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

Another work in progress; any thoughts/feedback appreciated.

Atherosclerotic cardiovascular disease (ASCVD) is ubiquitous and a leading cause of death globally, while a cornerstone of dietary guidelines for prevention 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 from the fact that atherosclerosis is widely acknowledged to involve lipid oxidation, among which PUFAs are most susceptible, supporting a theoretical basis of some concern, particularly with seed oils ^2,5–8 (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 ^9,10, anti-inflammatory ^11–15 and antioxidant activity ^16,17, suggesting context matters and opportunity for harmonisation.

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) ¹⁸, in the presence of antioxidants and with positional specificity, thereby producing metabolites of physiological relevance (e.g. Fig. 1). Indeed, both early and late-stage PUFA oxidation products exhibit signalling activity, as discussed herein. Hence it is important to consider the full scope and context of lipid oxidation in vivo for pathological interpretation, and this article is particularly concerned with disentangling physiology.

Lipid oxidation and atherogenesis

Already in the 1950s lipid peroxidation was detected in human plaque, and much subsequent research supports the involvement of lipid and protein oxidation in atherosclerosis ¹⁹. 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 ²⁰. Oxidised LDL (oxLDL) can also be detected in plasma and associates with CVD ^21,22, although not always independently of apoB (e.g. CHD ²³ and MetS ²⁴), potentially due to 4E6 antibody cross-reactivity ²⁵. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB), which normally represent a very small fraction of LDL ²⁶, are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ²⁵. Conversely, 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 ^27,28. While the results of large RCTs with high-dose (i.e. supra-physiological) antioxidant supplements in general populations have mostly failed to show benefit ^19,29 (unlike general lipid-lowering), there is scant outcome data on more physiological approaches targeting specific deficits and underlying redox biology ^29,30.

Notably, plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^25,31. The high antioxidant capacity of plasma also suggests lipoprotein oxidation may occur elsewhere, such as within the arterial wall. 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 ¹⁹. 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 ²². Further, unlike native LDL, macrophage uptake of oxLDL results in lipid trapping within lysosomes ³², cholesterol crystallisation and NLRP3 activation ^33,34. Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ³⁵. These changes involve advanced lipid peroxidation. MDA in particular reacts with lysine residues of apoB₁₀₀ (more than 4-HNE) resulting in recognition by scavenger receptors ³⁶; and similarly impairs apoA-I efflux activity (vs. other reactive carbonyls) ³⁷. Further, CE aldehydes have reduced macrophage hydrolysis ³⁸ and may be converted to 7-ketocholesterol ³⁹, which inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ³², and dose-dependently induces cholesterol crystals ⁴⁰. Supporting the relevance of these mechanisms, such aldehydes are detected in human arterial lesions (e.g. LDL ²⁰, HDL ³⁷ and CEs ^38,39). Further, in human tracer studies with autologous native 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 most abundant lipophilic antioxidant ⁴².

Despite such data apparently supporting a causal role of lipid oxidation in atherogenesis, the general failure of high-dose vitamin E/antioxidant trials to improve hard outcomes suggests more complexity ¹⁹. Foremost, several early studies found plaque lipid oxidation occurred despite normal levels of ascorbate, α-tocopherol and ubiquinone ⁴³, and was similar in T2D ⁴⁴. Moreover, oxidation by free transition metals in vitro may have limited pathophysiological analogy in plaque, where at least heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ⁴⁵. On the other hand, early interest turned to 15-LOXs (i.e. non-heme iron-dependent dioxygenases) ⁴⁶, since they can initiate PUFA oxidation and lipoprotein modification ^47,48, which is not blocked by vitamin E ⁴⁹. Human plaques express 15-LOXs (i.e. ALOX15 ^47,50 and ALOX15B ⁴⁸) and COXs ⁵¹ within specific macrophage populations. Further, increased iNOS ^52,53 and MPO ⁵⁴, along with lipoprotein enrichment in their protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^55,56, also implicates immuno-oxidative activity ¹⁹. These 2-electron pathways are also not blocked by vitamin E ⁴³ (or serum ⁵⁷), and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptors ^57,58, while MPO-modified tryptophan residues within apoA-I/HDL inactivate its ABCA1-dependent acceptor activity ^56,59.

