Search

Various lipoxygenases (LO) oxidize low density lipoprotein (LDL) in vitro and 15-LO has been implicated in the development of atherosclerosis in vivo. Direct oxidation of phospholipids (PL) and cholesteryl esters (CE) by LO has been proposed as a mechanism whereby these enzymes cause or contribute to LDL lipid peroxidation. Herein we show that the extent to which recombinant human 15-LO (rhLO) caused peroxidation of LDL's esterified core and surface lipids depended on, and directly related to, the alpha-tocopherol (alpha-TOH) content of the lipoprotein. Thus, CE and PL of in vivo alpha-TOH-depleted LDL, isolated from a patient with familial isolated vitamin E deficiency, were resistant to oxidation by rhLO, whereas those in alpha-TOH-containing LDL from the same patient receiving vitamin E supplements readily oxidized. The extent to which rhLO caused oxidation of CE and PL directly and linearly correlated with LDL's content of vitamin E, as demonstrated by studies with in vitro alpha-TOH-depleted lipoproteins. The geometric isomers of oxidized cholesteryl linoleate formed in LDL during oxidation initiated by rhLO, matched those obtained during non-enzymic, peroxyl radical-initiated oxidation of LDL whilst alpha-TOH was present. Ascorbate, an efficient co-antioxidant for alpha-TOH, completely prevented rhLO-initiated oxidation of LDL's CE, but did not inhibit rhLO-mediated oxidation of unesterified linoleate. These results are inconsistent with direct oxidation of LDL esterified lipids by rhLO. Isolated LDL contained free fatty acids (FFA), and its exposure to rhLO caused a rapid formation of linoleate hydroperoxide. To reconcile these data, we propose that during rhLO-initiated oxidation of LDL, enzymic oxidation of FFA preceeds the oxidation of CE and PL, which occurs largely via a tocopherol-dependent process.

We report a rapid and convenient method for screening potential inhibitors of the initiation of low density lipoprotein (LDL) lipid peroxidation. The method uses positively and negatively charged micelles of either cetyltrimethyl ammonium chloride or sodium dodecyl sulfate with added alpha-tocopherol. It is based on the capacity of an antioxidant to attenuate alpha-tocopheroxyl radicals, generated by irradiation of the alpha-tocopherol-containing micelles with UV light, and measured directly by electron spin resonance spectroscopy. To establish the reliability of the method, we compared the alpha-to-copheroxyl radical attenuating ability (TRAA) of 53 natural and synthetic potential antioxidants with their respective ability to inhibit the early stages of LDL lipid peroxidation initiated by a low flux of water-soluble peroxyl radicals. The relationship between the measured TRAA and corresponding LDL antioxidation activity was highly significant (P < 0.00005, Rank test). Thus, the potency of a co-antioxidant for LDLs alpha-tocopherol could be predicted with > 98% probability by the TRAA test alone. The results suggest that this relatively simple method represents an effective and simple screening test that can be used for large numbers of potential inhibitors of the early stages of LDL lipid oxidation.

This study examines the cholesteryl ester transfer protein (CETP)-mediated exchange of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) and cholesteryl linoleate hydroxide (Ch18:2-OH) between low density lipoprotein (LDL) and high density lipoprotein (HDL). When [3H]Ch18:2-OOH- and [3H]18:2-OH-labeled LDL were incubated at 37 degrees C for 0-24 h with unoxidized HDL and purified CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the HDL. Similarly, when incubations were carried out with [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled HDL, unoxidized LDL, and CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the LDL. Comparable results were obtained for the CETP-mediated transfer of [3H]Ch18:2-OH alone from LDL to HDL. Transfer to HDL of oxidized cholesteryl linoleate from [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled LDL was comparable to that of unoxidized cholesteryl linoleate (Ch18:2). However, the rate of transfer of [3H]Ch18:2-OOH and [3H]Ch18:2-OH from LDL to HDL increased linearly as the molar ratio of acceptor (HDL) to donor (oxidized LDL) particles in the incubation increased from 0.5:1 to 10:1. This increased rate of exchange was accompanied by an increased proportion of the oxidized Ch18:2 being present as the hydroxide rather than hydroperoxide. Further increases in the molar ratio of HDL to oxidized LDL particles neither affected the transfer rate nor the extent of reduction of Ch18:2-OOH to Ch18:2-OH. We therefore conclude that i) CETP mediates bidirectional transfers of Ch18:2-OOH and Ch18:2-OH between HDL and LDL; ii) CETP does not distinguish between Ch18:2-OOH, Ch18:2-OH, and Ch18:2 as it mediates their exchange between HDL and LDL; and iii) association with HDL hastens the reduction of Ch18:2-OOH to Ch18:2-OH.

