Do changes in the cell membrane structure induce the generation of lipid peroxidation products which serve as first signalling molecules in cell to cell communication?

Evidence is presented that mammalian and plant cells respond equally to any event which changes their cell membrane structure. Proliferation, wounding or aging induces generation of lipidhydroperoxides from cell wall phospholipids. These are transformed to signalling compounds, some of these induce apoptosis. If the exerted impact exceeds a certain level, the original enzymic reaction switches to a non-enzymic one which produces peroxylradicals. The latter are not liberated enzymically. Peroxylradicals generate a second set of signalling compounds, but cause also severe damage: they epoxidize double bonds, and oxidize proteins, sugars and nucleic acids. Such reactions occur in all inflammatory diseases. Lipidhydoperoxides and their degradation products are incorporated in fat. Apparently, these compounds are transferred partly to LDL. Such LDL is still recognized by the cell LDL receptor. Toxic lipid peroxidation products are therefore introduced into cells and might be able to damage cells from inside long before the typical signs of atherosclerosis and other chronic diseases become visible.

Oxidized products of linoleic acid stimulate adrenal steroidogenesis.

Adrenal steroidogenesis is under complex control, and clinical observations suggest that not all regulators have been identified. We postulated that fatty acid oxidation products found in the diet or formed in the body could affect steroidogenesis. Linoleic acid is a prominent constituent of animal fat and is readily oxidized. We found that several products of linoleic acid oxidation affect production of aldosterone and corticosterone by isolated cells from rat adrenals. We characterized one linoleic acid derivative by gas chromatography/mass spectrometry. It is 12,13-epoxy-9-oxo-10(trans)-octadecenoic acid ("EKODE"). At concentrations between 1 and 30 microM, EKODE stimulated production of aldosterone by zona glomerulosa cells, but at concentrations above 50 microM, it was inhibitory. In zona fasciculata cells, EKODE stimulated corticosterone production at concentrations of 5 microM or greater, and there was no evidence of inhibition at high concentrations. Stimulation of steroidogenesis was observed after 15 min of incubation and continued for at least 2 hrs. The potential relevance of our findings to the hypertension of obesity is discussed.

Lipid peroxidation in aging and age-dependent diseases.

Aging is related with an increase in oxidation products derived from nucleic acids, sugars, sterols and lipids. Evidence will be presented that these different oxidation products are generated by processes induced by changes in the cell membrane structure (CMS), and not by superoxide, as commonly assumed. CMS activate apparently membrane bound phospholipases A2 in mammals and plants. Such changes occur by proliferation, aging and especially by wounding. After activation of phospholipases, influx of Ca2+ ions and activation of lipoxygenases (LOX) is induced. The LOX transform polyunsaturated fatty acids (PUFAs) into lipid hydroperoxides (LOOHs), which seem to be decomposed by action of enzymes to signalling compounds. Following severe cell injury, LOX commit suicide. Their suicide liberates iron ions that induce nonenzymic lipid peroxidation (LPO) processes by generation of radicals. Radicals attack all compounds with the structural element -CH=CH-CH(2)-CH=CH-. Thus, they act on all PUFAs independently either in free or conjugated form. The most abundant LPO products are derived from linoleic acid. Radicals induce generation of peroxyl radicals, which oxidise a great variety of biological compounds including proteins and nucleic acids. Nonenzymic LPO processes are induced artificially by the treatment of pure PUFAs with bivalent metal ions. The products are separable after appropriate derivatisation by gas chromatography (GC). They are identified by electron impact mass spectrometry (EI/MS). The complete spectrum of LPO products obtained by artificial LPO of linoleic acid is detectable after wounding of tissue, in aged individuals and in patients suffering from age-dependent diseases. Genesis of different LPO products derived from linoleic acid will be discussed in detail. Some of the LPO products are of high chemical reactivity and therefore escape detection in biological surrounding. For instance, epoxides and highly unsaturated aldehydic compounds that apparently induce apoptosis.

Peroxidation of linoleic acid and its relation to aging and age dependent diseases.

