A unique pool of free arachidonate serves as substrate for both cyclooxygenase and lipoxygenase in platelets

Stimulation of platelets induces a rapid release of arachidonate from specific phospholipids and subsequent remodeling of arachidonate-containing phospholipids. This process is accompanied by transformation of released arachidonate by cyclooxygenase and lipoxygenase enzymes. We addressed the question of whether the cyclooxygenase and the lipoxygenase products originated from the same arachidonate-containing phospholipids. [¹⁴C]Arachidonate prelabeled platelets were stimulated by thrombin or by ionophore A 23187. We monitored the cyclooxygenase pathway by following 12-hydroxy-5,8,10-heptadecatrienoic acid [12(S)-HHT] formation and the lipoxygenase pathway by following 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] formation and compared specific activities. The data showed that the same pool of released arachidonate can be utilized by either cyclooxygenase or by lipoxygenase. Indeed, the specific activity of both products was identical when both enzymes were acting. Since cyclooxygenase was rapidly deactivated while lipoxygenase continued to be active, the specific activity of 12(S)-HETE became lower than the specific activity of 12(S)-HHT when large amounts of 12(S)-HETE were synthesized. Based on comparison of specific activity between phospholipids and oxygenated products, the pools of arachidonate-containing phospholipids involved in the synthesis of oxygenated products are dependent on the amount of arachidonate released. Based on a paper presented at the Third International Conference of Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

1-O-alkyl-linked phosphoglycerides of human platelets: Distribution of arachidonate and other acyl residues in the ether-linked and diacyl species

In this study, the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine content of human platelets was determined. The distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alk-l′-enyl-2-acyl classes of choline- and ethanolamine-containing phosphoglycerides was also assessed. The major platelet phospholipids were choline-containing phosphoglycerides (38%), ethanolamine-containing phosphoglycerides (25%) and sphingomyelin (18%), with smaller amounts of phosphatidylserine (11%) and phosphatidylinositol (4%). In addition to the diacyl class, the choline-linked fraction was found to contain both 1-O-alkyl-2-acyl (10%) and 1-O-alk-l′-enyl-2-acyl (9%) species. The ethanolamine-linked fraction, on the other hand, had an elevated level of the 1-O-alk-l′-enyl-2-acyl (60%) species and a small amount of the 1-O-alkyl-2-acyl component (4%). The major fatty acyl residues found in all classes of the choline and ethanolamine phospholipids were 16∶0, 18∶0, (Δ⁹), 18∶2(n−6) and 20∶4(n−6). The 1-O-alk-l and 1-O-alk-l′-enyl fraction of the ethanolamine-linked phospholipids also contained substantial amounts of 22∶4(n−6), 22∶5(n−3) and 22∶6(n−3) acyl chains. Arachidonate comprised 44% of the acyl residues in thesn-2 position of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. Corresponding values for the diacyl and 1-O-alk-l′-enyl-2-acyl species were 23% and 25%, respectively, based on all 20∶4(n−6) being linked to thesn-2 position of all classes. In the ethanolamine-linked phosphoglycerides, arachidonate constituted 60%, 20% and 68% of the acyl groups in thesn-2 position of the 1,2-diacyl, 1-O-alkyl-2-acyl and 1-O-alk-l′-enyl-2-acyl classes, respectively. The content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine appears sufficient to support the synthesis of platelet activating factor by a deacylation-reacylation pathway in platelets. Our findings also demonstrate that human platelets contain a significant amount of 1-O-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine that could possibly serve as a precursor of both platelet activating factor and bioactive arachidonate metabolites.     

Inhibitory effect of stearidonic acid (18∶4 n−3) on platelet aggregation and arachidonate oxygenation

The effect of stearidonic acid (18∶4n−3) present in fish and some plant oils, such as black currant seed oil, was studied on human platelets. When added to platelets simultaneously with collagen, arachidonic acid or endoperoxide mimetic U46619, 18∶4n−3 appeared as a weak inhibitor of platelet aggregation. In addition, 18∶4n−3 did not alter the metabolism of exogenous arachidonic acid. In contrast, when preincubated with platelets after precoating onto albumin, 18∶4n−3 inhibited platelet aggregation induced by thrombin, collagen, arachidonic acid or U46619, and was as potent as eicosapentaenoic acid (20∶5n−3) tested under similar conditions. Stearidonic acid also altered the endogenous arachidonate oxygenation stimulated by low doses of thrombin, but to a significantly lesser extent than did 20∶5n−3. It seems therefore that, in addition to competing with endogenous arachidonate metabolism, 18∶4n−3 may affect platelet aggregation by another mechanism.     

