Mass spectral behavior and HPLC of some unusual molecular phospholipid species

The molecular species of the major phospholipids from the marine sponges Parasperella psila and Microciona prolifera were studied using chemical hydrolysis, enzymatic degradation and capillary gas chromatography (GC), high performance liquid chromatography (HPLC), desorption chemical ionization (DCI), fast atom bombardment (FAB) combined with collisionally activated decomposition (CAD) mass spectrometry. Two new solvent systems were developed for the isolation of these species from the sponges. Our investigations indicated the existence of unusual symmetrical phospholipids as major components. 1,2-Di-(5Z,9Z)-5,9-hexacosadienoyl-sn-glycero-3-phosph oethanolamine was found in both organisms, while 1,2-di(5Z,9Z,19Z)-5,9,19-hexacosatrienoyl-sn-gly cero-3-phosphoethanolamine was present in M. prolifera, 1,2-Di-(4Z,7Z,10Z,13Z,16Z,19Z)-4,7,1 0,13,16,19-docosahexaenoyl-sn-glycero-3- phosphocholine was the major molecular species in the PC fraction of M. prolifera.     

Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers

To analyze proteins interacting at the membrane interface, a phospholipid analogue was used with a photoactivatable headgroup (ASA-DLPE, N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) for selective cross-linking. The peripheral membrane protein cytochrome c from the inner mitochondrial membrane was rendered carbonate wash-resistant by cross-linking to ASA-DLPE in a model membrane system, validating our approach. Cross-link products of cytochrome c and its precursor apocytochrome c were demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), taking advantage of the intrinsic UV absorbance of the cross-linker. Application of the method to inner mitochondrial membranes from Saccharomyces cerevisae revealed cross-link products of both exogenously added apocytochrome c and endogenous proteins with molecular weights around 34 and 72 kDa. Liquid chromatograpy (LC)-MS/MS was performed to identify these proteins, resulting in a list of candidate proteins potentially cross-linked at the membrane interface. The approach described here provides methodology for capturing phospholipid-protein interactions in their native environment of the biomembrane using modern proteomics techniques.     

Evidence for the release of arachidonic acid through the selective action of phospholipase A2 in thrombin-stimulated human platelets

The release of arachidonic acid from thrombin-stimulated platelets can be attributed to the action of phospholipase A2 on membrane phospholipid. Previously, analysis of individual subclasses of phospholipid demonstrated that 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphocholine and to a lesser degree 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphoethanolamine were the main source of [3H]arachidonic acid in thrombin-stimulated cells. In the present work, 1,2-diacyl phospholipid subclasses were analyzed as 1,2-diacylglycerobenzoates by high-pressure liquid chromatography in order to analyze arachidonate release as mass changes in individual molecular species of phospholipid. Following thrombin stimulation (5 U/ml, 5 min, 37 degrees C) all arachidonoyl-containing molecular species of 1,2-diacyl-sn-glycerophosphocholine decreased in mass and [3H]arachidonate content by almost 50%, while those of 1,2-diacyl-sn-glycerophosphoethanolamine decreased by 20%. The mass change was substantial and indicated that these phospholipids are a major source of arachidonate in stimulated cells. No variation was seen in the other non-arachidonate-containing molecular species of either subclass. Thus, deacylation of membrane 1,2-diacylglycerophosphocholine and 1,2-diacylglycerophosphoethanolamine by phospholipase A2 is selective for those molecular species of phospholipid containing arachidonic acid, suggesting that a certain proportion of arachidonoyl-containing molecular species of phospholipid are compartmentalized with the platelet membrane proximal to the site of action of this enzyme. These studies demonstrate that the human platelet is a cell poised and specialized to release rapidly substantial amounts of arachidonic acid upon stimulation.     