Juvenile fish are known to be the most impacted during hydropeaking events due to stranding or uncontrolled drift resulting from changes to water depth and flow velocity. To shed light on their response to such hydraulic alterations, we coupled flume experiments with image-based fish tracking and quantified the fine-scale movement behavior of wild (n = 30) and hatchery-reared (n = 38) brown trout (Salmo trutta) parr. We exposed fish to two distinct hydropeaking treatments in a laterally inclined (14 %) flume section stocked with real cobbles to create refuge and heterogeneous hydraulic conditions. Fish were individually acclimated (20 min) to baseflow (Q = 1.6 L s-1) and then exposed to three consecutive hydropeaking events, reaching peakflows tenfold larger than baseflow (Q = 16 L s-1). We found that, within just minutes, fish exhibited fine-scale movement responses to cope with the change of hydrodynamic conditions. Fish moved perpendicular to the main flow direction to shallow areas as these became submerged during discharge increase, holding position at low velocity zones. This resulted in a significant difference (p < 0.001) in lateral occupancy of the experimental section between baseflow and peakflow. During peakflow, fish occupied specific positions around cobbles and exhibited swimming behaviors, including bow-riding and entraining, that allowed them to hold position while likely minimizing energy expenditure. As a result, swimming distance reduced 60-70 % compared to baseflow. During the decrease in discharge following peakflow, fish abandoned areas falling dry by moving laterally. In the treatment with the larger down-ramping rate, the time to initiate relocation was lower while the relocation speed was higher. This study shows that, for the conditions investigated here, brown trout parr is capable of swiftly deploying multiple behavioral responses to navigate rapid changes in hydrodynamic conditions. These findings can be incorporated into habitat modeling and improve our capacity to inform hydropeaking mitigation efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Protecting healthcare workers (HCWs) from coronavirus disease-19 (COVID-19) is critical to preserve the functioning of healthcare systems. We therefore assessed seroprevalence and identified risk factors for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) seropositivity in this population., Methods: Between 22 June 22 and 15 August 2020, HCWs from institutions in northern/eastern Switzerland were screened for SARS-CoV-2 antibodies. We recorded baseline characteristics, non-occupational and occupational risk factors. We used pairwise tests of associations and multivariable logistic regression to identify factors associated with seropositivity., Results: Among 4664 HCWs from 23 healthcare facilities, 139 (3%) were seropositive. Non-occupational exposures independently associated with seropositivity were contact with a COVID-19-positive household (adjusted OR 59, 95% CI 33-106), stay in a COVID-19 hotspot (aOR 2.3, 95% CI 1.2-4.2) and male sex (aOR 1.9, 95% CI 1.1-3.1). Blood group 0 vs. non-0 (aOR 0.5, 95% CI 0.3-0.8), active smoking (aOR 0.4, 95% CI 0.2-0.7), living with children <12 years (aOR 0.3, 95% CI 0.2-0.6) and being a physician (aOR 0.2, 95% CI 0.1-0.5) were associated with decreased risk. Other occupational risk factors were close contact to COVID-19 patients (aOR 2.7, 95% CI 1.4-5.4), exposure to COVID-19-positive co-workers (aOR 1.9, 95% CI 1.1-2.9), poor knowledge of standard hygiene precautions (aOR 1.9, 95% CI 1.2-2.9) and frequent visits to the hospital canteen (aOR 2.3, 95% CI 1.4-3.8)., Discussion: Living with COVID-19-positive households showed the strongest association with SARS-CoV-2 seropositivity. We identified several potentially modifiable work-related risk factors, which might allow mitigation of the COVID-19 risk among HCWs. The lower risk among those living with children, even after correction for multiple confounders, is remarkable and merits further study., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nutrition therapy, by enteral, parenteral, or both routes combined, is a key component of the management of critically ill, surgical, burns, and oncology patients. Established evidence indicates overfeeding (provision of excessive calories) results in increased risk of infection, morbidity, and mortality. This has led to the practice of "permissive underfeeding" of calories; however, this can often lead to inadequate provision of guideline-recommended protein intakes. Acutely ill patients requiring nutritional therapy have high protein requirements, and studies demonstrate that provision of adequate protein can result in reduced mortality and improvement in quality of life. However, a significant challenge to adequate protein delivery is the current lack of concentrated protein solutions. Patients often have fluid administration restrictions and existing protein solutions are frequently not sufficiently concentrated to deliver a patient's protein requirements. This has led to the development of new enteral and parenteral nutrition solutions incorporating higher levels of protein in smaller volumes. This review article summarizes current evidence supporting the role of higher protein intakes, especially during the early phases of nutrition therapy in acute illness, methods for assessing protein requirements, as well as, the currently available high-protein enteral and parenteral nutrition solutions. There is sufficient evidence (albeit limited from true randomized, controlled studies) to indicate that earlier provision of guideline-recommended protein intakes may be key to improving patient outcomes and that nutritional therapy that tailors caloric and protein intake to the patients' needs should be considered a desired standard of care., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recently we reported that Rearranged L-Myc Fusion, RLF, acts as an epigenetic modifier maintaining low levels of DNA methylation at CpG island shores and enhancers across the genome. Here we focus on the phenotype of Rlf null mutant mice generated via an ENU mutagenesis screen, to identify genes required for epigenetic regulation. RLF is expressed in a range of fetal mouse tissues, including the fetal heart. Comprehensive timed-mating studies are consistent with our previously reported findings that Rlf homozygous mutant mice rarely survive to adulthood, with the majority dying shortly after birth. Histological analysis of two independent Rlf ENU mutant lines at E11.5-E14.5 showed heart defects resembling those present in humans with Left Ventricular Non-Compaction (LVNC). In situ hybridisation analysis localized expression of Rlf to the endocardium and epicardium of embryonic and postnatal hearts, and transiently to cardiomyocytes during heart looping and early chamber formation stages. RNA-seq analysis of Rlf mutant hearts highlighted defective NOTCH pathway signalling, recently describe as one cause of LVNC. This study provides the first evidence that RLF is required for normal heart development in the mouse. The heart morphological defects present at high penetrance in Rlf mutants are consistent with features of LVNC in humans, and molecular analysis identified attenuated JAGGED 1 expression and NOTCH signalling as likely contributors to these defects. Our study highlights the importance of RLF-dependent epigenetic modifications to DNA for maintaining correct gene regulatory network and intercellular signalling interactions during heart chamber and septal development. Further investigations are needed to define the biochemical role of RLF in the developing heart, and whether RLF mutations are a cause of heart defects in humans., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

I had the fortune to be introduced to Helmut Sies during the mid 1980s, while working as a post-doctoral scientist at the University of California, Berkeley. At that time, Helmut was a frequent visitor of the Bruce Ames' laboratory and a leading authority in antioxidants and oxidative stress. His concepts, ideas and willingness to listen and make constructive suggestions have been far-reaching and visionary. Moreover, they have also been highly infectious, so much so that much of my research to this day has been on the same topic. The following is a personal recount on how the field of antioxidants has evolved since those exciting days in Berkeley., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study, we use direct numerical simulations (DNS) to investigate the response of chemotactic bacteria to an isolated patch of chemoattractant in a turbulent environment. Previous work has shown that by stirring nutrients that are chemoattractants into a network of thin, elongated filaments, turbulence directly influences the rate at which chemotactic bacteria consume nutrients. However, the quantitative outcome of this process is influenced by a host of physical and biological factors, and many of these remain unexplored. Here, we analyse the sensitivity of nutrient uptake by chemotactic bacteria on a wide range of physical and biological parameters using a series of controlled DNS. Starting with uniformly distributed populations of motile and non-motile bacteria in a fully developed homogeneous, isotropic turbulent flow, we inject a patch of dissolved nutrients. We then assess the chemotactic advantage, defined as the difference between the nutrients consumed by motile and non-motile bacteria over the lifetime of the patch. We find that the chemotaxis can enhance the total uptake rate by a factor of 1.6 and allows the population of chemotactic bacteria to absorb nutrients 2.2 times faster than non-motile bacteria Results show that chemotactic bacteria are subject to a trade-off between swimming to leave regions devoid of nutrients and, once a nutrient gradient is detected, staying in regions of large nutrient concentration. These findings could help explain how the physical characteristics of turbulent marine ecosystems influence the optimal biological traits of bacteria through the competition for limited resources., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The development of immune cytopenias is a well-recognized side effect of many drugs. Quinine- and quinidine-dependent antibodies are classic examples of drug-induced effects that cause severe, life-threatening thrombocytopenia. Whereas the effects of drug-dependent antibodies on platelets have been well documented, their effects on megakaryocyte (Mk) biology are still unclear. We analyzed sera from several quinine-induced thrombocytopenia (QITP) patients on highly pure Mks (98% glycoprotein IIb-positive [GPIIb(+)]; 92% GPIX(+)) derived from human CD34(+) cells cultured with human thrombopoietin. We demonstrate by flow cytometry and confocal microscopy that QITP IgGs bind Mks efficiently in the presence of quinine. Incubation of day-4 Mks with QITP sera or purified IgG resulted in induction of apoptosis, a significant decrease in cell viability, and an increase in cell death. Furthermore, QITP sera preferentially reduced the number of late GPIX(+)/GPIbα(+) Mks and the number of receptors per cell in the surviving population. Ploidy distribution, lobularity, and average cell size of Mks remained unchanged after treatment. In addition, treated Mks showed a marked decrease in their proplatelet production capacity, suggesting that drug-dependent antibodies hinder platelet production. Therefore, QITP antibodies considerably reduce the proplatelet production capabilities of Mks despite undetectable effects on DNA content, morphology, and cell size.

