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 pervasive finding in humans and underlies most cardiovascular disease (CVD)—itself a leading cause of death globally. A long-standing cornerstone of many dietary guidelines for prevention of such diseases 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 the totality of evidence from observational and interventional studies, both of which are susceptible to confounding; 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: atherogenesis is commonly thought to involve lipid peroxidation ⁵, 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 ^2,6–11 (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 ^12,13, anti-inflammatory ^14–18 and antioxidant activity ^19,20, suggesting context matters and opportunity for harmonisation.

PUFAs are defined chemically 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), 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 and modulate gene expression as ligands for transcription factors, such as PPARs ²¹ and Nrf2 ²²; 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 for context.

Oxidation and atherogenesis

Atherosclerosis essentially involves the accumulation of lipids (esp. cholesterol) and leukocytes (esp. macrophages) in the arterial wall at susceptible sites due to imbalanced influx/efflux. At onset retention of plasma lipoproteins ²³ may precede macrophage infiltration ²⁴, while later cholesterol supersaturation ²⁵ and inefficient clearance of apoptotic cells (via efferocytosis) ²⁶ may underlie development of a lipid-rich necrotic core. Already in the 1950s lipid peroxidation was also detected in human plaque, with vast subsequent research supporting the involvement of both lipid and protein oxidation in atherosclerosis and inspiring causal hypotheses, as comprehensively reviewed elsewhere ^27,28. Both early and advanced PUFA peroxidation products are present in lesions; native and MDA/HNE-modified low-density lipoprotein (LDL) were even found in fetal aortas with/without macrophages, suggesting an early event ²⁹. Oxidised LDL (oxLDL) can also be detected in plasma and associates with CVD ^30,31, although not always independently of apoB (e.g. CHD ³² and MetS ³³), potentially due to 4E6 antibody cross-reactivity ³⁴. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB), which normally represent a very small fraction of LDL ³⁵, are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ³⁴.

Plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^34,36. The high antioxidant capacity of plasma also suggests LDL oxidation may occur elsewhere. Initial studies incubated LDL with arterial cells, beyond which many methods exist, but most typically LDL is incubated with copper (e.g. as CuSO₄), a transition metal mediating 1-electron oxidations ^27,28. In turn, incubation of oxLDL with various cells has many seemingly pro-atherogenic and pro-thrombotic effects. Most characteristically, extensive LDL oxidation results in macrophage recognition and uptake via scavenger receptors, which promotes cholesterol-loading (i.e. foam cell formation); similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ³⁷. Further, unlike native LDL, uptake of oxLDL results in lipid trapping within lysosomes ³⁸, cholesterol crystallisation and NLRP3 activation ^39,40. This can result in pyroptosis, while the presence of oxLDL (or peroxynitrite) also inhibited efferocytosis ²⁶. 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), but also detected more frequently (at 1hr) in areas of carotid lesions ⁴¹. 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 ⁴². Even in the pre-plaque stage, human native LDL injected into rodents appears as oxLDL in blood (after 30mins) ⁴³ and the arterial wall with endothelial activation (within 6hrs) ^44,45, which were suppressed by antioxidants; whereas direct oxLDL injection was rapidly cleared ⁴³ by the liver and did not appear in the arterial wall ^44,45. As such most endogenous oxLDL detected in plasma is likely mildly oxidised ²⁸.

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) ²⁵. The PUFA/linoleate content of LDL is largely responsible for its oxidative susceptibility; artificially ‘saturating’ human LDL with a reducing agent prior to injection into mice blocks its conversion to oxLDL in blood ⁴³. The characteristic atherogenic effects seen in vitro also involve lipid peroxidation ²⁸; MDA in particular reacts with lysine residues of apoB₁₀₀ (more than 4-HNE) resulting in recognition by scavenger receptors ⁴⁶, and similarly impairs apoA-I efflux activity (vs. other reactive carbonyls) ⁴⁷. CE aldehydes also have reduced macrophage hydrolysis ⁴⁸ and may be converted to 7-ketocholesterol ⁴⁹, which inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ³⁸, and dose-dependently induces cholesterol crystals ⁵⁰. Supporting the relevance of these mechanisms, such aldehydes are detected in human arterial lesions (e.g. LDL ²⁹, HDL ⁴⁷ and CEs ^48,49). In comparing (LOX-derived) LDL hydroperoxides to direct LDL–MDA modification, only the latter induced macrophage uptake ⁴⁶. However, oxLDL may induce the CD36 scavenger receptor via the content of n-6 PUFA hydroxides (i.e. HODES and HETEs) ⁵¹ and 4-HNE ⁵²; CE hydroperoxides also induced TLR4-dependant macropinocytosis and bulk LDL uptake ⁵³, later attributed to oxidised arachidonate ⁵⁴. Further, VLDL is rich in triglycerides which may be released by lipolysis in the arterial wall ⁵⁵, and among fatty acids free linoleic acid can particularly induce endothelial activation ⁵⁵ and barrier disruption, which are inhibited by vitamin E ^56,57. This may involve linoleic peroxidation (via peroxisomes) ⁵⁸ and epoxidation (via CYP2C9) ⁵⁹, with further generation of superoxide and peroxynitrite (via eNOS) ¹¹. Thus, all such observations can support a more specific oxidised linoleic acid hypothesis of atherosclerosis ⁶.

