Tumor necrosis factor release by human monocytes stimulated with platelet-activating factor

Platelet-activating factor (PAF) is a low molecular weight phospholipid which enhances human monocyte cytotoxicity for tumor cells. In the current studies, the capacity of PAF to stimulate release of tumor necrosis factor α (TNFα) by human monocytes was assessed. PAF induced maximal TNFα synthesis 2–3 hr after monocyte stimulation as assessed by dot blotting of cell-associated TNFα using polyclonal anti-TNF antibody. Maximal net release of TNFα occurred 5–16 hr after monocyte stimulation, as assessed by a specific ELISA. Dose-response studies revealed that a maximal two- to three-fold increase in release of TNFα occurs at 10–100 pM PAF. LysoPAF and the optical isomer of PAF did not stimulate release of TNFα, suggesting that stimulation is mediated by specific PAF receptors. Scatchard analysis of [³H]PAF binding to monocyte membranes revealed 651±495 binding sites/monocyte with a Kd of 4.7±4.2×10⁻¹⁰M. PAF is a structurally unique activator of monocytes whose interactions with TNFα and other cytokines may be critical to host defense against tumors. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Quantitation by radioimmunoassay of PAF in human saliva

Approximately 75% of the PAF present in saliva is recovered on extraction of whole saliva (0.8 vol) with chloroform/methanol/water (2∶2∶1 v/v/v). PAF levels, determined by our recently developed radioimmunoassay, in saliva extracts ranged from 0.5–21 ng/mL with 59% between 2–6 ng/mL. These figures, for apparently healthy subjects, are higher than previously reported levels obtained by platelet assays. The validity of our radioimmunoassay results was checked by isolating and quantitating the PAF fraction from whole saliva. In addition, when we examined our saliva samples by platelet aggregation, low levels of PAF, comparable with the values found in the literature, were detected. Investigations revealed the presence of a substance(s) which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.     

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.     

Chemical synthesis and physiological activity of sulfonium analogues of platelet activating factor

Phosphatidylsulfocholine (PSC), the sulfonium analogue of phosphatidylcholine (PC), occurs naturally in some diatoms. The replacement of the −N⁺(CH₃)₃ group by a −S⁺(CH₃)₂ results in an increase in the polar head group size in PSC relative to that of PC, consistent with the observed increase in permeability of PSC bilayers towards urea. It was of interest to see whether replacement of the −N⁺(CH₃)₃ group in platelet activating factor (PAF) by an −S⁺(CH₃)₂ group leads to any change in platelet aggregation or other physiological activity. Synthesis of the sulfonium analogue of PAF was carried out by suitable modifications of known procedures. The PAF-sulfonium analogue was found to have almost the same platelet aggregating activity as PAF itself, in the concentration range 1–20 μM, but a much lower activity in the range 0.01–1 μM. The analogue had little or no effect on the platelet aggregation activity of PAF when added in the concentration range 0.01–1 μM and had about half the hypotensive activity of PAF towards hypertensive CDF male rats. The sulfonium analogue, however, was much more cytotoxic to HL-60 cells than PAF itself, in the concentration range 0–15 μM; replacement of the acetate group by a benzyl group increased the cytotoxicity to the level of that of the methoxy analogue of PAF. Thus, replacement of the −N⁺(CH₃)₃ group by a −S⁺(CH₃)₂ group in the polar head group region of PAF results in a relatively small change in its platelet aggregation activity and a decrease in its hypotensive activity, but greatly increases its antitumor activity. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 8–12, 1989.     

Platelet-activating factor may participate in signal transduction processes in rabbit leukocytes

The bacterial chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), induces the generation of platelet-activating factor (PAF), the mobilization of arachidonic acid and generation of superoxide anion (O₂ ⁻) in rabbit polymorphonuclear leukocytes (PMNs). The PAF receptor antagonists, WEB 2086 (10–100 μM) and CV 6209 (1–10 μM), reduced the mobilization of arachidonic acid and the O₂ ⁻ generation in response to fMLP but not that in response to A23187. Pretreatment of PMNs with the phospholipase A₂ inhibitor, chloroquine, or the serine protease inhibitor, tosyl-phenylalanine chloromethyl ketone, reduced the fMLP-stimulated generation of PAF and also reduced the generation of O₂ ⁻. The respiratory burst induced by a submaximal concentration of phorbol myristate acetate was not affected by these compounds. These data are consistent with the suggestion that endogenous PAF may contribute to the signal transduction cascade initiated by fMLP. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May, 1989.     