Cell proliferation, cell injury and aging are connected with changes in the cell membrane structure. Apparently these changes activate, in mammalian as well as in plant cells, lipases which liberate polyunsaturated fatty acids (PUFAs). PUFAs are the substrates for lipoxygenases which convert them to corresponding hydroperoxides (LOOHs). Lipoxygenases commit suicide by releasing iron ions. LOOHs react with iron ions to generate radicals. Thus, a nonenzymic lipid peroxidation process (LPO) is induced. It is speculated that the change from enzymic to nonenzymic LPO is connected with the switch from apoptosis to necrosis and that LOOHs produced in enzymic reactions are degraded specifically to signal compounds which induce physiological responses, while nonenzymic reactions seem to induce generation of reactive oxygen species, cell death and age related diseases. Enzymic and nonenzymic LPO processes concern all PUFAs not only arachidonic acid. The main PUFA in mammals is linoleic acid. Since these products serve signalling functions, different degradation paths of linoleic-hydroperoxides are described in detail and the physiological properties of LPO products are discussed in relation to aging and age related diseases.

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.

Ceramide-epoxides.

Previously unknown 4,5-epoxy-N-acetyl-sphingosine (1) was synthesized by epoxidation of N-acetyl-sphingosine with 1,1-dimethyldioxirane. A by-product generated by HPLC purification is the tetrahydrofuryl derivative of acetamide (2). Mainly allylic oxidation was observed when natural ceramides were reacted with dimethyldioxirane.

Synthesis of 9,12-dioxo-10(Z)-dodecenoic acid, a new fatty acid metabolite derived from 9-hydroperoxy-10,12-octadecadienoic acid in lentil seed (Lens culinaris Medik.)

The previously unknown linoleic acid peroxidation product 9,12-dioxo-10(Z)-decenoic acid (Z5) was detected in lentil seed flour (Lens culinaris Medik.) by electron impact mass spectrometry (EI-MS) after derivatization with pentafluorobenzyl-hydroxylamine-hydrochloride, methylation of acidic groups with diazomethane, and protection of hydroxylic groups with N-methyl-N-trimethylsilyl-trifluoroacetamide. The structure of the natural product was confirmed by synthesis of Z5, 9,12-dioxo-l0(E)-decenoic acid, and derivatives. EI-MS, nuclear magnetic resonance and gas chromatographic data of these compounds and synthetic intermediates are discussed.

Oxidation of plasmalogens produces highly effective modulators of macrophage function.

Model derivatives of plasmalogens and chemically synthesized oxidative degradation products as found e.g. during oxidation of low density lipoproteins show strong effects on phagocytosis induced secretion of reactive oxygen species of macrophages which was measured by luminol-enhanced chemiluminescence. Whereas a plasmalogen epoxide showed enhancing effects in submicromolar range, inhibition was found with higher concentrations as well as with alpha-hydroxyaldehydes. The substances showed only little effects on the non-cellular ROS-dependent chemiluminescence of the reaction between hydrogen peroxide and opsonized zymosan and no cytotoxic effects under the assay conditions used. These results show that oxidative modification and degradation of plasmalogens occuring also under pathophysiological situations in vivo produces effective modulators of macrophage function which could be important; e.g. during inflammation or atherogenesis.

Identification of Toxic 2,4-Decadienal in Oxidized, Low-Density Lipoprotein by Solid-Phase Microextraction.

The oxidation of low-density lipoprotein (LDL) in vitro was studied by a combination of solid-phase microextraction and GC/MS. 2-trans,4-cis-2,4-Decadienal, which is strongly toxic in vitro, was detected as the early oxidation product. This compound is degraded further to hexanal and (by cyclization of 4-hydroxy-2-nonenal) to 2-pentylfuran.

Oxidation of Linoleic Acid in Low-Density Lipoprotein: An Important Event in Atherogenesis.

Contrary to earlier views the main oxidation products of low-density lipoprotein (LDL) are derived from linoleic acid and not arachidonic acid, as determined by GC/MS investigations of the in vitro oxidation of LDL samples. A similar product spectrum, in which epoxyhydroxyoctadecenoic acids such as 1 and 2 have been identified for the first time, is obtained from minimally oxidized (that is, aged) LDL. Since this is still recognized by the LDL receptor, it is concluded that toxic oxidation products are introduced in endothelial cells in vivo and cause damage there.

10-Hydroxystearic acid--identified after homogenization of tissue--is derived from bacteria.

