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.     

Oxidation and reduction of 4-hydroxyalkenals catalyzed by isozymes of human alcohol dehydrogenase

4-Hydroxyalkenals, natural cytotoxic products of lipid peroxidation, are substrates for human alcohol dehydrogenases (ADH). Class I and II ADHs reduce aliphatic 4-hydroxyalkenals with chain lengths of from 5 to 15 carbons at pH 7 with kcat and Km values comparable to simple aliphatic aldehydes of the same chain length. Class II is particularly effective in the reduction with kcat values as high as 3300 min-1 for 4-hydroxyundecenal. Class III ADH is essentially inactive toward all of these substrates. The class I and II isozymes also catalyze the oxidation of the 4-hydroxy group at pH 10. However, during the reaction, an NAD(+)-dependent irreversible partial inactivation of the alpha beta 1 isozyme is observed which is attributed, with the aid of computer graphics modeling, to selective modification of the alpha subunit. Both ethanol and 1,10-phenanthroline, known to compete with conventional substrates, instantaneously, reversibly, and competitively inhibit 4-hydroxyalkenal reduction and oxidation, indicating that 4-hydroxyalkenals bind at the same site as do conventional substates. The fact that the class II enzyme pi pi-ADH so far is found only in the liver and that the 4-hydroxyalkenals are the best substrates known for this isozyme suggest that it may play a significant role in cellular defenses in the conversion of the cytotoxic aldehydes to the less reactive alcohols.     

Lipid Peroxidation and Biogenic Aldehydes: From the Identification of 4-Hydroxynonenal to Further Achievements in Biopathology

The formation, reactivity and toxicity of aldehydes originating from lipid peroxidation of cellular membranes are reviewed. Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals are formed in biological conditions, i.e. during lipid peroxidation of liver microsomes incubated in the NADPH-Fe systems. Our studies carried out in collaboration with Hermann Esterbauer which led to the identification of 4-hydroxynonenal (4-HNE) are reported. 4-HNE was the most cytotoxic aldehyde and was then assumed as a model molecule of oxidative stress. Many other aldehydes (alkanals, alk-2-enals and dicarbonyl compounds) were then identified in peroxidizing liver microsomes or hepatocytes. The in vivo formation of aldehydes in liver of animals intoxicated with agents that promote lipid peroxidation was shown in further studies. In a first study, evidence was forwarded for aldehydes (very likely alkenals) bound to liver micro-somal proteins of CCl₄ or BrCCl₃-intoxicated rats. In a second study, 4-HNE and a number of other aldehydes (alkanals and alkenals) were identified in the free (non-protein bound) form in liver extracts from bromoben-zene or ally-1 alcohol-poisoned mice. The detection of free 4-HNE in the liver of CCl₄ or BrCCl₃-poisoned animals was obtained with the use of an electrochemical detector, which greatly increased the sensitivity of the HPLC method. Furthermore, membrane phospho-lipids bearing carbonyl groups were demonstrated in both in vitro (incubation of microsomes with NADPH-Fe) and in vivo (CCl₄ or BrCCl₃ intoxication) conditions. Finally, the results concerned with the histochemical detection of lipid peroxidation are reported. The methods used were based on the detection of lipid peroxidation-derived carbonyls. Very good results were obtained with the use of fluorescent reagents for carbonyls, in particular with 3-hydroxy-2-naphtoic acid hydrazide (NAH) and analysis with confocal scanning fluorescence microscopy with image video analysis. The significance of formation of toxic aldehydes in biological membranes is discussed.     

Aliphatic aldehydes inhibit the proliferative response of human peripheral blood lymphocytes to phytohemagglutinin and alloantigens

The effect of methyl glyoxal (MG) and various 4-hydroxyalkenals on the response of human peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) or allogeneic cells has been investigated. Pretreatment of PBL with aldehydes significantly reduced the percentage of blast-transformed cells and [3H]thymidine incorporation into DNA in both PHA- and alloantigen-stimulated cultures, hydroxyalkenals being more effective than MG. Further experiments showed that these aldehydes also affected the proliferation of pre-activated lymphocytes. The percentage of blasts as well as [3H]thymidine incorporation into DNA were significantly decreased when the aldehydes were added until 72 h after application of the mitogenic stimulus.     

Routes to 4-hydroxynonenal: fundamental issues in the mechanisms of lipid peroxidation

Although investigation of the toxicological and physiological actions of alpha/beta-unsaturated 4-hydroxyalkenals has made great progress over the last 2 decades, understanding of the chemical mechanism of formation of 4-hydroxynonenal and related aldehydes has advanced much less. The aim of this review is to discuss mechanistic evidence for these non-enzymatic routes, especially of the underappreciated intermolecular pathways that involve dimerized and oligomerized fatty acid derivatives as key intermediates. These cross-molecular reactions of fatty acid peroxyls have also important implications for understanding of the basic initiation and propagation steps during lipid peroxidation and the nature of the products that arise.     

