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Biomedical subjects

Robert C Murphy

Publications and source records attributed to Robert C Murphy.

53 records · Page 3Linked to original sources
PubMed2003

Endotoxins stimulate neutrophil adhesion followed by synthesis and release of platelet-activating factor in microparticles.

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.

Blood Platelets↗
PubMed2003

Ligand-receptor binding revealed by the TNF family member TALL-1.

The tumour necrosis factor (TNF) ligand TALL-1 and its cognate receptors, BCMA, TACI and BAFF-R, were recently identified as members of the TNF superfamily, which are essential factors contributing to B-cell maturation. The functional, soluble fragment of TALL-1 (sTALL-1) forms a virus-like assembly for its proper function. Here we determine the crystal structures of sTALL-1 complexed with the extracellular domains of BCMA and BAFF-R at 2.6 and 2.5 A, respectively. The single cysteine-rich domain of BCMA and BAFF-R both have saddle-like architectures, which sit on the horseback-like surface formed by four coil regions on each individual sTALL-1 monomer. Three novel structural modules, D2, X2 and N, were revealed from the current structures. Sequence alignments, structural modelling and mutagenesis revealed that one disulphide bridge in BAFF-R is critical for determining the binding specificity of the extracellular domain eBAFF-R to TALL-1 instead of APRIL, a closely related ligand of TALL-1, which was confirmed by binding experiments in vitro.

Amino Acid Sequence↗
PubMed2003

Urinary metabolites of leukotriene B4 in the human subject.

Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB4, four human subjects were injected with 150 nmol/kg LTB4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0-6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB4. In the current investigation, 11 different metabolites of LTB4 were identified in the urine from those subjects injected with LTB4, and none were present in the urine from the placebo-injected subject. The unconjugated LTB4 metabolites found in urine were structurally characterized as 18-carboxy-LTB4, 10,11-dihydro-18-carboxy-LTB4, 20-carboxy-LTB4, and 10,11-dihydro-20-carboxy-LTB4. Several glucuronide-conjugated metabolites of LTB4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB4, 10-hydroxy-4,6,12-octadecatrienoic acid, LTB4, and 10,11-dihydro-LTB4. The amount of LTB4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB4 (18.9-30.6 pmol/ml) present in the urine of subjects injected with LTB4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with beta-glucuronidase. The urinary metabolites of LTB4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.

Chromatography, High Pressure Liquid↗
PubMed2003

Biosynthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid from 5-hydroperoxyeicosatetraenoic acid in the murine macrophage.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid shown to possess important biological activities within different cell types. In the neutrophil, a specific NADP(+)-dependent dehydrogenase utilizes 5-lipoxygenase-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) as the required substrate. In the present study, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE), rather than 5-HETE, was found to be the biosynthetic precursor of 5-oxo-ETE in the murine macrophage. The macrophage was not able to convert 5-HETE into 5-oxo-ETE even when preincubated with phorbol ester or with other lipid hydroperoxides. The factor responsible for the conversion of 5-HpETE into 5-oxo-ETE was found predominantly in the cytosolic fraction of the macrophage, with an approximate molecular weight of 50,000-60,000, as assessed by size exclusion chromatography. Formation of 5-oxo-ETE was rapid and the catalytic protein was found to have an apparent K(m) of 5.3 microM for the eicosanoid. Furthermore, the protein could efficiently utilize 5(R,S)-HpETE as substrate and was heat and protease labile. This novel pathway of 5-oxo-ETE biosynthesis in the murine macrophage was consistent with reduction of a 5-hydroperoxy group to an intermediate alkoxy radical that could be subsequently oxidized to the 5-oxo product. Such a mechanism would enable racemic 5-HpETE, derived from free radical oxidation of arachidonic acid, to be efficiently converted into this potent chemotactic eicosanoid.

Animals↗
PubMed2003

Covalent binding of leukotriene A4 to DNA and RNA.

