Background Acute Lung Injuries (ALI) are a severe consequence of influenza-induced cytokine storm that can cause respiratory failure and death. It has been demonstrated that Toll-like Receptor 4 (TLR4) is involved in cytokine storm and that TLR4 −/− mice are protected against ALI. Therefore, TLR4 is a prime target for protection against ALI. FP12 is a known TLR4 antagonist that reduces TLR4-dependent immune activation and it is a promising lead compound for the treatment of innate immunity related pathologies. Objectives We present here the preclinical development of FP12 as an anti-inflammatory lead compound acting on influenza-induced ALI. Methods In vitro: We pre-treated THP-1 cells with FP12 (10 μM) for 0.5 h, then exposed to LPS (100 ng/ml) for 0 to 16 h. In some experiments, cells were simultaneously incubated with FP12 and LPS, or FP12 was added 30 min after LPS. Cytokine levels were measured by Western blot and ELISA assays. In vivo: WT C57BL/6J mice were infected with mouse-adapted influenza virus (PR8). Two days after infection, mice received either vehicle, FP7 (200 µg/mouse), or FP12 (200 µg/mouse) once daily (Day 2 to Day 6). Mice were monitored daily for survival for 14 days. Data were collected through histological staining, qRT-PCR, and ELISA assay. Results FP12 treatment inhibited both LPS- and HMGB1-induced TLR4 intracellular pathways (MyD88 and TRIF) leading to significantly reduced levels of a variety of proinflammatory cytokines including Type I interferon (IFN-β), highlighting its effectiveness in controlling proinflammatory protein production and reducing inflammation. FP12 protected mice therapeutically from influenza virus-induced lethality and reduced both cytokine gene expression and High Mobility Group Box 1 (HMGB1) levels in the lungs as well as ALI. Conclusion FP12 can antagonize TLR4 activation in vitro and protects mice from severe influenza infection, most likely by reducing the TLR4-dependent cytokine storm mediated by danger-associated molecular patterns (DAMPs).

LDL can be oxidised in the lysosomes of macrophages. Cysteamine, a thiol antioxidant that accumulates in lysosomes, inhibits the oxidation of LDL by iron at lysosomal pH (pH 4.5) and protects against atherosclerosis in mice. We have investigated the effects of cysteamine and its related thiol cysteine and their disulfides on LDL oxidation by iron or copper at both pH 4.5 and 7.4. The oxidation of LDL by ferrous iron (5 µM) at pH 4.5 was delayed 12.9-fold by 100 µM cysteamine and 5.6-fold by 100 µM cysteine. Cystamine and cystine (the disulfide oxidation products of cyteamine and cysteine, respectively) did not inhibit LDL oxidation by ferrous iron at pH 4.5. LDL oxidation by 5 µM copper at pH 4.5 was delayed about 2-fold by 100 µM of the thiols cysteamine and cysteine, but there was little effect of the disulfides cystamine and cystine. Cysteamine and cystine did not inhibit the oxidation of LDL by ferrous iron at pH 7.4 in a MOPS buffer and even accelerated LDL oxidation later in the incubation. Cysteine initially inhibited the oxidation of LDL by ferrous iron at pH 7.4, but increased it later. LDL oxidation by copper at pH 7.4 was delayed 7.8-fold by 100 µM cysteamine. Cysteine delayed LDL oxidation by copper at pH 7.4 to a similar extent as cysteamine but, unlike cysteamine, continued to decrease the rate of oxidation even after the period of total inhibition had ended. Cystamine had no effect on LDL oxidation by copper at pH 7.4, but cystine partially inhibited LDL oxidation. The effects of thiols and disulfides on LDL oxidation, therefore, depend not only on the metal ion catalysing the oxidation but also on the pH of the environment.

Endothelial dysfunction is a risk factor for atherosclerosis and includes impaired endothelium-dependent vasodilatation. We have shown previously that low density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Macrophage lysis in atherosclerotic lesions might expose endothelial cells to this oxidized LDL and adversely affect their function. LDL was oxidized by ferrous sulfate (5 mu M) for 24 h at pH 4.5 at 37 degrees C. Aortas from male Wistar rats were cut into rings and subjected to wire myography for isometric tension recording. The rings were incubated with or without oxidized LDL (50 mu g protein/ml) for one hour, constricted with 100 nM phenylephrine and relaxation to acetylcholine (1 nM - 3 mu M) was measured. There was about 50% less relaxation in the presence of this oxidized LDL. Endothelial-independent vasodilatation induced by sodium nitroprusside was less affected by oxidized LDL. Oxidized LDL increased the formation of reactive oxygen species by the aortic rings and by cultured human aortic and dermal microvascular endothelial cells, which might have inactivated nitric oxide. Acetylcholine increased the activatory phosphorylation of eNOS (ser-1177), but oxidized LDL had little effect on this activation in cultured human aortic endothelial cells. These findings raise the possibility that LDL oxidized in lysosomes and released from lysed macrophages might decrease vasodilatation in atherosclerotic arteries.

