High performance liquid chromatography of platelet-activating factors

Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.     

Influence of alkyl ether chain length of acetyl glyceryl ether phosphorylcholine and its ethanolamine analog on biological activity toward rabbit platelets

The preparation of the potent new lipid chemical mediator, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), through a facile semisynthetic procedure utilizing the vinyl ether-containing lecithins (plasmalogens) of bovine heart muscle as the starting material results in a derivative with mixed chain alkyl residues. In order to focus more closely on the importance of the various components of AGEPC relative to its biological activity, it was of importance to develop a method for isolation of molecules rich in a specific chain length of the alkyl residue. In the current study it was found that a simple thin-layer chromatographic technique, using a solvent system of methanol water (2:1, $\text{v}\text{v}$), afforded an excellent separation of semisynthetic AGEPC into two species, one containing over 95 mol% 16:0 and the other 95 mol% 18:0 alkyl chain species. The same procedure allowed a comparable separation of the species of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylethanolamine (AGEPE) prepared from AGEPC by phospholipase D action. On the basis of their effectiveness in releasing serotonin from washed rabbit platelets, the 16:0-rich derivatives of AGEPC and AGEPE exhibited significantly higher specific activities, from three to six-fold, on a molar basis, respectively, than the corresponding 18:0-rich derivatives. These findings are discussed in relation to the importance of the chain length of the alkyl ether group in expression of the biological activity of AGEPC.     

Alkyl chain homologs of platelet-activating factor and their effects on the mammalian heart

Platelet-activating factor (PAF; AGEPC) is a potent negative inotropic and coronary-vasoconstricting agent. Minor structural alterations in the PAF molecule are known to greatly affect its biological activity; thus, we have investigated the effects of selected synthetic saturated and unsaturated alkyl chain PAF homologs on the isolated guinea pig heart. The rank order of potency for the negative inotropic effect was C16:0- greater than C18:1- greater than beef-heart AGEPC greater than C15:0- greater than C18:0- greater than C14:0-AGEPC; the rank order for the coronary-vasoconstricting effect was C16:0- approximately C18:1- approximately beef-heart AGEPC greater than C15:0- greater than C18:0- approximately C14:0-AGEPC. With the exception of C16:0- and C18:1-AGEPC, the relative potencies for the cardiac and coronary effects of the alkyl chain AGEPC homologs did not correlate well with their relative potencies in stimulating rabbit platelets and human neutrophils. The differences in the rank order of potency for these AGEPC homologs suggest the presence of species and/or target cell PAF receptor heterogeneity.     

Occurrence of platelet-activating factor in rabbit spermatozoa

Spermatozoa obtained from rabbit ejaculate were analyzed for the presence of platelet-activating factor [PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)] by using standard HPLC and TLC procedures. Fractions corresponding to synthetic PAF (AGEPC) revealed PAF-like activity amounting to 0.35 +/- 0.06 pmol/10(8) cells (mean +/- SE) as determined by bioassays based on the release of [3H]serotonin from washed rabbit platelets. This activity was lost upon base-catalyzed methanolysis, but was restored to the original level after reacetylation. Analysis of the phosphatidylcholine (PC) fraction by GC-MS subsequent to base-catalyzed methanolysis showed that 1-O-alkyl-2-acylphosphocholine comprises about 12% of the PC fraction with alkyl chain lengths of 16:0 (88%) and 18:0 (12%).     

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Molecular conformation of prostaglandin A1

The crystal and molecular structure of prostaglandin A₁ (PGA₁) has been determined by X-ray diffraction. (Space group P2₁2₁2₁, $\text{a}\text{= 18.10}\text{A}\text{?}\text{A}$, $\text{b}\text{= 21.09}\text{A}\text{?}\text{A}\text{,}\text{c}\text{= 5.42,}\text{Z}\text{= 4)}$. Comparison of the structure of PGA₁ with that of PGF_1β (determined previously as the tribromobenzoate) indicates significant differences in the relative intramolecular side chain orientations. However, conformation and torsional angles within each side chain of PGA₁ are retained with little or no change in comparison with PGF_1β, indicating the presence of the C₁₅-bromobenzoate groups in the latter have had little effect in altering internal side chain conformation in the solid state. The overall differences in relative side chain orientation in PGA₁ are attributable to chemical and conformational changes in the substituted cyclopentane moiety (C₈-C₁₂).     

