Susceptible to oxidation yet resistant to atherosclerosis—reconciling the PUFA paradox via PPARs and Nrf2

Another work in progress; any thoughts/feedback appreciated.

Atherosclerosis is a ubiquitous and major cause of cardiovascular disease (CVD)—itself a leading cause of death globally—while a long-standing cornerstone of many dietary guidelines has been replacing saturated fats (SFAs) with unsaturated fats (UFAs), especially plant-based PUFAs (e.g. from seed oils), and consuming more oily fish/n-3 PUFAs (FAO). Such public health recommendations are based in evidence from observational and interventional studies, both of which are susceptible to confounding and uncertainty; with diet–heart trials being particularly old, heterogenous and debatable ^1–4. Mechanistic data can inform variables to aid interpretation and support biological plausibility, although here too a potential paradox arises: atherosclerosis is widely acknowledged to involve lipid oxidation, yet PUFAs are most susceptible. This underlies the theoretical basis of some concern, particularly with the post-industrial increase in seed oil/n-6 intake ^2,5–10 (incl. the 2026 DGA report). Thus, a simple dichotomous discourse could weigh any putative benefits, such as lipid-lowering or anti-inflammatory activity, against susceptibility to oxidation. However, PUFA oxidation can take many paths and produce many molecules with diverse effects, including lipid-lowering ^11,12, anti-inflammatory ^13–17 and antioxidant activity ^18,19, suggesting context matters and greater opportunity for harmonisation.

Chemically, PUFAs are defined by their multiple double bonds, which confer fluidity and susceptibility to oxidation via adjacent bisallylic hydrogens with low dissociation energy. At one extreme, radical-mediated autoxidation eventually degrades and fragments PUFAs into reactive aldehydes, such as n-6-derived 4-hydroxynonenal (4-HNE), n-3-derived 4-hydroxyhexenal (4-HHE) and malondialdehyde (MDA), which can covalently bind proteins and exert toxicity. On the other hand, PUFA oxidation can initially generate various full-chain oxygenated metabolites (aka. oxylipins), and these reactions can be explicitly catalysed by enzymes, such as cyclooxygenases (COXs), lipoxygenases (LOXs) and cytochrome P450s (CYPs) ²⁰, in the presence of antioxidants and with positional specificity, thereby producing metabolites of physiological relevance. Indeed, both early and late-stage PUFA oxidation products exhibit signalling activity; hence it is important to consider the full scope and context of lipid oxidation in vivo for pathological interpretation. To this end, this article seeks to understand the role of PUFA oxidation in atherosclerosis by disentangling adaptive physiology, with a central focus on linoleic acid/n-6 as the major dietary/tissue PUFA, and with comparisons to n-3s.

The oxidative hypothesis

Already in the 1950s lipid peroxidation was detected in human plaque, with much subsequent research supporting the involvement of lipid and protein oxidation in atherogenesis and inspiring causal hypotheses, as comprehensively reviewed elsewhere ²¹. Both early and advanced PUFA peroxidation products are present in lesions; native and MDA/HNE-modified LDL were even found in fetal aortas with/without macrophages, suggesting an early event ²². Oxidised LDL (oxLDL) can also be detected in plasma and associates with CVD ^23,24, although not always independently of apoB (e.g. CHD ²⁵ and MetS ²⁶), potentially due to 4E6 antibody cross-reactivity ²⁷. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB), which normally represent a very small fraction of LDL ²⁸, are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ²⁷. Plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^27,29. Even in the pre-plaque stage, human native LDL injected into rodents appears as oxLDL in blood (after 30mins) ³⁰ and the arterial wall with endothelial activation (within 6hrs) ^31,32, which is suppressed by antioxidants. The high antioxidant capacity of plasma suggests oxidation may occur elsewhere; various catalysts are used in vitro, but most typically LDL is incubated with copper (e.g. as CuSO₄), a transition metal mediating 1-electron oxidations ²¹. In turn, incubation of oxLDL with various cells has many seemingly pro-atherogenic and thrombotic effects, and most characteristically induces macrophage uptake and cholesterol loading via scavenger receptors ²⁴. Further, unlike native LDL, macrophage uptake of oxLDL results in lipid trapping within lysosomes ³³, cholesterol crystallisation and NLRP3 activation ^34,35. Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ³⁶. OxLDL injected into healthy rodents is rapidly cleared by the liver and did not appear in the arterial wall ^31,32. Similarly, in human tracer studies with autologous and copper-oxidised LDL, the latter was cleared more quickly from plasma (T_1/2=85.8 vs. 124mins), although was also detected more frequently (at 1hr) in areas of carotid lesions ³⁷. In a subsequent study advanced carotid plaques (AHA type VI) were excised (at 24–72hrs) post-injection of labelled native LDL and revealed accumulation in foam cells specifically, which was suppressed in those on 4 weeks of high-dose α-tocopherol (aka. vitamin E)—the major endogenous lipophilic antioxidant ³⁸.

Of lipids both UFAs and cholesterol are susceptible to oxidation at their double bonds, of which PUFAs have many and linoleic acid (C18:2n-6) is most abundant in plasma and plaque, particularly cholesteryl esters (CEs) ³⁹. The PUFA/linoleate content of LDL is largely responsible for its oxidative susceptibility; artificially enriching human LDL in SFAs prior to injection into mice blocks its conversion to oxLDL in blood ³⁰. The characteristic atherogenic effects seen in vitro also involve lipid peroxidation; MDA in particular reacts with lysine residues of apoB₁₀₀ (more than 4-HNE) resulting in recognition by scavenger receptors ⁴⁰, and similarly impairs apoA-I efflux activity (vs. other reactive carbonyls) ⁴¹. CE aldehydes also have reduced macrophage hydrolysis ⁴² and may be converted to 7-ketocholesterol ⁴³, which inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ³³, and dose-dependently induces cholesterol crystals ⁴⁴. Supporting the relevance of these mechanisms, such aldehydes are detected in human arterial lesions (e.g. LDL ²², HDL ⁴¹ and CEs ^42,43). In comparing lipoxygenase-derived hydroperoxides to direct MDA–LDL modification, only the latter induced macrophage uptake ⁴⁰. Elsewhere however, such hydroperoxides induced TLR4-dependant macropinocytosis and LDL uptake (incl. native LDL) ⁴⁵, later attributed to oxidised arachidonate ⁴⁶. Further, VLDL is rich in triglycerides which may be released by lipolysis in the arterial wall ⁴⁷, and among fatty acids free linoleic acid can particularly induce endothelial activation ⁴⁷ and barrier disruption, which are inhibited by vitamin E ^48,49. This may involve linoleic peroxidation (via peroxisomes) ⁵⁰ and epoxidation (via CYP2C9) ⁵¹, with further generation of superoxide and peroxynitrite (via eNOS) ¹⁰.

Besides oxidation, in multiple prospective cohorts dietary and plasma antioxidant nutrients (esp. carotenoids and vitamins C/E—largely reflecting fruit/veg and seed oil intake) are inversely associated with CVD risk and mortality, up to present ^52,53. However, the results of large RCTs with high-dose antioxidant supplements in general populations have mostly failed to show benefit ^21,54 (unlike general lipid-lowering), although there is scant outcome data on more physiological and targeted approaches ^54,55. Regardless, this general failure to improve hard outcomes in humans (and animals) with ‘frontline’ antioxidants suggests more complexity ²¹. Foremost, early studies found human plaque lipid oxidation occurs despite no deficiency of antioxidant nutrients, such as α-tocopherol and ascorbate ^21,56—later extended to T2D ⁵⁷. Moreover, oxidation by free transition metals in vitro may have limited analogy in plaque, where at least heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ⁵⁸. On the other hand, early interest turned to 15-LOXs (i.e. non-heme iron-dependent dioxygenases) ⁵⁹, since they can initiate PUFA oxidation and lipoprotein modification ^60,61, which is not blocked by vitamin E ⁶². Accordingly, human plaques express 15-LOXs (i.e. ALOX15 ^60,63 and ALOX15B ⁶¹) and COXs ⁶⁴ within specific macrophage populations. Further, increased iNOS ^65,66 and MPO ⁶⁷, along with lipoprotein enrichment in their protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^68,69, also implicates immuno-oxidative activity ²¹. These 2-electron pathways are also not blocked by vitamin E ⁵⁶ (or serum ⁷⁰), and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptors ^70,71, while MPO-modified tryptophan residues within apoA-I/HDL inactivate its ABCA1-dependent acceptor activity ^69,72.

However, other data present more fundamental challenges ^21,73. For instance, in early studies LDL isolated from plaques was not always sufficiently oxidised for receptor-mediated uptake; rather LDL from human aortic fatty streaks and plaques exhibited increased macrophage uptake in a non-saturable manner attributable to aggregates ⁷⁴. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^74,75 and CE accumulation beyond native or oxLDL ^76,77. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ³³. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁷⁸ and induce macropinocytosis ⁴⁵, lysosomal crystals and NLRP3 activation ^34,35, so may contribute in these ways ⁷⁴. On the other hand, a systematic study of LDL oxidised with copper for 0.5–24hr showed that mild oxidation (>30min) initially inhibits macrophage selective CE uptake and native LDL-induced foam cell formation in relation to apoB fragmentation, before more extensive oxidation (>3hr) induces aggregation, CE oxidation and particle uptake ⁷⁹. Moreover, mild oxidation of HDL by copper, ALOX15 or HOCl (i.e. the product of MPO) actually increases efflux capacity by promoting formation of pre-β-migrating particles ⁸⁰. And contrasting earlier studies ⁸¹, copper-oxidation can induce HDL anti-platelet activity ^82,83 and suppress LDL and VLDL inflammatory signalling ^31,47. Of enzymatic pathways, human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ⁸⁴, consistent with ALOX15 overexpression increasing reverse cholesterol transport ⁸⁵. Thus, the extent and type of oxidation may be important.

Lipid oxygenation and signalling

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some context. Of PUFA oxidation products, linoleic hydro(pero)xides dominate ²¹. In particular, comprehensive analysis of CEs from human peripheral vascular plaques revealed a substantial proportion are oxidised (avg. 21%), with cholesteryl linoleate to the greatest extent (i.e. C18:2 > C20:4 > C22:6), and the most abundant species being full-chain mono- and dioxygenated derivatives (e.g. HODEs, KODEs, EpOMEs, etc.) ⁸⁶. The HODE-CE profile exhibited no regio- or stereo-specificity suggesting a dominance of non-enzymatic peroxidation, although triglyceride PUFAs were not oxidised indicating some specificity ⁸⁶. 15-LOX in particular can directly initiate CE oxidation ⁵⁹, while subsequent radical reactions may erode product specificity ^87,88; and at least in several earlier reports 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ⁸⁹. Conversely, mouse models can lack oxidised CEs despite LOX activity ⁹⁰. Recent high-resolution imaging of advanced human carotid plaques also found oxidised CEs and sphingomyelin concentrate in the necrotic core, while a metabolite resembling 7-ketocholesterol was uncorrelated ⁹¹. Further, from a temporal perspective, a seminal analysis of intimal lipoprotein-containing fractions of aortic lesions from early to late-stage disease found accumulation of cholesterol (AHA types II–III) and CEs (types IV–V) preceded their major oxidised derivatives (i.e. 27-hydroxychoesterol and CE hydro(pero)xides, respectively), while 7-ketocholesterol only increased at late stages (types V–VI), and α-tocopherol and CoQ₁₀ levels remained relatively stable throughout ⁹².

These observations in vivo notably contrast the typical situation in vitro ²¹, where LDL oxidation generates hydroperoxides immediately followed by MDA ^40,71,93, before depletion of CEs with accumulation of 7-ketocholesterol ^43,94. Under such strong oxidising conditions α-tocopherol acts as a chain-breaking antioxidant and its depletion underlies the lipid oxidation lag phase and formation of secondary/advanced oxidation products (e.g. isoprostanes, aldehydes, etc.) ⁹². Conversely, the profile in vivo implies more mildly oxygenating conditions where enzymes and/or the α-tocopherol radical can initiate lipid peroxidation ⁹⁵; the latter being favoured by insufficient regenerative co-antioxidants ⁹⁶ (e.g. CoQ₁₀ and carotenoids) ⁹⁷. In particular, another stage-dependent analysis of whole aortic lesions also included tocopherol oxidation products, which exceeded lipid oxidation in early stages with a pattern implicating 2-electron oxidants and activated monocytes ⁹⁶. Accordingly, MPO/iNOS-derived oxidants can directly induce protein modification and initiate lipid peroxidation, while in mice aortic lesions may lack MPO ⁶⁹.

Of oxysterols, human plaque is generally dominated by 27-hydroxychoesterol (27-HC), followed by 7-ketocholesterol ²¹, although some reports suggested the opposite in human macrophages ^98,99 and absence of 27-HC in animals ¹⁰⁰. 27-HC is produced by mitochondrial sterol 27-hydroxylase (CYP27A1), which is also increased in plaque, particularly macrophages ^101–103. In the liver this enzyme initiates the ‘acidic pathway’ of bile acid synthesis, while elsewhere it can sequentially metabolise cholesterol to water-soluble cholestenoic acid ¹⁰⁴. Moreover, sterol 27-hydroxylase has even greater activity on 7-ketocholesterol (i.e. macrophages ¹⁰⁵ and isolated enzyme ¹⁰⁶) and 27-hydroxylated 7-ketocholesterol was also detected at low levels in human plaque ¹⁰⁵. In extrahepatic tissues this pathway may facilitate efflux by increasing the polarity of cholesterol ¹⁰⁴ and generating ligands for LXR—an oxysterol sensor. In particular, LDL/cholesterol loading of macrophages induces 27-HC and LXR, which may interact directly ¹⁰⁷ and within a feed-forward loop with autophagy ¹⁰⁸; whereas human CTX disorder ^105,107, 7-hydroperoxy-cholesterol ¹⁰⁹ (i.e. 7-ketocholesterol precursor) and isoLGE₂ (i.e. PGH₂ oxidation) ¹¹⁰ can inhibit sterol 27-hydroxylase and efflux. Accumulation of cholesterol in plaque suggests this pathway is insufficient in vivo ¹⁰⁵. At least initially, incubation of human macrophages with human plaque (for 48hrs) induced LXR-dependent efflux transporters, whereas LXR inhibition increased intracellular free cholesterol, CE-SFAs/MUFAs and 27-HC, and endothelial inflammation via IL-6 ⁹⁹. In the absence of LXR inhibition, 27-HC induced ABCA1/IL-1β and lowered IL-6/IL-18BP ⁹⁹, and drives IL-10/M2 polarisation ¹¹¹. On the other hand, recent studies find 27-HC can also induce ROS ¹¹² and inflammation ¹¹³ in human pro-monocytes, and mediate plaque macrophage accumulation in APOE^–/– mice ¹⁰¹; although apoE is actually a target of LXR and required for efficient efflux (i.e. via secreted ¹¹⁴ and exogenous apoE ¹¹⁵).

