Analytical methods for (oxidized) plasmalogens: Methodological aspects and applications.

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Analytical methods for (oxidized) plasmalogens – methodological aspects and applications Beate Fuchs 10.3109/10715762.2014.999675

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Abstract Plasmalogens are a unique class of glycerophospholipids (GPLs) containing a fatty alcohol linked by a vinyl-ether moiety at the sn-1position of the glycerol backbone. At the sn-2 position there is normally a polyunsaturated fatty acyl residue. These two features provide interesting properties to the plasmalogen GPL. Their physiological roles have been challenging to elucidate although plasmalogens represent up to 20% of the total membrane GPLs in humans. Recent studies have revealed plasmalogen deficiencies associated with several human disorders and, therefore, plasmalogens are likely to be specific of different tissues, metabolic processes and developmental stages. The first chapter of this review will discuss the molecular structure and chemistry of plasmalogens, their biological roles and their distributions in cells and tissues in different species. In the second chapter currently used methods of analyzing plasmalogens and their degradation products are described. Although chromatographic methods will be also discussed, special attention will be given to (31P) nuclear magnetic resonance (NMR) spectroscopy and soft ionization mass spectrometry (MS) techniques such as electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF) MS. Finally, in the third chapter of this review selected human diseases and disorders, which are presumable characterized by changes in plasmalogen contents and compositions are described and the used analytical methods discussed.

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Analytical methods for (oxidized) plasmalogens – methodological aspects and applications

Beate Fuchs University of Leipzig, Medical Faculty, Institute of Medical Physics and Biophysics,


Härtelstraße 16-18, D-04107 Leipzig, Germany

Corresponding author: Dr. Beate Fuchs, Institute of Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Tel.: +49-341-9715769, Fax: +49-341-9715709, E-mail: [email protected]

Short title: Methods for plasmalogen analysis Abstract Plasmalogens are a unique class of glycerophospholipids (GPLs) containing a fatty alcohol linked by a vinyl-ether moiety at the sn-1-position of the glycerol backbone. At the sn-2 position there is normally a polyunsaturated fatty acyl residue. These two features provide interesting properties to the plasmalogen GPL. Their physiological roles have been challenging to elucidate although plasmalogens represent up to 20% of the total membrane GPLs in humans. Recent studies have revealed plasmalogen deficiencies associated with several human disorders and, therefore, plasmalogens are likely to be specific of different tissues, metabolic processes and developmental stages. The first chapter of this review will discuss the molecular structure and chemistry of plasmalogens, their biological roles and their distributions in cells and tissues in different species. In the second chapter currently used methods of analyzing plasmalogens and their degradation products are described. Although chromatographic methods will be also discussed, special attention will be given to (31P) nuclear magnetic resonance (NMR) spectroscopy and soft ionization mass spectrometry (MS) techniques such as electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF) MS. Finally, in the third chapter of this review selected human diseases and disorders, which are presumable characterized by changes in plasmalogen contents and compositions are described and the used analytical methods discussed.

Key Words: mass spectrometry, NMR spectroscopy, vinyl-ether glycerophospholipids, oxidation, chromatography Abbrevations AA 9-AA

Arachidonic acid 9-Aminoacridine


AD CHO CID 2-ClHDA CRF DHA DHB DHSM DNPH EI ESI FT GC GPC GPCplasm GPE GPEplasm GPI GPIplasm GPL GPS HDL HOBr HOCl HPLC HR-MAS IMS ICR IRD LC LDL LPC LPL MALDI MS MS/MS NMR PAF PH3 PLA2 PMN PNA PSD PUFA Q-TOF RCDP RCS ROS SM TFA TLC TOF

Alzheimer‟s disease Chinese hamster ovary cells Collision-induced dissociation 2-Chlorohexadecanal Chronic renal failure Docosahexaenoic acid 2,5-Dihydroxybenzoic acid Dihydrosphingomyelin 2,4-Dinitro-phenylhydrazine Electron (impact) ionization Electrospray ionization Fourier transformation Gas chromatography Glycerophosphatidylcholine Glycerophosphocholine-Plasmalogen Glycerophosphatidylethanolamine Glycerophosphoethanolamine-Plasmalogen Glycerophosphatidylinositol Glycerophosphoinositol-Plasmalogen Glycerophospholipid Glycerophosphatidylserine High density lipoproteins Hypobromous acid Hypochlorous acid High performance liquid chromatography High-resolution magic angle spinning Imaging mass spectrometry Ion cyclotron resonance Infantile refsum disease Liquid chromatography Low density lipoproteins Lysophosphatidylcholine Lysophospholipids Matrix-assisted laser desorption and ionization Mass spectrometry Tandem mass spectrometry Nuclear magnetic resonance Platelet activating factor Phosphine Phospholipase A2 Polymorphonuclear leukocytes para-Nitroaniline Post source decay Polyunsaturated fatty acyl Quadrupole time-of-flight Rhizomelic chondrodysplasia punctata Reactive chlorinating species Reactive oxygen species Sphingomyelin Trifluoroacetic acid Thin-layer chromatography Time-of-flight

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INTRODUCTION Plasmalogens are a special class of glycerophospholipids (GPL) which are structurally similar to other ("normal") GPL, except that plasmalogens have a vinyl ether linkage at the the sn-1 position of the glycerol backbone. Plasmalogen GPL obtained particular relevance upon the investigation of diseases with very low plasmalogen contents such as the Zellweger syndrome. The Zellweger syndrome is characterized by absent peroxisomes contributing to the pathology. Plasmalogens are synthesized in the peroxisomes and therefore, the plasmalogen deficiency in peroxisome-absent pathologies can be explained [1]. Another key


relevance of plasmalogens in biological systems, their potential antioxidative function in biomembranes, is also often considered [2].

Structure and biological roles of plasmalogens Plasmalogens with a choline or ethanolamine (polar) head group at the sn-3 position of the glycerol backbone predominate in the majority of biological membranes, however, to a minor extent inositol or serine plasmalogens are also known. More obvious diversity is coming from the apolar residues at the sn-1 and sn-2 positions, leading to diacyl and ether GPL. Ether GPL species differ from the more common diacyl GPL in having a fatty alcohol or aldehyde, rather than a fatty acyl chain, bound at the sn-1 position. The related fatty alcohols are normally restricted to saturated C16 (C16:0), or mono-unsaturated (C18:1) chains and are linked by an 1-O-alkyl ether bond, also termed a plasmanyl GPL, or contain a vinyl ether, or 1-O-(1Z-alkenyl) bond, termed a plasmenyl GPL or plasmalogen (Fig. 1) [3]. At the sn-2 position, plasmalogens are often enriched in polyunsaturated fatty acyl (PUFA) residues, specifically docosahexaenoic, C22:6 ω−3 (DHA), or arachidonic acid, C20:4 ω−6 (AA). In general, 1-O-alkyl groups are more prominent in choline GPL (GPC) and particularly represented by platelet activating factor, a potent inflammatory mediator having the structure 1-O-alkyl-2-acetyl-sn-GPC. 1-O-(1Z-alkenyl) groups are found primarily in ethanolamine GPL (GPE). As plasmalogens are located in the cellular membrane, organelles and lipid rafts and represent (at least in selected cases) even major constituents of membrane lipids, their presence is responsible for characteristic biophysical properties. The perpendicular orientation of the sn-2 acyl chain and the lack of a carbonyl group at the sn-1 position affect

