Liquid chromatography/tandem mass spectrometry characterization of nitroso, nitrated and nitroxidized cardiolipin products.

Cardiolipins (CL) are anionic dimeric phospholipids bearing four fatty acids, found in inner mitochondrial membrane as structural components and are involved in several processes as oxidative phosphorylation or apoptotic signalling. As other phospholipids, CL can be modified by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can modulate various cellular functions. Modifications of CL by RNS remain largely unstudied although other nitrated lipids are emerging as bioactive molecules. In this work, we developed a C30-LC-HRMS/MS methodology to identify the nitrated and nitroxidized tetralinoleoyl-cardiolipin (TLCL), using a biomimetic model of nitration, and to disclose specific fragmentation pathways under HCD MS/MS. Using this lipidomics approach, we were able to separate and identify nitro, nitroso, nitronitroso, and nitroxidized TLCL derivatives, comprising 11 different nitrated compounds. These products were identified using accurate mass measurements and the fragmentation pattern acquired in higher-energy collision dissociation (HCD)-tandem MS/MS experiments. These spectra showed classifying fragmentation pathways, yielding phosphatidic acid (PA-), lysophosphatidic acid (LPA-), and carboxylate fragment ions with the modifying moiety. Remarkably, the typical neutral losses associated with the added moieties were not observed. In conclusion, this work has developed a new method for the identification of nitroso, nitrated and nitroxidized cardiolipin products by using a C30LC-MS platform method, potentially allowing their detection in biological samples.

The cellular lipids of Romboutsia.

We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (>95% sequence identity).

[Lipid Composition in Cell Walls and in Mycelial and Spore Cells of Mycelial Fungi].

Qualitative and quantitative differences were found between the lipids of cell walls (CW) and of whole mycelial cells and dormant cells of mucoraceous and ascomycete fungi. Thus, whole mycelial cells (WC) contained more lipids than CW. Unlike sporangiospores and conidia (exogenous dormant spores), zygotes were found to have the highest content of triacylglycerol lipids (70%). Cell walls of mucoraceous fungi contained more triacylglycerols (TAG) and less polar lipids than ascomycete lipids. While all CW and WC studied were similar in fatty acid (FA) composition, their ratio was specific for each structure: linoleic acid predominated in mycelial CW and WC, while oleic acid was predominant in the spores; this difference was especially pronounced in conidial WC. Unlike WC, in CW massive lipids may be represented not by phosphatidylethanolamine (PEA) and phosphatidylcholine (PC), but by free fatty acids (FFA), free (FSt) and etherified sterols (ESt), phosphatidic acid (PA), fatty acid methyl esters (FAME), and glycolipids (GL), which is an indication of a special functional role of CW.

The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer.

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS) is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500-1000 was performed on optimal cutting temperature (OCT) compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS) showed that these molecules included 14 phosphatidylinositols (PIs), 3 phosphatidylethanolamines (PEs) and 3 phosphatidic acids (PAs). Among the PIs, the expression of PI(18:0/18:1), PI(18:0/20:3) and PI(18:0/20:2) were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA). The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column.

A hydrophilic interaction liquid chromatography (HILIC) - ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain well-shaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R(2), 0.990-0.999), retention time stability (RSD<1%), within day repeatability (RSD, 5-13%), between day variation (RSD, 7-14%) and recoveries (99.6-115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.

Molecular species of phosphatidylethanolamine from continuous cultures of Saccharomyces pastorianus syn. carlsbergensis strains.

