Susceptible to oxidation yet resistant to atherosclerosis—reconciling the PUFA paradox via signalling

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 ^1–3. 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 ^2,4–7 (as in the 2026 DGA report). At first glance, a simple dichotomous reconciliation is that the putative benefits of PUFAs, such as lipid lowering or anti-inflammatory activity, may outweigh any negative effects. However, PUFA oxidation can take many paths and produce many molecules with diverse effects, including lipid lowering ^8,9 and anti-inflammatory activity ^10–15, suggesting context matters and opportunity for harmonisation.

At one extreme, radical-mediated autoxidation eventually degrades 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, before later cleavage and fragmentation, and these reactions are explicitly catalysed by enzymes, such as lipoxygenases (LOXs), cyclooxygenases (COXs) and cytochrome P450s (CYPs), with positional specificity and in the presence of antioxidants, thereby producing stable metabolites of physiological relevance (e.g. Fig. 1). Indeed, both early and late-stage PUFA peroxidation products exhibit signalling activity, as discussed herein. Hence it is important to consider the full scope and context of lipid peroxidation 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 ¹⁶. 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 ¹⁷. Oxidised LDL (oxLDL) can also be detected in plasma and associates with CVD ^18,19, although not always independently of apoB (e.g. CHD ²⁰ and MetS ²¹), potentially due to 4E6 antibody cross-reactivity ²². On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB), which normally represent a very small fraction of LDL ²³, are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ²². 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 ^24,25, although the results of large RCTs with high-dose (i.e. supra-physiological) supplements in general populations have mostly failed to show benefit ^16,26 (unlike general lipid-lowering). However, there is scant outcome data on more physiological and targeted antioxidant approaches, including for other associated redox modulators (e.g. glycine ²⁷ and polyphenols ²⁶).

Notably, plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^22,28. 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 ¹⁶. 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 ¹⁹. Further, unlike native LDL, macrophage uptake of oxLDL results in lipid trapping within lysosomes ²⁹, cholesterol crystallisation and NLRP3 activation ^30,31. Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ³². 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 ³³; and similarly impairs apoA-I efflux activity (vs. other reactive carbonyls) ³⁴. Further, CE aldehydes have reduced macrophage hydrolysis ³⁵ and may be converted to 7-ketocholesterol ³⁶, which inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ²⁹, and dose-dependently induces cholesterol crystals ³⁷. Supporting the relevance of these mechanisms, such aldehydes are detected in human arterial lesions (e.g. LDL ¹⁷, HDL ³⁴ and CEs ^35,36). 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 ³⁸. 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 ³⁹.

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 ¹⁶. Foremost, oxidation by free transition metals may have limited physiological analogy; for instance, heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ⁴⁰. On the other hand, early interest turned to 15-LOXs (i.e. non-heme iron-dependent dioxygenases) ⁴¹, since they can initiate PUFA oxidation and lipoprotein modification ^42,43, which is not blocked by vitamin E ⁴⁴. Human plaques may particularly express ALOX15B, in association with macrophages and HIF-1α ⁴³. Further, expression of COX-2 ⁴⁵, along with iNOS ^46,47 and MPO ⁴⁸, and lipoprotein enrichment in their protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^49,50, collectively implicates immuno-oxidative activity ¹⁶. These 2-electron pathways are also not blocked by vitamin E ⁵¹ (or serum ⁵²), and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptors ^52,53, while MPO-modified tryptophan residues within apoA-I/HDL inactivate its ABCA1-dependent acceptor activity ^50,54.

However, other data present more fundamental challenges ^16,55. 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 ⁵⁶. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^56,57 and CE accumulation beyond native or oxLDL ^58,59. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ²⁹. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁶⁰ and induce lysosomal crystals and NLRP3 activation ^30,31, so may contribute in these ways ⁵⁶. 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 ⁶¹. Further, 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 ⁶². And contrasting earlier studies ⁶³, HDL anti-platelet activity correlates oxylipin content in vivo and is induced by copper-oxidation in vitro ^64–66, while human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ⁶⁷, consistent with ALOX15 overexpression increasing reverse cholesterol transport ⁶⁸. Thus, the extent and type of oxidation may be important.