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, from past to present ^60,61. However, the results of large RCTs with high-dose antioxidant supplements in general populations have mostly failed to show benefit ^27,62 (unlike general lipid-lowering), although there is scant outcome data on more physiological and targeted approaches ^62,63. Regardless, this general failure to improve hard outcomes in humans (and animals) with ‘frontline’ antioxidants suggests more complexity ²⁷. Foremost, early studies found human plaque lipid oxidation occurs despite no deficiency of antioxidant nutrients, such as α-tocopherol and ascorbate ^27,64—later extended to T2D ⁶⁵. Moreover, oxidation by free transition metals in vitro may have limited analogy in plaque ²⁸, where particularly heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ⁶⁶. On the other hand, early interest turned to 15-LOXs (i.e. non-heme iron-dependent dioxygenases) ⁶⁷, since they can initiate PUFA oxidation and lipoprotein modification ^68,69, which is not blocked by vitamin E ⁷⁰. Accordingly, human plaques express 15-LOXs (i.e. ALOX15 ^68,71 and ALOX15B ⁶⁹) and COXs ⁷² within specific macrophage populations. Further, increased iNOS ^73,74 and MPO ⁷⁵, along with lipoprotein enrichment in their protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^76,77, also implicates immuno-oxidative activity ²⁷. These 2-electron pathways are also not blocked by vitamin E ⁶⁴ (or serum ⁷⁸) and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptors ^78,79, while MPO-modified tryptophan residues within apoA-I/HDL associate with lipid-poor particles in plaque and inactivate ABCA1-dependent acceptor activity ^77,80.

However, other data present more fundamental challenges ^27,28,81. 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 ⁸². Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^82,83 and CE accumulation beyond native or oxLDL ^84,85. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ³⁸. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁸⁶ and induce macropinocytosis ⁵³, lysosomal crystals and NLRP3 activation ^39,40, so may contribute in these ways ⁸². 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 ⁸⁷. 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 ⁸⁸. The early linoleic oxidation product 13-HODE also induces macrophage efflux ⁸⁹. And contrasting earlier studies ⁹⁰, copper-oxidation can induce HDL anti-platelet activity ^91,92 and suppress LDL and VLDL inflammatory signalling ^44,55. Of enzymatic pathways, human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ⁹³, consistent with ALOX15 overexpression increasing reverse cholesterol transport ⁹⁴. Thus, the extent and type of oxidation may be important.

Lipid oxygenation and efflux

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some context. Of PUFA oxidation products, linoleic hydro(pero)xides dominate ²⁷. 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.) ⁹⁵. 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 ⁹⁵. 15-LOX in particular can directly initiate CE oxidation ⁶⁷, while subsequent radical reactions may erode product specificity ^96,97; and at least in several earlier reports 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ⁹⁸. Conversely, mouse models can lack oxidised CEs despite LOX activity ⁹⁹. 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 ¹⁰⁰. Further, from a temporal perspective, an 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 ¹⁰¹.

These observations in vivo notably contrast the typical situation in vitro ²⁷, where LDL oxidation generates hydroperoxides immediately followed by MDA ^46,79,102, before depletion of CEs with accumulation of 7-ketocholesterol ^49,103. 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.) ¹⁰¹. Conversely, the profile in vivo implies more mildly oxygenating conditions where enzymes and/or the α-tocopherol radical can initiate lipid peroxidation ¹⁰⁴; the latter being favoured by insufficient regenerative co-antioxidants ¹⁰⁵ (e.g. CoQ₁₀ and carotenoids) ¹⁰⁶. 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 ¹⁰⁵. Accordingly, MPO/iNOS-derived oxidants can directly induce protein modification and initiate lipid peroxidation, while in mice aortic lesions may lack MPO ⁷⁷.