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the hydrophobicity of these lipids causing stronger intermolecular hydrogen bonding between the individual phospholipid molecules [4]. Plasmalogens are usually found in the membranes of anaerobic bacteria, vertebrate and invertebrate animal species. They are mostly absent in aerobic bacteria, fungi and plants which have distinct biosynthesis than most animals. This suggests the appearance, disappearance and re-appearance of plasmalogens during evolution. This "interrupted" evolution might be explained by the lability of the vinyl-ether bond to oxidation and the


ability of higher organisms to utilize this in an advantageous manner [5]. Although details of the functions of plasmalogens in lipoproteins and cell membranes are not yet well understood, they are anyway supposed to play a crucial role [6]. Plasmalogens are not only structural membrane components and a reservoir of lipid-derived second messengers; they are also known to facilitate membrane fusion, play an important role during differentiation, are involved in ion transport, cholesterol efflux and store longchain PUFA. However, the function of plasmalogens as endogenous antioxidants in defending cellular membranes and lipoproteins from reactive oxygen species (ROS) is controversially discussed [2,6,7]. Comprehensive surveys of ROS-induced oxidation reactions particularly relevant in inflammatory processes are available in [8,9,10,11,12].

The controversial antioxidative function of plasmalogens The acid-labile vinyl ether moiety at the sn-1 position of the glycerol backbone of the plasmalogen causes the unique susceptibility of these compounds to oxidative damage [7,13,14,15,16]. The hydrogen atoms adjacent to the vinyl ether bond have relatively low disassociation energies and are preferentially oxidized in comparison to common diacyl GPL when exposed to ROS [17]. Therefore, it was proposed that plasmalogens reduce the oxidation of PUFAs and other oxidation-sensitive membrane lipids, suggesting a role of plasmalogens as antioxidants [18]. For instance hypochlorous/hypobromous acid (HOCl/ HOBr) which can be generated under inflammatory conditions by granulocytes reacts about 10-times faster with the alkenyl-ether of plasmalogens than with unsaturated acyl residues [19]. In [20] it was shown that the saturation degree of diacyl GPLs increases in the absence of plasmalogen GPE during oxidation. In contrast, the rate of GPL destruction decreased in the presence of plasmalogen GPE indicating a protective function of plasmalogens. However, similar studies with plasmalogen GPCs are needed because the ethanolamine

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headgroup of plasmalogen GPE is highly reactive itself [21]. Sindelar et al. [22] showed that the products of plasmalogen oxidation are unable to further propagate lipid peroxidation; thus plasmalogens may terminate lipid oxidations by free radicals. However, it remains to be elucidated whether the plasmalogen oxidation products themselves - for instance free aldehydes, 1-hydroxy (or lyso)-2-acyl-sn-GPL, 1-formyl-2-acyl-sn-GPL - are harmful (Fig. 2). In contrast, Jansen & Wanders [23] found only a marginal influence of plasmalogens on the superoxide anion radical-induced oxidation of lipids. However, it must be emphasized


that the investigation of superoxide anion radicals under in vivo conditions is extremely difficult because these species possess only a very moderate reactivity but are easily converted into much higher reactive species in the presence of traces of low-valent transition metal ions such as Cu+ or Fe2+. The first in vivo study of plasmalogen GPE indicated more pronounced effects regarding energy metabolism disruption and only a moderate lipid peroxidative insult after phosphine (PH3) administration in brain regions, such as brainstem, which is known to contain high basal levels of plasmalogen GPE. In contrast, regions such as the brain cortex with the lowest plasmalogen GPE concentrations are characterized by deleterious PH3 effects [24]. The majority of the studies dealing with plasmalogen oxidation focused on the influence of the vinyl ether bond at the sn-1 position but the highly reactive PUFAs typically esterified at the sn-2 position were often neglected [25]. In [26] two major classes of oxidized plasmalogen products could be identified: oxidation products of the sn-1 position (cf. Fig. 2) and oxidation products of the fatty acyl residue at the sn-2-position [27]. Oxidation of the PUFA at the sn-2 position by addition of one or two oxygen atoms seems to play a particular role, whereas the vinyl ether in the sn-1 position is partially retained [26]. According to recent data [28] the oxidation products of the polyunsaturates affect the vinyl ether rather than the vinyl ether protects the unsaturates from oxidation. However, the suggested protective role of the unsaturated residue in the sn-2 position is supported by the slow rate of lysoplasmenylethanolamine oxidation and a delayed oxidative degradation of sn-2 arachidonoyl diacyl glycerophospholipids in the presence of plasmalogens [18]. This is in partial agreement with our own studies were it was shown that the sn-1 lyso-product formation is followed by the cleavage of the sn-2 polyunsaturated fatty acyl group [29,30]. Oxidized plasmalogens seem to be converted into harmless products that can be re-utilized

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by the organism [29]. Lower levels of lipid peroxides exist in brain tissue after DHA supplementation [31]. Two possible mechanisms are likely to account for the antioxidant protection which can obtained when brain tissue is enriched with DHA-containing plasmalogens. Firstly, an increase in plasmalogen biosynthesis may take place after supplementation with DHA. Secondly, enrichment with DHA i.e. n-6 species may enhance the antioxidant capacity of the existing plasmalogen and diacyl-GPE species [32].

Plasmalogens as lipid mediators


Plasmalogens represent a major storage depot for AA supply [7,33,34]. The release of AA and also DHA from the sn-2 position is assumed to play an important role in signal transduction [7]. This applies for oxidation products of AA such as thromboxanes or leukotrienes and DHA may be involved in the vesicle formation during neurotransmitter release [7]. A plasmalogen-selective Ca2+-independent phospholipase A2 (PLA2), e.g. the 40 kDa myocardial cytosolic PLA2 [35] as well as the 39 kDa bovine brain cytosolic PLA2 [36,37,38] induce plasmalogen degradation into lysoplasmalogens and free AA [36], whereby the latter product can be subsequently metabolized to eicosanoids [7]. The eicosanoid formation constitutes the first wave of second messenger generation, the immediate phase [7]. Both, physiological and pathophysiological processes are associated with the concentration of free AA [39]. The lysoplasmalogens will be reacylated or degraded by lysoplasmalogenase [36]. The generation of lysoplasmalogen can induce changes in membrane permeability, membrane fluidity and allows the influx of external Ca 2+ ions via plasma channels. This enables the translocation of Ca2+-dependent enzymes and induces a subsequent wave of second messenger generation, the late phase of signal transduction [7]. Low levels of these mediators have trophic effects, whereas high concentrations cause cytotoxic effects, and may be involved in allergic response, inflammation and trauma [7]. The availability of lysoplasmalogens can induce the biosynthesis of another lipid mediator: the platelet activating factor (PAF) which contains an acetyl residue in sn-2 position by CoA independent transacylases [40,41,42].

