Differences in erythrocyte phospholipid membrane long-chain polyunsaturated fatty acids and the prevalence of fatty acid desaturase genotype among African Americans and European Americans.

Numerous studies have reported an association between genetic variants in fatty acid desaturases (FADS1 and FADS2) and plasma or erythrocyte long chain polyunsaturated fatty acid (PUFA) levels. Increased levels of n-6 PUFAs have been associated with inflammation and several chronic diseases, including diabetes and cancer. We hypothesized that genetic variants of FADS that more efficiently convert precursor n-6 PUFA to arachidonic acid (AA) may explain the higher burden of chronic diseases observed in African Americans. To test this hypothesis, we measured the level of n-6 and n-3 PUFAs in erythrocyte membrane phospholipids and genotyped the rs174537 FADS variants associated with higher AA conversion among African American and European American populations. We included data from 1,733 individuals who participated in the Tennessee Colorectal Polyp Study, a large colonoscopy-based case-control study. Erythrocyte membrane PUFA percentages were measured using gas chromatography. Generalized linear models were used to estimate association of race and genotype on erythrocyte phospholipid membrane PUFA levels while controlling for self-reported dietary intake. We found that African Americans have higher levels of AA and a higher prevalence of GG allele compared to whites, 81% vs 43%, respectively. Homozygous GG genotype was negatively associated with precursor PUFAs (linoleic [LA], di-homo-γ-linolenic [DGLA]), positively associated with both product PUFA (AA, docosahexaenoic acid [DHA]), product to precursor ratio (AA to DGLA), an indirect measure of FADs efficiency and increased urinary isoprostane F2 (F2-IsoP) and isoprostane F3 (F3-IsoP), markers of oxidative stress. Increased consumption of n-6 PUFA and LA resulting in increased AA and subsequent inflammation may be fueling increased prevalence of chronic diseases especially in African descent.

Smoking and red blood cell phospholipid membrane fatty acids.

Smoking is associated with lower n-3 long chain polyunsaturated fatty acids (LCPUFA) concentrations; however, limited studies have accounted for dietary PUFA intake or whether tobacco dose or smoking duration influences this association. We measured red blood cell phospholipid (RBC) membrane concentrations of fatty acids in 126 current smokers, 311 former smokers, and 461 never smokers using gas liquid chromatography and tandem mass spectrometry. Smokers had lower RBC membrane percentages of total n-3 LCPUFAs compared to former smokers or never smokers (median percent: 5.46, [interquartile range (IQR) 4.52, 6.28] versus 6.39; [IQR: 5.18, 7.85] versus 6.59; [IQR 5.34, 8.01]) (p<0.001) and this association remained after adjusting for dietary PUFA intake. Duration of smoking and cigarettes per day were not associated with RBC membrane n-3 LCPUFA differences. Smoking is associated with lower n-3 LCPUFA RBC membrane percentages and this association was not influenced by diet or smoking dose or duration.

Free fatty acids are associated with metabolic syndrome and insulin resistance but not inflammation in systemic lupus erythematosus.

Free fatty acids (FFAs) are implicated in the pathogenesis of insulin resistance and atherosclerosis. Inflammatory cytokines promote lipolysis and increase FFAs, a cause of endothelial dysfunction and increased atherosclerosis risk. We hypothesized that increased inflammation is associated with increased FFAs, resulting in insulin resistance and atherosclerosis in patients with systemic lupus erythematosus (SLE). We measured clinical variables, serum FFAs, homeostasis model assessment for insulin resistance (HOMA), inflammatory cytokines, markers of endothelial activation, cholesterol concentrations and coronary artery calcium in 156 patients with SLE and 90 controls. We compared FFAs in patients with SLE and controls using Wilcoxon rank sum tests and further tested for the independent association between FFAs and disease status with adjustment for age, race and sex using multivariable regression models. We assessed the relationship between FFAs and continuous variables of interest using Spearman correlation and multivariable regression analysis. Levels of FFAs were higher in patients with SLE than controls (0.55 mmol/l (0.37-0.71) vs 0.44 mmol/l (0.32-0.60), P = 0.02). Levels of FFAs remained significantly higher among patients with SLE after adjustment for age, race and sex (P = 0.03) but not after further adjustment for body mass index (P = 0.13). FFA levels did not differ according to the usage of current immunosuppressive medications in univariate and adjusted analysis (all P > 0.05). Among patients with SLE, concentrations of FFAs were higher among those with metabolic syndrome compared to those without (0.66 mmol/l (0.46-0.81) vs 0.52 mmol/l (0.35-0.66), P < 0.001). FFAs were positively correlated with insulin resistance (HOMA) (rho = 0.23, P = 0.004, P adjusted = 0.006) and triglyceride levels (rho = 0.22, P = 0.01, P adjusted = 0.004). FFAs were not associated with inflammatory cytokines (IL-6, TNF-α) (all P > 0.05) but were positively associated with levels of E-selectin (rho = 0.33, P = < 0.001, P adjusted = 0.001) and ICAM-1 (rho = 0.35, P < 0.001, P adjusted = 0.001). FFAs were correlated with coronary artery calcium score (rho = 0.20, P = 0.01) but this was attenuated after adjustment for age, race and sex (P = 0.33). From our study we concluded that FFAs are elevated in patients with SLE, particularly those with metabolic syndrome. FFAs in patients with SLE are not associated with markers of generalized inflammation but are associated with insulin resistance and markers of endothelial activation.

