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 is replacing saturated fats (SFAs) with unsaturated fats (UFAs), especially plant-based PUFAs (e.g. from seed oils) ¹, and consuming more oily fish/n-3 PUFAs (FAO). Such public health recommendations are supported by the totality of evidence from observational and interventional studies (i.e. the mean of heterogeneous populations). Mechanistically however, this creates a potential paradox, since atherosclerosis is widely acknowledged to involve lipid peroxidation, and among lipids PUFAs are most susceptible, forming the basis of some concern ^2–6. At first glance, a simple dichotomous reconciliation is the benefits of PUFAs, such as lipid lowering or anti-inflammatory activity, may outweigh any putative negative effects. However, PUFA peroxidation can take many paths and produce many molecules with diverse effects, including lipid lowering ⁷ and anti-inflammatory activity ⁸, suggesting context matters and opportunity for harmonisation.

At one extreme, free radical mediated autoxidation eventually degrades PUFAs into reactive aldehydes, such as n-6-derived 4-hydroxynonenal (4-HNE; C₉H₁₆O₂), n-3-derived 4-hydroxyhexenal (4-HHE; C₆H₁₀O₂), malondialdehyde (MDA; C₃H₄O₂) and acrolein (C₃H₄O), which can covalently bind proteins and exert toxicity. However, initial stages of peroxidation generate full-chain oxygenated metabolites (i.e. hydroperoxides), such as linoleic-derived HpODEs (C₁₈H₃₂O₄), arachidonic-derived HpETEs (C₂₀H₃₂O₄) and DHA-derived HpDHAs (C₂₂H₃₂O₄), before later cleavage and fragmentation. Further, these reactions are explicitly catalysed by enzymes, such as lipoxygenases (LOXs) and cyclooxygenases (COXs), with positional specificity and in the presence of antioxidants, thereby producing stable metabolites of physiological relevance. 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.

The lab vs. plaque

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 ⁹. Native and oxLDL were even found in fetal aortas with and without macrophages, suggesting an early event ¹⁰. OxLDL can also be detected in plasma where it normally represents a very small fraction of LDL ¹¹ and associates with CVD ^12,13, although not always independently of apoB (e.g. CHD ¹⁴ and MetS ¹⁵), likely due to 4E6 antibody cross-reactivity ¹⁶. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB) are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ¹⁶. Conversely, dietary and plasma antioxidant nutrients (esp. vitamins C, E and carotenoids—largely reflecting fruit/veg and seed oil intake) are inversely associated with CVD ^17,18, although the results of RCTs with high-dose (i.e. supra-physiological) supplements in general populations have mostly failed to show benefit ^9,19 (unlike general lipid-lowering). However, there is far less outcome data on more physiological antioxidant repletion/optimisation approaches and other phytochemicals (e.g. polyphenols) ¹⁹.

Interestingly, plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^16,20. 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 (Cu²⁺) sulfate, a transition metal mediating 1-electron oxidations ⁹. LDL oxidation induces many pro-atherogenic endothelial/inflammatory effects (e.g. eNOS ²¹, CCL20 ²², EPCs ²³ and HSPCs ²⁴), and most characteristically, macrophage uptake and cholesterol loading via scavenger receptors ¹³. Further, unlike native LDL, macrophage uptake of oxLDL results in lipid trapping within lysosomes ²⁵, cholesterol crystallisation and NLRP3 activation ^26,27. Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ²⁸. 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 ²⁹. 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) ³⁰.

However, despite much data apparently supporting a causal role of oxidative stress in atherogenesis, the general failure of high-dose vitamin E/antioxidant trials to improve hard outcomes suggests more complexity ⁹. Foremost, oxidation by free transition metals may have limited physiological analogy; although heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ³¹. On the other hand, human lesions express LOXs (non-heme iron-dependent dioxygenases), which oxidise PUFAs and can induce similar lipoprotein modification as copper oxidation ^32,33, but are not blocked by vitamin E ³⁴. Human plaques also express iNOS ^35,36 and MPO ³⁷ (both heme-dependent enzymes), while recovered LDL and apoA-I/HDL are highly enriched in their 2-electron protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^38,39, implicating immune-dependant redox modifications ⁹. These pathways are also not blocked by vitamin E ⁴⁰ (or serum ⁴¹), and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptor CD36 ^41,42, while MPO-modified tryptophan residues within apoA-I/HDL inactivate its ABCA1-dependent acceptor activity ^39,43. However, other data present yet further challenges ^9,44. 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 ⁴⁵. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^45,46 and CE accumulation beyond native or oxLDL ^47,48. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ²⁵. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁴⁹ and induce lysosomal crystals and NLRP3 activation ^26,27, so may contribute in these ways ⁴⁵. 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 ⁵⁰. Furthermore, 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 ⁵¹. Also, human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ⁵², consistent with ALOX15 overexpression increasing reverse cholesterol transport ⁵³.