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.     

Reactive aldehydes – second messengers of free radicals in diabetes mellitus

Abstract

Elevated levels of pro-oxidants and various markers of oxidative tissue damage were found in diabetic patients, indicating involvement of oxidative stress in the pathogenesis of diabetes mellitus (DM). On one side, physiological levels of reactive oxygen species (ROS) play an important role in redox signaling of various cells, while on the other, excessive ROS production can jeopardize the integrity and physiological functions of cellular macromolecules, in particular proteins, thus contributing to the pathogenesis of DM. Reactive aldehydes, especially 4-hydroxynonenal (HNE), are considered as second messengers of free radicals that act both as signaling molecules and as cytotoxic products of lipid peroxidation causing long-lasting biological consequences, in particular by covalent modification of macromolecules. Accordingly, the HNE and related reactive aldehydes may play important roles in the pathophysiology of DM, both in the development of the disease and in its progression and complications due to the following: (i) exposure of cells to supraphysiological levels of 4-hydroxyalkenals, (ii) persistent and sustained generation of 4-hydroxyalkenals that progressively affect vulnerable cells that lack an efficient bioactive aldehyde neutralization system, (iii) altered redox signaling influenced by reactive aldehydes, in particular by HNE, and (iv) induction of extracellular generation of similar aldehydes under secondary pathological conditions, such as low-grade inflammation.     

An efficient synthesis of γ-hydroxy-α,β-unsaturated aldehydic esters of 2-lysophosphatidylcholine

The diverse biological activities of ??-hydroxyalkenal phospholipids and their involvement in disease are the subject of intense study. Phospholipid aldehydes, such as the 4-hydroxy-7-oxohept-5-enoic acid ester of 2-lyso-phosphatidylcholine (HOHA-PC), the 5-hydroxy-8-oxo-6-octenoic acid ester of 2-lyso-PC (HOOA-PC), and the 9-hydroxy-12-oxododec-10-enoic acid ester of 2-lyso-PC (HODA-PC), are generated by oxidative cleavage of polyunsaturated fatty acyl phospholipids. To facilitate investigations of their chemistry and biology, we now report efficient total synthesis of HOOA, HODA, and HOHA phospholipids. Because the target ??-hydroxyalkenals readily decompose through oxidation of the aldehyde group to a carboxylic acid or through cyclization to furans, these synthesis generate the sensitive functional array of the target phospholipids under mild conditions from acetal derivatives that are suitable for long-term storage.     

Lipid peroxidation of poly-unsaturated fatty acids in normal and obese adipose tissues

Adipose tissues function as the primary storage compartment of fatty acids and as an endocrine organ that affects peripheral tissues. Many of adipose tissue-derived factors, often termed adipokines, have been discovered in recent years. The synthesis and secretion of these factors vary in different depots of adipose tissues. Excessive lipid accumulation in adipocytes induces inflammatory processes by up-regulating the expression and release of pro-inflammatory cytokines. In addition, activated macrophages in the obese adipose tissue release inflammatory cytokines. Adipose tissue inflammation has also been linked to an enhanced metabolism of polyunsaturated fatty acids (PUFAs). The non-enzymatic peroxidation of PUFAs and of their 12/15-lipoxygenase-derived hydroperoxy metabolites leads to the generation of the reactive aldehyde species 4-hydroxyalkenals. This review shows that 4-hydroxyalkenals, in particular 4-hydroxynonenal, play a key role in lipid storage homeostasis in normal adipocytes. Nonetheless, in the obese adipose tissue an increased production of 4-hydroxyalkenals contributes to the inflamed phenotype.     

Evidence for 4-hydroxyalkenals in rat renal cortex peroxidized by N2-methyl-9-hydroxyellipticinium acetate or Celiptium

The antitumor drug celiptium is an ellipticine derivative whose nephrotoxic pathogenesis implicates a lipid peroxidation process. It has been shown that hydrophobic lipid deposits overload the proximal tubular cells. Histochemistry with Holczinger's technique has demonstrated that these deposits are free fatty acids. In this study, the fatty acid analysis of phospholipids and neutral lipids was performed in rat renal cortex 4 and 8 days following a single i.v. dose of 20 mg/kg celiptium and showed: (1) a loss of polyunsaturated fatty acids within total phospholipids and a loss of phosphatidylethanolamine with a preferential decrease of arachidonic (20:4) and docosahexaenoic (22:6) acids; (2) an increase of free fatty acid levels with an increase in oleic (18:1) and linoleic (18:2) acids; (3) an increase of thiobarbituric acid-reactive substances or aldehydes. The analysis of these aldehydes showed significant amounts of 4-hydroxyalkenals, mainly the presence of 4-hydroxynonenal on day 4 and of a hydroxyaldehyde with a chromatographic behavior very similar to 4-HNE on day 8. We conclude that celiptium induced a preferential decrease of phosphatidylethanolamine linked to the formation of unsaturated free fatty acids and of 4-hydroxyalkenals. The toxic side-effects of these breakdown products produced in the proximal tubular cell are discussed in light of the lipid peroxidation process involved in the renal toxicity of celiptium.     