We have previously demonstrated that low-density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Some of the iron content of lysosomes might be delivered through autophagy of ferritin (the main iron-storage protein in the body). We have now investigated the effects of ferritin-mediated LDL oxidation on macrophage function. The addition of ferritin to human THP-1 cells and human monocyte-derived macrophages increased lysosomal lipid peroxidation, as shown by LPO-Foam, a fluorescent probe targeted to lysosomes. Incubating THP-1 cells with ferritin and native LDL or LDL aggregated by sphingomyelinase, to allow their endocytosis and delivery to lysosomes, led to the formation of lysosomal ceroid (an advanced lipid oxidation product), indicative of lysosomal LDL oxidation. Incubating THP-1 cells with ferritin and LDL caused metabolic activation of the cells, as shown by increased extracellular acidification and oxygen consumption measured by a Seahorse analyzer. LDL oxidized by ferritin in lysosomes might be released from macrophages when the cells die and lyse and affect neighboring cells in atherosclerotic lesions. Adding LDL oxidized by ferritin at lysosomal pH (pH 4.5) to macrophages increased their intracellular reactive oxygen species formation, shown using dihydroethidium, and increased apoptosis. Ferritin might therefore contribute to LDL oxidation in the lysosomes of macrophages and have atherogenic effects.

Atherosclerosis, the major cause of vascular disease, is an inflammatory process driven by entry of blood monocytes into the arterial wall. LDL normally enters the wall, and stimulates monocyte adhesion by forming oxidation products such as oxidised phospholipids (oxPLs) and malondialdehyde. Adhesion molecules that bind monocytes to the wall permit traffic of these cells. CD14 is a monocyte surface receptor, a cofactor with TLR4 forming a complex that binds oxidised phospholipids and induces inflammatory changes in the cells, but data have been limited for monocyte adhesion. Here, we show that under static conditions, CD14 and TLR4 are implicated in adhesion of monocytes to solid phase oxidised LDL (oxLDL), and also that oxPL and malondialdehyde (MDA) adducts are involved in adhesion to oxLDL. Similarly, monocytes bound to heat shock protein 60 (HSP60), but this could be through contaminating lipopolysaccharide. Immunohistochemistry on atherosclerotic human arteries demonstrated increased endothelial MDA adducts and HSP60, but endothelial oxPL was not detected. We propose that monocytes could bind to MDA in endothelial cells, inducing atherosclerosis. Monocytes and platelets synergized in binding to oxLDL, forming aggregates; if this occurs at the arterial surface, they could precipitate thrombosis. These interactions could be targeted by cyclodextrins and oxidised phospholipid analogues for therapy.

BACKGROUND: We have shown previously that low-density lipoprotein (LDL) can be oxidized in the lysosomes of macrophages, that this oxidation can be inhibited by cysteamine, an antioxidant that accumulates in lysosomes, and that this drug decreases atherosclerosis in LDL receptor-deficient mice fed a high-fat diet. We have now performed a regression study with cysteamine, which is of more relevance to the treatment of human disease. METHODS AND RESULTS: LDL receptor-deficient mice were fed a high-fat diet to induce atherosclerotic lesions. They were then reared on chow diet and drinking water containing cysteamine or plain drinking water. Aortic atherosclerosis was assessed, and samples of liver and skeletal muscle were analyzed. There was no regression of atherosclerosis in the control mice, but cysteamine caused regression of between 32% and 56% compared with the control group, depending on the site of the lesions. Cysteamine substantially increased markers of lesion stability, decreased ceroid, and greatly decreased oxidized phospholipids in the lesions. The liver lipid levels and expression of cluster of differentiation 68, acetyl-coenzyme A acetyl-transferase 2, cytochromes P450 (CYP)27, and proinflammatory cytokines and chemokines were decreased by cysteamine. Skeletal muscle function and oxidative fibers were increased by cysteamine. There were no changes in the plasma total cholesterol, LDL cholesterol, high-density lipoprotein cholesterol, or triacylglycerol concentrations attributable to cysteamine. CONCLUSIONS: Inhibiting the lysosomal oxidation of LDL in atherosclerotic lesions by antioxidants targeted at lysosomes causes the regression of atherosclerosis and improves liver and muscle characteristics in mice and might be a promising novel therapy for atherosclerosis in patients.