However, other data present more fundamental challenges ^19,60. 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 ⁶¹. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^61,62 and CE accumulation beyond native or oxLDL ^63,64. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ³². More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁶⁵ and induce lysosomal crystals and NLRP3 activation ^33,34, so may contribute in these ways ⁶¹. 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 ⁶⁶. 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 ⁶⁷. And contrasting earlier studies ⁶⁸, HDL anti-platelet activity can also be induced by copper-oxidation in vitro ^69,70, while human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ⁷¹, consistent with ALOX15 overexpression increasing reverse cholesterol transport ⁷². Thus, the extent and type of oxidation may be important.

Lipid-specific oxidation and signalling 

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some context. 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 CEs ⁷³. Of PUFA oxidation products, linoleic hydro(pero)xides dominate ¹⁹. For instance, 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.) ⁷⁴. 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 ⁷⁴. 15-LOX in particular can directly initiate CE oxidation ⁴⁶, while subsequent radical reactions may erode product specificity ^75,76; and at least in several earlier reports 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ⁷⁷. Conversely, mouse models can lack oxidised CEs despite LOX activity ⁷⁸. Of oxysterols, human plaque is generally dominated by 27-hydroxychoesterol (27-HC), followed by 7-ketocholesterol ¹⁹, although some reports suggest the opposite in human macrophages ^79,80 and absence of 27-HC in animals ⁸¹. 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 ⁸². Furthermore, an earlier 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-HC 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 ⁸³. 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 ⁸⁴. Accordingly, MPO/iNOS-derived oxidants can directly induce protein modification and initiate lipid peroxidation, while in mice aortic lesions may lack MPO ⁵⁶.

These observations in vivo contrast the typical situation in vitro ¹⁹, where LDL oxidation generates hydroperoxides immediately followed by MDA ^36,58,85, before depletion of CEs with accumulation of 7-ketocholesterol ^39,86. 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.) ⁸³. Conversely, the profile in vivo implies more mildly oxidising conditions where enzymes and/or the α-tocopherol radical can initiate lipid peroxidation ⁸⁷; the latter being favoured by insufficient regenerative co-antioxidants ⁸⁴ (e.g. CoQ₁₀ and carotenoids) ⁸⁸. In particular, 27-HC is produced by mitochondrial sterol 27-hydroxylase (CYP27A1), indicating a dominance of enzymatic sterol oxidation ⁸³. 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 ⁸⁹. Moreover, sterol 27-hydroxylase has even greater activity on 7-ketocholesterol (i.e. macrophages ⁹⁰ and isolated enzyme ⁹¹) and 27-hydroxylated 7-ketocholesterol was also detected at low levels in human plaque ⁹⁰. In extrahepatic tissues this pathway may facilitate efflux by increasing the polarity of cholesterol ⁸⁹ and generating ligands for LXR—an oxysterol sensor. In particular, LDL/cholesterol loading of macrophages induces 27-HC and LXR, which may interact directly ⁹² and within a feed-forward loop with autophagy ⁹³; whereas human CTX disorder ^90,92, 7-hydroperoxy-cholesterol ⁹⁴ (i.e. 7-ketocholesterol precursor) and isoLGE₂ (i.e. PGH₂ oxidation) ⁹⁵ can inhibit sterol 27-hydroxylase and efflux. Accumulation of cholesterol in plaque suggests this pathway is insufficient in vivo ⁹⁰. 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 ⁸⁰. Of note, in the absence of LXR inhibition, 27-HC induced ABCA1/IL-1β and lowered IL-6/IL-18BP ⁸⁰. On the other hand, recent studies find 27-HC can also induce ROS ⁹⁶ and inflammation ⁹⁷ in human pro-monocytes, and mediate plaque macrophage accumulation in APOE^–/– mice ⁹⁸; although apoE is actually a target of LXR and required for efficient efflux (i.e. via secreted ⁹⁹ and exogenous apoE ¹⁰⁰).

