Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.     

Synthetically prepared Aamadori-glycated phosphatidylethanolaminecan trigger lipid peroxidation via free radical reactions

This study for the first time confirmed the peroxidative role of the Amadori product derived from the glycation of phosphatidylethanolamine (PE), namely Amadori-PE. The product was synthesized from the reaction of dioleoyl PE with D-glucose, and then purified by a solid-phase extraction procedure, which was a key step in the next HPLC technique for the isolation of essentially pure Amadori-PE. When the synthetically prepared Amadori-PE was incubated with linoleic acid in the presence of Fe(3+) in micellar system, a remarkable formation of thiobarbituric acid reactive substances was observed together with increases in lipid hydroperoxides. In addition, the lipid peroxidation caused by Amadori-PE was effectively inhibited by superoxide dismutase, mannitol, catalase and metal chelator. These results indicated that Amadori-PE triggers oxidative modification of lipids via the generation of superoxide, and implied the involvement of 'lipid glycation' along with membrane lipid peroxidation in the pathogenesis of diabetes and aging.     

LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma

An amino group of phosphatidylethanolamine (PE) is considered as a target for nonenzymatic glycation, and the potential involvement of lipid glycation in the pathogenesis of diabetic complications has generated interest. However, unlike an early glycation product of PE (Amadori-PE), the occurrence and roles of advanced glycation end products of PE (AGE-PE) in vivo have been unclear. Here, we developed an LC-MS/MS method for the analysis of AGE-PE [carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE)]. Collision-induced dissociation of CM-PE and CE-PE produced characteristic ions, permitting neutral loss scanning (NLS) and multiple reaction monitoring (MRM) of AGE-PE. By NLS analysis, a series of AGE-PE molecular species was detected in human erythrocytes and blood plasma. In LC-MS/MS analysis, MRM enabled the separation and determination of the predominant AGE-PE species. Between healthy subjects and diabetic patients, no significant differences were observed in AGE-PE concentrations in erythrocytes and plasma, whereas Amadori-PE concentrations were higher in diabetic patients. These results provide direct evidence for the presence of AGE-PE in human blood, and indicated that, compared with Amadori-PE, AGE-PE is less likely to be accumulated in diabetic blood. The presently developed LC-MS/MS method appears to be a powerful tool for understanding in vivo lipid glycation and its pathophysiological consequence.     

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

Lipid glycation and protein glycation in diabetes and atherosclerosis

Recent instrumental analyses using a hybrid quadrupole/linear ion trap spectrometer in LC-MS/MS have demonstrated that the Maillard reaction progresses not only on proteins but also on amino residues of membrane lipids such as phosphatidylethanolamine (PE), thus forming Amadori-PE (deoxy-d-fructosyl PE) as the principal products. The plasma Amadori-PE level is 0.08 mol% of the total PE in healthy subjects and 0.15–0.29 mol% in diabetic patients. Pyridoxal 5′-phosphate and pyridoxal are the most effective lipid glycation inhibitors, and the PE-pyridoxal 5′-phosphate adduct is detectable in human red blood cells. These findings are beneficial for developing a potential clinical marker for glycemic control as well as potential compounds to prevent the pathogenesis of diabetic complications and atherosclerosis. Glucose and other aldehydes, such as glyoxal, methylglyoxal, and glycolaldehyde, react with the amino residues of proteins to form Amadori products and Heynes rearrangement products. Because several advanced glycation end-product (AGE) inhibitors such as pyridoxamine and benfotiamine inhibit the development of retinopathy and neuropathy in streptozotocin (STZ)-induced diabetic rats, AGEs may play a role in the development of diabetic complications. In the present review, we describe the recent progress and future applications of the Maillard reaction research regarding lipid and protein modifications in diabetes and atherosclerosis.     

