Acyl Composition Research Articles

Cardiolipin linoleic acid content and mitochondrial cytochrome c oxidase activity are associated in rat skeletal muscle

Cardiolipin (CL) is an inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function. Specifically, CL and CL linoleic (18:2ω6) content are known to be positively associated with cytochrome c oxidase (COX) activity. However, this association has not been examined in skeletal muscle. In this study, rats were fed high-fat diets with a naturally occurring gradient in linoleic acid (coconut oil [CO], 5.8%; flaxseed oil [FO], 13.2%; safflower oil [SO], 75.1%) in an attempt to alter both mitochondrial CL fatty acyl composition and COX activity in rat mixed hind-limb muscle. In general, mitochondrial membrane lipid composition was fairly resistant to dietary treatments as only modest changes in fatty acyl composition were detected in CL and other major mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). As a result of this resistance, CL 18:2ω6 content was not different between the dietary groups. Consistent with the lack of changes in CL 18:2ω6 content, mitochondrial COX activity was also not different between the dietary groups. However, correlational analysis using data obtained from rats across the dietary groups showed a significant relationship (p=0.009, R2=0.21). Specifically, our results suggest that CL 18:2ω6 content may positively influence mitochondrial COX activity thereby making this lipid molecule a potential factor related to mitochondrial health and function in skeletal muscle.

A mass spectrometry-based method for the assay of ceramide synthase substrate specificity

The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.

Understanding Molecular Complexity in Protein and Peptide-Lipid Systems

Peptide addition to lipid membranes has been shown in some cases to lead to significant molecular complexity, principally as a consequence of the chemical processes that occur subsequent to the addition (http://dx.doi.org/10.1016/j.jmb.2013.07.013). Foremost amongst these is acyl transfer from lipid constituents of the membrane to acceptor sites on the peptide, generating lipidated peptides and lyso-lipids. As an example, addition of the peptide melittin to a two-component lipid membrane leads to the potential formation of up to 20 different peptide species and 8 different lyso-lipids, many of which have been detected, some in significant quantities. The lipidation by-products, lyso-lipids, may also serve as acyl group donors for further lipidation. This process is not restricted to peptides; some proteins and low molecular weight compounds exhibit the hallmarks of this lipidation activity. In a proof-of-principle study, we have examined the lipidation profiles of candidate proteins and shown that they closely resemble the fatty acyl composition of the membrane in which they are generated, supporting the hypothesis that their lipidation arises from membrane lipid precursors. At the other extreme, low molecular weight compounds have been found to undergo similar lipidation processes. Current work to understand the factors that dictate this reactivity, and the consequences of lipidation, will be discussed.

Profiling and relative quantitation of phosphoinositides by multiple precursor ion scanning based on phosphate methylation and isotopic labeling.

Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP3), phosphatidylinositol bisphosphate (PtdInsP2), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called "charged diacylglycerol fragment ion-specific multiple precursor ion scanning" (DAG(+)-specific MPIS), coupled with prior phosphate methylation. Using DAG(+)-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP2, and 3 PtdInsP3 molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian samples. We also developed a method that involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD2N2) for quantitation of phosphoinositides. CD2N2 was generated in situ through acid-catalyzed H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane). Phosphoinositides, extracted from a PC3 prostatic cancer cell line, were labeled either with CH2N2 or CD2N2 and mixed in known proportions for DAG(+)-specific MPIS-based mass spectrometry (MS) analysis. The results indicate that isotopic labeling is capable of providing accurate quantitation of PtdInsP3, PtdInsP2, and PtdInsP with adequate linearity as well as high reproducibility with an average coefficient variation of 18.9%. More importantly, this new methods excluded the need for multiple phosphoinositide internal standards. DAG(+)-specific MPIS and isotopic labeling based MS analysis of phosphoinositides offers unique advantages over existing approaches and presents a powerful tool for research of phosphoinositide metabolism.

