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.     

Steric requirements for the stimulation of glycosyltransferase activity by lysophosphatidylcholine

1-Palmitoyl-sn-glycerol-3-phosphocholine and 3-palmitoyl-sn-glycerol-1-phosphocholine have been found to be equipotent in the stimulation of membrane-bound glycosyltransferases in microsomes of rat intestinal villus cells. This indicates that the stimulatory effect of lysophosphatidylcholine is not stereospecific, but that it may be related to a specific detergent property dependent upon the peculiar balance of hydrophilic and hydrophobic components in the molecule.     

Availability of lysophosphatidylcholine in single bilayer vesicles for hydrolysis by lysophospholipase

Single bilayer vesicles were prepared from total rat liver microsomal lipids to which 5 mol% lysophosphatidylcholine had been added. The availability of lysophosphatidylcholine for enzymatic hydrolysis by lysophospholipase (EC 3.1.1.5) was found to be higher in vesicles prepared by the cholate dispersion technique when compared with sonicated vesicles. Sepharose 4 B chromatography showed that the vesicles prepared by the cholate technique were smaller than those prepared by sonication. This is in contrast to previous observations for egg phosphatidylcholine vesicles. Total rat liver microsomal extracts were found to contain proteolipid, which could be removed by ether precipitation. Cholate vesicles prepared from proteolipid-free extracts were still smaller than sonicated vesicles from this extract. Experiments with [¹⁴C] dextran entrapped in the vesicles indicate that there is no loss of the permeability barrier of the vesicles for high molecular weight solutes during vesicle treatment with lysophospholipase. The high availability of lysophosphatidylcholine in cholate vesicles of total rat liver microsomal lipids is discussed in terms of a highly asymmetric distribution of lysophosphatidylcholine over the inner and outer monolayer of the bilayer.     

Metabolism of alkane by yeast

We demonstrated two NAD⁺-linked alcohol dehydrogenases in cell free extracts ofCandida tropicalis grown onn-tetradecane. Comparative studies of localization, properties and regulation indicate that these enzymes are involved in two different pathways ofn-alkane metabolism, one cytoplasmic and the other mitochondrial. Kinetic properties, such as the variation of the K_m and V_max as a function of substrate chain length of the soluble NAD⁺-linked alcohol dehydrogenase, might involve hydrophobic interactions between the substrate and the enzyme. One of five papers being published from the Symposium “Biochemistry of Hydrocarbon Degradation,” presented at the AOCS Meeting, Chicago, September 1970.     

The association of lysophosphatidylcholine with isolated cardiac myocytes

The ability of exogenous lysophosphatidylcholine to produce electrophysiological derangements and cardiac arrhythmias in the heart has been documented. The action of lysophosphatidylcholine is thought to be mediated via its association with the membrane. The present study examined the nature of the association of lysophosphatidylcholine with isolated rat myocyte membrane. The association was studied by incubating myocytes in a lysophosphatidylcholine-containing medium. The association of lysophosphatidylcholine with the myocyte sarcolemma was not affected by palmitic acid and glycerophosphocholine but was reduced by platelet-activating factor (PAF). The addition of albumin (5 mg/mL) at the end of the incubation period effectively removed the lysophosphatidylcholine from the myocytes. Our results suggest that most of the lysophosphatidylcholine in isolated myocytes was associated preferentially with the outer leaflet of the myocyte sarcolemma. This type of association might be responsible for the lysophosphatidylcholine-induced electrophysiological alterations in the heart.     

Mechanism of lysophosphatidylcholine accumulation in the ischemic canine heart

The metabolism of lysophosphatidylcholine (LPC) in non-ischemic and ischemic canine heart was investigated byin vitro enzyme analysis. Selected subcellular fractions were assayed for the LPC-producing enzyme phospholipase A and the LPC-eliminating enzymes LPC:acyl-CoA acyltransferase, LPC:LPC transacylase and lysophospholipase. The canine heart was found to contain all enzymes differing, however, in subcellular distribution and specific activity. Phospholipase A activity did not change significantly in any of the fractions prepared from the ischemic tissue of hearts rendered ischemic for 1, 3 or 5 hr when compared to non-ischemic tissue. Changes in the activity of the microsomal LPC:acyl-CoA acyltransferase over the course of 5 hr of ischemia were observed. Significant decreases in the activity of the cytosolic and microsomal lysophospholipases were detected especially after 3 and 5 hr of ischemia. Similarly, a decrease in the activity of the microsomal LPC:LPC transacylase was noted after 3 and 5 hr of ischemia. Our results suggest that impaired catabolism of LPC rather than an enhanced production of LPC is the principal mechanism for the increase in LPC levels in the ischemic canine heart.     

