Decrease and structural modifications of phosphatidylethanolamine plasmalogen in the brain with Alzheimer disease.

Several lipid modifications, some of which were attributed to oxidative stress, have been reported in the brains of patients with Alzheimer disease (AD). To evaluate this possibility, all phospholipids and their ether subclasses from the frontal cortex, hippocampus, and the white matter of AD brain were analyzed by high performance liquid chromatography and gas chromatography. The total phospholipid in the frontal cortex and hippocampus decreased on a DNA basis by about 20% and this change was essentially explained by a selective decrease in phosphatidylethanolamine and phosphatidylcholine. The lower content of phosphatidylethanolamine was due to a specific decrease in the plasmalogen subclass. Phosphatidylethanolamine plasmalogen was also the only lipid exhibiting major structural modifications: a significant decrease in polyunsaturated fatty acids and oleic acid as well as a shift of the aldehyde pattern from 18:1 to 18:0. The only modification observed in the other phospholipids was a decrease in oleic acid in diacyl-phosphatidylethanolamine and diacyl-phosphatidylcholine. None of these changes were observed in the white matter. Both the vinyl ether bond of phosphatidylethanolamine plasmalogen and polyunsaturated fatty acids are major targets in oxidative stress; thus, these specific lipid modifications strongly support the involvement of free radicals in the pathogenesis of AD.

Regulation of ubiquinone metabolism.

Interest in ubiquinone (UQ) has increased during recent years, mainly because of its antioxidant function and its use as a dietary supplement. However, our knowledge of the biosynthesis, catabolism, and regulation of this lipid in mammalian tissues is quite limited. UQ exhibits a high rate of turnover in all tissues indicating that cells possess efficient metabolic pathways for handling this compound and controlling its tissue levels. Besides reviewing the generally accepted metabolic pathway, alternative synthetic mechanisms are described. The lack of data concerning catabolism and regulation of this compound is emphasized. Reasons for the rather limited uptake of dietary UQ are discussed and alternative mechanisms for its beneficial effects on organ function are suggested. Since appropriate tissue uptake of dietary UQ probably only occurs in deficient states, the definition of partial UQ deficiency and its consequences is urgently needed. The possibility of raising tissue UQ levels by drug treatment or natural metabolites is raised as a choice of preference for the future.

The protective role of plasmalogens in iron-induced lipid peroxidation.

The role of plasmalogens in iron-induced lipid peroxidation was investigated in two liposomal systems. The first consisted of total brain phospholipids with and without plasmalogens, and the second of phosphatidylethanolamine/phosphatidylcholine liposomes with either diacyl- or alkenylacyl-phosphatidylethanolamine. By measuring thiobarbituric acid reactive substances, oxygen consumption, fatty acids and aldehydes, we show that plasmalogens effectively protect polyunsaturated fatty acids from oxidative damage, and that the vinyl ether function of plasmalogens is consumed simultaneously. Furthermore, the lack of lag phase, the increased antioxidant efficiency with time, and the experiments with lipid- and water-soluble azo compounds, indicate that plasmalogens probably interfere with the propagation rather than the initiation of lipid peroxidation, and that the antioxidative effect cannot be related to iron chelation.

Content and fatty acid composition of cardiolipin in the brain of patients with Alzheimer's disease.

The frontal, temporal and occipital cortex from human brains affected by Alzheimer's disease were analyzed for their contents and fatty acid compositions of cardiolipin. Phospholipids were purified using an HPLC system and cardiolipin was found to be present in the same amount (on a protein basis) as in age-matched controls. One-third of the total fatty acyl moieties of this phospholipid were saturated, one-third monounsaturated and one-third polyunsaturated. In affected brain regions the levels of certain polyunsaturated fatty acids displayed moderate decreases, not exceeding 10-15%. However, the total amount of polyunsaturated fatty acids decreased by only 9%. These results demonstrate that the amount and structure of brain cardiolipin are not modified to any great extent in connection with Alzheimer's disease.