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some reconciliation. 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 ⁵⁴. 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 mono- and di-oxygenated derivatives of linoleate (i.e. HODEs and HpODEs, respectively) ⁵⁵. The HODE-CE profile exhibited no regio- or stereo-specificity suggesting a dominance of non-enzymatic peroxidation (vs. 15-LOXs), although triglyceride PUFAs were not oxidised indicating some specificity ⁵⁵. In several earlier studies 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ⁵⁶, while more recent studies on carotid plaques found increased expression of ALOX15B only, and in association with macrophages and HIF-1α ³³. 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 (representing 1-electron cholesterol oxidation) was uncorrelated ⁵⁷. Several earlier studies also found plaque lipid oxidation occurred despite normal levels of α-tocopherol ⁴⁰, and was similar in T2D ⁵⁸. 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 ⁵⁹. Another stage-dependent analysis of whole aortic lesions included tocopherol oxidation products and implicated 2-electron (enzymatic) oxidants ⁶⁰.

These observations contrast typical conditions in vitro ⁹, where under strong copper-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.) ⁵⁹. Conversely, under more mild conditions the α-tocopherol radical can initiate lipid peroxidation ⁶¹, especially when there are insufficient regenerative co-antioxidants (e.g. CoQ₁₀ and carotenoids) ⁶². Further, copper-oxidation of LDL generates substantial 7-ketocholesterol, which in macrophages inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ²⁵, and dose-dependently induces cholesterol crystals ⁶³, thereby mediating key effects of oxLDL. However, in vivo 7-ketocholesterol is quantitatively and temporally overshadowed by 27-hydroxycholesterol, which is produced by sterol 27-hydroxylase, indicating a dominance of enzymatic oxidation ⁵⁹. In the liver this enzyme initiates the acidic pathway of bile acid synthesis, while elsewhere it may increase the polarity of cholesterol and facilitate efflux, before return to the liver for further conversion and excretion ⁶⁴. In this regard, in human lesion macrophages increased expression of sterol 27-hydroxylase is accompanied by the expression of genes functionally linked in vitro; specifically, RXR and PPARg ligands induce sterol 27-hydroxylase ⁶⁵, which in turn induces 27-hydroxycholesterol and LXR—an oxysterol sensor mediating efflux ⁶⁶. Further, the early linoleate peroxidation metabolite 13-HODE is a natural PPAR ligand ⁵⁶ and also induces macrophage efflux to apoA-I via a PPARα/g–LXRα pathway ⁷. Upstream, 15-LOXs can directly oxidise CE-PUFAs, which are also preferred substrates for hydrolysis, and reincorporated into phospholipids ³³; while ALOX15 specifically is induced by Th2/M2 cytokines and apoptotic cells (via LXR ⁶⁷), consistent with a role in lipid/tissue homeostasis ^32,33. Therefore, considering all the above, mild enzymatic oxidation of trapped lipids may support cholesterol clearance ^56,59,68, whereas excessive oxidation may favour lipid trapping via 7-ketocholesterol ²⁵ and inactivation of apoA-I/HDL ^39,43,51, perhaps in relation to advanced disease and inflammation ⁹.