Platelet-activating factor (PAF) receptor antagonists inhibit arachidonic acid induced platelet aggregation in rabbit whole blood

The potency of several platelet-activating factor (PAF) receptor antagonists was measured by observing their inhibitory effects against PAF induced platelet aggregation. Their selectivity was assessed by monitoring their effect on platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP). The antagonists inhibited platelet aggregation induced at the PAF EC₅₀ (0.023 μM) with the following rank order of potency: WEB 2086> WEB 2170> SRI 64–412> SRI 63–675> BN 52021>kadsurenone> SRI 63–441> alprazolam. While the antagonists had no inhibitory effect at the EC₅₀ for ADP (10 μM), they did inhibit platelet aggregation induced at the EC₅₀ for AA (55 μM). However, there was considerable variability in the slope of the inhibitory response and the relative potency of each antagonist against PAF induced platelet aggregation as compared to AA induced platelet aggregation. The antagonist IC₅₀ (μM) against PAF and AA were as follows, with those that showed significantly different (p<0.01) slopes indicated by an asterisk: SRI 63-441^* (3.8, 15.1); SRI 63-675 (1.4, 36.2); SRI 64-412 (0.5, 10.5); BN 52021^* (2.4, 58.9); kadsurenone^* (2.8, 28.3); alprazolam^* (10, 25); WEB 2086 (0.055, 0.220), and WEB 2170 (0.107, 0.534). Therefore, in rabbit whole blood the antagonists were potent, although not completely selective, inhibitors of PAF induced platelet aggregation. These results suggest that the mode of action of PAF and AA induced platelet aggregation may share some common features. However, since the slope of the inhibitory response against PAF and AA for some antagonists differed, mechanistic differences in their action appear to exist. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effect of the hetrazepinoic platelet-activating factor antagonist bepafant (WEB 2170) in models of active and passive anaphylaxis in mice and guinea pigs

The selective hetrazepinoic platelet-activating factor (PAF) antagonist WEB 2170 (Bepafant) was used to study the pathophysiological role of PAF in several models of anaphylaxis in mice and guinea pigs. In actively sensitized mice, the PAF antagonist WEB 2170 (1.0–10 mg/kgp.o.) protected mice from anaphylactic death in a dosedependent manner when the anaphylactic response was potentiated by the beta-receptor antagonist propranolol. When active anaphylaxis in guinea pigs was induced intravenously by 100 mg/kg ovalbumin (OA) in the presence of small doses of the antihistamine mepyramine, additional treatment with oral or intravenous WEB 2170 protected the guinea pigs from anaphylactic death. Also, the remaining anaphylactic bronchoconstriction and blood pressure changes (including anaphylactic hypotension) were attenuated. When guinea pigs were passively sensitized with a heterologous antibodyvia the tracheal route and then challenged by ovalbumin (100 mg/kgi.v.) 24 hr after sensitization in the presence of 0.003 mg/kgi.v. mepyramine, additional treatment with tracheal WEB 2170 at 0.1–1 mg/kg protected the guinea pigs dosedependently not only from anaphylactic death but also from a further decrease of respiratory flow and changes of blood pressure. Increased levels of PAF-like activity (20–50 ng PAF/whole lung) were detected in lungs removed from antigen-challenged animals. The results suggest a causative role for PAF in active and passive anaphylaxis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor (PAF) stimulates the lysoPAF acetyltransferase in leukocyte-rich plasma: Use in PAF antagonist studies