Responses of insulin-like growth factor (IGF)-I and IGF-binding proteins to nutritional status in peroxisome proliferator-activated receptor-alpha knockout mice.

Peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in glucose and lipid homeostasis. Mice lacking PPARalpha(-/-) have a sexually dimorphic phenotype. We have characterized the IGF system in wild type and PPARalpha-/- mice. In normal mice fasting IGF-I and the IGFBP-3 ternary complex were 2-fold higher in males than in females. PPARalpha influenced the IGF/IGFBP response to feeding, particularly in males. Compared to wild type, male PPARalpha-/- mice had 40% lower total fasting IGF-I concentrations, decreased ALS and less IGFBP-3 ternary complex formation, but within 4 h of refeeding there was an increase in IGF-I and IGFBP-3 ternary complex to values similar to controls. Circulating IGFBP protease activity was induced in male PPARalpha-/- mice during refeeding. IGFBP-1 and insulin concentrations were higher in males than females, and were increased by PPARalpha knockout, suggesting significant hepatic insulin resistance. We speculate that gender differences in the IGF system contribute to the PPARalpha-/- phenotype.

Regulatory aspects of coenzyme Q metabolism.

A number of factors are involved in the regulation of the amount and distribution of coenzyme Q in cells and tissues. These factors modify preferentially the biosynthetic mechanism in order to keep up an optimal tissue concentration of the lipid. The amount of substrate provided by the mevalonate pathway is able to both up- and down-regulate the velocity of synthesis. At the translation level, regulation occurs by receptor-mediated ligand binding and appears most clearly upon treatment with hormones and peroxisomal inducers. There are a number of pathophysiological conditions when these mechanisms of regulation are modified and explain the decreased coenzyme Q tissue concentrations. It is of considerable interest to establish appropriate physiological, hormonal and drug-mediated conditions in order to counteract disturbed cellular functions caused by coenzyme Q deficiency

Influence of chronic fluorosis on membrane lipids in rat brain.

Brain membrane lipid in rats were analyzed after being fed either 30 or 100 ppm fluoride for 3, 5, and 7 months. The protein content of brain with fluorosis decreased, whereas the DNA content remained stable during the entire period of investigation. After 7 months of fluoride treatment, the total brain phospholipid content decreased by 10% and 20% in the 30 and 100 ppm fluoride groups, respectively. The main species of phospholipid influenced by fluorosis were phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. The fatty acid and aldehyde compositions of individual phospholipid classes were unchanged. No modifications could be detected in the amounts of cholesterol and dolichol. After 3 months of fluoride treatment, ubiquinone contents in brain were lower; however, at 7 months they were obviously increased in both groups of fluoride treatment. The results demonstrate that the contents of phospholipid and ubiquinone are modified in brains affected by chronic fluorosis and these changes of membrane lipids could be involved in the pathogenesis of this disease.

Induction of endogenous coenzyme Q biosynthesis by administration of peroxisomal inducers.

A large number of chemical compounds have been identified which cause peroxisomal proliferation and induce a number of enzymes, mainly those participating in lipid metabolism. Administration of these drugs/chemicals to rats increased coenzyme Q levels in the blood and most of the organs. Levels were raised in all cellular membranes of such organs. The extent of induction of this lipid was 8-fold in young animals but decreased during aging and was absent at 1.5 year of age. One of the regulating factors of the mevalonate pathway is farnesol, which is produced by dephosphorylation of farnesyl-PP and eliminated by phosphorylation including two kinases. Future research will involve a search for modified intermediary metabolites, which increase coenzyme Q synthesis and thereby efficiently elevate the level of this lipid in conditions of deficiency.

Direct instruction in math word problems: students with learning disabilities.