Cell & tissue distribution of plasmalogens in different species Individual human and animal tissues and cells can differ significantly regarding their plasmalogen contents. In human cells the plasmalogen GPL amount accounts for

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approximately 8–20 % of the total GPL mass [4]. Human heart contains 36-41% plasmalogen GPCs, whereas human polymorphonuclear leukocytes (PMN) possess only about 9.4% plasmalogen GPC related to the total GPC content. Human heart (32-50%) [7], brain (20-50%) [7] and spermatozoa (about 55%) [43] contain the highest amounts of plasmalogens. The same applies for other vertebrate species such as cattle. Spermatozoa from different species are characterized by different plasmalogen contents and compositions. The plasmalogen content of porcine spermatozoa constitutes


about 23%, whereby these alkenyl-acyl compounds constitute nearly 37% of the total GPC content in bull spermatozoa [44]. An extraordinarily high contribution of plasmalogens in the GPC and GPE fractions has been established in ram spermatozoa [45]. High amounts of PUFA can be found in the plasmalogen fractions of human and boar spermatozoa [46]. The PUFA chains (22:5 and 22:6) at the sn-2 position of the glycerol backbone represent an important prerequisite for the fertilizing capacity because they enhance the sperm membrane fluidity which is crucial for successful fusion with the female oocyte [28,43,47]. Additionally, plasmalogens contribute to the formation of macro- and microdomains which are required for spermatogenesis and are assumed to obtain the maintenance during the passage of the sperm cells through the female reproductive tract [48,49]. The predominance of ether lipids including essential amounts of plasmalogens in ruminantia spermatozoa is obvious but this is not always the case: the more complex GPL composition of feloideae spermatozoa is clearly dominated by diacyl GPL with only marginal amounts of plasmalogens - as demonstrated in [50]. In the same way as spermatozoa some heart tissues also contain high levels of plasmalogens. Human, bovine, rabbit and guinea pig heart contain between 32 and 52% plasmalogens compared to the total GPL content [7,51], whereas rat heart tissue contains much smaller plasmalogen amounts (less than 10%). The plasmalogen distribution in the brain of different species differs regarding GPE and GPC plasmalogen contents. Human, rat, and guinea pig brain contains about 20-22% plasmalogen GPE [51], whereas koala brain contains 55% in the grey and 70% in the white matter [52]. In contrast, the plasmalogen GPC concentration may be also very small (human, rat, guinea pig) or even negligible (koala) [51]. During the myelination, the plasmalogen GPE concentration increases rapidly. This rise can be observed up to an age of 30 to 40 years

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followed by a decline during subsequent aging. The ethanolamine GPLs of the myelin sheath contain up to 70% plasmalogen GPE [7]. Large amounts of these lipids are esterified with DHA at the sn-2 position [32]. Plasmalogens, in particular plasmalogen GPE, also have been identified as abundant GPLs of eye lenses, whereby the distribution is inhomogeneous in different regions [28,53,54,55,56]. Plasmalogen GPEs are enriched in the epithelial layer exhibiting an important function in epithelial cell polarity, i.e. the control of cell migration and


differentiation as well as lens transparency. The tremendous age-dependent decrease of plasmalogens finally causes cataract [28]. However, it is speculated that in the rodent with the highest life span (more than 28 years), the naked mole-rat, the high levels of plasmalogens enhance the membrane antioxidant protection and contribute to the longevity of this animal [57,58].

The role of (oxidized) plasmalogens in selected diseases The vinyl ether bond and the corresponding lyso-compounds of plasmalogens are considered as sensitive surrogate markers of oxidative stress [6]. Beside lyso-compounds, however, the long chain aldehydes released upon plasmalogen oxidation are also studied and possible injurious or harmless effects (for instance by their reaction with the amino groups of proteins) are examined [59]. Elevated levels of aldehydes or their reaction products with amino groups (of proteins but as well phosphatidylethanolamines and -serines), respectively, are involved in numerous cellular conditions, some of which are potentially toxic [60]. The oxidation of phospholipids in biological membranes has been implicated in a variety of human diseases, such as atherosclerosis [61], ischemia [62], carcinogenesis [63], aging of brain in general and Alzheimer‟s disease [64]. In particular, the plasmalogen GPL decline in some tissues upon normal aging and at some pathological conditions is often considered to be associated with oxidative stress. Plasmalogens are highly positively correlated with the integrity of high-density lipoproteins (and particularly the integrity of the apolipoprotein A1) and experience a significant reduction (40 %) upon aging which is presumably caused by the reduced function of the peroxisomes in the liver [65]. Plasmalogens are linearly correlated with metastases spreading in vivo. Therefore, the plasmalogen concentration can be useful in the prognosis of the most frequently observed human cancers, particularly in pathological breast, lung and prostate tissue. The significantly increased content of monoenoyl fatty acyl

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residues (particularly oleic acid) in the plasmalogen subfraction is also very indicative. The determination of the monoenoic/saturated fatty acyl ratio facilitates the discrimination between normal tissues, benign and neoplastic tissues with high sensitivity. This makes plasmalogens suitable as reliable biomarkers [66].

Analytical methods to detect plasmalogens and their oxidation derived products


Each of the available analytical techniques for plasmalogen analysis has its specific advantages and drawbacks. Generally, most of the analytical methods used for the analysis of GPLs are also useful for the detection of plasmalogens and/or their oxidation products [67]. In order to remove watersoluble metabolites, the sample lipids should be extracted, i.e. enriched prior to further analysis by established procedures, normally by using mixtures of chloroform and methanol [68]. It is important to know, that any addition of acid (for instance in order to improve the extraction of acidic GPLs by screening the negative charges) must be strictly avoided as plasmalogens are extremely sensitive to even traces of acids and decompose under the generation of lysophosphatidylcholine (LPC) and the corresponding aldehyde [44]. The obtained chloroform layer can be used for further analysis without the necessity of further purification. In Tab. 1 several selected techniques of lipid/plasmalogen analysis are listed. This table provides a very general overview of various methods and the underlying principles as well as the corresponding advantages and disadvantages. All the methods for plasmalogen GPL analysis can be grouped into techniques based on chromatography, mass spectrometry, and other spectroscopic techniques.

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Thin-Layer Chromatography Thin-layer chromatography (TLC) is the most frequently applied technique for the separation of GPL, since this method has low costs and can be performed in a faster and more convenient way than high performance liquid chromatography (HPLC). Touchstone published in [69] a comprehensive review on this topic and there is another updated review available [70].


Particular advantages of TLC are that several samples can be simultaneously investigated on a single TLC sample plate and that there are no memory effects at all since a TLC plate is discarded after its single use. The selective staining of a certain GPL can be achieved by spray reagents that are commercially available [69]. One particular powerful approach is the treatment of plasmalogen containing samples by acidic DNPH (2,4-dinitrophenylhydrazine): under these conditions plasmalogens are hydrolyzed and the corresponding aldehyde is released [71]. The aldehyde reacts with the DPNH and the resulting orange color can be used densitometrically to determine the plasmalogen content. Beside these major advantages one serious disadvantage of TLC is, that the determination of the fatty acyl composition of (plasmalogen) GPLs and also the differentiation between diacyl and alkyl-acyl linked species within one GPL class is difficult. However, if TLC is combined with other techniques, the rather limited resolution can be overcome [72,73]. Compounds separated by TLC can be directly analyzed, i.e. when they are still bound to the stationary phase by high-resolution magic angle spinning (HR-MAS) NMR [74]. This means that there is no need for the elution of the GPL from the sample plate which is necessary for many other methods. Thus, HR-MAS NMR reduces the risk of 'loosing' certain lipid fractions during the elution process. On the other hand,

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there are indications that not all lipid classes can be released to the same extent from the TLC plate. This applies for TLC as well as HPLC, but the effect seems more striking in the case of TLC [75]. However, in contrast Teuber et al. found out that there is no lipid class-dependent loss for TLC analysis of phospholipids [76]. Another elegant method to avoid losses during the elution process subsequent to TLC is TLC-MALDI (imaging) [77]


that allows the detection of all relevant GPL classes without the need of previous staining. Even minor lipid classes – including alkenyl-acyl GPLs – can be easily detected with much higher sensitivity in comparison to common TLC staining protocols. However, it should be noted that a significant part of GPLs present on the silica surface is exposed to atmospheric oxygen, which can facilitate autoxidation reactions. This is indeed particularly true when highly unsaturated lipid species are of interest. Furthermore, the silica surface is slightly acidic and may also lead to unwanted chemical reactions, in particular, the hydrolysis of plasmalogen species into the corresponding lysophospholipids (LPLs) [78]: the detection of LPC in the PC fraction is always an indication that there is a certain plasmalogen moiety.