In human lesion macrophages expression of sterol 27-hydroxylase was accompanied by genes functionally linked in vitro, where RXR/PPARg ligands induced sterol 27-hydroxylase ¹⁰², 27-HC and LXR ¹⁰³. Plaque PPARg expression was also specifically associated with M2 macrophage markers distant from the lipid core ¹¹⁶; although these macrophages actually had suppressed LXRα/ABCA1-dependent efflux, whereas PPARg supported phagocytosis ⁶³. In genetic mouse models macrophage PPARg–LXR signaling is athero-protective ¹¹⁷. From a temporal perspective, tracer studies in APOE^–/– mice revealed accumulation of M2 markers with PPARg and ABCA1 in early stages, whereas M1 markers with HIF-1α and NLRP3 in the advanced plaque microenvironment; suggesting an initial PPARg-based efflux attempt is followed by development of a hypoxic core favouring a shift to glycolysis, inflammation and pyroptosis ¹¹⁸. Conversely, murine athero-regression may involve a shift to M2 and PPARg/LXR activity ^119,120. Regarding natural PPAR ligands, healthy arteries produce various oxylipins, of which COX-derived prostanoids are most abundant ²⁰. Indeed, the first natural PPARg ligand discovered was 15d-PGJ₂, which was subsequently found in human plaque foam cells with COX-2, where it may mediate negative feedback ¹²¹. However, other oxylipins may also be relevant.

As above, accumulation of 27-HC is paralleled by CE hydro(pero)xides ^92,96, which may also have a divergent fate in vivo. While hydroperoxides may induce TLR4-dependant LDL uptake ⁴⁵, macrophages may preferentially hydrolyse oxygenated CEs ¹²² liberating natural PPAR ligands ^89,123,124. In particular, similar to plaque ⁸⁶, plasma LDL from patients with atherosclerosis contained various n-6 oxylipins (i.e. HODEs > HETEs) which activated PPARg at physiological levels in vitro ¹²⁵; although later 15-HETE was shown to prefer PPARβ/δ ¹²³. PPARg primes M2 polarisation in human monocytes, not plaque macrophages ¹¹⁶, but where it may still induce both scavenger receptor CD36 ¹¹⁶ and LXRα/ABCA1-dependent efflux ¹¹⁷, similar to 13-HODE ¹²⁶. Further, while ALOX15B is induced by hypoxia ⁶¹ and mediates cholesterol biosynthesis ¹²⁷, ALOX15 is specifically induced by Th2/M2 cytokines and efferocytosis of apoptotic cells (via LXR ¹²⁸), consistent with a role in lipid/tissue homeostasis ⁶¹. Here IL-4 induced ALOX15 may suppress LXRα/ABCA1-dependent efflux, while PPARg mediates phagocytosis ⁶³ and anti-inflammation ¹²⁹. Conversely, in naive RAW macrophages ALOX15 overexpression increased CE hydrolysis and cholesterol efflux, but not via 15/13S-HETE (and 13S-HODE was undetectable) ⁸⁵. 15-LOX-derived hydroxides undergo reincorporation into specific phospholipids ¹³⁰, with specific functional implications ⁶¹; in particular, macrophage oxidation of CEs from intra- and extracellular sources resulted in 13-HODE–oxPC ⁸⁸. Moreover, macrophages overexpressing 15-LOX also oxidised LDL via (LRP-dependent) selective uptake and efflux of CE linoleate ¹³¹. In the extracellular context, LDL oxidation favoured net transfer of CEs to HDL (via CETP) ¹³², while in rats HDL-associated CE hydro(pero)xides (i.e. [³H]Ch-18:2-O(O)H) were more rapidly removed by liver ¹³³ and excreted in bile (with the radioactivity in bile acids) ¹³⁴, suggesting increased reverse cholesterol transport.

Oxylipins are hydrolysed and reduced by enzymes and exchanges with HDL ¹³⁵, linking lipid oxidation and flux to endogenous antioxidant metabolism. In human carotid plaque linoleate hydroperoxides directly correlated blood HbA1c, while inversely with HDL-C and paraoxonase-1 ¹³⁶, itself an anti-atherogenic enzyme induced by hepatic PPARg ¹³⁷. In addition, low arterial glutathione and related enzyme activity (i.e. GR, GPx and GST) was associated with 4-HNE-related markers and plaque severity ^138,139, while in plasma oxidation of glutathione redox (i.e. GSH/GSSG ratio) was associated with carotid intima–media thickening ¹⁴⁰ independent of traditional markers ¹⁴¹. Furthermore, in various human cohorts lower plasma glycine (a glutathione precursor) associates with metabolic and coronary disease, while causality was shown in APOE^–/– mice on low and high glycine diets ⁵⁵. Specifically, dietary glycine induced glutathione biosynthesis and effector enzymes (incl. GR, GPxs and GSTs), while lowering aortic/macrophage superoxide and atherosclerosis independent of plasma lipids ⁵⁵. Depletion of glutathione and related enzymes precedes plaque formation in APOE^–/– mice ^142,143, and in many studies augmenting glutathione via genes or supplements has a protective effect beyond lipid-lowering and involving macrophages ^30,144–146. Mechanistically, the glutathione system mediates reduction of ascorbate (and consequently tocopherol) and linoleic hydroperoxides (to HODEs) ⁸⁷, as well as conjugation of KODEs ¹⁴⁷ and aldehydes ¹⁴⁸, and as such may regulate PUFA oxidation, signalling and clearance. In macrophages glutathione deficiency increased ROS and CD36 expression independent of PPARg ¹⁴⁹, whereas glutathione supplementation induced efflux and PPARα ¹⁵⁰, and selenium supported IL-4 induced M2 polarisation via GPx1, PPARg and PGD₂ ¹⁵¹. Further, LDL and HDL also contain GPx activity ¹⁴⁵, while oxidised glutathione can inhibit HDL efflux activity via glutathionylation of paraoxonase-1 ¹⁵².

In summary, while advanced lipid oxidation and protein modifications may favour arterial retention, the preponderance of mild cholesterol and PUFA oxidation (i.e. hydroxylation) in plaque might be secondary ²¹ and even support clearance ^{89,92,117,153}. Critically however, this may depend on functional antioxidant networks and transport, or else failed clearance may promote further pathology ^101,113.

Dietary PUFAs on plaque and redox

Given the diversity of linoleate oxidation products in vivo, their differential effects in vitro and the potential involvement of whole-body physiology in CVD, it seems important to consult holistic human data on the impact of diet. As an essential fat, linoleic acid intake moderately correlates tissue levels and in meta-analyses of prospective cohorts higher dietary and blood/adipose linoleate is associated with reduced CVD incidence ¹⁵⁴ and all/CVD/cancer mortality ¹⁵⁵; and notably, some stronger associations were reported with blood CEs ¹⁵⁴—the major lipid in plaque ³⁹. Similarly, circulating long-chain n-3s are also inversely associated with all/CVD/cancer mortality ^156,157. Among diet–heart trials, in the Cochrane meta-analysis the most favourable effect was seen in replacing SFAs with PUFAs (which may include some n-3s ²), and meta-regression implicated reductions in serum cholesterol as a source of heterogeneity ¹⁵⁸. Accordingly, dietary fat saturation has well-characterised quantitative effects on plasma lipids: replacing typical C12–16 SFAs with C18 MUFAs/PUFAs lowers apoB and total/LDL cholesterol, and to a smaller extent triglycerides, with PUFAs having the largest effect ¹⁵⁹, and without significantly affecting lipoprotein(a) ¹⁶⁰. Whereas fish oil/long-chain n-3 PUFA supplementation preferentially lowers triglycerides and non-HDL-cholesterol, particularly in those with hyperlipidemia or overweight/obesity ¹⁶¹. In addition, recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas C18 UFAs lower LDL proteoglycan binding in vitro ^162,163. Clearly all these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite of atherogenesis ¹⁶⁴.

On the other hand, effects on lipoprotein oxidation-related markers seem more contentious. For instance, Lp-PLA₂ hydrolyses oxidised PUFAs, mostly travels with LDL and associates with CVD. In a subset of the healthy MESA cohort (n=2246) plasma phospholipid n-3 and n-6 fatty acids negatively and positivity correlated Lp-PLA₂ mass/activity, respectively ¹⁶⁵. Consistent with this, in a meta-analysis of RCTs n-3s decreased Lp-PLA₂ mass ¹⁶⁶, whereas a trial with soy oil capsules (replacing carbohydrates) increased Lp-PLA₂ activity in association with oxLDL (i.e. 4E6 antibody) and apoB ⁹; although an increase in the latter seems atypical ¹⁵⁹. Moreover, since the early 90s various short-term trials showed MUFA-rich diets (vs. n-6 PUFAs or oily fish/n-3) can lower LDL/HDL lipid oxidation markers, and susceptibility to copper oxidation (i.e. lag time and/or rate) and monocyte adhesion in vitro, which correlated lipoprotein phospholipid oleate/linoleate ratios ^167–169. Thus, competition between C18:1/MUFAs and C18:2/PUFAs for membrane incorporation may modulate substrate for oxidation; similarly, fish oil/long-chain n-3 PUFAs may displace respective long-chain n-6 PUFAs (i.e. C20/22 species) with less double bonds ¹³. Despite this, there are examples in men ¹⁷⁰, monkeys ¹⁷¹ and mice ¹⁷² of n-6 PUFA-rich diets increasing linoleate/oleate ratios in plasma and plaques, and oxidation in vitro ¹⁷¹ and in vivo ^172,173, yet being protective. In humans the long-chain n-3 content of advanced carotid plaques was also increased by supplementation and correlated greater stability and lower inflammatory markers ^174,175. And a transgenic mouse model with deuterium-reinforced PUFAs (d-PUFAs), where deuterium replaced bisallylic hydrogens in C18:2 and C18:3, markedly lowered non-enzymatic isoprostane markers, yet lowered the aortic lesion area (mean –26%) in proportion to plasma non-HDL-C (–28%) and cholesterol absorption ¹⁷⁶ (see below). These PUFA studies are consistent with other dissociations between oxidation and atherosclerosis ²¹.

Importantly, whether dietary PUFAs have an oxidative effect at all depends on the food matrix, and seeds are particularly rich in tocopherols, among other antioxidants. Accordingly, in the long-term LA Veterans trial the experimental seed oil group (vs. SFA-rich control) had much higher intake and blood levels of α-tocopherol, concomitant with a lower erythrocyte susceptibility to peroxide ¹⁷⁰. Further, a 3-week diet of 31% sunflower oil/n-6 PUFAs (vs. olive oil/MUFAs) also lowered LDL levels, oxidation susceptibility and proteoglycan binding, in relation to LDL antioxidant content and size ¹⁷⁷. And in healthy adults an n-6/n-3 PUFA-rich walnut meal (i.e. 59g fat, 42g PUFAs) increased postprandial antioxidant capacity and lowered MDA (5hr AUC) and oxLDL (at 2hrs) ¹⁷⁸, while longer trials show enrichment of PUFAs with preservation of oxidation status ^179,180, alongside many other cardio-protective effects (reviewed in ¹⁸¹). Conversely, food storage and processing can oxidise lipids prior to ingestion. For instance, prolonged heating (i.e. 195°C for 9hrs) of refined tocopherol-depleted soybean oil induced a gradual increase in peroxides before a decline (at 6hrs), while secondary aldehydes continue to increase ¹⁸². When fed to humans oxidised linoleic acid (i.e. conjugated dienes) could be detected in chylomicrons/remnants for 8hrs, with an exaggerated response in diabetics with poor glycaemic control, whereas oxidised cholesterol appeared in all major lipoproteins and persisted for 72hrs; tested in vitro oxidised cholesterol was transferred to LDL and HDL, potentially via CETP ⁶. As such a bolus of oxidised lipids may overwhelm intestinal detoxification, while in T2D baseline glutathione is already low and hydroperoxides elevated, which was ameliorated by glycine-cysteine supplementation ¹⁸³. In obese adults thermo-oxidised sunflower oil/n-6 PUFAs also acutely increased protein carbonyls and lowered plasma glutathione redox (i.e. GSH/GSSG ratio) compared to oils rich in MUFAs and polyphenols ¹⁸⁴. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁶, but also lower blood lipids ¹² and atherosclerosis ¹²⁶, suggesting context may be important. In this regard, 13-HODE was shown to elevate blood lipids and atherosclerosis only in the presence of dietary cholesterol, possibly due to its increased solubilisation and absorption ¹⁸⁵. Interestingly, in a unique RCT on healthy adults (with low TGs), comparing high quality to oxidised fish oil (both 1.6g/day of EPA+DHA) or control (high-oleic sunflower oil/MUFAs) for 7 weeks, only the former lowered IDL/LDL particles and cholesterol content, which correlated CETP ¹⁸⁶. Thus, dietary PUFA oxidation may affect blood lipid responses.