A randomized, double-blind, negatively controlled pilot study to determine whether the use of emollients or calcipotriol alters the sensitivity of the skin to ultraviolet radiation during phototherapy with narrowband ultraviolet B.

BACKGROUND: There is contradictory evidence suggesting that emollients increase, decrease or have no effect on minimal erythema dose (MED) or minimal phototoxic dose values prior to phototherapy. Few studies have looked at the in vivo use of emollients or calcipotriol prior to narrowband ultraviolet (UV) B (NB-UVB) treatment. OBJECTIVES: To investigate whether emollients or calcipotriol alter MED readings of skin on the back of healthy subjects prior to NB-UVB irradiation. METHODS: Topical agents were applied to the backs of 20 healthy volunteers for 30 min prior to MED testing. These agents were aqueous cream, 50:50 white soft paraffin and liquid paraffin, Diprobase(®) (Schering-Plough, Welwyn Garden City, U.K.), Epaderm(®) (Medlock, Oldham, U.K.) and calcipotriol ointment and cream. A control MED strip was used with no topical agent applied prior to testing. MED readings were recoded as integer steps between 1 and 9 (one is lowest MED dose for skin type; eight is highest; nine is no response, i.e. a higher MED). RESULTS: The median MED was between step 5 and 6 for all treatments and control. There was no significant difference at the 5% level between control and each topical agent. The study was powered to detect a median difference of approximately 0·4-0·6 steps. CONCLUSIONS: This has important implications at a practical level when advising patients not to apply creams prior to treatment with NB-UVB. Studies where agents are applied immediately prior to phototherapy have been more likely to show that emollients block transmission of UV radiation. If they are applied at least 30 min prior to treatment, they have no effect.

The current and future role of the dietetic support worker.

BACKGROUND: The role of the dietetic support worker (DSW) was developed to provide cost-effective support to dietetic services in the National Health Service (NHS). However, there is little evidence about how the role is perceived or the impact of the introduction of Agenda for Change in 2004 (a guide to pay terms and conditions for NHS staff) on role definition. The present study aimed to gather evidence from DSWs and dietitians on the current role of the DSW to help inform the future development of the role. METHODS: A questionnaire survey was conducted on DSWs and dietitians in 10 trusts in the East of England. Issues included qualifications, experience, training, satisfaction with Agenda for Change, supervision and responsibility. Themes were further explored by semi-structured telephone interviews conducted on a subsample. RESULTS: Eighteen out of 24 DSWs and 62 out of 98 dietitians responded to the questionnaire. The role of the DSW is highly valued by dietitians. Over three-quarters of DSWs and over 90% of dietitians agree that the employment of DSWs improved the working lives of dietitians. Only 50% of DSWs were happy with their Agenda for Change banding, although this did not adversely affect their job satisfaction. Both groups saw the role of the DSW becoming more specialised, despite concerns about access to appropriate training and the lack of a structured career pathway. CONCLUSIONS: This study identified issues relating to the future development of the role of the DSW from the viewpoint of DSWs and dietetic assistants.

Barminomycin, a model for the development of new anthracyclines.