Leukotriene A(4) (LTA(4)) is a highly reactive electrophilic intermediate formed during the biosynthesis of the lipid mediators leukotriene B(4) and leukotriene C(4). Deoxynucleosides were found to react as nucleophiles with LTA(4) in aqueous solutions as assessed by UV spectroscopy and electrospray ionization mass spectrometry. Aqueous solutions of native DNA and RNA were also found to react with LTA(4) as assessed by mass spectrometric analysis of the constituent nucleosides derived from enzymatic hydrolysis of the nucleic acids. The most abundant adducts were observed for guanine- and adenine-containing deoxynucleosides and nucleosides. At neutral pH, these reactions led to an overall modification of deoxyguanosine/guanosine residues in DNA and RNA at 15 +/- 1 adducts/10(7) bases and 230 +/- 20 adducts/10(7) bases, respectively, determined by quantitative assay using stable isotope-labeled LTA(4)-nucleoside adduct. An estimation of the relative reactivity of LTA(4) with each of the purine and pyrimidine bases in DNA and RNA was carried out by comparisons of the mass spectral ion abundance of the different adducts (LTA(4)-dAdo, LTA(4)-dCyd, LTA(4)-Thd, LTA(4)-Ado, LTA(4)-Cyd, and LTA(4)-Urd) to the ion signal of known amounts of LTA(4)-dGuo and LTA(4)-Guo standards. The data were corrected for different mass spectrometric response factors that were experimentally determined for each adduct product. The structures of the two most abundant LTA(4)-Guo products were determined by NMR, UV spectroscopy, and mass spectrometry to be 5-hydroxy,12-[Guo-N(2)-yl]-6,8,11,14-eicosatetraenoic acid. Stimulation of human neutrophils with calcium ionophore led to the covalent modification of DNA within the cell as determined by mass spectrometric analysis of lipophilic nucleosides obtained after hydrolysis of extracted DNA. These observations, combined with the intracellular site of 5-lipoxygenase translocation and LTA(4) biosynthesis at the nuclear envelope, suggest that LTA(4) may have access to DNA and RNA within cells and furthermore modify nucleic acids in situ following the activation of 5-lipoxygenase and initiation of LTA(4) biosynthesis.

Calcimycin↗
PubMed2002

Glutathione adducts of oxyeicosanoids.

Glutathione (GSH) is a major cellular antioxidant, which can conjugate chemically reactive, electrophilic molecules and thus, prevent unwanted reactions with important cell constituents. A large number of electrophilic eicosanoids, in particular alpha/beta-unsaturated ketones, are synthesized during arachidonic acid oxidative metabolism which can participate in the Michael addition reaction with GSH catalyzed by the GSH-S-transferase (GST) family. The structures of these adducts have been determined primarily using mass spectrometry techniques in the past after degradation to volatile products, but more recently by electrospray ionization. GSH-adducts have been observed with molecules synthesized through the 5-lipoxygenase (LTB4, LTC4, and 5-oxo-ETE), 12-lipoxygenase (hepoxilin A3), 15-lipoxygenase (13-oxo-ODE), PGH synthase (PGA1, PGA2, PGD2, PGE2, and PGJ2), and cytochrome P450-epoxygenase (14,15-EET) pathways of arachidonic acid metabolism. It has also been demonstrated that these oxyeicosanoid GSH-adducts do not represent just inactivation products, but they can both retain (GSH-adduct of hepoxilin A3) or show novel biological activities (LTC4 and FOG7).

Antioxidants↗
PubMed2002

Mass spectrometric analysis of leukotriene A4 and other chemically reactive metabolites of arachidonic acid.

The biosynthesis of prostaglandins and leukotrienes proceeds through the formation of chemically reactive intermediates leukotriene A4 (LTA4) and prostaglandin H2 (PGH2) which in aqueous solutions have chemical half-lives of 3 s and 3 min, respectively. Prostacyclin (PGI2) is another chemically reactive prostanoid that has a chemical half-life of 3-4 min. The recent development of reversed phase HPLC stationary phases that are stable to elevated pH (pH 10-12) without significant column damage has permitted direct analysis of these acid-sensitive eicosanoids. Using electrospray ionization, molecular anions [M - H]- of these compounds were observed at m/z 317 for LTA4 and m/z 351 for both PGH2 and PGI2. The mechanism of formation of ions derived from collisional activation of LTA4 was studied using stable isotope labeled and chemical analogs of LTA4 and found to involve formation of highly conjugated anions at m/z 261 and 163. The collisional activation of the molecular anion of PGH2 yielded a product ion spectrum identical to that observed for the isomeric prostaglandins PGE2 and PGD2. However, it was possible to baseline separate PGE2, PDG2, and PGH2 by reversed phase HPLC using basic HPLC mobile phases. The collisional activation of PGI2 led to a family of abundant ions including highly conjugated carbon-centered and oxygen-centered radical species (m/z 245 and 205) likely derived from the attack of the carboxylate anion on the cyclic enolether of PGI2 as well as the most abundant product ion (m/z 215) which formed following loss of neutral hexanal and water. The structures of these product ions were consistent with high resolution measurements measured in a quadrupole time-of-flight mass spectrometer.