Regarding other dietary pro-oxidants, in humans and mice red meat ingestion also induced postprandial lipid peroxidation and plasma LDL–MDA modification, which was greatly inhibited by polyphenols ^187,188. Animal and in vitro models have localised this oxidative activity to the stomach, which has been conceptualised as a bioreactor that denatures foods and facilitates redox chemistry ¹⁸⁷. Indeed under simulated conditions in vitro, incubation of red meat, metmyoglobin (contains heme-iron as Fe³⁺) or free iron exhibit pro-oxidant activity and can deplete antioxidant vitamins and induce advanced lipid peroxidation (like copper oxidation in vitro), whereas catalysis is inverted to antioxidant activity by polyphenols ¹⁸⁷. The activity of many plant foods in this model has been indexed and correlates polyphenol content ¹⁸⁹; additionally, peroxidation was inhibited by oleate/MUFAs, opposite to fish oil/n-3 PUFAs ¹⁹⁰. Further, in APOE^–/– mice on a high fat diet (60% kcal) with red meat, addition of sunflower oil/n-6 PUFAs induced digestive and plasma 4-HNE and oxLDL, and worsened endothelial dysfunction and atherosclerosis (esp. necrotic core size), all of which was prevented by apple puree or polyphenol extract ⁸. Importantly, supplements containing free transition metals (i.e. iron and copper) can also induce gastrointestinal oxidation in humans ¹⁹¹ and animals ¹⁹², which might particularly confound rodent formula diets where antioxidants lower cholesterol.

A simple PUFA-driven peroxidation paradigm is further challenged by other data. For instance, in a healthy Japanese cohort (n=130, median age=55) plasma oxPC–apoB was modestly and independently associated with LDL-C ²⁸. Plasma PUFAs can also inversely associate with CRP/inflammation; further, in a young Finnish cohort (n=2196, age=24–39) serum PUFAs, particularly n-6 PUFAs, were negatively associated with LDL lipid oxidation and CRP, opposite to SFAs/MUFAs, which withstood adjustment for CVD risk factors and red meat intake ¹⁹³. Moreover, despite long-chain n-3 PUFAs (e.g. DHA/C22:6) being most susceptible to oxidation, meta-analysis of 39 RCTs shows they can improve some peripheral redox markers (i.e. TAC, GPx and MDA) ¹⁸. Conversely, despite the oxidative stability of SFAs and the responsiveness of serum stearate to diet ¹⁹⁴, in short-term trials SFA-rich diets (vs. carbohydrates or MUFAs) can also increase LDL susceptibility to oxidation in relation to MUFA/PUFA ratios ¹⁶⁹, vitamin E ¹⁶⁸, apoB/LDL-C ^195,196 and APOE promoter variants ¹⁹⁶. Excess dairy fat can also favour oxidation in vivo. For instance, in healthy adults a high fat milkshake (vs. low fat) induced pathological RBC remodelling and foamy monocytes, while elevating plasma and RBC-bound MPO in association with impaired flow-mediated dilation (FMD) and chlorination of HDL; tested in vitro major cow milk fatty acids (i.e. oleic or palmitic acid) induced MPO release by monocytes and uptake by porcine arteries ¹⁹⁷. In mice a diet rich in dairy fat/SFAs (i.e. 21% weight) also elevated oxidised HDL and LDL, while replacement with soybean oil/PUFAs (i.e. ~5:1 of n-6:n-3) enhanced HDL antioxidant and Lp-PLA₂ activity, without affecting macrophage–faeces reverse transport (or paraoxonase-1) ¹⁹⁸. Elsewhere however, a diet rich in palm oil/SFAs (i.e. 45% kcal) vs. sunflower oil/MUFAs enriched the liver and HDL in acute-phase proteins and lowered paraoxonase-1 and faecal cholesterol excretion ¹⁹⁹.

Mechanistically, SFAs may affect lipid oxidation indirectly. For instance, in an animal model lipoprotein susceptibility to oxidation increased with particle age (i.e. plasma residence) ²⁰⁰, while in human tracer studies PUFAs (vs. SFAs) lower plasma lipids and increase LDL catabolism ²⁰¹. LDL susceptibility to oxidation is also associated with small particle size (i.e. pattern B), which is itself increased by insulin resistance ²⁰²; in people with pattern B a SFA-rich diet (vs. MUFAs) increased small–medium LDL particles ²⁰³, while a meta-analysis of RCTs suggests exchanging SFAs for n-6 PUFAs may particularly improve glucose-insulin homeostasis ²⁰⁴. Regarding inflammation, human carotid plaque contains LPS from E.coli, which at similar levels in vitro induced TLR4/NOX2-dependent LDL oxidation; plaque LPS was also associated with plasma LPS and zonulin, implicating the gut microbiome as a source ²⁰⁵. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ²⁰⁶, which at similar levels in vitro also induces LDL oxidation ²⁰⁷. Moreover, the bioactivity of LPS is actually mediated by acylated SFAs, which at high levels in vitro can directly induce TLR2/4 signalling, opposite to n-3 PUFAs ²⁰⁸. Dietary fats may also modulate glycine-dependent redox via the gut microbiome. For instance, in twins an 8-week vegan diet (vs. omnivorous with a higher SFA/PUFA ratio) lowered faecal Bilophila wadsworthia in association with fasting insulin and increased serum glycine; tested in vitro B. wadsworthia consumed glycine (in Stickland fermentation) and its removal from mice increased serum glycine and hepatic Gstt2 expression, while decreasing body weight and LDL-C ²⁰⁹. Accordingly, SFA intake is associated with Bilophila abundance ^210–212. In mice a milk fat/SFA-rich diet (vs. safflower oil/n-6 PUFAs) induced B. wadsworthia by favouring secretion of taurine-conjugated bile acids to fuel sulfite-based respiration, which was offset by supplementation of fish oil/n-3 PUFAs ²¹³. Of note, in people with moderate hypercholesterolemia exchanging SFAs (6.5% kcals) for mostly n-6 PUFAs for 8 weeks did not significantly increase serum glycine, although in multivariate analysis it was a highly ranked variable of importance ²¹⁴. In other trials the individual response to SFAs was related to baseline Bilophila ²¹⁰ and diet ²¹¹, which may be sources of heterogeneity.

In summary, PUFA-rich diets increase the major lipid substrate for peroxidation in general, although how and where this occurs depends on the food matrix and dietary pattern. For instance, even prior to absorption non-enzymatic peroxidation is modulated by the balance of pro-oxidants (e.g. heat and heme-iron) vs. antioxidants (e.g. vitamins and polyphenols) during food processing and digestion. Moreover, dietary fats may also modulate systemic redox indirectly via metabolism (e.g. lipoprotein turnover and phenotype) and microbiome (e.g. LPS and glycine), where SFAs may particularly lower antioxidant status and favour immune-mediated oxidation.

PUFA oxidation-dependant homeostasis?

While increased PUFA intake and tissue content, especially of linoleate, is typically considered an oxidative liability ^2,5–9,176, could there be some more favourable effects? For instance, in healthy human trials replacing SFAs with mostly n-6 PUFAs lowers plasma cholesterol, while inducing serum bile acids and PBMC transcripts related to cholesterol uptake (e.g. LDLR/TLR4) and efflux (e.g. LXRα/ABCG1) ^214,215. Elsewhere, lowering dietary linoleic acid/n-6 PUFA (vs. mostly SFAs) also lowered respective plasma oxylipins (i.e. HODEs and KODEs) ²¹⁶. Linking these effects, linoleate oxygenation may induce PPARg, 27-HC and reverse transport, as above. Upon return to the liver, these oxidised lipids may similarly induce LXRα to increase bile output and plasma cholesterol uptake (i.e. LDLR expression), underlying clinical effects of n-6 PUFAs (incl. lower HDL-C) ^214,217. Regarding major sources of linoleate hydro(pero)xides in vivo, isomer analyses of both human plaque ⁸⁶ and healthy fasting plasma ²¹⁸ suggest a dominance of radical-mediated oxidation, while preclinical studies implicate cell-mediated oxidation, as earlier. From a physiologic perspective, the specificity of LCAT for linoleate ²¹⁹ may support CE fluidity ³⁹ and substrate for LOX ⁸⁸, while oxygenation (i.e. hydroxylation) of CEs/cholesterol may increase polarity/solubility to facilitate protein interactions and ‘fast-track’ transport to the liver ^104,105,134, before more extensive oxygenation to bile acids. Within the arterial wall, LDL accumulation and oxidation may induce endothelial activation and monocyte recruitment ^31,32, wherein PPARg–LXR signalling might then orchestrate lipid clearance ¹¹⁷. Further, plasma linoleate ¹⁹³ and HODEs ²²⁰ have been inversely associated with inflammatory markers in humans, while PPARg ¹¹⁶ and 27-HC ¹¹¹ may favour M2 polarisation in vitro, possibly supporting immune resolution.

By comparison, in women with obesity supplementation of n-3 PUFAs (~5:1 DHA:EPA) for 3 months lowered triglycerides (not cholesterol), insulin and inflammatory markers, while inducing PPARα and Nrf2-related antioxidant genes (incl. HO-1) ¹⁹; similar to mice on a high fat diet supplemented with DHA (alone/with EVOO) ²²¹. In human trials fish oil/n-3 PUFAs also increase plasma early peroxidation products (e.g. HDHAs) ^13,14,16,17 and downstream pro-resolving mediators (e.g. resolvins) ^15,19, at the expense of arachidonic-derived oxylipins ^13,15. Early cell studies found (ambient) oxidation of EPA was required for inhibition of (cytokine-induced) NF-κB, which also required PPARα ²²²; more recently, 7-HDHA (formed via ALOX5) was identified as a high-affinity PPARα ligand regulating brain morphology ²²³. In the periphery n-3 PUFA induction of PPARα may also support triglyceride lowering ²¹⁷ via suppression of SREBP-1 (which mediates hepatic lipogenesis) ²²⁴ and apoC-III (which inhibits VLDL lipolysis) ²²⁵. Further, in animal models dietary oxidised linoleate can also lower hepatic and plasma triglycerides via PPARα ¹². Presumably differences in n-6 and n-3-related lipid-lowering may relate to their differential lipid distribution, oxidative metabolism and signalling specificity. Also, in T2D ⁸³ and coronary syndrome ²²⁶ the anti-platelet activity of HDL was associated with content of several n-6 and n-3 hydroxides and causality was similarly shown in vitro (i.e. for HODEs, HEPEs and HDHAs).

Notably, the protective effects of linoleic acid/n-6 PUFAs (vs. MUFAs) on atherosclerosis in LDLR^–/– mice persisted even when switching to a cholesterol/SFA-rich diet, and lesions negatively correlated plasma isoprostanes (i.e. 8-iso-PGF_2α) and aortic catalase (decomposes H₂O₂), implicating oxidative stress-induced hormesis ¹⁷³. Accordingly, in vitro 13-HpODE/HODE induced catalase in several arterial cells ²²⁷; catalase is also regulated by PPARg ²²⁸ and blocks MPO-induced oxidation ⁷¹. As above, the ability of n-3 PUFAs to improve redox markers ¹⁸ may involve Nrf2 ^19,221, which induces 100s of genes supporting redox homeostasis (incl. glutathione, HO-1, catalase, etc.). Several n-3 oxylipins can activate Nrf2, such as 17-oxo-DHA, resolvins and maresins ²²⁹. In addition, early cell studies found radical-mediated oxidation of EPA and DHA was required for induction of Nrf2–HO-1 (in contrast to sulforaphane), and implicated formation of J₃-isoprostanes ²³⁰. Later, in mice fish oil/n-3 PUFAs increased aortic HO-1 expression and vasodilation, which were abolished by Nrf2 deletion; tested in vitro DHA-derived 4-HHE induced Nrf2–HO1 ²³¹. Fish oil/n-3 PUFAs were further shown to induce 4-HHE and HO-1 in multiple organs, while safflower oil/n-6 PUFAs did not ²³². However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ²³³. LPS induces various oxylipins ²⁰ sensitive to n-3 status ¹⁶; and n-6 series Nrf2-inducers include EKODE ²³⁴, 15d-PGJ₂ ²³⁵ and LXA₄ ²²⁹. Moreover, low-level 4-HHE and 4-HNE (from n-6 PUFAs) similarly induce Nrf2 in endothelial cells ²³⁶, and in APOE^–/– mice on a high fat diet endothelial inflammation and 4-HNE precede Nrf2 activation, which then appears to exert negative feedback regulation and restrain atherosclerosis ²³⁷.

Turning to macrophages, efferocytosis of apoptotic cells is impaired in human atherosclerosis and vital for limiting secondary necrosis, inflammation and plaque progression ²³⁸. In human plaque Nrf2 expression was highest in macrophages in the lipid core and paralleled Myh9, while in preclinical studies macrophage Nrf2 deficiency increased plaque severity, necrotic core size and accumulation of apoptotic cells, whereas Nrf2–Myh9 binding supported efferocytosis via the actin cytoskeleton ²³⁹. Similarly, 15d-PGJ₂ and 17-oxo-DHA (both formed via COX-2) augmented efferocytosis via Nrf2/HO1-dependent expression of CD36 ²⁴⁰, LOX/COX-2 and pro-resolving mediators ²⁴¹, which may themselves activate Nrf2 ²²⁹. Furthermore, oxLDL/4-HNE ²⁴² and 15d-PGJ₂ ²⁴⁰ induce Nrf2-dependent CD36 expression independent of PPARg ^242,243 (similar to GSH depletion ¹⁴⁹), while phytochemicals induce Nrf2-dependent efflux via suppression of NF-κB signalling ²⁴⁴ and induction of SR-B1 and ABCA1/G1 transporters ^245–247; thus, Nrf2 may support cholesterol clearance in parallel to PPARg ¹¹⁷. Of note, macrophage Nrf2 status may have even broader impact: foam cell-derived exosomes were shown to propagate redox imbalance to brain microglia via Nrf2 exacerbating white matter injury and cognitive impairment ²⁴⁸.