Barminomycin is a member of the anthracycline class of anticancer agents and was originally discovered as a pink/red complex with DNA and RNA and named SN-07. The chromophore was subsequently separated from the nucleic acids by nuclease digestion and contained the four-membered anthraquinone ring system characteristic of anthracyclines, but with an unusual eight membered ring that contained a carbinolamine which readily interconverted to an imine. The imine form is analogous to the formaldehyde-activated form of other anthracyclines such as doxorubicin. The imine form confers exceptional activity to barminomycin which is 1,000-fold more cytotoxic than doxorubicin. Barminomycin rapidly forms adducts with DNA, reacting with the exocyclic amino group of guanine residues and with high selectivity for 5'-GC-3' sequences. The coupling to DNA appears to be identical to the N-C-N aminal linkage formed between doxorubicin and DNA where the carbon derives from formaldehyde for doxorubicin-DNA adducts, whereas this "activated carbon" is an inherent component of the imine group in the eight membered ring of barminomycin. Although the linkage of both drugs to DNA appears to be identical, barminomycin-DNA complexes are essentially irreversible compared to the labile doxorubicin-DNA adducts which have an in vitro (purified DNA) half-life of 25 h at 37 degrees C. A 3D model of the barminomycin-DNA complex has been defined from 307 NOE distance constraints. The enhanced stability of barminomycin-DNA adducts appears to be due primarily to protection of the aminal linkage from hydrolysis and this has provided insight into the design of new anthracycline derivatives with enhanced stability and activity. Strategies for harnessing the extreme reactivity and activity of barminomycin are also presented.

Effect of water hardness on cardiovascular mortality: an ecological time series approach.

BACKGROUND: Numerous studies have suggested an inverse relationship between drinking water hardness and cardiovascular disease. However, the weight of evidence is insufficient for the WHO to implement a health-based guideline for water hardness. This study followed WHO recommendations to assess the feasibility of using ecological time series data from areas exposed to step changes in water hardness to investigate this issue. METHOD: Monthly time series of cardiovascular mortality data, subdivided by age and sex, were systematically collected from areas reported to have undergone step changes in water hardness, calcium and magnesium in England and Wales between 1981 and 2005. Time series methods were used to investigate the effect of water hardness changes on mortality. RESULTS: No evidence was found of an association between step changes in drinking water hardness or drinking water calcium and cardiovascular mortality. The lack of areas with large populations and a reasonable change in magnesium levels precludes a definitive conclusion about the impact of this cation. We use our results on the variability of the series to consider the data requirements (size of population, time of water hardness change) for such a study to have sufficient power. Only data from areas with large populations (>500,000) are likely to be able to detect a change of the size suggested by previous studies (rate ratio of 1.06). CONCLUSION: Ecological time series studies of populations exposed to changes in drinking water hardness may not be able to provide conclusive evidence on the links between water hardness and cardiovascular mortality unless very large populations are studied. Investigations of individuals may be more informative.

Recent advances in understanding and exploiting the activation of anthracyclines by formaldehyde.

The anthracycline group of compounds are amongst the most effective chemotherapy agents currently in use for cancer treatment. They are generally classified as topoisomerase II inhibitors but also have a variety of other targets in cells. It has been known for some years that the anthracyclines are capable of forming DNA adducts, but the relevance and extent of these DNA adducts in cells and their role in causing cell death has remained obscure. When the adduct structure was solved, it became clear that formaldehyde was an absolute requirement for adduct formation. This led to a renewed interest in the capacity of anthracyclines to form DNA adducts, and there are now several ways in which adduct formation can be facilitated in cells. These involve strategies to provide the requisite formaldehyde in the form of anthracycline-formaldehyde conjugates, and the use of formaldehyde-releasing drugs in combination with anthracyclines. Of particular interest is the new therapeutic compound AN-9 that releases both butyric acid and formaldehyde, leading to efficient anthracycline-DNA adduct formation, and synergy between the two compounds. Targeted formation of adducts using anthracycline-formaldehyde conjugates tethered to cell surface targeted molecules is now also possible. Some of the cellular consequences of these adducts have now been studied, and it appears that their formation can overcome anthracycline-resistance mechanisms, and that they are more efficient at inducing apoptosis than when functioning primarily through impairment of topoisomerase II. The clinical application of the use of anthracyclines as DNA adduct forming agents is now being explored.

A national evaluation of school breakfast clubs: evidence from a cluster randomized controlled trial and an observational analysis.