Characterization of polar lipids of oral isolates of Candida, Pichia and Saccharomyces by Fast Atom Bombardment Mass Spectrometry (FAB MS)

AIMS: To characterize fatty acid and phospholipid analogue profiles of oral yeasts. METHODS AND RESULTS: Twenty-seven strains of oral yeasts were cultured on SDA and lipids of freeze-dried cells were extracted and analysed by FAB MS. The most abundant carboxylate anion was m/z 281 (C18 : 1). The most intense phospholipid analogue ions were of PE, PG, PA and PI. Pichia etchellsii contained molecular species of PG and PE, whereas Saccharomyces cerevisiae had PA, PG and PE analogues. Mass spectra revealed that S. cerevisiae and Candida glabrata were distinct from one another and from the other species tested. CONCLUSION: Oral yeasts largely differ with respect to their polar lipids. It is concluded that oral yeast species have distinctive fatty acid and phospholipid analogue anion profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: FAB MS provided novel chemotaxonomic information.     

Phospholipid analogues of Porphyromonas gingivalis

Porphyromonas has lipids containing hydroxy acids and C16:0 and iso-C15:0 major monocarboxylic acids among others. Nothing is known of its individual phospholipid molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species extracted from Porphyromonas gingivalis (seven strains), P. asaccharolytica (one strain) and P. endodontalis (two strains). Cultures on Blood-Fastidious Anaerobe Agar were harvested, washed and freeze-dried. Phospholipids were extracted and separated by fast atom bombardment mass spectrometry (FAB MS) in negative-ion mode. Phospholipid classes were also separated by thin layer chromatography (TLC). The major anions in the range m/z 209-299 were consistent with the presence of the C13: 0, C15: 0, C16: 0 and C18: 3 mono-carboxylate anions. Major polar lipid anion peaks in the range m/z 618-961 were consistent with the presence of molecular species of phosphatidylethanolamine, phosphatidylglycerol and with unidentified lipid analogues. Porphyromonas gingivalis differed from comparison strains of other species by having major anions with m/z 932, 946 and 960. Unusually, a feline strain of P. gingivalis had a major peak of m/z 736. Selected anions were studied by tandem FAB MS which revealed that peaks with m/z 653 and 946 did not correspond to commonly occurring classes of polar lipids. They were however, glycerophosphates. It is concluded that the polar lipid analogue profiles obtained with Porphyromonas are quite different from those of the genera Prevotella and Bacteroides but reveal heterogeneity within P. gingivalis.     

Fast atom bombardment and tandem mass spectrometry for structure determination of cysteine, N-acetylcysteine, and glutathione adducts of xenobiotics

Fast atom bombardment mass spectrometry (FAB/MS) and FAB combined with tandem mass spectrometry (MS/MS) were examined for their applicability to the structure determination of xenobiotics conjugated with the members of the glutathione family (glutathione, cysteine, and N-acetylcysteine). Comparisons between FAB/MS and thermospray MS data are made. FAB/MS is generally successful at generating molecular ion species under both positive and negative ion conditions. Upon collisional activation the adducts undergo characteristic cleavages around the sulfur providing structural information about the conjugate. The analysis of the N-acetylcysteine conjugate isolated from rat urine is presented as an example of the application of FAB/MS/MS to biological problems.     

Phospholipid molecular species distribution of some medically important Candida species analysed by fast atom bombardment mass spectroscopy

The aim of this study was to obtain detailed information on phospholipids (PL) of the medically important Candida species and to determine their possible chemotaxonomic significance. Lipids were extracted from 22 strains representing 8 Candida species and their PL molecular species distributions were determined by Fast Atom Bombardment Mass Spectroscopy (FAB MS) in negative ion mode. Fifteen major lower mass peaks (m/z 221 to 289) were attributable to the expected presence of carboxylate anions and 24 major higher mass peaks (m/z 557 to 837) were attributable to phospholipid anions. Major carboxylate peaks were of the following m/z and identities : 253, C16:1; 255, C16:0; 277, C18:3; 279, C18:2; 281, C18:1; and 283, C18:0. The most abundant peaks consistent with the presence of phospholipid molecular species anions include those of m/z 673, 743, 833, 834 and 836 tentatively identified as phosphatidic acid (PA) (34:1), phosphatidylglycerol (PG) (34:3), phosphtidylinositol (PI) (34:2) and two unknown molecular species. This profile is diagnostic for the genus Candida. Quantitative differences were observed between different Candida species. Thus, polar lipid molecular species distribution in Candida spp. has chemotaxonomic significance, especially so in the case of carboxylate anions.     