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.     

Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes

Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress and results in the generation of various bioactive compounds, of which 4-hydroxyalkenals are prominent. During the progression of type 2 diabetes mellitus, the ensuing hyperglycemia promotes the generation of reactive oxygen species (ROS) that contribute to the development of diabetic complications. It has been suggested that ROS-induced lipid peroxidation and the resulting 4-hydroxyalkenals markedly contribute to the development and progression of these pathologies. Recent findings, however, also suggest that noncytotoxic levels of 4-hydroxyalkenals play important signaling functions in the early phase of diabetes and act as hormetic factors to induce adaptive and protective responses in cells, enabling them to function in the hyperglycemic milieu. Our studies and others′ have proposed such regulatory functions for 4-hydroxynonenal and 4-hydroxydodecadienal in insulin secreting β-cells and vascular endothelial cells, respectively. This review presents and discusses the mechanisms regulating the generation of 4-hydroxyalkenals under high glucose conditions and the molecular interactions underlying the reciprocal transition from hormetic to cytotoxic agents.     

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.     

4-Hydroperoxy-2-nonenal is not just an intermediate but a reactive molecule that covalently modifies proteins to generate unique intramolecular oxidation products

α,β-Unsaturated aldehydes generated during lipid peroxidation, such as 4-oxoalkenals and 4-hydroxyalkenals, can give rise to protein degeneration in a variety of pathological states. Although the covalent modification of proteins by these end products has been well studied, the reactivity of unstable intermediates possessing a hydroperoxy group, such as 4-hydroperoxy-2-nonenal (HPNE), with protein has received little attention. We have now established a unique protein modification in which the 4-hydroperoxy group of HPNE is involved in the formation of structurally unusual lysine adducts. In addition, we showed that one of the HPNE-specific lysine adducts constitutes the epitope of a monoclonal antibody raised against the HPNE-modified protein. Upon incubation with bovine serum albumin, HPNE preferentially reacted with the lysine residues. By employing N(α)-benzoylglycyl-lysine, we detected two major products containing one HPNE molecule per peptide. Based on the chemical and spectroscopic evidence, the products were identified to be the N(α)-benzoylglycyl derivatives of N(ε)-4-hydroxynonanoic acid-lysine and N(ε)-4-hydroxy-(2Z)-nonenoyllysine, both of which are suggested to be formed through mechanisms in which the initial HPNE-lysine adducts undergo Baeyer-Villiger-like reactions proceeding through an intramolecular oxidation catalyzed by the hydroperoxy group. On the other hand, using an HPNE-modified protein as the immunogen, we raised a monoclonal antibody against the HPNE-modified protein and identified one of the HPNE-specific lysine adducts, N(ε)-4-hydroxynonanoic acid-lysine, as an intrinsic epitope of the monoclonal antibody. Furthermore, we demonstrated that the HPNE-specific epitopes were produced not only in the oxidized low density lipoprotein in vitro but also in the atherosclerotic lesions. These results indicated that HPNE is not just an intermediate but also a reactive molecule that could covalently modify proteins in biological systems.     

Detection of short-chain carbonyl products of lipid peroxidation from malaria-parasite (Plasmodium vinckei)-infected red blood cells exposed to oxidative stress.

Reversed-phase h.p.l.c. was used to detect 2,4-dinitrophenylhydrazine-reactive carbonyl products, which excludes malonaldehyde, in malaria-parasite (Plasmodium vinckei)-infected murine red blood cells (RBCs). A number of alkanals, 4-hydroxyalk-2-enals and alka-2,4-dienals were tentatively identified by comparison with authentic standards. The formation of 4-hydroxynon-2-enal, deca-2,4-dienal and hexanal was greater in P. vinckei-infected RBCs than in their uninfected counterparts and was increased by the presence of t-butyl hydroperoxide. Several of these aldehydes have previously been shown to be toxic to various types of cells, including P. falciparum, in vitro. The iron chelator desferrioxamine and the free-radical scavenger butylated hydroxyanisole inhibited the formation of these aldehydes. These experiments demonstrate that products of lipid peroxidation other than malonaldehyde are formed during the exposure of malaria-infected RBCs in vitro to drugs that generate reactive oxygen species and have anti-parasitic activity. The formation of products of this type during the natural course of malaria infection may have implications for the mechanisms underlying intra-RBC parasite death and the tissue damage associated with the disease.     