STUDY OBJECTIVE: To measure the health, educational and social impacts of breakfast club provision in schools serving deprived areas across England. DESIGN: A cluster randomized controlled trial and an observational analysis. SETTING: England, the UK. INTERVENTION: funding to establish a school-based breakfast club vs. control (no funding). MAIN RESULTS: Intention to treat analysis showed improved concentration (Trail Making Test Part A) amongst the intervention group at 3 months. Fewer pupils within the intervention group reported having skipped classes within the last month and fewer pupils within the intervention group reported having skipped 1 or more days of school within the last month at 1 year. Observational analysis at 1 year showed a higher proportion of primary-aged breakfast club attendees reported eating fruit for breakfast in comparison to non-attendees. A higher proportion of breakfast club attendees had borderline or abnormal conduct and total difficulties scores (primary-aged pupils) and prosocial score (secondary-aged pupils). CONCLUSIONS: Analyses revealed a mixed picture of benefit and apparent disbenefit. This study illustrated the challenges of evaluating a complex intervention in which the evaluators had less control than is usual in randomized trials over recruitment, eligibility checking and implementation. If the impact of new policy initiatives is to be assessed using the most robust forms of evaluation, social policy needs to be organized so that evaluations can be constructed as experiments. This is likely to prove most difficult where the perceived value of implementing an intervention rapidly is high.

Shared patients: multiple health and social care contact.

The paper describes results from the 'Tracking Project', a new method for examining agency overlap, repeat service use and shared clients/patients amongst social and health care agencies in the community. This is the first project in this country to combine total population databases from a range of social, health care and criminal justice agencies to give a multidisciplinary database for one county (n = 97,162 cases), through standardised anonymisation of agency databases, using SOUNDEX, a software programme. A range of 20 community social and health care agencies were shown to have a large overlap with each other in a two-year period, indicating high proportions of shared patients/clients. Accident and Emergency is used as an example of major overlap: 16.2% (n = 39,992) of persons who attended a community agency had attended Accident and Emergency as compared to 8.2% (n = 775,000) of the total population of the county. Of these, 96% who had attended seven or more different community agencies had also attended Accident and Emergency. Further statistical analysis of Accident and Emergency attendance as a characteristic of community agency populations (n = 39,992) revealed that increasing frequency of attendance at Accident and Emergency was very strongly associated with increasing use of other services. That is, the patients that repeatedly attend Accident and Emergency are much more likely to attend more other agencies, indicating the possibility that they share more problematic or difficult patients. Research questions arising from these data are discussed and future research methods suggested in order to derive predictors from the database and develop screening instruments to identify multiple agency attenders for targeting or multidisciplinary working. It is suggested that Accident and Emergency attendance might serve as an important predictor of multiple agency attendance.

Interactions between apolipoprotein E gene and dietary alpha-tocopherol influence cerebral oxidative damage in aged mice.

Cerebral oxidative damage is a feature of aging and is increased in a number of neurodegenerative diseases. We pursued the gene-environment interaction of lack of apolipoprotein E (apoE) and modulation of dietary alpha-tocopherol on cerebral oxidative damage in aged male and female mice by quantifying the major isomers of cerebral isoprostanes, derived from arachidonic acid (AA) oxidation, and neuroprostanes, derived from docosahexaenoic acid (DHA) oxidation. Mice fed alpha-tocopherol-deficient, normal, or -supplemented diet had undetectable, 4486 +/- 215, or 6406 +/- 254 ng of alpha-tocopherol per gram of brain tissue (p < 0.0001), respectively. Two factors, male gender and lack of apoE, combined to increase cerebral AA oxidation by 28%, whereas three factors, male gender, lack of apoE, and deficiency in alpha-tocopherol, combined to increase cerebral DHA oxidation by 81%. alpha-Tocopherol supplementation decreased cerebral isoprostanes but not neuroprostanes and enhanced DHA, but not AA, endoperoxide reduction in vivo and in vitro. These results demonstrated that the interaction of gender, inherited susceptibilities, and dietary alpha-tocopherol contributed differently to oxidative damage to cerebral AA and DHA in aged mice.

Clinical concentrations of doxorubicin inhibit activity of myocardial membrane-associated, calcium-independent phospholipase A(2).

