Hydrolysis of polyhydroxy polyunsaturated fatty acid-glycerophosphocholines by Group IIA, V, and X secretory phospholipases A.

It is widely accepted that unesterified polyunsaturated ω-6 and ω-3 fatty acids (PUFA) are converted through various lipoxygenases, cyclooxygenases, and cytochrome P450 enzymes to a range of oxygenated derivatives (oxylipins), among which the polyhydroxides of unesterified PUFA have recently been recognized as cell signaling molecules with anti-inflammatory and pro-resolving properties, known as specialized pro-resolving mediators (SPMs). This study investigates the mono-, di-, and trihydroxy 16:0/PUFA-GPCs, and the corresponding 16:0/SPM-GPC, in plasma lipoproteins. We describe the isolation and identification of mono-, di-, and trihydroxy AA, EPA, and DHA-GPC in plasma LDL, HDL, HDL3, and acute phase HDL using normal phase LC/ESI-MS, as previously reported.

Destruction of polyunsaturated alkyl/acyl and alkenyl/acyl glycerophosphocholine of plasma lipoproteins during incubation with group V and X secretory phospholipase A s.

Plasma lipoproteins are carriers of various glycerophospholipids including diacyl, alkenyl/acyl, and alkyl/acyl glycerophosphocholines (GPCs), which become distributed among cells and tissues during metabolism. For metabolic function, these phospholipids require hydrolysis by phospholipases, but the responsible enzymes have not been identified. We had previously shown that after complete digestion of lipoprotein diacyl- and oxo-diacyl-GPCs, degradation of residual alkyl/acyl and alkenyl/acyl GPCs continues, despite the fact that ether lipids are resistant to hydrolysis by Ca -activated secretory PLA s and require the presence of the Ca -independent PLA .

Hydrolysis of glycerophosphocholine epoxides by human group IIA, V, and X secretory phospholipases A.

This study was prompted by recent reports that epoxyeicosatrienoic (EET) and epoxyeicosatetraenoic (EEQ) acids accelerate tumor growth and metastasis by stimulation of angiogenesis, while eicosapentaenoic (EPA) and epoxydocosapentaenoic (EDP) acids inhibit angiogenesis, tumor growth, and metastasis. Cytochrome P450 epoxygenases convert arachidonic to EET, eicosapentaenoic acid to EEQ, and docosahexaenoic acid to EDP, which are found both in free form and esterified to glycerophosphocholine (GPC). Both free and esterified epoxy (EP) acids are also formed during lipid autoxidation.

Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA, V and X Secretory Phospholipases A (sPLA).

Biologically active F- and E/D-type-prostane ring isomers (F-IP and E/D-IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA, which also have a wide peripheral tissue distribution.

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl-γ-cyclodextrin-modified micellar electrokinetic chromatography.

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cyclodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C.

Hydrolysis of lipoproteins by sPLA2's enhances mitogenesis and eicosanoid release from vascular smooth muscle cells: Diverse activity of sPLA2's IIA, V and X.

Mitogenesis of Vascular Smooth Muscle Cells (VSMC) plays an important role in atherogenesis. Until recently, the effect of lipid subfractions has not been clarified. Secretory phospholipases A2 (sPLA2's) hydrolyse glycerophospholipids and release pro-inflammatory lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.

Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells.

Objective: Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC).

Material: We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC.

Methods: Recombinant human sPLA2's and Boyden's chamber method were applied.

Effects of antioxidants on rapeseed oil oxidation in an artificial digestion model analyzed by UHPLC-ESI-MS.

A normal diet contains large quantities of oxidized fatty acids, glycerolipids, cholesterol, and their cytotoxic degradation products because many foods in the diet are fried, heated, or otherwise processed and consumed often after long periods of storage. There is also evidence that the acid medium of the stomach promotes lipid peroxidation and that the gastrointestinal tract is a major site of antioxidant action, as demonstrated by various colorimetric methods. The identity and yields of specific products of lipid transformation have seldom been determined.

Liquid chromatography-light scattering detector-mass spectrometric analysis of digested oxidized rapeseed oil.

Rapeseed oil was oxidized chemically and thermally to produce two distinct oxidized oils. These oils, along with unoxidized oils, were subjected to an artificial digestion model to simulate the digestive processes in humans. Lipid digestion involves lipases that break down the intact triacylglycerol (TAG) molecules first to diacylglycerols, and eventually to sn-2-monoacylglycerols (MAG) and free fatty acids.

Use of lipidomics for analyzing glycerolipid and cholesteryl ester oxidation by gas chromatography, HPLC, and on-line MS.

Various analytical techniques have been adopted for the isolation and identification of the oxolipids and for determining their functionality. Gas chromatography in combination with mass spectrometry (MS) has been specifically utilized in analysis of isoprostanes and other low molecular weight oxolipids, although it requires derivatization of the solutes. In contrast, liquid chromatography (LC) in combination with on-line MS has proven to be well suited for analysis of intact oxolipids without (or minimal) derivatization.

Lipidomic analysis of glycerolipid and cholesteryl ester autooxidation products.

Thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) in combination with mass spectrometry (MS) have been adopted for the isolation and identification of oxolipids and for determining their functionality. TLC provides a rapid separation and access to most oxolipids as intact molecules and has recently been effectively interfaced with time-of-flight (TOF) MS (TOF-MS). GC with flame ionization (FI) (GC/FI) and electron impact (EI) MS (GC/EI-MS) has been extensively utilized in the analysis of isoprostanes and other low-molecular-weight oxolipids, although these methods require derivatization of the analytes.