High performance liquid chromatography Silica gel and aluminum oxide are the most established stationary phases for the separation of GPL mixtures by HPLC [79]. GPL can be separated according to their tendency to interact with certain polymeric compounds (termed „stationary phases‟) or the solvents. At present, chemically-modified silica gels are also increasingly commercially available. In this context, the terms „normal phase‟ (silica gel) and „reversed phase‟ (chemically-modified

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silica gel - often with C18 residues) chromatography are often used. As a very general rule, „normal phase‟ columns are used for the separation of GPL mixtures into the individual GPL subclasses, whereas „reversed phase‟ columns are used for the separation of GPL according to differences in their fatty acyl compositions [80]. More difficult than the stationary phase is the selection of an appropriate solvent system. Again, as a general rule,


acetonitrile/ methanol/ water mixtures are most suitable for the separation of GPL with choline headgroups, whereas hexane/isopropanol/water mixtures are more useful for other GPL classes. Many applications of HPLC separations for GPL analysis exist and are well documented in the literature [79,81,82,83]. HPLC is rather time-consuming if many different samples have to be investigated, however, nowadays there are also "multiplexing" systems available allowing the analysis of several samples in parallel. In the case of UV detection, which is the most frequently used method in routine applications of HPLC [80], only GPL with unsaturated fatty acyl residues can be detected. Plasmalogen GPLs normally contain highly unsaturated fatty acyl residues and are, thus, sensitively detectable by UV.

(Liquid Chromatography/) Mass Spectrometry based methods Mass spectrometry (MS) is often used in lipid research [84], especially in combination with gas chromatography (GC) - particularly in the past [85]. If MS is used in combination with GC, only the free fatty acid produced in a first derivatization step from the intact GPL, e.g. by acidic or alkaline hydrolysis can be analyzed [85]. Thus, the fatty acyl and long chain fatty aldehyde compositions of given GPLs can be obtained by using 'normal'

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GC/MS. A recent review about MS of fatty aldehydes is available in [86]. Strategies based on GC-MS and LC-MS for the analysis of chlorinated plasmalogen lipids (which are generated in the presence of activated chlorine, for instance, if there is a reaction with HOCl) were summarized in a recently published review [87]. Electron impact ionization (EI) MS (which was nearly exclusively available in the past) [72,88,89] is capable of ionizing


intact lipids but structural information is normally obtained from typical fragmentation patterns and the intensity of the molecular ion is often very weak or is even completely absent. Nevertheless, with the invention of more sophisticated ionization techniques, the analysis of intact GPLs has become possible by the so-called 'soft-ionization' techniques [72]. Although a number of different ionization techniques are currently available in lipid research, only two of them play a major role: electrospray ionization (ESI) [90,91] and matrix-assisted laser desorption and ionization (MALDI) [92,93]. Of course, the determination of the molecular weight alone does not provide structural information and tandem mass spectrometry (MS/MS) is normally required. Tandem MS is the process of selecting an ion, causing it to fragment and obtaining a mass spectrum of the resulting fragment ions. In [94] linear iontrap ESI mass spectrometric approaches applying MS3 and MS4 were used regarding the structural characterization of alkenyl-, alkyl- and diacyl-GPLs. This method surely has surely broad applications in structural identifications of plasmalogens. In [95] collision-induced dissociation (CID) ESI tandem MS was used to study the collision-induced dissociation of various GPE plasmalogens and these authors could provide evidence that both, the ether residue and the acyl residue in sn-2 position can be unequivocally assigned by this approach.

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One very important strategy - called „shotgun lipidomics‟ - was developed by Richard W. Gross and Xianlin Han by employing ESI intrasource separation techniques [96]. Individual molecular species of most major and many minor lipid classes can be fingerprinted and quantitated directly from biological lipid extracts without the need for chromatographic purification prior to MS characterization. Shotgun lipidomics is fast, highly sensitive, and can help to identify hundreds of lipids missed by other methods – all with a very small tissue sample so that specific cell or minute biopsy samples can be examined.


However, sophisticated MS instruments with excellent sensitivities and mass analyzers with a significant dynamic range (to be able to detect major and minor species in a single mass spectrum) are required. Therefore, this approach is rather expensive.

MALDI-TOF mass spectrometry based methods Although MALDI devices with real MS/MS (or TOF/TOF) are nowadays commercially available, they are rather seldom because they are expensive. However, if MALDI-TOF devices with a reflectron are available, it is possible to record 'post source decay' (PSD) spectra that are also helpful to obtain some structural information on the compounds of interest: TOF/TOF instruments may be additionally combined with "Collision Induced Dissociation" (CID): an inert gas (Ar or He at a certain pressure are commonly used) is introduced into a dedicated collision cell between both TOF analyzers. The parent ion of interest is selected and transmitted into the collision cell, where collisions between the gas molecules and the parent ions will occur. By this energy transfer, fragmentations of the parent ions occur leading to the generation of charged and neutral fragments that may help structural elucidation [97,98]. Using MALDI MS, a special "matrix" enabling the cationization of the analyte is

required.

As these

are

normally organic

acids

(2,5-

dihydroxybenzoic acid (DHB) is highly established for positive ion detection

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while 9-aminoacridine (9-AA) is increasingly used to record negative ion spectra) lipids are normally detected as H+ (from the matrix) and Na+ (from salts which are normally present in the sample) adducts. This complicates the shape of the spectra. Trifluoroacetic acid (TFA) is a frequently used additive in MALDI MS in order to enhance the intensities of the H+ adducts. While TFA is used only in rather small amounts (about 0.1 vol-%) this amount is


already sufficient to induce the partial hydrolysis of the plasmalogens in lipid samples [44]. Therefore, analysis of samples containing alkenyl-acyl GPLs has to be performed with great caution regarding their acid-labile group in the sn-1 position. On the other hand, the extreme sensitivity of the alkenyllinked GPL against acid can be also used to estimate the plasmalogen content in lipid mixtures by MALDI-TOF MS, TLC or 31P NMR spectroscopy [46]. When problems with peak overlapping (H+, Na+ and differences in acyl compositions) have to be solved, the use of Cs+ is advisable [99,100]. By the typical mass differences in comparison to the diacyl GPL species alkenyl( = -16 u) or alkyl-acyl ( = -14 u) GPL species can be differentiated. Typical spectral patterns of biological GPL mixtures (extracted from two different stem cell types) containing different amounts of plasmalogens are shown in Fig. 3. The mass spectrum (recorded in the presence of an excess of CsCl) of the murine stem cells (a) indicates a more pronounced diversity of GPL, particularly of ether-linked GPCs (cf. the peaks at m/z = 878.5, 900.5 and 928.5) [101]. Furthermore, murine stem cells contain high amounts of highly unsaturated fatty acyl chains (the peak at m/z = 942.5, for instance, represents GPC 18:0/20:4) [101]. It is obvious that such compounds are nearly completely absent in the spectrum of ovine mesenchymal stem cells (b). In contrast, the mass spectrum of the ovine mesenchymal stem cell