This investigation was conducted to test the effectiveness of strategy teaching and sequencing practice problems in teaching students with learning disabilities to identify the correct algorithm for solving addition and subtraction word problems. Sixty-two students were assigned to one of three experimental groups: strategy plus sequence, strategy only, and sequence only. The results indicated that students in the strategy-plus-sequence group, as well as those in the strategy-only group, scored significantly higher than did students in the sequence-only group. Findings indicated that strategy teaching was the more effective of the two instructional components. Implications are discussed in terms of instructional design for students with learning disabilities.

Hepatic lipase acylates dolichol in the presence of a plasma cofactor in vitro.

Phosphatidylethanolamine:dolichol acyltransferase (PEDAT), an enzyme previously partially purified and characterized in rat liver [Sindelar, P., Chojnacki, T., & Valtersson, C. (1992) J. Biol. Chem. 267, 20594-20599], is here purified to homogeneity from a heparin perfusate of rat liver and is shown to be identical to hepatic lipase. However, in contrast to triglyceride hydrolysis by hepatic lipase, the PEDAT activity is strongly dependent on a heat-stable plasma cofactor. This cofactor stimulates the activity up to 15-fold and shifts the pH optimum for the reaction from 8.5 to 7.5. Upon gel filtration on Bio-Gel A-1.5, the factor is heterogeneously distributed, with a major peak at 220 kDa. The dolichol-acylating activity can also be detected in rat adrenals and ovaries, and evidence is presented that the PEDAT assay shows a higher degree of specificity for hepatic lipase than the standard assay with triolein-gum arabic emulsion in 1 M NaCl.

A dolichol acyltransferase present in rat and human postheparin plasma.

Incubation of small unilamellar vesicles consisting of dioleoyl phosphatidylcholine-dioleoyl phosphatidylethanolamine (3:1) and 2 mol% [3H]dolichol-19 with postheparin plasma from rat resulted in the formation of dolichyl oleate. Normal plasma or heat-treated postheparin plasma contained no activity and, hence, the results indicate the presence of a cell surface associated dolichol acyltransferase that can be released into the blood by heparin. The reaction is strongly stimulated by phosphatidylethanolamine and Ca2+, whereas no stimulation with triglycerides or acyl-CoA was observed. Together with the fact that the only product formed was dolichyl oleate, these results strongly suggest that a transacylation mechanism from the phospholipids to dolichol is operative in the liposomes. Gel chromatography of postheparin plasma yielded a molecular mass of about 350 kilodaltons for the active enzyme and density gradient centrifugation indicated that this high molecular mass complex consists mainly of proteins. Finally, we conclude that this enzyme is not unique to the rat, but is also present in human postheparin plasma.

Phosphatidylethanolamine:dolichol acyltransferase. Characterization and partial purification of a novel rat liver enzyme.

Incubation of rat or human post-heparin plasma with [3H]dolichol incorporated in liposomes consisting of dioleoyl phosphatidylcholine:dioleoyl phosphatidylethanolamine (3:1) resulted in the formation of radioactive dolichyl oleate. Non-heparinized plasma did not esterify dolichol, and, hence, the enzyme involved is probably associated with the cell surface and released into the blood by heparin. The major location of this activity was the liver, and, therefore, a partial purification of the enzyme from heparinized rat liver perfusates was performed using DEAE-Sephacel and heparin-Sepharose chromatography. The dolichol acyltransferase activity copurified with hepatic lipase activity in a lipid-protein complex of 350 kDa. Optimal acylation is achieved at pH 7.5 in the presence of 5% plasma and 20 mM Ca2+. Esterification can only be obtained when dolichol is present in a phospholipid bilayer, and the reaction is strongly stimulated by unsaturated phosphatidylethanolamine or phosphatidylserine. Radiolabeling experiments demonstrated that the primary acyl donor is phosphatidylethanolamine from which the fatty acid is transferred exclusively from position 1. Neither cholesterol nor retinol are esterified by the enzyme, and the reaction is not stimulated by acyl-CoA. Both the extracellular localization and the mechanism of transacylation clearly distinguish this new enzyme from the acyl-CoA:dolichol acyltransferase described earlier in microsomes.

