A simple and precise method for the routine determination of platelet-activating factor in blood and urine

A simple and precise method is described for the measurement of platelet-activating factor (PAF) in blood and urine. The method involves the isolation of PAF from blood samples by two successive steps. In the first step, blood proteins are precipitated with ethanol and the "free" PAF, i.e. the PAF which is extractable with ethanol, is recovered. In the second step, "bound" PAF, i.e., PAF not extractable with ethanol, is extracted from the protein precipitate with chloroform/methanol/water. The extraction of PAF from urine samples requires only the ethanol extraction step. "Free" and "bound" PAF are then each fractionated by silicic acid column chromatography, and the methanol/water eluent containing PAF is then further fractionated by high-performance liquid chromatography using an isocratic solvent system of acetonitrile/methanol/water. PAF is then quantitated by measuring its ability to induce platelet aggregation in an aggregometer. Application of the method to blood and urine samples from twenty-three healthy volunteers revealed PAF levels in blood of 140-480 pg/mL (630-254.4 pg "free" PAF/mL and 64-225.6 pg "bound" PAF/mL), and of 1.2-4.0 pg PAF/mL in urine. The method overcomes various technical problems and was shown to be very precise. It should prove useful for monitoring PAF levels in various disease conditions. © 1994 American Oil Chemists' Society

Application of a TCA-precipitation method for the determination of 1-alkyl-sn-glycero-3-phosphate: Acetyl-CoA acetyltransferase in human renal tissue

The activity of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase, which catalyses the first step of the de novo biosynthesis of PAF, was determined and characterised in cortical and medullary human renal tissues. A novel thin-layer chromatographic system as well as a trichloroacetic acid precipitation method, were utilised in order to determine the enzyme's activity. The acetyltransferase activity was associated with the membranous fractions of the renal tissue, it showed an optimum pH of 8.4 and it had a bell-shaped dependence on BSA concentration. One or more disulphide bonds were necessary for the action of acetyltransferase while the enzyme seemed to be independent from divalent cations. Two assay products were extracted from the incubation mixture namely alkylacetylphosphatidic acid, produced by the acetylating action of the acetyltransferase on alkyllyso-phosphatidic acid and alkylacetyl-glycerol, which is produced by the action of a phosphohydrolase on alkylacetylphosphatidic acid. The presence of NaF in the assay mixture resulted to a decreased degradation of alkylacetylphosphatidic acid, as well as to an increased overall product formation. Cortical and medullary acetyltransferases share similar biochemical properties and there is no statistical difference between the two activities. © 2004 Elsevier Inc. All rights reserved

Platelet-activating factor detection, metabolism, and inhibitors in the ethanologenic bacterium Zymomonas mobilis

Platelet-activating factor (PAF) is a signaling phospholipid with a significant physiological role in multicellular and unicellular organisms, including fermentative organisms such as yeast. Zymomonas mobilis is an ethanologenic α-proteobacterium currently studied for bioethanol production. In order to examine the presence of PAF and/or PAF inhibitors in Z. mobilis, a new one-step high performance liquid chromatography (HPLC) separation procedure of total lipids was performed, using a C8 reversed-phase semi-preparative column. According to this method and to bioassays based on washed rabbit platelet aggregation, two lipid molecules with PAF-like activity and same retention times as those of standard PAF were detected; electron-spray ionization MS and MS/MS analysis revealed that they share similar structure with 16:0 and 18:0 PAF. Furthermore, other lipids extracted from Z. mobilis were found to exhibit a potent anti-PAF activity. Enzyme activities indicative of key PAF biosynthetic enzymes, such as dithiothreitol-insensitive cholinephosphotransferase (PAF-CPT) and lyso-PAF acetyltransferase were detected in Z. mobilis homogenates. As for PAF degradation, activity similar to that of PAF acetylhydrolase was also discovered. Overall, the presence of PAF, PAF-specific inhibitors, and enzyme activities relating to PAF metabolism, suggests that PAF may play an intrinsic role in this biotechnological organism. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Phosphono-platelet activating factor I. synthesis of 1-O-hexadecyl-2-O-acetyl-glyceryl-3-(2-trimethyl ammonium-methyl) phosphonate and its platelet activating potency