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extracts indicates the presence of a significant amount of GPC 16:0/18:1 (m/z = 892.5) i.e. a monounsaturated GPL. The comparison between these two cell types is exclusively done using the ratios of the intensities of the different GPL peaks and no absolute data analysis is attempted. To be able to detect GPE and GPE plasmalogens in the negative ion mode and to avoid suppression effects of further negatively charged GPLs


(e.g. glycerophosphatidylinositol (GPI) 18:0/20:4 at m/z = 885.6) or GPL with quaternary ammonia groups (choline headgroups) [102,103], pnitroaniline (PNA) [104] or 9-AA [105] is often used as matrix. From Fig. 4 it is obvious that the negative ion MALDI spectra allow a very fast identification of GPE and ether-linked GPE as well as the GPI and etherlinked GPI species in even complex lipid mixtures: For instance, the murine stem cells (a) [101] possess a relatively high portion of GPE and ether-linked GPE, whereas the ovine mesenchymal stem cells (b) have only a very small GPE and ether-linked GPE content. In contrast, the ovine mesenchymal and the murine stem cells contain both ether-linked GPIs - beside the base peak GPI 18:0/20:4. Recently, a very simple method to identify plasmalogens in crude lipid extracts was suggested [106]. As illustrated in Fig. 5 the reaction of plasmalogens with acidic dinitrophenylhydrazine (DNPH) directly leads to the hydrolysis of the plasmalogen and the subsequent conversion of the released aldehyde into a 2,4-dinitrophenylhydrazone that is easily detectable in the negative ion MALDI spectrum. The reader should note, however, that the applied conditions are not sufficiently acidic to induce also the hydrolysis of alkyl-acyl or diacyl lipids. Therefore, the discrimination between plasmalogens and other lipids can be unequivocally made.

16

MALDI/TOF MS analysis is not limited to organic extracts but tissue slices may be also directly analyzed [107,108]. In [109] MALDI imaging MS (IMS) of GPLs was reviewed. This led to an increased interest in lipids: lipids are relatively abundant in all tissues and lead to high ion yields, i.e. they can be sensitively detected. One additional approach to provide more detailed compositional


information without resorting to tandem MS/MS is exact mass measurement. Such analysis is based on the observation that the individual atoms within a biological sample will have masses that are not exact integers. This is especially relevant for hydrogen, with a mass of 1.008. Consequently, species with the same nominal mass but comprising different numbers of H, O or 13C atoms will have mass differences of 0.1 amu (atomar mass units) or less. Separating such small mass differences is normally beyond the maximum resolution of triple quadrupole (Q) or Q-TOF instruments, but is possible using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [110]. Nowadays, however, mass spectrometers with high resolving power and high mass accuracy are normally equipped with an orbitrap mass analyzer [111]. By using such an FT-ICR or orbitrap mass spectrometer e.g. GPE a diacyl species with m/z = 752.5230 (GPE 17:0/20:5) can be clearly differentiated from an alkyl-acyl species (containing one oxygen atom less) with m/z = 752.5587 (GPE 18:0alk/20:5) [110]. Of course, however, this is only feasible when isobaric species are of interest - "real" isomers cannot be differentiated without using fragment ion information. For instance, GPC 18:0/18:2 and GPC 218:1 can be exclusively differentiated by means of MS/MS.

17

31

P Nuclear Magnetic Resonance Spectroscopy

Among all the nuclei present in plasmalogen GPLs, phosphorus is (besides the proton) the most widely used NMR-active nucleus. The high magnetogyric ratio of the (spin-1/2) P−31 nucleus in combination with its high natural abundance (100%) leads to good detectability and highlyresolved spectra even at lower field strengths [118]. Although the proton is


still more sensitive, the 31P NMR the resonances (even in complex mixtures) show less overlap and there is also no need of solvent suppression [112] which may lead to distortions of the baseline. Individual GPL classes and even the fatty acyl composition and the linkage type in sn-1 position of a given GPL can be differentiated by

31

P NMR. For optimization of the

resolution and sensitivity the interested reader is referred to [113] and [114], where the careful selection of most suitable solvent systems are described. Finally,

31

P NMR seems superior for the determination of absolute amounts

of different GPL classes in mixtures and most of the other methods so far described do not provide optimum results [115] - even if internal standards (such as deuterated lipids) are used. A set of typical

31

P NMR spectra of an

artificial GPL mixture (a) and two organic extracts of biological relevance (b and c) recorded in water in the presence of a large excess of the detergent sodium cholate is shown in Fig. 6. The detergent sodium cholate is used to suppress the aggregation of lipids which would be accompanied by increased line-widths. All major GPL classes can be simultaneously detected and further information about fatty acyl composition and the linkage type in the sn-1 position of a given GPL can be readily obtained. By shifting pH or changing temperature the resonance overlap of e.g. (diacyl) GPC and plasmalogen GPC can be additionally improved [116] and some fine-tuning

18

of the measuring conditions are normally required in dependence on the system of interest. Hopefully, it is obvious from Fig. 6 that both, boar spermatozoa (b) and murine stem cells (c), have a more complex peak pattern in comparison to the spectrum shown in (a). Please also note that boar spermatozoa (b) and murine stem cells (c) contain plasmalogen GPE, but only boar spermatozoa (b) contain plasmalogen GPC.


1

H/13C Nuclear Magnetic Resonance Spectroscopy

Proton and carbon NMR spectroscopy are traditionally used for studies of biological systems [117,118,119]. However, due to the large number of chemically non-equivalent protons within a GPL sample and the relatively small chemical shift range of 1H NMR spectra (only 10 ppm), confusion arises already if relatively simple lipid mixtures consisting of only two or three individual GPL classes have to be analyzed. Therefore, 1H NMR (even in combination with 2D) is no longer the lipid analysis method of choice because considerable signal overlap occurs [118]. Therefore, 1H NMR is increasingly replaced by 13C NMR, if a sufficient amount of sample is available [117], and/or if 'inverse' methods can be applied [118]. 'Inverse' techniques do not detect sample the bound to

13

13

C nuclei directly, but

C chemical shift information by observing the protons directly

13

C. This advanced approach is explained in more details in the

textbook by Braun et al. [118]. However, even if this method is much more sensitive in comparison to the direct

13

C observation, it provides only about

one percent of 1H NMR: the natural abundance of 13C is about 1% only and this limits the achievable sensitivity even if sophisticated methods are used.

