Quinones as free-radical fragmentation inhibitors in biologically important molecules

Effects of a number of quinones and diphenols of various structures on free-radical fragmentation processes taking place in alpha-diols, glycerol, 2-aminoethanol, glycero-1-phosphate, ethylene glycol monobutyrate, maltose, and some lipids were investigated. Quinone additions have been found to change the direction of free-radical transformations of the compounds cited above by inhibiting formation of the respective fragmentation products owing to oxidation of radicals of the starting compounds. The results obtained and literature data available allow a suggestion to be made that the system quinone/diphenol is able to not only deactivate or generate such active species as O2.- but also control the realization probability of free-radical processes of peroxidation and fragmentation in biologically important molecules.     

Quinones as Free-radical Fragmentation Inhibitors in Biologically Important Molecules

Effects of a number of quinones and diphenols of various structures on free-radical fragmentation processes taking place in &#102 -diols, glycerol, 2-aminoethanol, glycero-1-phosphate, ethylene glycol monobutyrate, maltose, and some lipids were investigated. Quinone additions have been found to change the direction of free-radical transformations of the compounds cited above by inhibiting formation of the respective fragmentation products owing to oxidation of radicals of the starting compounds. The results obtained and literature data available allow a suggestion to be made that the system quinone/diphenol is able to not only deactivate or generate such active species as O 2 &#148 &#109 but also control the realization probability of free-radical processes of peroxidation and fragmentation in biologically important molecules.     

Lipid analysis of human HDL and LDL by MALDI-TOF mass spectrometry and (31)P-NMR

The analysis of HDL and LDL is important for the further understanding of atherosclerosis because changes of the protein and lipid moieties occur under pathological conditions. Because destruction of lipids leads to the formation of well-defined products such as lysophospholipids or chlorohydrins, methods that allow their fast and reliable determination would be useful. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for the analysis of the lipid composition of human lipoproteins. These data were compared with high resolution (31)P-NMR spectroscopy. Differences between LDL and HDL in sphingomyelin and phosphatidylcholine content could be monitored by NMR and mass spectrometry, and differences with respect to the extraction efficiency were found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A(2) resulted in marked changes (formation of chlorohydrines and lysolipids). Lysophosphatidylcholines were detectable by both methods, whereas MALDI-TOF MS failed to detect chlorohydrines of phospholipids.We conclude that MALDI-TOF MS provides rapidly a reliable lipid profile of lipoproteins. However, a previous lipid separation must be performed to detect lipid oxidation products. NMR can be directly applied, but suffers from lower sensitivity, and provides only limited information on fatty acid composition.     

Properties of liposomes adsorbed by montmorillonite

Adsorption of phosphatidylcholine liposomes by montmorillonite was studied for its effect on peroxidation of phospholipids and structure of the bilayer. The UV spectroscopy has shown that adsorption decreases peroxidation of lipids. A parameter is suggested for estimating an oxidation degree of lipids at the stage of the intensive formation of secondary products of their peroxidation. Fluorescent probes (1-anilinonaphthalene-8-sulphonate and pyrene) are used to establish that structural changes of the bilayer take place in adsorbed liposomes being followed by a decrease in the mobility of surface zones of phospholipid "heads" and by an increase in the polarity of hydrophobic parts of the bilayer.     

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.     

Comparison of different procedures for the lipid extraction from HL-60 cells: a MALDI-TOF mass spectrometric study

A human leukaemia cell line--HL-60--can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.     

Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury

Lipids, particularly phospholipids, are fundamental to CNS tissue architecture and function. Endogenous polyunsaturated fatty acid chains of phospholipids possess cis-double bonds each separated by one methylene group. These phospholipids are very susceptible to free-radical attack and oxidative modifications. A combination of analytical methods including different versions of chromatography and mass spectrometry allows detailed information to be obtained on the content and distribution of lipids and their oxidation products thus constituting the newly emerging field of oxidative lipidomics. It is becoming evident that specific oxidative modifications of lipids are critical to a number of cellular functions, disease states and responses to oxidative stresses. Oxidative lipidomics is beginning to provide new mechanistic insights into traumatic brain injury which may have significant translational potential for development of therapies in acute CNS insults. In particular, selective oxidation of a mitochondria-specific phospholipid, cardiolipin, has been associated with the initiation and progression of apoptosis in injured neurons thus indicating new drug discovery targets. Furthermore, imaging mass-spectrometry represents an exciting new opportunity for correlating maps of lipid profiles and their oxidation products with structure and neuropathology. This review is focused on these most recent advancements in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury.     

Liposomes alter thermal phase behavior and composition of red blood cell membranes

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.     

On the position of the hydro-phobic/philic boundary in lipid bilayers

The sensitivity of calculated structural dimensions of hydrated lipids to the position of the hydrophobic/hydrophilic boundary is reviewed. The position of this boundary is critical in determining the extent of hydration and location of water in the bilayer. A calculation of the dimensions of the hydrophilic and hydrophobic parts of the phosphatidylcholine and ethanolamine bilayer from literature values of the x-ray long spacing shows that the choice of boundary in phospholipids is not arbitrary and is best placed at the average position of the first CH₂ group in the hydrocarbon chains. Calculated dimensions of the hydrocarbon core and the water accessible regions agree with neutron and x-ray diffraction measurements. Hydration differences between phosphatidylcholines and phosphatidylethanolamines are readily explained from derived estimates of the layers of water which cover these headgroups.     

The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31P NMR

The analysis of beef lipids is normally based on chromatographic techniques and/or gas chromatography in combination with mass spectrometry (GC/MS). Modern techniques of soft-ionization MS were so far scarcely used to investigate the intact lipids in muscle tissues of beef. The objective of the study was to investigate whether matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry and ³¹P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef. For the MALDI-TOF MS and ³¹P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. Beside the triacylglycerols (TAGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) species the MALDI-TOF mass spectra of total muscle lipids gave also intense signals of cardiolipin (CL) species.The application of different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), leads to completely different mass spectra: 9-AA is particularly useful for the detection of (polar) phospholipids, whereas apolar lipids, such as cholesterol and triacylglycerols, are exclusively detected if DHB is used. Finally, the quality of the negative ion mass spectra is much higher if 9-AA is used.     

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic “fingerprints” are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.     

A Theoretical Model for the Association Probabilities of Saturated Phospholipids from Two-Component Bilayer Lipid Membranes

The non-random mixing of biomembrane components, especially saturated phospholipids, exhibits important consequences in molecular biology. Particularly, the distribution of lipids within natural and model membranes is strongly determined by the selective association processes. These processes of phospholipids take place due to the cooperative modes in multiparticle systems as well as the specific lipid-lipid interactions both in the hydrophobic core and in the region of the polar headgroups. We demonstrated that the investigation of the selective association processes of saturated phospholipids might contribute to the insight of the lipid domains appearance inside the bilayer membranes. The association probabilities of like-pairs and cross-pairs from a binary mixture of saturated phospholipids were tested for both parallel and anti-parallel alignments of the polar headgroups. The present model confirms the experimental evidence for saturated phospholipids to have a high tendency for association in parallel configuration of the electric dipole moments of the polar headgroups whether the cross-sectional area of the polar headgroup is in an usual range of 25-55 2. There are three major lipid domains in a binary mixture of saturated phospholipids: (i) lipid domains in non-mixed phase of the first mixture component, in parallel alignment of the polar headgroups; (ii) lipid domains in non-mixed phase of the second mixture component, in anti-parallel alignment of the polar headgroups; (iii) lipid domains in mixed phase. We think that the selective association processes of phospholipids are neither exclusively, nor only involved in promoting the lipid domains appearance through bilayer phospholipid membranes.     