Metabolism of plant sterols by nematodes

Parasitic nematodes do not biosynthesize sterolsde novo and therefore possess a nutritional requirement for sterol, which must be obtained from their hosts. Consequently, the metabolism of phytosterols by plant-parasitic nematodes is an important process with potential for selective exploitation. The sterol compositions of several species of plant-parasitic nematodes were determined by capillary gas chromatography-mass spectrometry and compared with the sterol compositions of their hosts. Saturation of the phytosterol nucleus was the major metabolic transformation performed by the root-knot nematodesMeloidogyne arenaria andM. incognita and the corn root lesion nematode,Prytalenchus agilis. In addition to saturation, the corn cyst nematode,Heterodera zeae, dealkylated its host sterols at C-24. Because free-living nematodes can be cultured in sterol-defined artificial medium, they have been successfully used as model organisms for investigation of sterol metabolism in plant-parasitic nematodes. Major pathways of phytosterol metabolism inCaenorhabditis elegans, Turbatrix aceti andPanagrellus redivivus incleded C-24 dealkylation and 4α-methylation (a pathway unique to nematodes).C. elegans andT. aceti introduced double bonds at C-7, andT. aceti andP. redivivus saturated the sterol nucleus similarly to the plant-parasitic species examined. Several azasteroids and long-chain dimethylalkylamines inhibited growth and development ofC. elegans and also the Δ²⁴-sterol reductase enzyme system involved in the nematode C-24 dealkylation pathway. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, heald at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Metabolism of sterols by anaerobicSaccharomyces cerevisiae

Anaerobically grownSaccharomyces cerevisiae retained the ability to transfer a C₁-group to the C-24 position of a Δ²⁴⁽²⁵⁾-sterol and to reduce the Δ²⁴⁽²⁸⁾-bond of a 24-methylenesterol. Both desmosterol and 24-methylenecholesterol yielded 24β-methylcholesterol. However, when the substituent at C-24 was enlarged to a 24-ethylidene group (fucosterol), reduction of the Δ²⁴⁽²⁸⁾-bond did not occur. In no cases was a Δ⁷ a Δ²²-bond introduced. Because the Δ²⁴⁽²⁸⁾-bond was, reduced in the absence of the Δ²²-bond the Δ²² is not an obligatory requirement for reduction.     

Metabolism of trans fatty acids by hepatocytes

The present work was undertaken to study the metabolism of fatty acids with trans double bonds by rat hepatocytes. In liver mitochondria, elaidoyl-CoA was a poorer substrate for carnitine palmitoyltransferase I (CPT-I) than oleoyl-CoA. Likewise, incubation, of hepatocytes with oleic acid produced a more pronounced stimulation of CPT-I than incubation with trans fatty acids. This was not due to a differential effect of cis and trans fatty acids on acetyl-CoA carboxylase (ACC) activity and malonyl-CoA levels. Elaidic acid was metabolized by hepatocytes at a higher rate than oleic acid. Surprisingly, compared to oleic acid, elaidic acid was a better substrate for mitochondrial and, especially, peroxisomal oxidation, but a poorer substrate for cellular and very low density lipoprotein triacylglycerol synthesis. Results thus show that trans fatty acids are preferentially oxidized by hepatic peroxisomes, and that the ACC/malonyl-CoA/CPT-I system for coordinate control of fatty acid metabolism is not responsible for the distinct hepatic utilization of cis and trans fatty acids.     