6-Ketoprostaglandin F1 alpha↗
PubMed2002

Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability.

Ozone is known to be a highly toxic gas present in the urban air which exerts its effect on pulmonary tissue through its facile chemical reactions with target molecules in the airway. One of the first barriers encountered by ozone is epithelial lining fluid which contains pulmonary surfactant rich in glycerophosphocholine lipids. The reaction of ozone with calf lung surfactant extract was found to result in the production of 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho) as an expected product of the ozonolysis of abundant unsaturated phospholipids containing unsaturated fatty acyl groups with a double bond at carbon-9. This oxidized phospholipid was identified as a biologically active product in that it reduced elicited macrophage viability by necrosis with an ED(50) of 6 microM. Further studies of the biological activity of 16:0a/9-al-GPCho revealed that concentrations from 100 to 200 nM initiated apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity. The release of IL-8, a neutrophil chemokine, from A549 cells was also stimulated by 50-100 nM 16:0a/9-al-GPCho. Exposure of calf lung surfactant to low levels of ozone (62.5, 125, and 250 ppb) for various time periods from 2 to 48 h in a feedback-regulated ozone exposure chamber resulted in a dose- and time-dependent increase in the formation of 16:0a/9-al-GPCho as measured by a specific and sensitive LC/MS/MS assay. The quantity of this biologically active chain-shortened glycerophosphocholine lipid generated even at 125 ppb ozone for 2-4 h (50-100 nM) was consistent with this product mediating the toxic effects of ozone on cells in contact with surfactant.

Animals↗
PubMed2002

Enhanced cutaneous inflammatory reactions to Aspergillus fumigatus in a murine model of chronic granulomatous disease.

Chronic granulomatous disease is the manifestation of genetic defects of the leukocyte NADPH oxidase resulting in the absence of a respiratory burst. Patients with chronic granulomatous disease can develop chronic granulomas in many locations of the body, including the skin. Using an established murine model of X-linked chronic granulomatous disease (X-CGD) created by homologous recombinant disruption of the gene encoding the gp91phox component of the NADPH oxidase, in this study we examined cutaneous reactivity to sterile Aspergillus fumigatus hyphae. Injection of Aspergillus fumigatus into the dorsal ears of X-CGD mice resulted in an enhanced inflammatory response by 24 h, consisting of neutrophils, which developed into suppurative granulomas by 10 d. Intradermal injection of Aspergillus fumigatus into wild-type mice only resulted in a transient inflammatory response that resolved by 10 d. Injection of Aspergillus fumigatus into female carrier mice resulted in an acute inflammatory response that was similar to that of wild-type mice, but, at higher doses of Aspergillus fumigatus, many carriers subsequently developed granulomatous lesions that were qualitatively similar but smaller than those seen in X-CGD mice by 30 d. Consistent with the ability of X-CGD mice to mount an enhanced neutrophil-rich inflammatory response to Aspergillus fumigatus, significant levels of the potent neutrophil activator/chemoattractant leukotriene B4 were measured by mass spectrometry in skin biopsies at 24 and 72 h. In contrast to the exaggerated inflammatory response to intradermal Aspergillus fumigatus in X-CGD mice compared to their wild-type counterparts, similar levels of inflammation were seen in a model of delayed-type hypersensitivity using 2,4-dinitrofluorobenzene. This study represents the first report of a cutaneous granuloma model in mice with X-CGD, which may also prove useful as a functional test to evaluate the efficacy of gene therapy protocols being developed for chronic granulomatous disease.