Perhaps cellular PUFA status could lower the threshold for Nrf2 induction under oxidative conditions, favouring earlier feedback and pleiotropic regulation of redox, immune and lipid homeostasis, thereby limiting plaque growth and instability. On the other hand, excess unsaturated aldehydes are ultimately toxic; indeed while low-level 4-HNE induces Nrf2 and supports homeostasis, high levels block Nrf2 and favour apoptosis ²⁴⁹. In women supplemented with n-6 or n-3, LDL isolated and oxidised (via copper) from the latter group had less apoptotic activity on pro-monocytes, mirroring differential effects of HNE vs. HHE, respectively ²⁵⁰. However, in LPS-treated mice 4-HNE inhibited inflammasome activation and pyroptosis; tested in vitro this was independent of its effects on Nrf2/NF-κB signalling, but may involve direct binding to NLRP3 ²⁵¹—another major pathway mediating experimental atherosclerosis ³⁵. In mammalian cells 4-HNE is normally cleared extremely rapidly, especially by hepatocytes and enterocytes, and via several pathways including glutathione conjugation ²⁵². Thus, induction of Nrf2/glutathione would provide feedback inhibition and moderate oxylipin/aldehyde levels, which could be reinforced by glycine availability, as earlier. This may also support hormetic effects; for instance, 27-HC induces autophagy via ROS/Nrf2 favouring cell survival ¹¹². Conversely, in metabolic disorders and highly stressed cells Nrf2/glutathione exhaustion may favour apoptosis, which itself could indirectly exert homeostatic pressure via oxPL-dependent efferocytosis and subsequent induction of ALOX15-dependent pro-resolving mediators ^60,61. In particular, enriching neutrophil-like cells and their phospholipids in linoleic acid did not affect peroxide-induced apoptosis per se, but increased efferocytosis of intrinsic apoptosis via surface display of oxPS, which was enriched in di-oxygenated linoleate species, and subject to hydrolysis and abrogation by Lp-PLA₂ ²⁵³.

Further homeostatic insight may lie in other perspectives; for instance, the effects of PUFAs may somewhat overlap with exercise ¹⁷³. Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ²⁵⁴. In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ¹⁰⁰, as well as hepatic LXR and reverse cholesterol transport ²⁵⁵. Further, in LDLR^–/– mice on a high fat diet exercise training reversed endothelial (vasodilatory) dysfunction by increasing eNOS/nitric oxide and nNOS/hydrogen peroxide, while lowering NOX2/superoxide and inducing superoxide dismutase ²⁵⁶. And in a similar mouse model exercise induced aortic catalase activity and eNOS, and lowered plasma cholesterol and atherosclerosis, all of which was thwarted by high-dose vitamin E (human equivalent 1000iu) ²⁵⁷. This aligns with the more general finding that high-dose antioxidants (typically vitamins C and E) can block beneficial metabolic and functional adaptations to exercise in humans and mice ²⁵⁴, which may be mediated in large part by ROS/Nrf2 ²⁵⁸. Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ²²⁰, isoprostanes and aldehydes ²⁵⁹), and preferentially in HDL ²⁶⁰, while some recent studies suggest a synergistic effect of exercise training and n-3 PUFA supplementation on antioxidant status, lipids and performance, among other factors ^261,262. Further, both cold exposure and moderate exercise induce release of linoleic-derived 12,13-diHOME (an epoxide synthesised via CYP) from brown adipose to stimulate fatty acid uptake and reduce serum triglycerides ¹¹.

In considering optimal PUFA intake for tissue homeostasis, post-industrial dietary and tissue linoleic acid/n-6 may seem high ^2,5, although still has favourable health associations ^154,155, but long-chain n-3s more so by mass ^156,157. Besides seed oils, many nuts are also rich in linoleate (e.g. walnuts, pinenuts, brazils, pecans and mongongo) and of potential native/ancestral relevance. Among fatty acids, linoleic acid offers unique lipid-lowering effects, even in healthy individuals. In the diet C18:2/n-6 competes with SFAs/MUFAs, while in the body with C18:1/MUFAs for esterification and C18:3/n-3 for elongation. However, in some individuals dietary linoleate may also increase arachidonic acid and inflammation via FADS1 variants affecting n-6 desaturases ²⁶³—here the dietary n-6/n-3 balance may become more important. Long-chain n-3s also appear to have substantial plaque ^174,175 and cell membrane incorporation in humans (e.g. the omega-3 index) and greater susceptibility to non-enzymatic oxidation, so may support earlier Nrf2 activation and feedback inhibition ²³², and moderate effects of 4-HNE ²⁵⁰. Of note, despite the relative oxidative stability of MUFAs, extra virgin olive oil may also support antioxidant/Nrf2 activity via its polyphenol content ²²¹, which is much higher in whole olives ²⁶⁴ (along with sodium).

Importantly, the ability of PUFA oxidation to favour homeostasis may also rest upon the site of oxidation. In the body oxidation products induce antioxidant and detoxification systems, thereby limiting further oxidation and maintaining spatiotemporal control over subcellular/organelle-specific ROS/RNS generation and transient/gradient redox signalling. In contrast, during food processing and digestion the extent of PUFA oxidation essentially depends entirely on the chemistry of the food matrix and stomach before absorption by the body, wherein resulting peroxidation products can apparently overwhelm intestinal detoxification and incorporate into plasma lipoproteins for delivery to tissues ^6,187,188. Dietary thermo-oxidised oils can induce PPARs ¹² and Nrf2 ^184,265,266, but alongside signs of inflammation, antioxidant depletion and DNA damage ²⁶⁷. Sufficient lipoprotein modification/damage may also result in rapid clearance by phagocytes, largely in the liver ^31,32, but also potentially arterial plaque ^8,37. Thus, excessive oxidation ‘ex vivo’ may eventually overwhelm homeostasis and promote pathogenesis in vivo. Notably, dietary oil oxidation typically involves prolonged storage ²⁶⁸ or heating ^182,184,265, whereas red meat may induce significant advanced oxidation within the normal digestive/postprandial phase ^187,188, which is exaggerated by addition of PUFA-rich oils ^8,190, suggesting it may be particularly relevant and a potential confounder in PUFA studies. For instance, many old CVD trials had heterogeneous outcomes and reduced saturated fat via replacement with isolated seed oils to be used for cooking and incorporation into provided ‘filled’ foods ⁴, which included sausage products (i.e. Veterans study ¹⁷⁰), filled beef (i.e. Minnesota study ²), and more recently liver pâté ²¹⁴, suggesting direct contact with heme-iron. However, this could represent a small proportion of oil consumed and be offset by other dietary components.

Conclusions

PUFA peroxidation is associated with human atherosclerosis and induces toxic effects in vitro, supporting a pathogenic view and implicating the post-industrial increase in tissue linoleate. However, this clashes with much outcome and experimental data, hence this review sought to explore reconciliation through a more physiological perspective by attention to oxidative specificity and signalling in vivo. The theory arose that enzymatic and non-enzymatic oxidation products of both n-6 and n-3 may couple to adaptive responses, particularly via PPARs and Nrf2 signalling; consequently, even advanced peroxidation products might initially induce hormesis before toxicity. This could have some analogy and synergy with exercise, which also generally benefits cardiovascular health. As such, the inherent susceptibility of PUFAs to oxidation may not be dichotomous with their health benefits, but even underlie favourable modulation of redox, immune and lipid homeostasis—and opposite to typical SFAs—ultimately supporting efflux and efferocytosis to limit plaque growth and instability. However, in acknowledging the negative effects of excessive peroxidation, adaptive responses may be undermined by sufficient redox dysregulation due to antioxidant (e.g. glutathione) deficiency and exogenous oxidation, especially in those with metabolic disorders. In particular, lipid peroxidation during food processing and/or digestion effectively bypasses the opportunity for physiologic signalling and feedback inhibition, thereby allowing accumulation of end products and increasing the potential for toxicity. Notably, this situation seems most analogous to the typical oxidative conditions used to create atherogenic lipoproteins in vitro, and thus most harmonious with the traditional oxidative stress hypothesis—i.e. here a susceptibility to oxidation may confer a susceptibility to atherosclerosis.

This review has notable limitations. Its mechanistic and theoretical nature make it susceptible to the reductionist trap; an attempt was made to follow human data, oxidative specificity and systems-level interaction, although other pathways will exist. Its narrative synthesis is also inevitably limited by personal bias and incomplete research; for instance, future studies could particularly investigate the role of endogenous linoleic oxidation on lipid-lowering and immune polarisation, among other areas. Moreover, heterogeneity and inconsistencies within the current research literature can challenge this and any coherent perspective at all; as such, some potential counterviews and reconciliations are summarised and listed in the supplement. Regarding practical implications, this qualitative review subserves more quantitative human outcome data and simply suggests oxidative metabolism of n-6 and n-3 may underlie complementary and overlapping health benefits, but which could be modified by dysregulated oxidation. In the natural context, wholefood plant-based PUFAs may be least susceptible to oxidation ex vivo, while still providing substrate for favourable oxidation in vivo; whereas in the post-industrial era the extent of any isolated oil peroxidation will be highly dependent on the degree of processing and dietary context. Controlling for these factors in human and animal studies may help refine and homogenise the evidence base; nonetheless, dietary guidelines already typically favour whole plant foods over processed foods and red meat, which may help safeguard PUFA quality.

Supplementary

• Diet–heart trials have inconsistent outcomes—This may be explained by methodological heterogeneity introducing many confounding factors (e.g. trans fats, demographics, adherence, duration, etc.) as discussed by others ^1–4 and herein (e.g. filled foods).
• Observational and interventional studies with antioxidant vitamins are discordant ^21,54—The trials typically target non-deficient antioxidants with synthetic forms and/or supra-physiological doses, which may displace other fat-soluble nutrients and disrupt redox networks, so do not necessarily invalidate the epidemiology or oxidative hypothesis. Other approaches seem more promising ^54,55.
• In many cell studies lipoprotein and PUFA oxidation seem atherogenic—The typical extreme oxidative conditions in vitro may not model the typical situation in vivo ²¹, except for some specific areas addressed herein (e.g. hemolysis and digestion). The extreme isolation in vitro may also not capture systemic physiology. For instance, free linoleic acid induces endothelial activation, but this is offset by PPARg ²⁶⁹ and involves epoxidation ⁵¹, which can lower systemic triglycerides ¹¹. Beyond endothelial cells, typical SFAs can also induce cytotoxicity and inflammation ²⁰⁸.
• Recent preclinical studies suggest the CYP27A1/27-HC pathway is mainly pro-atherogenic ^101,113—This view does not reconcile increased CYP27A1 expression with hyperlipidemia, PPARg ¹⁰³ or exercise ¹⁰⁰; CYP27A1 mediated efflux of cholesterol ^104,107 and 7-ketocholesterol ^105,106; 27-HC-induced cell survival (via Nrf2/autophagy) ¹¹² and efflux/immune modulation (via LXR) ^99,111; nor human CTX pathophysiology ^105,107. On the other hand, since apoE supports LXR-dependent efflux ^114,115 typical APOE^–/– mouse models ¹⁰¹ may have an artificial bottleneck. By analogy, in human macrophages exposed to human plaque, LXR inhibition reduced apoE secretion and increased intracellular 27-HC (not in supernatant), while favouring endothelial inflammation via IL-6 ⁹⁹.
• In mouse studies the role of Nrf2 is inconsistent—Specifically, global Nrf2 knockout ameliorates atherosclerosis, but in relation to suppression of hepatic and blood lipids ²³⁹, whereas arterial cell-specific modulation suggests Nrf2 has a protective effect (e.g. endothelium ²³⁷ and macrophages ²³⁹). Dietary PUFAs may actually enable the best of both by lowering lipids and inducing Nrf2.
• In a transgenic mouse model d-PUFAs lowered the aortic lesion area ¹⁷⁶—This was more in proportion to a reduction in plasma non-HDL-C and cholesterol absorption, rather than isoprostanes, and is therefore more consistent with the lipid hypothesis. As a possible mechanism, rodent formulas often contain ‘free’ transition metals which may catalyse digestive oxidation ¹⁹², while hydroperoxides may increase cholesterol absorption and atherosclerosis ¹⁸⁵.

References

1.           Sacks, F. M. et al. Dietary Fats and Cardiovascular Disease: A Presidential Advisory From the American Heart Association. Circulation 136, e1–e23 (2017).

2.           Ramsden, C. E. et al. Re-evaluation of the traditional diet-heart hypothesis: analysis of recovered data from Minnesota Coronary Experiment (1968-73). BMJ 353, i1246 (2016).

3.           Yamada, S. et al. Saturated Fat Restriction for Cardiovascular Disease Prevention: A Systematic Review and Meta-analysis of Randomized Controlled Trials. JMA J. 8, 395–407 (2025).

4.           Hamley, S. The effect of replacing saturated fat with mostly n-6 polyunsaturated fat on coronary heart disease: a meta-analysis of randomised controlled trials. Nutr. J. 16, 30 (2017).

5.           DiNicolantonio, J. J. & O’Keefe, J. H. Omega-6 vegetable oils as a driver of coronary heart disease: the oxidized linoleic acid hypothesis. Open Hear. 5, e000898 (2018).

6.           Staprans, I., Pan, X.-M., Rapp, J. H. & Feingold, K. R. The role of dietary oxidized cholesterol and oxidized fatty acids in the development of atherosclerosis. Mol. Nutr. Food Res. 49, 1075–82 (2005).