Dietary fats and lipid oxidation

Dietary fat saturation has well recognised effects on plasma lipids: replacing typical C12–16 SFAs with C18 MUFAs or PUFAs lowers apoB and total/LDL cholesterol, and to a smaller extent triglycerides, with PUFAs having the largest effect ⁶⁹. In addition, more recent trials find dietary SFAs can increase LDL sphingolipids and aggregation in vitro, whereas UFAs lower LDL proteoglycan binding in vitro ^70,71 and increase PBMC LXRα/ABCG1 expression ^72,73. Clearly these effects of UFAs may be beneficial by lowering arterial lipoprotein and lipid retention—the major prerequisite to atherogenesis ⁷⁴. On the other hand, the effect of dietary fats on lipoprotein oxidation has been tested since the early 90s and is much more controversial. In short-term trials MUFA-rich diets (vs. n-6/n-3 PUFAs) 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 ^75–77. Thus competition between C18 MUFAs and PUFAs for membrane incorporation may modulate substrate for oxidation. 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 ⁷⁸, monkeys ⁷⁹ and mice ⁸⁰ n-6 PUFA-rich diets increase linoleate/oleate ratios in plasma and plaques, and oxidation in vitro ⁷⁹ and in vivo ^80,81, yet are protective. The long-chain n-3 content of advanced carotid plaques is also increased by supplementation and correlates greater stability and lower inflammation, consistent with anti-inflammatory effects ^82,83.

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 ⁸⁴. 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 (5-hour AUC) and oxLDL (at 2 hours) ⁸⁵, while longer trials show enrichment of PUFAs with preservation of oxidation status ^86,87, alongside many other cardio-protective effects (reviewed in ⁸⁸). Conversely, food storage and processing can oxidise lipids prior to ingestion. For instance, prolonged exposure of soybean oil to high cooking temperatures induces a gradual increase in peroxides before a decline (at 6hrs), while secondary aldehydes continue to increase ⁸⁹. When fed to humans oxidised linoleic acid could be detected in chylomicrons/remnants for 8 hours (esp. in diabetics with poor glycaemic control), whereas oxidised cholesterol appeared in all major lipoproteins and persisted for 72 hours; tested in vitro oxidised cholesterol was transferred to LDL and HDL, potentially via CETP ⁴. In animal models dietary oxidised linoleic acid can promote atherosclerosis ⁴, although has also been reported to lower blood lipids and atherosclerosis ⁷. 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 ⁹⁰. Also, in a unique RCT comparing high quality to oxidised fish oil (approximating some commercial supplements), only the former lowered apoB-lipoproteins ⁹¹, despite being more well-known for triglyceride lowering.

The gut is another potentially important site of redox activity ⁹². For instance, in humans and mice red meat ingestion induced postprandial lipid peroxidation and plasma LDL–MDA modification, which was greatly inhibited by polyphenols ^93,94; in gastric models this was also inhibited by olive oil/MUFAs, opposite to fish oil/n-3 PUFAs ⁹⁵. Indeed the stomach has been conceptualised as a bioreactor, which denatures foods and facilitates redox chemistry, and where heme-iron can deplete antioxidant vitamins and induce advanced lipid peroxidation ⁹², as with copper-oxidation in vitro. Accordingly, in APOE^–/– mice on a western diet 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 ⁶.

Of note, despite the oxidative stability of SFAs, and the responsiveness of serum stearate to diet ⁹⁶, in the short-term SFA-rich diets (vs. carbohydrates or MUFAs) may also increase LDL susceptibility to oxidation in vitro in relation to LDL MUFA/PUFA ratios ^76,77 and APOE promoter variants ⁹⁷. Further, excess dairy fat may 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 ⁹⁸. In mice a diet rich in dairy fat/SFAs also elevated oxidised HDL and LDL, while replacement with soybean oil/PUFAs (i.e. ~5:1 of n-6:n-3) enhanced HDL antioxidant activity ⁹⁹; and in another study replacement with olive oil and nuts lowered monocyte oxLDL uptake and CD36 expression, which was modulated correspondingly by TRLs from each diet ¹⁰⁰.

Dietary SFAs may affect lipid oxidation indirectly. For instance, 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 ¹⁰¹. In a systematic review of RCTs dietary SFAs (vs. UFAs) induced postprandial LPS ¹⁰², which at similar levels in vitro also induces LDL oxidation ¹⁰³. 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 ¹⁰⁴. The glutathione system is also implicated in human atherosclerosis by associations with low plasma glutathione redox (i.e. GSH:GSSG) ^105,106 and arterial glutathione-related enzyme activity (i.e. GR, GPx and GSTs) ^107,108. Of interest here, in various human cohorts lower plasma glycine (a glutathione precursor) associates with metabolic and coronary disease, while in mice dietary glycine favourably modulated atherosclerosis, alongside lipids, glutathione and superoxide ¹⁰⁹. Further, host glycine availability is itself dependent on the gut microbiome: 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 ¹¹⁰. Accordingly, 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 ¹¹¹. Of note however, in people with moderate hypercholesterolemia exchanging SFAs (6.5% kcals) for mostly n-6 PUFAs for 8 weeks did not significantly increase serum glycine ⁷².