Saccharomyces pastorianus syn. carlsbergensis strain 34/70 is well known to be the most used strain for lager beer production. The difference between this strain and very closely related strain 34/78 is the latter's greater flocculating character. This single physiological trait can cause technical difficulties in beer production. The aim of this study was to determine whether lipid analysis by a combination of thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI-MS) could be used as a strain-typing technique in order to distinguish S. pastorianus syn. carlsbergensis strain 34/70 from strain 34/78. Both strains (34/70 and 34/78) were harvested after continuous culture under standard conditions. Polar lipids were then extracted from lyophilized cultures and analysed by TLC in order to separate phospholipid families. Phosphatidylethanolamine (PE) was extracted and investigated using ESI-MS, to gain further information on individual molecular species. Using TLC analysis, lipids were separated corresponding to standards for PE, phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). ESI-MS of the PE band, separated by TLC, showed that electrospray mass spectra were highly reproducible for repeat cultures. Novel findings were that both brewing strains displayed major phospholipid peaks with m/z 714, PE (34 : 2) m/z 742, PE (36 : 2) and m/z 758, PE (37 : 1). However, strain 34/78 had additional peaks of m/z 700, PE (33 : 2) and m/z 728, PE (35 : 2). Strain 34/70 had an extra peak with m/z 686 PE (32 : 2). We conclude that combined TLC/ESI-MS can distinguish between S. pastorianus syn. carlsbergensis 34/70 and 34/78 and may be a useful typing technique for differentiation of closely related yeast strains. This novel approach may aid quality assurance and could be suitable for yeast collections and larger industrial companies.

Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes.

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.

Isolation and identification of phosphatidic acid targets from plants.

Phosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress-signalling pathways and is formed in response to wounding, osmotic stress, cold stress, pathogen elicitors, Nod factors, ethylene and abscisic acid. How PA exerts its effects is still unknown, mainly because of the lack of characterized PA targets. In an approach to isolate such targets we have used PA-affinity chromatography. Several PA-binding proteins were present in the soluble fraction of tomato and Arabidopsis cells. Using mass spectrometric analysis, several of these proteins, including Hsp90, 14-3-3 proteins, an SnRK2 serine/threonine protein kinase and the PP2A regulatory subunit RCN1 could be identified. As an example, the binding of one major PA-binding protein, phosphoenolpyruvate carboxylase (PEPC), was characterized further. Competition experiments with different phospholipids confirmed specificity for PA. Hypo-osmotic treatment of the cells increased the amount of PEPC that bound the PA beads without increasing the absolute amount of PEPC. This suggests that PEPC's affinity for PA had increased. The work shows that PA-affinity chromatography/mass spectrometry is an effective way to isolate and identify PA-binding proteins from plants.

Biological functions of a novel lipid mediator, cyclic phosphatidic acid.

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was isolated originally from myxoamoebae of a true slime mold, Physarum polycephalum, and has now been detected in a wide range of organisms from slime molds to humans. It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. This substance shows specific biological functions, including antimitogenic regulation of the cell cycle, regulation of actin stress fiber formation and rearrangement, inhibition of cancer cell invasion and metastasis, regulation of differentiation and viability of neuronal cells, and mobilization of intracellular calcium. Although the structure of cPA is similar to that of lysophosphatidic acid (LPA), its biological activities are apparently distinct from those of LPA. In the present review, we focus mainly on the enzymatic formation of cPA, the antimitogenic regulation of the cell cycle, the inhibition of cancer cell invasion and metastasis, and the neurotrophic effect of cPA.

Chemical structure of a novel aminophospholipid from Hydrogenobacter thermophilus strain TK-6.

The phospholipid composition of Hydrogenobacter thermophilus strain TK-6, an obligately chemolithoautotrophic, extremely thermophilic hydrogen bacterium, was analyzed. Two of four phospholipids detected from the strain were assumed to be phosphatidylinositol and phosphatidylglycerol. An aminophospholipid named PX, whose content among the phospholipids was 65%, was found to have a novel chemical structure by analysis of the dilyso form with nuclear magnetic resonance and fast atom bombardment-mass spectrometry (FAB-MS) and by analysis of the intact PX with FAB-MS as 1,2-diacyl-3-O-(phospho-2'-O-(1'-amino)-2',3',4',5'-pentanetetrol)-sn-glycerol. Structurally similar phospholipids have been identified in Methanospirillum hungatei, Methanolacinia paynteri, and Methanogenium cariaci, which all belong to the Archaea.

Characterization of N-acylphosphatidylethanolamine and acylphosphatidylglycerol in oats.