Addition of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to leukocyte-rich plasma from several species resulted in the rapid and pronounced activation of the PAF biosynthetic enzyme acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67). Activation of acetyltransferase by PAF occurred in leukocyte-rich plasma from human, chimpanzee, rhesus monkey, and dog. The neutrophil was indicated to be the major cellular source of the activabable acetyltransferase in leukocyte-rich plasma. The induction of acetyltransferase was substantial with 10 nM PAF, and maximal at 10–30 seconds. Measurable acetyltransferase activation was significantly greater when the PAF-activated cells were separated from the plasma by centrifugation before the acetyltransferase assay. This may be due in part to the removal of the PAF-specific acetylhydrolase present in plasma which can cleave the acetyl group from PAF. Measuring PAF activation of acetyltransferase in leukocyte-rich plasma can be useful to determine the potency of PAF antagonists with neutrophils in plasma compared to isolated neutrophils in aqueous buffer, and as anex vivo assay to determine the efficacy and plasma concentration equivalents of antagonists administered to whole animals. The PAF antagonist L-659,989 was shown to be 3–5 times more potent in inhibiting PAF induction of acetyltransferase in isolated human neutrophils than in human leukocyte-rich plasma, with IC₅₀ values of 10 nM and 40 nM, respectively. In theex vivo assay, oral administration of the PAF antagonist L-667, 131 to dogs resulted in very substantial inhibition of PAF induction of acetyltransferase in the leukocyte-rich plasma. Utilizing theex vivo assay, oral administration of 1 mg/kg L-659,989 to rats was found to result in plasma concentration equivalents of approximately 200–300 nM L-659,989. Our findings offer a new approach for charagerizing thein vitro andin vivo efficacy of PAF receptor antagonists and demonstrate that PAF may be able to activate neutrophils in the bloodin vivo, further enhancing PAF synthesis. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet-activating factor type activity in plasma from patients with septicemia and other diseases

The purpose of the present study was to determine whether increased levels of platelet-activating factor (PAF) type activity can be detected in plasma from patients with septicemia and other diseases. A level of PAF below 0.5 ng/mL of plasma was considered normal. We found that plasma from a patient with adverse anaphylactoidic reaction to intravenous analgetics contained 2.1 ng PAF/mL. In seven patients with septicemia, including urosepsis, endocarditis and peritonitis, and with positive blood culture, increased plasma PAF levels (1–20 ng PAF/mL) were observed. Other patients with clinical indications of septicemia had negative blood cultures and/or increased levels of C-reactive protein (CRP). Yet, in the plasma from these patients, no increased PAF levels were detected under the assay conditions used. Two patients with allergic asthma, requiring treatment with steroids, had no measurable plasma PAF. In the plasma from a patient with idiopathic thrombocytopenic purpura (ITP) only an “endogenous” inhibitor of PAF induced platelet aggregation was initially observed. In spite of this, the patient responded to treatment with the PAF antagonist WEB 2086 with a dramatic increase in platelet count (Lohmannet al., Lancet ii, 1147, 1988). Thereafter, also increased PAF levels (3.3 ng PAF/mL) were detected in plasma, although some “endogenous” inhibitor of PAF was still present. In conclusion, increased PAF levels in plasma from patients support a role of PAF in certain human disease states, such as in anaphylactoid reaction, sepsis and septic shock. The type, relevance and specificity of endogenous inhibitors of PAF deserve further study. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Preparation and physical properties of some C18 unsaturated fatty esters containingL-amino acid residues and methyl esters ofN-stearoylamino acids

A series of five C₁₈ unsaturated fatty esters (1–5) containing anL-amino acid residue (glycine, alanine, valine, leucine, phenylalanine) was prepared from methyl 12-amino-9-cis-octadecenoate and five methylN-stearoyl-amino acid ester derivatives (6–10) from stearoyl chloride and the sameL-amino acids. The infrared analysis of compounds 1–5 showed characteristic absorption bands at 3300 and 1665 cm⁻¹ for the amino and amido functions, while the amido function in compounds 6–10 gave absorption bands at 3300 and 1680 cm⁻¹. The position of the amido group (peptide linkage) in compounds 1–5 was readily determined by mass spectral analysis.¹H nuclear magnetic resonance (NMR) analysis showed a doublet at 7.15 ppm for the amide proton (NHCO) in compounds 1–5, while in compounds 6–10 the amide proton signal appeared at 6.0 ppm. In¹³C NMR, the amido carbonyl resonance appeared at 171–175 ppm. In compounds 1–5 the effect of the amide function on the ethylene carbon (C-9) gave a signal at 124.1–125.3 ppm, while the remaining ethylenic carbon appeared at 131.9–132.5 ppm. The methine carbon (C-12) of the alkenyl chain was shifted to 48.4–49.8 ppm, and the assignment of the various carbon nuclei in the amino acid residue was readily achieved. In the methylN-stearoylamino acid ester derivatives (6–10), the methine carbon adjacent to the amido system and alpha to the carbomethoxy group appeared between 41.2–57.0 ppm depending on the type of alkyl group present in the amino acid moiety. The phenyl system in compounds 5 and 10 was confirmed by the¹³C signals in the 127–136 ppm range and by the proton signals in the 7.0–7.27 ppm region.     