10-Hydroxystearic acid seems to be widely distributed in nature: Bacteria generate it by hydroxylation of oleic acid, but it was found also as constituent of plants, in cancer cell cultures and in mammalian tissue homogenates. Investigation of 10-hydroxystearic acid, obtained from mammalian tissue homogenates, revealed its identity with that of bacteria. Thus not 10-hydroxystearic acid is widely distributed in nature but its producers: bacteria. When biological material is processed in aqueous media, lipases are activated, these cleave membrane phospholipids. Thus liberated oleic acid is the substrate for widespread bacteria which are introduced into the media when the work up procedure is done in not sterile surrounding. The bacteria transform then oleic acid to 10R-hydroxystearic acid.

Transformations of 12,13-epoxy-11-hydroxy-9-octadecenoic acid and 4,5-epoxy-N-acetylsphingosine by incubation with liver homogenate and liver microsomes.

Transformation of 12,13-epoxy-11-hydroxy-9-octadecenoic acid and 4,5-epoxy-N-acetylsphingosine by addition of porcine liver homogenate and human liver microsomes, respectively was investigated. Both epoxides were converted to corresponding dioles by porcine liver homogenate, but not by human liver microsomes, suggesting location of the hydrolyzing enzymes not in the microsomes, but within the cell wall.

The reaction of hyaluronic acid and its monomers, glucuronic acid and N-acetylglucosamine, with reactive oxygen species.

Synovial fluid is a approximately 0.15% (w/v) aqueous solution of hyaluronic acid (HA), a polysaccharide consisting of alternating units of GlcA and GlcNAc. In synovial fluid of patients suffering from rheumatoid arthritis, HA is thought to be degraded either by radicals generated by Fenton chemistry (Fe2+/H2O2) or by NaOCl generated by myeloperoxidase. We investigated the course of model reactions of these two reactants in physiological buffer with HA, and with the corresponding monomers GlcA and GlcNAc. meso-Tartaric acid, arabinuronic acid, arabinaric acid and glucaric acid were identified by GC-MS as oxidation products of glucuronic acid. When GlcNAc was oxidised, erythronic acid, arabinonic acid, 2-acetamido-2-deoxy-gluconic acid, glyceric acid, erythrose and arabinose were formed. NaOCl oxidation of HA yielded meso-tartaric acid; in addition, arabinaric acid and glucaric acid were obtained by oxidation with Fe2+/H2O2. These results indicate that oxidative degradation of HA proceeds primarily at glucuronic acid residues. meso-Tartaric acid may be a useful biomarker of hyaluronate oxidation since it is produced by both NaOCl and Fenton chemistry.

Linoleic acid peroxidation--the dominant lipid peroxidation process in low density lipoprotein--and its relationship to chronic diseases.

Modern separation and identification methods enable detailed insight in lipid peroxidation (LPO) processes. The following deductions can be made: (1) Cell injury activates enzymes: lipoxygenases generate lipid hydroperoxides (LOOHs), proteases liberate Fe ions--these two processes are prerequisites to produce radicals. (2) Radicals attack any activated CH2-group of polyunsaturated fatty acids (PUFAs) with about a similar probability. Since linoleic acid (LA) is the most abundant PUFA in mammals, its LPO products dominate. (3) LOOHs are easily reduced in biological surroundings to corresponding hydroxy acids (LOHs). LOHs derived from LA, hydroxyoctadecadienoic acids (HODEs), surmount other markers of LPO. HODEs are of high physiological relevance. (4) In some diseases characterized by inflammation or cell injury HODEs are present in low density lipoproteins (LDL) at 10-100 higher concentration, compared to LDL from healthy individuals.

Strong increase of 9-hydroxy-10,12-octadecadienoic acid in low density lipoprotein after a hemorrhagic shock.

9-hydroxy-10,12-octadecadienoic acid (9-HODE) is generated by lipid peroxidation (LPO) processes in comparison to other marker compounds in at least 10 fold amount. A 10-25 fold increase of this new marker compound in relation to age matched healthy individuals was observed in the low density lipoprotein (LDL) fraction of ten patients suffering from a hemorrhagic shock. The 9-HODE values dropped to normal levels after recovery. Similarly the 9-HODE content in LDL of patients which had to undergo orthopedic surgery--replacement of their arthritic hip joints by endoprosthesis--were investigated. The rather high HODE values dropped also after recovery reflecting obviously the disappearance of inflammatory processes associated with arthritis.

The course of enzymatically induced lipid peroxidation in homogenized porcine kidney tissue.