Transbilayer distribution of aminophospholipids and the oxidative stability of their component polyunsaturated fatty acids

We examined the relationship between the transbilayer distribution of aminophospholipids, such as phosphatidylethanolamine (PE), PE plasmalogen and phosphatidylserine, and the oxidative stability of polyunsaturated fatty acids (PUFAs) in the aminophospholipids. To modulate the transbilayer distribution of aminophospholipid in liposomes, we used phosphatidylcholine (PC) with two types of acyl chain region: dipalmitoyl (PC16:0) or dioleoyl (PC18:1). In the smaller-sized liposomes, the proportions of aminophospholipid in the liposomal external layer were significantly higher in liposomes containing PC18:1 than in those containing PC16:0. Additionally, aminophospholipids in the external layer of smaller-sized liposomes were able to protect their component PUFAs from 2,2'-azobis(2-amidinopropane)dihydrochloride-mediated lipid peroxidation.     

UV analysis of Amadori-glycated phosphatidylethanolamine in foods and biological samples

Maillard reactions are among the most important of the chemical and oxidative changes occurring in food and biological samples that contribute to food deterioration and to the pathophysiology of human disease. Although the association of lipid glycation with this process has recently been shown, the number of lipid glycation products in food and biological materials has not been clear. In this study, we synthesized the Amadori products derived from the glycation of phosphatidylethanolamine (PE), i.e., Amadori-PEs. Dioleoyl PE was incubated with glucose and lactose for 15 days, and the resultant Amadori-PEs were purified and isolated using solid phase extraction followed by HPLC. With this procedure, essentially pure (>98% purity) Amadori-PEs glycated with glucose (Glc-PE) and with lactose (Lac-PE) were obtained and used as standards in the subsequent studies.To determine the presence of Amadori-PEs in food and biological samples, the carbonyl group of Amadori-PEs was ultraviolet (UV)-labeled with 3-methyl-2-benzothiazolinone hydrazone, and the labeled Amadori-PEs were analyzed with normal phase HPLC-UV (318 nm). The detection limit was 4.5 ng (5 pmol) for Glc-PE and 5.3 ng (5 pmol) for Lac-PE. Among the several food samples examined, infant formula and chocolate contained a high amount of both Glc-PE and Lac-PE over wide concentration ranges, such as 1.5-112 microg/g. Testing biological materials showed Amadori-PE (Glc-PE) was detectable in rat plasma.     

Formation of N-(hexanoyl)ethanolamine, a novel phosphatidylethanolamine adduct, during the oxidation of erythrocyte membrane and low-density lipoprotein

The primary amino groups of biomolecules such as aminophospholipids, as well as proteins, are the potential targets of covalent modifications by lipid peroxidation products; however, little attention has been paid to the modification of aminophospholipids such as phosphatidylethanolamine (PE). The purpose of this study is to characterize the formation of a novel modified phospholipid, N-(hexanoyl)phosphatidylethanolamine (HEPE), in the reaction of PE with lipid hydroperoxides using mass spectrometric analyses. Upon reaction of egg PE with 13-hydroperoxyoctadecadienoic acid or other oxidized polyunsaturated fatty acids followed by phospholipase D-mediated hydrolysis, the formation of N-(hexanoyl)ethanolamine (HEEA), a head group of HEPE, was confirmed by isotope dilution liquid chromatography/tandem mass spectrometry. Moreover, increasing HEEA was detected in the hydrolysates of oxidized erythrocyte ghosts and low-density lipoprotein with their increasing lipid peroxidation levels. Collectively, these results suggest that the N-hexanoylated product of phospholipid, HEPE, can be generated during lipid peroxidation and may serve as one mechanism for the covalent modification of aminophospholipids in vivo.     

Docosahexaenoic acid-containing phosphatidylethanolamine in the external layer of liposomes protects docosahexaenoic acid from 2,2'-azobis(2-aminopropane)dihydrochloride-mediated lipid peroxidation

We have proposed that incorporation of docosahexaenoic acid (DHA) into phosphatidylethanolamine (PE) might enhance resistance to lipid peroxidation in vivo. In this study, we examined the relationship between the transbilayer distribution of PE and the oxidative stability of DHA in PE. Liposomes composed of a phospholipid mixture were used as models for biological membranes. To modulate the transbilayer distribution of PE obtained from the liver of rats fed DHA (PE-DHA), we used phosphatidylcholine (PC) with two types of acyl chain region: dipalmitoyl (PC16:0) or dioleoyl (PC18:1). The proportion of PE-DHA in the liposomal external layer was significantly higher in liposomes containing PC18:1 than in those containing PC16:0. This tendency was more pronounced in liposomes extruded using a polycarbonate filter with smaller pore sizes. Additionally, PE-DHA in the external layer of liposomes prepared using a filter with smaller pore sizes could protect DHA itself from 2,2(')-azobis(2-aminopropane)dihydrochloride-mediated lipid peroxidation.     