Lifestyle- and genetically induced disorders related to disturbances in cholesterol metabolism have shown the detrimental impact of excessive cholesterol levels on a plethora of pathological processes such as inflammation. In this context, two-hydroxypropyl-β-cyclodextrin (CD) is increasingly considered as a novel pharmacological compound to decrease cellular cholesterol levels due to its ability to increase cholesterol solubility. However, recent findings have reported contra-indicating events after the use of CD questioning the clinical applicability of this compound. Given its potential as a therapeutic compound in metabolic inflammatory diseases, in this study, we evaluated the inflammatory effects of CD administration in the context of cholesterol-induced metabolic inflammation in vivo and in vitro . The inflammatory and cholesterol-depleting effects of CD were first investigated in low-density lipoprotein receptor knockout ( Ldlr -/ ) mice that were transplanted with Npc1 nih or Npc1 wt bone marrow and were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks, thereby creating an extreme model of lysosomal cholesterol-induced metabolic inflammation. In the final three weeks, these mice received daily injections of either control (saline) or CD subcutaneously. Subsequently, the inflammatory properties of CD were investigated in vitro in two macrophage cell lines and in murine bone marrow-derived macrophages (BMDMs). While CD administration improved cholesterol mobilization outside lysosomes in BMDMs, an overall pro-inflammatory profile was observed after CD treatment, evidenced by increased hepatic inflammation in vivo and a strong increase in cytokine release and inflammatory gene expression in vitro in murine BMDMs and macrophages cell lines. Nevertheless, this CD-induced pro-inflammatory profile was time-dependent, as short term exposure to CD did not result in a pro-inflammatory response in BMDM. While CD exerts desired cholesterol-depleting effects, its inflammatory effect is dependent on the exposure time. As such, using CD in the clinic, especially in a metabolic inflammatory context, should be closely monitored as it may lead to undesired, pro-inflammatory side effects.

Background and Aims: We have shown previously that low density lipoprotein (LDL) can be oxidized in the lysosomes of macrophages, that this oxidation can be inhibited by cysteamine, an antioxidant which accumulates in lysosomes, and that this drug decreases atherosclerosis in LDL receptor-deficient mice fed a high-fat diet. We have now carried out a regression study with cysteamine, which is of more relevance to the treatment of human disease.

Low density lipoprotein (LDL) might be oxidized by iron in the lysosomes of macrophages in atherosclerotic lesions. We have shown previously that the iron-storage proteinferritin can oxidize LDL at lysosomal pH. We have now investigated the roles of the most important antioxidant contained in LDL, alpha-tocopherol (the main form of vitamin E) and of ascorbate (vitamin C), a major water-soluble antioxidant, on LDL oxidation by ferritin at lysosomal pH (pH 4.5). We incubated LDL with ferritin at pH 4.5 and 37 degrees C and measured its oxidation by monitoring the formation of conjugated dienes at 234 n min a spectrophotometer. alpha-Tocopherol is well known to inhibit LDL oxidation at pH 7.4, but enrichment of LDL with alpha-tocopherol was unable to inhibit LDL oxidation by ferritin at pH 4.5. Ascorbate had a complex effect on LDL oxidation by ferritin at lysosomal pH and exhibited both antioxidant and pro-oxidant effects. It had no antioxidant effect on partially oxidized LDL, only a pro-oxidant effect. Ascorbate completely inhibited LDL oxidation by copper at pH 7.4 for a long period, but in marked contrast did not inhibit LDL oxidation by copper at lysosomal pH. Dehydroascorbate, the oxidation product of ascorbate, had a pronounced pro-oxidant effect on LDL incubated with ferritin at pH 4.5. The inability of alpha-tocopherol and ascorbate to effectively inhibit LDL oxidation by ferritin at lysosomal pH might help to explain why the large clinical trials with these vitamins failed to show protection against cardiovascular diseases.

Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. alpha-Tocopherol has been shown to inhibit LDL oxidationin vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of alpha-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with alpha-tocopherol by incubating human plasma with alpha-tocopherol followed by LDL isolation by ultracentrifugation. The alpha-tocopherol content of LDL was increased from 14.4 +/- 0.2 to 24.3 +/- 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with alpha-tocopherol was oxidised more slowly than control LDL by Cu2+ at pH 7.4, but was not protected against oxidation by Cu2+ or Fe(3+)or a low concentration of Fe2+ at pH 4.5 (it was sometimes oxidised faster by alpha-tocopherol with Cu2+ or Fe3+ at pH 4.5). alpha-Tocopherol-enriched LDL reduced Cu(2+)and Fe3+ into the more pro-oxidant Cu+ and Fe2+ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of alpha-tocopherol did not protect against cardiovascular disease.

Background and Aims: We have shown that LDL aggregated either mechanically by vortexing or enzymatically by sphingomyelinase (SMase-LDL) is internalised by macrophages and oxidised in lysosomes. We have studied the effects of lysosomal oxidation of LDL on various lysosome-associated functions and also shown how the lysosomotropic antioxidant cysteamine is able to prevent these effects. We also investigated the effect of cysteamine on atherosclerosis in mice.

Aims: The lifestyle and physical activity (PA) habits of young people play a key role in the prevention of cardiovascular and metabolic diseases at older ages. The current generation of biological science students at university holds promise for better future medicine and medical technology. However, their physical fitness and lifestyle are often ignored. Methods: Lifestyle, PAs and common risk factors for cardiovascular disease before, and at, university were collected from 408 students using self-completed, anonymous surveys between the academic years of 2017 and 2019 from the School of Biological Sciences, University of Reading. Statistical analysis was performed using SAS® 9.4 software. Results: Among the 408 participants, 134 were male and 274 were female with a mean (SD) age of 19.6 (2.24). Approximately 19% of participants consumed alcohol beyond the safe limit of <14 units/week (112 g/week). Among them, 65% were males. Before university, 47% of students failed to meet the UK National Physical Activity Guidelines (NPAG) which increased to 56% during university with males exhibiting a steeper incline. Compared to their lifestyles before university, more students had insufficient sleep and displayed greater sedentariness during university. Moreover, 16% of students declared no engagement in PA which was greater than the value of 12% before university. Fitness perceptions worsened by 11% during university particularly for females. Statistical analysis revealed that gender, BMI and fitness perceptions were significantly correlated with PA levels. The most prevalent explanation for inadequacy in meeting NPAG was insufficient time. Conclusions: Compared to their pre-university lifestyles, biological science students at university are more likely to adopt unhealthier behaviours with less time for exercise and prolonged sedentary behaviours, which increases the risk for cardiovascular diseases. It is important to raise awareness of their fitness perceptions and to encourage health-promoting programmes at university.

We have shown that aggregated LDL is internalized by macrophages and oxidized in lysosomes by redox-active iron. We have now investigated to determine whether the lysosomal oxidation of LDL impairs lysosomal function and whether a lysosomotropic antioxidant can prevent these alterations. LDL aggregated by SMase (SMase-LDL) caused increased lysosomal lipid peroxidation in human monocyte-derived macrophages or THP-1 macrophage-like cells, as shown by a fluorescent probe, Foam-LPO. The pH of the lysosomes was increased considerably by lysosomal LDL oxidation as shown by LysoSensor Yellow/Blue and LysoTracker Red. SMase-LDL induced senescence-like properties in the cells as shown by β-galactosidase staining and levels of p53 and p21. Inflammation plays a key role in atherosclerosis. SMase-LDL treatment increased the lipopolysaccharide-induced secretion of TNF-α, IL-6, and MCP-1. The lysosomotropic antioxidant, cysteamine, inhibited all of the above changes. Targeting lysosomes with antioxidants, such as cysteamine, to prevent the intralysosomal oxidation of LDL might be a novel therapy for atherosclerosis.

Background and aims: We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol), which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. Methods: LDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19-22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. Results: Confocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. Conclusions: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease.