Of oxysterols, human plaque is generally dominated by 27-hydroxychoesterol (27-HC), followed by 7-ketocholesterol ²⁷, although some reports suggested the opposite in human macrophages ^107,108 and absence of 27-HC in animals ¹⁰⁹. As above, accumulation of cholesterol precedes 27-HC ¹⁰¹ and at the fatty streak stage they are highly correlated ¹¹⁰. 27-HC is produced by mitochondrial sterol 27-hydroxylase (CYP27A1), which is also increased in plaque, particularly macrophages ^111–113. 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 ¹¹⁴. Moreover, sterol 27-hydroxylase has even greater activity on 7-ketocholesterol (i.e. macrophages ¹¹⁵ and isolated enzyme ¹¹⁶) and 27-hydroxylated 7-ketocholesterol was also detected at low levels in human plaque ¹¹⁵. In extrahepatic tissues this pathway may facilitate efflux by increasing the polarity of cholesterol ¹¹⁴ and generating ligands for LXR—a nuclear oxysterol sensor. In particular, LDL/cholesterol loading of macrophages induces 27-HC and LXR, which may interact directly ¹¹⁷ and within a feed-forward loop with autophagy ¹¹⁸; whereas human CTX disorder ^115,117, 7-hydroperoxy-cholesterol ¹¹⁹ (i.e. 7-ketocholesterol precursor) and isoLGE₂ (i.e. PGH₂ oxidation) ¹²⁰ can inhibit sterol 27-hydroxylase and efflux. Accumulation of cholesterol in plaque suggests this pathway is insufficient in vivo ¹¹⁵. 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-6 ¹⁰⁸. In the absence of LXR inhibition, 27-HC induced ABCA1/IL-1β and lowered IL-6/IL-18BP ¹⁰⁸, and drives IL-10/M2 polarisation ¹²¹. On the other hand, recent studies find 27-HC can also induce ROS ¹²² and inflammation ¹²³ in human pro-monocytes, and mediate plaque macrophage accumulation in APOE^–/– mice ¹¹¹; although apoE is actually a target of LXR and required for efficient efflux (i.e. via secreted ¹²⁴ and exogenous apoE ¹²⁵).

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 ¹¹², 27-HC and LXR ¹¹³. Plaque PPARg expression was also specifically associated with M2 macrophage markers distant from the lipid core ¹²⁶; although these macrophages actually had suppressed LXRα/ABCA1-dependent efflux, whereas PPARg supported phagocytosis ⁷¹. 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 ¹²⁷. Accordingly, in mouse models macrophage PPARg/LXR signaling is athero-protective ¹²⁸ and involved in athero-regression ^129,130. Regarding natural PPAR ligands, healthy arteries produce various oxylipins, of which COX-derived prostanoids are most abundant ²¹. 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 ¹³¹. Some years later, oxLDL was shown to induce a PPARg–CD36 uptake pathway via n-6 oxylipins (i.e. 13-KODE > 9/13-HODE > 15-HETE) ⁵¹, and subsequently a counterpoise PPARg–LXRα/ABCA1 efflux pathway ¹²⁸, similar to 13-HODE ⁸⁹. Such oxylipins may be largely in the CE fraction ⁵¹ and macrophages may preferentially hydrolyse oxygenated CEs ¹³². Further, plasma LDL from patients with atherosclerosis was also shown to contain n-6 hydroxides (i.e. HODEs > HETEs), but mainly in the phospholipid fraction, and which activated PPARg at physiological levels in vitro ¹³³. The potency of such PPARg ligands varies ^51,98,134 and 15-HETE may prefer PPARβ/δ ¹³⁵. In humans PPARg agonists induced an M2 marker in PBMCs, but not plaque macrophages, where it did still induce CD36 ¹²⁶.