Simultaneous treatment of methane and swine slurry by biofiltration

BACKGROUND: The piggery industry is important both worldwide and in Canada, but localized production of large quantities of swine slurry causes severe environmental problems such as aquatic pollution and greenhouse gas emissions. The main objective of this study was to determine whether it is possible to simultaneously treat methane (CH₄) and swine slurry using an inorganic biofilter. RESULTS: A novel biofilter was designed to overcome the inhibition of CH₄ biodegradation by swine slurry. The CH₄ elimination capacity increased with the inlet load and a maximum value of 18.8 ± 1.0 g m^?3 h^?1 was obtained at an inlet load of 46.7 ± 0.9 g m^?3 h^?1 and a CH₄ concentration of 3.3 g m^?3. Four pure strains of fungi were used in an attempt to improve the removal of CH₄, but no significant effect was observed. Between 0.35 and 3.4 g m^?3, the CH₄ concentration had no effect on swine slurry treatment with removal efficiencies of 67 ± 10% for organic carbon and 70 ± 7% for ammonium. The influence of the slurry supply was analyzed and the best results were obtained with a supply method of six doses of 50 mL per day. CONCLUSION: Even though the results were lower than those obtained for the biofiltration of CH₄ alone, this study demonstrated the feasibility of treating CH₄ and swine slurry with the same biofilter using a novel design. Copyright © 2012 Society of Chemical Industry     

Reinforcement of poly(dimethylsiloxane) networks by montmorillonite platelets

A PDMS network, synthesized from a vinyl‐terminated precursor, was reinforced by plate‐like montmorillonite (volclay) particles with different surface cations. The optimal ratio of crosslinker‐to‐PDMS precursor was ascertained from the mechanical properties of networks prepared with different crosslinker concentrations. The elastic modulus of the polymer was enhanced by the montmorillonite particles. The increase in modulus was higher in the Li– than in the Na–volclay composites. The ultimate strength of the composites was also strongly enhanced by the small platelets, especially in presence of surface Li⁺. The stronger influence of Li–volclay on the mechanical properties of the composites can be attributed to the partial formation of an intercalated structure, which leads to thinner particles with a high aspect ratio. Both composite strength and modulus were proportional to the filler‐volume‐fraction, but the increase in strength was limited by rising particle agglomeration at high loading. In contrast to organic‐modified montmorillonite, the inorganic surface of volclay catalyzed the thermal degradation of PDMS. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 2175–2183, 2002     

Suppressive effect of docosahexaenoyl‐lysophosphatidylcholine and 17‐hydroxydocosahexaenoyl‐lysophosphatidylcholine on levels of cytokines in spleen of mice treated with lipopolysaccharide

Lysophosphatidylcholine (lysoPC) with polyunsaturated fatty acyl chains has been known to be anti‐inflammatory in vivo. In the present study, we examined the effect of docosahexaenoyl‐lysophosphatidylcholine (DHE‐lysoPC) and 17‐hydroxydocosahexaenoyl‐lysophosphatidylcholine (17‐HDHE‐lysoPC) on spleen weight and cytokine level in spleen of mice treated with lipopolysaccharide (LPS). For this purpose, mice were administrated i.p. with DHE‐lysoPC or 17‐HDHE‐lysoPC 1 h before i.p. injection of LPS. First, DHE‐lysoPC (50–400 µg/kg) was found to suppress the LPS‐induced increase of spleen weight dose‐dependently, and such a suppressive effect was greater for 17‐HDHE‐lysoPC, compared to DHE‐lysoPC. Next, in an attempt to see the effect of DHE‐lysoPC on cytokine levels in spleen of mice treated with LPS, DHE‐lysoPC was found to suppress LPS‐induced increase in the levels of cytokines such as TNF‐α, IL‐1β, or IL‐6 in a dose dependent manner (50–400 µg/kg), in contrast to DHA showing a significant action at a high dose (400 µg/kg) only. The greater suppressive effect of 17‐HDHE‐lysoPC (15–150 µg/kg) than DHE‐lysoPC suggested that action of DHE‐lysoPC may be enhanced through lipoxygenation process. Presumably in support of this, when the interval time between 17‐HDHE‐lysoPC administration and LPS challenge was varied, the cytokine‐suppressing effect was found to be augmented in a time‐dependent manner. Taken all together, it is suggested that DHE‐lysoPC and 17‐HDHE‐lysoPC may be beneficial in suppressing the inflammation in spleen tissue.     