Fast atom bombardment-mass spectrometric identification of molecular species of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

Fast atom bombardment mass spectrometry was used to identify molecular species of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes. Normal and reverse-phase high performance liquid chromatography were employed to separate the individual regions with PAF activity prior to mass spectrometric analysis. The following alkyl chain homologs of acetyl glyceryl ether phosphorylcholine (AGEPC) were found: C16:0, C17:0, C18:0 and C18:1. There was also evidence for the presence of the C15:0 homolog, as well as other species which have not yet been identified.     

Sex pheromone of the Asian corn borer moth

By 1975, insect sex pheromones had been identified from 60 species (1) of moths (Lepidoptera), and nearly all of the 40 compounds involved had been found to be mono- or di-unsaturated C₁₀ to C₁₈ straight-chain aldehydes, alcohols, or acetates, and the sites of olefinic unsaturation were, with one exception (2), at odd-numbered positions in the carbon chain. Since that time, many more lepidopteran pheromone components have been identified, and mono-unsaturated compounds with unsaturation at even-numbered positions in the carbon chain remain extremely rare (3–6). However, in females of $\text{Ostrinia furnacalis}$ (Guenee), called the Asian corn borer, we have discovered another mono-olefinic lepidopteran sex pheromone that has the site of unsaturation at ab even carbon position. Females of this corn-pest species produce a $\text{ca}$. 1:1 geometric mixture of ($\text{Z}$)- and ($\text{E}$)- 12-tetradecen-1-o1 acetate to attract and sexually stimulate males. On the other hand, the nearest taxonomic relative (7) of the Asian corn borer, the European corn borer, $\text{Ostrinia nubilalis}$ (Hübner), uses a mixture of ($\text{Z}$)- and ($\text{E}$)-11-tetradecen-1-o1 acetate as its sex pheromone (8). Biochemical explanation of the paucity of monounsaturated moth sex pheromones with olefinic sites at even numbered carbon positions remains to be elucidated.     

Hydroxylation of n-alkanes to secondary alcohols and their esterification in the grasshopper Melanoplus sanguinipes

Labeled n-alkanes administered to the grasshopper $\text{Melanoplus}$$\text{sanguinipes}$ are hydroxylated at or near the middle of the carbon chain. The secondary alcohols formed are then esterified. Chain length specificity is evident in both the hydroxylation of n-alkanes and the esterification of secondary alcohols, with the shorter chain C₂₃, C₂₁, C₁₉, and C₂₅ compounds converted to secondary alcohol wax esters more readily than the longer chain C₂₇, C₂₉, and C₃₁ compounds. Secondary alcohols and ketones are not reduced to alkanes.     

Neocarzinostatin chromophore: Presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C₁₂-subunit of the previously determined partial structure $\text{2a}$ (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C₁₂-substructure, whereas a parallel two-step reduction occurs on NaBH₄ treatment. Both reactions occur with rearrangement of the C₁₂-substructure and the implication for the mechanism of action of NCS-Chrom $\text{A}$ in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom $\text{A}$ as well as minor components $\text{B}$ and $\text{C}$ by two protons.     

Marine sterols. V. Isolation and structure of occelasterol, a new 27-norergostane-type sterol, from an annelida, Pseudopotamilla occelata

Occelasterol, a new marine C₂₇ sterol, has been isolated from an annelida, $\text{Pseudopotamilla occelata}$ and its structure was confirmed as 22-$\text{trans}$-27-nor-(24S)-24-methylcholesta-5, 22-dien-3β-ol (IIa) from the spectral data and by synthesis. Thissterol, the second member of a class of sterols having 27-norergostane-type side chain, had been formerly regarded as 22-$\text{cis}$-cholesta-5, 22-dien-3β-ol (Va). Gas-liquid Chromatographic studies have shown that occelasterol is distributed in various amounts in most of marine invertebrates.     