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 ⁶⁹. Accordingly, 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 di-oxygenated derivatives (e.g. HODEs, KODEs, EpOMEs, etc.) ⁷⁰. 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 ⁷⁰. 15-LOX in particular can directly initiate CE oxidation ⁴¹, while subsequent radical reactions may erode product specificity ^71,72; and at least in several earlier reports 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ⁷³. Recent high-resolution imaging of advanced carotid plaques also found oxidised CEs co-localise with sphingomyelin in the necrotic core, while a metabolite resembling 7-ketocholesterol was uncorrelated ⁷⁴. Several earlier studies also found plaque lipid oxidation occurred despite normal levels of α-tocopherol ⁵¹, and was similar in T2D ⁷⁵. In particular, an analysis of intimal lipoprotein-containing fractions of human 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-hydroxycholesterol 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 ⁷⁶. 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 ⁷⁷. For instance, MPO/iNOS-derived oxidants can directly induce protein modification and initiate lipid peroxidation ⁵⁰; and notably, in mouse models aortic lesions lack MPO ⁵⁰ and oxidised CEs ⁷⁸.

These observations in vivo contrast the typical situation in vitro ¹⁶, where LDL oxidation generates hydroperoxides immediately followed by MDA ^33,53,79, before depletion of CEs with accumulation of 7-ketocholesterol ^36,80. 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.) ⁷⁶. Conversely, the profile in vivo implies more mildly oxidising conditions where enzymes and/or the α-tocopherol radical can initiate lipid peroxidation ⁸¹; the latter being favoured by insufficient regenerative co-antioxidants ⁷⁷ (e.g. CoQ₁₀ and carotenoids) ⁸². 7-ketocholesterol is also quantitatively and temporally overshadowed by 27-hydroxycholesterol, which is produced by sterol 27-hydroxylase, indicating a dominance of enzymatic sterol oxidation ⁷⁶. In the liver this enzyme initiates the acidic pathway of bile acid synthesis, while elsewhere it may facilitate efflux ⁸³. In particular, LDL/cholesterol loading of macrophages induces 27-hydroxycholesterol and LXR ⁸⁴ (an oxysterol sensor mediating efflux), which may involve a feed-forward loop with autophagy ⁸⁵. Further, in human lesion macrophages increased expression of sterol 27-hydroxylase was accompanied by that of genes functionally linked in vitro, where RXR and PPARg ligands induce sterol 27-hydroxylase ⁸⁶, 27-hydroxycholesterol and LXR ⁸⁷. Plaque PPARg expression was also shown to associate specifically with M2 macrophage markers (not M1) ⁸⁸, whereas the PPARg ligand 15d-PGJ₂ associated with COX-2 in the cytoplasm of foam cells, suggesting a source of negative feedback signalling ⁸⁹. From a temporal perspective, intimal lipoprotein accumulation precedes macrophage infiltration ⁹⁰ and immunometabolism evolves with plaque development ⁹¹; in particular, recent 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 hypoxic microenvironment ⁹². Thus, an initial adaptive PPARg-driven programme may support cholesterol efflux ⁹².

As above, accumulation of 27-hydroxycholesterol is paralleled by CE hydro(pero)xides, 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 ³³. Elsewhere however, 15-LOX-derived hydroperoxides induced TLR4-dependant macropinocytosis and LDL uptake, which can also be interpreted pathologically (in the absence of HDL) ⁹³. On the other hand, macrophages may preferentially hydrolyse such oxygenated CEs ⁹⁴ and free oxylipins are natural PPAR ligands ^73,95,96. For instance, LDL from patients with atherosclerosis contained various HODEs and HETEs which at similar levels in vitro activated PPARg ⁹⁷. In human monocytes PPARg primes M2 polarisation ⁸⁸, while in macrophages it may induce both scavenger receptor CD36 and LXRα/ABCA1-dependent efflux ⁹⁸, similar to 13-HODE ⁹⁹. Further, while ALOX15B is induced by hypoxia ⁴³ and mediates cholesterol accumulation ¹⁰⁰, ALOX15 is specifically induced by Th2/M2 cytokines and efferocytosis of apoptotic cells (via LXR ¹⁰¹), consistent with a role in lipid/tissue homeostasis ⁴³. Specifically, IL-4 induction of ALOX15 and PPARg mediates expression of CD36 and suppression of iNOS ¹⁰²; while ALOX15 overexpression increased CE hydrolysis and cholesterol efflux, but not via 15/13S-HETE (and 13S-HODE was undetectable) ⁶⁸. Free oxylipins undergo reincorporation into specific phospholipids ¹⁰³ and macrophage 15-LOX-dependent oxidation of CEs from intra- and extracellular sources resulted in 13-HODE–oxPC ⁷². Moreover, macrophages overexpressing 15-LOX also oxidised LDL via (LRP-dependent) selective uptake and efflux of CE linoleate ¹⁰⁴. In the extracellular context, LDL oxidation favoured net transfer of CEs to HDL (via CETP) ¹⁰⁵, while in rats HDL-associated CE hydro(pero)xides (i.e. [³H]Ch-18:2-O(O)H) were more rapidly removed by liver ¹⁰⁶ and excreted in bile (with the radioactivity in bile acids) ¹⁰⁷, suggesting increased reverse cholesterol transport. Therefore, while advanced lipid oxidation and protein modifications may favour retention, the preponderance of mild lipid oxidation (i.e. oxygenation) in vivo might be secondary ¹⁶ and even support clearance ^73,76,98,108.