Oxylipin-induced homeostasis

Could the divergent effects of PUFAs on oxidation and atherosclerosis be further reconciled by oxylipin signalling? For instance, dietary linoleic acid/n-6 PUFA (vs. SFAs) increases plasma levels of early peroxidation metabolites (i.e. HODEs and oxo-ODEs) ¹¹², which may mediate macrophage efflux via PPAR–LXR signalling ⁷, as above. In addition, 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 ⁸¹. Further, despite long-chain n-3 PUFAs being most susceptible to oxidation, meta-analysis of 39 RCTs suggests they can actually improve some peripheral redox markers (i.e. TAC, GPx and MDA) ¹¹³, which might also involve anti-inflammatory activity and hormesis. For instance, fish oil/n-3 PUFA supplementation can increase plasma early peroxidation products (i.e. HDHAs) ⁸, as well as downstream pro-resolving DHA derivatives ¹¹⁴, while suppressing inflammatory markers and inducing PPARα and Nrf2-related antioxidant genes (incl. HO-1) ¹¹⁴. 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 ¹¹⁵. 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 ¹¹⁶. 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 ¹¹⁷. However, in the context of inflammation, prior injection of linoleic acid alleviated LPS-induced liver injury via Nrf2 ¹¹⁸. Moreover, low-level 4-HHE and 4-HNE similarly induce Nrf2 in endothelial cells ¹¹⁹, 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 ¹²⁰. 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 ¹²¹—another major pathway mediating experimental atherosclerosis ²⁷.

Importantly, while low-level 4-HNE induces Nrf2 and supports homeostasis, high levels also block Nrf2 and favour apoptosis ¹²²; indeed accumulation of unsaturated aldehydes is eventually toxic. Regardless, given the benefits of PUFAs even in advanced human disease (e.g. seed oils ¹ and fish oil ^82,83), an overall beneficial/homeostatic effect seems likely. Initial 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 ¹²³ and induction of SR-B1 and ABCA1/G1 transporters ^124–126, and thus may also support lipid-lowering and prevent cholesterol sequelae. Conversely, eventual Nrf2/glutathione exhaustion may favour aldehyde accumulation and apoptosis, which itself could exert another round of homeostatic pressure via induction of ALOX15-derived SPMs ^32,33. A macrophage Nrf2 deficit may also have broader impact: foam cell-derived exosomes were shown to propagate redox imbalance to brain microglia via Nrf2 exacerbating white matter injury and cognitive impairment ¹²⁷. Therefore, timely activation of Nrf2 may limit systemic pathology. In this regard, perhaps cellular PUFA status may lower the threshold for Nrf2 induction under oxidative conditions, favouring earlier feedback and pleiotropic regulation of redox, immune and lipid homeostasis, which ultimately limits atherosclerosis.

Further hormetic insight may lie in other perspectives; for instance, the effects of PUFAs may somewhat overlap with exercise ⁸¹. Firstly, exercise is well-documented to induce ROS/oxidative stress and adaptive/hormetic antioxidant responses ¹²⁸. In mice exercise training also induces aortic catalase and sterol 27-hydroxylase ¹²⁹, as well as hepatic LXR and reverse cholesterol transport ¹³⁰. 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 (SOD) ¹³¹. 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) ¹³². 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, which may be mediated in large part by ROS/Nrf2 ¹²⁸. Intriguingly, exercise can also induce lipid peroxidation (incl. plasma HODEs ¹³³, isoprostanes and aldehydes ¹³⁴), 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 ^135,136. 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 ¹³⁷.

In summary, the general susceptibility of PUFAs to oxidation may not be inherently bad and dichotomous with their health benefits, rather it is intrinsic to formation of oxylipins which support lipid and immune homeostasis; and even non-enzymatic and advanced peroxidation products may support redox homeostasis and hormesis. In this regard, the effects of PUFAs may have some analogy in exercise, which also generally benefits cardiovascular health. However, the site and extent of PUFA oxidation may be determinate; excessive peroxidation during food processing and/or digestion would presumably bypass the opportunity for physiologic signalling and instead favour accumulation of fragmented end products, with increasing 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. 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 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 and discourage red meat, which may help safeguard PUFA quality.

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