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 ¹⁰¹, 27-HC and LXR ¹⁰². Plaque PPARg expression was also specifically associated with M2 macrophage markers distant from the lipid core ¹⁰³; although these macrophages actually had suppressed LXRα/ABCA1-dependent efflux, whereas PPARg supported phagocytosis ⁵⁰. In genetic mouse models macrophage PPARg–LXR signaling is athero-protective ¹⁰⁴. 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 ¹⁰⁵. Conversely, murine athero-regression may involve a shift to M2 and PPARg/LXR activity ^106,107. Regarding natural PPAR ligands, healthy arteries produce various oxylipins, of which COX-derived prostanoids are most abundant ¹⁸. 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 ¹⁰⁸. However, other oxylipins may also be relevant.

As above, accumulation of 27-HC is paralleled by CE hydro(pero)xides ^83,84, which may also have a divergent fate in vivo. For instance, in comparing 15-LOX-derived hydroperoxides to direct MDA–LDL modification, only the latter induced macrophage uptake ³⁶. Elsewhere however, 15-LOX-derived hydroperoxides induced TLR4-dependant macropinocytosis and LDL uptake, which can also be interpreted pathologically (in the absence of HDL) ¹⁰⁹. On the other hand, macrophages may preferentially hydrolyse such oxygenated CEs ¹¹⁰ liberating natural PPAR ligands ^77,111,112. In particular, similar to plaque ⁷⁴, plasma LDL from patients with atherosclerosis contained various n-6 oxylipins (i.e. HODEs > HETEs) which activated PPARg at physiological levels in vitro ¹¹³; although later 15-HETE was shown to prefer PPARβ/δ ¹¹¹. PPARg primes M2 polarisation in human monocytes, not plaque macrophages ¹⁰³, but where it may still induce both scavenger receptor CD36 ¹⁰³ and LXRα/ABCA1-dependent efflux ¹⁰⁴, similar to 13-HODE ¹¹⁴. Further, while ALOX15B is induced by hypoxia ⁴⁸ and mediates cholesterol biosynthesis ¹¹⁵, ALOX15 is specifically induced by Th2/M2 cytokines and efferocytosis of apoptotic cells (via LXR ¹¹⁶), consistent with a role in lipid/tissue homeostasis ⁴⁸. Here IL-4 induced ALOX15 may suppress LXRα/ABCA1-dependent efflux, while PPARg mediates phagocytosis ⁵⁰ and anti-inflammation ¹¹⁷. Conversely, in naive RAW macrophages ALOX15 overexpression increased CE hydrolysis and cholesterol efflux, but not via 15/13S-HETE (and 13S-HODE was undetectable) ⁷². 15-LOX-derived hydroxides undergo reincorporation into specific phospholipids ¹¹⁸, with specific functional implications ⁴⁸; in particular, macrophage oxidation of CEs from intra- and extracellular sources resulted in 13-HODE–oxPC ⁷⁶. Moreover, macrophages overexpressing 15-LOX also oxidised LDL via (LRP-dependent) selective uptake and efflux of CE linoleate ¹¹⁹. In the extracellular context, LDL oxidation favoured net transfer of CEs to HDL (via CETP) ¹²⁰, while in rats HDL-associated CE hydro(pero)xides (i.e. [³H]Ch-18:2-O(O)H) were more rapidly removed by liver ¹²¹ and excreted in bile (with the radioactivity in bile acids) ¹²², suggesting increased reverse cholesterol transport. Therefore, while advanced lipid oxidation and protein modifications may favour retention, the preponderance of mild lipid oxidation (i.e. hydroxylation) in vivo might be secondary ¹⁹ and even support clearance ^{77,83,104,123}. On the other hand, failed clearance may promote further pathology; for instance, via 27-HC ^97,98.