Animals↗
PubMed2002

A novel class of microbial phosphocholine-specific phospholipases C.

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.

Amino Acid Sequence↗
PubMed2002

Leukotriene B4 production in human mononuclear phagocytes is modulated by interleukin-4-induced 15-lipoxygenase.

The aim of this study was to evaluate the consequences of interleukin (IL)-4-induced 15-lipoxygenase (15-LO) expression on leukotriene B4 (LTB4) synthesis in human monocytes. Human monocytes incubated for 24, 48, and 72 h with IL-4 (10 ng/ml) were stimulated with Ca2+-ionophore A23187 (calcimycin; 5 microM) or opsonized zymosan. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], LTB4, and arachidonic acid (AA) release were measured by high-performance liquid chromatography/radioimmunoassay, liquid chromatography/tandem mass spectrometry (LC/MS/MS), or gas chromatography/mass spectrometry. 15-LO activity was evaluated in AA-treated monocytes. 15-LO, 5-lipoxygenase (5-LO) and 5-LO activating protein (FLAP) expression were analyzed by reverse transcription-polymerase chain reaction. Neutrophil chemotactic activity was evaluated using a microtaxis chamber assay. A23187-induced synthesis of 15(S)-HETE was significantly increased after treatment with IL-4 (10 ng/ml) for 48 and 72 h (p < 0.001). Concomitant decrease of LTB4 release was observed after 72 h of incubation with IL-4 (p < 0.001). LC/MS/MS analysis confirmed the production of 15(S)-HETE and the significant inhibition of LTB4 synthesis in IL-4-treated monocyte after challenge with opsonized zymosan. IL-4 treatment induced 15-LO enzymatic activity as well as 15-LO mRNA, but did not affect either 5-LO or FLAP mRNA expression in monocytes. Supernatant from IL-4-treated monocytes showed significantly lower neutrophil chemotactic activity than controls. 15(S)-HETE significantly inhibited LTB4 production induced by A23187-stimulated human monocytes without affecting AA release. IL-4-induced expression of 15-LO in monocytes caused a significant reduction of LTB4 production. Whereas this effect did not reflect changes in 5-LO and FLAP mRNA expression, synthetic 15(S)-HETE was able to significantly inhibit the synthesis of LTB4, without affecting AA release.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2002

Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidylcholine synthase pathway.

Phosphatidylcholine (PC) is a ubiquitous membrane lipid in eukaryotes but has been found in only a limited number of prokaryotes. Both eukaryotes and prokaryotes synthesize PC by methylating phosphatidylethanolamine (PE) by use of a phospholipid methyltransferase (Pmt). Eukaryotes can synthesize PC by the activation of choline to form choline phosphate and then CDP-choline. The CDP-choline then condenses with diacylglycerol (DAG) to form PC. In contrast, prokaryotes condense choline directly with CDP-DAG by use of the enzyme PC synthase (Pcs). PmtA was the first enzyme identified in prokaryotes that catalyzes the synthesis of PC, and Pcs in Sinorhizobium meliloti was characterized. The completed release of the Pseudomonas aeruginosa PAO1 genomic sequence contains on open reading frame predicted to encode a protein that is highly homologous (35% identity, 54% similarity) to PmtA from Rhodobacter sphaeroides. Moreover, the P. aeruginosa PAO1 genome encodes a protein with significant homology (39% amino acid identity) to Pcs of S. meliloti. Both the pcs and pmtA homologues were cloned from PAO1, and homologous sequences were found in almost all of the P. aeruginosa strains examined. Although the pathway for synthesizing PC by use of Pcs is functional in P. aeruginosa, it does not appear that this organism uses the PmtA pathway for PC synthesis. We demonstrate that the PC synthesized by P. aeruginosa PAO1 localized to both the inner and outer membranes, where it is readily accessible to its periplasmic, PC-specific phospholipase D.

Amino Acid Sequence↗
PubMed2001

Formation of murine macrophage-derived 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) is catalyzed by leukotriene C4 synthase.