Metabolism of platelet-activating factor by blood platelets and plasma

High performance liquid chromatography in combination with a radioactivity detector was used to study the metabolism of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by washed platelets, platelet-free plasma and platelet-rich plasma obtained from rabbits and humans. Degradation of platelet-activating factor in plasma was completely inhibited by diisopropylfluorophosphate and was partially inhibited by ethylenediamine tetraacetic acid. Washed platelets metabolized platelet-activating factor not only to the 2-lyso compound but also, by reacylation of this lyso intermediate, to an analogue of platelet-activating factor probably containing a long-chain acyl group at thesn-2 position. These transformations occurred, but to a lesser extent, in platelet-rich plasma.     

Epoxy/graphene platelets nanocomposites with two levels of interface strength

Graphene platelets (GP) are a novel class of nanofillers due to its good compatibility with most polymers, high aspect ratio, high absolute strength and cost-effectiveness. We in this study synthesised two types of epoxy/GP nanocomposites with different interface strength using the combination of sonication and chemical modification. Although the surface-modified graphene platelets (m-GP) formed clusters, a higher degree of dispersion and exfoliation of graphene was observed in each cluster owning to the improved interface by modification. The scrolling of graphene was found predominantly in the interface-modified nanocomposite. At 4 wt%, the modified nanocomposite shows fracture energy release rate G_1c 613.4 J m⁻², while the unmodified nanocomposite indicates 417.3 J m⁻², in comparison with neat epoxy G_1c 204.2 J m⁻². The interface modification enhanced the glass transition temperature of neat epoxy from 94.7 to 108.6 °C, 14.7% increment. Toughening mechanisms are attributed to the voiding, microcracking and breakage of GP, while matrix may not consume as much fracture energy as m-GP do.     

微孔曝气充氧性能的影响因素研究

曝气量、孔径和水深是影响微孔曝气充氧性能的3个重要因素。通过试验研究了曝气量、孔径和水深对氧总传质系数、氧利用率及理论动力效率的影响,并通过曲线分析优化出最佳参数组合。结果表明,安装水深为2.0 m时采用200μm孔径的曝气器和0.5 m3/h的曝气量具有最佳的充氧性能。     

Artificial reconstruction of two cryptic angucycline antibiotic biosynthetic pathways

Genome-sequencing projects have revealed that Streptomyces bacteria have the genetic potential to produce considerably larger numbers of natural products than can be observed under standard laboratory conditions. Cryptic angucycline-type aromatic polyketide gene clusters are particularly abundant. Sequencing of two such clusters from Streptomyces sp. PGA64 and H021 revealed the presence of several open reading frames that could be involved in processing the basic angucyclic carbon skeleton. The pga gene cluster contains one putative FAD-dependant monooxygenase (pgaE) and a putatively bifunctional monooxygenase/short chain alcohol reductase (pgaM), whereas the cab cluster contains two similar monooxygenases (cabE and cabM) and an independent reductase (cabV). In this study we have reconstructed the biosynthetic pathways for aglycone synthesis by cloning and sequentially expressing the angucycline tailoring genes with genes required for the synthesis of the unmodified angucycline metabolite-UWM6-in Streptomyces lividans TK24. The expression studies unequivocally showed that, after the production of UWM6, the pathways proceed through the action of the similar monooxygenases PgaE and CabE, followed by reactions catalysed by PgaM and CabMV. Analysis of the metabolites produced revealed that addition of pgaE and cabE genes directs both pathways to a known shunt product, rabelomycin, whereas expression of all genes from a given pathway results in the production of the novel angucycline metabolites gaudimycin A and B. However, one of the end products is most probably further modified by endogenous S. lividans TK24 enzymes. These experiments demonstrate that genes that are either inactive or cryptic in their native host can be used as biosynthetic tools to generate new compounds.     

Analysis of alpha tocopherol in blood plasma and platelets by gas liquid chromatography

A method is described for the analysis of α-tocopherol by gas liquid chromatography (GLC) with 0.3% Apiezon L as liquid phase. Impurities that interfere with GLC are removed by saponification. Cholesterol and other nonsaponifiables are separated from α-tocopherol by GLC.     

Characterization of the volatile decomposition products of oxidized methyl arachidonate

The volatile thermal and oxidative decomposition products of methyl arachidonate were separated by capillary gas chromatography and identified by mass spectrometry. Various aldehydes, ketones, aldehyde esters, hydrocarbons and alcohols were identified. The major products included hexanal, methyl 5-oxopentanoate, pentane, methyl butanoate and 2,4-decadienal, which could be important to off-odor development in oxidized food systems containing arachidonate.     