Cellular senescence is a mechanism to inhibit the growth of mammalian cells after oncogenic activation, or in response to damage or stress. We describe here the identification of a novel gene, SENEX, that regulates stress induced premature senescence pathways in endothelial cells (ECs) involving p16(INK4a) and retinoblastoma protein activation. Endogenous levels of SENEX remain unchanged during replicative senescence but are regulated by H(2)O(2)-mediated stress. In contrast to that previously described for senescence in other cell types, the SENEX induced senescent ECs are profoundly anti-inflammatory. The cells are resistant to tumor necrosis factor (TNF)α-induced apoptosis, adhesion of neutrophils and mononuclear cells, and the surface (but not cytoplasmic) expression of endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. Furthermore they are resistant to thrombin induced vascular leak. Senescent ECs such as those lining atherosclerotic lesions may therefore function to limit the inflammatory response. SENEX is also essential for EC survival since depletion either ectopically by siRNA or by high- dose H(2)O(2) treatment causes apoptosis. Together, these findings expand our understanding of the role of senescence in the vasculature and identify SENEX as a fulcrum for driving the resultant phenotype of the endothelium after activation.

Introduction: Post-prandial hypertriglyceridaemia is a risk factor for cardiovascular disease, although the underlying mechanisms remain unclear. High density lipoproteins (HDL) have been shown to be atheroprotective, in part through attenuation of vascular inflammation. In this study, the influence of acute hypertriglyceridaemia on the composition and anti-inflammatory properties of HDL was investigated., Methods: Eight fasting healthy male subjects (34+/-2 years) received 20% Intralipid (15 mg/kg/h) or saline, on separate occasions in random order. At baseline and 60 min post-infusion, the total HDL fraction was isolated and its chemical composition determined. HDL were added to TNF-alpha stimulated human coronary artery endothelial cells and VCAM-1 and ICAM-1 expression was determined by flow cytometry., Results: Serum triglyceride (97.4+/-8.5mg/dL baseline, 283.2+/-35.4 mg/dL post-infusion, p<0.001) and HDL triglyceride content (3.8+/-0.5% HDL mass baseline, 5.3+/-0.9% HDL mass post-infusion, p<0.05) increased significantly after Intralipid infusion. HDL post-Intralipid were significantly less anti-inflammatory compared with control (e.g. at 8 microM apoA-I, %VCAM-1 expression 54+/-5 post-saline, 73+/-4 post-Intralipid, p=0.01; %ICAM-1 expression 94+/-1 post-saline, 99.4+/-0.6 post-Intralipid, p<0.01). There was also a significant correlation between HDL triglyceride content and VCAM-1 expression (R=0.70, p=0.005); as well as between plasma triglyceride levels and both VCAM-1 (R=0.71, p<0.005) and ICAM-1 expression (R=0.80, p<0.005)., Conclusion: Acute hypertriglyceridaemia, simulating the post-prandial state, results in triglyceride-rich HDL with impaired anti-inflammatory capacity.

Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO(86) and MetO(112) (termed apoA-I(+32)) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I(+32), apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.

Background: Evidence suggests that delayed re-endothelialization is responsible for in-stent thrombosis. Probucol inhibits neointimal thickening in animals via enhanced re-endothelialization and is the only oral drug that consistently inhibits restenosis after coronary angioplasty in humans. Here, we examined the effects of probucol on re-endothelialization and neointimal formation in a stent model., Methods and Results: New Zealand White rabbits were fed a hypercholesterolemic diet with probucol (1%) or without (control) (n=11 each) for 6 weeks. At 2 weeks, endothelial denudation and stenting of the iliac artery was performed. Iliac arteries were harvested at week 6, and stented segments sectioned and analyzed. Compared with control, probucol increased in-stent re-endothelialization (74+/-6% in controls versus 93+/-3% in probucol-treated; P=0.008), and decreased average luminal stenosis (58+/-27 versus 31+/-16%; P=0.01) and stent depth (619+/-310 versus 314+/-158 microm; P=0.009). Compared with control, probucol also decreased accumulation of macrophages in the neointima. Furthermore, none of the probucol-treated rabbits had in-stent thrombosis, whereas four of eleven control rabbits showed thrombosis (P=0.04)., Conclusions: Probucol demonstrates anti-restenotic and appears to have anti-thrombotic properties that are likely related to its ability to promote in-stent re-endothelialization.

Statins decrease LDL cholesterol and the risk of atherosclerotic cardiovascular disease (CVD). They also decrease coenzyme Q10 (CoQ10), an effect that may negate some of the statin benefit on CVD. We examined the relationship between plasma CoQ10 concentration and CVD in a prospective case-control study of the effect of pravastatin. Plasma samples from 250 LIPID trial patients who over 6 years suffered a recurrent CVD event (CVD death, nonfatal MI or stroke) and 250 matched controls who remained event-free for the same duration of follow-up were assayed for CoQ10 and lipids (cholesterol and cholesterylesters). Mean plasma CoQ10 concentrations were significantly lower in pravastatin-treated patients than in those assigned placebo (0.51 versus 0.60 micromol/L, P = 0.006), and there was a moderate correlation between CoQ10 and common cholesterylesters (Pearson correlation coefficients in patients randomised to placebo, range r = 0.42-0.63). Univariate conditional logistic regression did not suggest any relationship between plasma CoQ10 and the risk of future CVD events (odds ratio 1.18; 95% CI 0.74-1.87; P = 0.49). Instead, we observed a reduction in the rate of recurrent CVD events with increasing ratio of plasma cholesterylarachidonate to cholesteryllinoleate. This study confirms that pravastatin lowers plasma CoQ10 concentrations, but this does not appear to predict the risk of recurrent CVD events.

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.

Hypoxia increases and hyperoxia decreases experimental atherosclerosis, but it is unclear if repetitive hypoxic and hyperoxic insults affect intimal thickening after arterial injury. Rabbits on 2% cholesterol diet for 6 weeks underwent balloon injury to the abdominal aorta (AA) after week 3, and were then exposed to normoxia (n = 6), or 12 h daily of intermittent repetitive hypoxia (n = 6) or hyperoxia (n = 6). After week 6, damaged AA and undamaged thoracic aorta (TA) were assessed for intimal thickening and lipid content. Compared with normoxia, hypoxia and hyperoxia did not alter the rise in serum cholesterol related to cholesterol feeding. However, compared to normoxia, hypoxia markedly increased the intima-to-media ratio in AA (1.18 +/- 0.09 versus 1.96 +/- 0.14, P < 0.01) and TA (0.15 +/- 0.02 versus 0.41 +/- 0.01, P < 0.01) whereas hyperoxia had no effect on AA disease and increased intimal thickening in TA (0.26 +/- 0.03, P < 0.01). Hyperoxia promoted positive arterial remodeling in both TA and AA, resulting in larger luminal size. The cholesterol content in AA was increased by hypoxia and decreased by hyperoxia, but decreased by both treatments in TA. Lipophilic antioxidants and the proportion of arterial lipids that was oxidized were not altered by hypoxia or hyperoxia. These results suggest that intermittent repetitive hyperoxia is not protective and intermittent repetitive hypoxia promotes arterial disease in normal and injured arteries independent of lipid peroxidation.

