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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, which have long been studied in relation to rancidity in foods because they produce off-flavours and have unpleasant odours. Now much research is focussed upon are their biological properties as they 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, including acrolein and crotonaldehyde, 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. While 4-hydroxy-2-nonenal and 4-oxo-2-nonenal are the most active of the γ-substituted aldehydes, there is increasing interest in 4-hydroxy-2-hexenal and 4-hydroxy-2,6-dodecadienal, the latter derived from the breakdown of 12-hydroperoxy-eicosatetraenoic acid from arachidonic acid. 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 other non-radical mechanisms, including catabolism of sphingolipids via sphingosine-1-phosphate from which the α,β-unsaturated aldehyde trans-2-hexadecenal is generated, and the action of myeloperoxidase and hypochlorous acid (HOCl) on plasmalogens (2-chloro-aldehydes in this instance), but these processes are discussed elsewhere on this web site.

Catabolism: α,β-Unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal can be oxidized to carboxylic acids by aldehyde dehydrogenases of which three isoforms have been identified before the molecule is further oxidized by the introduction of a 9-hydroxyl group for elimination as a conjugate in urine. Aldehyde dehydrogenases are believed to have protective effects in a number of pathologies, including Alzheimer's disease and various heart conditions. In addition, aldehydes can be reduced to alcohols by alcohol dehydrogenase or aldo-keto reductase enzymes. While aldehydes are also removed from tissues by conjugation to the thiol group of the antioxidant glutathione by glutathione-S-transferases, the products are highly pro-inflammatory so this is considered to be a secondary detoxification mechanism, although the conjugates can also be oxidized and reduced and are eventually eliminated in urine and bile.

2.   Biological Effects

Under normal physiological conditions, cells are in a stable state of redox homeostasis, which is maintained by continuous generation of reactive oxygen species (ROS) in balance with mechanisms involved in antioxidant activity. Oxidative stress results from ROS overproduction or a reduction in the antioxidant defenses of cells leading to alterations in the redox homeostasis that promote oxidative damage to major components of the cell, including the biomembrane phospholipids, 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 promotes oxidative damage to major components of the cell, including the biomembrane phospholipids and 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 than in animals, 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). Aldehyde production tends to increase with ageing.

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 peptides such as glutathione and 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'. The reaction does not require enzyme catalysis. 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. The levuglandins and isolevuglandins are also important in this context but are discussed elsewhere.

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 unique free cysteine (Cys34) of human albumin shows a remarkable reactivity towards 4-hydroxy-trans-2-nonenal, while 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 most widely described roles for 4-hydroxy-trans-2-nonenal in signalling pathways are associated with its activation of kinases and of transcription factors that are responsible for redox homeostasis. 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.

A further important metabolite of 4-hydroxy-trans-2-nonenal is formed by conjugation with glutathione catalysed by an enzyme of the glutathione S-transferase family to give 3-(S-glutathionyl)-4-hydroxynonanal. In this instance, the Michael addition reaction removes the trans C2-C3 double bond and the product exists in equilibrium between the free aldehyde and its cyclic hemiacetal. Further enzymic reactions occur to produce many different metabolites, including mercapturic acids, with varying biological activities.

Glutathione adduct of hydroxynonenal

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. As the Michael addition reaction is reversible, it has been suggested that this may be a protective mechanism under conditions of especially high stress. 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 can be 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.

α-Chloro fatty aldehydes: 2-Chlorohexadecanal and related aldehydes formed by the action of myeloperoxidase and hypochlorous acid on plasmalogens have been detected in clinical samples or animal models of disease. They have a number of potentially pro-inflammatory effects ranging from direct toxicity to inhibition of nitric oxide synthesis.

Ester-bound oxidation products: Much of the above discussion has been concerned with the volatile products of oxidative scission, but a fragment of the oxidized molecule remains esterified to the original phospholipid. These are sometimes termed 'core aldehydes' and are discussed briefly in our web page dealing with platelet-activating factor. In addition, phospholipids containing isoprostanes and other oxylipins are discussed elsewhere in this web site. While they were once simply considered as waste products, it is now evident that the lipid-bound fragments resulting from oxidative cleavage can exert profound biological effects towards inflammation, infection and the immune response in animal tissues. While much research has been concentrated on oxidation of the unsaturated acyl moieties in phospholipids, the polar head groups can also be affected. It should be noted that hundreds of such oxidized lipids are formed in tissues of all kinds under innumerable physiological conditions, but the biological properties of only a handful of model compounds have been studied in any detail. On the other hand, phosphatidylcholine metabolites tend to predominate in vivo, often in fewer molecular species than might be expected from experiments in vitro.

Many effects of oxidized phospholipids may be exerted through the perturbation of the structures of cellular membranes. In addition, oxidatively modified acyl chains in oxidized phospholipids are believed to protrude into the aqueous medium so rendering them accessible physically for participation in signalling events including macrophage recognition. These interactions are often mediated via specific 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).

Formula of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine

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 by induction of chemokines and proinflammatory cell adhesion molecules. They are considered to be biomarkers of atherosclerosis, but it has been argued that there is insufficient information on their concentrations in other tissues under varying physiological conditions. 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, complete inhibition of the proinflammatory action of lipopolysaccharides can result thereby inhibiting the worst effects of TLR4-mediated inflammatory signalling and the expression of cytokines. Inflammatory pathways can also be inhibited by activation of the peroxisome proliferator-activated receptor (PPARγ) where the sn-1 alkyl phospholipid hexadecyl-azelaoyl-phosphatidylcholine is a specific and high-affinity ligand.

3.  Analysis

Free aldehydes are analysed after formation of stable derivatives by liquid or gas-liquid chromatography and mass spectrometry. Liquid chromatography and modern mass spectrometric methods are favoured for intact oxidized phospholipids, but selective antibody assays are also available. Analysis of the protein adducts is more daunting technically, but new HPLC-mass spectrometry methods that detect the specific product ions from positively ionized adducts in a selected reaction monitoring mode hold promise. 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: August 17th, 2017 Author: William W. Christie LipidWeb icon