Phase transition characteristics of diphosphatidylglycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers: Mono- and divalent metal ion effects

Synthesis and phase transition characteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>m</mtext>}}$) and the enthalpy of transition ($\text{ΔH}$) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric $\text{sn-3,3-}\text{and}\text{sn-1,1-}\text{phosphatidyldiacylglycerol}$ and for the diastereomeric, $\text{meso}\text{-sn-1,3-}\text{phosphatidyldiacylglycerol}$ are approximately the same. The molar enthalpy for the transition of cardiolipin-NH₄⁺ bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C_{1 6}-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e. the highest $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ is observed for Ca²⁺- cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca²⁺. The lowest $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ is observed for Rb⁺-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.     

High pressure liquid chromatographic separation of the metabolites of 3-methylcholanthrene

All the known metabolites of 3-methylcholanthrene can be separated by high pressure liquid chromatography on a μBondapak C₁₈ column. The separation is accomplished with a reverse phase linear gradient of 40% acetonitrile water to 100% acetonitrile at room temperature. The addition of 0.2% acetic acid to the 40% acetonitrile chamber is required for resolution of the $\text{cis}$ 1,2-dihydrodiol.     

Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine model membrane structure as studied by 31P-NMR

The interaction of |C_nH_2n+1N⁺(CH₃)₃| · I^? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by ³¹P-NMR spectroscopy. It is shown that the effective anisotropy of ³¹P chemical shift (?Δσ_eff) of the lamellar phospholipid liquid-crystalline phase L_α increases with increasing concentration and alkyl chain length of the drug. Addition of $\text{|}\text{C}{}_6\text{H}{}_{13}\text{N}{}^{\mathrm{+}}\text{(CH}{}_3\text{)}{}_3\text{| ·I}{}^{\mathrm{?}}$ or |C₉H₁₉N⁺(CH₃)₃|·I^? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n  12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells.     

Influence of local and neutral anaesthetics on the polymorphic phase preferences of egg yolk phosphatidylethanolamine

(1) The polymorphic phase preferences of egg phosphatidylethanolamine have been examined in the presence of normal alcohols and alkanes of varying chain length, as well as charged amine anaesthetics. (2) It is shown that the charged anaesthetics, ethanol and butanol can stabilize a bilayer arrangement for egg phosphatidylethanolamine. In contrast, longer chain ($\text{C}\text{?6}$) normal alcohols and alkanes induce the hexagonal (H_II) phase. (3) The relative potency of local anaesthetics in vitro (chlorpromazine, dibucaine, tetracaine and procaine) is mirrored by their relative ability to stabilize bilayer structure for hydrated egg phosphatidylethanolamine. Further, the aqueous concentrations of anaesthetic required to affect phospholipid polymorphism is sensitive to the lipid composition. For example, the inclusion of 20 mol% egg phosphatidylserine in egg phosphatidylethanolamine dispersions can reduce the aqueous concentrations of dibucaine required to induce appreciable bilayer stabilization effects from 5.0 mM to 0.5 mM. (4) It is suggested that the ability of amphipatic molecules such as anaesthetics to influence phosphatidylethanolamine polymorphism arises from their molecular shape. The possibility that anaesthetic molecules may exert their effects by virtue of this shape property is raised.     

Elasnin,a new human granulocyte elastase inhibitor produced by a strain of Streptomyces

Elasnin, a new human granulocyte elastase inhibitor, has been isolated from $\text{Streptomyces}\text{noboritoensis}$ KM-2753. Elasnin is a neutral, lipophilic colorless and viscous oil ($\text{n}{}_{\mathrm{D}}{}^{17}\text{=1.4983}$, $\text{[α]}{}_{\mathrm{D}}{}^{18}\text{?0.9°}$, λ_max^EtOH 291 nm (ε, 7760)). The molecular formula was C₂₄H₄₀O₄ (M.W.: 392) as determined by its elemental analysis and mass spectrum. Elasnin inhibits markedly human granulocyte elastase, but is almost ineffective for pancreatic elastase, trypsin, chymotrypsin, thermolysin and papain.     