Atherosclerosis and its resultant cardiovascular events represent a state of heightened oxidative stress that is commonly thought to contribute to atherogenesis. The aim of this review is to summarize the data linking oxidative events to the pathogenesis of atherosclerosis. Despite abundant data supporting the presence of lipid and protein oxidation in the vascular wall, the poor performance of antioxidant strategies in limiting either atherosclerosis or cardiovascular events from atherosclerosis remain a fundamental problem for implicating oxidative stress as pathophysiologically important. Direct evidence that oxidative stress in general, and the oxidative modification of low-density lipoprotein in particular, is both necessary and sufficient for atherosclerosis has been difficult to find. There are many potential reasons for this difficulty, not the least of which is our lack of sufficient knowledge delineating the precise molecular events that beget oxidative stress in the vessel wall, and the precise mediators involved. Further investigation elucidating these oxidative events are required to provide us with the tools to limit oxidative stress at its source and ameliorate all of its secondary phenomena. Only then will we know what components of atherosclerosis are directly due to oxidative stress.

MRC CRASH is a randomised controlled trial (ISRCTN74459797) of the effect of corticosteroids on death and disability after head injury. We randomly allocated 10,008 adults with head injury and a Glasgow Coma Scale score of 14 or less, within 8 h of injury, to a 48-h infusion of corticosteroid (methylprednisolone) or placebo. Data at 6 months were obtained for 9673 (96.7%) patients. The risk of death was higher in the corticosteroid group than in the placebo group (1248 [25.7%] vs 1075 [22.3%] deaths; relative risk 1.15, 95% CI 1.07-1.24; p=0.0001), as was the risk of death or severe disability (1828 [38.1%] vs 1728 [36.3%] dead or severely disabled; 1.05, 0.99-1.10; p=0.079). There was no evidence that the effect of corticosteroids differed by injury severity or time since injury. These results lend support to our earlier conclusion that corticosteroids should not be used routinely in the treatment of head injury.

Background: Inflammation and oxidative stress are processes that mark early metabolic abnormalities in vascular diseases., Objectives: We explored the effects of a high-fat, high-cholesterol (HFHC) diet on vascular responses in baboons and the potential response-attenuating effects of vitamin E and coenzyme Q(10) (CoQ(10)) supplementation., Design: We used a longitudinal design by subjecting 21 baboons (Papio hamadryas) to sequential dietary challenges., Results: After being maintained for 3 mo on a baseline diet (low in fat and cholesterol), 21 baboons were challenged with an HFHC diet for 7 wk. The serum C-reactive protein (CRP) concentrations did not change. Subsequent supplementation of the HFHC diet with the antioxidant vitamin E (250, 500, or 1000 IU/kg diet) for 2 wk reduced serum CRP concentrations from 0.91 +/- 0.02 to 0.43 +/- 0.06 mg/dL. Additional supplementation with CoQ(10) (2 g/kg diet) further reduced serum CRP to approximately 30% of baseline (0.28 +/- 0.03 mg/dL; P = 0.036 compared with the HFHC diet). Introduction of the HFHC diet itself significantly decreased serum P-selectin (from 48.8 +/- 7.2 to 32.9 +/- 3.7 ng/dL, P = 0.02) and von Willebrand factor (from 187.0 +/- 10.1 to 161.9 +/- 9.0%, P = 0.02) concentrations. However, neither vitamin E alone nor vitamin E plus CoQ(10) significantly altered the serum concentrations of P-selectin or von Willebrand factor., Conclusions: Dietary supplementation with vitamin E alone reduces the baseline inflammatory status that is indicated by the CRP concentration in healthy adult baboons. Cosupplementation with CoQ(10), however, significantly enhances this antiinflammatory effect of vitamin E.

Background: Oxidation of LDL is thought to be important in the development of atherosclerosis. Effective protection against lipoprotein oxidation is achieved by the use of alpha-tocopherol plus coantioxidants, ie. compounds that prevent the prooxidant activity of the vitamin. Wines contain a large number of polyphenols, micronutrients that may act as coantioxidants and may enhance the in vivo antioxidant activity of vitamin E., Objective: We examined whether wines and wine-derived fractions are able to act synergistically with vitamin E in vitro and whether dealcoholized red wine (DRW) retards the development of atherosclerosis., Design: Synergy with vitamin E was assessed in vitro by the ability of red and white wines to both attenuate alpha-tocopheroxyl radicals and inhibit in vitro oxidation of LDL in the presence of vitamin E. Female, 6-8-wk-old apolipoprotein E gene-deficient mice were fed a normal nonpurified stock diet for 24 wk to assess the effect on atherosclerosis of DRW at a dose equivalent to 200 mL x 80 kg body wt(-1) x d(-1)., Results: DRW synergized with vitamin E as effectively as did red and white wine, and phenolic acids accounted for most of this activity. Administration of DRW increased plasma and aortic antioxidants concentrations and the resistance of plasma lipoproteins to ex vivo oxidation. Whereas lipoprotein oxidation in the artery wall was not affected, DRW significantly decreased atherosclerosis in the aortic arch, but not in the root, as assessed by morphometry., Conclusions: DRW contains polyphenolic compounds capable of synergizing with vitamin E, and long-term moderate consumption of DRW can decrease atherosclerosis in apolipoprotein E gene-deficient mice.

Study Objectives: To investigate the long-term outcome of lung function in survivors of severe ARDS after modern treatment strategies including lung protective mechanical ventilation and prone positioning maneuvers., Design: Follow-up cohort study., Setting: University hospital pulmonary division and level 1 trauma center., Patients: Sixteen survivors of severe ARDS (from 1992 to 1994) with a lung injury score > or = 2.5., Measurements: The follow-up study (from 1995 to 1996) included interview, physical examination, chest radiographs, static and dynamic lung volumes, diffusion capacity of the lung for carbon monoxide (DLCO), blood gas analysis, and cardiopulmonary exercise testing (CPET)., Results: The mean +/- SD interval between hospital discharge and functional assessment was 29.5 +/- 8.7 months (range, 15.0 to 40.7 months). In approximately one half of the patients, mild abnormalities in static and dynamic lung volumes were found. In 25% (4 of 16 patients), lung function was obstructive; in 25% (4 of 16 patients), lung function was restrictive; and in 6.3% (1 of 16 patients), a combined obstructive-restrictive pattern was revealed. DLCO was impaired in 12.5% (2 of 16 patients); gas exchange during exercise was impaired in 45.5% (5 of 11 patients)., Conclusions: Residual obstructive and restrictive defects as well as impaired pulmonary gas exchange remain common after severe ARDS. CPET is a very sensitive measure to evaluate residual impairment of lung function after ARDS. Using CPET, reduced pulmonary gas exchange can be detected in many patients with normal DLCO.

Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at -20 degrees C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues. Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis.

Monocyte adhesion to and transmigration across the endothelium are initiating steps in atherogenesis. Cytokine-induced adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) has been reported to be inhibited by either native HDL or reconstituted discoidal HDL (rHDL). In the present study we investigated these putative anti-atherosclerotic effects of HDL and rHDL in a more physiologically relevant cell type, i.e. human aortic endothelial cells (HAEC). HDL isolated by ultracentrifugation from eleven healthy subjects or rHDL made with apoA-I and either 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), or 1,2-dimyristoyl-sn-glycero-3-phosphocholine was incubated for 16 h with HAEC prior to stimulation with tumor necrosis factor-alpha (TNFalpha, 100 U/ml). Expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was measured by cell ELISA and Northern blot analysis. HDL (0.25, 0.5, 1.0 and 2.0 mgprotein/ml) failed to significantly inhibit TNFalpha-induced mRNA and protein expression of all three adhesion molecules. Furthermore, of the three rHDL preparations (16 micromol/l apoA-I) only that containing the polyunsaturated PLPC significantly reduced TNFalpha-induced VCAM-1 expression (by 29.9+/-9.1%). These data contrast with previously reported results using plasma HDL and HUVEC, and show that human HDL and rHDL, except for PLPC-rHDL, are ineffective inhibitors of TNFalpha-induced adhesion molecule expression in HAEC. The ability of polyunsaturated phospholipids in HDL to affect endothelial activation remains to be further investigated.

There is clear evidence of lipoprotein oxidation in atherosclerotic lesions. Animal studies and observational prospective human cohort studies have been interpreted as supporting a role for antioxidants in the prevention of coronary heart disease (CHD). However, firm recommendations to take antioxidant supplements to treat or prevent CHD require evidence derived from randomised controlled studies. In primary prevention studies, low dose alpha-tocopherol does not reduce the incidence of coronary events (ATBC study), and beta-carotene either has no effect or increases the incidence of coronary events and cancer death (ATBC, CARET, Physician's Health studies). Secondary preventions, those with smaller populations and shorter duration of follow up have shown some benefit from alpha-tocopherol (CHAOS, SPACE), but larger randomised studies indicate no benefit from treatment with alpha-tocopherol (HOPE, GISSI, PPP). Recent studies with antioxidant combinations also show no benefit (HATS, MPS). On the basis of these data, supplements of alpha-tocopherol and beta-carotene cannot be recommended for the treatment or prevention of CHD. Fundamental and applied research may yet find a role for antioxidant supplements in the treatment of coronary disease. However, this will require positive results from combined antioxidant studies currently in progress, and the targeting of oxidative processes that operate in the artery wall and cause or contribute to disease.

Oxidative modification of lipoproteins may trigger and maintain atherogenesis. We compared the effects of different antioxidants (alpha-tocopherol, probucol, ubiquinone-10) at doses similar to those used in humans in Watanabe Heritable Hyperlipidemic (WHHL) rabbits for 12 months. Aortic lesions were analyzed for their extent and cellular composition of lesions, mean thickness of fibrous caps and density of smooth muscle cells therein, content of antioxidants, non-oxidized and oxidized lipids. Compared to controls, probucol significantly lowered the extent and macrophage content of lesions and increased the existence and smooth muscle cell density of fibrous caps. alpha-Tocopherol supplementation increased the aortic content of vitamin E, but had no decreasing effect on either the accumulation of macrophage-specific antigen in the aorta or lesion size. Nevertheless, both probucol and alpha-tocopherol significantly decreased in vitro LDL oxidizability, measured under typically strong oxidative conditions. Ubiquinone-10 supplement increased lesion size and the fraction of lesions containing fibrous caps; however, LDL oxidizability remained unaffected by ubiquinone-10 treatment. None of the antioxidants tested lowered oxidized lipids within aortic tissue; however, long-term treatment with probucol provided the most effective anti-atherosclerotic effect, while alpha-tocopherol may be pro-atherogenic and ubiquinone-10 exerts ambivalent effects. Our data suggest that (i) widely used oxidation measures, such as ex-vivo LDL oxidizability, do not reflect the degree of atherosclerosis; and (ii) long-term beneficial effects of relatively low doses of antioxidants may be outweighed by high levels of plasma cholesterol in WHHL rabbits.

Serious haemodynamic instability occurred during emergency surgery for a perforated duodenal ulcer in a 72-year-old man with acute myocardial infarction. Intraoperative transoesophageal echocardiography was crucial for diagnosis of the location of myocardial infarction in the right ventricle and the subsequent haemodynamic management. Postoperatively, a thrombus in the right coronary artery was removed by coronary angiography. The patient's trachea was extubated on the fourth postoperative day. Another 4 days later a leak in the lower oesophagus was suspected because of pleural empyema, and verified. The patient's trachea had to be re-intubated and an oesophageal stent was inserted. The patient was discharged, fully recovered, 2 months after the operation.

Oxidative modification of low-density lipoprotein is thought to promote arterial lipid accumulation and atherogenesis. Previous studies reported on the presence of certain lipid or protein oxidation products in lesions, although a systematic investigation measuring several oxidation parameters and the accumulation of nonoxidized lipids and antioxidants at various stages of atherosclerosis has not been performed in the same tissue. Using the intimal lipoprotein-containing fraction of human aortic lesions, we demonstrate here that cholesterol accumulated with lesion development and that this increase was already significant at the fatty streak stage. By comparison, cholesterylesters increased significantly only in fibro-fatty and more complex lesions that also contained significantly increased amounts of cholesterylester hydro(pero)xides and 27-hydroxycholesterol. Cholesterylester hydroxides were the major lipid oxidation product detected. Despite accumulation of oxidized lipid, alpha-tocopherol was also present and maintained at a comparable level over the disease process. Of the oxidized protein moieties measured only o,o-dityrosine increased with disease, although chlorotyrosines were present at relatively high levels in all lesions compared to healthy vessels. Our data show that accumulation of nonoxidized lipid precedes that of oxidized lipid in human aortic lesions.

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.

Sequestration of parasitized erythrocytes in the central nervous system microcirculation and increased cerebrospinal fluid lactate are prominent features of cerebral malaria (CM), suggesting that sequestration causes mechanical obstruction and ischemia. To examine the potential role of ischemia in the pathogenesis of CM, Plasmodium berghei ANKA (PbA) infection in CBA mice was compared to infection with P. berghei K173 (PbK) which does not cause CM (the non-CM model, NCM). Cerebral metabolite pools were measured by (1)H nuclear magnetic resonance spectroscopy during PbA and PbK infections. Lactate and alanine concentrations increased significantly at the terminal stage of CM, but not in NCM mice at any stage. These changes did not correlate with parasitemia. Brain NAD/NADH ratio was unchanged in CM and NCM mice at any time studied, but the total NAD pool size decreased significantly in the CM mice on day 7 after inoculation. Brain levels of glutamine and several essential amino acids were increased significantly in CM mice. There was a significant linear correlation between the time elapsed after infection and small, progressive decreases in the cell density/cell viability markers glycerophosphocholine and N-acetylaspartate in CM, indicative of gradual loss of cell viability. The metabolite changes followed a different pattern, with a sudden significant alteration in the levels of lactate, alanine, and glutamine at the time of terminal CM. In NCM, there were significant decreases with time of glutamate, the osmolyte myo-inositol, and glycerophosphocholine. These results are consistent with an ischemic change in the metabolic pattern of the brain in CM mice, whereas in NCM mice the changes were more consistent with hypoxia without vascular obstruction. Mild obstructive ischemia is a likely cause of the metabolic changes during CM, but a role for immune cell effector molecules cannot be ruled out.