Turnover Rates of the Molecular Species of Ethanolamine Plasmalogen of Rat Brain

1,2-Diradyl-3-acetylglycerols prepared from 1-O-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (alkenylacyl-GPE, ethanolamine plasmalogen) and 1-alkyl-2-acylglycero-3-phosphoethanolamine (alkylacyl-GPE) of rat brain at 18 days of age were subfractionated into six species by AgNO3-impregnated TLC. The percent compositions of substractions were compared with that of 1,2-diacylglycero-3-phosphoethanolamine (diacyl-GPE). The incorporation rate of [1-3H]glycerol into each molecular species was also estimated to examine the turnover rate and selective synthesis of molecular species of ethanolamine phosphoglycerides (EPG). Among the molecular species of EPG, a major proportion contained polyunsaturated fatty chains, and the sum of tetraene-, pentaene-, and hexaene-containing species was greater than 65% in common with three classes of EPG. It was possible to calculate the turnover time, synthesis rate, and synthesis rate constant of ethanolamine plasmalogen in myelinating rat brain by the equation of Zilversmit et al. since the time-dependent change of specific activity and the distribution of molecular species indicated that each molecular species of alkenylacyl-GPE is synthesized from the corresponding species of alkylacyl-GPE. The observed turnover time of ethanolamine plasmalogen was about 5 h. The observed turnover times of the various molecular species were of the order: tetraene greater than or equal to hexaene greater than pentaene greater than or equal to monoene greater than or equal to diene. The synthesis rate constants of each molecular species, in the formation of alkenylacyl-GPE from alkylacyl-GPE, were of the order: hexaene greater than tetraene greater than pentaene greater than diene greater than or equal to monoene.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: I. Structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer

A method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of glycosphingolipids was developed, which involves a frit interface between the HPLC and the MS. The molecular species of glucosylceramide (GlcCer) purified from the spleen of a patient with Gaucher's disease and galactosylglobotetraosylceramide (IV3 beta Gal-Gb4Cer) from mouse kidney were analyzed using this system on a reversed-phase column, with methanol containing 1% glycerol as the elution solvent. The injection of 1 microgram of GlcCer gave the mass spectra of seven major molecular species, the pseudo-molecular ion for each of the seven molecular species being observed at m/z 698, 726, 754, 782, 808, 796, and 810, respectively. The injection of 200 pg of synthetic N-stearoyl glucosylsphingosine (d18:1) gave a clear peak with the single ion monitoring method detecting the pseudo-molecular ion at m/z 726. The injection of 5 micrograms of IV3 beta Gal-Gb4Cer gave the mass spectra of six major molecular species, the pseudo-molecular ions being observed at m/z 1,489, 1,471, 1,515, 1,497, 1,517, and 1,499. This report deals with a new HPLC/FAB/MS system, which was successfully applied to the structural characterization of the molecular species of neutral glycosphingolipids, and the system is a quite promising for development into a quantitative method for glycosphingolipids with high sensitivity and specificity.     

Cholesterol interactions with tetracosenoic acid phospholipids in model cell membranes: role of the double-bond position

The synthesis and thermotropic properties of 1,2-di-(9Z)-9-tetracosenoylphosphatidylcholine [delta 9-PC(24:1,24:1), 1], 1,2-di-(5Z)-5-tetracosenoylphosphatidylcholine [delta 5-PC(24:1,24:1), 2], and 1,2-di-(15Z)-15- tetracosenoylphosphatidylcholine [delta 15-PC(24:1,24:1), 3] are reported. Liposomes prepared from these phospholipids differ from those of the natural sponge phospholipids, 1,2-di-(5Z,9Z)-5,9-hexacosadienoylphosphatidylcholine (4a) and the corresponding ethanolamine (4b), both of which virtually exclude cholesterol from their bilayers. The behavior of 1 and 2 is similar to that of 1,2-di-(6Z,9Z)-6,9-hexacosadienoylphosphatidylcholine (5), which exhibits a partial molecular interaction with cholesterol. In the case of 3, cholesterol appears to interact with the saturated acyl chain regions of this phospholipid in a manner similar to that of its interaction with DPPC acyl chains. This study delineates the effect of the double-bond location in long fatty acyl chains of phospholipids on their interactions with cholesterol.     

