Bioactive Aldehydes

1.  Introduction

Formula of 4-hydroxy-trans-2-nonenalDuring conditions of oxidative stress and redox imbalance like inflammation or environmental toxicity, reactive oxygen species (ROS), such as the superoxide anion (O2•-), hydroxyl radical (OH), nitric oxide (NO) and peroxyl radical (LOO), are generated in cells by the action of enzymes that include NADPH oxidases, xanthine oxidase and the mitochondrial electron-transport chain, and exposure to external factors (xenobiotics, UV light, pollutants, etc) are also important. Although some of these ROS have important beneficial functions, an imbalance can have consequences that are not benign.

Unsaturated fatty acids in animals and plants are vulnerable to many types of oxidation by these ROS in tissues by non-enzymatic mechanisms (autoxidation), although enzymatic oxidation occurs also. This leads to the formation initially of many different hydroperoxides, the primary peroxidation products, which can react further to produce innumerable secondary oxygenated metabolites, often with reactive electrophilic carbonyl groups. Lipoxidation is a term used to describe non-enzymatic reactions of electrophilic carbonyl species produced during the oxidation of lipids with proteins and other important macromolecules such as DNA, leading to a loss of functionality.

Included among these oxidized lipids 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 focused upon their biological properties as they are considered to be good markers for oxidative stress in relation to disease states in animals. 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. Oxidized metabolites can have signalling functions, and in addition, it is increasingly being recognized that lipid-bound oxidation fragments remaining after aldehyde formation can have profound biological effects in animal tissues especially. 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 esterified hydroperoxy- and hydroxy-eicosatetraenoic acids (HETE) produced enzymatically, oxidized phospholipids and oxidized sterols.

2.  Structures and Formation of Hydroperoxides and Bioactive Aldehydes

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 fewer 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 oxidized phospholipids, and also with regard to the antioxidant properties of tocopherols and coenzyme Q. In brief, autoxidation 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, or more often when an anti-oxidant molecule such as tocopherol reacts with a radical.

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.

As a final detoxification step, reduction to the chemically less reactive lipid hydroxide can be catalysed by a reductase such as glutathione reductase 4 (GPX4) to generate a hydroxy-eicosatetraenoic acid (HETE) esterified to a phospholipid.

Instead of the reduction step, aldehyde generation can occur by oxidative cleavage via a variety of mainly non-enzymatic mechanisms, and via hydroperoxy, epoxy and dioxetane intermediates to give many different aldehydes from each hydroperoxide. While the hydroperoxide cleavage reaction has been studied in some detail in relation to food spoilage, some details of the mechanisms in living tissues appear uncertain. A number of possible chemical reaction mechanisms are known and may indeed occur, but there are doubts as to how far these can be extrapolated to conditions in vivo. For example, the Hock rearrangement is a well-known chemical reaction in which hydroperoxide acidification is followed by hydrolysis, but in the test tube it requires strong acids or elevated temperatures so its importance under biological conditions has been questioned. As an alternative, intramolecular cyclization of a peroxyl radical may occur to yield a highly strained dioxetane, which undergoes fission in a concerted rearrangement. Other suggested mechanisms include cleavage of dihydroperoxides, or cross-chain reactions between hydroperoxides in adjacent acyl chains of lipids such as cardiolipin, the oxidation products of which function as apoptotic signalling molecules.

Mechanisms for hydroperoxide cleavage and aldehyde formation

In the example illustrated next for one precursor and set of products only, 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 together 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, and the relative proportions of each in a given tissue will vary with the fatty acid composition. In human plasma , the HNE concentration 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

Of the many different aldehydes formed, α,β-unsaturated aldehydes, including acrolein and crotonaldehyde, are especially important because of their electrophilic 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

Photo-oxidation of lipids with singlet oxygen is a somewhat less studies aspect of autoxidation in animal cells, but it is important for skin metabolism and in relation to photodynamic therapies used clinically to treat such diseases as cancer and bacterial infections (see our web page on oxidized phospholipids); it is especially important in photosynthetic organelles in plants. The process may be different but the end results are similar to other aspects of autoxidation, including formation of aldehydes and oxidized phospholipids, and resulting in membrane disruption and cytotoxic effects.