Mixtures of human myoglobin (Mb) (or the Y103F variant of human Mb), authentic peroxynitrite (ONOO(-), ONOO(-):protein 2 mol/mol), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave radicals adducts at cysteine-110 (DMPO-C110) that are detected directly by electron paramagnetic magnetic spectroscopy (EPR). DMPO-C110 was detected exclusively over a range of DMPO concentrations (DMPO:protein ratios 25-100 mol/mol). Treatment of human Mb (or Y103F Mb) with the ONOO(-) generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) chloride (ONOO(-):protein 5 mol/mol) yielded a cross-linked Mb dimer as judged by SDS-PAGE analyses. Addition of DMPO or carbonate effectively eliminated the cross-linked product. Mass analyses of samples containing human Mb (or Y103F Mb), carbonate, and ONOO(-) indicated that nitration occurs exclusively at Y103. Thus, reaction of human Mb and ONOO(-) yields specific products that depend on the presence or absence of physiological concentrations of carbonate. These products may serve as biomarkers for the participation of Mb-derived radicals in the oxidative damage associated with myocardial reperfusion injury., (Copyright 2001 Academic Press.)

15-Lipoxygenase (15-LO)-induced oxidation of lipids in human LDL may be pro-atherogenic. However, the extent to which 15-LO promotes enzymatic oxidation of esterified (i.e., major) lipids in LDL may depend on various factors. Here, we show that overall, LDL lipid oxidation was favored with high activity of human 15-LO, that phospholipids were the preferred esterified substrate, and that low temperature maintained a higher proportion of enzymatic product. However, under all conditions, 15-LO induced alpha-tocopherol consumption and the accumulation of nonenzymatic products that predominated with increasing time of incubation and inactivation of the enzyme. Lysates prepared from cells overexpressing human 15-LO oxidized linoleic acid readily and in an almost exclusive enzymatic manner. In sharp contrast, such lysates failed to oxidize LDL lipids unless linoleic acid was added, in which case nonenzymatic oxidation of LDL lipids occurred. We conclude that although purified 15-LO can oxidize isolated LDL lipids in vitro, such oxygenation always includes nonenzymatic reactions that likely play a major role in the more extensive oxidation of LDL by cell-derived 15-LO.

L-Arginine, the physiologic substrate of nitric oxide synthase, has antiatherogenic properties in animal models of atherosclerosis, and improves endothelial function in hypercholesterolemic humans. Some of these effects may be mediated by increased production of nitric oxide; however, some investigators have postulated a direct antioxidant action related to its aminoguanidine moiety. We aimed therefore, to assess the antioxidant properties of L-arginine. The antioxidant properties of 200 microM L-arginine. 200 microM D-arginine and 200 microM L-glutamate were compared with the powerful antioxidant ascorbate and a control (phosphate-buffered saline). Compounds were tested using four in vitro methods: (1) the Esterbauer technique (which tests the ability of the compounds to scavenge free radicals or chelate transition metals); (2) the effect on the autoxidation of ascorbate; (3) anti-tocopherol mediated peroxidation (which tests the compound's ability to synergize with alpha-tocopherol to prevent mild chemically induced LDL oxidation); and (4) the ability of the compounds to attenuate alpha-tocopherol radical in micellular emulsions (TRAA). The above methods were repeated using the metabolites of the test compounds after incubation with human endothelial cells. Ex vivo studies were then carried out by measuring levels of lipid peroxide production (using HPLC with UV and chemiluminescence detection) in three healthy volunteers before and 2 h after a single 7-g oral dose of L-arginine. By the Esterbauer technique, L-arginine increased lag time by 45% compared to control, as did D-arginine by 50%; L-glutamate had no effect and ascorbate increased lag time by 325%. Neither L-arginine, D-arginine or L-glutamate had significant effects on the autoxidation of ascorbate or anti-tocopherol mediated peroxidation. By the TRAA method, L-arginine had a small effect on preventing the decay of tocopherol. The results were similar for the studies of the compound's metabolites. In ex vivo studies, no changes were seen in lipid peroxide levels following acute dosage with L-arginine. L-Arginine has only weak and non-specific antioxidant effects, suggesting that its major cardioprotective benefits occur through other mechanisms, such as via the nitric oxide pathway.

Oxidation of lipoproteins is thought to be an early event in atherogenesis. To evaluate whether aortic lipoprotein lipid (per)oxidation contributes to atherosclerosis, we investigated the time-dependent changes to lipids and antioxidants in plasma and aortas of apolipoprotein E gene knockout (apoE-/-) mice receiving a high fat diet, and compared these changes with lesion development. Circulating buoyant lipoproteins and associated cholesterol (C), cholesteryl esters (CE), and alpha-tocopherol (alpha-TOH) increased within 1 month then remained largely constant up to 6 months. Coenzyme Q (CoQ) remained unchanged for the first 3 months and increased marginally after 6 months. With increasing duration of the diet, plasma lipids showed an increased propensity to undergo peroxyl radical-induced (per)oxidation. Absolute concentrations of aortic C, hydroperoxides and hydroxides of CE (CE-O(O)H) and alpha-TOH increased gradually while aortic CE increased more markedly with changes to cholesteryl linoleate being most pronounced. Aortic CoQ remained largely unchanged. Overall, the extent of aortic CE (per)oxidation remained low (

Endothelial dysfunction is thought to be an important early event in atherogenesis, related in part to reduced bioavailability of nitric oxide in the arterial wall. Endothelial function may be impaired in the presence of oxidised low density lipoprotein. The use of vitamin E as an anti-oxidant might enhance the bioavailability of nitric oxide in this situation. The effect of vitamin E 1000 IU/day on arterial endothelial physiology was studied in 20 asymptomatic older subjects, aged 45-70 years, who showed evidence of age-related endothelial dysfunction. Endothelial function was assessed non-invasively using brachial ultrasound and the primary outcome measure was flow-mediated endothelium-dependent dilatation (FMD) in response to reactive hyperaemia. A double-blind, placebo-controlled, randomised crossover design was employed. After 3 weeks of stabilisation on a standard fat-reduced diet, subjects received vitamin E or placebo for 10 weeks in random order, separated by a washout period of 8 weeks. There were no significant changes in blood pressure, plasma lipid or lipoprotein concentrations. Plasma alpha-tocopherol increased from 50+/-3 (mean+/-S.E.M.) to 91+/-6 micromol/l (P < 0.001) with vitamin E ingestion. Total plasma F2alpha-isoprostanes, a measure of free radical-induced lipid peroxidation, were not altered by vitamin E ingestion (0.86+/-0.26 versus 0.82+/-0.25 nmol/l, P > 0.6). FMD was not significantly different between the placebo and vitamin E periods (2.7+/-0.6% versus 2.4+/-0.4%). Variation in FMD was not correlated with change in plasma alpha-tocopherol (r = - 0.03, P > 0.8). The study was powered to detect a minimum change in FMD of 2%. Glyceryl trinitrate endothelium-independent dilatation was not significantly changed with vitamin E (13.7+/-1.3% versus 13.6+/-1.4%,). These results exclude a major impact of medium-term supplementation with vitamin E on arterial endothelial function when age-related dysfunction is already present.