Analysis of mammalian fatty acyl-coenzyme A species by mass spectrometry and tandem mass spectrometry

Acyl-CoAs are intermediates of numerous metabolic processes in eukaryotic cells, including beta-oxidation within mitochondria and peroxisomes, and the biosynthesis/remodeling of lipids (e.g. mono-, di-, and triglycerides, phospholipids and sphingolipids). Investigations of lipid metabolism have been advanced by the ability to quantitate acyl-CoA intermediates via liquid chromatography coupled to electrospray ionization-tandem mass spectrometric detection (LC-ESI-MS/MS), which is presently one of the most sensitive and specific analytical methods for both lipids and acyl-CoAs. This review of acyl-CoA analysis by mass spectrometry focuses on mammalian samples and long-chain analytes (i.e. palmitoyl-CoA), particularly reports of streamlined methodology, improved recovery, or expansion of the number of acyl chain-lengths amenable to quantitation.     

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

MALDI-TOF MS of phosphatidylethanolamines: Different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described.In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an established tool for the analysis of proteins, whereas it gained by far less interest in the field of lipid analysis. This method works well with phospholipids as well as organic cell extracts and provides high sensitivity and reproducibility. The aim of the present paper is to extend our previous studies to the analysis of lysophospholipids and phospholipid mixtures. To study the suitability of MALDI-TOF mass spectrometry for the analysis of lysophospholipids, different phospholipids like phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylinositol as well as their mixtures were digested with phospholipase A(2). Positive and negative ion mass spectra of all phospholipids before and after digestion were recorded. In all these cases, the molecular ions of the expected digestion products could be detected and only a very small extent of further fragmentation was observed. On the other hand, spectra of phospholipid mixtures containing phosphatidylcholine were strongly dominated by phosphatidylcholine and lysophosphatidylcholine signals, which prevented the detection of further phospholipids even if those lipids were present in comparable amounts. This is of paramount interest for the analysis of tissue and cell extracts.     

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.     

Practical considerations in BIA/MS: optimizing the biosensor-mass spectrometry interface

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multiplexed analytical technique that utilizes a unique combination of surface plasmon resonance (SPR) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection and analysis of small amounts of proteins residing in complex biological systems. In order to achieve high sensitivity during BIA/MS, certain experimental parameters and sequences of events need to be optimized and maintained. Immobilized ligand density, flow rate and biosensor control (in SPR-BIA) and matrix choice and application (in MALDI-TOF MS) have significant influence on the final outcome of the BIA/MS analysis and, consequently, need to be optimized and carefully controlled. In addition, chip washing and cutting are essential in converting the SPR-active sensor chips into target surfaces amenable to MALDI-TOF MS. Reviewed here are the prerequisites for successfully interfacing SPR-BIA with MALDI-TOF MS.     

Structural characterization of oxidized phospholipid products derived from arachidonate-containing plasmenyl glycerophosphocholine

Plasmenyl phospholipids are a structurally unique class of lipids that contain a vinyl ether substituent at the sn-1 position of the glycerol backbone, imparting unique susceptibility to oxidative reactions that may take place at the cell membrane lipid bilayer. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanism for the antioxidant properties of plasmenyl phospholipids is not fully understood, the oxidation of arachidonate-containing plasmalogen-glycerophosphocholine (GPC) was studied using electrospray tandem mass spectrometry after exposure to the free radical initiator 2, 2'-azobis(2-amidinopropane)hydrochloride (AAPH). Various oxidized GPC products involving the sn-1 position alone (1-formyl-2-arachidonyl lipids and lysophospholipid), oxidation products involving the sn-2 position alone (chain-shortened omega-aldehyde radyl substituents at sn-2) as well as products oxidized both at the sn-1 and sn-2 positions were observed and structurally identified.The results of these experiments suggest that oxidation of plasmenyl phospholipids esterified with polyunsaturated fatty acid groups at sn-2 likely undergo unique and specific free radical oxidation at the 1'-alkenyl position as well as oxidation of the double bond closest to the ester moiety at sn-2.