Arterial lipid oxidation and flux may also depend on redox metabolism. For instance, while lipophilic antioxidants may not be depleted in plaque ⁷⁶, their regeneration depends upon the glutathione–ascorbate redox cycle and ultimately reducing equivalents from central metabolism. Notably, 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 ^109,110, while in plasma oxidation of glutathione redox (i.e. GSH/GSSG ratio) was associated with carotid intima–media thickening ¹¹¹ independent of traditional markers ¹¹². 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 ²⁷. Specifically, dietary glycine induced glutathione biosynthesis and related enzymes (incl. GR, GPxs and GSTs), while lowering aortic/macrophage superoxide and atherosclerosis independent of plasma lipids ²⁷. Glutathione supplementation also reduced lesion area and macrophage oxLDL uptake while increasing efflux ¹¹³. Mechanistically, in macrophages glutathione deficiency increased ROS and CD36 expression independent of PPARg ¹¹⁴, while glutathione supplementation induced efflux and PPARα ¹¹⁵, and selenium supported IL-4 induced M2 polarisation via GPx1, PPARg and PGD₂ ¹¹⁶. Further, LDL and HDL also contain glutathione and GPx activity ¹¹³, which mediates reduction of hydroperoxides to HODEs ⁷¹, while oxidised glutathione can inhibit HDL efflux activity via glutathionylation of paraoxonase-1 ¹¹⁷.

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 ¹¹⁸. Whereas fish oil/long-chain n-3 PUFA supplementation preferentially lowers triglycerides and non-HDL-cholesterol, particularly in those with hyperlipidemia or overweight/obesity ¹¹⁹. In addition, recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas C18 UFAs lower LDL proteoglycan binding in vitro ^120,121. Clearly all these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite of atherogenesis ¹²². 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 ^123–125. 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 ¹⁰. 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 ¹²⁶, monkeys ¹²⁷ and mice ¹²⁸ n-6 PUFA-rich diets increase linoleate/oleate ratios in plasma and plaques, and oxidation in vitro ¹²⁷ and in vivo ^128,129, 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 ^130,131.

The effect of PUFAs also depends on the food matrix. For instance, a 3-week diet of 31% sunflower oil/n-6 PUFAs (vs. olive oil/MUFAs) lowered LDL levels, oxidation susceptibility and proteoglycan binding, in relation to LDL antioxidant content and size ¹³². Further, 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) ¹³³, while longer trials show enrichment of PUFAs with preservation of oxidation status ^134,135, alongside many other cardio-protective effects (reviewed in ¹³⁶). Conversely, food storage and processing can oxidise lipids prior to ingestion. For instance, prolonged heating (i.e. 195°C for 9hrs) of refined soybean oil induces a gradual increase in peroxides before a decline (at 6hrs), while secondary aldehydes continue to increase ¹³⁷. 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 ⁵. 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 ¹³⁸. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁵, although has also been reported to lower blood lipids ⁹ and atherosclerosis ⁹⁹. 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 ¹³⁹. 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 ¹⁴⁰.