Regarding enzymatic oxygenation, while ALOX15B is induced by hypoxia ⁶⁹ and mediates cholesterol biosynthesis ¹³⁶, ALOX15 is specifically induced by Th2/M2 cytokines and efferocytosis of apoptotic cells (via LXR ¹³⁷), consistent with a role in lipid/tissue homeostasis ⁶⁹. IL-4 induced ALOX15 may induce HODEs/HETEs, PPARg and CD36, while suppressing iNOS ¹³⁸; and may also suppress LXRα/ABCA1-dependent efflux (not PPARg-dependent efferocytosis) ⁷¹. Conversely, in naive mouse macrophages ALOX15 overexpression increased CE hydrolysis and cholesterol efflux, but not via 15/13S-HETE (and 13S-HODE was undetectable) ⁹⁴; and also oxidised LDL via (LRP-dependent) selective uptake and efflux of CE linoleate ¹³⁹. 15-LOX-derived hydroxides also undergo reincorporation into specific phospholipids ¹⁴⁰, with specific functional implications ⁶⁹. In particular, macrophage oxidation of CEs from intra- and extracellular sources resulted in 13-HODE–oxPC ⁹⁷; this may involve LPCAT3 which mediates sn-2 incorporation of PUFAs ¹⁴⁰. LPCAT3 is also a target of LXR and hemopoietic knockout in LDLR^–/– mice impairs cholesterol efflux and exacerbates atherosclerosis ¹⁴¹. Further, in human endothelial cells LXR induced both PUFA synthesis (i.e. 18:2–20:4 and 18:3–20:5/22:6) and phospholipid enrichment, while suppressing 9/13-HODE and 15-HETE but inducing 5-HETE (i.e. 5-LOX) ¹⁴². Therefore, induction of PPARg–LXR may exert negative feedback on natural ligands and redirect linoleate toward elongation. In parallel, enrichment of PC in PUFAs may fine-tune substrate for CE synthesis by HDL-associated LCAT, which has specificity for sn-2 linoleate ¹⁴³ and may also act on hydro(pero)xides ¹⁴⁴. LDL oxidation also favoured net transfer of CEs to HDL (via CETP) ¹⁴⁵, while in rats HDL-associated CE hydro(pero)xides (i.e. [³H]Ch-18:2-O(O)H) were more rapidly removed by liver ¹⁴⁶ and excreted in bile (with the radioactivity in bile acids) ¹⁴⁷. As earlier, lipoprotein(a) is a major carrier of oxPLs ³⁴ and induced PPARg/LXR/efflux in hepatocytes by selective uptake of oxPLs via SR-B1 ¹⁴⁸, suggesting a novel physiological function at low levels ¹⁴⁹.

Antioxidant metabolism and regulation

In human carotid plaque linoleate hydroperoxides were increased in symptomatic cases and directly correlated blood HbA1c, while inversely with HDL-C and paraoxonase-1 activity ¹⁵⁰. This may suggest impaired reduction/clearance. Accordingly, lipid hydroperoxides are reduced by HDL/apoA-I and associated enzymes, including paraoxonase-1 ¹⁵¹, which is itself induced by hepatic PPARg ¹⁵², thereby reinforcing reduction. HDL, paraoxonase-1 and MPO may also exist within a ternary complex and reciprocally inhibit one another ¹⁵³, which could regulate acceptor/efflux activity, as above. Additionally, paraoxonase-1 may also protect LCAT from oxidative inactivation ¹⁵⁴.

In human atherosclerosis low arterial glutathione and related enzyme activity (i.e. GR, GPx and GST) was associated with 4-HNE-related markers and plaque severity ^155,156, while in plasma oxidation of glutathione redox (i.e. GSH/GSSG ratio) was associated with carotid intima–media thickening ¹⁵⁷ independent of traditional markers ¹⁵⁸. An RCT of n-acetyl cysteine (a glutathione precursor) for 1 week on 10 patients with CAD and hyperlipidemia selectively lowered oxLDL (not other lipids) ⁴³. Further, in various human cohorts lower plasma glycine (another glutathione precursor) associates with metabolic and coronary disease, while causality was shown in APOE^–/– mice on low and high glycine diets ⁶³. Here dietary glycine induced glutathione biosynthesis and effector enzymes (incl. GR, GPxs and GSTs), while lowering aortic/macrophage superoxide and atherosclerosis independent of plasma lipids ⁶³. Accordingly, depletion of glutathione and biosynthetic enzymes precedes plaque formation in APOE^–/– mice ¹⁵⁹, and in many studies augmenting glutathione via genes or supplements has a protective effect beyond lipid-lowering and involving macrophages ^43,160–162. Mechanistically, the glutathione system mediates reduction of ascorbate (and consequently tocopherol) and linoleic hydroperoxides (to HODEs) ⁹⁶, as well as conjugation of KODEs ¹⁶³ and aldehydes ¹⁶⁴, and as such may regulate PUFA oxidation, signalling and clearance. In macrophages glutathione deficiency increased ROS and CD36 expression independent of PPARg ¹⁶⁵, whereas glutathione supplementation induced efflux and PPARα ¹⁶⁶, and selenium supported IL-4 induced M2 polarisation via GPx1, PPARg and PGD₂ ¹⁶⁷. Further, LDL and HDL also contain GPx activity ¹⁶¹, while oxidised glutathione can inhibit HDL efflux activity via glutathionylation of paraoxonase-1 ¹⁶⁸.