Role of apolipoprotein A-IV in hepatic lipase-catalyzed dolichol acylation and phospholipid hydrolysis.

Hepatic lipase catalyzes the hydrolysis of phospholipids and neutral glycerides as well as transacylation reactions between several of these lipids. We have previously reported that this enzyme also transacylates the sn-I fatty acid of phosphatidylethanolamine to dolichol and that this reaction requires a plasma cofactor. In this study, we have purified the cofactor from the lipoprotein-free fraction of human plasma and present evidence demonstrating that it is identical to apolipoprotein A-IV. The effect of apolipoprotein A-IV on hepatic lipase-catalyzed dolichol acylation and phospholipid hydrolysis was studied in model membranes and compared with the effects of apolipoprotein A-I and E. Apolipoprotein A-IV strongly stimulated dolichol acylation and phosphatidylethanolamine hydrolysis but partly inhibited phosphatidylcholine hydrolysis. Apolipoprotein A-I had only a minor influence on the various activities studied and could not replace apolipoprotein A-IV. Apolipoprotein E stimulated the hydrolysis of both phospholipids but had no effect on dolichol acylation. The effect of apolipoprotein A-IV on hepatic lipase activity was then studied with the gum arabic-stabilized triglyceride emulsion. The apolipoprotein neither stimulated nor inhibited triglyceride hydrolysis in the emulsion. Finally, human high-density lipoprotein-2 and very low-density lipoprotein were also used as substrates. Apolipoprotein A-IV strongly stimulated the hydrolysis of phosphatidylcholine and phosphatidylethanolamine in both lipoproteins, while the hydrolysis of triglycerides was completely inhibited. These results demonstrate that apolipoprotein A-IV is an important cofactor to hepatic lipase affecting both catalytic rates and the substrate specificity of the enzyme. We therefore suggest that apolipoprotein A-IV-rich high-density lipoprotein is the preferred substrate for hepatic lipase.

Separation and quantitation of phospholipids and their ether analogues by high-performance liquid chromatography.

The common mobile phase hexane/isopropanol/water used for separation of phospholipids on high-performance liquid chromatography silica columns poses several problems, such as incomplete separation and rapid column deterioration. By inclusion of 5 mM ammonium sulfate in the aqueous phase, we were able to substantially improve the chromatographic resolution and obtain complete separation of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, and sphingomyelin. In addition, ammonium sulfate prevented column degeneration and greatly improved reproducibility. A new quantitation method for alkenylacyl, alkylacyl, and diacyl forms of phospholipids was also developed based on derivatization with [(3)H]acetic anhydride. Separation and quantitation of the radioactive acetyl diradylglycerols were performed by straight-phase HPLC coupled to a radioactive flow detector and enabled detection of the various ether analogues at the picomole level with high reproducibility. The described methods are mild and nondestructive and can therefore be easily combined with analysis of either molecular species or fatty acid and aldehyde composition of the individual phospholipids.

Blood concentration of coenzyme Q(10) increases in rats when esterified forms are administered.

Coenzyme Q levels decrease during aging in most tissues and in the target organs of a number of diseases. The uptake of this lipid into the blood and other tissues was investigated in 6-wk-old male Sprague-Dawley rats after 3 wk of dietary supplementation. In addition to the natural form of coenzyme Q(10), acetylated and succinylated forms were also administered. Coenzyme Q(10) was taken up into the blood, but uptake was significantly greater in rats given the succinylated ( approximately 40%), and particularly, the acetylated forms ( approximately 70%). All three forms increased significantly the total coenzyme Q concentration in both the liver ( approximately 100%) and spleen ( approximately 130%). Coenzyme Q(10) and its esterified forms were not taken up into kidney, heart, muscle or brain. Intraportal and intraperitoneal administration of succinylated coenzyme Q(10) gave results similar to those obtained in the dietary experiments. Uptake of the dietary coenzyme Q(10) into the liver and spleen did not down-regulate the endogenous synthesis, i.e., the amounts of isolated coenzyme Q(9) did not change in these tissues. Thus, esterification of coenzyme Q increases the uptake of dietary lipid into the blood; however, the derivatization does not contribute to the elevation of coenzyme Q levels in various organs.