Lipid oxidation and flux may also critically depend on intermediary redox metabolism. In particular, in human arteries low glutathione and related enzyme activity (i.e. GR, GPx and GST) was associated with 4-HNE-related markers and plaque severity ^124,125, while in plasma oxidation of glutathione redox (i.e. GSH/GSSG ratio) was associated with carotid intima–media thickening ¹²⁶ and independent of traditional markers ¹²⁷. 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 ³⁰. 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 ³⁰. Direct glutathione supplementation also reduced lesion development and macrophage oxLDL uptake while increasing efflux ¹²⁸. Mechanistically, the glutathione system mediates reduction of ascorbate (and consequently tocopherol) and linoleic hydroperoxides (to HODEs) ⁷⁵, as well as conjugation of KODEs ¹²⁹ and aldehydes ¹³⁰, and as such may regulate PUFA oxidation, signalling and clearance. In macrophages glutathione deficiency increased ROS and CD36 expression independent of PPARg ¹³¹, whereas glutathione supplementation induced efflux and PPARα ¹³², and selenium supported IL-4 induced M2 polarisation via GPx1, PPARg and PGD₂ ¹³³. Further, LDL and HDL also contain GPx activity ¹²⁸, while oxidised glutathione can inhibit HDL efflux activity via glutathionylation of paraoxonase-1 ¹³⁴.

Dietary fat and host redox

Dietary fat saturation has well recognised 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 ¹³⁵. Whereas fish oil/long-chain n-3 PUFA supplementation preferentially lowers triglycerides and non-HDL-cholesterol, particularly in those with hyperlipidemia or overweight/obesity ¹³⁶. In addition, recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas C18 UFAs lower LDL proteoglycan binding in vitro ^137,138. Clearly all these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite of atherogenesis ¹³⁹. On the other hand, the effect of dietary fats on lipoprotein oxidation is much more controversial. Tested since the early 90s in short-term trials, MUFA-rich diets (vs. n-6 PUFAs or oily fish/n-3) typically lower LDL and HDL oxidation, and susceptibility to copper oxidation (i.e. lag time and/or rate) and monocyte adhesion in vitro, which correlates lipoprotein phospholipid oleate/linoleate ratios ^140–142. 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 ¹¹. This has fuelled some concern, although doesn’t mirror hard outcome data or reflect the more nuanced redox biology in vivo, as above. Accordingly, in men ¹⁴³, monkeys ¹⁴⁴ and mice ¹⁴⁵ n-6 PUFA-rich diets increase linoleate/oleate ratios in plasma and plaques, and oxidation in vitro ¹⁴⁴ and in vivo ^145,146, yet are protective. The long-chain n-3 content of advanced carotid plaques in humans is also increased by supplementation and correlates greater stability and lower inflammation, consistent with anti-inflammatory effects ^147,148.

However, an oxidative effect of PUFAs depends on the food matrix, and seed oils are naturally 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 lower erythrocyte susceptibility to peroxide ¹⁴³. 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 ¹⁴⁹. 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) ¹⁵⁰, while longer trials show enrichment of PUFAs with preservation of oxidation status ^151,152, alongside many other cardio-protective effects (reviewed in ¹⁵³). 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 ¹⁵⁴. When fed to humans oxidised linoleic acid could be detected in chylomicrons/remnants for 8hrs (esp. 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. 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 ¹⁵⁵. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁶, or lower blood lipids ¹⁰ and atherosclerosis ¹¹⁴, implicating context. More specifically, 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 ¹⁵⁶. Also, 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 ¹⁵⁷.

Regarding 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 ^158,159. 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 ¹⁵⁸. 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 ¹⁵⁸. The activity of many plant foods in this model has been indexed and correlates polyphenol content ¹⁶⁰; additionally, peroxidation was inhibited by oleate/MUFAs, opposite to fish oil/n-3 PUFAs ¹⁶¹. 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. Of note, iron deficiency is common and supplementation can also induce gastrointestinal lipid peroxidation ¹⁶².