7.           Lawrence, G. D. Perspective: The Saturated Fat–Unsaturated Oil Dilemma: Relations of Dietary Fatty Acids and Serum Cholesterol, Atherosclerosis, Inflammation, Cancer, and All-Cause Mortality. Adv. Nutr. 1–10 (2021). doi:10.1093/advances/nmab013

8.           Bolea, G. et al. Digestive n-6 Lipid Oxidation, a Key Trigger of Vascular Dysfunction and Atherosclerosis in the Western Diet: Protective Effects of Apple Polyphenols. Mol. Nutr. Food Res. 65, e2000487 (2021).

9.           Kim, M. et al. Impact of 8-week linoleic acid intake in soy oil on Lp-PLA2 activity in healthy adults. Nutr. Metab. (Lond). 14, 32 (2017).

10.        Saraswathi, V., Wu, G., Toborek, M. & Hennig, B. Linoleic acid-induced endothelial activation: role of calcium and peroxynitrite signaling. J. Lipid Res. 45, 794–804 (2004).

11.        Stanford, K. I. et al. 12,13-diHOME: An Exercise-Induced Lipokine that Increases Skeletal Muscle Fatty Acid Uptake. Cell Metab. 27, 1111-1120.e3 (2018).

12.        Ringseis, R. & Eder, K. Regulation of genes involved in lipid metabolism by dietary oxidized fat. Mol. Nutr. Food Res. 55, 109–21 (2011).

13.        Ramsden, C. E. et al. Dietary alteration of n-3 and n-6 fatty acids for headache reduction in adults with migraine: randomized controlled trial. BMJ 374, n1448 (2021).

14.        Zamora, D. et al. A High Omega-3, Low Omega-6 Diet Reduces Headache Frequency and Intensity in Persistent Post-Traumatic Headache: A Randomized Trial. J. Neurotrauma 42, 1719–1731 (2025).

15.        Oakes, E. G. et al. Joint effects of one year of marine omega-3 fatty acid supplementation and participant dietary fish intake upon circulating lipid mediators of inflammation resolution in a randomized controlled trial. Nutrition 123, 112413 (2024).

16.        Walker, R. E. et al. Effect of omega-3 ethyl esters on the triglyceride-rich lipoprotein response to endotoxin challenge in healthy young men. J. Lipid Res. 64, 100353 (2023).

17.        So, J. et al. EPA and DHA differentially modulate monocyte inflammatory response in subjects with chronic inflammation in part via plasma specialized pro-resolving lipid mediators: A randomized, double-blind, crossover study. Atherosclerosis 316, 90–98 (2021).

18.        Heshmati, J. et al. Omega-3 fatty acids supplementation and oxidative stress parameters: A systematic review and meta-analysis of clinical trials. Pharmacol. Res. 149, 104462 (2019).

19.        Polus, A. et al. Omega-3 fatty acid supplementation influences the whole blood transcriptome in women with obesity, associated with pro-resolving lipid mediator production. Biochim. Biophys. Acta 1861, 1746–1755 (2016).

20.        Edin, M. L. et al. Vascular Lipidomic Profiling of Potential Endogenous Fatty Acid PPAR Ligands Reveals the Coronary Artery as Major Producer of CYP450-Derived Epoxy Fatty Acids. Cells 9, (2020).

21.        Stocker, R. & Keaney, J. F. Role of oxidative modifications in atherosclerosis. Physiol. Rev. 84, 1381–478 (2004).

22.        Napoli, C. et al. Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions. J. Clin. Invest. 100, 2680–90 (1997).

23.        Gao, S. & Liu, J. Association between circulating oxidized low-density lipoprotein and atherosclerotic cardiovascular disease. Chronic Dis. Transl. Med. 3, 89–94 (2017).

24.        Ishigaki, Y., Oka, Y. & Katagiri, H. Circulating oxidized LDL: a biomarker and a pathogenic factor. Curr. Opin. Lipidol. 20, 363–9 (2009).

25.        Wu, T. et al. Is plasma oxidized low-density lipoprotein, measured with the widely used antibody 4E6, an independent predictor of coronary heart disease among U.S. men and women? J. Am. Coll. Cardiol. 48, 973–9 (2006).

26.        Koskinen, J. et al. Apolipoprotein B, oxidized low-density lipoprotein, and LDL particle size in predicting the incidence of metabolic syndrome: the Cardiovascular Risk in Young Finns study. Eur. J. Prev. Cardiol. 19, 1296–303 (2012).

27.        Tsimikas, S. & Witztum, J. L. Oxidized phospholipids in cardiovascular disease. Nat. Rev. Cardiol. 21, 170–191 (2024).

28.        Shoji, T. et al. Inverse relationship between circulating oxidized low density lipoprotein (oxLDL) and anti-oxLDL antibody levels in healthy subjects. Atherosclerosis 148, 171–7 (2000).

29.        Itabe, H., Obama, T. & Kato, R. The Dynamics of Oxidized LDL during Atherogenesis. J. Lipids 2011, 418313 (2011).

30.        Cui, Y. et al. N-acetylcysteine inhibits in vivo oxidation of native low-density lipoprotein. Sci. Rep. 5, 16339 (2015).

31.        Niemann-Jönsson, A. et al. Accumulation of LDL in rat arteries is associated with activation of tumor necrosis factor-alpha expression. Arterioscler. Thromb. Vasc. Biol. 20, 2205–11 (2000).

32.        Calara, F. et al. An animal model to study local oxidation of LDL and its biological effects in the arterial wall. Arterioscler. Thromb. Vasc. Biol. 18, 884–93 (1998).

33.        Maor, I., Mandel, H. & Aviram, M. Macrophage uptake of oxidized LDL inhibits lysosomal sphingomyelinase, thus causing the accumulation of unesterified cholesterol-sphingomyelin-rich particles in the lysosomes. A possible role for 7-Ketocholesterol. Arterioscler. Thromb. Vasc. Biol. 15, 1378–87 (1995).

34.        Sheedy, F. J. et al. CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation. Nat. Immunol. 14, 812–20 (2013).

35.        Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–61 (2010).

36.        La Ville, A. E., Sola, R., Balanya, J., Turner, P. R. & Masana, L. In vitro oxidised HDL is recognized by the scavenger receptor of macrophages: implications for its protective role in vivo. Atherosclerosis 105, 179–89 (1994).

37.        Iuliano, L. et al. Preparation and biodistribution of 99m technetium labelled oxidized LDL in man. Atherosclerosis 126, 131–41 (1996).

38.        Iuliano, L., Mauriello, A., Sbarigia, E., Spagnoli, L. G. & Violi, F. Radiolabeled native low-density lipoprotein injected into patients with carotid stenosis accumulates in macrophages of atherosclerotic plaque : effect of vitamin E supplementation. Circulation 101, 1249–54 (2000).

39.        Small, D. M. George Lyman Duff memorial lecture. Progression and regression of atherosclerotic lesions. Insights from lipid physical biochemistry. Arteriosclerosis 8, 103–29 (1988).

40.        Lankin, V. Z., Tikhaze, A. K. & Melkumyants, A. M. Malondialdehyde as an Important Key Factor of Molecular Mechanisms of Vascular Wall Damage under Heart Diseases Development. Int. J. Mol. Sci. 24, (2022).

41.        Shao, B. et al. Modifying apolipoprotein A-I by malondialdehyde, but not by an array of other reactive carbonyls, blocks cholesterol efflux by the ABCA1 pathway. J. Biol. Chem. 285, 18473–84 (2010).

42.        Hoppe, G., Ravandi, A., Herrera, D., Kuksis, A. & Hoff, H. F. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions. J. Lipid Res. 38, 1347–60 (1997).

43.        Kawai, Y. et al. Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis. J. Biol. Chem. 278, 21040–9 (2003).

44.        Geng, Y.-J., Phillips, J. E., Mason, R. P. & Casscells, S. W. Cholesterol crystallization and macrophage apoptosis: implication for atherosclerotic plaque instability and rupture. Biochem. Pharmacol. 66, 1485–92 (2003).

45.        Choi, S.-H. et al. Lipoprotein accumulation in macrophages via toll-like receptor-4-dependent fluid phase uptake. Circ. Res. 104, 1355–63 (2009).

46.        Choi, S.-H. et al. Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages. PLoS One 8, e83145 (2013).

47.        Dichtl, W. et al. Very low-density lipoprotein activates nuclear factor-kappaB in endothelial cells. Circ. Res. 84, 1085–94 (1999).

48.        Hennig, B. et al. Linoleic acid activates nuclear transcription factor-kappa B (NF-kappa B) and induces NF-kappa B-dependent transcription in cultured endothelial cells. Am. J. Clin. Nutr. 63, 322–8 (1996).

49.        Hennig, B., Boissonneault, G. A. & Wang, Y. Protective effects of vitamin E in age-related endothelial cell injury. Int. J. Vitam. Nutr. Res. 59, 273–9 (1989).

50.        Hennig, B. et al. Effect of vitamin E on linoleic acid-mediated induction of peroxisomal enzymes in cultured porcine endothelial cells. J. Nutr. 120, 331–7 (1990).

51.        Viswanathan, S. et al. Involvement of CYP 2C9 in mediating the proinflammatory effects of linoleic acid in vascular endothelial cells. J. Am. Coll. Nutr. 22, 502–10 (2003).

52.        Jayedi, A., Rashidy-Pour, A., Parohan, M., Zargar, M. S. & Shab-Bidar, S. Dietary and circulating vitamin C, vitamin E, β-carotene and risk of total cardiovascular mortality: a systematic review and dose-response meta-analysis of prospective observational studies. Public Health Nutr. 22, 1872–1887 (2019).

53.        Aune, D. et al. Dietary intake and blood concentrations of antioxidants and the risk of cardiovascular disease, total cancer, and all-cause mortality: a systematic review and dose-response meta-analysis of prospective studies. Am. J. Clin. Nutr. 108, 1069–1091 (2018).

54.        An, P. et al. Micronutrient Supplementation to Reduce Cardiovascular Risk. J. Am. Coll. Cardiol. 80, 2269–2285 (2022).

55.        Rom, O. et al. Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis. Redox Biol. 52, 102313 (2022).

56.        Kontush, A., Chapman, M. J. & Stocker, R. Vitamin E is not deficient in human atherosclerotic plaques. Arterioscler. Thromb. Vasc. Biol. 24, e139-40; author reply e141-2 (2004).

57.        Kathir, K. et al. Equivalent lipid oxidation profiles in advanced atherosclerotic lesions of carotid endarterectomy plaques obtained from symptomatic type 2 diabetic and nondiabetic subjects. Free Radic. Biol. Med. 49, 481–6 (2010).

58.        Cornelissen, A., Guo, L., Sakamoto, A., Virmani, R. & Finn, A. V. New insights into the role of iron in inflammation and atherosclerosis. EBioMedicine 47, 598–606 (2019).

59.        Takahashi, Y., Zhu, H. & Yoshimoto, T. Essential roles of lipoxygenases in LDL oxidation and development of atherosclerosis. Antioxid. Redox Signal. 7, 425–31 (2005).

60.        Singh, N. K. & Rao, G. N. Emerging role of 12/15-Lipoxygenase (ALOX15) in human pathologies. Prog. Lipid Res. 73, 28–45 (2019).

61.        Snodgrass, R. G. & Brüne, B. Regulation and Functions of 15-Lipoxygenases in Human Macrophages. Front. Pharmacol. 10, 719 (2019).

62.        Ganini, D. & Mason, R. P. Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase. Free Radic. Biol. Med. 76, 61–8 (2014).

63.        Chinetti-Gbaguidi, G. et al. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways. Circ. Res. 108, 985–95 (2011).

64.        Cuccurullo, C., Fazia, M. L., Mezzetti, A. & Cipollone, F. COX-2 expression in atherosclerosis: the good, the bad or the ugly? Curr. Med. Chem. 14, 1595–605 (2007).

65.        Ohishi, M. et al. Increased expression and co-localization of ACE, angiotensin II AT(1) receptors and inducible nitric oxide synthase in atherosclerotic human coronary arteries. Int. J. Physiol. Pathophysiol. Pharmacol. 2, 111–24 (2010).

66.        Perrotta, I. et al. iNOS induction and PARP-1 activation in human atherosclerotic lesions: an immunohistochemical and ultrastructural approach. Cardiovasc. Pathol. 20, 195–203 (2011).

67.        Nadel, J., Jabbour, A. & Stocker, R. Arterial myeloperoxidase in the detection and treatment of vulnerable atherosclerotic plaque: a new dawn for an old light. Cardiovasc. Res. (2022). doi:10.1093/cvr/cvac081

68.        Marsche, G., Stadler, J. T., Kargl, J. & Holzer, M. Understanding Myeloperoxidase-Induced Damage to HDL Structure and Function in the Vessel Wall: Implications for HDL-Based Therapies. Antioxidants (Basel, Switzerland) 11, (2022).

69.        Nicholls, S. J. & Hazen, S. L. Myeloperoxidase, modified lipoproteins, and atherogenesis. J. Lipid Res. 50, (2009).

70.        Podrez, E. A. et al. Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species. J. Clin. Invest. 105, 1095–108 (2000).

71.        Podrez, E. A., Schmitt, D., Hoff, H. F. & Hazen, S. L. Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro. J. Clin. Invest. 103, 1547–60 (1999).

72.        Huang, Y. et al. An abundant dysfunctional apolipoprotein A1 in human atheroma. Nat. Med. 20, 193–203 (2014).

73.        Kruth, H. S. Fluid-phase pinocytosis of LDL by macrophages: a novel target to reduce macrophage cholesterol accumulation in atherosclerotic lesions. Curr. Pharm. Des. 19, 5865–72 (2013).