Selected biological applications

19

Many oxidation products of plasmalogens, for instance, induced by addition of HOCl, were studied by combinations of different analytical methods. For instance, the oxidative degradation of plasmalogen GPLs was analyzed by 1H NMR and TLC [18] and 1H NMR, TLC, GC and ESI-MS [120]. In [121] chlorohydrin generation from plasmalogens was verified by LC-ESI MS using CID MS/MS of the molecular ion of the LPC-chlorohydrin and


additionally by GC-MS analysis of the resulting -chloro fatty aldehyde derivatives. The subsequent conversion of the initially generated LPC to glycerophosphocholine was also investigated in plasmalogen samples by combined MALDI-TOF MS and

31

P NMR spectroscopy [122]. In [123]

plasmalogens were identified as the major GPEs in rat peritoneal surfaces by LC-ESI MS and their fragmentation patterns by CID using an ion trap MS. By an adapted Girard derivatizing reagent (4-Hydrazino-N,N,N-trimethyl-4oxobutanaminium iodide with a propyl chain) long-chain aldehydes released from plasmalogens present in the plasma extracts from children with metabolic defects were modified. This was done because it was found that the de novo generated derivatives have superior ESI-MS/MS fragmentation characteristics [124]. The examination of oxidized plasmalogens, which was primarily performed by ESI MS in the past [125,126], is nowadays increasingly performed by MALDI-TOF MS due to the simplicity of this method and the capacity to record spatially resolved spectra when biological samples are of interest. MALDI-TOF MS analyses were, for instance, performed to detect typical oxidation products in spermatozoa (a rich source of plasmalogens) upon storage [127].

20

Performing comparative measurements prior and subsequent to hydrolysis of plasmalogens is a commonly used method and was applied, for instance, to monitor the changes of the lipid composition of neutrophilic granulocytes in dependence on stimulation conditions by

31

P NMR [128].

Many further plasmalogen containing cell and tissue extracts such as human spermatozoa [129], animal spermatozoa [44,46] and lens tissue [100] have


been also investigated by MALDI MS. By using shotgun lipidomics and multi-dimensional MS techniques, new roles for plasmalogens in cerebral function have been accrued [96]. In [58] the plasmalogen contents of skeletal muscle, heart, liver, and liver mitochondria were compared in two different animal species (rat/mice) with reference to their maximum lifespan. It became evident that the plasmalogen content correlates closely with the lifespan of the corresponding species [58]. Recently, the role of plasmalogens in maintaining oxidative stability in selected liposomal systems was studied by 31P-NMR and it was concluded that plasmalogen GPE is not an antioxidant but rather a pro-oxidant because it has absolutely no antioxidative effect towards GPL oxidation. In this study there was also no different effect of ethanolamine plasmalogen extracted from bovine brain and soy lecithin on lipid oxidation [130].

Aging Recently, Maeba et al. [65] developed a highly sensitive and convenient determination method of plasmalogens by HPLC using radioactive iodine (125I), which is capable of reacting with the vinyl ether bond of plasmalogens. Plasmalogen GPE and GPC in human plasma were about three orders of magnitude more sensitively detectable than by the classical determination with non-radioactive iodine and it was obvious that the plasmalogen concentration decreases with age.

21

Aging processes promote oxidative imbalances and simultaneously confer a decline of the endogeneous antioxidative functions [131,132]. In different mammalian tissues an age-dependent plasmalogen decrease could be observed [2]. A gradual linear decline of plasmalogen GPE content with age in normal human brain, starting with 30 years, was reported [133]. An 80 year old person reaches the actual plasmalogen content value of a one year old child. The decreasing plasmalogen content in the aging brain is associated with an increasing ratio of plasmalogen epoxide to native plasmalogen suggesting a potential role of


lipid peroxidation [134]. In [135] age-related diseases were investigated and the serum levels of different human subjects quantified by the

125

I-HPLC method (vide supra) and

characterized by LC-MS/MS. Oxidized products of plasmalogens, free aldehydes and hydroxyaldehydes accumulate in 30-times higher concentrations in aged brains indicating an increased turnover of plasmalogens, their increased hydrolysis and a more significant extent of oxidation reactions [136]. Nevertheless, the mild stress occurring during normal aging can be nearly completely tolerated by the up-regulation of the synthesis of antioxidant defense systems. These antioxidant defense systems, such as tocopherols and ascorbic acid, are able to restore the oxidative balance [7].

Neurological disorders In [137] a high correlation between increased plasmalogen GPE levels (quantified by

31

P NMR spectroscopy and high-performance TLC) and the

effects of myo-inositol on the rat brain membrane GPL metabolism could be established. Further myo-inositol and plasmalogen metabolism studies of rat brain were performed using 13C and 31P NMR spectroscopy. It was suggested that an increased plasmalogen GPE synthesis [138,139] is capable of protecting the neurons against oxidative stress [140]. Very recently, a detailed one-dimensional

and two-dimensional

1

H and

13

C

NMR

spectroscopic study of brain lipid extracts was performed to clarify the role of lipids in the disease progression of human intracranial tuberculomas in comparison to healthy brain tissue. It was shown that specific resonances of

22

cholesterol ester, signals for plasmalogens and phenolic glycolipids detected in the spectra of intracranial tuberculoma extracts were absent in the control samples [141]. LC coupled to FT-ICR MS revealed in [142] that mouse brain plasmalogens are important targets for HOCl-mediated modification in vitro and in vivo. Furthermore, a novel glycosphingolipid containing a long chain aldehyde conjugated to galactose and glycerol has been isolated from brain


and characterized by combined 1H and 13C NMR spectroscopy [143,144]. Besides Alzheimer‟s disease (AD) other neurological disorders such as multiple sclerosis [145] and fetal alcohol syndrome [7] have been successfully investigated by TLC and correlated with decreased plasmalogen levels. Gerstl et al. [146] investigated already in 1965 the alterations in myelin fatty acids and plasmalogens in multiple sclerosis by GC and HPLC. This obviously emphasizes the long-term interest in plasmalogens.

Spinal Cord Ischemia and Reperfusion Plasmalogen content alterations in neurological disorders may result from reduced biosynthesis or an increased degradation. Neuropathologic conditions (such as such as spinal cord ischemia and reperfusion) which are associated with oxidative stress cause an inversely variation of the plasmalogen levels according to the oxidative burden [2] and are accompanied by neurodegeneration. Plasmalogen decrease in the myelin sheath caused by the stimulation of the plasmalogen-selective PLA2 was characterized by TLC and a 10% reduction after 1 minute compression (spinal cord trauma) and even 18% after 30 minutes compression is obvious [147]. The consequences due to the shear stress are manifested in changes of the membrane fluidity and permeability, increased Ca 2+-influx, impaired mitochondrial function as well as the formation of ROS accompanied by subsequent lipid peroxidation.

