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.

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.

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.

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.

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.

Cerebrospinal fluid lipoproteins in Alzheimer's disease.

Interest in cerebrospinal fluid (CSF) lipoproteins has been stimulated by the association of certain alleles of the human apolipoprotein E gene (APOE) with an increased risk of Alzheimer's disease (AD), and because apolipoprotein E (apoE) is one of the major apolipoproteins in CSF. CSF lipoproteins (d < 1.210 g/ml fraction) are distinct from their plasma counterparts, and in AD patients CSF may contain novel particles. The protein concentration of CSF lipoproteins is reduced in AD patients. Moreover, the molecular distribution of apoE- and apoAII-containing apolipoproteins in CSF is dictated by APOE. The lipid composition suggests that CSF lipoproteins from AD patients may have undergone increased free radical-mediated damage; experimental data support the possibility that this may occur both before and after lipoprotein assembly. Finally, human CSF lipoproteins oxidized ex vivo are neurotoxic to neuronal cells in culture and disrupt microtubule structure, an activity not observed with oxidized bovine CSF lipoproteins. CSF lipoproteins may represent a means whereby apoE influences the outcome of free radical-mediated damage to brain.

The cell biology and physiologic relevance of ApoE recycling.

Apolipoprotein E (apoE) is unique among the different apoproteins because of its many functions in the assembly, processing, and removal of plasma lipoproteins. Some of these functions require the presence of apoE inside the cell or in close association with its outer membrane. For example, the presence of apoE increases triglyceride content in newly formed very low-density lipoprotein (VLDL), whereas the fast removal of chylomicrons requires the presence of a cellular pool of apoE ready to surface and bind to membrane proteoglycans and possibly to lipoprotein receptors such as the LDL receptor-related protein (LRP). Our recent discovery that VLDL-apoE internalized by hepatocytes is partially protected from lysosomal degradation and recycles through the Golgi apparatus suggests that the biologic cycle of apoE may not be concluded when the lipoprotein is taken up by the cell, and that recycling apoE may have a physiologic role in lipoprotein assembly, remnant removal, and cholesterol efflux.

Increased cerebral cortical lipid peroxidation and abnormal phospholipids in aged homozygous apoE-deficient C57BL/6J mice.

Aged homozygous apolipoprotein E gene-deficient (apoE -/-) mice have been proposed as an experimental model for the role of human apoE isoforms in Alzheimer's disease (AD). However, results from different laboratories have been in conflict regarding the presence or absence of neurodegeneration in these mice. Moreover, despite apoE being the major lipid trafficking molecule in the central nervous system, there has been no investigation of brain lipid levels in apoE -/- mice. Here we have examined male and female apoE -/- and control mice aged 10 to 12 months, testing the hypothesis that lack of apoE leads to some of the neuropathological changes seen in AD. Our results failed to demonstrate significant neurodegeneration, histopathological changes, or reduction in cerebral cortical synaptophysin in apoE -/- mice. However, we did observe a significant reduction in cerebral cortical phospholipids and their constituent fatty acids, as well as elevated lipid peroxidation products, in apoE -/- mice compared to apoE +/+ mice with the same genetic background. Our results suggest that the brains of aged apoE -/- mice display some of the lipid abnormalities associated with AD; however, these changes alone, at the magnitudes achieved in the apoE -/- mice, do not directly lead to the major neurodegenerative changes of AD.

Determination of the lower threshold of apolipoprotein E resulting in remnant lipoprotein clearance.

Apolipoprotein E (apoE) is the ligand for receptor-mediated clearance of remnant lipoproteins. ApoE at concentrations only 10% of normal, achieved through transplantation of wild-type marrow into apoE(-/-) mice, is sufficient for the maintenance of normal serum lipid and lipoprotein levels. The goal of the present study was to identify the minimal concentration of serum apoE still affecting cholesterol levels, and to determine whether any effects on remnant clearance below this level of apoE were detectable. ApoE(+/+) marrow was mixed with apoE(-/-) marrow in proportions of 1, 5, 10, and 25% to make chimeric mice with serum levels of apoE ranging from 0.005 to 0.46 mg/dl. Analysis of serum cholesterol and apoE levels demonstrated a positive correlation between apoE levels and cholesterol reduction (r = 0.83), with levels of 0.04 mg/dl representing the functional threshold level. There were no differences in lipoprotein profiles and clearance between apoE(-/-) mice and mice with serum apoE of less than 0.04 mg/dl, as assessed by FPLC, non-denaturing gel electrophoresis, and turnover studies. However, electron microscopy of negative stains showed fewer lipoprotein particles with a diameter of <30 nm in the serum of these mice compared to apoE(-/-) mice. These data demonstrate that the threshold of serum apoE resulting in cholesterol reduction is 0. 04 mg/dl, and indicate that apoE below this level affects lipoprotein size distribution possibly by accelerating the clearance of smaller remnants.