The total synthesis of 1-O-alkyl-2-acetyl-3-glyceryl-(2-trimethyl ammoniummethyl) phosphonate, the phosphono analogue of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, is described. The phosphonolipid shows much lower activity than the phospholipid stimulating serotonin release from rabbit platelets. © 1983

Antithrombotic lipid minor constituents from vegetable oils. Comparison between olive oils and others

Many epidemiological studies suggest that vegetable oils and especially olive oil present a protective effect against atherosclerosis. In this study, total lipids (TL) of Greek olive oils and seed oils of four kinds, namely, soybean, corn, sunflower, and sesame oil, were separated into total polar lipids (TPL) and total neutral lipids (TNL) via a novel extraction procedure. TPL and TNL of olive oil were fractionated by HPLC for further study. Each lipid fraction from HPLC separation along with TL, TPL, and TNL lipid samples from oils were tested in vitro for their capacity to induce or to inhibit washed rabbit platelet aggregation. Comparison between olive and seed oils supports the superiority of olive oil as high levels of platelet activating factor (PAF) antagonists have been detected, mainly in TPL. In addition, the structure of the most active fraction from olive oil was elucidated, as a glycerol-glycolipid. Because it has already been reported that PAF plays a pivotal role in atherogenesis, the existence of PAF agonists and antagonists in vegetable oils may explain their protective role against atherosclerosis

Triacylglycerol metabolism

Apart from being the main energy reserves of the human body, triacylglycerols take part in metabolic processes that determine the rate of fatty acid oxidation, the plasma levels of free fatty acids, the biosynthesis of other lipid molecules and the metabolic fate of lipoproteins. Allosteric, hormonal, nutritional and transcriptional signals activate short-term and long-term regulatory mechanisms that assure the storage of triacylglycerols (TAGs) under states of excess energy and their mobilization under conditions of metabolic stress. New enzymes and novel regulatory mechanisms, involved in triacylglycerol metabolism, have been recently discovered and new details on the fine tuning of their metabolic reactions are coming to light. This knowledge will help us understand the biochemical basis of several diseases for the pathogenesis of which triacylglycerols play a role. © 2009 Bentham Science Publishers Ltd

Lipid Separation from Urtica dioica: Existence of Platelet-Activating Factor

The common wild plant nettle, especially Urtica dioica, is one of the most potent plants in producing direct irritation to the skin (urticaria). In this study, total lipids of Urtica dioica were separated into neutral and polar lipids, which were further fractionated by high-performance liquid chromatography (HPLC). Triglycerides, sterol-esters, fatty acids, fatty acid methyl esters, glyceryl ethers, sterols, tocopherols, diglycerides, and galactosyldiglycerides were identified as the main neutral lipid classes by comparing their retention times on an HPLC column and their migration following spraying with specific reagents on thin-layer chromatography (TLC) with standards. Four main classes of phospholipids (i.e., phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and lysophosphatidylcholine) were also identified. A phospholipid that induced platelet aggregation was identified as platelet-activating factor on the basis of biological, chemical, and spectral methods

Identification and Study of Gangliosides from Scomber scombrus Muscle

Scomber scombrus has been implicated in the disease known as scombroid food poisoning (histamine intoxication). However, some investigators claim that scombroid food poisoning is not caused by histamine only. Gangliosides have been found in different type fishes, but in all cases the sources are brain, melanoma, retina, and optic nerve. These complex lipids as well as their derivatives, exhibit important biological activities. In this study, gangliosides (8 x 10-4 % w/w in muscle) were isolated from S. scombrus muscle for the first time, and the major one was found to be monosialoganglioside. Gangliosides existed as a proteolipid type complex combined with a protein consisting of high amounts of iron and copper and which seemed to be the red protein. After fractionation of gangliosides onto cation exchange, silica, and G18 HPLC columns, we detected two compounds which induced aggregation through platelet-activating factor (PAF) way. Both of them were eluted onto HPLC in the region of gangliosides and from the results of the biological experiments as well as from chemical determinations, they seemed to be O-acetyl derivatives of gangliosides. These molecules could partly contribute to scombroid food poisoning since the main symptoms of this disease are well-known actions of PAF