An improved method for the detection of myeloperoxidase chlorinating activity in biological systems using the redox probe hydroethidine.

Conversion of the redox probe hydroethidine (HE) to 2-chloroethidium (2-Cl-E+) by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) provides comparable specificity and superior sensitivity to measurement of 3-chlorotyrosine (3-Cl-Tyr), the gold standard biomarker for MPO chlorinating activity in biological systems. However, a limitation of the former method is the complex mixture of products formed by the reaction of HE with reagent HOCl, coupled with the difficult purification of 2-Cl-E+ from this mixture for analytical purposes. This limitation prompted us to test whether 2-Cl-E+ could be formed by reaction of HE with the strong and widely used chlorinating agent, N-chlorosuccinimide (NCS). Unexpectedly, such reaction yielded 2-chlorohydroethidine (2-Cl-HE) as the major product in addition to 2-Cl-E+, as assessed by high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). 2-Cl-HE was also observed to be the major chlorination product formed from HE with both reagent and enzymatically generated HOCl, just as it was formed ex vivo in different healthy and diseased mouse and human tissues upon incubation with glucose/glucose oxidase to generate a flux of hydrogen peroxide (H2O2). Quantification of 2-Cl-HE plus 2-Cl-E+ improved the sensitivity of the HE-based method compared with measurement of only 2-Cl-E+. Moreover, 2-chlorodimidium (2-Cl-D+) was developed as a practical internal standard instead of the previously used internal standard, deuterated 2-Cl-E+ (d5-2-Cl-E+). Overall, the present study describes an improved method for the detection of MPO/chlorinating activity in biological systems of health and disease.

Bilirubin deficiency renders mice susceptible to hepatic steatosis in the absence of insulin resistance.

BACKGROUND & AIMS: Plasma concentrations of bilirubin, a product of heme catabolism formed by biliverdin reductase A (BVRA), inversely associate with the risk of metabolic diseases including hepatic steatosis and diabetes mellitus in humans. Bilirubin has antioxidant and anti-inflammatory activities and may also regulate insulin signaling and peroxisome proliferator-activated receptor alpha (PPARα) activity. However, a causal link between bilirubin and metabolic diseases remains to be established. Here, we used the global Bvra gene knockout (Bvra-/-) mouse as a model of deficiency in bilirubin to assess its role in metabolic diseases. APPROACH & RESULTS: We fed mice fat-rich diets to induce hepatic steatosis and insulin resistance. Bile pigments were measured by LC-MS/MS, and hepatic lipids by LC-MS/MS (non-targeted lipidomics), HPLC-UV and Oil-Red-O staining. Oxidative stress was evaluated measuring F2-isoprostanes by GC-MS. Glucose metabolism and insulin sensitivity were verified by glucose and insulin tolerance tests, ex vivo and in vivo glucose uptake, and Western blotting for insulin signaling. Compared with wild type littermates, Bvra-/- mice contained negligible bilirubin in plasma and liver, and they had comparable glucose metabolism and insulin sensitivity. However, Bvra-/- mice exhibited an inflamed and fatty liver phenotype, accompanied by hepatic accumulation of oxidized triacylglycerols and F2-isoprostanes, in association with depletion of α-tocopherol. α-Tocopherol supplementation reversed the hepatic phenotype and observed biochemical changes in Bvra-/- mice. CONCLUSIONS: Our data suggests that BVRA deficiency renders mice susceptible to oxidative stress-induced hepatic steatosis in the absence of insulin resistance.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds.