Effect of asymmetric distribution of phospholipids in ghost membrane from rat blood on peroxidation induced by ferrous ion

This study used rat erythrocyte ghost membrane to investigate the effect of aminophospholipid distribution in biological membranes on oxidative susceptibility. Aminophospholipids, lipid peroxidation, and carbonyl compounds were quantified; plasma membrane structure was examined using atomic force microscopy (AFM) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Inside-out vesicles (IOVs) had significantly more aminophospholipids and greater lipid peroxidation than right-side-out vesicles (ROVs). Spectrin bands in IOVs disappeared obviously than in ROVs as shown in SDS-PAGE. In both systems vesicle protein size increased significantly with oxidation. Proteins aggregated much more in IOVs than ROVs at 48 h. These observations suggest that IOVs were more susceptible to ferrous ion-induced peroxidation than ROVs and that asymmetric phospholipid distribution affects biomembranes' oxidative susceptibility.     

Lipid requirement of the branched-chain amino acid transport system of Streptococcus cremoris

The role of the membrane lipid composition on the transport protein of branched-chain amino acids of the homofermentative lactic acid bacterium Streptococcus cremoris has been investigated. The major membrane lipid species identified in S. cremoris were acidic phospholipids (phosphatidylglycerol and cardiolipin), glycolipids, and glycerophosphoglycolipids. Phosphatidylethanolamine (PE) was completely absent. Protonmotive force-driven and counterflow transport of leucine was assayed in fused membranes of S. cremoris membrane vesicles and liposomes composed of different lipids obtained by the freeze/thaw-sonication technique. High transport activities were observed with natural S. cremoris and Escherichia coli lipids, as well as with mixtures of phosphatidylcholine (PC) with PE or phosphatidylserine. High transport activities were also observed with mixtures of PC with monogalactosyl diglyceride, digalactosyl diglyceride, or a neutral glycolipid fraction isolated from S. cremoris. PC or mixtures of PC with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activities. In mixtures of PC and methylated derivatives of PE, both counterflow and protonmotive force-driven transport activities decreased with increasing degree of methylation of PE. The decreased transport activity in membranes containing PC could be restored by refusion with PE-containing liposomes. These results demonstrate that both aminophospholipids and glycolipids can be activators of the leucine transport system from S. cremoris. It is proposed that aminophospholipids in Gram-negative bacteria and glycolipids in Gram-positive bacteria have similar functions with respect to solute transport.     

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.     

A New Role of Phosphoglucose Isomerase. Involvement of the Glycolytic Enzyme in Aldehyde Metabolism

Lipid peroxidation in biological membranes is accompanied by malonic dialdehyde (MDA) formation, but the problem of its further metabolism in cytoplasm remains unsolved. The experimental data obtained in this work showed that the liver fraction prepared by centrifugation at 10,000g contained phosphoglucose isomerase and enzymes of the glyoxalase system. In this fraction in the presence of GSH there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes (MDA conversion to methylglyoxal and further to neutral D-lactate). This means that MDA is slowly accumulated because it is a substrate of aldehyde isomerase (MDA <--> methylglyoxal). Most probably, phosphoglucose isomerase serves as this enzyme. We concluded that D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Phosphatidylethanolamines Glycation, Oxidation, and Glycoxidation: Effects on Monocyte and Dendritic Cell Stimulation