Alzheimer’s disease (AD) Pettegrew et al. studied the brain membrane phospholipid alterations in AD by 31P NMR spectroscopy, and found amongst other differences a significant elevation in the plasmalogen GPEs [148]. However, the opposite turned out

23

in the majority of similar studies. One reason for the decreased ethanolamine plasmalogen level in AD is the decreasing peroxisomal pathway of ethanolamine synthesis in elderly people. A correlation of the decreased plasmalogen GPE levels with the severity of the dementia could be established by LC-MS/MS [149]. By means of a linear regression models it can be predicted that the plasmalogen levels decrease years before the


clinical symptoms are manifested. In contrast to normal aging, in AD severe oxidative stress can be observed [7]. The plasmalogen reduction in AD is poorly understood, however, the plasmalogen deficiency is correlated with different clinical dementia ratings of AD patients. The increased oxidative damage in AD brain due to several factors such as age-related decrements in energy

availability,

mitochondrial

dysfunction,

glutamatergic

neurotransmission and accumulation of amyloid-beta correlates with membrane instability resulting in synapse loss and neurodegeneration [150,151]. Ischemia, Reperfusion, Arteriosclerosis Myocardial ischemic injury [152] has attracted interest because of the predominant existence of plasmalogens in the sarcolemma, the electrophysiologically active membrane in the myocardium. Plasmalogen hydrolysis has been investigated by FAB MS and GC-MS [153]. Oxidation products of plasmalogens as well as unsaturated fatty acids, that accumulated in infarcted tissue were detected in [154] by GC-MS. The key step in atherosclerosis development is most likely the macrophage transformation into foam cells by the uptake of oxidized LDL [155]. The plasmalogen decrease and subsequent plasmalogen epoxide and -hydroxyaldehyde formation was studied by TLC and plays a major role in this respect by causing an NADPH-oxidase activity increase associated with the increased ROS generation in stimulated macrophages [156]. LDL plasmalogens are oxidized nearly completely after 180 minutes while HDL plasmalogens suffer from oxidation only to about 5% resulting in the release of -chloro

24

fatty aldehyde species, i.e. 2-chlorohexadecanal (2-ClHDA) and the concomitant production of sn-1 LPL through an PLA2-independent mechanism [157]. As plasmalogens do not only reduce the lipoprotein oxidation as endogenous antioxidants but are also required for the process of HDL-mediated cellular cholesterol-efflux, serum plasmalogens can be regarded as beneficial factors as well as an enhanced HDL/LDL ratio [65]. Therefore, the determination of the serum plasmalogen concentrations could be very useful in clinical diagnosis [65].

Chronic renal failure (CRF)


A reduced content of serum plasmalogen GPL and elevated levels of lipid peroxidation products in uraemic patients suffering from chronic renal failure (CRF) suggests increased oxidative stress in haemodialysis [158]. Both, the excessive ROS generation and the low antioxidant levels potentially contribute to atherogenesis and consequently to the high incidence of premature cardiovascular disease in these patients. The progression of CRF can be associated with an enhanced production of oxidized LDL whereby LDL-particles with an increased plasmalogen content could be detected by TLC and are characterized by an enhanced oxidative resistance [2,159] emphasizing the protective role of plasmalogens in lipoproteins [160,161,162]. This impaired antioxidant defense system and the disturbed lipid metabolism contribute to the pathogenesis of cardiovascular diseases and cancer observed in CRF patients receiving long-term haemodialysis [163,164]. Recently, the -oxidation of α-chlorinated fatty acids was studied by LC-MS/MS and ESI-MS/MS and it turned out that α-chlorinated dicarboxylic acids are generated that are excreted in the urine [165].

Peroxisomal disorders In [166] the amount of plasmalogen-linked PL was determined and compared in mutated and control cells on the basis of the acid-lability of plasmalogens directly on a TLC plate, which was exposed to HCl vapors releasing the corresponding LPLs that can be easily identified by their characteristic chromatographic properties as well as their typical free hydroxyl group. It turned out that plasmalogen GPE was absent in the mutated cells. Recently, the

specific

brain

metabolic

changes

associated

with

rhizomelic

25

chondrodysplasia punctata – a genetic peroxisomal disorder, characterized by delayed myelination, which is related to the inadequacy of plasmalogens biosynthesis - were successfully examined by proton magnetic resonance (MR) spectroscopy [167]. Peroxisomal

disorders

such

as

the

Zellweger

Syndrome,

neonatal

adrenoleukodystrophy (NALD), Infantile Refsum disease (IRD) - autosomal recessive diseases - are characterized by an enzyme deficiency of dihydroxyacetone phosphate


acyltransferase and alkyldihydroxyacetone phosphate synthase and the resulting deficiency of plasmalogens [7,168,169]. A survey of the plasmalogen ratios between healthy controls and patients with different peroxisomal disorders determined by GC-MS is available in [170]. Rhizomelic Chondrodysplasia Punctata (RCDP) is classified also as „peroxisomal‟ disorder and its severity depends on the plasmalogen levels in the blood. In [171] different methods based on GC or GC-MS to characterize peroxisomal disorders are reviewed. To clarify cellular functions a mutant CHO (Chinese hamster ovary) cell line with a dramatically reduced rate (90%) of plasmalogen biosynthesis and reduced levels of plasmalogens may serve as a suitable cellular model [172]. Although the peroxisomes of this cell line are intact, it represents a representative model for complications associated with peroxisomal disorders such as RCDP or Zellweger syndrome resulting from the dysfunction of one or more metabolic functions. In a mouse model the importance of plasmalogens was also studied and plasmalogen levels of different cells and tissues measured by GC [173]. Plasmalogen deficiency is a severe problem in both diseases but only in the Zellweger syndrome it can be normalized by enhancement of plasmalogens by diet and circulating lipoproteins [174]. Chronic feeding of 1-O-octadecyl-sn-glycerol to patients suffering from deficiency in tissue ether glycerolipids showed an increase in the plasmalogens content of their erythrocytes. However, very little is so far known about the ether lipid content of other tissues in these patients. Nevertheless, feeding 1-O-heptadecyl-sn-glycerol to young rats showed that this ether lipid is incorporated to a high extent into the plasmalogens of all tissues except brain [175].

Conclusions

26

Hopefully, this review could provide some information on structures, functions and diseaserelated in vivo and in vitro changes of plasmalogens. Methods of plasmalogen analysis and their specific oxidation product detection were also discussed. In this author´s opinion the development of suitable analytical methods is urgently required to shed light on the important functional roles of plasmalogens and their contributions to different pathologies. Beside their possible role as endogenous antioxidants, plasmalogens possess many different


functions under in vivo conditions, for instance, 

Mediators of membrane dynamics



Source of lipid second messengers



Storage of polyunsaturated fatty acids



Mediators of membrane signaling

Due to these different tasks, it is not surprising that changes in plasmalogen contents and/or structure and/or compositions are of considerably relevance in many diseases, for instance, atherosclerosis, Alzheimer´s disease and peroxisomal disorders. Nevertheless, the effects of plasmalogen depletion or reduction still require detailed additional studies and further improvements of suitable analytical methods in order to unravel the underlying biochemical processes. Acknowledgements The author wishes to thank Dr. Jürgen Schiller for his continuous support and many helpful hints. Declaration of interest This work was supported by the German Research Council (DFG FU 771/1-2). The author declares that she does not have any financial, consulting, or personal relationships with other people or organizations that could influence the author‟s work. It is also declared that this manuscript was not written with the help of any scientific writing assistance (use of an agency or freelance writer) and there was no grant support other than the above mentioned one. Finally, the author declares that she is employed at a non-profit institute at the Medical Faculty of the University of Leipzig.

27

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Table 1: Overview of selected techniques of plasmalogen and/or glycerophospholipid analysis. The advantages and drawbacks of the various methods are listed.

