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.     

Effect of docosahexaenoic acid intake on lipid peroxidation in diabetic rat retina under oxidative stress

Docosahexaenoic acid (DHA) plays an important role in visual function but has a highly oxidation-prone chemical structure. Therefore, we investigated how dietary DHA affects the generation of lipid peroxides in rat retina under oxidative stress in diabetes with/without vitamin E (VE) deficiency. Streptozotocin-induced (50 mg i.p./kg B.W.) diabetic Sprague-Dawley (SD) rats were assigned to four groups: (i) control/VE(+), (ii) DHA/VE(+), (iii) control/VE( - ) and (iv) DHA/VE( - ), and raised for 28 days. We then measured lipid peroxide levels in the retina, serum and liver. With a normal intake of VE, dietary DHA increased only the retinal level of thiobarbituric acid-reactive substances (TBARS) slightly. In contrast, in rats with VE deficiency, dietary DHA increased serum and liver lipid peroxide levels but not in the retina. These results suggest that dietary DHA does not necessarily promote lipid peroxidation in the retina even under high oxidative stress.     

Inhibition of lipid peroxidation by heme-nonapeptide derived from cytochrome c

Heme-nonapeptide, derived from cytochrome c, inhibited both the NADPH- and NADH-dependent lipid peroxidation of brain microsomes but, in the case of liver microsomes, this inhibitory effect manifested itself in the presence of SKF-525A (a specific blocker of cytochrome P-450) only. Heme-nonapeptide prevented the transient accumulation of lipid peroxides in microsomes during lipid peroxidation. The oxygen consumption of microsomes in the presence of NADPH or NADH was stimulated by heme-nonapeptide. From these results we concluded that, in vitro, there are two independent mechanisms of lipid peroxidation in liver microsomes. It is suggested that, in vivo, the heme-peptide-sensitive mechanism, observed in brain microsomes, is more important.     

Binding of the lipid peroxidation product 4-hydroxynonenal to human polymorphonuclear leukocytes

4-Hydroxynonenal (HNE) is produced during peroxidation of polyunsaturated fatty acids. It exerts a chemokinetic effect on human polymorphonuclear leukocytes (PMN). Investigations of this mechanism were performed. The results indicate that [³H]-HNE binding to PMN results both in non-specific bonds to the numerous SH groups of the cells and in binding to a saturable, reversible and specific HNE site. Scatchard analysis revealed that this is a single site with an apparent affinity constant of 319 nM and a density of 1·57 pmol (10⁶)^?1 cells. This specific binding site may be involved in the chemokinetic effect of HNE.     

Chemotactic activity of the lipid peroxidation product 4-hydroxynonenal and homologous hydroxyalkenals

The effect of the lipid peroxidation product 4-hydroxynonenal and homologous aldehydes (4-hydroxyoctenal, 4-hydroxyundecenal, 4-hydroxytetradecenal and 4-hydroxypentadecenal) on migration and polarization of rat neutrophils was examined. The most effective aldehydes were 4-hydroxyoctenal and 4-hydroxypentadecenal, which stimulated oriented migration at ED50 = 1.4 X 10(-12) M and 1.3 X 10(-12) M, resp., whereas the other aldehydes had ED50 between 1 X 10(-7) and 6 X 10(-11) M. The peptides fMet-Phe and fMet-Leu-Phe used as positive controls had ED50 values of 4.2 X 10(-7) M and 4.5 X 10(-10) M resp. The 4-hydroxyalkenals induced only a small increase of the percentage of polarized cell and did not enhance the random migration. The effects of 4-hydroxyalkenals were only observed when the incubation buffer contained bovine serum albumin (BSA), in the absence of BSA neither the aldehydes nor the peptides exhibited chemotactic properties. Since the aldehydes easily react with the sulfhydryl groups of the BSA to form the S-alkylated BSA in an equilibrium reaction, the chemotactic substance could either be the free aldehyde or the BSA-aldehyde adduct. The adduct prepared from BSA and 4-hydroxynonenal was chemotactic at doses of 0.65 to 0.0065 mg/ml, when tested in the presence of unmodified BSA. Since the adduct released free 4-hydroxyalkenal during the assay in the reverse reaction, it can not be decided whether the active principle is the aldehyde itself or the aldehyde attached to the BSA. From the effective doses of the aldehydes (10(-7) to 10(-12)M) and the BSA-aldehyde adduct it appears very unlikely that the BSA itself gained chemotactic properties through the alkylation of its sulfhydryl groups by the aldehyde.     

Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal

The effects of the lipid peroxidation product 4-hydroxynonenal on freshly prepared human low-density lipoprotein (LDL) were studied. At a fixed LDL concentration (5.7 mg/ml) the amount of 4-hydroxynonenal incorporated into the LDL increased with increasing aldehyde concentration from 28-30 (0.2 mM) to 140 (1 mM) mol per mol LDL, whereas at a fixed aldehyde concentration (0.2 mM) its incorporation into LDL decreased with increasing LDL concentration from 48 (1 mg LDL/ml) to 26 (12 mg LDL/ml) mol 4-hydroxynonenal bound per mol LDL. Of the total hydroxynonenal taken up 78% was bound to the protein and 21% to the lipid moiety; the remaining 1% was dissolved as free aldehyde in the lipid fraction. Amino acid analysis of the apolipoprotein B revealed that 4-hydroxynonenal attacks mainly the lysine and tyrosine residues and to a lesser extent also serine, histidine and cysteine. Treatment of LDL with 4-hydroxynonenal results in a concentration-dependent increase of the negative charge of the LDL particle as evidenced by its increased electrophoretic mobility. Moreover, 4-hydroxynonenal treatment leads to a partial conversion of the apolipoprotein B-100 into higher molecular weight forms most probably apolipoproteins B-126 and B-151. Compared to malonaldehyde, 4-hydroxynonenal exhibits a much higher capacity to modify LDL and it is therefore believed that this aldehyde is a more likely candidate for being responsible for LDL modification under in vivo lipid peroxidation conditions.     

The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response.

An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system. We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002. SOS-inducing potencies varied widely among the carbonyl compounds tested. 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM. Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression. Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM. Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity. Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency. Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.     

Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid

Isoeicosanoids are free radical-catalyzed isomers of the enzymatic products of arachidonic acid. They are formed in situ in cell membranes, are cleaved, circulate, and are excreted in urine. Isomers of prostaglandin F(2alpha), the F(2)-isoprostanes, have emerged as sensitive indices of lipid peroxidation in vivo. Analogous compounds formed from docosahexaenoic acid (DHA) are termed neuroprostanes and are more abundant than isoprostanes (iPs) in brain. Isofurans are another class of isoeicosanoids characterized by a substituted tetrahydrofuran ring. They are preferentially formed, relative to iPs, under conditions of elevated oxygen tension. Here, we report the discovery of neurofurans (nFs), the analogous family of compounds formed from DHA. Formation of nFs is characterized by mass spectrometry and confirmed by oxidation of DHA in vitro and following CCl(4) administration in liver in vivo. It is demonstrated that the levels of nFs are elevated in the brain cortex of a mouse model of Alzheimer disease and are depressed in mouse brain cortex by deletion of p47(phox), an essential component of the phagocyte NADPH oxidase. Measurement of the nFs may ultimately prove useful in diagnosis, timing, and selection of dose in the treatment and chemoprevention of neurodegenerative disease.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

Detection of 4-hydroxynonenal as a product of lipid peroxidation in native Ehrlich ascites tumor cells

4-Hydroxynonenal, which is a major product of lipid peroxidation in rat liver microsomes, was detected in native Ehrlich ascites tumor cells. Its formation was stimulated either by ferrous ions or by Fe(II)-histidinate. The identification was based on chromatographic (TLC/HPLC) and ultraviolet-spectroscopic evidence using synthetic 4-hydroxynonenal as reference. Highest values of 4-hydroxynonenal concentration (about 0.1 microM in the cell suspension) after 30 min of incubation were observed with Fe(II)-histidinate as stimulant. Saturation was already reached after an incubation period of 10 min. The results confirm the expectation by Schauenstein and Esterbauer (in Submolecular Biology and Cancer, Ciba Foundation Series 67 (1979) pp. 225-244, Excerpta Medica, Amsterdam) that endogenous lipid peroxidation gives rise to a distinct intracellular level of alpha, beta-unsaturated aldehydes. A simple hypothetical mechanism for the formation of 4-hydroxynonenal from n-6-polyunsaturated fatty acids is presented.     

