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Bioactive Aldehydes Derived from Fatty Acids

Formula of 4-hydroxy-trans-2-nonenalUnsaturated fatty acids in animals and plants are vulnerable to many types of oxidation by reactive oxygen species in tissues, by both enzymatic and non-enzymatic mechanisms. This leads to the formation initially of many different hydroperoxides that in turn can react further to produce a wide variety of oxygenated metabolites. Included among these are scission products of which the most important are volatile short-chain aldehydes that are considered to be good markers for oxidative stress in relation to disease states in animals. In particular, 4-hydroxy-trans-2-nonenal derived from the n-6 family of polyunsaturated fatty acids has a special position because of its appreciable biological activity in both animals and plants. For example, it is cytotoxic in the low micromolar range. The remaining lipid-bound oxidation products can also have profound biological effects, and the isoprostanes formed by oxidation reactions to fatty acids in esterified form must be considered in this context (they have their own web page). Similarly, the formation and properties of oxidized sterols are discussed separately.

1.   Structures and Formation

Fatty acids are oxidized to hydroperoxy fatty acids in animal and plant tissues by several different lipoxygenases and cytochrome P450 enzymes, which produce products with a high degree of positional and stereospecificity. These reaction are discussed in our web page dealing with hydroxyeicosatetraenes (HETE). In addition, all polyunsaturated fatty acids can undergo autoxidation by free radical chain reaction mechanisms as discussed in greater detail in our web page dealing with isoprostanes.

In brief, the latter process consists of three main steps: initiation, propagation and termination. The initiation step begins with abstraction of a hydrogen atom on a bis-allylic carbon of a 1,4-cis,cis-pentadiene moiety of a polyunsaturated fatty acid, illustrated here for one only of the possible reactions of arachidonic acid with formation of an alkyl radical, which tends to be stabilized by a molecular rearrangement to form a conjugated diene. This initial step is followed by the propagation step in which the unstable fatty acid radical reacts with molecular oxygen to generate a peroxyl radical; this propagates the reaction by abstracting a hydrogen atom from another unsaturated fatty acid to produce a reactive hydroperoxide and a further alkyl radical; such reactions have limited positional and no stereospecificity. Termination of the process occurs when two radicals interact to produce dimeric products.

Autoxidation of arachidonic acid

The example illustrated is for one only of the many possible mono-hydroperoxy isomers of arachidonic acid that can be formed. If the comparable reactions by enzymatic and non-enzymatic mechanisms in animals of linoleate, linolenate, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are also taken into consideration, together with the probable formation of dihydroperoxides, the range of possible hydroperoxy-products is enormous.

Aldehyde generation then occurs by oxidative cleavage via a variety of mainly non-enzymatic mechanisms, including the Hock reaction, and via hydroperoxy, epoxy and dioxetane intermediates to give many different aldehydes from each hydroperoxide, although it is illustrated for one precursor and one possible reaction as an example below. In this instance, a hydroperoxide derived from linoleate, 13-hydroperoxy-9c,11t-octadecadienoate, is the precursor and 4-hydroxy-trans-2-nonenal (HNE) is the volatile aldehyde formed with 9-oxo-nonanoic acid, which remains esterified to the lipid backbone. C9 and C6 aldehydes are the main volatile products from the n-6 and n-3 families of polyunsaturated fatty acids, respectively. The relative proportions of each product in a given tissue will vary with the fatty acid composition, but the HNE concentration in human plasma is reportedly in the range of 0.28 to 0.68 μM while in rat hepatocytes, it is in the range of 2.5 to 3.8 μM.

Oxidative cleavage of 13-hydroperoxy-9c,11t-octadecadienoate

In plants, there is a more restricted range of unsaturated fatty acid precursors and enzymic mechanisms are more important. In particular, a fatty acid hydroperoxide lyase can react with a hydroperoxy fatty acid to form an unstable hemiacetal, which rearranges to from an enol and then trans-3-alcohols and aldehydes. Further modification by an isomerase to the trans-2 forms, or by hydrogenation via reductases can then occur. As linolenate is the main fatty acid in leaf tissue, C6 products predominate; trans-2-hexenal and trans-3-hexenol are sometimes termed the 'leaf aldehyde and alcohol', respectively. These are discussed further in a separate web page on plant oxylipins.