Homogenization of mammalian tissue--exemplified by porcine kidney-- causes enzymatically induced lipid peroxidation (LPO) processes proven by measuring the amounts of the typical lipid peroxidation products 9- and 13-hydroxy-octadecadienoic acid (HODE) either after homogenization in aqueous (activation of enzymes) or an organic (inactivated enzymes) solvent. A kinetic study revealed that the level of the 9- and 13-isomer reached maximum values 6 hours after tissue injury. Within one day the amount of these primary oxidation products was reduced fast, indicating that they undergo degradation in their biological environment. In contrast, the level of 10-hydroxy-octadecanoic acid--obviously derived from LPO of oleic acid--increased continuously even after one day. These observations reflect that the generation and degradation of hydroperoxides occurs at different rates which might be of interest in pathological processes connected with tissue injury, e.g. myocardial infarction.

Strong dependence of the lipid peroxidation product spectrum whether Fe2+/O2 or Fe3+/O2 is used as oxidant.

Catalytic amounts of Fe2+ or Fe3+ ions are widely applied to induce simulated biological lipid peroxidation reactions. Independently, whether Fe2+ or Fe3+ were used, similar products were obtained. We show in this paper that the product spectrum is indeed very different, whether one ion species, either Fe2+ or Fe3+, is present in excess; thus, decomposition of (13S,9Z,11E) 13-hydroxyperoxy-9, 11-octadecadienoic acid (13S-HPODE) generates in the presence of equimolar amounts of Fe2+ ions mainly the corresponding alcohol (13S, 9Z,11E) 13-hydroxy-9,11-octadecadienoic acid besides 12,13-epoxy-11-hydroxy-9-octadecenoic acid (12,13-epHOD) and 13-oxo-9,11-octa-decadienoic acid (13-KODE), while decomposition of 13S-HPODE with equimolar amounts of Fe3+ produces mainly 12,13-epHOD, hydrolysis products thereof and other oxidized products, e.g., hydroxyoxo acids. In addition, unusually large amounts of aldehydes are formed, e.g., the amount of 4-hydroxy-nonenal was found to exceed that obtained by Fe2+ induced air oxidation for a factor of about 100. Since these further oxidation products are suspected to cause cell damage, liberated Fe3+ ions seem to be responsible for generation of toxic products in inflammatory diseases, e.g., atherosclerosis.

Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients.

Linoleic acid is the most abundant fatty acid in human low density lipoproteins (LDL). Oxidation of LDL transforms linoleic acid to hydroperoxyderivatives. These are converted to 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODE). 9-HODE is much more abundant in oxidized LDL than other lipid peroxidation products and therefore an indicator of lipid peroxidation (LPO). In this study the 9-HODE content in the LDL of 19 obviously healthy volunteers and 17 atherosclerotic patients was investigated. The level of 9-HODE obtained from LDL of young atherosclerotic patients (aged 36-47 years) was increased by a factor of 20 when compared with samples from healthy volunteers of the same age group. The content of 9-HODE in the LDL of atherosclerotic patients aged between 69 and 94 years increased 30-100 fold when compared with young healthy individuals, but when compared with 'healthy' individuals of the same age group it was only 2-3 fold increased. Obviously, as individuals grow older LDL becomes more and more oxidized. Consequently, assuming that LDL oxidation is a precondition for atherosclerosis--older individuals will suffer from atherosclerosis, even if no easy detectable visible signs of this disease are recognizable. According to 9-HODE determination, the onset of the disease starts slowly in most individuals at around 50 years of age.

Lignans interfering with 5 alpha-dihydrotestosterone binding to human sex hormone-binding globulin.

The natural lignans (-)-3,4-divanillyltetrahydrofuran (1), (-)-matairesinol (2), (-)-secoisolariciresinol (3), (+/-)-enterolactone (4), (+/-)-enterodiol (5), and nordihydroguaiaretic acid (NDGA) (6) reduce the binding of 3H-labeled 5 alpha-dihydrotestosterone (DHT) to human sex hormone-binding globulin (SHBG). (-)-3,4-Divanillyltetrahydrofuran (1) has the highest binding affinity (Ka = 3.2 +/- 1.7 x 10(6)M-1) of all lignans investigated so far; the reversibility of its binding and a double reciprocal plot suggest a competitive inhibition of the SHBG-DHT interaction. Increasing hydrophobity in the aliphatic part of the lignans (butane-1,4-diol-butanolide-tetrahydrofuran structures) leads to higher binding affinity. In the aromatic part, a 3-methoxy-4-hydroxy substitution pattern is most effective for binding to SHBG.