A new member of the family of di-iron carboxylate proteins. Coq7 (clk-1), a membrane-bound hydroxylase involved in ubiquinone biosynthesis.

Ubiquinone (UQ) is an essential cofactor for respiratory metabolism. In yeast, mutation of the COQ7 gene results in the absence of UQ biosynthesis and demonstrates a role for this gene in the step leading to the hydroxylation of 5-demethoxyubiquinone. Intriguingly, the disruption of the corresponding gene in Caenorhabditis elegans, clk-1, results in a prolonged life span and a slowing of development. Because of the pleiotropic effect of this disruption, the small size of the protein, and the lack of obvious homology to other known hydroxylases, it has been suggested that Coq7 may be a regulatory or structural component in UQ biosynthesis, rather than acting as the hydroxylase per se. Here we identify Coq7 as belonging to a family of a di-iron containing oxidases/hydroxylases based on a conserved sequence motif for the iron ligands, supporting a direct function of Coq7 as a hydroxylase. We have cloned COQ7 from Pseudomonas aeruginosa and Thiobacillus ferrooxidans and show that indeed this gene complements an Escherichia coli mutant that lacks an unrelated 5-demethoxyubiquinone hydroxylase. Based on the similarities to other well studied di-iron carboxylate proteins, we propose a structural model for Coq7 as an interfacial integral membrane protein.

Peroxisomal impairment in Niemann-Pick type C disease.

Niemann-Pick type C disease (NPC) belongs to the group of lysosomal storage diseases characterized by an accumulation of cholesterol and sphingomyelin. Using a mutant mouse strain, enzymatic markers for lysosomes, mitochondria, microsomes, and peroxisomes were investigated in the liver and brain. Aside from lysosomal changes, we found a sizable decrease of peroxisomal beta-oxidation of fatty acids and catalase activity in the brain and liver. Isolated peroxisomes displayed a significant decrease of these enzyme activities. Furthermore, the only phospholipid change in brain was a decreased content of the plasmalogen form of phosphatidylethanolamine, and the dimethylacetal pattern was also modified. The electron microscopical appearance of peroxisomes did not display any large changes. The defect of peroxisomal enzymes was already present 18 days before the onset of the disease. In contrast, the lysosomal marker enzyme increased in activity only 6 days after appearance of the symptoms. The events of the studied process have previously been considered to be elicited by a lysosomal deficiency, but this study demonstrates disturbances similar to those in a number of peroxisomal diseases. It appears that the peroxisomal impairment is an early event in the process and could be a factor in the development of Niemann-Pick type C disease.

Lipid composition in scrapie-infected mouse brain: prion infection increases the levels of dolichyl phosphate and ubiquinone.

The neutral and phospholipid composition of mouse brain infected with scrapie prions was investigated. During the later stages of this disease, the level of dolichol decreased by 30% whereas the level of dolichyl phosphate increased by 30%. In terminally ill mice, there was also a 2.5-fold increase in both total ubiquinone and its reduced form. Furthermore, alpha-tocopherol was elevated at this stage by 50%. In contrast, no changes were observed in phospholipid amount, in phospholipid composition, and in phosphatidylethanolamine plasmalogen content during the entire disease process. The fatty acid and aldehyde composition of individual phospholipids remained unaltered as well. No modifications could be detected in cholesterol content. Thus, the majority of membrane lipids in scrapie-infected mouse brain are modified in neither quantity nor structure, but specific changes occur to a few polyisoprenoid lipids. This specificity indicates that, although prions accumulate in lysosomes, the infection process is not associated with a general membrane destruction caused by lysosomal enzyme leakage.