Insect-parasitic nematodes: from lab curiosities to model organisms

Interest in studying insect-parasitic nematodes was originally focused on their potential as biological control agents of insects and other arthropod pests. Now, after 30 years of intense basic and applied research, realization of the practical use of insect-parasitic nematodes, particularly of entomopathogenic nematodes and their symbiotic bacteria, has spurred developments across a far broader scientific front. We are now entering a new era of discovery in which tools of molecular genetics are being increasingly used to address a range of biological questions. The knowledge gained from these efforts will directly benefit the practical application of insect-parasitic nematodes as more effective biopesticides. Moreover, these studies will advance these nematodes as unique and intrinsically interesting biological model systems not only for basic research but also in applied fields such as plant health, human medicine, pharmaceutical bioprospecting, and genetic engineering. In this review, the past and current state of insect-parasitic nematode research is summarized. Future research priorities and goals are identified and discussed.     

Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes

The alteration of structural and biological properties of human plasma low density lipoprotein (LDL) exposed to oxidative conditions is in part ascribed to lipid peroxidation. The objective of this investigation was to measure quantitatively several parameters in oxidizing LDL indicative for lipid peroxidation. Exposure of freshly prepared EDTA-free LDL to an oxygen-saturated buffer led to a complete depletion of alpha- and gamma-tocopherol within 6 hr, thereafter lipid peroxidation commenced as indicated by the kinetics of the loss of linoleic (18:2) and arachidonic (20:4) acids, the formation of aldehydic lipid peroxidation products and fluorescent apoB. Within 24 hr of oxidation, on average 79 nmol of 18:2 (initial 345) and 12.8 nmol of 20.4 (initial 25.6) were oxidized per mg of LDL and the sample contained in total 7.1 nmol of aldehydes with the following molar distribution: 36.6% malonaldehyde, 25% hexanal, 8.9% propanal, 8.2% 4-hydroxynonenal, 7.6% butanal, 4.1% 2.4-heptadienal, 3.4% pentanal, 3.4% 4-hydroxyhexenal, and 2.5% 4-hydroxyoctenal. Malonaldehyde was predominantly (93%) in the aqueous phase, whereas the other aldehydes remained mostly (34-98%) within the LDL particle, where the total aldehyde concentration was in the range of 12 mM. Oxidized LDL exhibited a 1.6-fold enhanced electrophoretic mobility. Similarily, native LDL incubated for 5 hr with aldehydes showed increased electrophoretic mobility. At equal concentrations (5 mM) 4-hydroxynonenal was most effective, followed by 2,4-heptadienal, hexanal, and malonaldehyde. This study reports for the first time the rate and extent of the change of LDL constituents occurring during lipid peroxidation.     

Inhibition of calcium sequestration activity of liver microsomes by 4-hydroxyalkenals originating from the peroxidation of liver microsomal lipids

Aldehydes released during peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (4-hydroxynonenal being quantitatively the most significant) strongly inhibited the calcium sequestration activity of liver microsomes. The ID50 for 4-hydroxynonenal was 42 microM. The inhibition appeared to be correlated with the amount of the aldehyde bound to the microsomal protein. In rats intoxicated with BrCCl3 significant amounts of protein-bound aldehydes were formed at only 5 min after poisoning, a time at which the calcium sequestring capacity is markedly inhibited.     

Synthetic biology of minimal systems

“Synthetic biology” is a concept that has developed together with, or slightly after, “systems biology”. But while systems biology aims at the full understanding of large systems by integrating more and more details into their models, synthetic biology phrases different questions, namely: what particular biological function could be obtained with a certain known subsystem of reduced complexity; can this function be manipulated or engineered in artificial environments or genetically modified organisms; and if so, how? The most prominent representation of synthetic biology has so far been microbial engineering by recombinant DNA technology, employing modular concepts known from information technology. However, there are an increasing number of biophysical groups who follow similar strategies of dissecting cellular processes and networks, trying to identify functional minimal modules that could then be combined in a bottom-up approach towards biology. These modules are so far not as particularly defined by their impact on DNA processing, but rather influenced by core fields of biophysics, such as cell mechanics and membrane dynamics. This review will give an overview of some classical and some quite new biophysical strategies for constructing minimal systems of certain cellular modules. We will show that with recent advances in understanding of cytoskeletal and membrane elements, the time might have come to experimentally challenge the concept of a minimal cell.