74.        Steinbrecher, U. P. & Lougheed, M. Scavenger receptor-independent stimulation of cholesterol esterification in macrophages by low density lipoprotein extracted from human aortic intima. Arterioscler. Thromb.  a J. Vasc. Biol. 12, 608–25 (1992).

75.        Meyer, J. M., Ji, A., Cai, L. & van der Westhuyzen, D. R. High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation. J. Lipid Res. 53, 2081–2091 (2012).

76.        Sanda, G. M. et al. Aggregated LDL turn human macrophages into foam cells and induce mitochondrial dysfunction without triggering oxidative or endoplasmic reticulum stress. PLoS One 16, e0245797 (2021).

77.        Asmis, R. & Jelk, J. Large variations in human foam cell formation in individuals: a fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis 148, 243–53 (2000).

78.        Oörni, K., Pentikäinen, M. O., Ala-Korpela, M. & Kovanen, P. T. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions. J. Lipid Res. 41, 1703–14 (2000).

79.        Meyer, J. M., Ji, A., Cai, L. & van der Westhuyzen, D. R. Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation. J. Lipid Res. 55, 1648–56 (2014).

80.        Pirillo, A., Uboldi, P. & Catapano, A. L. Dual effect of hypochlorite in the modification of high density lipoproteins. Biochem. Biophys. Res. Commun. 403, 447–51 (2010).

81.        Siess, W. Platelet interaction with bioactive lipids formed by mild oxidation of low-density lipoprotein. Pathophysiol. Haemost. Thromb. 35, 292–304 (2006).

82.        Valiyaveettil, M. et al. Oxidized high-density lipoprotein inhibits platelet activation and aggregation via scavenger receptor BI. Blood 111, 1962–71 (2008).

83.        Lê, Q. H. et al. Glycoxidized HDL, HDL enriched with oxidized phospholipids and HDL from diabetic patients inhibit platelet function. J. Clin. Endocrinol. Metab. 100, 2006–14 (2015).

84.        Hersberger, M. et al. No association of two functional polymorphisms in human ALOX15 with myocardial infarction. Atherosclerosis 205, 192–6 (2009).

85.        Weibel, G. L. et al. Overexpression of human 15(S)-lipoxygenase-1 in RAW macrophages leads to increased cholesterol mobilization and reverse cholesterol transport. Arterioscler. Thromb. Vasc. Biol. 29, 837–42 (2009).

86.        Hutchins, P. M., Moore, E. E. & Murphy, R. C. Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions. J. Lipid Res. 52, 2070–83 (2011).

87.        Belkner, J., Stender, H. & Kühn, H. The rabbit 15-lipoxygenase preferentially oxygenates LDL cholesterol esters, and this reaction does not require vitamin E. J. Biol. Chem. 273, 23225–32 (1998).

88.        Hutchins, P. M. & Murphy, R. C. Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine. J. Lipid Res. 53, 1588–97 (2012).

89.        Vangaveti, V., Baune, B. T. & Kennedy, R. L. Hydroxyoctadecadienoic acids: novel regulators of macrophage differentiation and atherogenesis. Ther. Adv. Endocrinol. Metab. 1, 51–60 (2010).

90.        Shewale, S. V et al. Botanical oils enriched in n-6 and n-3 FADS2 products are equally effective in preventing atherosclerosis and fatty liver. J. Lipid Res. 56, 1191–205 (2015).

91.        Moerman, A. M. et al. Lipid signature of advanced human carotid atherosclerosis assessed by mass spectrometry imaging. J. Lipid Res. 62, 100020 (2021).

92.        Upston, J. M. et al. Disease stage-dependent accumulation of lipid and protein oxidation products in human atherosclerosis. Am. J. Pathol. 160, 701–10 (2002).

93.        Darley-Usmar, V. M., Severn, A., O’Leary, V. J. & Rogers, M. Treatment of macrophages with oxidized low-density lipoprotein increases their intracellular glutathione content. Biochem. J. 278 ( Pt 2, 429–34 (1991).

94.        Maor, I. & Aviram, M. Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesteryl ester. J. Lipid Res. 35, 803–19 (1994).

95.        Thomas, S. R. & Stocker, R. Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis. Free Radic. Biol. Med. 28, 1795–805 (2000).

96.        Terentis, A. C., Thomas, S. R., Burr, J. A., Liebler, D. C. & Stocker, R. Vitamin E oxidation in human atherosclerotic lesions. Circ. Res. 90, 333–9 (2002).

97.        Ohkawa, S. et al. Pro-oxidative effect of alpha-tocopherol in the oxidation of LDL isolated from co-antioxidant-depleted non-diabetic hemodialysis patients. Atherosclerosis 176, 411–8 (2004).

98.        Maor, I. et al. Oxidized monocyte-derived macrophages in aortic atherosclerotic lesion from apolipoprotein E-deficient mice and from human carotid artery contain lipid peroxides and oxysterols. Biochem. Biophys. Res. Commun. 269, 775–80 (2000).

99.        Leleu, D. et al. Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation. Arterioscler. Thromb. Vasc. Biol. 45, 910–927 (2025).

100.      Ferreira, G. S. et al. Aerobic Exercise Training Selectively Changes Oxysterol Levels and Metabolism Reducing Cholesterol Accumulation in the Aorta of Dyslipidemic Mice. Front. Physiol. 8, 644 (2017).

101.      Yu, L. et al. Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice. Nat. Commun. 14, 4101 (2023).

102.      Quinn, C. M., Jessup, W., Wong, J., Kritharides, L. & Brown, A. J. Expression and regulation of sterol 27-hydroxylase (CYP27A1) in human macrophages: a role for RXR and PPARgamma ligands. Biochem. J. 385, 823–30 (2005).

103.      Szanto, A. et al. Transcriptional regulation of human CYP27 integrates retinoid, peroxisome proliferator-activated receptor, and liver X receptor signaling in macrophages. Mol. Cell. Biol. 24, 8154–66 (2004).

104.      Björkhem, I., Diczfalusy, U. & Lütjohann, D. Removal of cholesterol from extrahepatic sources by oxidative mechanisms. Curr. Opin. Lipidol. 10, 161–5 (1999).

105.      Brown, A. J., Watts, G. F., Burnett, J. R., Dean, R. T. & Jessup, W. Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture. J. Biol. Chem. 275, 27627–33 (2000).

106.      Heo, G.-Y. et al. Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells. J. Lipid Res. 52, 1117–1127 (2011).

107.      Fu, X. et al. 27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J. Biol. Chem. 276, 38378–87 (2001).

108.      Saliba-Gustafsson, P. et al. Subclinical atherosclerosis and its progression are modulated by PLIN2 through a feed-forward loop between LXR and autophagy. J. Intern. Med. 286, 660–675 (2019).

109.      Korytowski, W. et al. Impairment of Macrophage Cholesterol Efflux by Cholesterol Hydroperoxide Trafficking: Implications for Atherogenesis Under Oxidative Stress. Arterioscler. Thromb. Vasc. Biol. 35, 2104–13 (2015).

110.      Salomon, R. G. & Bi, W. Isolevuglandin adducts in disease. Antioxid. Redox Signal. 22, 1703–18 (2015).

111.      Marengo, B. et al. Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages. Biofactors 42, 80–92 (2016).

112.      Vurusaner, B. et al. The role of autophagy in survival response induced by 27-hydroxycholesterol in human promonocytic cells. Redox Biol. 17, 400–410 (2018).

113.      Gargiulo, S. et al. Up-regulation of COX-2 and mPGES-1 by 27-hydroxycholesterol and 4-hydroxynonenal: A crucial role in atherosclerotic plaque instability. Free Radic. Biol. Med. 129, 354–363 (2018).

114.      Yancey, P. G., Yu, H., Linton, M. F. & Fazio, S. A pathway-dependent on apoE, ApoAI, and ABCA1 determines formation of buoyant high-density lipoprotein by macrophage foam cells. Arterioscler. Thromb. Vasc. Biol. 27, 1123–31 (2007).

115.      Horiuchi, Y. et al. Characterization of the cholesterol efflux of apolipoprotein E-containing high-density lipoprotein in THP-1 cells. Biol. Chem. 400, 209–218 (2019).

116.      Bouhlel, M. A. et al. PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metab. 6, 137–43 (2007).

117.      Chawla, A. et al. A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol. Cell 7, 161–71 (2001).

118.      Shen, L. et al. NIR-II Imaging for Tracking the Spatiotemporal Immune Microenvironment in Atherosclerotic Plaques. ACS Nano 18, 34171–34185 (2024).

119.      Chistiakov, D. A., Myasoedova, V. A., Revin, V. V., Orekhov, A. N. & Bobryshev, Y. V. The phenomenon of atherosclerosis reversal and regression: Lessons from animal models. Exp. Mol. Pathol. 102, 138–145 (2017).

120.      Zimmer, S. et al. Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming. Sci. Transl. Med. 8, 333ra50 (2016).

121.      Shibata, T. et al. 15-deoxy-delta 12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. J. Biol. Chem. 277, 10459–66 (2002).

122.      Belkner, J., Stender, H., Holzhütter, H. G., Holm, C. & Kühn, H. Macrophage cholesteryl ester hydrolases and hormone-sensitive lipase prefer specifically oxidized cholesteryl esters as substrates over their non-oxidized counterparts. Biochem. J. 352 Pt 1, 125–33 (2000).

123.      Naruhn, S. et al. 15-hydroxyeicosatetraenoic acid is a preferential peroxisome proliferator-activated receptor beta/delta agonist. Mol. Pharmacol. 77, 171–84 (2010).

124.      Umeno, A. et al. Comprehensive analysis of PPARγ agonist activities of stereo-, regio-, and enantio-isomers of hydroxyoctadecadienoic acids. Biosci. Rep. 40, (2020).

125.      Wigren, J. et al. Differential recruitment of the coactivator proteins CREB-binding protein and steroid receptor coactivator-1 to peroxisome proliferator-activated receptor gamma/9-cis-retinoic acid receptor heterodimers by ligands present in oxidized low-density lipoprote. J. Endocrinol. 177, 207–14 (2003).

126.      Kämmerer, I., Ringseis, R., Biemann, R., Wen, G. & Eder, K. 13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages. Lipids Health Dis. 10, 222 (2011).

127.      Benatzy, Y. et al. ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2. Redox Biol. 72, 103149 (2024).

128.      Snodgrass, R. G. et al. Efferocytosis potentiates the expression of arachidonate 15-lipoxygenase (ALOX15) in alternatively activated human macrophages through LXR activation. Cell Death Differ. 28, 1301–1316 (2021).

129.      Ricote, M., Welch, J. S. & Glass, C. K. Regulation of macrophage gene expression by the peroxisome proliferator-activated receptor-gamma. Horm. Res. 54, 275–80 (2000).

130.      Carpanedo, L. et al. Substrate-dependent incorporation of 15-lipoxygenase products in glycerophospholipids: 15-HETE and 15-HEPE in PI, 17-HDHA in plasmalogen PE, and 13-HODE in PC. J. Lipid Res. 66, 100841 (2025).

131.      Takahashi, Y. et al. Selective uptake and efflux of cholesteryl linoleate in LDL by macrophages expressing 12/15-lipoxygenase. Biochem. Biophys. Res. Commun. 338, 128–35 (2005).

132.      Castilho, L. N. et al. Oxidation of LDL enhances the cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester transfer rate to HDL, bringing on a diminished net transfer of cholesteryl ester from HDL to oxidized LDL. Clin. Chim. Acta. 304, 99–106 (2001).

133.      Christison, J., Karjalainen, A., Brauman, J., Bygrave, F. & Stocker, R. Rapid reduction and removal of HDL- but not LDL-associated cholesteryl ester hydroperoxides by rat liver perfused in situ. Biochem. J. 314 ( Pt 3, 739–42 (1996).

134.      Fluiter, K. et al. Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells. Biochem. J. 319 ( Pt 2, 471–6 (1996).

135.      Brites, F., Martin, M., Guillas, I. & Kontush, A. Antioxidative activity of high-density lipoprotein (HDL): Mechanistic insights into potential clinical benefit. BBA Clin. 8, 66–77 (2017).

136.      Cohen, E. et al. Increased Levels of Human Carotid Lesion Linoleic Acid Hydroperoxide in Symptomatic and Asymptomatic Patients Is Inversely Correlated with Serum HDL and Paraoxonase 1 Activity. J. Lipids 2012, 762560 (2012).

137.      Khateeb, J., Gantman, A., Kreitenberg, A. J., Aviram, M. & Fuhrman, B. Paraoxonase 1 (PON1) expression in hepatocytes is upregulated by pomegranate polyphenols: a role for PPAR-gamma pathway. Atherosclerosis 208, 119–25 (2010).

138.      Lapenna, D. et al. Glutathione-related antioxidant defenses in human atherosclerotic plaques. Circulation 97, 1930–4 (1998).

139.      Lapenna, D., Ciofani, G., Calafiore, A. M., Cipollone, F. & Porreca, E. Impaired glutathione-related antioxidant defenses in the arterial tissue of diabetic patients. Free Radic. Biol. Med. 124, 525–531 (2018).

140.      Huang, Y. et al. Elevated peroxidative glutathione redox status in atherosclerotic patients with increased thickness of carotid intima media. Chin. Med. J. (Engl). 122, 2827–32 (2009).

141.      Ashfaq, S. et al. The relationship between plasma levels of oxidized and reduced thiols and early atherosclerosis in healthy adults. J. Am. Coll. Cardiol. 47, 1005–11 (2006).

142.      Biswas, S. K., Newby, D. E., Rahman, I. & Megson, I. L. Depressed glutathione synthesis precedes oxidative stress and atherogenesis in Apo-E(-/-) mice. Biochem. Biophys. Res. Commun. 338, 1368–73 (2005).

143.      ’t Hoen, P. A. C. et al. Aorta of ApoE-deficient mice responds to atherogenic stimuli by a prelesional increase and subsequent decrease in the expression of antioxidant enzymes. Circ. Res. 93, 262–9 (2003).

