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Bioactive Aldehydes and Oxidized Phospholipids

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, which can react further to produce innumerable 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 as a cytotoxic agent in the low micromolar range. On the other hand, it is increasingly being recognized that oxidized phospholipids, including the lipid-bound oxidation fragments remaining after aldehyde formation, can also have profound biological effects. The isoprostanes formed by non-enzymatic oxidation reactions of fatty acids in esterified form should perhaps be considered in this context, but they have their own web page, as do the formation and properties of oxidized sterols.

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 reactions in animals are discussed in our web page dealing with hydroxyeicosatetraenes (HETE), and while they occur mainly on unesterified polyunsaturated fatty acids such as arachidonate, most of the oxidation products can be esterified subsequently by the enzymes of the Lands' cycle, mainly to phosphatidylethanolamine. However, the 15- and 12,15-lipoxygenases are able to oxidize phospholipid-bound fatty acids directly. The result is a relatively restricted range of products that are formed through controlled pathways, especially in innate immune cells. Even so, at least 100 unique oxidized phospholipids of this type have been identified, mainly of phosphatidylethanolamine, but also of phosphatidylcholine with much less of phosphatidylinositol.

In addition, all polyunsaturated fatty acids can undergo autoxidation by free radical chain reaction mechanisms as discussed in greater detail in our web pages dealing with isoprostanes and the antioxidant properties of tocopherols. 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 stereo specificity. Termination of the process occurs when two radicals interact to produce dimeric products. Reduction to the chemically less reactive hydroxide is catalysed by a reductase such as glutathione reductase 4 (GPX4) to generate a hydroxy-eicosatetraenoic acid (HETE) esterified to a phospholipid.

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 autoxidation reactions by such mechanisms of linoleate, linolenate, eicosapentaenoate (EPA) and docosahexaenoate (DHA) are also taken into consideration, together with the lack of stereochemical control and the probable formation of dihydroperoxides, the range of possible hydroperoxy-products is enormous.

Instead of the reduction step, aldehyde generation can occur 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. While the reaction has been studied in some detail in relation to food spoilage, the cleavage mechanism in living tissues appears uncertain. 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 the enzymic mechanisms for aldehyde production are better characterized. In particular, a fatty acid hydroperoxide lyase (more accurately termed a hemiacetal synthase) 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. The reaction mechanism and products 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 in animals. 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, as discussed below, 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-oxo-hept-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 of Aldehydes

Under normal physiological conditions, cells are in a stable state of redox homeostasis, which is maintained by continuous generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in balance with mechanisms involved in antioxidant activity (for example, see our web page on tocopherols). Oxidative stress results from ROS overproduction or a reduction in the antioxidant defenses of cells leading to alterations in the redox homeostasis that promotes oxidative damage to major components of the cell, including the biomembrane phospholipids (see below), 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 oxidation of polyunsaturated fatty acids 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 and there may be effects upon age-related neurological disorders, such as Alzheimer's disease.

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 on this website.

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. Generation of such adducts enables 4-hydroxy-nonenal to transfer information on oxidative stress from the cell membrane to the cytoplasm to initiate cell signalling. Often the result is dysregulation of NADPH levels or of the cellular redox status and stress signalling but especially the amplification of oxidative stress by inhibiting antioxidant enzymes, such as catalase, glutathione peroxidase, and thioredoxin reductase by binding to the cysteine residue at the enzyme’s active site. Signalling pathways associated with its activation of kinases and of transcription factors that are responsible for redox homeostasis are also affected, leading to inflammation and apoptosis. Among innumerable other 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. 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.

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

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

Similarly, 4-hydroxy-2-nonenal and products of further oxidation such as 4-oxo-2-alkenals have been shown to form adducts rapidly with DNA bases. As cellular mechanisms exist to ensure that DNA is repaired efficiently, very little of the total DNA damage is believed to result in permanent mutations. On the other hand, 4-oxo-2-alkenal-derived DNA adducts associated with age-related diseases has been detected in rodents and humans, and it seems dangerous to assume that such mutagenic properties have no link to increased cancer risk.

Reaction of an aldehydes with a DNA base

Malondialdehyde is a further reactive end-product of lipid peroxidation, which exhibits a high affinity for the formation of adducts with DNA but also with proteins by interaction with lysine residues. Such adducts are characteristic of cellular stress, especially that caused by exposure to cigarette smoke or alcohol, and they are often associated with a pro-inflammatory reaction throughout the body. Collagen is especially vulnerable to modification by malondialdehyde, which forms adducts with cysteine residues and favors glycation reactions. This can affect the regeneration and reorganization of tissues leading to a loss of elasticity and disturbance to tissue remodelling. Acrolein is a relatively simple unsaturated aldehyde, which forms Michael adducts with thiol groups of cysteines, which affect the activity of many proteins. These adducts have been implicated in a number of pathological conditions, and they are also apoptotic signals.