Lipid glycation is a non-enzymatic reaction between glucose and the free amino group of aminophospholipids, particularly in chronic hyperglycemia. Glycated phosphatidylethanolamine have been found in plasma and atherosclerotic plaques of diabetic patients and was correlated with increased oxidative and inflammatory stress in diabetes. However, the biological roles of glycated lipids are not fully understood. In this study, we evaluated the effect of palmitoyl-oleoyl-phosphatidylethanolamine (POPE) oxidation, glycation, and glycoxidation products on monocyte and myeloid dendritic cell stimulation. Flow cytometry analysis was used to evaluate the capability of each modified PE to induce the expression of different cytokines (IL-1β, IL-6, IL-8, MIP-1β, and TNF-α) in monocytes or myeloid dendritic cells (mDC). Our results showed that PE modifications induced different effect on the stimulation of cells producing cytokines. All PE modifications induced higher frequencies of cytokine-producing cells than basal state. Higher stimulation levels were obtained with glycated POPE, followed by glycoxidized POPE. In contrast, oxidized POPE negatively regulated the frequency of monocytes and mDC producing cytokines, when compared with non-modified POPE. In conclusion, we verified that PE glycation, compared with oxidation and glycation plus oxidation, had higher ability to stimulate monocytes and mDC. Thus detection of increased levels of PE glycation in diabetes could be considered a predictor of a inflammatory state.     

Implications of a non-lamellar lipid phase for the tight junction stability. Part I: Influence of basic amino acids, pH and protamine on the bilayer-hexagonal II phase behaviour of PS-containing PE membranes.

Inverted lipid micelles have been proposed, among other biological functions, to constitute the structural basis of the so-called tight junctions, a special cell cell contact found in epithelia and endothelial, which act as a barrier for the paracellular solute passage. As a model system for the opening and closing of this gate, we investigated the formation of the inverted hexagonal phase (HII phase) in lipid bilayer systems consisting of egg phosphatidylethanolamine (egg PE) and mixed egg PE/bovine brain phosphatidylserine (BBPS) membranes. The formation of the HII phase was modulated by Ca2+ ions, pH, basic amino acids and protamine. The lamellar-HII phase transition temperature TH of pure egg PE membranes at pH 7.0 was lowered with increasing Ca2+ concentration. This effect was attenuated by the presence of 50 mM lysine methyl ester. In the mixed lipid system, this effect was also observed, but even more pronounced. However this effect could be compensated for by raising the Ca2+ concentration from 2 to 10 mM. This was not observed in the pure PE system. In the absence of Ca2+, lysine methyl ester and protamine lowered TH in both monocomponent and mixed lipid systems, whereas lysine caused the opposite effect. The pH-dependence of mixed lipid systems, which were investigated up to a BBPS content of 20 mol%, clearly shows that increasing PS content stabilizes the lamellar phase even at low pH. The results obtained with model membranes are discussed with respect to biological implications of the lamellar-HII phase transition for the modulation of tight junction stability.     

Membrane-rigidifying effects of anti-cancer dietary factors

Since several anti-cancer drugs interact with cell membrane lipids, the effects of anti-cancer dietary factors on liposomal membranes with different lipid composition were comparatively studied by measuring fluorescence polarization. Fluidity was imparted on both hydrophobic and hydrophilic regions of lipid bilayers by decreasing cholesterol and increasing unsaturated phosphatidylcholine in membranes. At 0.625-10 microM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-cancer drug, doxorubicin, rigidified the tumor cell model membranes consisting of 20 mol% cholesterol and 80 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 1.0, but not daidzein. They were more effective on the membrane core than the membrane surface. Quercetin showed a biphasic effect on the hydrophobic regions of membrane lipid bilayers to rigidify above 5 microM and fluidize below 2.5 microM. In contrast, anti-cancer dietary factors and doxorubicin were not or much less effective in rigidifying the normal cell model membranes consisting of 40 mol% cholesterol and 60 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 0.5. The membrane-rigidifying effects were greater depending on a decrease of the cholesterol/phosphatidylcholine ratio and an increase of the phosphatidylcholine unsaturation degree. Membrane-active dietary factors and doxorubicin inhibited the growth of mouse myeloma cells at 10-100 microM, while the growth inhibition by membrane-inactive daidzein was relatively weak. Anti-cancer dietary factors appear to act on more fluid membranes like tumor cells as well as doxorubicin to induce rigidification, especially in the hydrocarbon core of membrane lipids, which is determined by the composition of cholesterol and unsaturated phospholipids.     