In humans and mice red meat ingestion also induced postprandial lipid peroxidation and plasma LDL–MDA modification, which was greatly inhibited by polyphenols ^141,142. 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 ¹⁴¹. 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 ¹⁴¹. The activity of many plant foods in this model has been indexed and correlates polyphenol content ¹⁴³; additionally, peroxidation was inhibited by oleate/MUFAs, opposite to fish oil/n-3 PUFAs ¹⁴⁴. 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 ⁷. Of note, iron deficiency is common and supplementation can also induce gastrointestinal lipid peroxidation ¹⁴⁵.

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 ²³. 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 ¹⁴⁶. 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) ¹⁴⁷. Conversely, despite the oxidative stability of SFAs and the responsiveness of serum stearate to diet ¹⁴⁸, in short-term trials SFA-rich diets (vs. carbohydrates or MUFAs) can also increase LDL susceptibility to oxidation in relation to MUFA/PUFA ratios ¹²⁵, vitamin E ¹²⁴, apoB/LDL-C ^149,150 and APOE promoter variants ¹⁵⁰. 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 ¹⁵¹. In mice a diet rich in dairy fat/SFAs (i.e. 21% wt) 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) ¹⁵². 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 ¹⁵³.

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) ¹⁵⁴, while in human tracer studies PUFAs (vs. SFAs) lower plasma lipids and increase LDL catabolism ¹⁵⁵. LDL susceptibility to oxidation is also associated with small particle size (i.e. pattern B), which is itself increased by insulin resistance ¹⁵⁶; in people with pattern B a SFA-rich diet (vs. MUFAs) increased small–medium LDL particles ¹⁵⁷, while a meta-analysis of RCTs suggests exchanging SFAs for n-6 PUFAs may particularly improve glucose-insulin homeostasis ¹⁵⁸. 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 ¹⁵⁹. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ¹⁶⁰, which at similar levels in vitro also induces LDL oxidation ¹⁶¹. 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 ¹⁶². 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 ¹⁶³. Accordingly, SFA intake is associated with Bilophila abundance ^164–166. 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 ¹⁶⁷. 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 ¹⁶⁸. In other trials the individual response to SFAs was related to baseline Bilophila ¹⁶⁴ and diet ¹⁶⁵, 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) ^168,169. Elsewhere, lowering dietary linoleic acid/n-6 PUFA (vs. mostly SFAs) also lowered respective plasma oxylipins (i.e. HODEs and KODEs) ¹⁷⁰. Linking these effects, linoleate oxygenation may induce PPARg, 27-hydroxycholesterol 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) ^168,171. From a biophysical perspective, LCAT specificity for linoleate ¹⁷² may support CE fluidity ⁶⁹ and substrate for LOX ⁷², while oxygenation (i.e. hydroxylation) of CEs and cholesterol may increase polarity/solubility to facilitate protein interactions and ‘fast-track’ transport to the liver ^83,107, 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) ¹⁴; similar to mice on a high fat diet supplemented with DHA (alone/with EVOO) ¹⁷³. In human trials fish oil/n-3 PUFAs also increase plasma early peroxidation products (e.g. HDHAs) ^10,11,13,15 and downstream pro-resolving mediators (e.g. resolvins) ^12,14, at the expense of arachidonic-derived oxylipins ^10,12. Early cell studies found (ambient) oxidation of EPA was required for inhibition of (cytokine-induced) NF-κB, which also required PPARα ¹⁷⁴; more recently, 7-HDHA (formed via ALOX5) was identified as a high-affinity PPARα ligand regulating brain morphology ¹⁷⁵. In the periphery n-3 PUFA induction of PPARα may also support triglyceride lowering ¹⁷¹ via suppression of SREBP-1 (which mediates hepatic lipogenesis) ¹⁷⁶ and apoC-III (which inhibits VLDL lipolysis) ¹⁷⁷. Further, in animal models dietary oxidised linoleate can also lower hepatic and plasma triglycerides via PPARα ⁹. 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 ⁶⁵ and coronary syndrome ⁶⁶ 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, implicating oxidative stress-induced hormesis ¹²⁹. Accordingly, in vitro catalase was induced by 13-HpODE/HODE in several arterial cells ¹⁷⁸, and is also regulated by PPARg, decomposes H₂O₂ ¹⁷⁹ and blocks MPO-induced oxidation ⁵³. As above, the ability of n-3 PUFAs to improve redox markers ¹⁴⁷ may involve Nrf2 ^14,173, 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 ¹⁸⁰. 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 ¹⁸¹. 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 ¹⁸². 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 ¹⁸³. However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ¹⁸⁴. LPS induces various oxylipins ¹⁸⁵ sensitive to n-3 status ¹³; and n-6 series Nrf2-inducers include EKODE ¹⁸⁶, 15d-PGJ₂ ¹⁸⁷ and LXA₄ ¹⁸⁰. Moreover, low-level 4-HHE and 4-HNE (from n-6 PUFAs) similarly induce Nrf2 in endothelial cells ¹⁸⁸, 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 ¹⁸⁹. 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 ¹⁹⁰—another major pathway mediating experimental atherosclerosis ³¹.