144.      Callegari, A. et al. Gain and loss of function for glutathione synthesis: impact on advanced atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 31, 2473–82 (2011).

145.      Rosenblat, M., Volkova, N., Coleman, R. & Aviram, M. Anti-oxidant and anti-atherogenic properties of liposomal glutathione: studies in vitro, and in the atherosclerotic apolipoprotein E-deficient mice. Atherosclerosis 195, e61-8 (2007).

146.      Kader, T. et al. Ribose-cysteine protects against the development of atherosclerosis in apoE-deficient mice. PLoS One 15, e0228415 (2020).

147.      Boeglin, W. E., Stec, D. F., Noguchi, S., Calcutt, M. W. & Brash, A. R. The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation. J. Biol. Chem. 300, 107293 (2024).

148.      Singhal, S. S. et al. Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling. Toxicol. Appl. Pharmacol. 289, 361–70 (2015).

149.      Yang, X. et al. Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake. J. Biol. Chem. 290, 21788–99 (2015).

150.      Rosenblat, M. et al. Reduced glutathione increases quercetin stimulatory effects on HDL- or apoA1-mediated cholesterol efflux from J774A.1 macrophages. Free Radic. Res. 48, 1462–72 (2014).

151.      Nelson, S. M., Lei, X. & Prabhu, K. S. Selenium levels affect the IL-4-induced expression of alternative activation markers in murine macrophages. J. Nutr. 141, 1754–61 (2011).

152.      Rozenberg, O. & Aviram, M. S-Glutathionylation regulates HDL-associated paraoxonase 1 (PON1) activity. Biochem. Biophys. Res. Commun. 351, 492–8 (2006).

153.      Kühn, H., Heydeck, D., Hugou, I. & Gniwotta, C. In vivo action of 15-lipoxygenase in early stages of human atherogenesis. J. Clin. Invest. 99, 888–93 (1997).

154.      Marklund, M. et al. Biomarkers of Dietary Omega-6 Fatty Acids and Incident Cardiovascular Disease and Mortality. Circulation 139, 2422–2436 (2019).

155.      Li, J., Guasch-Ferré, M., Li, Y. & Hu, F. B. Dietary intake and biomarkers of linoleic acid and mortality: systematic review and meta-analysis of prospective cohort studies. Am. J. Clin. Nutr. 112, 150–167 (2020).

156.      Harris, W. S. et al. Blood n-3 fatty acid levels and total and cause-specific mortality from 17 prospective studies. Nat. Commun. 12, 2329 (2021).

157.      O’Keefe, E. L. et al. Circulating Docosahexaenoic Acid and Risk of All-Cause and Cause-Specific Mortality. Mayo Clin. Proc. 99, 534–541 (2024).

158.      Hooper, L. et al. Reduction in saturated fat intake for cardiovascular disease. Cochrane database Syst. Rev. 5, CD011737 (2020).

159.      Mensink, R. P. Effects of saturated fatty acids on serum lipids and lipoproteins: a systematic review and regression analysis. (World Health Organization., 2016).

160.      Riley, T. M., Sapp, P. A., Kris-Etherton, P. M. & Petersen, K. S. Effects of saturated fatty acid consumption on lipoprotein (a): a systematic review and meta-analysis of randomized controlled trials. Am. J. Clin. Nutr. 120, 619–629 (2024).

161.      Wang, T. et al. Association Between Omega-3 Fatty Acid Intake and Dyslipidemia: A Continuous Dose-Response Meta-Analysis of Randomized Controlled Trials. J. Am. Heart Assoc. 12, e029512 (2023).

162.      Ruuth, M. et al. Overfeeding Saturated Fat Increases LDL (Low-Density Lipoprotein) Aggregation Susceptibility While Overfeeding Unsaturated Fat Decreases Proteoglycan-Binding of Lipoproteins. Arterioscler. Thromb. Vasc. Biol. 41, 2823–2836 (2021).

163.      Jones, P. J. H. et al. High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans. Atherosclerosis 238, 231–8 (2015).

164.      Borén, J. & Williams, K. J. The central role of arterial retention of cholesterol-rich apolipoprotein-B-containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity. Curr. Opin. Lipidol. 27, 473–83 (2016).

165.      Steffen, B. T. et al. n-3 and n-6 Fatty acids are independently associated with lipoprotein-associated phospholipase A2 in the Multi-Ethnic Study of Atherosclerosis. Br. J. Nutr. 110, 1664–71 (2013).

166.      Simental-Mendía, L. E., Simental-Mendía, M., Barragán-Zúñiga, L. J. & Aguillón-Marín, P. Impact of omega-3 supplementation on lipoprotein-associated phospholipase A2 mass and activity: A systematic review and meta-analysis of randomized controlled trials. J. Clin. Lipidol. (2025). doi:10.1016/j.jacl.2025.12.013

167.      Solà, R. et al. Oleic acid rich diet protects against the oxidative modification of high density lipoprotein. Free Radic. Biol. Med. 22, 1037–45 (1997).

168.      Mata, P. et al. Effect of dietary fat saturation on LDL oxidation and monocyte adhesion to human endothelial cells in vitro. Arterioscler. Thromb. Vasc. Biol. 16, 1347–55 (1996).

169.      Hargrove, R. L., Etherton, T. D., Pearson, T. A., Harrison, E. H. & Kris-Etherton, P. M. Low fat and high monounsaturated fat diets decrease human low density lipoprotein oxidative susceptibility in vitro. J. Nutr. 131, 1758–63 (2001).

170.      DAYTON, S., PEARCE, M. L., HASHIMOTO, S., DIXON, W. J. & TOMIYASU, U. A Controlled Clinical Trial of a Diet High in Unsaturated Fat in Preventing Complications of Atherosclerosis. Circulation 40, (1969).

171.      Rudel, L. L., Parks, J. S. & Sawyer, J. K. Compared with dietary monounsaturated and saturated fat, polyunsaturated fat protects African green monkeys from coronary artery atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 15, 2101–10 (1995).

172.      Sato, M. et al. Linoleic acid-rich fats reduce atherosclerosis development beyond its oxidative and inflammatory stress-increasing effect in apolipoprotein E-deficient mice in comparison with saturated fatty acid-rich fats. Br. J. Nutr. 94, 896–901 (2005).

173.      Penumetcha, M., Song, M., Merchant, N. & Parthasarathy, S. Pretreatment with n-6 PUFA protects against subsequent high fat diet induced atherosclerosis--potential role of oxidative stress-induced antioxidant defense. Atherosclerosis 220, 53–8 (2012).

174.      Cawood, A. L. et al. Eicosapentaenoic acid (EPA) from highly concentrated n-3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and increased stability. Atherosclerosis 212, 252–9 (2010).

175.      Thies, F. et al. Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomised controlled trial. Lancet (London, England) 361, 477–85 (2003).

176.      Berbée, J. F. P. et al. Deuterium-reinforced polyunsaturated fatty acids protect against atherosclerosis by lowering lipid peroxidation and hypercholesterolemia. Atherosclerosis 264, 100–107 (2017).

177.      Carmena, R. et al. Effect of olive and sunflower oils on low density lipoprotein level, composition, size, oxidation and interaction with arterial proteoglycans. Atherosclerosis 125, 243–55 (1996).

178.      Haddad, E. H., Gaban-Chong, N., Oda, K. & Sabaté, J. Effect of a walnut meal on postprandial oxidative stress and antioxidants in healthy individuals. Nutr. J. 13, 4 (2014).

179.      McKay, D. L. et al. Chronic and acute effects of walnuts on antioxidant capacity and nutritional status in humans: a randomized, cross-over pilot study. Nutr. J. 9, 21 (2010).

180.      Zambón, D. et al. Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women. A randomized crossover trial. Ann. Intern. Med. 132, 538–46 (2000).

181.      Kris-Etherton, P. M. Walnuts decrease risk of cardiovascular disease: a summary of efficacy and biologic mechanisms. J. Nutr. 144, 547S-554S (2014).

182.      Skinner, J., Arora, P., McMath, N. & Penumetcha, M. Determination of Oxidized Lipids in Commonly Consumed Foods and a Preliminary Analysis of Their Binding Affinity to PPARγ. Foods (Basel, Switzerland) 10, (2021).

183.      Sekhar, R. V. et al. Glutathione synthesis is diminished in patients with uncontrolled diabetes and restored by dietary supplementation with cysteine and glycine. Diabetes Care 34, 162–7 (2011).

184.      Perez-Herrera, A. et al. The antioxidants in oils heated at frying temperature, whether natural or added, could protect against postprandial oxidative stress in obese people. Food Chem. 138, 2250–9 (2013).

185.      Khan-Merchant, N., Penumetcha, M., Meilhac, O. & Parthasarathy, S. Oxidized fatty acids promote atherosclerosis only in the presence of dietary cholesterol in low-density lipoprotein receptor knockout mice. J. Nutr. 132, 3256–62 (2002).

186.      Rundblad, A., Holven, K. B., Ottestad, I., Myhrstad, M. C. & Ulven, S. M. High-quality fish oil has a more favourable effect than oxidised fish oil on intermediate-density lipoprotein and LDL subclasses: a randomised controlled trial. Br. J. Nutr. 117, 1291–1298 (2017).

187.      Kanner, J., Gorelik, S., Roman, S. & Kohen, R. Protection by polyphenols of postprandial human plasma and low-density lipoprotein modification: the stomach as a bioreactor. J. Agric. Food Chem. 60, 8790–6 (2012).

188.      Sirota, R., Gorelik, S., Harris, R., Kohen, R. & Kanner, J. Coffee polyphenols protect human plasma from postprandial carbonyl modifications. Mol. Nutr. Food Res. 57, 916–9 (2013).

189.      Kanner, J. et al. Redox homeostasis in stomach medium by foods: The Postprandial Oxidative Stress Index (POSI) for balancing nutrition and human health. Redox Biol. 12, 929–936 (2017).

190.      Tirosh, O., Shpaizer, A. & Kanner, J. Lipid Peroxidation in a Stomach Medium Is Affected by Dietary Oils (Olive/Fish) and Antioxidants: The Mediterranean versus Western Diet. J. Agric. Food Chem. 63, 7016–23 (2015).

191.      Bertuccelli, G. et al. Iron supplementation in young iron-deficient females causes gastrointestinal redox imbalance: protective effect of a fermented nutraceutical. J. Biol. Regul. Homeost. Agents 28, 53–63 (2014).

192.      Rabovsky, A. B., Buettner, G. R. & Fink, B. In vivo imaging of free radicals produced by multivitamin-mineral supplements. BMC Nutr. 1, 1–9 (2015).

193.      Kaikkonen, J. E. et al. High serum n6 fatty acid proportion is associated with lowered LDL oxidation and inflammation: the Cardiovascular Risk in Young Finns Study. Free Radic. Res. 48, 420–6 (2014).

194.      Senyilmaz-Tiebe, D. et al. Dietary stearic acid regulates mitochondria in vivo in humans. Nat. Commun. 9, 3129 (2018).

195.      Yu-Poth, S. et al. Lowering dietary saturated fat and total fat reduces the oxidative susceptibility of LDL in healthy men and women. J. Nutr. 130, 2228–37 (2000).

196.      Moreno, J. A. et al. Apolipoprotein E gene promoter -219G->T polymorphism increases LDL-cholesterol concentrations and susceptibility to oxidation in response to a diet rich in saturated fat. Am. J. Clin. Nutr. 80, 1404–9 (2004).

197.      Benson, T. W. et al. A single high-fat meal provokes pathological erythrocyte remodeling and increases myeloperoxidase levels: implications for acute coronary syndrome. Lab. Invest. 98, 1300–1310 (2018).

198.      Cedó, L. et al. Consumption of polyunsaturated fat improves the saturated fatty acid-mediated impairment of HDL antioxidant potential. Mol. Nutr. Food Res. 59, 1987–96 (2015).

199.      O’Reilly, M. et al. High-Density Lipoprotein Proteomic Composition, and not Efflux Capacity, Reflects Differential Modulation of Reverse Cholesterol Transport by Saturated and Monounsaturated Fat Diets. Circulation 133, 1838–50 (2016).

200.      Walzem, R. L., Watkins, S., Frankel, E. N., Hansen, R. J. & German, J. B. Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease. Proc. Natl. Acad. Sci. U. S. A. 92, 7460–4 (1995).

201.      Shepherd, J. et al. Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man. J. Lipid Res. 21, 91–9 (1980).

202.      Ohmura, H. et al. Lipid compositional differences of small, dense low-density lipoprotein particle influence its oxidative susceptibility: possible implication of increased risk of coronary artery disease in subjects with phenotype B. Metabolism. 51, 1081–7 (2002).

203.      Chiu, S., Williams, P. T. & Krauss, R. M. Effects of a very high saturated fat diet on LDL particles in adults with atherogenic dyslipidemia: A randomized controlled trial. PLoS One 12, 1–14 (2017).

204.      Imamura, F. et al. Effects of Saturated Fat, Polyunsaturated Fat, Monounsaturated Fat, and Carbohydrate on Glucose-Insulin Homeostasis: A Systematic Review and Meta-analysis of Randomised Controlled Feeding Trials. PLoS Med. 13, e1002087 (2016).

205.      Carnevale, R. et al. Localization of lipopolysaccharide from Escherichia Coli into human atherosclerotic plaque. Sci. Rep. 8, 3598 (2018).

206.      Cândido, T. L. N. et al. Effects of dietary fat quality on metabolic endotoxaemia: a systematic review. Br. J. Nutr. 124, 654–667 (2020).

207.      Carnevale, R. et al. Gut-derived lipopolysaccharides increase post-prandial oxidative stress via Nox2 activation in patients with impaired fasting glucose tolerance: effect of extra-virgin olive oil. Eur. J. Nutr. 58, 843–851 (2019).