A sensitive and specific radioimmunoassay for platelet-activating factor

A platelet-activating factor (PAF) analog with a reactive ω-aldehyde group at thesn-1 position was synthesized. The hapten-thyroglobulin conjugate was used to immunize rabbits to produce specific antibodies to PAF. The purified immunoglobulin G (IgG) fraction was found to bind stereo-specifically to tritiated PAF and to crossreact minimally with lysoPAF, plasmalogens, and other phospholipids. The radioimmunoassay detected as little as 20 pg of PAF per assay tube and was used to explore agonist-induced synthesis of PAF in rabbit neutrophils. Calcium ionophore A23187 at 1 μM induced PAF synthesis peaking at 2 min and reaching basal levels after 5 min.N-Formyl-Met-Leu-Phe (FMLP) at 0.1 μM also stimulated rapid synthesis and degradation of PAF with a peak at 5 min. Both A23187 and FMLP stimulated PAF synthesis in a dose-dependent manner. The radioimmunoassay should be applicable to the quantitation of PAF in biological samples. Based on a paper originally presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Biological response of guinea pig peritoneal macrophages to platelet-activating factor

We investigated the effects of platelet-activating factor (PAF) on guinea pig peritoneal macrophages. Specific and high-affinity binding sites for PAF were detected on guinea pig peritoneal macrophages. Scatchard analysis of PAF binding revealed high affinity binding sites (7.9×10⁴/cell) with a dissociation constant of 2.3×10⁻¹⁰ M. When treated with 10⁻⁹−10⁻⁵ M PAF, guinea pig peritoneal macrophages released hydrogen peroxide into the medium in a time-dependent manner. The release reaction upon stimulation with 10⁻⁵ M PAF reached a plateau within 30 min and the extent of release was twice as high as that when stimulated byN-formyl-L-methionyl-leucyl-L-phenylalanine (fMLP; 2 μM)-treated cells. Neither lysoPAF nor the PAF enantiomer was effective. PAF-induced H₂O₂ release was inhibited specifically by PAF antagonists, suggesting that PAF activated macrophages through binding to specific sites. Lysosomal enzyme (N-acetyl-β-D-glucosaminidase) was released from guinea pig peritoneal macrophages upon treatment with 10⁻⁵ M PAF for 60 min. Guinea pig peritoneal macrophages were treated with PAF for 8 hr and the conditioned medium was examined for cytokines. The medium exhibited cytocidal activity against mouse fibroblast L929 cells [tumor necrosis factor (TNF) activity], and this activity was comparable to that detected after treatment of cells with the bacterial lipopolysaccharide (LPS). Furthermore, the same conditioned medium also showed colony-stimulating factor (CSF) activity. Generation of these cytokines was stereospecific. Our findings suggest that PAF is a unique macrophage activator that potentiates both respiratory burst/lysosomal enzyme release (early-phase response) and monokine production/glucose consumption (late-phase response). Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Synthesis of platelet activating factor by cholinephospho-transferase in developing fetal rabbit lung