5-Oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG(7)), a biologically active glutathione (GSH) adduct of the eicosanoid 5-oxo-eicosatrienoic acid (5-oxoETE), is the major metabolite formed within the murine peritoneal macrophage. The conjugation of GSH to electrophilic 5-oxoETE in vitro was found to be catalyzed by both soluble glutathione S-transferase and membrane-bound leukotriene C(4) (LTC(4)) synthase. The cytosolic glutathione S-transferase-catalyzed products were not biologically active; however, the adduct formed from recombinant LTC(4) synthase had identical mass spectrometric properties and biological activity to the macrophage-derived FOG(7). The biosynthesis of FOG(7) in the macrophage was inhibited by MK-886, a known inhibitor of LTC(4) synthase, suggesting that this nuclear membrane-bound enzyme might be responsible for GSH conjugation to 5-oxoETE in the intact cell. Subcellular fractionation revealed that the microsomal fraction from the murine macrophage contained the enzyme responsible for FOG(7) biosynthesis. Western blot analysis confirmed the presence of LTC(4) synthase in the microsomal fraction that did not catalyze conjugation of GSH to 1-chloro-2,4-dinitrobenzene, indicating an absence of microsomal glutathione S- transferase activity. These results suggest that LTC(4) synthase, thought to be specific for the conjugation of GSH to LTA(4), can also recognize 5-oxoETE as an electrophilic substrate.

5-Lipoxygenase-Activating Proteins↗
PubMedDate not supplied

Electrospray mass spectrometry of phospholipids.

Phospholipids play a central role in the biochemistry of all living cells. These molecules constitute the lipid bilayer defining the outer confines of a cell, but also serve as the structural entities which confine subcellular components. Mass spectrometry has emerged as a powerful tool useful for the qualitative and quantitative analysis of complex phospholipids, including glycerophospholipids and the sphingolipid, sphingomyelin. Collision induced decomposition of both positive and negative molecular ion species yield rich information as to the polar head group of the phospholipid and the fatty-acyl substituents esterified to the glycerophospholipid backbone. This review presents the current level of understanding of the mechanisms involved in the formation of various product ions following collisional activation of molecular ion species generated by electrospray ionization of the common glycerophospholipids, including phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, cardiolipin, and sphingomyelin. Recent advances in the application of matrix assisted laser desorption ionization is also considered. Several applications of mass spectrometry applied to phospholipid analysis are presented as they apply to physiology as well as pathophysiology.

Animals↗
PubMedDate not supplied

Free radical oxidation of plasmalogen glycerophosphocholine containing esterified docosahexaenoic acid: structure determination by mass spectrometry.

Plasmalogen phospholipids have a vinyl ether substituent at the sn-1 position that is susceptible to oxidative reactions that occur at cell membranes. However, the mechanism by which this oxidation occurs and the effect of the polyunsaturated fatty acid at the sn-2 position have not been established. To gain insight into these mechanisms, the oxidized phospholipid products resulting from the exposure of 1-O-hexadec-1'-enyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (16:0p/22:6-GPCho) to the free radical initiator 2, 2'-azobis (2- amidinopropane) hydrochloride were examined. Electrospray ionization tandem mass spectrometry, UV spectroscopy, and electron ionization-gas chromatography/mass spectrometry were used to structurally characterize the oxidized glycerophosphocholine (GPCho) products. The radical-induced peroxidation of 16:0p/22:6-GPCho revealed two major classes of oxidized phospholipids. The first class of products was formed by oxidation at the sn-1 position and included 1-lyso-2-docosahexaenoyl-GPCho and 1-formyl-2-docosahexaenoyl-GPCho. Additionally, the second class of oxidized products where oxidation occurred at the sn-2 position was classified into three categories that included chain-shortened omega-aldehydes, terminal gamma-hydroxy-alpha,beta-unsaturated aldehydes, and the addition of one or two oxygen atoms onto the sn-2 position of 16:0p/22:6-GPCho. These results clearly indicate that free radical-induced oxidation of plasmalogen phospholipids with esterified docosahexaenoic acid at the sn-2 position underwent oxidation at both the sn-1 and sn-2 positions.

Aldehydes↗