Recycling of apolipoprotein E in mouse liver.

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.

Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer's disease and are neurotoxic when oxidized ex vivo.

Brain regional oxidative damage is thought to be a central mechanism in the pathogenesis of Alzheimer's disease (AD). Recent studies of cerebrospinal fluid (CSF) have suggested that increased lipid peroxidation of CSF and CSF lipoproteins also may occur in AD patients. In the present study, we determined the susceptibility of human CSF to ex vivo lipid peroxidation and tested the hypothesis that oxidized CSF lipoproteins may be neurotoxic. Whole CSF or a CSF lipoprotein fraction (d < 1.210 g/mL) was oxidized with 2,2'-azobis(2-amidino-propane)dihydrochloride (AAPH), a hydrophilic free-radical generator. Kinetics of CSF lipid peroxidation were followed by a standard fluorescence product accumulation assay. Oxidation of AD CSF yielded significantly shorter fluorescent lag times than controls, indicating reduced antioxidant capacity. Electrophoretic mobilities of CSF apolipoproteins were specifically reduced upon oxidation of CSF with AAPH, suggesting that lipoproteins are primary targets of CSF lipid peroxidation. Cultured neuronal cells were exposed to physiological concentrations of isolated CSF lipoproteins oxidized with increasing concentrations of AAPH; the resulting neurotoxicity showed a significant linear AAPH concentration-response relationship. These results suggest that oxidized CSF lipoproteins may contribute to the pathogenesis of neurodegeneration in AD.

Apolipoprotein E allelic influence on human cerebrospinal fluid apolipoproteins.

The major apolipoproteins (apo) on human cerebrospinal fluid lipoproteins are apoA-I and apoE. Given the association between inheritance of the varepsilon4 allele of the apoE gene (APOE4) and increased susceptibility to Alzheimer's disease, we tested the hypothesis that cerebrospinal fluid apolipoproteins may be influenced by APOE genotype and Alzheimer's disease. Lipoprotein fractions (d < 1.210 g/ml) were isolated from cerebrospinal fluid obtained from individuals with different APOE genotypes and with or without pathologically verified Alzheimer's disease. Apolipoproteins were separated by SDS-polyacrylamide gel electrophoresis and identified by silver nitrate staining, Western blotting, and N-terminal amino acid sequencing. Four protein species were detected by silver nitrate staining in subjects with an APOE3 allele: apoA-I, apoE monomer, apoE-apoA-II heterodimer, and apoE homodimer. In APOE4 homozygotes, only apoA-I and apoE monomer were detected. ApoA-II homodimer was demonstrated in all subjects by Western blotting. The relative levels of apoE- and apoA-II-containing apolipoproteins correlated with APOE genotype but were not altered by Alzheimer's disease. In contrast to apoE, no apoA-II immunoreactivity was observed with pathological structures in Alzheimer's disease brain. These differences in cerebrospinal fluid apolipoproteins may influence lipoprotein trafficking and may be an element in the stratification of risk for Alzheimer's disease with APOE genotype.

Comparison of formation of D2/E2-isoprostanes and F2-isoprostanes in vitro and in vivo--effects of oxygen tension and glutathione.