Core aldehydes: 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 in the web page dealing with oxidized phospholipids and also in relation to biological activities that resemble those of platelet-activating factor. The oxidized lipid fragment can take part in biological reactions as the aldehydo-acid after hydrolysis by lipases. In particular, oxidation of docosahexaenoic acid (DHA) esterified into a phospholipid can lead to the generation of 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.

Scottish thistleLong-chain fatty aldehydes: Longer-chain aldehydes are 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. This is reported to form adducts with glutathione, deoxyguanosine in DNA and various proteins. Enzymatic or non-enzymatic hydrolysis of plasmalogens can also generate aldehydes, and in particular, 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 that range from direct toxicity to inhibition of nitric oxide synthesis.

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 in human tissues, before the molecule is further oxidized by the introduction of a 9-hydroxyl group for elimination as a conjugate in urine. These enzymes 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. Finally, aldehydes are removed from tissues by conjugation to the thiol group of the antioxidant glutathione by glutathione-S-transferases, and they are eventually eliminated in urine and bile; these enzymes are now recognized as key regulators of the signalling as opposed to toxic roles of endogenous bioactive aldehydes.

3.  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 redox homeostasis that promote oxidative damage to major components of the cell, including the membrane phospholipids. In some cases, this is beneficial as in the stimulation of ROS production by macrophages as an innate immune response to eliminate bacterial infection; ROS are also important for the maintenance of vascular tone, cardiovascular functions, and cell proliferation and differentiation. On the other hand, dysregulation of ROS levels promotes oxidative damage to major components of the cell, and it has been associated with a number of inflammatory and age-associated disease states. Then, oxidative stress leads to the oxidation of cellular fatty acids with excessive formation of oxidized lipids including aldehydes and ultimately to the modification of other lipids, DNA, proteins and carbohydrates. The levuglandins, isolevuglandins and cyclopentenone prostaglandins, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), are important in this context, but they are discussed elsewhere on this website. Similar reactions occur in plants.

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 cell viability and function. They can affect tissue metabolism directly or after reaction with other tissue components to produce further bioactive mediators. 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). 4-Hydroxy-2-nonenal is involved in the oxidation of low-density lipoprotein, so inducing macrophage activation and foam cell formation in atherosclerotic plaques. Aldehyde production tends to increase with ageing and there may be effects upon age-related neurological disorders, such as Alzheimer's disease.

The nature of the products formed by reaction with lipids such as phosphatidylethanolamine are discussed in relation to that lipid elsewhere on this website, but lipoproteins and cellular proteins can also be attacked. With the bio-active aldehydes, such as 4-oxo-2-nonenal and 4-hydroxy-2-nonenal, 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 carbonyl formation' (or less accurately 'carbonylation'). With cysteine residues, and the protonated form especially, the reaction can be up to two orders of magnitude faster than for the reaction of histidine or lysine analogues. The reaction does not require enzyme catalysis, and the products can be stabilized somewhat by formation of acetals. After Schiff base formation with lysine residues, further cyclization reaction can lead to the formation of ethylpyrrole or carboxyethylpyrroles from the aldehyde or aldehydo-carboxylic acid products of oxidative fission, respectively. In model systems, it has been demonstrated that the vast majority of proteins modified in this way retain a free carbonyl group.

Michael addition (protein carbonylation) and Schiff base formation

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, and 4-oxo-2-nonenal is of particular importance in this regard. It is also possible for 4-hydroxy-2-nonenal to react first with a lysine residue in one protein followed by a reaction with the thiol group in a cysteine residue of a second protein to effect cross-linking.

Cross-linking of proteins

Bifunctional aldehydes such as malondialdehyde and methylglyoxal can react with arginine to form Schiff bases, which rearrange to form stable adducts through an intramolecular reaction of the second carbonyl group. Malondialdehyde exists as an enolic anion of low chemical reactivity at pHs above a pKa of 4.46, but at lower pH an equilibrium mixture of the protonated enol and dialdehyde forms of high reactivity is formed. As the last, it exhibits a high affinity for the formation of adducts with DNA but also with proteins by interaction with arginine 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. Similarly, 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. The brain in the elderly is especially vulnerable to adduct formation by such aldehydes with the mitochondrial ATP-synthase.