Two polar lipid classes, both with three acyl groups, were isolated from an extract of oats and characterized by nuclear magnetic resonance spectroscopy, electrospray mass spectrometry (MS), and electron ionization MS (EIMS). Distortionless enhancement by polarization transfer (DEPT) and the two-dimensional correlation experiments 1H-detected heteronuclear multiple quantum coherence spectroscopy, heteronuclear multiple bond correlation spectroscopy, double quantum filtered correlation spectroscopy, and total correlation spectroscopy provided sufficient information for determination of the structure of the two lipid classes. The polar lipid classes were found to be N-acylphosphatidylethanolamine [1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-1'-ethanolamine; N-acyl-PE] and acylphosphatidylglycerol [1,2-diacyl-sn-glycero-3-phospho-(3'-acyl)-1'-sn-glycerol]. High-performance liquid chromatography with electrospray ionization MS (HPLC-ESMS) and with electrospray ionization tandem MS (HPLC-MS/MS) were utilized for the separation and subsequent determination of molecular species. With HPLC-ESMS, ions of deprotonated molecules were obtained and with HPLC-MS/MS carboxylate ions (representing acyl groups) were obtained as well as other structurally significant ions. Fifty molecular species of N-acylphosphatidylethanolamine and 24 molecular species of acylphosphatidylglycerol were found, with a molecular mass range of 924-1032 Da and 959-1035 Da, respectively. Identification of the fatty acid isomers, as picolinyl ester derivatives, was done with gas chromatography with EIMS. Three isomers of 16:1 fatty acids were found in N-acyl-PE, and their double bond positions were determined to 6, 9, and 11 with a relative abundance of 4:10:1.

Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum albumin.

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was identified in lipids bound to human serum albumin. A cPA fraction was extracted and purified from human serum albumin by use of a combination of preparative TLC and HPLC. Electrospray ionization mass spectrometry of the purified fraction showed molecular ions corresponding to cPA, which was composed of some different fatty acid species. The most abundant component was identified as palmitoyl-cPA by tandem mass spectrometry using collision-induced dissociation. These data have established that cPA is a naturally occurring lipid bound to human serum albumin.

Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry.

Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion at m/z 319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs with tert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.

Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices.

Noradrenaline- and ionomycin-stimulated as well as basal phospholipase D activity from rat hippocampus produced, in the presence of ethanol, two different classes of [32P]phosphatidylethanol (designated I and II), which were separated by thin layer chromatography. Endogenous labeling experiments using 3H-fatty acids showed that two different classes of phosphatidylcholine, separated by two-dimensional TLC, one enriched with high incorporation of [3H]arachidonic acid (B) and the other with [3H]myristic acid (A), were the most likely sources for the two classes of phosphatidylethanol. Experiments where individual 32P-phospholipids extracted from [32P]Pi-labeled hippocampal slices were incubated with cabbage phospholipase D, in the presence of ethanol, showed that each class of [32P]phosphatidylcholine, i.e. A and B, produced a different band of [32P]phosphatidylethanol, with the same mobility in TLC as phosphatidylethanol II and I, respectively.

Quantitation of the fatty acid composition of phosphatidic acid by capillary gas chromatography electron-capture detection with picomole sensitivity.

We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in sensitivity compared to the most sensitive of the previously described methods. An example is provided of quantitating phosphatidic acid from minute amounts of biological samples such as islets of Langerhans.

Synthesis and antiviral activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112-6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with 31P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC50 values of AZTTP-DSG were 0.33 and 0.79 microM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 microM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.

pH-dissociation characteristics of cardiolipin and its 2'-deoxy analogue.