208.      Hwang, D. H., Kim, J.-A. & Lee, J. Y. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid. Eur. J. Pharmacol. 785, 24–35 (2016).

209.      Carter, M. M. et al. A gut pathobiont regulates circulating glycine and host metabolism in a twin study comparing vegan and omnivorous diets. medRxiv  Prepr. Serv. Heal. Sci. (2025). doi:10.1101/2025.01.08.25320192

210.      Jian, C., Luukkonen, P., Sädevirta, S., Yki-Järvinen, H. & Salonen, A. Impact of short-term overfeeding of saturated or unsaturated fat or sugars on the gut microbiota in relation to liver fat in obese and overweight adults. Clin. Nutr. 40, 207–216 (2021).

211.      Zhu, C. et al. Human gut microbiome composition and tryptophan metabolites were changed differently by fast food and Mediterranean diet in 4 days: a pilot study. Nutr. Res. 77, 62–72 (2020).

212.      Wolf, P., Cummings, P., Shah, N., Gaskins, H. R. & Mutlu, E. Sulfidogenic Bacteria Abundance in Colonic Mucosa is Positively Correlated with Milk and Animal Fat Intake and Negatively Correlated with Mono and Polyunsaturated Fatty Acids. FASEB J. 29, (2015).

213.      Devkota, S. & Chang, E. B. Interactions between Diet, Bile Acid Metabolism, Gut Microbiota, and Inflammatory Bowel Diseases. Dig. Dis. 33, 351–6 (2015).

214.      Ulven, S. M. et al. Using metabolic profiling and gene expression analyses to explore molecular effects of replacing saturated fat with polyunsaturated fat-a randomized controlled dietary intervention study. Am. J. Clin. Nutr. 109, 1239–1250 (2019).

215.      Koutsos, A. et al. Variation of LDL cholesterol in response to the replacement of saturated with unsaturated fatty acids: a nonrandomized, sequential dietary intervention; the Reading, Imperial, Surrey, Saturated fat Cholesterol Intervention (‘RISSCI’-1) study. Am. J. Clin. Nutr. 120, 854–863 (2024).

216.      Ramsden, C. E. et al. Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans. Prostaglandins. Leukot. Essent. Fatty Acids 87, 135–41 (2012).

217.      Fernandez, M. L. & West, K. L. Mechanisms by which dietary fatty acids modulate plasma lipids. J. Nutr. 135, 2075–8 (2005).

218.      Mashima, R., Onodera, K. & Yamamoto, Y. Regioisomeric distribution of cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans provides evidence for free radical-mediated lipid peroxidation in vivo. J. Lipid Res. 41, 109–15 (2000).

219.      Subbaiah, P. V. & Liu, M. Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates. J. Lipid Res. 37, 113–22 (1996).

220.      Nieman, D. C., Meaney, M. P., John, C. S., Knagge, K. J. & Chen, H. 9- and 13-Hydroxy-octadecadienoic acids (9+13 HODE) are inversely related to granulocyte colony stimulating factor and IL-6 in runners after 2h running. Brain. Behav. Immun. 56, 246–52 (2016).

221.      Hernández-Rodas, M. C. et al. Supplementation with Docosahexaenoic Acid and Extra Virgin Olive Oil Prevents Liver Steatosis Induced by a High-Fat Diet in Mice through PPAR-α and Nrf2 Upregulation with Concomitant SREBP-1c and NF-kB Downregulation. Mol. Nutr. Food Res. 61, (2017).

222.      Mishra, A., Chaudhary, A. & Sethi, S. Oxidized omega-3 fatty acids inhibit NF-kappaB activation via a PPARalpha-dependent pathway. Arterioscler. Thromb. Vasc. Biol. 24, 1621–7 (2004).

223.      Liu, J. et al. The omega-3 hydroxy fatty acid 7(S)-HDHA is a high-affinity PPARα ligand that regulates brain neuronal morphology. Sci. Signal. 15, eabo1857 (2022).

224.      Xu, J., Nakamura, M. T., Cho, H. P. & Clarke, S. D. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats. J. Biol. Chem. 274, 23577–83 (1999).

225.      Sahebkar, A. et al. Effect of omega-3 supplements on plasma apolipoprotein C-III concentrations: a systematic review and meta-analysis of randomized controlled trials. Ann. Med. 50, 565–575 (2018).

226.      Garcia, C. et al. Acute coronary syndrome remodels the antiplatelet aggregation properties of HDL particle subclasses. J. Thromb. Haemost. 16, 933–945 (2018).

227.      Meilhac, O., Zhou, M., Santanam, N. & Parthasarathy, S. Lipid peroxides induce expression of catalase in cultured vascular cells. J. Lipid Res. 41, 1205–13 (2000).

228.      Okuno, Y. et al. Human catalase gene is regulated by peroxisome proliferator activated receptor-gamma through a response element distinct from that of mouse. Endocr. J. 57, 303–9 (2010).

229.      Kang, G. J., Kim, E. J. & Lee, C. H. Therapeutic Effects of Specialized Pro-Resolving Lipids Mediators on Cardiac Fibrosis via NRF2 Activation. Antioxidants (Basel, Switzerland) 9, (2020).

230.      Gao, L. et al. Novel n-3 fatty acid oxidation products activate Nrf2 by destabilizing the association between Keap1 and Cullin3. J. Biol. Chem. 282, 2529–37 (2007).

231.      Ishikado, A. et al. 4-Hydroxy hexenal derived from docosahexaenoic acid protects endothelial cells via Nrf2 activation. PLoS One 8, e69415 (2013).

232.      Nakagawa, F. et al. 4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs. Biochem. Biophys. Res. Commun. 443, 991–6 (2014).

233.      Zhang, Q. et al. Linoleic Acid Alleviates Lipopolysaccharide Induced Acute Liver Injury via Activation of Nrf2. Physiol. Res. 73, 381–391 (2024).

234.      Wang, R., Kern, J. T., Goodfriend, T. L., Ball, D. L. & Luesch, H. Activation of the antioxidant response element by specific oxidized metabolites of linoleic acid. Prostaglandins. Leukot. Essent. Fatty Acids 81, 53–9 (2009).

235.      Shibata, T. 15-Deoxy-Δ¹²,¹⁴-prostaglandin J₂ as an electrophilic mediator. Biosci. Biotechnol. Biochem. 79, 1044–9 (2015).

236.      Ishikado, A. et al. Low concentration of 4-hydroxy hexenal increases heme oxygenase-1 expression through activation of Nrf2 and antioxidative activity in vascular endothelial cells. Biochem. Biophys. Res. Commun. 402, 99–104 (2010).

237.      He, L. et al. Activation of Nrf2 inhibits atherosclerosis in ApoE-/- mice through suppressing endothelial cell inflammation and lipid peroxidation. Redox Biol. 74, 103229 (2024).

238.      Schrijvers, D. M., De Meyer, G. R. Y., Kockx, M. M., Herman, A. G. & Martinet, W. Phagocytosis of apoptotic cells by macrophages is impaired in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 25, 1256–61 (2005).

239.      Xu, X. et al. Nrf2 deficiency in myeloid cells accelerates atherosclerosis by promoting the inflammatory response and impairing efferocytosis. J. Adv. Res. (2026). doi:10.1016/j.jare.2026.01.005

240.      Kim, W. et al. 15-Deoxy-Δ12,14-Prostaglandin J2 Exerts Proresolving Effects Through Nuclear Factor E2-Related Factor 2-Induced Expression of CD36 and Heme Oxygenase-1. Antioxid. Redox Signal. 27, 1412–1431 (2017).

241.      Isot, I. et al. 17-Oxo-DHA Potentiates Macrophage Efferocytosis via Nrf2/HO-1-Mediated Biosynthesis of Specialized Pro-Resolving Mediators. IUBMB Life 77, e70057 (2025).

242.      Ishii, T. et al. Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: activation by oxidatively modified LDL and 4-hydroxynonenal. Circ. Res. 94, 609–16 (2004).

243.      Olagnier, D. et al. Nrf2, a PPARγ alternative pathway to promote CD36 expression on inflammatory macrophages: implication for malaria. PLoS Pathog. 7, e1002254 (2011).

244.      Jiang, J. et al. Epigallocatechin-3-gallate prevents TNF-α-induced NF-κB activation thereby upregulating ABCA1 via the Nrf2/Keap1 pathway in macrophage foam cells. Int. J. Mol. Med. 29, 946–56 (2012).

245.      Liu, S. et al. Sulforaphane Inhibits Foam Cell Formation and Atherosclerosis via Mechanisms Involving the Modulation of Macrophage Cholesterol Transport and the Related Phenotype. Nutrients 15, (2023).

246.      Yang, X.-J. et al. Berberine Attenuates Cholesterol Accumulation in Macrophage Foam Cells by Suppressing AP-1 Activity and Activation of the Nrf2/HO-1 Pathway. J. Cardiovasc. Pharmacol. 75, 45–53 (2020).

247.      Zhong, Y., Feng, J., Fan, Z. & Li, J. Curcumin increases cholesterol efflux via heme oxygenase‑1‑mediated ABCA1 and SR‑BI expression in macrophages. Mol. Med. Rep. 17, 6138–6143 (2018).

248.      Zhang, H. et al. The foam cell-derived exosomes exacerbate ischemic white matter injury via transmitting metabolic defects to microglia. Cell Metab. 37, 1636-1654.e10 (2025).

249.      Łuczaj, W., Gęgotek, A. & Skrzydlewska, E. Antioxidants and HNE in redox homeostasis. Free Radic. Biol. Med. 111, 87–101 (2017).

250.      Lee, Y.-S. & Wander, R. C. Reduced effect on apoptosis of 4-hydroxyhexenal and oxidized LDL enriched with n-3 fatty acids from postmenopausal women. J. Nutr. Biochem. 16, 213–21 (2005).

251.      Hsu, C. G. et al. The lipid peroxidation product 4-hydroxynonenal inhibits NLRP3 inflammasome activation and macrophage pyroptosis. Cell Death Differ. 29, 1790–1803 (2022).

252.      Siems, W. & Grune, T. Intracellular metabolism of 4-hydroxynonenal. Mol. Aspects Med. 24, 167–75 (2003).

253.      Tyurin, V. A. et al. Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me’ signals: cleavage and inhibition of phagocytosis by Lp-PLA2. Cell Death Differ. 21, 825–35 (2014).

254.      Merry, T. L. & Ristow, M. Do antioxidant supplements interfere with skeletal muscle adaptation to exercise training? J. Physiol. 594, 5135–47 (2016).

255.      Pinto, P. R. et al. Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta. Lipids Health Dis. 14, 109 (2015).

256.      Guizoni, D. M. et al. Aerobic exercise training protects against endothelial dysfunction by increasing nitric oxide and hydrogen peroxide production in LDL receptor-deficient mice. J. Transl. Med. 14, 213 (2016).

257.      Meilhac, O., Ramachandran, S., Chiang, K., Santanam, N. & Parthasarathy, S. Role of arterial wall antioxidant defense in beneficial effects of exercise on atherosclerosis in mice. Arterioscler. Thromb. Vasc. Biol. 21, 1681–8 (2001).

258.      Done, A. J. & Traustadóttir, T. Nrf2 mediates redox adaptations to exercise. Redox Biol. 10, 191–199 (2016).

259.      Thirupathi, A. et al. Effect of Running Exercise on Oxidative Stress Biomarkers: A Systematic Review. Front. Physiol. 11, 610112 (2020).

260.      Ahotupa, M. Oxidized lipoprotein lipids and atherosclerosis. Free Radic. Res. 51, 439–447 (2017).

261.      Okut, S. et al. The Effects of Omega-3 Supplementation Combined with Strength Training on Neuro-Biomarkers, Inflammatory and Antioxidant Responses, and the Lipid Profile in Physically Healthy Adults. Nutrients 17, (2025).

262.      Paduchová, Z. et al. Synergistic Effects of Omega-3 Fatty Acids and Physical Activity on Oxidative Stress Markers and Antioxidant Mechanisms in Aged Rats. Nutrients 17, (2024).

263.      Vaittinen, M. et al. The FADS1 genotypes modify the effect of linoleic acid-enriched diet on adipose tissue inflammation via pro-inflammatory eicosanoid metabolism. Eur. J. Nutr. 61, 3707–3718 (2022).

264.      Pérez-Jiménez, J., Neveu, V., Vos, F. & Scalbert, A. Identification of the 100 richest dietary sources of polyphenols: An application of the Phenol-Explorer database. Eur. J. Clin. Nutr. 64, S112–S120 (2010).

265.      Varady, J., Gessner, D. K., Most, E., Eder, K. & Ringseis, R. Dietary moderately oxidized oil activates the Nrf2 signaling pathway in the liver of pigs. Lipids Health Dis. 11, 31 (2012).

266.      Varady, J., Eder, K. & Ringseis, R. Dietary oxidized fat activates the oxidative stress-responsive transcription factors NF-κB and Nrf2 in intestinal mucosa of mice. Eur. J. Nutr. 50, 601–9 (2011).

267.      Rangel-Zuñiga, O. A. et al. Frying oils with high natural or added antioxidants content, which protect against postprandial oxidative stress, also protect against DNA oxidation damage. Eur. J. Nutr. 56, 1597–1607 (2017).

268.      Staprãns, I., Rapp, J. H., Pan, X. M., Kim, K. Y. & Feingold, K. R. Oxidized lipids in the diet are a source of oxidized lipid in chylomicrons of human serum. Arterioscler. Thromb.  a J. Vasc. Biol. 14, 1900–5 (1994).

269.      Meerarani, P., Reiterer, G., Toborek, M. & Hennig, B. Zinc modulates PPARgamma signaling and activation of porcine endothelial cells. J. Nutr. 133, 3058–64 (2003).