Exposure of plasma from apolipoprotein E gene knockout (apoE-/-) and control (CBA or C57BL/6J) mice plasma to a constant rate of aqueous peroxyl radicals (ROO.) resulted in the depletion of ascorbate, urate and alpha-tocopherol (alpha-TOH), with substantial and little lipid peroxidation, respectively. Alpha-TOH levels were 3-times higher in plasma from apoE-/- than control mice and its addition enhanced the oxidizability of control mouse plasma. In apoE-/- mouse plasma, alpha-TOH was associated primarily with very low density lipoprotein (VLDL), whereas in plasma from control mice, the vitamin was located largely in high density lipoproteins. Oxidation of isolated lipoproteins by ROO. resulted in the accumulation of lipid hydroperoxides to an extent that reflected the plasma concentration and alpha-TOH content of the different lipoprotein fractions. Oxidation of 'plasma' reconstituted from components of apoE-/- mice and/or human plasma showed that human and apoE-/- mouse lipoproteins peroxidized with similar kinetics, although the initiation of lipid peroxidation was greater in the presence of mouse than human lipoprotein-deficient plasma. Also, the chain length of lipid peroxidation in apoE-/- mouse plasma after ascorbate depletion appeared to be independent of the rate of ROO. generation. Together, these results show that the ROO. induced peroxidation of plasma lipoproteins in atherogenesis-susceptible apoE-/- mice exhibits some, though not all, features of tocopherol-mediated peroxidation (TMP). Therefore, apoE-/- mice may represent a suitable animal model to test a role for TMP in atherogenesis and the prevention of this disease by anti-TMP agents.

The pathogenesis of human cerebral malaria (CM) remains unresolved. In the most widely used murine model of CM, the presence of T lymphocytes and/or interferon (IFN)-gamma is a prerequisite. IFN-gamma is the key inducer of indoleamine 2,3-dioxygenase (IDO), which is the catalyst of the first, and rate-limiting, step in the metabolism of tryptophan (Trp) along the kynurenine (Kyn) pathway. Quinolinic acid (QA), a product of this pathway, is a neuro-excitotoxin, like glutamic acid (Glu) and aspartic acid (Asp). Kynurenic acid (KA), also produced from the Kyn pathway, antagonizes the neuro-excitotoxic effects of QA, Glu, and Asp. We therefore examined the possible roles of IDO, metabolites of the Kyn pathway, Glu, and Asp in the pathogenesis of fatal murine CM. Plasmodium berghei ANKA infection was studied on days 6 and 7 post-inoculation (p.i.), at which time the mice exhibited cerebral symptoms such as convulsions, ataxia, coma, and a positive Wooly/White sign and died within 24 hours. A model for noncerebral malaria (NCM), P. berghei K173 infection, was also studied on days 6 and 7 and 13 to 17 p.i. to examine whether any changes were a general response to malaria infection. Biochemical analyses were done by high-pressure liquid chromatography and gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS). IDO activity was low or absent in the brains of uninfected mice and NCM mice (days 6 and 7 p.i.) and was induced strongly in the brains of fatal murine CM mice (days 6 and 7 p.i.) and NCM animals (days 13 to 17 p.i.). This induction was inhibited greatly by administration of dexamethasone, a treatment that also prevented CM symptoms and death. Furthermore, IDO induction was absent in IFN-gamma gene knockout mice, which were also resistant to CM. Brain concentrations of Kyn, 3-hydroxykynurenine, and the neuro-excitotoxin QA were significantly increased in both CM mice on days 6 and 7 p.i. and NCM mice on days 13 to 17 p.i., whereas an increase in the ratio of brain QA to KA occurred only in the CM mice at the time they were exhibiting cerebral symptoms. Brain concentrations of Glu and Asp were significantly decreased in CM and NCM mice (days 13 to 17 p.i.). The results imply that neuro-excitation induced by QA may contribute to the convulsions and neuro-excitatory signs observed in CM.

Study Objectives: Investigating the effect of low-volume pressure-limited ventilation and repeated prone positioning on the short-term course and outcome in patients with severe ARDS., Setting: Level 1 trauma center of a university hospital., Patients: Twenty-five patients suffering from ARDS with a lung injury score (LIS) > or = 2.5 admitted consecutively to our ICU from January 1992 to December 1994., Methods: Mechanical ventilation with peak inspiratory pressure limitation to 35 mbar, irrespective of hypercapnia and prone positioning to achieve adequate oxygenation. SCORING AND MEASUREMENTS: Patient assessment with LIS, APACHE (acute physiology and chronic health evaluation) II score, injury severity score, and multiple organ failure score. Blood gas analyses and estimation of static compliance were repeated at least every 4 h during the treatment period. PaO2/FIO2 (fraction of inspired oxygen) ratio, alveolo-arterial oxygen difference, and intrapulmonary shunt were calculated according to standard equations. The best values taken from each 4-h period during the investigation were used to evaluate the best possible performance of the lung within this interval and to investigate the entire course., Results: Mean predicted mortality based on the APACHE II score was 35.4+/-15.2%. Three of the 25 patients (12%) died. However, none was related to respiratory failure. No pneumothorax occurred. Sixteen patients, lacking any contraindication for prone positioning, responded positively to this change in position, each to a different individual degree., Conclusion: We assume that our low mortality in patients with severe ARDS might be due mainly to low-volume pressure-limited ventilation and prone positioning. This simple strategy seems to allow successful treatment for patients with severe ARDS.