Fuchs Table 1 PRINCIPLE

ADVANTAGES


- separation of Thin-layer GPLs on a - well established chromatography "stationary phase" - not expensive (TLC) due to differences in - fast polarity Highperformance liquid chromatography (HPLC)

- separation of GPLs on a stationary phase under high pressure by evolution with different solvents - separation of Gas volatile compounds chromatography on a carrier gas. (GC) - detection often by mass spectrometry

Soft ionization mass spectrometry (MS)

31

P nuclear magnetic resonance (NMR)

1

H/13C NMR

- nowadays often used as modern MS techniques to avoid fragmentation of the analyte: e.g. ESI and MALDI

- differences in electron densities lead to different chemical shifts of the nuclei within a given compound

- differences in electron densities lead to different chemical shifts of the nucleus

- well established - can be used also for the preparative scale

DRAWBACKS

REMARKS

- rather low resolution, only GPL classes can be separated (normal phase) - fatty acyl composition of a given plasmalogen can be hardly determined (reversed phase)

- standards are required - frequently used as the very first step in the analysis of an unknown lipid sample

- widely empirical - rather time-consuming - high amounts of solvents are needed

- highly automated devices available - most important technique in lipid research

- derivatization steps are required - not the plasmalogen but only free fatty acids are detected

- extremely sensitive - low fragmentation - molecular ions (or “quasimolecular ions”) detectable

- semiquantitative method - sensitivity differs for different plasmalogen GPL classes - impurities should be strictly avoided (ESI)

- only phosphoruscontaining groups are detectable - most relevant GPL classes can be easily differentiated - good sensitivity (at least regarding other NMR active nuclei) - all compounds detectable - not affected by ion composition - correlation (2D) experiments can be performed

- in most lipid laboratories available - frequently used as the first step in the analysis of an unknown GPL sample - changes of the fatty acyl composition of plasmalogens can be investigated, e.g. in diseases or under oxidative stress - this field is currently fastly developing - ESI is so far most frequently used in plasmalogen analysis, but applications of MALDI are increasing

- resolution depends strongly on experimental conditions - expensive equipment - some overlap between plasmalogen and diacyl species occur depending of experimental conditions

- recording a 31P NMR spectrum takes some time, but is user-friendly - work-up of samples is minimal

- expensive equipment - low sensitivity (13C) - solvent suppression is required (1H)

- one major advantage of 1 H NMR is that it can be applied under in vivo conditions.

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Fig. 1: Schematic sketch of the molecular architecture of diacyl GPL, plasmanyl and plasmenyl GPL (commonly termed "plasmalogens"). Plasmalogens are characterized by a vinyl ether bond at the sn-1-position of the glycerol backbone that is formed from plasmanyl GPL by plasmenylethanolamine desaturase and further enzymes. Further details are available in [175]. "X" denotes the polar head group, which is typically ethanolamine or choline. R 1 denotes the carbon chain at the sn-1 position, and R2 the fatty acyl residue at the sn-2 position. The fatty acyl chains at the sn-2-position of plasmalogens are often highly


unsaturated residues such as arachidonoyl or docosahexaenoyl.

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Fig. 2: Schematic sketch of the degradation products of plasmalogens. Vinyl ether containing plasmalogens are highly sensitive to ROS and reactive chlorinating species (RCS). RCS cleave the vinyl ether bond generating -chloro fatty aldehydes and LPLs [29,175,175]. A secondary attack of RCS causes the formation of chlorohydrins at the polyunsaturated fatty acyl chain. Finally, the chlorine atom destabilizes (due to its high electronegativity and the negative inductive (-I) effect) the ester bond and glycerophosphocholine (GPC) is generated. The reaction with other ROS leads similarly to


the formation of LPLs and aldehydes as well as the corresponding formyl-compound. For abbreviations, cf. Fig. 1.

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Figure 3: Selected positive ion MALDI-TOF mass spectra of two organic extracts of murine stem cells (a) and ovine mesenchymal stem cells (b). The spectra were recorded with PNA as matrix and were saturated with CsCl prior to MS characterization in order to minimize peak overlapping. Spectra are scaled with reference to the peak of the highest intensity. Selected mass assignments are shown in the spectra. In the upper part of the figure, specific GPL classes are assigned to different mass regions. A more detailed assignment is available in [101]. Reproduced with modifications and permissions from Elsevier [101] and


Bentham Science [175].

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Figure 4: Negative ion MALDI-TOF mass spectra of organic extracts of murine stem cells (a) and ovine mesenchymal stem cells (b). All spectra were recorded with para-nitroaniline (PNA) as matrix and scaled with reference to the peak of the highest intensity. Selected mass assignments are shown in the spectra. In the upper part specific GPL classes are assigned to different mass regions. A more detailed assignment is available in [101]. Reproduced with


modifications and permission from Bentham Science [Error! Bookmark not defined.].

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Figure 5: Scheme how plasmalogens can be identified in complex lipid mixtures. Plasmalogens are hydrolyzed under acidic conditions into lysolipids and a characteristic aldehyde, that is subsequently derivatized with DNPH into a product that is easily detectable by negative ion (ESI or MALDI) MS. For abbreviations please see Fig. 1. Reproduced with


modifications and permission from Springer [106].

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Figure 6: 31P NMR spectra of an artificial phospholipid mixture (a) as well as of organic extracts of boar spermatozoa (b) and murine stem cells (c) recorded in aqueous sodium cholate (as a detergent to suppress the generation of supramolecular structures) at 310 K. Abbreviations used in peak assignment: GPEplasm – alkenyl-acyl glycerophosphoethanolamine; GPEether – alkyl-acyl glycerophosphoethanolamine; GPCplasm – alkenyl-acyl glycerophosphocholine; GPCether – alkyl-acyl glycerophosphocholine; SM – sphingomyelin; DHSM – dihydrosphingomyelin; GPS –


phosphatidylserine; GPI – phosphatidylinositol; GPC – phosphatidylcholine; GPE – phosphatidylethanolamine. Reproduced with modifications and permission from Bentham Science [Error! Bookmark not defined.].

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Analytical methods in lipidomics and their applications.

Platelet-rich plasma (PRP): methodological aspects and clinical applications.

Special issue on "Analytical methods for the detection of oxidized biomolecules and antioxidants".

Comparison of five analytical methods for the determination of peroxide value in oxidized ghee.

Analytical methods for Staphylococcus aureus.

PP089. Analytical aspects of marinobufagenin and its applications in the diagnosis of preeclampsia.

Methodological aspects of perfusion SPECT.

Adriamycin cellular transport: methodological aspects.

Correlation 2D-NMR experiments involving both 13C and 2H isotopes in oriented media: methodological developments and analytical applications.

Reticulated platelets: analytical aspects and clinical utility.

The hemocompatibility of oxidized diamond nanocrystals for biomedical applications.

New insights into perfluorinated adsorbents for analytical and bioanalytical applications.

Some methodological aspects in the psychosomatic gynaecology.

Radiosurgery with a linear accelerator. Methodological aspects.

Methodological aspects on drug receptor binding analysis.

Active control equivalence trials: some methodological aspects.

Typing for MLC determinants: methods and applications.

Recombinant horseradish peroxidase: production and analytical applications.

Analytical methods for Bacillus cereus and other Bacillus species.

Polymeric vehicles for topical delivery and related analytical methods.

Current initiatives for the validation of analytical methods for botanicals.

Analytical methods in biotechnology: introduction.

Safety, Health, and Methodological Aspects of Plant Sterols and Stanols.

[Pharmacokinetics, metabolism, and analytical methods of ethanol].