Activation of AMP-kinase by Policosanol Requires Peroxisomal Metabolism

Policosanol, a well-defined mixture of very long chain primary alcohols that is available as a nutraceutical product, has been reported to lower blood cholesterol levels. The present studies demonstrate that policosanol promotes the phosphorylation of AMP-kinase and HMG-CoA reductase in hepatoma cells and in mouse liver after intragastric administration, providing a possible means by which policosanol might lower blood cholesterol levels. Treatment of hepatoma cells with policosanol produced a 2.5-fold or greater increase in the phosphorylation of AMP-kinase and HMG-CoA reductase, and increased the phosphorylation of Ca⁺⁺/calmodulin-dependent kinase kinase (CaMKK), an upstream AMP-kinase kinase. Intragastric administration of policosanol to mice similarly increased the phosphorylation of hepatic HMG-CoA reductase and AMP-kinase by greater than 2-fold. siRNA-mediated suppression of fatty aldehyde dehydrogenase, fatty acyl-CoA synthetase 4, and acyl-CoA acetyltransferase expression in hepatoma cells prevented the phosphorylation of AMP-kinase and HMG-CoA reductase by policosanol, indicating that metabolism of these very long chain alcohols to activated fatty acids is necessary for the suppression of cholesterol synthesis, presumably by increasing cellular AMP levels. Subsequent peroxisomal β-oxidation probably augments this effect.     

Inhibition of theClostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle-bound ammonium and sulfonium cations

Cetyltrimethylammonium andn-octadecyldimethylsulfonium bromides inhibit theClostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37°C, μ=0.15 with KCl. Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but nonlinear function of the mole fraction of inhibitor. Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor. Inhibition constants for cetyltrimethylammonium andn-octadecyldimethylsulfonium bromides are 0.66±0.04 and 0.25±0.02 mM, respectively. The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K ₅₀ for dodecamethylene-bis(dimethylsulfonium) bromide, 27μM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.     

The effect of lysophosphatidylcholine on coronary and renal circulation in the rabbit

It has been shown that different lysophosphatidylcholines (LPC) possess relaxing properties in vascular strips. We report on the effect of intraatrial injection of LPC micelles into white New Zealand rabbits. Changes in renal and coronary flow were determined by radioactive microspheres. Coronary blood flow increased significantly following administration of LPC, while no significant changes were observed in renal blood flow, cardiac output and myocardial contractility. Coronary, renal and total vascular resistance, as well as mean arterial pressure, dropped significantly. Cardiac arrhythmia and hemolysis were not noted. The results demonstrate the vasorelaxant effect of LPCin vivo. This work was presented in part at a meeting of the Association of American Physicians in 1988.     

Metabolism of labeled isomeric octadecenoates by the laying hen

Discrimination between octadecenoic acid isomers by the laying hen has been studied using tritium (³H), carbon-14 (¹⁴C) and deuterium (d) labeled oleate and elaidate esters. Hydrogen isotopes were positioned at the double bond, whereas¹⁴C was located in the 1-, carboxyl carbon. The egg acted as a biological trap, providing an automatic daily biopsy with which to study the metabolism of the fed isomers. Monitoring the incorporation of isomers was facilitated by dual label feeding experiments, and³H/¹⁴C, d₂/d_o and d₂/d₁ ratios were determined on the isomeric mixtures fed, on the total egg lipids extracted and on the isolated neutral lipid and phospholipid fractions. Comparison of isotopic ratios of the fed mixture and of the lipid fractions provided an evaluation of discrimination by the hen during the transport of isomeric octadecenoates into the egg lipids. Radioactive and stabl isotope ratios determined for the neutral lipid indicated a preferential incorporation of thecis isomer. Stable isotope ratios determined for the phospholipid showed that thetrans isomer is preferentially incorporated. The³H/¹⁴C ratios for the phospholipid recovered in each experiment increased greatly whichever isomer was labeled with³H, indicating an elimination of the 1-¹⁴C-label. Gas liquid radiochromatographic separation of the methyl esters from the neutral lipids and phospholipids showed that the isotopic labels were present almost exclusively in the octadecenoic acid constituent. Presented at the AOCS Meeting, Houston, May 1971. No. Mktg. and. Nutr. Res. Div., ARS, USDA.