Cardiolipin (CL) is found in inner mitochondrial membranes and the plasma membrane of aerobic prokaryotes. CL is tightly bound to those transmembrane enzymes associated with oxidative phosphorylation. CL has earlier been reported to have a single pK at low pH. We have titrated CL in aqueous suspension (bilayers) and in solution in methanol/water (1:1, vol/vol) and found it to display two different pK values, pK1 at 2.8 and pK2 initially at 7.5 but shifting upwards to 9.5 as the titration proceeds. The unusually high pK2 might be explained by the formation of a unique hydrogen bond in which the free hydroxyl on the central glycerol forms a cyclic intramolecular hydrogen-bonded structure with one protonated phosphate (P-OH group). We have therefore chemically synthesized the 2'-deoxycardiolipin analogue, which lacks the central free hydroxyl group, and measured its pH-dissociation behavior by potentiometric titration, under the same conditions as those for CL. The absence of the hydroxyl group changes the titration dramatically so that the deoxy analogue displays two closely spaced low pK values (pK1 = 1.8; pK2 = 4.0). The anomalous titration behavior of the second dissociation constant of CL may be attributed to the participation of the central glycerol OH group in stabilizing the formation of a cyclic hydrogen-bonded monoprotonated form of CL, which may function as a reservoir of protons at relatively high pH. This function may have an important bearing on proton pumping in biological membranes.

Deacylation-reacylation cycle: a possible absorption mechanism for the novel lymphotropic antitumor agent dipalmitoylphosphatidylfluorouridine in rats.

Dipalmitoylphosphatidylfluorouridine (DPPF) is a potent antitumor agent that selectively gains access to the lymphatic system. To determine whether DPPF enters the lymph in an unmodified form, we administered DPPF orally to rats and analyzed lymph collected from a cannula in the thoracic duct. Although lymph was found to contain only very low levels of DPPF, two congeners of DPPF were detected at high levels. Instrumental analysis demonstrated that these congeners are 1-palmitoyl-2-arachidonoylphosphatidylfluorouridine (PAPF) and 1-palmitoyl-2-linoleoyl-phosphatidylfluorouridine (PLPF). PAPF and PLPF levels in thoracic lymph were shown to be approximately 30 times higher than those in plasma. These results suggest that DPPF is absorbed from the intestinal tract via the deacylation-reacylation cycle for the uptake of phospholipids and is selectively delivered to the lymphatic route after oral administration. DPPF is a candidate drug for the treatment of tumor metastasis, especially in cases where metastasis has occurred via the lymphatic route.

Molecular species composition of glycerophospholipids in rat sciatic nerve and its alteration in streptozotocin-induced diabetes.

The molecular species composition of glycerophospholipid classes in nerves of normal and experimentally diabetic rats was determined. sn-1,2-Diacylglycerol (DAG) moieties of purified phospholipids were liberated enzymatically and analyzed as the benzoate derivatives by high-performance liquid chromatography. The most abundant molecular species in phosphatidylinositol (PI) from normal nerve were 18:0/20:4 (54%) and 16:0/18:1 (17%), whereas in phosphatidylcholine (PC), 16:0/18:1 (52%), 16:0/16:0 (12%) and 18:0/18:1 (11%) predominated. In phosphatidylethanolamine and ethanolamine plasmalogen, 18:1/18:1, 16:0/18:1 and 18:0/18:1 comprised more than 60% and 75% of the molecular species, respectively. Phosphatidylserine was characterized by a high content of 18:0/18:1 (38%) and a relative abundance of the 18:1/20:0, 18:1/22:0 and 18:1/24:0 molecular species, which together accounted for over 30% of the total. The molecular species profile of phosphatidic acid did not closely resemble that of any other phospholipids or DAG. In diabetic nerve, the molecular species composition of all diacylphospholipids showed a significant decline in the content of one or more arachidonoyl-containing molecular species. The largest decline occurred in PC and the least in PI. Except in PC, 16:0/20:4 was more depressed than 18:0/20:4. In combination with previous analyses of DAG molecular species which showed a similar decline in the content of arachidonoyl-containing molecular species in nerve from experimentally diabetic rats (Zhu, X. and Eichberg, J. (1990) J. Neurochem. 55, 1087-1090), the results suggest that nerve DAG arises largely, but not entirely, from phosphoinositides and that PC could be a significant precursor, especially in diabetic nerve.