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 ²⁶. 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/inflammation, opposite to SFAs/MUFAs, which withstood adjustment for CVD risk factors and red meat intake ¹⁶³. 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) ¹⁶. Conversely, despite the oxidative stability of SFAs and the responsiveness of serum stearate to diet ¹⁶⁴, in short-term trials SFA-rich diets (vs. carbohydrates or MUFAs) can also increase LDL susceptibility to oxidation in relation to MUFA/PUFA ratios ¹⁴², vitamin E ¹⁴¹, apoB/LDL-C ^165,166 and APOE promoter variants ¹⁶⁶. 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 ¹⁶⁷. 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 platelet activating factor acetyl-hydrolase activity (which can hydrolyse oxidised lipids), without affecting macrophage–faeces reverse transport (or paraoxonase-1) ¹⁶⁸. 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 (also hydrolyses oxidised lipids) and faecal cholesterol excretion ¹⁶⁹.

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) ¹⁷⁰, while in human tracer studies PUFAs (vs. SFAs) lower plasma lipids and increase LDL catabolism ¹⁷¹. LDL susceptibility to oxidation is also associated with small particle size (i.e. pattern B), which is itself increased by insulin resistance ¹⁷²; in people with pattern B a SFA-rich diet (vs. MUFAs) increased small–medium LDL particles ¹⁷³, while a meta-analysis of RCTs suggests exchanging SFAs for n-6 PUFAs may particularly improve glucose-insulin homeostasis ¹⁷⁴. 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 ¹⁷⁵. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ¹⁷⁶, which at similar levels in vitro also induces LDL oxidation ¹⁷⁷. 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 ¹⁷⁸. 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 ¹⁷⁹. Accordingly, SFA intake is associated with Bilophila abundance ^180–182. 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 ¹⁸³. 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 ¹⁸⁴. In other trials the individual response to SFAs was related to baseline Bilophila ¹⁸⁰ and diet ¹⁸¹, 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

Could the discordant effects of PUFAs on oxidation and atherosclerosis be further reconciled by signalling? For instance, in 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) ^184,185. Elsewhere, lowering dietary linoleic acid/n-6 PUFA (vs. mostly SFAs) also lowered respective plasma oxylipins (i.e. HODEs and KODEs) ¹⁸⁶. 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) ^184,187. From a biophysical perspective, LCAT specificity for linoleate ¹⁸⁸ may support CE fluidity ⁷³ and substrate for LOX ⁷⁶, while oxygenation (i.e. hydroxylation) of CEs and cholesterol may increase polarity/solubility to facilitate protein interactions and ‘fast-track’ transport to the liver ^89,90,122, before more extensive oxygenation to bile acids. Thus, dietary linoleate might actively support cholesterol clearance (e.g. vs. carbs or oleate). 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) ¹⁷; similar to mice on a high fat diet supplemented with DHA (alone/with EVOO) ¹⁸⁹. In human trials fish oil/n-3 PUFAs also increase plasma early peroxidation products (e.g. HDHAs) ^11,12,14,15 and downstream pro-resolving mediators (e.g. resolvins) ^13,17, at the expense of arachidonic-derived oxylipins ^11,13. Early cell studies found (ambient) oxidation of EPA was required for inhibition of (cytokine-induced) NF-κB, which also required PPARα ¹⁹⁰; more recently, 7-HDHA (formed via ALOX5) was identified as a high-affinity PPARα ligand regulating brain morphology ¹⁹¹. In the periphery n-3 PUFA induction of PPARα may also support triglyceride lowering ¹⁸⁷ via suppression of SREBP-1 (which mediates hepatic lipogenesis) ¹⁹² and apoC-III (which inhibits VLDL lipolysis) ¹⁹³. Further, in animal models dietary oxidised linoleate can also lower hepatic and plasma triglycerides via PPARα ¹⁰. 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 ⁷⁰ and coronary syndrome ¹⁹⁴ 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 ¹⁴⁶. Accordingly, in vitro 13-HpODE/HODE induced catalase in several arterial cells ¹⁹⁵; catalase is also regulated by PPARg ¹⁹⁶ and blocks MPO-induced oxidation ⁵⁸. As above, the ability of n-3 PUFAs to improve redox markers ¹⁶ may involve Nrf2 ^17,189, 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 ¹⁹⁷. 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 ¹⁹⁸. 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 ¹⁹⁹. 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 ²⁰⁰. However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ²⁰¹. LPS induces various oxylipins ¹⁸ sensitive to n-3 status ¹⁴; and n-6 series Nrf2-inducers include EKODE ²⁰², 15d-PGJ₂ ²⁰³ and LXA₄ ¹⁹⁷. Moreover, low-level 4-HHE and 4-HNE (from n-6 PUFAs) similarly induce Nrf2 in endothelial cells ²⁰⁴, 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 ²⁰⁵. In addition, in LPS-treated mice 4-HNE inhibited inflammasome activation; tested in vitro this was independent of its effects on Nrf2/NF-κB signalling, but may involve direct binding to NLRP3 ²⁰⁶—another major pathway mediating experimental atherosclerosis ³⁴.