Various lipoxygenases (LO) oxidize low density lipoprotein (LDL) in vitro and 15-LO has been implicated in the development of atherosclerosis in vivo. Direct oxidation of phospholipids (PL) and cholesteryl esters (CE) by LO has been proposed as a mechanism whereby these enzymes cause or contribute to LDL lipid peroxidation. Herein we show that the extent to which recombinant human 15-LO (rhLO) caused peroxidation of LDL's esterified core and surface lipids depended on, and directly related to, the alpha-tocopherol (alpha-TOH) content of the lipoprotein. Thus, CE and PL of in vivo alpha-TOH-depleted LDL, isolated from a patient with familial isolated vitamin E deficiency, were resistant to oxidation by rhLO, whereas those in alpha-TOH-containing LDL from the same patient receiving vitamin E supplements readily oxidized. The extent to which rhLO caused oxidation of CE and PL directly and linearly correlated with LDL's content of vitamin E, as demonstrated by studies with in vitro alpha-TOH-depleted lipoproteins. The geometric isomers of oxidized cholesteryl linoleate formed in LDL during oxidation initiated by rhLO, matched those obtained during non-enzymic, peroxyl radical-initiated oxidation of LDL whilst alpha-TOH was present. Ascorbate, an efficient co-antioxidant for alpha-TOH, completely prevented rhLO-initiated oxidation of LDL's CE, but did not inhibit rhLO-mediated oxidation of unesterified linoleate. These results are inconsistent with direct oxidation of LDL esterified lipids by rhLO. Isolated LDL contained free fatty acids (FFA), and its exposure to rhLO caused a rapid formation of linoleate hydroperoxide. To reconcile these data, we propose that during rhLO-initiated oxidation of LDL, enzymic oxidation of FFA preceeds the oxidation of CE and PL, which occurs largely via a tocopherol-dependent process.

We report a rapid and convenient method for screening potential inhibitors of the initiation of low density lipoprotein (LDL) lipid peroxidation. The method uses positively and negatively charged micelles of either cetyltrimethyl ammonium chloride or sodium dodecyl sulfate with added alpha-tocopherol. It is based on the capacity of an antioxidant to attenuate alpha-tocopheroxyl radicals, generated by irradiation of the alpha-tocopherol-containing micelles with UV light, and measured directly by electron spin resonance spectroscopy. To establish the reliability of the method, we compared the alpha-to-copheroxyl radical attenuating ability (TRAA) of 53 natural and synthetic potential antioxidants with their respective ability to inhibit the early stages of LDL lipid peroxidation initiated by a low flux of water-soluble peroxyl radicals. The relationship between the measured TRAA and corresponding LDL antioxidation activity was highly significant (P < 0.00005, Rank test). Thus, the potency of a co-antioxidant for LDLs alpha-tocopherol could be predicted with > 98% probability by the TRAA test alone. The results suggest that this relatively simple method represents an effective and simple screening test that can be used for large numbers of potential inhibitors of the early stages of LDL lipid oxidation.

The oxidation of low-density lipoproteins (LDLs) is now commonly implicated as an important early event in atherogenesis. The resulting interest in LDL antioxidation has focused on alpha-tocopherol, the biologically and chemically most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. We review advances made in our understanding of the molecular action of alpha-tocopherol in radical-mediated oxidation of isolated human LDL and how the vitamin's antioxidant activity is enhanced or even dependent on the presence of suitable reducing species, which are referred to as coantioxidants.

This study examines the cholesteryl ester transfer protein (CETP)-mediated exchange of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) and cholesteryl linoleate hydroxide (Ch18:2-OH) between low density lipoprotein (LDL) and high density lipoprotein (HDL). When [3H]Ch18:2-OOH- and [3H]18:2-OH-labeled LDL were incubated at 37 degrees C for 0-24 h with unoxidized HDL and purified CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the HDL. Similarly, when incubations were carried out with [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled HDL, unoxidized LDL, and CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the LDL. Comparable results were obtained for the CETP-mediated transfer of [3H]Ch18:2-OH alone from LDL to HDL. Transfer to HDL of oxidized cholesteryl linoleate from [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled LDL was comparable to that of unoxidized cholesteryl linoleate (Ch18:2). However, the rate of transfer of [3H]Ch18:2-OOH and [3H]Ch18:2-OH from LDL to HDL increased linearly as the molar ratio of acceptor (HDL) to donor (oxidized LDL) particles in the incubation increased from 0.5:1 to 10:1. This increased rate of exchange was accompanied by an increased proportion of the oxidized Ch18:2 being present as the hydroxide rather than hydroperoxide. Further increases in the molar ratio of HDL to oxidized LDL particles neither affected the transfer rate nor the extent of reduction of Ch18:2-OOH to Ch18:2-OH. We therefore conclude that i) CETP mediates bidirectional transfers of Ch18:2-OOH and Ch18:2-OH between HDL and LDL; ii) CETP does not distinguish between Ch18:2-OOH, Ch18:2-OH, and Ch18:2 as it mediates their exchange between HDL and LDL; and iii) association with HDL hastens the reduction of Ch18:2-OOH to Ch18:2-OH.

The reaction of phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Ebselen with phospholipid and cholesterylester hydroperoxides associated with HDLox and LDLox was investigated using specific HPLC assays for the hydroperoxides of phosphatidylcholine (PCOOH) and cholesteryllinolate (Ch18:2-OOH) and for cholesteryllinolate hydroxides (Ch18:2-OH). HDLox and LDLox were formed from the corresponding isolated native lipoproteins by controlled and limited oxidation initiated by aqueous peroxyl radicals. Incubation of HDLox or LDLox in the presence of PHGPx/GSH or Ebselen/GSH resulted in rapid degradation of both classes of lipid hydroperoxides, with equimolar amounts of Ch18:2-OH formed from Ch18:2-OOH. No pronounced differences were observed between PCOOH and Ch18:2-OOH in terms of substrate specificity, whereas HDLox-associated PCOOH and Ch18:2-OOH appeared to be slightly better substrates for PHGPx/GSH as compared to those in LDLox. Also, Ch18:2-OOH associated with HDLox but not LDLox were reduced by Ebselen or GSH alone. These in vitro findings indicate that the enzymatic PHGPx/GSH and the nonenzymatic Ebselen/GSH systems can efficiently reduce hydroperoxides of phospholipids and cholesterylesters associated with intact lipoproteins.

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

Oxidative stress has been implicated in the adult respiratory distress syndrome (ARDS). In this study, we determined the levels of selected antioxidants in the plasma of 25 patients with ongoing ARDS and 16 healthy control subjects. We also examined these plasmas and pulmonary edema fluid of ARDS patients for lipid hydroperoxides. Both ascorbate and ubiquinol-10 concentrations in ARDS plasma were significantly lower than in normal plasma. alpha-Tocopherol concentrations, when standardized to total plasma cholesterol, were not lower in ARDS patients than in normal subjects. A pattern of antioxidant levels virtually identical to that observed in ARDS plasma was obtained after in vitro incubation of healthy plasma with stimulated polymorphonuclear leukocytes: very low ascorbate, decreased ubiquinol-10, and unchanged alpha-tocopherol concentrations. Nanomolar concentrations of lipid hydroperoxides were found in pulmonary edema fluid of ARDS patients, but not in plasma, nor in the plasma of healthy individuals, when a sensitive and selective chemiluminescence assay for hydroperoxides was used. ARDS patients also showed significant decreases in plasma levels of cholesterol esters in conjunction with discoidal high-density lipoprotein profiles, indicating a decrease in lecithin-cholesterol acyltransferase activity. We conclude that ARDS is associated with oxidative stress, possibly exerted by oxidants released from activated phagocytic leukocytes, and major changes in plasma cholesterol metabolism.