ROS in insect diets is discussed.     

Comparison of fluorescence characteristics of products of peroxidation of membrane phospholipids with those of products derived from reaction of malonaldehyde with glycine as a model of lipofuscin fluorescent substances

The fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to product (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorescent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.