Effect of 4-hydroxynonenal,a lipid peroxidation product,on exocytosis in HL-60 cells

Our work analysed the effect of 4-hydroxynonenal (HNE), a chemotactic aldehydic end-product of lipid peroxidation, on exocytosis in HL-60 cells. We measured the release of beta-glucuronidase, an enzyme of azurophil granules, from the cells incubated at 37 degrees C for 10 min in the presence of HNE concentrations ranging between 10(-8) and 10(-5) M. The release of lactate dehydrogenase was assayed to test cell viability. HNE (1 microM) was able to induce a significant and strong stimulation of beta-glucuronidase secretion without leading to cytotoxic effects. The finding that HNE could increase the exocytotic secretion from HL-60 cells together with its known chemotactic property supports the hypothesis that this lipid peroxidation product may play an important role as a chemical mediator of inflammation; moreover it is noteworthy that micromolar concentrations of HNE have actually been found in exudates from acute and chronic inflammations.     

Conjugated docosahexaenoic acid inhibits lipid accumulation in rats

Conjugated linoleic acid (CLA), which contains a conjugated double-bond system, and n-3 highly unsaturated fatty acids such as docosahexaenoic acid (DHA) are widely known to improve lipid metabolism. To examine the possibility that a fatty acid with a combination of these structural features might have stronger physiological effects, we prepared conjugated DHA (CDHA) by alkaline isomerization of DHA and examined its effects on lipid and sugar metabolism in rats. Rats were force fed with 200 mg of test oils [linoleic acid (LA), DHA, CLA or CDHA] everyday for 4 weeks. Compared with the animals from the other groups, those in the CDHA group showed a significant weight loss in white adipose tissue (57% of adipose tissue weight in the LA group) and significant decreases in the levels of liver triacylglycerol (TG; 65% of TG level in the LA group) as well as total cholesterol (TC; 88% of TC level in the LA group), indicating suppression of lipid accumulation in the liver and adipose tissue. In addition, plasma TG and TC levels significantly decreased (69% of TG level and 82% of TC level in the LA group), indicating improved lipid metabolism. In the liver, the fatty acid synthesis system was inhibited and the fatty acid beta-oxidation system was activated, whereas the free fatty acid, glucose and tumor necrosis factor alpha levels in the plasma were lowered following CDHA administration. Hence, intake of CDHA appears to suppress the accumulation of fat in the liver and epididymal adipose tissue and improves lipid and sugar metabolism in rats.     

Docosahexaenoic acid-containing phosphatidylethanolamine in the external layer of liposomes protects docosahexaenoic acid from 2,2'-azobis(2-aminopropane)dihydrochloride-mediated lipid peroxidation

We have proposed that incorporation of docosahexaenoic acid (DHA) into phosphatidylethanolamine (PE) might enhance resistance to lipid peroxidation in vivo. In this study, we examined the relationship between the transbilayer distribution of PE and the oxidative stability of DHA in PE. Liposomes composed of a phospholipid mixture were used as models for biological membranes. To modulate the transbilayer distribution of PE obtained from the liver of rats fed DHA (PE-DHA), we used phosphatidylcholine (PC) with two types of acyl chain region: dipalmitoyl (PC16:0) or dioleoyl (PC18:1). The proportion of PE-DHA in the liposomal external layer was significantly higher in liposomes containing PC18:1 than in those containing PC16:0. This tendency was more pronounced in liposomes extruded using a polycarbonate filter with smaller pore sizes. Additionally, PE-DHA in the external layer of liposomes prepared using a filter with smaller pore sizes could protect DHA itself from 2,2(')-azobis(2-aminopropane)dihydrochloride-mediated lipid peroxidation.     

Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets

The stimulation by ADP or arachidonic acid of the aggregation of human platelets in plasma was inhibited by 4-hydroxynonenal (HNE). This reduction of aggregation was time related, and was increased by prolonged preincubation of the platelets with the aldehyde. HNE was more potent than its homologue 4-hydroxypentenal (HPE). HNE was less active in decreasing the aggregation induced by calcium ionophore A23187 or collagen in comparison with ADP. HNE was inactive against aggregation of platelet-rich plasma (PRP) stimulated by thrombin whereas it potently inhibited the aggregation of washed platelets in response to both thrombin and collagen. Platelets were found to degrade HNE, and mechanisms additional to covalent binding to glutathione are indicated by the results obtained. The aldehydes, including HNE, generated by platelets originated principally from arachidonic acid metabolism.     

Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation

4-Hydroxynonenal (HNE) is the major diffusible toxic product generated by lipid peroxidation of cellular membranes. The level of lipid peroxidation and, consequently, the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. In the present paper the effects of HNE on the proliferation and differentiation of the HL-60 human promyelocytic cell line have been investigated. Repeated treatment at 45-min intervals with HNE (1 microM) was performed to maintain the cells in the presence of the aldehyde for 7 1/2 or 9 h. The effect of HNE on cell proliferation and differentiation was compared with dimethyl sulfoxide (DMSO)-treated cells. HNE causes a strong inhibition of cell growth without affecting cell viability. Moreover, HL-60 cells acquire the capability to produce chemiluminescence after soluble (phorbol myristate acetate) or corpuscolate (zymosan) stimulation. The phagocytic ability has also been calculated by counting the number of cells that phagocytize opsonized zymosan. Values were 43 and 55% after 10 or 12 HNE treatments, respectively, and 88% in DMSO-treated cells. Myeloperoxidase activity, 5 days after treatment, decreased by 85% in either HNE- or DMSO-treated cells while acid phosphatase activity increased with respect to untreated cells. Results obtained indicate that HNE at concentrations close to those found in the normal tissues can induce inhibition of proliferation and induction of differentiation in the HL-60 cell line.     

Regiochemistry of neuroprostanes generated from the peroxidation of docosahexaenoic acid in vitro and in vivo

Isoprostanes (IsoPs) are isomers of prostaglandins that are generated from the free radical-initiated peroxidation of arachidonic acid (C20.4 omega-6). IsoPs exert potent bioactivity and are regarded as the "gold standard" to assess oxidative stress in various human diseases. Analogously, autoxidation of docosahexaenoic acid (DHA, C22.6 omega-3) generates an array of IsoP-like compounds that are termed neuroprostanes (NPs). A major class of NPs identified in vitro and in vivo contains F-type prostane rings and are know as F4-NPs. A number of different F4-NP regioisomers are formed from the peroxidation of DHA. Among the eight possible regioisomeric groups, we hypothesize that 4- and 20-series NPs are generated in greater amounts than other classes because the precursors that lead to regioisomers other than those of the 4- and 20-series can be further oxidized to form novel dioxolane-IsoP-like compounds, analogous to those generated from arachidonate. Various mass spectrometric approaches, including electron capture atmospheric pressure chemical ionization mass spectrometry, were utilized to analyze NPs formed in vitro and in vivo based on their characteristic fragmentation in the gas phase. Experimental results were consistent with our hypothesis that 4- and 20-series NP regioisomers are preferentially generated. The discovery of regioselectivity in the formation of NPs will allow studies of the biological activities of NPs to focus on the more abundantly generated compounds to determine their role in modulating the pathophysiological consequences of DHA oxidation and oxidant stress.     

Micellar electrokinetic chromatography separation and laser-induced fluorescence detection of the lipid peroxidation product 4-hydroxynonenal

4-Hydroxnonenal (HNE) is a product of lipid peroxidation in biological systems that causes a variety of harmful biological effects. A method for identifying HNE based on derivatization with the fluorescent reagent dansylhydrazine (5-(dimethylamino)naphthalene-1-sulphonehydrazine (DNSH) followed by micellar electrokinetic chromatography separation laser-induced fluorescence detection has been developed. The derivatization reaction has also been investigated for significant experimental parameters and rat brain homogenates with induced lipid peroxidation have been analysed for HNE contents. The limit of detection (3 S/N) was 30 nM or 0.3 fmol in the injected sample.     

Dihydroxynonene mercapturic acid, a urinary metabolite of 4-hydroxynonenal, as a biomarker of lipid peroxidation

The objective of our study was to compare the information obtained through the use of three different urinary biomarkers of lipoperoxidation during the time course of a bromotrichloromethane (BrCCl3) induced oxidative stress in rats. These biomarkers were malondialdehyde (MDA) measured by LC/MS after derivatization, the isoprostane 8-iso-PGF2alpha measured by enzyme immunoassay and 1,4-dihydroxynonene mercapturic acid (DHN-MA), the major 4-hydroxynonenal urinary metabolite [1], measured by LC-MS. Male Wistar rats received a single dose of 100 microL/kg BrCCl3 per os and lipid peroxidation was estimated every day for a 4-day-period after treatment. MDA, 8-iso-PGF2alpha and DHN-MA significantly increased in response to BrCCl3 treatment for this period of time, and DHN-MA showed the main increase during the 24-48 h period after treatment.