Synthesis,enzyme inhibition,and antitumor activity of new 1,4-benzoquinone analogs of coenzyme Q10

A rationale based upon coenzyme Q₁₀ (CoQ₁₀, ubiquinone) for the synthesis of potential antitumor agents constitutes a new approach in the search toward chemotherapy of cancer. The antitumor activities of 38 alkyl-1,4-benzoquinones, analogs of coenzyme Q, 24 of which are new compounds, are described. The 10 best antitumor analogs of CoQ all showed long-term cures of Walker carcinosarcoma 256 in rats. Particularly impressive were the 6-n-octylmercapto-5-chloro-2,3-dimethoxy-1,4-benzoquinone (NSC 252188), which cured six out of six rats with $\text{\%}\text{T}\text{C}\text{= 584 at 3.13 mg/kg}$, 6-phytyl-5-hydroxy-2,3-dimethoxy-1,4-benzoquinone (NSC 277818) (four out of four cures, $\text{\%}\text{T}\text{C}\text{= 923 at 50 mg/kg}$), and 5-phytyl-2,3-dimethoxy-1,4-benzoquinone (NSC 276371) (three out of six cures, $\text{\%}\text{T}\text{C}\text{= 789 at 0.78 mg/kg}$). In general, a 5-chloro or 5-hydroxy group on the quinone nucleus or a side chain with unsaturation and branching, such as the phytyl side chain of NSC 277818 and NSC 276371, seemed to increase antitumor activity. Although a perfect correlation was not to be expected, many of the most potent antitumor analogs were also among the best in vitro inhibitors of the mitochondrial CoQ₁₀-enzymes, succinoxidase, and NAD oxidase.     

Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugates of cortisol and cortisone.

Superior antitumor activity of 1-β-D-arabinofuranosylcytosine (ara-C) conjugates of prednisolone and prednisone against L1210 leukemic mice, based on ara-C content, has encouraged us to synthesize 5′-(cortisone-21-phosphoryl)-1-β-D-arabinofuranosylcytosine (I) and 5′-(cortisone-21-phosphoryl)-1-β-d-arabinofuranosylcytosine (II) by condensation of N⁴,2′,3′-triacetyl-1-β-d-arabinofuranosylcytosine 5′-monophosphate with cortisol and cortisone in the presence of N,N′-dicyclohexylcarbodiimide at room temperature followed by removing the acetyl groups in 2 N methanolic ammonia in 20% yield. The conjugates I and II inhibited the $\text{in}\text{vitro}$ growth of L1210 by 50% (ED₅₀) at 0.25 μM and 0.07 μM, respectively, while ara-C showed ED₅₀ 0.1 μM. However, the conjugates I and II exhibited 287% and 238% of $\text{T}\text{C}$ at 50 mg/kg/day × 5 doses against L1210 leukemic mice, respectively, while ara-C at 25 mg and 50 mg/kg/day × 5 gave the respective 127% and 110% of $\text{T}\text{C}$.     

Quantitation of guanosine 5',3'-bisdiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography

A new method to determine the guanosine 5′,3′-bisdiphosphate (ppGpp) concentration in bacterial cultures has been developed. Cells in which the nucleotide pool was stabilized by treatment with formaldehyde were concentrated from samples of cultures and lysed by alkali. The ppGpp was separated from other nucleotides on a reverse phase C₁₈ column by ion-pair high performance liquid chromatography using an isocratic elution system. The area of the peak corresponding to ppGpp in elution profiles obtained by monitoring the absorbance of the eluant at 254 nm was used to quantitate ppGpp. The method allows detection of as little as 1 pmol of ppGpp per A₄₆₀ of culture, with $\text{2.5}\text{pmol}\text{A}{}_{460}$ being quantitated with greater than 90% accuracy.     

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine: A novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187

Rabbit peritoneal neutrophils incorporated [¹⁴C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[¹⁴C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the $\text{sn}$-1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1-$\text{O}$-alkyl-2-arachidonoyl-$\text{sn}$-glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1-$\text{O}$-alkyl-2-lyso-GPC is the metabolic precussor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.