Importantly, antioxidant systems are highly regulated and may increase in early stages. For instance, in transgenic mice expressing human apoB and lipoprotein(a) a hepatic proteomic analysis prior to atherosclerosis revealed an increase in several antioxidant and efflux proteins ¹⁶⁹. In APOE^–/– mice the development of plaque is also preceded by an initial increase in many arterial antioxidant enzymes before a decline, suggesting a coordinated induction of antioxidant systems before a collapse with atherogenesis ¹⁷⁰. A subsequent proteomic study found oxidation of 1-Cys peroxiredoxin correlated lesion formation ¹⁷¹. More recently, endothelial inflammation and 4-HNE were shown to precede Nrf2 activation, which then appeared to exert negative feedback regulation by restraining inflammation, peroxidation and atherosclerosis ¹⁷². Accordingly, low-level 4-HNE induces Nrf2 ²², which in turn induces 100s of genes encoding antioxidants (incl. the glutathione system) and intermediary pathways to rewire redox metabolism for homeostasis ¹⁷³. In human plaque Nrf2 expression was highest in macrophages of the lipid core and paralleled Myh9, while in APOE^–/– mice macrophage Nrf2 deficiency increased plaque severity, necrotic core size and accumulation of apoptotic cells, whereas Nrf2–Myh9 binding supported efferocytosis via the actin cytoskeleton ¹⁷⁴. Similarly, 15d-PGJ₂ and 17-oxo-DHA (both formed via COX-2) augmented efferocytosis via Nrf2/HO1-dependent expression of CD36 ¹⁷⁵, LOX/COX-2 and pro-resolving mediators ¹⁷⁶. Furthermore, oxLDL/4-HNE ⁵² and 15d-PGJ₂ ¹⁷⁵ induce Nrf2-dependent CD36 expression independent of PPARg ^52,177 (similar to GSH depletion ¹⁶⁵), while phytochemicals induce Nrf2-dependent efflux via suppression of NF-κB signalling ¹⁷⁸ and induction of SR-B1 and ABCA1/G1 transporters ^179–181. Thus, Nrf2 may support both apoptotic cell and cholesterol clearance in parallel to PPARg ^52,128; the intermediate 27-HC also induces autophagy via ROS/Nrf2 favouring cell survival ¹²². 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 ¹⁸².

To summarise all the above, while advanced lipid oxidation and protein modifications are present in human plaque and may favour arterial retention, the preponderance of cholesterol and PUFA oxygenation products might actually support efflux ^{98,101,128,183} and efferocytosis ⁷¹, as may low levels of advanced peroxidation products (e.g. 4-HNE). However, clearly the very presence of plaque and lipid-poor apoA-I/HDL ⁸⁰ suggests this is insufficient, and such adaptive responses could depend on functional endogenous antioxidant systems, or else failed clearance may promote further pathology ^111,123. Thus, oxidation per se may not be the issue, but rather dysregulated redox, perhaps as a result of excessive inflammation and Nrf2/glutathione deficiency.

Dietary PUFAs on lipids 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 recent meta-analyses of prospective cohorts higher dietary and blood/adipose linoleate is associated with reduced CVD incidence ¹⁸⁴ and all/CVD/cancer mortality ¹⁸⁵; and notably, some stronger associations were reported with blood CEs ¹⁸⁴—the major lipid in plaque ²⁵. Similarly, circulating long-chain n-3s are also inversely associated with all/CVD/cancer mortality ^186,187. 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 main source of heterogeneity ¹⁸⁸. 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 ¹⁸⁹, and without significantly affecting lipoprotein(a) ¹⁹⁰. Whereas fish oil/long-chain n-3 PUFA supplementation preferentially lowers triglycerides and non-HDL-cholesterol, particularly in those with hyperlipidemia or overweight/obesity ¹⁹¹. In addition, recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas C18 UFAs lower LDL proteoglycan binding in vitro ^192,193. Clearly all these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite of atherogenesis ²³.

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 ¹⁹⁴. Consistent with this, in a meta-analysis of RCTs n-3s decreased Lp-PLA₂ mass ¹⁹⁵, whereas a trial with soy oil capsules (replacing carbohydrates) increased Lp-PLA₂ activity in association with oxLDL (i.e. 4E6 antibody) and apoB ¹⁰; although an increase in the latter seems atypical ¹⁸⁹. 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 ^196–198. Thus, competition between C18:1 and C18:2 may determine substrate for oxidation, while fish oil/long-chain n-3 PUFAs may displace respective long-chain n-6 PUFAs (i.e. C20/22 species) with less double bonds ¹⁴. Despite this, there are examples in men ¹⁹⁹, monkeys ²⁰⁰ and mice ²⁰¹ of n-6 PUFA-rich diets increasing linoleate/oleate ratios in plasma and plaques, and/or oxidation susceptibility in vitro ²⁰⁰ and in vivo (i.e. MDA and isoprostanes ^201,202), 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 ^203,204. 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 only lowered the aortic lesion area (mean –26%) in proportion to plasma non-HDL-C (–28%) and cholesterol absorption ²⁰⁵ (see below). These PUFA trials are consistent with other dissociations between general oxidation markers and atherosclerosis ²⁷.