Enzymic regulation of arachidonate metabolism in brain membrane phosphoglycerides

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl-sn-glycero-3-phosphoinositols (GPI) and diacyl-sn-glycero-3-phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl-sn-glycerophosphoinositols (diacyl-GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [¹⁴C] arachidonoyl groups, diacyl-GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl-GPI in brain synaptosomes may be represented by two pools with half-lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP-dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A₂ and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A₂. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1-acyl-GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A₂ in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl-GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1-acyl-2-[¹⁴C] arachidonoyl-GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl-GPI. The arachidonate released from incubation of labeled diacyl-GPI may be the result of phospholipase A₂ action which is not specific for diacyl-GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl-GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.     

Metabolism of arachidonate and stearate injected simultaneously into the mouse brain

The metabolism of a polyunsaturated and a saturated fatty acid in brain membrane phosphoglycerides was examined by injecting simultaneously a mixture of¹⁴C-arachidonate and³H-stearate into the mouse brain and isolating the microsomal and synaptosomal fractions at 1–40 min after injections. Both types of labeled fatty acids were utilized more readily in the microsomal than the synaptosomal fractions in brain. However, labeled arachidonate was incorporated more rapidly into membrane phosphoglycerides than was stearate. In both subcellular fractions, the relative specific radioactivity (³H and¹⁴C) of diacyl-glycerophosphorylinositol (diacyl-GPI) was higher than other types of phosphoglycerides such as diacyl-glycerophosphorylcholine (diacyl-GPC) and diacyl-glycerophosphorylethanolamine (diacyl-GPE). Furthermore, the apparent rates of incorporation of radioactivity into diacyl-GPI was more rapid for the¹⁴C-arachidonate than for the³H-stearate. Results of the experiment have demonstrated obvious differences in metabolism between stearate and arachidonate in brain. The more rapid transfer of arachidonate to diacyl-GPI is probably due to the presence of an acyl transferase system specially active for the transfer of arachidonyl groups to diacyl-GPI.     

Chemical pathways for the polymerization of enines

AM1 calculations are reported for enines with aromatic substituents. It was found that the position of the unsaturated bonds in each molecule may account for the reactivity of each isomer via different polymerization pathways. The resonant structure of the charged form of one of the molecules allowed speculation about the observed inhibition of the polymerization reaction.     

Adhesion of blood platelets under flow to wettability gradient polyethylene surfaces made in a shielded gas plasma

Adhesion and activation of platelets are important steps in the thrombosis of blood after contact with a biomaterial surface and are governed, in part, by the wettability of the surface. Since most implanted devices are in contact with blood under flow conditions, it is important to study the effect of wettability of device surfaces on the behavior of platelets also under flow. To this end, wettability gradient polyethylene surfaces were prepared through glow discharge with partial shielding over a length of 5 cm, with advancing water contact angles varying from 95 to 45 degrees and a contact angle hysteresis of 30 degrees. The role of blood flow on the adhesion of platelets was examined by incubating these gradient surfaces in anticoagulated, whole human blood under static conditions or in blood under a flow of 10 or 40 ml/min through a 3 mm diameter circuit or for 5 or 15 min with either the hydrophobic or hydrophilic end upstream. Generally, more platelets adhered on the hydrophilic end of the wettability gradient than on the hydrophobic end, although the increment along the wettability gradient was dependent on both the flow conditions and direction. More platelets adhered under a flow of 10 ml/min than under static conditions, due to higher mass transport. Especially when the hydrophilic end was upstream, there was a more pronounced adhesion. This can be explained in terms of immediate platelet activation by shear stress imposed at the upstream end. During flow of 40 ml/min, platelet adhesion on an upstream hydrophilic end was less than on a downstream hydrophilic end. We conclude that platelets detach from the hydrophilic end at high shear stress due to the spherical form of adhered platelets. Platelets on the hydrophobic end could withstand detachment by strong, flat shaped platelet-material contact.     

A lack of correlation between linoleate and arachidonate in human breast milk

The levels of arachidonic acid and linoleic acid found in the lipid fraction of human milk samples were compared. No correlation was found between the level of precursor linoleate and product arachidonate in 80 samples of colostrum (day 3–5) or 60 samples of mature breast milk. We attempted to test the hypothesis that the absence of a precursor-product relationship was caused by a constant level of arachidonate being secreted in the phospholipids (PL) of breast milk cells. Examination of the fatty acid (FA) composition of the PL and triglyceride fractions revealed arachidonate in high concentration in PL but that most of the total arachidonate existed in triglycerides.     