Of the many different aldehydes formed, α,β-unsaturated aldehydes are especially important because of their electrophylic nature, which enables them to react readily with the sulfhydryl or amine groups of proteins and lipids, often with profound metabolic consequences as discussed below. Some of the more important of these are illustrated -

Formulae of alpha,beta-unsaturated aldehydes produced by oxidative fission

As aldehydes are formed by reaction with intact lipids, the other part of the initial fatty acid remains at first in esterified form as a so-called "core-aldehyde" with immediate changes in membrane structure, and these lipids are discussed below and also in this website in relation to activities that resemble those of platelet-activating factor. The oxidized lipid fragment with its free aldehyde group can take part in other biological reactions in intact form or as the aldehydo-acid after hydrolysis by lipases. In particular, oxidation of docosahexaenoic acid (DHA) esterified into a phospholipid can generate 4-hydroxy-7-oxohept-5-enoic acid with the potential to exert deleterious effects, especially in tissues rich in DHA such as the retina.

Longer-chain aldehydes are also produced in animals and plants by various mechanisms, including catabolism of sphingolipids via sphingosine-1-phosphate and the action of myeloperoxidase on plasmalogens, but these also are discussed elsewhere on this web site.

Catabolism: α,β-Unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal can be detoxified by conjugation to the thiol group of the antioxidant glutathione by glutathione-S-transferases, or the aldehyde group can be oxidized to a carboxyl group before the molecule is further oxidized by the introduction of a 9-hydroxyl group before elimination as a conjugate in urine.

2.   Biochemical Function

Oxidative stress is usually considered to refer to a state of elevated cellular levels of reactive oxygen species (ROS) as a result of conditions that stimulate their production or reduce the antioxidant defenses of cells. In some cases, this is beneficial as in the stimulation of ROS production by macrophages as an innate immune response to bacterial infection. On the other hand, dysregulation of ROS levels has been associated with a number of inflammatory and age-associated disease states, including macular degeneration, muscular dystrophy, atherosclerosis and type 2 diabetes. Then, oxidative stress leads to the oxidation of cellular fatty acids with formation of aldehydes and ultimately to the modification of DNA, proteins, lipids and carbohydrates. These processes are less well studied in plants, but similar reactions are known to occur.

α,β-Unsaturated aldehydes: As discussed briefly above, the most reactive of the aldehydes generated from polyunsaturated fatty acid oxidation are α,β-unsaturated aldehydes, including 4-hydroxy-2-nonenal, 4-oxo-2-nonenal and acrolein. They are defined as lipotoxic in that they can accumulate in cells and tissues that are not equipped to metabolize or store them adequately with profound effects on cellular viability and function. They can affect tissue metabolism directly or via reaction with other tissue components. For example, injection of 4-hydroxy-2-nonenal and 4 oxo-2-nonenal into mice causes inflammation and pain by activation of transient receptor potential ankyrin 1 (TRPA1).

Product of protein carbonylationThe nature of the products formed by reaction with lipids such as phosphatidylethanolamine are discussed in relation to that lipid elsewhere on this website, but cholesterol and lipoproteins can also be attacked. With these active aldehydes, the double bond serves as a site for Michael addition with proteins, and in particular with the sulfur atom of cysteine, the imidizole nitrogen of histidine and the amine nitrogen of lysine and other ε-amino acids; this is often termed 'protein carbonylation'. In model systems, it has been demonstrated that the vast majority of proteins modified in this way retain a free carbonyl group. After the formation of Michael adducts, the aldehyde moiety will often undergo Schiff base formation with amines of adjacent lysines to produce intra- and/or intermolecular cross-linked proteins. With 4-oxo-2-nonenal, ketoamide adducts can be formed by modification of lysine residues through 1,2-addition, i.e. Schiff base formation. A further cyclization reaction can lead to the formation of ethylpyrrole or carboxyethylpyrroles from the aldehyde or aldehydo-carboxylic acid products of oxidative fission, respectively.