The L-tryptophan-derived tricyclic hydroperoxide cis-WOOH was recently identified as a novel and biologically important factor for regulating vascular tone and blood pressure under inflammatory conditions and potentially other cellular redox signaling events. cis-WOOH is highly labile and currently not available commercially. In this protocol, we provide procedures for the synthesis, purification, quantification and characterization of cis-WOOH, its epimer trans-WOOH and their respective alcohols (cis-WOH and trans-WOH). Photo-oxidation of L-tryptophan (L-Trp) results in a mixture containing cis-WOOH and trans-WOOH, which are separated and purified by semi-preparative HPLC. cis-WOH and trans-WOH are then produced by sodium borohydride reduction and purified by semi-preparative HPLC. Characterization of cis-WOOH and trans-WOOH and the reduced alcohol variants is achieved using HPLC, fluorescence, NMR and liquid chromatography-tandem mass spectrometry. The protocol provides instructions for storage and quantification, as well as ways to test the stability of these hydroperoxides in commonly used buffers and media. Finally, we describe examples of how to monitor the formation of cis-WOOH in biological samples. The protocol ensures reasonable yield (11%) and purity (>99%) of cis-WOOH and control compounds in 5-6 d and outlines conditions under which cis-WOOH is stable for several months.

Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs.

The RBC storage lesion is a multiparametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of NADP(H) were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBC oxidative status after restoration of body temperature constitutes a sensitive method for detecting storage-related alterations that has the potential to improve the quality of stored RBCs for transfusion.

Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP.

ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, ß and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate 'half-up' state to a condensed 'down' state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory 'up' state.

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

Absence of the biliverdin reductase-a gene is associated with increased endogenous oxidative stress.

Bilirubin, a byproduct of heme catabolism, has been shown to be an effective lipid-soluble antioxidant in vitro. Bilirubin is able to inhibit free radical chain reactions and protects against oxidant-induced damage in vitro and ex vivo. However, direct evidence for bilirubin's antioxidant effects in vivo remains limited. As bilirubin is formed from biliverdin by biliverdin reductase, we generated global biliverdin reductase-a gene knockout (Bvra-/-) mice to assess the contribution of bilirubin as an endogenous antioxidant. Bvra-/- mice appear normal and are born at the expected Mendelian ratio from Bvra+/- x Bvra+/- matings. Compared with corresponding littermate Bvra+/+ and Bvra+/- animals, Bvra-/- mice have green gall bladders and their plasma concentrations of biliverdin and bilirubin are approximately 25-fold higher and 100-fold lower, respectively. Naïve Bvra-/- and Bvra+/+ mice have comparable plasma lipid profiles and low-molecular weight antioxidants, i.e., ascorbic acid, α-tocopherol and ubiquinol-9. Compared with wild-type littermates, however, plasma from Bvra-/- mice contains higher concentrations of cholesteryl ester hydroperoxides (CE-OOH), and their peroxiredoxin 2 (Prx2) in erythrocytes is more oxidized as assessed by the extent of Prx2 dimerization. These data show that Bvra-/- mice experience higher oxidative stress in blood, implying that plasma bilirubin attenuates endogenous oxidative stress.

Behavioral and cognitive data in mice with different tryptophan-metabolizing enzymes knocked out.

This article demonstrates behavioral changes in mice in response to free adaptation and drinking session adaptation modules implemented in their social home environment, the IntelliCage. These data complement the study "Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function" (Too LK, Li KM, Suarna C, Maghzal GJ, Stocker R, McGregor IS, et al., 2016) [1]. Prior to programmed drinking sessions, all mice were exposed to a home cage adaptation module during which there was no time limit on water access - the free adaptation module. The exploratory behaviors are here expressed as percentages of visits with nosepokes and of visits with licks. The measurements by percentage of exploratory activity showed minimal genotype effects. The number of nosepokes or licks per corner visit also was compared between WT and gene knockout (GKO) IDO1 mice, WT and GKO IDO2 mice and WT and GKO TDO2 mice and demonstrated unremarkable behavioral changes during the free adaptation module. Analysis of drinking session adaptation behavior showed no genotype effect between WT and GKO of IDO1, IDO2 or TDO2 background. Notwithstanding the absence of genotype differences, each IDO1, IDO2 or TDO2 animal group displayed a specific pattern of adaptation to the drinking session modules. Furthermore, IDO1 GKO mice showed a more rapid recovery of lick frequency to the baseline level compared to the WT equivalents in a simple patrolling task during the first complete testing cycle (R1). TDO2 GKO mice on the other hand did not differ from their WT equivalents in terms of lick frequency over the three test days of complex patrolling and discrimination reversal tasks. Lastly, IDO2 GKO mice reduced their visits to the permanently non-rewarding reference corners by the same degree as did the WT mice.

Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function.

Tryptophan, an amino acid involved in routine energy metabolism, is a key modulator of sickness behaviors associated with inflammatory states and also plays roles in some psychiatric disorders. Tissue concentrations of tryptophan are regulated primarily by the enzymes indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO, encoded by TDO2). Altered IDO1 and TDO activities have been linked to the perturbed serotonergic neurotransmission that may underlie certain psychopathologies. Here we assessed mice genetically modified to be deficient in IDO1, IDO2 or TDO2 for their behavior and cognitive function using an automated home cage system, the IntelliCage™. A well-established behavioural and cognitive test battery was applied during two periods (Runs 1 and 2, "R1" and "R2") separated by one month. Various tryptophan-related neurochemicals also were measured in brain extracts. IDO1(-/-) mice displayed remarkable reductions of early diurnal exploration in the IntelliCage and this persisted in R2. In contrast, early diurnal hyperactivity was observed in IDO2(-/-) mice in both R1 and R2. TDO2(-/-) mice displayed increased diurnal and nocturnal exploration, but only in R2. Cognitive assessment suggested enhanced reference memory in IDO2(-/-) mice in a complex patrolling task, while TDO deficiency was associated with enhanced performance in complex patrolling and discrimination reversal tasks. Neurochemical measures showed attenuated brain serotonin levels in IDO1(-/-) mice and augmented tryptophan and serotonin levels in TDO2(-/-) animals, respectively. No neurochemical alterations were detected in IDO2(-/-) mice. Taken together, these findings reveal complex and dissimilar patterns of behavioral and cognitive changes induced by knockout of three different tryptophan-metabolizing enzymes.

Heme oxygenase-1 deficiency alters erythroblastic island formation, steady-state erythropoiesis and red blood cell lifespan in mice.

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119(+)-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119(+)-erythroid cells in the spleen, although α4ß1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.

Assessment of myeloperoxidase activity by the conversion of hydroethidine to 2-chloroethidium.

Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.

Serum amyloid A in uremic HDL promotes inflammation.

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.

A phosphatidylcholine-BODIPY 581/591 conjugate allows mapping of oxidative stress in P. falciparum-infected erythrocytes.

The chromophore, BODIPY 581/591, has an extended conjugated system that reacts with oxygen centered-radicals leading to changes in its spectral characteristics. Fatty acid-conjugated BODIPY 581/591 transfers readily between membrane bilayers and can be used as a sensor of oxidative stress in cell populations. We report here the use of a phosphatidylcholine (PC) derivative of BODIPY 581/591, which transfers much less rapidly between membranes. This allows the analysis of oxidative stress in individual cells and in different compartments within cells. Quantitative imaging and flow cytometry were used to measure the ratio of fully conjugated to oxidized probe in model systems and in Plasmodium falciparum-infected erythrocytes. We observed an increase in the oxidation of the parasite-associated BODIPY 581/591-PC as the intraerythrocytic parasite matures. By contrast, BODIPY 581/591-PC associated with the erythrocyte membrane experiences a low level of oxidation even in the later stages of parasite development. Treatment with a pro-oxidant compound caused increased oxidation of the probe in the parasite compartment, but less so in the host cell membrane. Conversely, treatment with ferricyanide increases oxidation of the probe in the erythrocyte cell membrane but does not inhibit parasite growth. Chromatographic analysis of the lipids in infected erythrocytes shows no evidence for loss of alpha-tocopherol or the accumulation of lipid hydroperoxides indicating that, despite the increased oxidative stress, the parasite membranes remain protected from substantial lipid oxidation. We have established BODIPY 581/591-PC as a useful probe of the spatial distribution of oxidative stress in P. falciparum-infected erythrocytes; however, the probe appears to be more sensitive to oxidative damage than endogenous lipids.

Association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.