Excess unsaturated aldehydes are ultimately toxic; indeed while low-level 4-HNE induces Nrf2 and supports homeostasis, high levels block Nrf2 and favour apoptosis ¹⁹¹. Regardless, given the benefits of PUFAs even in advanced human disease (e.g. seed oils ¹ and fish oil ^130,131), an overall homeostatic effect seems likely. In healthy cells induction of Nrf2 supports antioxidant activity and aldehyde clearance in part via the glutathione system, which may be further reinforced by increased glycine availability, as above. In addition, in vitro studies with various phytochemicals show Nrf2 induces macrophage cholesterol efflux, via suppression of NF-κB signalling ¹⁹² and induction of SR-B1 and ABCA1/G1 transporters ^193–195, and thus may also support lipid-lowering and prevent cholesterol sequelae. Conversely, in highly stressed cells Nrf2/glutathione exhaustion may favour aldehyde accumulation and apoptosis, which itself could indirectly exert homeostatic pressure via subsequent macrophage efferocytosis and induction of ALOX15-dependent pro-resolving mediators ^42,43. Further, 17-oxo-DHA (formed via COX-2) augmented efferocytosis via Nrf2/HO1-dependent expression of LOX/COX-2 and pro-resolving mediators ¹⁹⁶, which may themselves activate Nrf2 ¹⁸⁰. 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 ¹⁹⁷. 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 ¹²⁹. Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ¹⁹⁸. In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ¹⁹⁹, as well as hepatic LXR and reverse cholesterol transport ²⁰⁰. 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 ²⁰¹. 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) ²⁰². 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 ¹⁹⁸, which may be mediated in large part by ROS/Nrf2 ²⁰³. Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ²⁰⁴, isoprostanes and aldehydes ²⁰⁵), and preferentially in HDL ²⁰⁶, 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 ^207,208. 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 ⁸.

In considering how differences between dietary UFAs may affect tissue homeostasis, long-chain n-3 PUFAs have the greatest susceptibility to oxidation and seemingly cell membrane incorporation, suggesting they may be particularly well poised to support antioxidant and anti-inflammatory activity. Whereas replacing SFAs with linoleic acid/n-6 PUFA may offer greater cholesterol-lowering activity in healthy individuals, but also potentially inflammatory activity in association with FADS1 variants affecting n-6 desaturases and metabolism to arachidonic acid ²⁰⁹. 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 ¹⁷³, which may be much higher in whole olives ²¹⁰ (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 ^5,141,142. Dietary thermo-oxidised oils can induce PPARs ⁹ and Nrf2 ^138,211,212, but alongside signs of inflammation, antioxidant depletion and DNA damage ²¹³. Sufficient lipoprotein modification/damage may also result in rapid clearance by phagocytes, largely in the liver, but also potentially arterial plaque ^7,38. Thus, excessive oxidation ‘ex vivo’ may eventually overwhelm homeostasis and promote pathogenesis. Notably, dietary oil oxidation typically involves prolonged storage ²¹⁴ or heating ^137,138,211, whereas red meat can apparently induce significant advanced oxidation within the normal digestive/postprandial phase ^141,142, which is exaggerated by addition of PUFA-rich oils ^7,144, 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, which were to be used for cooking and incorporation into provided foods, including sausage products (i.e. Veterans study ¹²⁶), filled beef (i.e. Minnesota study ²), and more recently liver pâté ¹⁶⁸, suggesting direct contact with heme-iron.

Conclusion

PUFA oxidation is associated with human and experimental atherosclerosis and induces toxic effects in vitro, supporting a pathogenic view; however, attention to specificity and signalling in vivo may unveil a precedent physiology. Indeed biology couples enzymatic and non-enzymatic oxidation products to adaptive responses via signalling; 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.

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