Moreover, 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 ¹⁹⁹. 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 ²⁰⁶. 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) ²⁰⁷, while longer trials show enrichment of PUFAs with preservation of oxidation status ^208,209, alongside many other cardio-protective effects (reviewed in ²¹⁰).

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 continued to increase ²¹¹. 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 ⁷. As such a bolus of oxidised lipids may overwhelm intestinal detoxification, particularly in diabetics, where a study in T2D reported baseline glutathione is already low and hydroperoxides elevated, which was ameliorated by glycine-cysteine supplementation ²¹². 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 ²¹³. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁷, but also lower blood lipids ¹³ and atherosclerosis ⁸⁹, 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 ²¹⁴. 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 ²¹⁵. 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 ^216,217. 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 ²¹⁶. 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 ²¹⁶. The activity of many plant foods in this model has been indexed and correlates polyphenol content ²¹⁸; additionally, peroxidation was inhibited by oleate/MUFAs, opposite to fish oil/n-3 PUFAs ²¹⁹. 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 ⁹. Importantly, supplements containing free transition metals (i.e. iron and copper) can also induce gastrointestinal oxidation in humans ²²⁰ and animals ²²¹, which might particularly confound rodent formula diets where antioxidants lower cholesterol.

Role of metabolism and microbiome

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 ³⁵. 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 ²²². 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) ¹⁹. Conversely, despite the oxidative stability of SFAs and the responsiveness of serum stearate to diet ²²³, in short-term trials SFA-rich diets (vs. carbohydrates or MUFAs) can also increase LDL susceptibility to oxidation in relation to MUFA/PUFA ratios ¹⁹⁸, vitamin E ¹⁹⁷, apoB/LDL-C ^224,225 and APOE promoter variants ²²⁵. 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 ²²⁶. 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) ²²⁷. 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 ²²⁸.

Mechanistically, SFAs may affect lipid oxidation indirectly. For instance, hyperlipidemia may induce arterial lipid oxidation and antioxidant dysfunction, as earlier. Also, in an animal model lipoprotein susceptibility to oxidation increased with particle age (i.e. plasma residence) ²²⁹, while in human tracer studies PUFAs (vs. SFAs) lower plasma lipids and increase LDL catabolism ²³⁰. A subsequent study on the Finnish cohort above ²²² found serum SFAs/MUFAs and PUFAs are positively and negatively associated with cardiometabolic outcomes, respectively ²³¹. Accordingly, such SFAs/MUFAs are products of lipogenesis and LDL susceptibility to oxidation is associated with small particle size (i.e. pattern B), which is itself increased by insulin resistance ²³². In people with pattern B a SFA-rich diet (vs. MUFAs) increased small–medium LDL particles ²³³, while a meta-analysis of RCTs suggests exchanging SFAs for n-6 PUFAs may particularly improve glucose-insulin homeostasis ²³⁴.

Regarding microbes, 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 ²³⁵. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ²³⁶, which at similar levels in vitro also induces LDL oxidation ²³⁷. 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 ²³⁸. Regarding specific microbes, 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 ²³⁹ (preprint). Accordingly, SFA intake is associated with Bilophila abundance ^240–242. 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 ²⁴³. This bile profile may be a result of the lower solubility of long-chain SFAs—another corollary of lipid saturation. 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 ²⁴⁴. In other trials the individual response to SFAs was related to baseline Bilophila ²⁴⁰ and diet ²⁴¹, 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.

Oxidation-dependant homeostasis?