Developing fetal lung is a possible source of the platelet activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) present in amniotic fluid of women in labor. We have assayed the microsomal activities of a specific enzyme for the de novo synthesis of PAF in developing fetal and neonatal rabbit lung, 1-alkyl-2-acetyl-glycerol-dependent dithiothreitol-insensitive cholinephosphotransferase. The specific activity of this enzyme increased from 0.92 to 3.60 nmol×min⁻¹×mg⁻¹ protein between day 21 and day 31 of gestation. In contrast, during this same period the activity of the PAF-biosynthetic cholinephos-photransferase in developing rabbit kidney did not change significantly. The specific activity of the diacyl-glycerol-dependent, dithiothreitol-sensitive cholinephos-photransferase that catalyzes the final step in phosphatidylcholine biosynthesis was not altered during late gestation in either fetal lung or kidney. Previously, increased amounts of pulmonary PAF had been found during the latter stages of gestation (Hoffman, Truong and Johnston (1986)Biochim. Biophys. Acta 879, 88–96) and may be attributed to increased activity of the PAF biosynthetic enzymes found in this investigation. This elevated level of PAF in fetal lung may serve to facilitate breakdown of glycogen that provides, in part, the carbon and energy source for surfactant biosynthesis. In addition, PAF may be secreted in association with surfactant into amniotic fluid in which it may interact with amnion tissue and subsequently participate in the events associated with the initiation of parturition.     

Preparation of radiolabeled tetracosa mono- and dienoic acid methyl esters from rat erythrocyte lipids by thin layer chromatography

An easy method of obtaining pure fatty acid methyl esters (FAME) of tetracosa mono- and dienoic acids (24∶1, 24∶2) using thin layer chromatography (TLC) is described. The total lipids isolated from rat erythrocytes were treated with methanolic-NaOH. Sphingomyelin was unaffected by this treatment and was separated from FAME of glycerolipids and cholesterol by TLC. FAME of sphingomyelin were then prepared by acid methanolysis. These esters migrated into 2 distinct bands on TLC. The slow moving band contained FAME of 16∶0, 16∶1, 18∶0, 18∶1, 19∶0 and 20∶0 whereas the fast moving band contained FAME of 22∶0, 23∶0, 24∶0, 24∶1 and 24∶2. After AgNO₃-TLC, the FAME of the fast moving band separated into 3 species; esters of saturated acids, 24∶1 and 24∶2, respectively. With erythrocyte lipids of rats fed a fat-free diet and injected with¹⁴C-18∶1, this method yielded¹⁴C-24∶1. From rats injected with¹⁴C-18∶2 and maintained on a corn oil diet,¹⁴C-24∶2 was obtained. Presented at the ISF-AOCS Congress, April 27–May 1, 1980, New York City.     

Platelet-activating factor detected in bronchoalveolar lavage fluids from an asthmatic patient

It recently has been recognized that platelet-activating factor (PAF) may be a mediator of asthma exacerbation. We had the opportunity to analyze bronchoalveolar lavage fluids from an asthmatic infant, which were characterized by neutrophil infiltration. The patient's lungs were washed on three occasions with saline during asthmatic attacks. PAF was found in each case on the basis of its ability to cause the immediate aggregation of washed rabbit platelets. The PAF detected was equivalent to 1–1.4 pmol of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, three quarters of which were recovered in cell-associated form. By contrast, we did not detect PAF in bronchoalveolar exudates from patients with larungeal stenosis or with respiratory distress syndrome. LysoPAF, the direct precursor as well as initial metabolite of PAF, was also analyzed after being converted to PAF by acetylation. There was a wide variation in the amount of lysoPAF present in individual patients, suggesting that lysoPAF levels cannot be taken as an indicator for the presence of PAF. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Vitamin E. Deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129–240, 131–227 and 248–354 pmol/10⁶ cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [³H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [³H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28±0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06±0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26±0.71 and 4.26±0.06 nmol/min/mg protein, respectively), measured as degradation of [³H]PAF to [³H]lysoPAF.In vitro addition of α-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, in-dicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by α-tocopherol. The acetyltransferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 μM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 μM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.     

Synthesis of trideuteratedO-alkyl platelet activating factor and lyso derivatives