Use of the anticancer antibiotic doxorubicin continues to be limited by its cumulative dose-related cardiotoxicity. Our study reports inhibition of myocardial intracellular calcium-independent phospholipase A(2) (iPLA(2)) activity by clinically relevant concentrations of the drug. The effect was first shown in vitro using suspensions of freshly isolated rat and rabbit cardiomyocytes. Addition of 0.1-10 microM doxorubicin to these cells led to a concentration- and time-dependent inhibition of total iPLA(2), as measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence or absence of calcium. Subcellular fractionation into cytosolic and membrane fraction revealed that the drug selectively inhibits membrane-associated iPLA(2) activity, without altering activity of the cytosolic enzyme. Doxorubicin treatment of cells prelabeled with [H(3)]arachidonic acid led to a depression of baseline arachidonic acid release levels, corroborating iPLA(2) inhibition. Reducing agents blocked PLA(2) inhibition in cardiomyocyte suspensions, suggesting that the doxorubicin effect is mediated by oxidation of susceptible cysteines. In vivo experiments, in which adults rats were i.v. injected with a bolus dose of 4 mg/kg doxorubicin, confirmed in vitro findings, revealing a 2-fold decrease in membrane-associated Ca(2+)-independent iPLA(2) activity in the heart tissue of treated animals. The observed phenomenon has important implications for myocyte signaling cascades and membrane remodeling.

A recycling pathway for resecretion of internalized apolipoprotein E in liver cells.

We have investigated the recycling of apoE in livers of apoE(-)/- mice transplanted with wild type bone marrow (apoE(+/+) --> apoE(-)/-), a model in which circulating apoE is derived exclusively from macrophages. Nascent Golgi lipoproteins were recovered from livers of apoE(+/+) --> apoE(-)/- mice 8 weeks after transplantation. ApoE was identified with nascent d < 1.006 and with d 1.006-1.210 g/ml lipoproteins at a level approximately 6% that of nascent lipoproteins from C57BL/6 mice. Hepatocytes from apoE(+/+) --> apoE(-)/- mice were isolated and cultured in media free of exogenous apoE. ApoE was found in the media primarily on the d < 1.006 g/ml fraction, indicating a resecretion of internalized apoprotein. Secretion of apoE from C57BL/6 hepatocytes was consistent with constitutive production, whereas the majority of apoE secreted from apoE(+/+) --> apoE(-)/- hepatocytes was recovered in the last 24 h of culture. This suggests that release may be triggered by accumulation of an acceptor, such as very low density lipoproteins, in the media. In agreement with the in vivo data, total recovery of apoE from apoE(+/+) --> apoE(-)/- hepatocytes was approximately 6% that of the apoE recovered from C57BL/6 hepatocytes. Since plasma apoE levels in the transplanted mice are approximately 10% of control levels, the findings indicate that up to 60% of the internalized apoE may be reutilized under physiologic conditions. These studies provide definitive evidence for the sparing of apoE and its routing through the secretory pathway and demonstrate that internalized apoE can be resecreted in a quantitatively significant fashion.

Localized injury in cardiomyocyte network: a new experimental model of ischemia-reperfusion arrhythmias.

We developed a new experimental approach to study the effects of local injury in a multicellular preparation and tested the ability of the method to induce reperfusion arrhythmias in cardiomyocyte monolayers. A small region of injury was created using geometrically defined flows of control and ischemia-like solutions. Calcium transients were acquired simultaneously from injured, control, and border zone cells using fluo 4. Superfusion with the injury solution rapidly diminished the amplitude of calcium transients within the injury zone, followed by cessation of cell beating. Reperfusion caused an immediate tachyarrhythmic response in approximately 17% of experiments, with a wave front propagating from a single cell or small cell cluster within the former injury zone. Inclusion of a gap junction uncoupler (1 mM heptanol) in the injury solution narrowed the functional border and sharply increased the number of ectopic foci and the incidence of reperfusion arrhythmias. The model holds a potential to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in the normal and diseased myocardium and to serve as a new tool to test antiarrhythmic protocols in vitro.

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus.

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.

Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages.

During atherogenesis, circulating macrophages migrate into the subendothelial space, internalize cholesterol-rich lipoproteins, and become foam cells by progressively accumulating cholesterol esters. The inhibition of macrophage acyl coenzyme A:cholesterol acyltransferase (ACAT), which catalyzes the formation of cholesterol esters, has been proposed as a strategy to reduce foam cell formation and to treat atherosclerosis. We show here, however, that hypercholesterolemic LDL receptor-deficient (LDLR(-/-)) mice reconstituted with ACAT1-deficient macrophages unexpectedly develop larger atherosclerotic lesions than control LDLR(-/-) mice. The ACAT1-deficient lesions have reduced macrophage immunostaining and more free cholesterol than control lesions. Our findings suggest that selective inhibition of ACAT1 in lesion macrophages in the setting of hyperlipidemia can lead to the accumulation of free cholesterol in the artery wall, and that this promotes, rather than inhibits, lesion development.