While increased tissue linoleate is typically considered an oxidative liability ^2,6–10,205, 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) ^244,245. Elsewhere, lowering dietary linoleic acid/n-6 PUFA (vs. mostly SFAs) also lowered respective plasma oxylipins (i.e. HODEs and KODEs) ²⁴⁶. Linking these effects, linoleate oxygenation within LDL may favour macrophage CE uptake, hydrolysis and efflux via PPARg–27-HC–LXR signalling, as above. Similarly, oxygenated lipids within HDL may favour hepatic uptake and LXRα signalling to increase bile output and plasma cholesterol uptake (i.e. LDLR expression), underlying clinical effects of n-6 PUFAs (incl. lower HDL-C) ^244,247. Conversely, free linoleic acid itself does not induce efflux ⁸⁹ and can even suppress it ²⁴⁸, but may be lowered by LXR activation ¹⁴². Regarding major sources of linoleate hydro(pero)xides in vivo, isomer analyses of both human plaque ⁹⁵ and healthy fasting plasma ²⁴⁹ suggest a dominance of radical-mediated oxidation, which could also be initiated by enzymes ⁹⁷, as earlier. From a biophysical perspective, the specificity of LPCAT3 and LCAT for PUFAs may support CE fluidity ²⁵, while oxygenation (i.e. hydroxylation) of CEs/cholesterol may increase polarity/solubility to facilitate protein interactions and ‘fast-track’ transport to the liver ^114,115,147, before more extensive oxygenation to bile acids. In the pathological context, LDL is initially retained within the arterial wall via extracellular proteoglycans, with further aggregation and fusion making egress back to blood impossible ²³, at least without metabolism. Even in healthy rodents, native LDL appears in the arterial wall as oxLDL with endothelial activation ^44,45; perhaps this could be adaptive if it recruits monocytes and enhances uptake/efflux to support lipid clearance ¹²⁸. Further, plasma linoleate ²²² and HODEs ²⁵⁰ have also been inversely associated with inflammatory markers in humans, while PPARg ¹²⁶ and 27-HC ¹²¹ may favour M2 polarisation, 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) ²⁰; similar to mice on a high fat diet supplemented with DHA (alone/with EVOO) ²⁵¹. In human trials fish oil/n-3 PUFAs also increase plasma early peroxidation products (e.g. HDHAs) ^14,15,17,18 and downstream pro-resolving mediators (e.g. resolvins) ^16,20, at the expense of arachidonic-derived oxylipins ^14,16. Early cell studies found (ambient) oxidation of EPA was required for inhibition of (cytokine-induced) NF-κB, which also required PPARα ²⁵²; more recently, 7-HDHA (formed via ALOX5) was identified as a high-affinity PPARα ligand regulating brain morphology ²⁵³. In the periphery n-3 PUFA induction of PPARα may also support triglyceride lowering ²⁴⁷ via suppression of SREBP-1 (which mediates hepatic lipogenesis) ²⁵⁴ and apoC-III (which inhibits VLDL lipolysis) ²⁵⁵. Further, in animal models dietary oxidised linoleate can also lower hepatic and plasma triglycerides via PPARα ¹³. 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 ⁹² and coronary syndrome ²⁵⁶ 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 ²⁰². In endothelial cells free linoleic acid induced β-oxidation and catalase activity, which were sensitive to vitamin E ⁵⁸, and hydroperoxides with a decline in glutathione before an increase above baseline ⁵⁶. Further, 13-HpODE/HODE induced catalase expression in several arterial cells ²⁵⁷, which is regulated by PPARg ²⁵⁸ and blocks MPO-induced oxidation ⁷⁹. As above, the ability of n-3 PUFAs to improve redox markers ¹⁹ may involve Nrf2 ^20,251. Several n-3 oxylipins can activate Nrf2, such as 17-oxo-DHA, resolvins and maresins ²⁵⁹. 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 ²⁶⁰. 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 ²⁶¹. 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 ²⁶². However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ²⁶³. LPS induces various oxylipins ²¹ which are sensitive to n-3 status ¹⁷; and n-6 series Nrf2-inducers include EKODE ²⁶⁴, 15d-PGJ₂ ²⁶⁵ and LXA₄ ²⁵⁹. Moreover, low-level 4-HHE and 4-HNE (from n-6 PUFAs) similarly induce Nrf2 in endothelial cells ²⁶⁶, and in APOE^–/– mice on a high fat diet 4-HNE precedes Nrf2 activation ¹⁷²; whereas the non-specific peroxidation product MDA may be less effective ⁵².

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 ²². 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 ²⁶⁷. 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 ²⁶⁸—another major pathway mediating experimental atherosclerosis ⁴⁰. In mammalian cells 4-HNE is normally cleared extremely rapidly, especially by hepatocytes and enterocytes, and via several pathways including glutathione conjugation ²⁶⁹. Thus, induction of Nrf2/glutathione would provide feedback inhibition and moderate oxylipin/aldehyde levels, which could be reinforced by glycine availability, as earlier. 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 ^68,69. In particular, enriching neutrophil-like cells and their phospholipids in linoleic acid displaced oleate and 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 ²⁷⁰.

Context-dependant homeostasis?