Michael addition (protein carbonylation) and Schiff base formation

Protein carbonylation has many different effects on cells, but because the side chains of cysteine, histidine, and lysine residues are often used in catalysis, the most common observation is enzyme inactivation. Of these, Cys is often preferred, and the reactivity tends to follow the order of Cys > His > Lys, although this depends on their accessibility on the protein surface. Among innumerable examples, the inactivation of several membrane transporters in the brain by lipid-derived aldehydes has been linked to neurodegenerative disorders, including Alzheimer's disease. Often the result is dysregulation of NADPH levels or of the cellular redox status and stress signalling but especially the amplification of oxidative stress. Ultimately, this can lead to irreversible cytotoxic injuries and cell death. The carboxyethylpyrroles derived from DHA especially,i.e. by reaction with 4-hydroxy-7-oxohept-5-enoic acid, have been implicated in many diseases associated with inflammation, including atherosclerosis, hyperlipidemia, thrombosis, age-related macular degeneration, and tumor progression, probably by interactions with toll-like or scavenger receptors. For example, TLR2 has been identified as a receptor on endothelial cells that recognizes proteins modified by carboxyethylpyrroles and mediates cellular activation and signalling.

Not all of the effects of aldehydes are deleterious, however, as both 4-hydroxy-2-nonenal and 4-hydroxy-2E,6Z-dodecadienal, the latter derived from 12-hydroperoxy-eicosatetraenoic acid (12-HpETE) - the 12-lipoxygenase metabolite of arachidonic acid, are endogenous activating ligands of peroxisome proliferator-activated receptor gamma (PPARγ) at low and noncytotoxic concentrations. It is believed that they regulate genes that control the oxidative capacity of the mitochondrion, stimulate detoxification mechanisms and repress inflammation. Similarly, 4-hydroxy-2-nonenal and products of further oxidation have been shown to form adducts with DNA bases, but because cellular mechanisms exist to ensure that DNA is repaired efficiently, very little of the total DNA damage results in permanent mutations.

It is generally believed that like other types of protein oxidation, HNE-modified proteins are degraded through proteasomes with lysosomes and autophagy playing key roles in the recycling of HNE-protein adducts .

The emission of C6 aldehydes and alcohols occurs rapidly in plants in response to wounding, and they contribute to protection against the invasion of fungi and insects to which they are toxic. They are also believed to be involved in abiotic stress responses by inducing the expression of stress-associated genes.

Ester-bound oxidation products: The lipid-bound fragments resulting from oxidative cleavage can also exert profound biological effects in animal tissues that are mediated via receptors. For example, phosphatidylcholine is the most common phospholipid, and it is not recognized by any pattern-recognition receptors in native low-density lipoproteins (LDL) or on the surface of cells. However, an oxidized species, such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine, in oxidized LDL and on apoptotic cells is a key ligand that mediates the binding of oxidized LDL to the receptor CD36 and scavenger receptor class B type I (SR-B1). A concept has been developed of the formation of damage-associated molecular patterns (DAMPs) that arise from the oxidative damage of lipids and lipoproteins. Those DAMPs derived by oxidation share common structural motifs with microbial pathogen-associated molecular patterns and so activate the same pattern-recognition receptors that are present on the surface of macrophages and of immune and vascular cells and so initiate many different inflammatory signalling processes. As discussed in our web page on phosphatidylserine, this is an important aspect of the mechanism of apoptosis (programmed cell death). Similar products are formed in oxidized glycosyldiacylglycerols in plants.

Oxidized cholesterol esters formed by reaction with 15-lipoxygenase have been detected in oxidized LDL human blood and atherosclerotic lesions, but such "minimally oxidized LDL" does not bind to CD36 but rather to CD14, a receptor that recognizes bacterial lipopolysaccharides. The result is stimulation of toll-like receptor 4 (TLR4), although the response differs from that to lipopolysaccharides. In addition, oxidized metabolites of cholesteryl arachidonate of this kind stimulate macrophages to express inflammatory cytokines, among other effects.

On the other hand, these effects need not always be deleterious, as it has been suggested that oxidized lipids may prime the receptors that respond to bacterial infection to activate dendritic and T cells, resulting in enhanced protection. By binding to the receptors that recognize bacterial toxins, they may inhibit the worst effects of TLR4-mediated inflammatory signalling and the expression of cytokines.

3.  Analysis

Free aldehydes are analysed after formation of stable derivatives by liquid or gas-liquid chromatography and mass spectrometry. Analysis of the protein adducts is more daunting technically, but new HPLC-mass spectrometry methods hold promise that detect the specific product ions from positively ionized adducts in a selected reaction monitoring mode. On the other hand, the detoxified aldehyde-conjugates with glutathione and mercapturic acid can be analysed by HPLC and mass spectrometry methods, and may serve as non-invasive biomarkers of oxidative stress.

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Lipid listings Credits/disclaimer Updated: May 28th, 2017 Author: William W. Christie LipidWeb icon