Brain regional quantification of F-ring and D-/E-ring isoprostanes and neuroprostanes in Alzheimer's disease.

Isoprostanes (IsoP) are produced exclusively from free radical damage to arachidonic acid, a fatty acid that is evenly distributed throughout white matter and gray matter, whereas neuroprostanes (NPs) are generated analogously from docosahexaenoic acid (DHA), a fatty acid enriched in gray matter where it is concentrated in neurons. IsoP and NPs derive from endoperoxide intermediates that isomerize to D/E-ring forms or that are reduced to F-ring compounds. We quantified F-ring and D/E-ring IsoP and NPs in temporal and parietal cortex, hippocampus, and cerebellum of nine definite Alzheimer's disease (AD) patients and 11 age-matched controls. Total NP levels (F-ring plus D/E-ring), but not total IsoP, were significantly greater in AD than controls (P: < 0.0001); only cerebral regions in AD patients had NPs greater than controls (P: < 0.05). The F-ring to D/E-ring ratio for NPs, but not IsoP, was 40 to 70% lower in all brain regions of AD patients compared to controls (P: < 0.005). These data extend results from in situ techniques, that have localized reactive products of lipid peroxidation primarily to neurons, by quantifying significantly greater free radical damage to the DHA-containing compartments in cerebrum in AD patients than controls, and suggest that one mechanism of increased oxidative stress may be diminished reducing capacity in DHA-containing compartments.

Oxidant-induced inhibition of myocardial calcium-independent phospholipase A2.

We discovered the acute inhibition of myocardial phospholipase A2 activity by micromolar concentrations of tert-butyl hydroperoxide and hydrogen peroxide. Specifically, freshly isolated adult rat cardiomyocytes were treated with the oxidants for 30 min, and phospholipase A2 activity was assessed in cell subcellular fractions using (16:0, [3H]18:1) plasmenylcholine and phosphatidylcholine substrates in the absence or presence of calcium. Calcium-independent phospholipase A2 activity was inhibited by approx 50% in both the cytosolic and membrane fractions by the oxidants. The inhibition of the phospholipase A2 activity was concentration dependent and preceded detectable changes in cell viability as assessed by lactate dehydrogenase release and rod-shaped morphology. Taking into account that oxidized sn-2 fatty acyl groups must first be hydrolyzed by phospholipase A2 to be repaired by glutathione peroxidase, we suggest that the observed inhibition of phospholipase A2 activity by oxidants compromises the myocyte's ability to deal with lipid peroxidation. This conclusion was further validated by the experiments in which pretreatment with the calcium-independent phospholipase A2 inhibitor bromoenol lactone exacerbated cardiotoxicity of tert-butyl hydroperoxide in myocyte cultures.

Human, but not bovine, oxidized cerebral spinal fluid lipoproteins disrupt neuronal microtubules.

Cerebral spinal fluid (CSF) lipoproteins have become a focus of research since the observation that inheritance of particular alleles of the apolipoprotein E gene affects the risk of Alzheimer's disease (AD). There is evidence of increased lipid peroxidation in CSF lipoproteins from patients with AD, but the biological significance of this observation is not known. A characteristic of the AD brain is a disturbance of the neuronal microtubule organization. We have shown previously that 4-hydroxy-2(E)-nonenal, a major product of lipid peroxidation, causes disruption of neuronal microtubules and therefore tested whether oxidized CSF lipoproteins had the same effect. We exposed Neuro 2A cells to human CSF lipoproteins and analyzed the microtubule organization by immunofluorescence. In vitro oxidized human CSF lipoproteins caused disruption of the microtubule network, while their native (nonoxidized) counterparts did not. Microtubule disruption was observed after short exposures (1 h) and lipoprotein concentrations were present in CSF (20 microg/mL), conditions that did not result in loss of cell viability. Importantly, adult bovine CSF lipoproteins, oxidized under identical conditions, had no effect on the microtubule organization of Neuro 2A cells. Comparison of human and bovine CSF lipoproteins revealed similar oxidation-induced modifications of apolipoproteins E and A-I as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Fatty acid analysis revealed substantially lower amounts of unsaturated fatty acids in bovine CSF lipoproteins, when compared to their human counterparts. Our data therefore indicate that oxidized human CSF lipoproteins are detrimental to neuronal microtubules. This effect is species-specific, since equally oxidized bovine CSF lipoproteins left the neuronal microtubule organization unchanged.