Transbilayer complementarity of phospholipids in cholesterol-rich membranes

Lipid-lipid interactions across cholesterol-rich phospholipid bilayers were investigated by measuring nearest-neighbor preferences of exchangeable phospholipids derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), in the presence of nonexchangeable dimers (i.e., templates) made from DMPE or DSPE. When homotemplates were present, a significant preference for homophospholipid association was observed. In contrast, when the corresponding heterotemplate was present, heterodimer formation was favored. These results support a model in which the longer phospholipid in one monolayer preferentially associates with the shorter one in the adjoining monolayer. In the absence of cholesterol, transbilayer complementarity was also observed but to a lesser degree. Transbilayer complementarity of phospholipids is likely to play an important role in stabilizing biological membranes and in promoting a compositional interdependence of their two lipid leaflets.     

Biophysical properties of unusual phospholipids and sterols from marine invertebrates

Liposomes composed of 1,2-di-(5Z,9Z)-5-9-hexacosadienoyl-sn-glycero-3-pho sph ocholine underwent an endothermic phase transition at 42 degrees C. Cholesterol or the marine sterols studied did not affect this transition to an appreciable extent, but rather were excluded from the phospholipid bilayers. Membranes composed of 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl-sn-glycero-3-pho sph ocholine displayed very similar phase properties. Effects of the marine sterols on dipalmitoylphosphatidylcholine bilayers were also investigated.     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: II. Application to neutral glycolipids and monosialogangliosides

We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829-833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3 alpha GalNAc-Gb4Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 micrograms of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 micrograms of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Phospholipid molecular species of Bacteroides

The fatty acid composition, and types of polar lipid (PL) present, have been well-studied in Bacteroides. Nothing is known, however, of the detailed structures of individual phospholipid and molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species present in Bacteroides. Thirteen culture collection strains were harvested, washed and freeze-dried. Polar lipids were extracted and separated by conventional fast atom bombardment mass spectrometry (FAB MS). For each strain, hundreds of polar lipid peaks were seen. Nineteen major anions in the range m/z 209–299 were separated. The most intense of these was consistent with the expected presence of the C_15:0 anion. Other anions were attributable to saturated, mono-unsaturated, di-unsaturated, tri-unsaturated and hydroxy-carboxylate ions. Twenty-two major anion peaks were recorded in the range m/z 505–722. These were consistent with the presence of analogues of phosphatidic acid (PA), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The most intense peaks included those consistent with the presence of PE(30:0), PG(29:1), PG(30:1), PG(28:1), PE(31:0), PE(OH-30:0), PG(31:1) and PE(OH-31:0). This combination of PL molecular species is unique to Bacteroides and has not been reported in other organisms so far examined.     

12(S)-Hydroperoxy-eicosatetraenoic acid increases arachidonic acid availability in collagen-primed platelets

Lipid hydroperoxides have been reported to regulate cell function and eicosanoid formation. The aim of the present study was to determine the effect of 12(S)-hydroperoxy-eicosatetraenoic acid [12(S)-HPETE], the platelet 12-lipoxygenase-derived hydroperoxide of arachidonic acid (AA), on the availability of nonesterified AA, which represents a rate-limiting step in the biosynthesis of eicosanoids. The coincubation of human platelets with concentrations of 12(S)-HPETE below 50 nM and subthreshold concentrations (STC) of collagen (less than 0.24 microg/ml) significantly enhanced platelet aggregation and the formation of thromboxane B(2), the stable catabolite of the potent aggregating agent thromboxane A(2). In addition, the nonesterified endogenous AA concentration increased by 3-fold. Arachidonoyl-containing molecular species concentrations of 1,2-diacyl-glycero-3-phosphocholine, 1-alkyl-2-acyl-glycero-3-phosphocholine, and 1-alkenyl-2-acyl-glycero-3-phosphoethanolamine decreased specifically in response to 12(S)-HPETE, whereas no significant changes were observed within 1,2-diacyl-glycero-3-phosphoethanolamine and 1,2-diacyl-glycero-3-phosphoinositol molecular species. The 12(S)-HPETE-induced increase in nonesterified AA was fully prevented by arachidonoyl trifluoromethyl ketone, an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), and cPLA(2) was translocated to membranes and phosphorylated in platelets incubated with 12(S)-HPETE.In conclusion, these results indicate that nanomolar concentrations of 12(S)-HPETE could play a significant role in controlling the level of endogenous AA and the formation of thromboxane, thereby potentiating platelet function.     