Phase composition of lipoprotein SM/cholesterol/PtdCho affects FA specificity of sPLA2s.

We have previously reported preferential release of polyunsaturated FAs during hydrolysis of lipoprotein phosphatidylcholine (PtdCho) by group X secretory phospholipase A2 (sPLA2) and preferential release of oligounsaturated FAs during hydrolysis of lipoprotein PtdCho by group V sPLA2, but the mechanism of this selectivity has remained unknown. We now show that the rate and specificity of hydrolysis are affected by relative increases in endogenous SM and free cholesterol (FC) during the lipase digestion. The highest preference for arachidonate release from LDL and HDL by group X sPLA2 was observed for residual SM/PtdCho molar ratio of 1.

Lipidomics in triacylglycerol and cholesteryl ester oxidation.

Although direct mass spectrometry is capable of identification the major molecular species of lipids in crude total lipid extracts, prior chromatographic isolation is necessary for detection and identification of the minor components. This is especially important for the analysis of the oxolipids, which usually occur in trace amounts in the total lipid extract, and require prior isolation for detailed analysis. Both thin-layer chromatography and adsorption cartridges provide effective means for isolation and enrichment of lipid classes, while gas-liquid chromatography and high performance liquid chromatography with on-line mass spectrometry permit further separation and identification of molecular species.

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects.

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined.

Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2.

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s.

1 -O-alkyl-2-(omega-oxo)acyl-sn-glycerols from shark oil and human milk fat are potential precursors of PAF mimics and GHB.

This study examines the feasibility that peroxidation and lipolysis of 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGE) found in shark liver oil and human milk fat constitutes a potential source of dietary precursors of platelet activating factor (PAF) mimics and of gamma-hydroxybutyrate (GHB). Purified DAGE were converted into 1-O-alkyl-2-acyl-sn-glycerols by pancreatic lipase, without isomerization, and transformed into 1-O-alkyl-2-oxoacyl-sn-glycerols by mild autooxidation. The various core aldehydes without derivatization, as well as the corresponding dinitrophenylhydrazones, were characterized by chromatographic retention time and diagnostic ions by online electrospray mass spectrometry.

Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2.

Human groups IIA, V and X secretory phospholipases A2 (sPLA2s) were incubated with human HDL3, total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho.

Covalent binding of acetone to aminophospholipids in vitro and in vivo.

We have determined the ions characteristic of acetone adducts of reference aminophospholipids and have used them as markers for identification of acetone adducts of aminophospholipids in commercial lecithin, acetone extracts of tissue lipids, and in plasma and red blood cells of diabetic subjects. The acetonation products were determined by normal-phase high-performance liquid chromatography (HPLC) with on-line electrospray-mass spectrometry, and electrospray/collision-induced dissociation in the negative ion mode. The major acetone complexes of PtdEtn and PtdSer were identified as the diacetone derivatives [PtdEtn+116-H2O]- and [PtdSer+116-H2O]-, respectively, although ions corresponding to monoacetone [PtdEtn+58-H2O]- and doubly dehydrated diacetone adducts [PtdSer+116-2 x 18]- were also observed.

Regio- and stereospecific analysis of glycerolipids.

In recent years researchers have recognized the potential value of comprehensive lipid profiling (lipidomics), which was invented and promoted by lipidologists who recognized the many valuable applications that grew out of the fields of DNA profiling (genomics) and protein profiling (proteonomics). Through lipid class-selective intrasource ionization and subsequent analysis of two-dimensional cross-peak intensities, the chemical identity and mass composition of individual molecular species of most lipid classes can now be determined in a chloroform extract. There remains, however, the necessity to distinguish the enantiomers and isobaric regioisomers resulting from enzymatic and chemical reactions, which conventional high performance liquid chromatography/mass spectrometry (HPLC/MS) has been slow to accommodate, and tandem MS unable to provide.

Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339.

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [J.J.

Simple synthesis of diastereomerically pure phosphatidylglycerols by phospholipase D-catalyzed transphosphatidylation.

A simple method for synthesizing diastereomerically pure phosphatidylglycerols (PtdGro), namely, 1,2-diacyl-sn-glycero-3-phospho-3'-sn-glycerol (R,R configuration) and 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol (R,S configuration), was established. For this purpose, diastereomeric 1,2-O-isopropylidene PtdGro were prepared from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho) and enantiomeric 1,2-O-isopropylideneglycerols by transphosphatidylation with phospholipase D (PLD) from Actinomadura sp. This species was selected because of its higher transphosphatidylation activity and lower phosphatidic acid (PtdOH) formation than PLD from some Streptomyces species tested.

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol.

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp.

Asymmetric in vitro synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D.

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S.

Regiospecific determination of short-chain triacylglycerols in butterfat by normal-phase HPLC with on-line electrospray-tandem mass spectrometry.

This study uses normal-phase HPLC with on-line positive ion electrospray mass spectrometry (ESI-MS) to obtain quantitative compositional data on both synthetic and butterfat short-chain TAG. The product ion tandem MS of standards averaged 11.1 times lower in abundance of the ion formed by cleavage of FA from the sn-2-position for the pairs of regioisomers in the TAG classes: L/L/S-L/S/L and L/S/S-S/L/S, where L denotes long and S short acyl chain (C2-C6).