Gerhard Spiteller

Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme

The structures of the fumonisins, a family of structurally related mycotoxins isolated from cultures of Fusarium moniliforme, were elucidated by mass spectrometry and 1H and 13C n.m.r. spectroscopy as the diester of propane-1,2,3-tricarboxylic acid and either 2-acetylamino- or 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane as well as in each case the C-10 deoxy analogue; in all cases both the C-14 and C-15 hydroxy groups are involved in ester formation with the terminal carboxy group of propane-1,2,3-tricarboxylic acid.

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.

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.

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.

The common occurrence of furan fatty acids in plants

The observation that F-acids (1) occur in rat chow initiated a search for F-acids in human diet. We observed that the amount of F-acids with a pentyl side chain in α-position taken up with a one-day diet correlates well with the amount of excreted degradation products, the pentyl urofuran acids (2), (3) and (4). Therefore it can be concluded that F-acids with a pentyl side chain are not produced in the human body but are introduced through the diet. The origin of F-acids carrying an α-propyl side chain is less clear. The amount of propyl-urofuran acids (2) and (3) excreted in urine was found in one case out of three to be five times higher than the amount of F-acids carrying a propyl group in α-position taken up by the diet. Therefore, it can presently not be excluded that a portion of the propyl F-acids is produced by the body.F-acids found in human food are mainly introduced into the body by vegetables and fruits. F-acids were found also in birch leaves in considerable amounts, as well as in grasses, dandelion and clover leaves. Thus, we can conclude that F-acids are common constituents of plants.

Furan fatty acids: occurrence, synthesis, and reactions. Are furan fatty acids responsible for the cardioprotective effects of a fish diet?

Furan FA (F-acids) are tri-or tetrasubstituted furan derivatives characterized by either a propyl or pentyl side chain in one of the α-positions; the other is substituted by a straight long-chain saturated acid with a carboxylic group at its end. F-acids are generated in large amounts in algae, but they are also produced by plants and microorganisms. Fish and other marine organisms as well as mammals consume F-acids in their food and incorporate them into phospholipids and cholesterol esters. F-acids are catabolized to dibasic urofuran acids, which are excreted in the urine. The biogenetic precursor of the most abundant F-acid, F6, is linoleic acid. Methyl groups in the β-position are derived from adenosylmethionine. Owing to the different alkyl substituents, synthesis of F-acids requires multistep reactions. F-acids react readily with peroxyl radicals to generate dioxoenes. The radical-scavenging ability of F-acids may contribute to the protective properties of fish and fish oil diets against mortality from heart disease.

Lipid oxidation products in ischemic porcine heart tissue

Infarcted porcine heart tissue and surrounding tissue were investigated for the content of plasmalogens and oxidatively derived corresponding alpha-hydroxyaldehydes as well as for products of lipid peroxidation, e.g. malondialdehyde, glyoxal, 2-hydroxyheptanal and oxygenated fatty acids. Oxidation products of unsaturated fatty acids and plasmalogens were accumulated in infarcted tissue compared to the surrounding one. Their amounts increased with time of ischemia. In addition leukotoxins (9, 10-epoxy-12-octadecenoic acid and 12,13-epoxy-9-octadecenoic acid) as well as other epoxides of unsaturated fatty acids were identified. These compounds are absent in healthy heart tissue. Some of the monohydroxy fatty acids, found in comparable high yield, can not be derived from LPO processes. They are obviously generated from epoxides. Their distribution pattern indicates that they originate by an enzymic rather than by an autocatalytic process. We assume that the enzymes are activated by cell injury due to infarction. Linoleic acid seems to be an as equally well-suited substrate for enzymic attack as arachidonic acid.

Increased levels of lipid oxidation products in low density lipoproteins of patients suffering from rheumatoid arthritis

9-Hydroxy-10,12-octadecadienoic acid (9-HODE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODE) are accumulated in the low density lipoproteins of patients suffering from rheumatoid arthritis for a factor of 20-50 compared to healthy individuals of the same age. Both acids, derived by lipid peroxidation of linoleic acid, induce the release of interleukin 1 beta. The latter induces bone degression. The genesis of 9- and 13-HODE seems therefore to be an important factor in the development and progression of rheuma; in addition 9-HODE was reported to be a stimulus of inflammation, comparable to leukotrienes.

Investigation of aldehydic lipid peroxidation products by gas chromatography–mass spectrometry

Abstract Lipid peroxidation (LPO) of polyunsaturated fatty acids is induced in injured or dying cells. The generated lipid hydroperoxides readily decompose to a great variety of aldehydic compounds, which are consequently useful markers for LPO processes in biological materials. Since aldehydes are produced in tiny amounts only, their detection requires efficient separation methods combined with unambigious and specific identification techniques – e.g. separation by gas chromatography followed by identification with electron impact mass spectrometry. A great number of LPO aldehydes contains polar groups, especially carboxylic or hydroxy functions. These groups may cause decomposition when a sample is heated up in the hot injector of a gas chromatograph. Therefore such aldehydes must be protected before analysis. Derivatization enhances also volatility. Advantages and disadvantages of different methods to prepare suitable derivatives of LPO aldehydes for GC separation are discussed. The mass spectra of different derivatives are compared in order to demonstrate which ones are most useful to gain maximum information on the structure of the original aldehydes.

Generation of α-hydroxyaldehydic compounds in the course of lipid peroxidation

Based on 18O-labeling experiments a general scheme for the generation of hydroxy aldehydic compounds in the course of lipid peroxidation of linoleic acid is developed. Key intermediates are obviously dioxygenated fatty acids, since after reduction with either NaBH4 or Rh/H2 1,2 and 1,6 dihydroxy fatty acids can be identified. The postulated mechanism not only explains the formation of 2-hydroxyalkanals but also supports earlier hypothesis concerning the generation of 4-hydroxyalkenals. In addition it predicted the occurrence of (n − 1)-hydroxy-n-oxo fatty acids as additional oxidation products. A search for these previously unknown autoxidation products of linoleic acid was indeed successful.