Racemic heavy isotope analogs of 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were prepared for use as internal standards to facilitate quantitative studies based on mass spectrometry. Starting from pentadencane-1,15-diol andrac-glycerol-1,2-acetonide, a convergent synthesis of 1-O-[16′-²H₃]hexadecyl and 1-O-[18′-²H₃]octadecylrac-glycero-3-phosphocholine and their acetyl derivatives is described. Three deuterium atoms were introduced at the terminal position of the 1-O-alkyl group by displacement of thep-toluensulfonyl group from 1-O-alkyl-15′-p-toluensulfonate and 1-O-alkyl-17′-p-toluensulfonate with [²H₃]-methylmagnesium iodide. The 1-O-alkyl-17′-p-toluensulfonate was obtained by reaction of the 1-O-alkyl-15′-p-toluensulfonate with allylmagnesium bromide, followed by reductive ozonolysis and treatment withp-toluenesulfonyl chloride. The hydroxyl group at C-2 was protected by a benzyl group and removed at a late stage in the synthesis. This provided the corresponding lysoderivatives or allowed preparation of racemic PAF by subsequent acetylation of the free hydroxy group. The phosphocholine moiety was introduced at glycerol C-3 by reaction with bromoethyldichlorophosphate and trimethylamine. The synthetic compounds were analyzed by FAB/MS and GC/NICIMS. They were shown to contain less than 0.6% protium impurity.     

Electrical conductivity in iodine-doped nonconjugated polyanilinefurfural

Polyanilinefurfural (PAF) has been prepared. Its chain contains aromatic and furan rings, but the backbone is nonconjugated. However, when treatment with iodine, the color of PAF turns metallic black, and PAF becomes electrically conductive. The electrical conductivity of I₂-doped PAF can reach 10⁻³ S ⋅ cm⁻¹ which is more than 10 orders of magnitude higher than what was observed at the pristine state. The effects of iodine content on the conductivity of PAF and the conductivity stability were investigated. FTIR spectra, U.v./vis absorption spectra, E.s.r. measurement and X.p.s. measurement of the undoped and doped PAF were studied. This paper will demonstrate that: if given appropriate substituents and dopants, significant charge transfer may be expected even for nonconjugated polymers and it may display electronic conductivity to a certain level. Received: 20 November 1996/Revised: 7 February 1997/Accepted: 10 February 1997     

Platelet activating factor induced respiratory mucosal damage

Human sinus mucosal specimens from eight normal individuals were exposed to platelet activating factor (PAF) at concentrations ranging from 10⁻⁶ M to 10⁻¹¹ M in a humidified CO₂ chamber at 37°C. The mucosal surface of the specimens was recorded on video tapes and magnified 2,500 times on a 19-inch television (TV) monitor. Ciliary activity of each ciliated cell was photoelectrically measured on the TV monitor in real time. PAF induced mucosal damage which resulted in a coarse profile including ciliostasis and exfoliation of epithelial cells. The length of the incubation period in which the initial coarse profile occurred on the mucosal surface inversely correlated with the concentration of exposed PAF ranging from 10⁻⁶ M to 10⁻¹⁰ M with r=−0.712 (p<2×10⁻⁴). Both the control medium and 10⁻⁸ M lysoPAF showed no effect on ciliary activity or mucosal surface alteration even after 24 hr of exposure. Significant ciliary inhibition was noted after 6 hr of exposure to PAF at concentrations of 10⁻⁸ M and 10⁻¹⁰ M (p<0.05). After 10 hr of exposure, significant ciliary inhibition (p<0.01) was noted at all concentrations. Inhibition occurred in a time- and dose-dependent manner. The length of the incubation period in which initial ciliostatis occurred and the level of PAF concentration showed an inverse correlation with r=−0.918 (p<10⁻⁶). These results indicate that PAF is cytotoxic to human respiratory mucosa. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Chemical and biochemical characterization of lignan analogs as novel PAF receptor antagonists

Various derivatives and isosteres of neolignans of the 2,5-diaryl tetrahydrofuran type have been synthesized as antagonists of platelet-activating factor (PAF). A detailed analysis of their structure-activity relationship (SAR) has revealed a clear preference for an asymmetrical molecular configuration with a high degree of stereo and chiral specificity associated with greater potency. Thetrans-2S,5S enantiomers are generally 10–200 times more potentin vitro than their correspondingcis ortrans-2R,5R isomers. A similar stereochemical preference is indicated by the recently reported PAF antagonist MK-287 which has undergone clinical evaluation. An azido derivative L-662,025 has been characterized as a photolabile irreversible antagonist of PAF for the investigation of solubilized and partially purified PAF binding proteins from cell membranes. The biological justification for concomitant inhibition of both PAF receptor and 5-lipoxygenase in inflammation is well recognized. The feasibility of developing such dual-functional agents has been demonstrated by a group of dithiolane analogs of neolignans and several derivatives of futoenone. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.