Excess unsaturated aldehydes are ultimately toxic; indeed while low-level 4-HNE induces Nrf2 and supports homeostasis, high levels block Nrf2 and favour apoptosis ²⁰⁷. Regardless, if increasing PUFA intake can have benefits in advanced human disease (e.g. seed oils ¹ and fish oil ^147,148), an overall homeostatic effect may be possible. In healthy cells induction of Nrf2 supports antioxidant activity, which may moderate oxylipin and aldehyde metabolism/signalling via the glutathione system and glycine availability, as earlier. In macrophages oxLDL/4-HNE ²⁰⁸ and 15d-PGJ₂ ²⁰⁹ induce Nrf2-dependent CD36 expression independent of PPARg ^208,210 (similar to GSH depletion ¹³¹), while phytochemicals induce Nrf2-dependent efflux via suppression of NF-κB signalling ²¹¹ and induction of SR-B1 and ABCA1/G1 transporters ^212–214; thus Nrf2 may support cholesterol clearance in parallel to PPARg ¹⁰⁴. 27-HC also induces autophagy via ROS/Nrf2 favouring cell survival ⁹⁶. Conversely, in highly stressed cells Nrf2/glutathione exhaustion may favour aldehyde accumulation and apoptosis, which itself could indirectly exert homeostatic pressure via oxPL-dependent efferocytosis and subsequent induction of ALOX15-dependent pro-resolving mediators ^47,48. In human plaque Nrf2 expression was highest in macrophages in the lipid core and paralleled Myh9, while in preclinical studies Nrf2–Myh9 binding supported efferocytosis via the actin cytoskeleton ²¹⁵. Accordingly, 15d-PGJ₂ and 17-oxo-DHA (both formed via COX-2) augmented efferocytosis via Nrf2/HO1-dependent expression of CD36 ²⁰⁹, LOX/COX-2 and pro-resolving mediators ²¹⁶, which may themselves activate Nrf2 ¹⁹⁷. Taken together, perhaps cellular PUFA status could determine the threshold for Nrf2 induction under oxidative conditions, favouring earlier feedback and pleiotropic regulation of redox, immune and lipid homeostasis, which ultimately limits plaque growth and instability. 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 ²¹⁷. Therefore, timely activation of Nrf2 may also limit systemic pathology.

Further hormetic insight may lie in other perspectives; for instance, the effects of PUFAs may somewhat overlap with exercise ¹⁴⁶. Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ²¹⁸. In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ⁸¹, as well as hepatic LXR and reverse cholesterol transport ²¹⁹. 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 ²²⁰. 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) ²²¹. 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 ²¹⁸, which may be mediated in large part by ROS/Nrf2 ²²². Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ²²³, isoprostanes and aldehydes ²²⁴), and preferentially in HDL ²²⁵, 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 ^226,227. 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 ⁹.

In considering how differences between dietary UFAs may affect tissue homeostasis, besides differential n-6/n-3 oxylipin signalling, long-chain n-3 PUFAs have greater susceptibility to non-enzymatic oxidation and seemingly cell membrane incorporation, suggesting they may be particularly well poised to support Nrf2-based antioxidant and anti-inflammatory activity. Further, while linoleic acid/n-6 PUFAs may offer cholesterol-lowering activity in healthy individuals, there may also be potential for inflammatory activity in association with FADS1 variants affecting n-6 desaturases and metabolism to arachidonic acid ²²⁸. In this respect, the dietary n-3/n-6 balance may become more important. Alternatively, despite the relative oxidative stability of MUFAs, extra virgin olive oil may also support antioxidant/Nrf2 activity via its polyphenol content ¹⁸⁹, which may be much higher in whole olives ²²⁹ (along with sodium).