Aside from their actions with macro-molecules, some of the effects of aldehydes are beneficial, 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, bioactive aldehydes can serve in effect as second messengers for regulation of oxidative/electrophilic stress through activation of the antioxidant defense system. Both 4-hydroxyhexenal and 4-hydroxynonenal at low concentrations have protect effects against oxidative stress by activating the Nrf2 pathway, which regulates the expression of antioxidant proteins including the production of heme oxygenase-1, a potent antioxidant enzyme. They can also activate other cytoprotective pathways and enhance expression of other detoxification genes.

Plants: 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. This is discussed further in the web page dealing with plant oxylipins.

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. α-Bromo fatty aldehydes are formed in the same way. All react with thiols and have a number of potentially pro-inflammatory effects ranging from direct toxicity to inhibition of nitric oxide synthesis.

3.   Phospholipid 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, i.e. as oxidized phospholipids with both enzymic and non-enzymic origins. These are sometimes termed 'core aldehydes' and are discussed briefly in our web page dealing with platelet-activating factor as they have some properties in common. 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. 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, fewer molecular species are often observed in vivo than might be expected from experiments in vitro. They have a tendency to accumulate in tissues of the elderly, especially in the lung.

For example, phosphatidylcholine is the most abundant phospholipid in most animal cells, 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). Such oxidized phospholipids can be degraded by the platelet-activating factor-acetylhydrolase and a lysosomal phospholipase A2.

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

Phospholipids containing intact oxylipins have great biological relevance also, and phospholipids linked to isoprostanes are discussed elsewhere in this web site. Similarly, oxidized phospholipids formed enzymatically, for example by lipoxygenase activity, including phosphatidylethanolamines and phosphatidylcholines containing both hydroperoxy- and hydroxy-eicosatetraenoic acids (HETE), are of special interest. On cellular activation, approximately 30% of the 12-HETE generated by human platelets is esterified into these lipids at pico- to nanomole levels at the same rate as for the synthesis of free 12-HETE. Some may be formed by direct oxidation of intact phospholipids, for example by the action of 15-LOX in human monocytes (or murine 12/15-LOX), but most is produced by subsequent esterification of free oxylipins. This has lead to the suggestion that the biosynthetic enzymes are colocalized and work cooperatively. Similarly, oxylipin-containing glycosyldiacylglycerols are formed in plants.

Many effects of oxidized phospholipids may be exerted through the perturbation of the structures of cellular membranes, as 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. The electrophilic characteristics of some of these molecules can promote adduct formation with membrane proteins leading to further disruption.

Oxidized forms of polyunsaturated fatty acids

Other biological effects of oxidized phospholipids may be mediated via specific receptors. A concept has been developed of the formation of damage-associated molecular patterns (DAMPs) that arise from the controlled oxidation 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. During oxidative stress in vivo, high-density lipoproteins (HDL) are enriched in oxidized phospholipids and these can react with the apoproteins, apo A1 especially, to compromise the athero-protective functions of HDL. Oxidized phospholipids in excess in the circulation 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. Some highly specific activities of oxidized phospholipids are discussed in relation to particular phospholipids elsewhere on this website. For example, oxidized phosphatidylserine is an important aspect of the mechanism of apoptosis (programmed cell death), oxidized phosphatidylethanolamine promotes ferroptosis, and oxidized cardiolipin acts as a required signal for the execution of the intrinsic apoptotic programme in mitochondria.

Scottish thistleOxidized cholesterol esters formed initially by a 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, the effects of oxidized phospholipids need not always be deleterious, as it has been suggested that oxidized lipids at lower concentrations may prime the receptors that respond to bacterial infection to activate dendritic and T cells resulting in enhanced protection. After acute activation by bacteria or bacterial products, neutrophils generate 5-HETE-containing phospholipids by 5-lipoxygenase (5-LOX) reaction, while macrophages and monocytes generate similar phospholipids through 15-LOX and 12/15-LOX activity. Binding to the receptors that recognize bacterial toxins can result in complete inhibition of the proinflammatory action of lipopolysaccharides, thereby eliminating the worst effects of TLR4-mediated inflammatory signalling and the expression of cytokines. Oxidized phospholipids of this kind can also promote coagulation following tissue injury to limit bleeding while simultaneously inhibiting infection. In addition, the phospholipid products of 15-LOX have a vial function towards the resolution of inflammation. 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.

Those oxidized phospholipids generated non-enzymatically by free radical mechanisms tend to be very different in structure from those formed by enzymic oxidation, and they are always considered harmful because they contribute to autoimmune and inflammatory diseases and cell death. While much research has been concentrated on oxidation of the unsaturated acyl moieties in phospholipids, the polar head groups can also be affected.

4.  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 in urine can be analysed by HPLC and mass spectrometry methods, and may serve as non-invasive biomarkers of oxidative stress. Similarly, oxidized phospholipids are now more easily analysed by modern lipidomics methodology.

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Lipid listings Credits/disclaimer Updated: August 23rd, 2019 Author: William W. Christie LipidWeb icon