Further homeostatic insight may lie in other perspectives; for instance, the effects of PUFAs may somewhat overlap with exercise ²⁰². Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ²⁷¹. In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ¹⁰⁹, as well as hepatic LXR and reverse cholesterol transport ²⁷². 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 ²⁷³. 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) ²⁷⁴. 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 ²⁷¹, which may be mediated in large part by ROS/Nrf2 ²⁷⁵. Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ²⁵⁰, isoprostanes and aldehydes ²⁷⁶), and preferentially in HDL ²⁷⁷, 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 ^278,279. 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 ¹².

In considering optimal PUFA intake for tissue homeostasis, post-industrial dietary and tissue linoleic acid/n-6 may seem high ^2,6, although still has favourable health associations ^184,185, but long-chain n-3s more so by mass ^186,187. Many seed oils are notoriously rich in linoleate, although as added fats can substitute animal sources which may already amplify dietary SFAs/palmitate and MUFAs/oleate (Table S1) and distort favourable ratios. Indeed, in the context of whole foods many nuts/seeds are naturally rich in fat and linoleate, where the PUFA/MUFA content is strongly inversely correlated, and olives represent the other extreme (Table S2). In the body C18:2/n-6 may also compete with C18:1/n-9 for esterification and C18:3/n-3 for elongation. In some individuals dietary linoleate may further increase arachidonic acid and inflammation via FADS1 variants affecting n-6 desaturases ²⁸⁰—perhaps here the dietary n-6/n-3 balance may become more important. Long-chain n-3s also appear to have substantial cell membrane (e.g. omega-3 index) and plaque incorporation in humans ^203,204, and with a greater susceptibility to non-enzymatic oxidation may support earlier Nrf2 activation and feedback inhibition ²⁶², and moderate effects of 4-HNE ²⁶⁷. In contrast to PUFAs, serum oleate has unfavourable cardiometabolic associations ²³¹, while in the diet virgin olive oil (vs. common varieties) has been associated with lower all/CVD mortality, implicating other components ²⁸¹. For instance, extra virgin olive oil may also support antioxidant/Nrf2 activity via its polyphenol content ²⁵¹, which is actually much higher in whole olives ²⁸² (along with added sodium).

Importantly, the ability of PUFAs to support 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 ^7,216,217. Dietary thermo-oxidised oils can induce PPARs ¹³ and Nrf2 ^213,283,284, but alongside signs of inflammation, antioxidant depletion and DNA damage ²⁸⁵. Extensively oxidised LDL is rapidly cleared by hepatocytes ^44,45, but may also appear in arterial plaque ⁴¹, whereas mildly modified lipoproteins could linger increasing the probability of entering the arterial wall, wherein they may get further oxidised and/or exacerbate atherosclerosis ⁹. Thus, oxidation ‘ex vivo’ might subvert homeostasis and promote pathogenesis in vivo. Notably, dietary oil oxidation typically involves prolonged storage ²⁸⁶ or heating ^211,213,283, whereas red meat may induce significant advanced oxidation within the normal digestive/postprandial phase ^216,217, which is exaggerated by addition of PUFA-rich oils ^9,219, suggesting it may be particularly relevant as 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 ¹⁹⁹), filled beef (i.e. Minnesota study ²), and more recently liver pâté ²⁴⁴, suggesting direct contact with heme-iron. However, this could represent a small proportion of oil consumed and be offset by other dietary components.

Conclusion

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. Indeed, enzymatic and non-enzymatic oxidation products of both n-6 and n-3 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. On the other hand, 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 negative effects. Notably, this situation seems most analogous to the typical oxidative conditions used to create atherogenic lipoproteins in vitro, which inform classic oxidative stress-based hypotheses. However, the actual effects of such exogenous oxidation may be heterogenous and depend on the extent of oxidation, dietary pattern and host; those with metabolic disorders perhaps being most susceptible to negative effects.

As such a context-dependant homeostatic hypothesis is suggested here, wherein 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, acknowledging the negative effects of excessive peroxidation, adaptive responses may be undermined by sufficient antioxidant (e.g. glutathione) deficiency and exogenous oxidation—i.e. here a susceptibility to oxidation may confer a susceptibility to disease.

This review has notable limitations. Its mechanistic and theoretical nature make it susceptible to the reductionist trap; despite attempts to follow a holistic evidence hierarchy, prioritise human data and consider systems-level interaction, other pathways will exist. The narrative synthesis is also inevitably limited by personal bias and incomplete research. Moreover, heterogeneity and inconsistencies within the current literature can challenge this and any coherent perspective at all; as such, some potential controversies (and reconciliations) and general areas for further research are listed in the supplement. Regarding practical implications, this is a qualitative review which 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 effect of any isolated oil peroxidation may be highly context-dependent. 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.

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