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H]⁺ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.     

Metabolomic identification of potential phospholipid biomarkers for chronic glomerulonephritis by using high performance liquid chromatography-mass spectrometry

Plasma phospholipids metabolic profile of chronic glomerulonephritis was investigated using high performance liquid chromatography/mass spectrometry (LC/MS) and principal component analysis. The plasma samples of 18 patients with chronic glomerulonephritis, 17 patients with chronic renal failure (CRF) without renal replacement therapy and 18 healthy persons were collected and analyzed. It was found that combination of the LC/MS technique with PCA can be successfully applied to phospholipid profile analysis. The results showed that primary chronic glomerulonephritis and CRF had phospholipids metabolic abnormality. Nineteen phospholipid species were identified as possible biomarkers in plasma samples of chronic glomerulonephritis and chronic renal failure. Moreover, the identification of the molecular structure of the potential phospholipid markers was obtained by ESI-MS/MS experiment. It suggests that phospholipids can be used as potential biomarkers on the progress of primary chronic glomerulonephritis.     

Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry

Two physicochemical methods have been developed for the quantitative analysis of lyso-platelet activating factor (lyso-PAF) based on gas-liquid chromatography-mass spectrometry (GLC/MS) and fast atom bombardment-mass spectrometry (FAB/MS) using stable isotope dilution. After addition of deuterated internal standards, lyso-PAF produced from neutrophils was purified by silicic acid chromatography and thin-layer chromatography (TLC). The GLC/MS assay employed phospholipase C or hydrofluoric acid for hydrolysis of the phosphocholine moiety to yield ether monoglycerides. Condensation of monoglycerides with acetone yielded the 1-O-alkyl-2,3-isopropylidene glycerol which could be analyzed by GLC/MS. The ions corresponding to M-15 fragments for both the labeled and unlabeled derivatives were monitored in a selected ion recording mode. Standard curves were found to be linear over the range tested (10-2000 ng) with a limit of detection found to be below 200 pg injected on column. For the FAB/MS assay, the unmodified lyso-PAF was well suited for direct analysis; however, the limit of detection (S/N greater than 3) using a glycerol matrix was found to be 5 ng placed on the probe tip. It was found that human neutrophils contain approximately 300 pg/10(6) cells which increased 2-3-fold during the 5-min period following challenge with 1.9 microM calcium ionophore, A23187. Two molecular species of lyso-PAF were identified as hexadecyl and octadecyl ethers at sn-1 with the octadecyl molecular species of lyso-PAF predominating in abundance after stimulation.     

Isolation and identification of ferrioxamine G and E inHafnia alvei

Summary Under conditions of iron-deprivationHafnia alvei (Enterobacteriaceae) produces ferrioxamine G as the principal siderophore. Maximum hydroxamate siderophore production occurred at medium iron limitation. The ferrioxamines were extracted, purified by gel filtration and chromatography on silica gel yielding a major and a minor siderophore fraction. The minor siderophore fraction contained three siderophores, among which ferrioxamine E could be identified by HPLC and FAB mass spectrometry. Reductive hydrolysis of the ferrioxamine G fraction yielded succinic acid and a mixture of diaminopentane and diaminobutane, as determined by gas-liquid chromatography and GLC/MS. HPLC and FAB mass spectrometry confirmed that the ferrioxamine G fraction consisted of two different species, G₁ and G₂, possessing molecular masses of 671 Da and 658 Da respectively.