Hypochlorous acid-derived modification of phospholipids: characterization of aminophospholipids as regulatory molecules for lipid peroxidation

Hypochlorous acid (HOCl), an inflammatory oxidant derived from neutrophil myeloperoxidase, can chlorinate cytosolic proteins and nuclear DNA bases of target cells by passing through the cell membrane. However, little is known about the consequences of HOCl-derived modification of cell membrane components, including phospholipids. In this study, we characterize the reaction of HOCl with phospholipid molecules and found that aminophospholipids are the key molecules that chemically regulate lipid peroxidation. Upon incubation with HOCl, the peroxidation of egg yolk phosphatidylcholine was significantly enhanced in the presence of phosphatidylethanolamine (PE). In contrast, the peroxidation was significantly inhibited in the presence of phosphatidylserine (PS). On the basis of mass spectrometric and electron paramagnetic resonance characterization, the initiator of the peroxidation was identified as the nitrogen-centered radical originating from PE-derived chloramines, especially N,N-dichlorinated PE, a major product in the HOCl-modified PE. Although PS was also chlorinated upon reaction with HOCl, the formed chloramine rapidly decomposed to phosphatidylglycolaldehyde, a novel class of lipid aldehyde. Formation of phosphatidylglycolaldehyde was also confirmed in the porcine brain PS and erythrocyte cell membrane ghost exposed to HOCl. These results provide a novel mechanism for the HOCl-induced oxidative damage and its endogenous protection in the cell membrane at the site of inflammation.     

Oleic and docosahexaenoic acid differentially phase separate from lipid raft molecules: a comparative NMR, DSC, AFM, and detergent extraction study

We have previously suggested that the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) may in part function by enhancing membrane lipid phase separation into lipid rafts. Here we further tested for differences in the molecular interactions of an oleic (OA) versus DHA-containing phospholipid with sphingomyelin (SM) and cholesterol (CHOL) utilizing (2)H NMR spectroscopy, differential scanning calorimetry, atomic force microscopy, and detergent extractions in model bilayer membranes. (2)H NMR and DSC (differential scanning calorimetry) established the phase behavior of the OA-containing 1-[(2)H(31)]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE-d(31))/SM (1:1) and the DHA-containing 1-[(2)H(31)]palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE-d(31))/SM (1:1) in the absence and presence of equimolar CHOL. CHOL was observed to affect the OA-containing phosphatidylethanolamine (PE) more than the DHA-containing PE, as exemplified by >2 x greater increase in order measured for the perdeuterated palmitic chain in 16:0-18:1PE-d(31)/SM (1:1) compared to 16:0-22:6PE-d(31)/SM (1:1) bilayers in the liquid crystalline phase. Atomic force microscopy (AFM) experiments showed less lateral phase separation between 16:0-18:1PE-rich and SM/CHOL-rich raft domains in 16:0-18:1PE/SM/CHOL (1:1:1) bilayers than was observed when 16:0-22:6PE replaced 16:0-18:1PE. Differences in the molecular interaction of 16:0-18:1PE and 16:0-22:6PE with SM/CHOL were also found using biochemical detergent extractions. In the presence of equimolar SM/CHOL, 16:0-18:1PE showed decreased solubilization in comparison to 16:0-22:6PE, indicating greater phase separation with the DHA-PE. Detergent experiments were also conducted with cardiomyocytes fed radiolabeled OA or DHA. Although both OA and DHA were found to be largely detergent solubilized, the amount of OA that was found to be associated with raft-rich detergent-resistant membranes exceeded DHA by almost a factor of 2. We conclude that the OA-PE phase separates from rafts far less than DHA-PE, which may have implications for cellular signaling.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine: two products of nonenzymatic modification of aminophospholipids formed in vivo.

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.