Finally, the ability of PUFA oxidation to favour homeostasis may also rest upon the site of oxidation. In the body low level oxidation products may initially induce signaling pathways favouring lipid clearance, anti-inflammatory and antioxidant activity, thereby limiting further oxidation and maintaining homeostasis. This may involve subcellular/organelle-specific ROS/RNS generation and transient or 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 incorporate into plasma lipoproteins for delivery to tissues ^6,158,159. Dietary thermo-oxidised oils can induce PPARs ¹⁰ and Nrf2 ^155,230,231, but alongside signs of inflammation, antioxidant depletion and DNA damage ²³². Sufficient lipoprotein modification/damage may also result in rapid clearance by phagocytes, largely in the liver, but also potentially arterial plaque ^8,41. Thus, excessive oxidation ‘ex vivo’ may eventually overwhelm homeostasis and promote pathogenesis. Notably, dietary oil oxidation typically involves prolonged storage ²³³ or heating ^154,155,230, whereas red meat can apparently induce significant advanced oxidation within the normal digestive/postprandial phase ^158,159, which is exaggerated by addition of PUFA-rich oils ^8,161, suggesting it may be particularly relevant and a potential confounder in PUFA studies. For instance, many old CVD trials had heterogenous 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 ¹⁴³), filled beef (i.e. Minnesota study ²), and more recently liver pâté ¹⁸⁴, suggesting direct contact with heme-iron.

Conclusion

PUFA oxidation is associated with human atherosclerosis and induces toxic effects in vitro, supporting a pathogenic view; however, attention to specificity in vivo may unveil a precedent physiology. Indeed, biology couples enzymatic and non-enzymatic oxidation products to adaptive responses via signalling (e.g. PPARs and Nrf2); even advanced peroxidation products may initially induce hormesis before toxicity. As such, the susceptibility of PUFAs to oxidation may not be dichotomous with their health benefits, but rather underlie favourable modulation of redox, immune and lipid homeostasis, and opposite to typical SFAs. This may have some analogy and synergy with exercise, which also generally benefits cardiovascular health. However, the PUFA balance and site of oxidation may be determinate; in particular, excessive peroxidation during food processing and/or digestion would presumably bypass the opportunity for physiologic signalling and instead favour accumulation of fragmented end products, increasing the potential for toxicity. This situation seems more 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.

From a natural and practical perspective, wholefood PUFAs may be least susceptible to oxidation ex vivo, while still providing substrate for favourable oxidation in vivo, whereas 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 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. Finally, this review encountered various inconsistencies in the research literature challenging a coherent perspective; some potential counterviews and reconciliations are summarised and listed below.

• 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 ^19,29—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 ^29,30.
• In many cell studies lipoprotein and PUFA oxidation are toxic via peroxidation products—The typical extreme isolation and oxidative conditions in vitro may not model the typical situation in vivo ¹⁹, except for some specific areas addressed herein (e.g. hemolysis and digestion).
• Recent preclinical studies suggest the CYP27A1/27-HC pathway is mainly pro-atherogenic ^97,98—This view does not reconcile increased CYP27A1 expression with hyperlipidemia, PPARg ¹⁰² or exercise ⁸¹; CYP27A1 mediated efflux of cholesterol ^89,92 and 7-ketocholesterol ^90,91; 27-HC-induced cell survival (via Nrf2/autophagy) ⁹⁶ and efflux/immune modulation (via LXR) ⁸⁰; nor human CTX pathophysiology ^90,92. On the other hand, since apoE supports LXR-dependent efflux ^99,100 typical APOE^–/– mouse models ⁹⁸ 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 ⁸⁰.
• 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 ²¹⁵, whereas arterial cell-specific modulation suggests Nrf2 has a protective effect (e.g. endothelium ²⁰⁵ and macrophages ²¹⁵). Dietary PUFAs may actually enable the best of both by lowering lipids and inducing Nrf2.

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