The role of oxidative damage in the aetiology of coronary disease remains controversial, as clinical trials investigating the effect of antioxidants have not generally been positive. In the present study, 227 coronary cases, identified from a cohort study, were matched, by age and gender, with 420 controls in a nested case-control design. Stored plasma samples were analysed for F2-isoprostanes by stable isotope dilution MS, and specifically oxidized forms of apoA-I (apolipoprotein A-I) by HPLC of HDL (high-density lipoprotein). Median values of F2-isoprostanes were higher in plasma samples that contained oxidized apoA-I compared with samples with undetectable oxidized apoA-I (1542 compared with 1165 pmol/l). F2-Isoprostanes were significantly correlated with variants of non-oxidized apoA-II (r=-0.15) and were associated with HDL-cholesterol (P<0.0001). F2-Isoprostanes in cases (median, 1146 pmol/l) were not different from controls (1250 pmol/l); the odds ratio (95% confidence interval) for a 1 S.D. increase in F2-isoprostanes was 1.08 (0.91-1.29). Similarly, there was no independent association between the presence of oxidized apoA-I, detected in approx. 20% of the samples, and coronary risk. In conclusion, we found no evidence of associations between markers of lipid (F2-isoprostanes) and protein (oxidized apoA-I) oxidation and the risk of fatal or non-fatal coronary heart disease in a general population. This may be due to a true lack of association or insufficient power.

Inhibition of atherosclerosis by the serine palmitoyl transferase inhibitor myriocin is associated with reduced plasma glycosphingolipid concentration.

Glycosphingolipids (GSL) have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels in the absence of changes in cholesterol or triglyceride (TG) concentration and this leads to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE(-/-)) mice. The possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma GSL concentration has not been examined and was the primary aim of this investigation. We show that intraperitoneal delivery of the SPT inhibitor myriocin for 9 weeks inhibits atherosclerosis in apoE(-/-) mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentration of 42% and 25%, as assessed by enzymatic and HPLC methods, respectively. Moreover, SM and GSL concentrations were significantly correlated, indicating that SPT inhibition suppresses the synthesis of both these sphingolipids concomitantly. The inhibition of atherosclerosis induced by myriocin was not associated with changes in plasma cholesterol or TG concentrations or lipoprotein profiles as determined by FPLC. These data indicate that therapeutic reduction of plasma SM and/or GSL concentrations may offer a novel treatment for atherosclerosis.

Protective effect of vitamin E supplements on experimental atherosclerosis is modest and depends on preexisting vitamin E deficiency.

Vitamin E has failed to protect humans from cardiovascular disease outcome, yet its role in experimental atherosclerosis remains less clear. A previous study (Proc. Natl. Acad. Sci. USA 97:13830-13834; 2000) showed that vitamin E deficiency caused by disruption of the alpha-tocopherol transfer protein gene (Ttpa) is associated with a modest increase in atherosclerosis in apolipoprotein E gene deficient (Apoe(-/-)) mice. Here we confirm this finding and report that in Apoe(-/-)Ttpa(-/-) mice dietary alpha-tocopherol (alphaT) supplements restored circulating and aortic levels of alphaT, and decreased atherosclerosis in the aortic root to a level comparable to that seen in Apoe(-/-) mice. However, such dietary supplements did not decrease disease in Apoe(-/-) mice, whereas dietary supplements with a synthetic vitamin E analog (BO-653), either alone or in combination with alphaT, decreased atherosclerosis in Apoe(-/-) and in Apoe(-/-)Ttpa(-/-) mice. Differences in atherosclerosis were not associated with changes in the arterial concentrations of F(2)-isoprostanes and cholesterylester hydro(pero)xides, nor were they reflected in the resistance of plasma lipids to ex vivo oxidation. These results show that vitamin E at best has a modest effect on experimental atherosclerosis in hyperlipidemic mice, and only in situations of severe vitamin E deficiency and independent of lipid oxidation in the vessel wall.

Characterization of the oxidation products of BO-653 formed during peroxyl radical-mediated oxidation of human plasma.

4,6-Di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 muM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of alpha-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di-tert.-butyl-2,3,5,7a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.