Reaction of malondialdehyde with arginine

It is evident that some proteins are more susceptible to lipoxidation than others. Sometimes it may simply be the relative abundance of particular proteins within cells, but this is not the only factor as in the lipoxidation of transcription factors and signalling proteins, which are minor cellular components. The nucleophilicity of amino acid side chains by Schiff’s base formation or Michael addition may determine their reactivity, so a cysteine thiol is more reactive than the imidazole in histidine or amino group in lysine, although these activities can be influenced by their chemical microenvironment. A further factor can be the relative accessibility of a given amino acid within the tertiary structure.

As lipoxidation by this means alters the structure of proteins and introduces relatively hydrophobic entities, it has many different effects in cells at a structural and functional level that are dependent on the structure of the target protein and the nature of the reactive aldehyde. 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. Loss of enzyme activity can be the result of unfolding or conformational change of the protein, alterations to gene expression and the defence response, oligomerization or aggregation, or increased proteosomal degradation. Some enzyme proteins are activated by lipoxidation-induced oligomerization resulting in increased downstream signalling. Ultimately, carbonylated proteins are degraded by the proteosome system, although heavily carbonylated proteins can accumulate as cytotoxic aggregates due to their increased hydrophobicity; these have been linked to age-related diseases in humans as a result of clogging of the proteasome system.

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 sometimes via specific receptors. Electrophilic aldehydes may associate with membrane proteins to produce of damage(or danger)-associated molecular patterns (DAMPs), which can 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. These can initiate signalling processes by induction of chemokines and proinflammatory cell adhesion molecules. Reaction with cysteine and arginine residues of NF-κB (Nuclear Factor kappa B) can down-regulate the proinflammatory gene expression induced by this transcription factor. Among innumerable further 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.

Scottish thistleSimilarly, the carboxyethylpyrroles derived from oxidized 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. Sometimes 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 active site of the enzyme.

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 δ (PPARδ) 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 protective 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. In consequence, 4-hydroxy-2-nonenal especially is considered to be important to the pathophysiology of various human disease states, including the metabolic syndrome, diabetes, and cardiovascular, neurological, and immunological and age-related diseases. Carnosine-based scavengers of advanced lipoxidation end-products are under investigation, and in animal models they improve inflammatory conditions associated with obesity.

There are many reports that oxidative stress and electrophilic lipid peroxidation, including protein carbonylation, are involved in the induction of cell cycle arrest, the differentiation process and apoptosis in cancer cells. However, it appears that lipoxidation adducts can have either anti- or pro-carcinogenic effects, depending on the cell type and the nature of the adduct that is formed.

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

This may be a protective or detoxification mechanism, especially under conditions of high stress. Other enzymes such as aldehyde reductases can reduce the aldehyde group of HNE to decrease its bioavailability and reactivity in both plants and humans. 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, although it is possible that there is a carcinogenic effect through the modulation of proteins involved in DNA repair. 4-Oxo-2-alkenal-derived DNA adducts associated with age-related diseases have 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

4.  Bioactive Aldehydes in Plants

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 α-linolenate is the main fatty acid in leaf tissue, C6 products predominate; trans-3-hexenal and trans‑3‑hexenol are sometimes termed the 'leaf aldehyde and alcohol', respectively. The reaction mechanism, products and biological activities are discussed further in a separate web page on plant oxylipins.

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. Malondialdehyde is generated in leaves non-enzymatically from hydroperoxides produced first by the reaction of ROS on α-linolenate or enzymatically by the activity of lipoxygenases. In excess, it is considered to be an indicator of cellular damage, but it may have a more positive role by activating regulatory genes involved in plant defense to provide cellular protection under conditions of oxidative stress.

5.  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.

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Lipid listings Credits/disclaimer Updated: August 10th, 2021 Author: William W. Christie LipidWeb icon