脂肪氧化酶在不对称氧化中的生物催化作用

介绍了脂肪氧化酶在不对称氧化中的生物催化作用。讨论了影响脂肪氧化敏生物催化作用的区域特异性和立体特异性因素。     

Modulation of phorbol ester-associated events in epidermal cells by linoleate and arachidonate

To elucidate the events elicited by the skin tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), which are modulated by linoleic acid (LA) and arachidonic acid (AA), the activity of these fatty acids in cultured mouse epidermal cells was compared. Approximately 94% of either exogenous radiolabelled fatty acid was incorporated into the total phospholipid pool over 15 h. The relative distribution among the phospholipid classes differed, however, such that approximately 70% of phospholipid-associated [¹⁴C]-LA was found in phosphatidylcholine, compared to approximately 30% for [¹⁴C]AA. Phosphatidylethanolamine and phosphatidylinositol/phosphatidylserine contained 17 and 13% of the phospholipid [¹⁴C]LA, and 34 and 30% of [¹⁴C]AA, respectively. Prostaglandin (PG) E₂ production was low but similar in unstimulated cultures prelabelled with either [¹⁴C]LA or [¹⁴C]AA. However, in cultures treated with TPA (1.6 μM), [¹⁴C]AA-prelabelling resulted in approximately three times the amount of [¹⁴C]PGE₂ compared with cultures prelabelled with [¹⁴C]LA. Cultured cells were found to contain significant δ6 desaturase activity, which may enable conversion of LA to AA, and thus may account for the observed PGE₂ production from [¹⁴C]LA treated cells. AA-Supplemented (1.6 μM) cultures supported approximately twice the induction of ornithine decarboxylase activity by TPA compared with cultures treated with 1.8 μM LA. Activation of partially purified protein kinase C was similar for either fatty acid tested over a 10–300 μM dose range. Overall, the results suggest that LA does not have the same biological activity as AA with regard to several TPA-associated events known to be important in skin tumor promotion. This reduced biological activity of LA may be partly responsible for the known inhibition of mouse skin tumor promotion by high dietary levels of LA [Leyton, J., Lee, M.L., Locniskar, M.F., Belury, M.A., Slaga, T.J., Bechtel, D., and Fischer, S.M. (1991)Cancer Res. 51, 907–915].     

Ethyl arachidonate is the predominant fatty acid ethyl ester in the brains of alcohol-intoxicated subjects at autopsy

The role of fatty acid ethyl esters (FAEE), the nonoxidative ethanol metabolites, as mediators of alcohol-induced organ damage is increasingly being recognized. FAEE are detectable in the blood and in liver and adipose tissue after ethanol ingestion, and on that basis, FAEE can be used as markers of ethanol intake. In this study, 10 samples of human brain were collected at autopsy at the Massachusetts Medical Examiner's Office and analyzed for FAEE. FAEE were isolated and quantified as mass per gram of wet weight. The blood ethanol level was also obtained in each case along with the other drugs detected in routine postmortem toxicology screening tests. Ethyl arachidonate was the predominant FAEE species in the brain, representing up to 77.4% of total FAEE in the brain. The percent age of ethyl arachidonate of the total FAEE in the brain was significantly higher than what has been found in all other organs and tissues previously analyzed. Linoleate, the precursor of arachidonate, was a poor substrate for FAEE synthesis, as the percentage of ethyl linoleate of the total FAEE content was extremely low. Thus, this reflects preferred incorporation of arachidonate into newly synthesized FAEE in the brain. Since arachidonate is derived from linoleate, which is depleted in FAEE while arachidonate is enriched, the synthesis of FAEE may be linked to the desaturation and elongation of linoleate to arachidonate.     

Comparison of two data interpretation programs in automated blood cell counters

Two data interpretation programs incorporated into two different automated blood cell counters were evaluated. The programs analysed size distribution histograms for screening for abnormal specimens. The messages given by the programs were ‘normal’, ‘abnormal’ and ‘suspect’. ‘Abnormal’ messages meant abnormalities in the values of the measured parameters. ‘Suspect’ messages were given when the histograms showed either a possibility of the existence of abnormal blood cells, or an abnormal level of such cells; re-examination by a manual method was necessary for such specimens. For reference, the manual differential was done. The false-negative rate for each kind of message was less than 10% and efficiency was about 90%. These programs were helpful in avoiding unnecessary manual observations in reducing the workload in a routine clinical laboratory.