Oxidised low density lipoprotein (LDL) was considered to be important in the pathogenesis of atherosclerosis, but the large clinical trials of antioxidants, including the first one using probucol (the PQRST Trial), failed to show benefit and have cast doubt on the importance of oxidised LDL. We have shown previously that LDL oxidation can be catalysed by iron in the lysosomes of macrophages. The aim of this study was therefore to investigate the effectiveness of antioxidants in preventing LDL oxidation at lysosomal pH and also establish the possible mechanism of oxidation. Probucol did not effectively inhibit the oxidation of LDL at lysosomal pH, as measured by conjugated dienes or oxidised cholesteryl esters or tryptophan residues in isolated LDL or by ceroid formation in the lysosomes of macrophage-like cells, in marked contrast to its highly effective inhibition of LDL oxidation at pH 7.4. LDL oxidation at lysosomal pH was inhibited very effectively for long periods by N,N'-diphenyl-1,4-phenylenediamine, which is more hydrophobic than probucol and has been shown by others to inhibit atherosclerosis in rabbits, and by cysteamine, which is a hydrophilic antioxidant that accumulates in lysosomes. Iron-induced LDL oxidation might be due to the formation of the superoxide radical, which protonates at lysosomal pH to form the much more reactive, hydrophobic hydroperoxyl radical, which can enter LDL and reach its core. Probucol resides mainly in the surface monolayer of LDL and would not effectively scavenge hydroperoxyl radicals in the core of LDL. This might explain why probucol failed to protect against atherosclerosis in various clinical trials. The oxidised LDL hypothesis of atherosclerosis now needs to be re-evaluated using different and more effective antioxidants that protect against the lysosomal oxidation of LDL.

Background: Endothelial dysfunction is a risk factor for atherosclerosis categorised by impaired endothelium-dependent vasodilatation. We have shown that low density lipoprotein (LDL) can be oxidised by iron in the lysosomes of macrophages. Macrophage lysis in atherosclerotic lesions might expose endothelial cells to this oxidised LDL and affect their function.

TLRs, including TLR4, have been shown to play a crucial role in cardiovascular inflammatory-based diseases. The main goal of this study was to determine the potential of FP7, a synthetic glycolipid active as a TLR4 antagonist, to modulate haematopoietic and non-haematopoietic vascular TLR4 pro-inflammatory signalling. HUVEC, human THP-1 monocytes, THP-1-derived macrophages, mouse RAW-264.7 macrophages and Angiotensin II-infused apolipoprotein E-deficient mice were in vitro and in vivo models, respectively. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 on TLR4 functional activity in response to bacterial LPS (in vitro) and endogenous ligands of sterile inflammation (in vitro and in vivo). Following activation of TLR4, in vitro and in vivo data revealed that FP7 inhibited p38 MAPK and p65 NF-kB phosphorylation associated with down-regulation of a number of TLR4-dependent pro-inflammatory proteins. In addition to inhibition of LPS-induced TLR4 signalling, FP7 negatively regulated TLR4 activation in response to ligands of sterile inflammation (hydroperoxide-rich oxidised LDL, in vitro and Angiotensin II infusion, in vivo). These results demonstrate the ability of FP7 to negatively regulate in vitro and in vivo haematopoietic and non-haematopoietic vascular TLR4 signalling both in humans and mice, suggesting the potential therapeutic use of this TLR4 antagonist for pharmacological intervention of vascular inflammatory diseases.

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 degrees C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05-0.2 mu M). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N'-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis.

The oxidation of low density lipoprotein (LDL) was considered to be important in atherogenesis. It is well known that α-tocopherol protects against the oxidation of LDL by cells or copper ions, but α-tocopherol did not protect against cardiovascular disease in large clinical trials, leading some to doubt the importance of oxidised LDL. We have previously shown that LDL is oxidised in the lysosomes of macrophages, due to their acidic pH and presence of redox-active iron, raising the possibility that this is the main site of LDL oxidation, rather than the extracellular space of the arterial intima. We have now enriched LDL with α-tocopherol (by adding α-tocopherol to human plasma followed by the isolation of LDL by ultracentrifugation) and measured its oxidation by monitoring conjugated diene formation in a spectrophotometer set to 37°C and 234 nm. α-Tocopherol-enriched LDL was oxidised much slower by Cu2+ (2, 5 or 20 µM) at pH 7.4, as expected, but was not protected against oxidation by these concentrations of Cu2+, Fe2+ or Fe3+ at pH 4.5 (lysosomal pH). The lack of protection of cardiovascular disease by α-tocopherol is therefore not proof that oxidised LDL is not important in atherosclerosis, if the oxidation of LDL occurs in lysosomes. We therefore need to retest the oxidised LDL hypothesis using antioxidatnts that accumulate in lysosomes and inhibit the oxidation of LDL at acidic pH. We thank the Iraqi Government for a PhD studentship (HKMA).