The isoprostanes (IsoPs) are bioactive prostaglandin-like compounds derived from the free-radical-catalyzed peroxidation of arachidonic acid in vitro and in vivo. IsoPs possessing either an F-type prostane ring (F2-IsoPs) or D/E-type prostane rings (D2/E2-IsoPs) are formed depending on whether IsoP endoperoxide intermediates undergo reduction or isomerization, respectively. Little, however, is known regarding factors influencing the formation of various classes of IsoPs, particularly D2/E2-IsoPs. Thus, studies were undertaken to examine the formation of D2/E2-IsoPs in relation to F2-Isops both in vitro and in vivo. In peroxidizing rat liver microsomes, the formation of D2/E2-IsoPs increased in a time- and oxygen-dependent manner and correlated with F2-IsoP generation and loss of precursor arachidonic acid, although the absolute amount of D2/E2-IsoPs formed exceeded by over 5-fold the levels of F2-IsoPs formed. Surprisingly, however, in liver tissue from rats exposed to an oxidant stress, levels of F2-IsoPs were up to 10-fold greater than those of D2/E2-IsoPs, suggesting that an endogenous process causes IsoP endoperoxide reduction in vivo. Addition of glutathione (GSH) to peroxidizing microsomes at concentrations from 0.01 to 5 mM increased the formation of F2-IsoPs at the expense of D2/E2-IsoPs. Boiling of microsomes did not alter the effect of GSH. Formation of D2/E2-IsoPs in liver tissue in vivo was greatly enhanced compared to F2-IsoPs in rats depleted of GSH. Thus, GSH modulates the formation of different classes of IsoPs in vitro and in vivo. Other thiols, including beta-mercaptoethanol, dithiothreitol, and cysteine, were able to substitute for GSH. These studies indicate that GSH promotes F2-IsoP formation and diminishes D2/E2-IsoP levels in vitro and in vivo by causing reduction of IsoP endoperoxides.

Central nervous system lipoproteins in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia in the United States and has been associated with APOE genotype. Apolipoprotein (apo) E along with apoAI serve as the major apolipoproteins in the central nervous system; however, we are unaware of any study addressing lipoprotein metabolism in AD. We tested the hypothesis that lipoprotein metabolism is altered in patients with AD by isolating and characterizing ventricular fluid (VF) lipoproteins obtained during a rapid autopsy protocol from patients with AD and age-matched nondemented control patients. Our results demonstrated abnormalities in the protein and lipid constituents of VF lipoproteins from AD patients. Apolipoprotein concentration was reduced by half in AD patients relative to controls; however, there was no selective reduction in apoE or apoAI. In addition, cholesteryl ester fatty acids, but not phospholipid fatty acids, from AD patients demonstrated a significant reduction in some polyunsaturated fatty acids (18:2 and 22:6) and an enrichment in 18:0. None of these changes were directly related to APOE genotype. Our data indicate that VF lipoprotein composition is altered, at least terminally, in AD patients, and that these changes are not associated with APOE. These findings suggest that altered VF lipoprotein metabolism may be a component of AD pathogenesis.

Discovery of 5R-lipoxygenase activity in oocytes of the surf clam, Spisula solidissima.

Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE) are reported to induce reinitiation of meiosis in oocytes of the surf clam Spisula sachalinensis from the Sea of Japan (Varaksin et al., Comp. Biochem. Physiol. 101C, 627-630 (1992). As the Atlantic surf clam Spisula solidissima is a commonly used model for the study of meiosis reinitiation, we examined these cells for the possible occurrence of lipoxygenases and for the bioactivity of the products. Incubation of [14C]arachidonic acid with homogenates of S. solidissima oocytes led to the formation of two major metabolites: 5R-HETE, a novel lipoxygenase product, and 8R-HETE. The products were identified by HPLC, uv spectroscopy, and GC-MS. The corresponding hydroperoxy fatty acids, the primary lipoxygenase products, were isolated from incubations of ammonium sulfate fractionated oocyte cytosol. Arachidonic and eicosapentaenoic acids were identified as constituents of S. solidissima oocyte lipids and the free acids were equally good lipoxygenase substrates. We examined the activity of C18 and C20 polyunsaturated fatty acids and their lipoxygenase products on meiosis reinitiation in Spisula solidissima oocytes, using serotonin and ionophore A23187 as positive controls. The fatty acids and their derivatives were inactive. We conclude that in the surf clam, (as in starfish), there are responding and non-responding species in regard to the maturation-inducing activity of the oocyte lipoxygenase products, and that the lipoxygenase has another, as yet uncharacterized, function in oocyte physiology.