Effects of synthetic alkamides on Arabidopsis fatty acid amide hydrolase activity and plant development

Alkamides and N-acylethanolamines (NAEs) are bioactive, amide-linked lipids that influence plant development. Alkamides are restricted to several families of higher plants and some fungi, whereas NAEs are widespread signaling molecules in both plants and animals. Fatty acid amide hydrolase (FAAH) has been described as a key contributor to NAE hydrolysis; however, no enzyme has been associated with alkamide degradation in plants. Herein reported is synthesis of 12 compounds structurally similar to a naturally occurring alkamide (N-isobutyl-(2E,6Z,8E)decatrienamide or affinin) with different acyl compositions more similar to plant NAEs and various amino alkyl head groups. These “hybrid” synthetic alkamides were tested for activity toward recombinant Arabidopsis FAAH and for their effects on plant development (i.e., cotyledon expansion and primary root length). A substantial increase in FAAH activity was discovered toward NAEs in vitro in the presence of some of these synthetic alkamides, such as N-ethyllauroylamide (4). This “enhancement” effect was found to be due, at least in part, to relief from product inhibition of FAAH by ethanolamine, and not due to an alteration in the oligomerization state of the FAAH enzyme. For several of these alkamides, an inhibition of seedling growth was observed with greater results in FAAH knockouts and less in FAAH over-expressing plants, suggesting that these alkamides could be hydrolyzed by FAAH in planta. The tight regulation of NAE levels in vivo appears to be important for proper seedling establishment, and as such, some of these synthetic alkamides may be useful pharmacological tools to manipulate the effects of NAEs in situ.

Cardiolipin remodeling: a regulatory hub for modulating cardiolipin metabolism and function.

Cardiolipin (CL), the signature phospholipid of mitochondria, is involved in a plethora of cellular processes and is crucial for mitochondrial function and architecture. The de novo synthesis of CL in the mitochondria is followed by a unique remodeling process, in which CL undergoes cycles of deacylation and reacylation. Specific fatty acyl composition is acquired during this process, and remodeled CL contains predominantly unsaturated fatty acids. The importance of CL remodeling is underscored by the life-threatening genetic disorder Barth syndrome (BTHS), caused by mutations in tafazzin, which reacylates monolysocardiolipin (MLCL) generated from the deacylation of CL. Just as CL-deficient yeast mutants have been instrumental in elucidating functions of this lipid, the recently characterized CL-phospholipase mutant cld1Δ and the tafazzin mutant taz1Δ are powerful tools to understand the functions of CL remodeling. In this review, we discuss recent advances in understanding the role of CL in mitochondria with specific focus on the enigmatic functions of CL remodeling.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Distribution of C16:0, C18:0, C24:1, and C24:0 sulfatides in central nervous system lipid rafts by quantitative ultra-high-pressure liquid chromatography tandem mass spectrometry

Sulfated galactosylceramides (sulfatides) are glycosphingolipids associated with cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) and are highly expressed in brain tissue. Although it is known that sulfatide species show heterogeneity in their fatty acid acyl group composition throughout brain development, their lipid raft distribution and biological relevance is poorly understood. We validated a fast and sensitive ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to measure developmentally regulated sulfatide species (C16:0, C18:0, C24:1, and C24:0) in central nervous system (CNS) lipid rafts isolated without using detergent. Our UHPLC–MS/MS assay showed good accuracy and precision with a linear range of 5 to 1000nM for C18:0 and C24:1 sulfatides and 10 to 1000nM for C16:0 and C24:0 sulfatides. We applied this quantitative analysis to detergent-free lipid rafts isolated from wild-type mice and arylsulfatase A-deficient (ASA knockout) mice that accumulate sulfatides. All four sulfatide species were more abundant in raft membranes than in non-raft membranes, with a significant increase in lipid rafts isolated from ASA knockout mice. This is the first description of an analytical method to study these sulfatide species in raft and non-raft membranes and has the potential to be applied to preparations from other tissues.

Cardiolipin metabolism and the role it plays in heart failure and mitochondrial supercomplex formation.

Cardiolipin is a major membrane phospholipid in the mitochondria and is essential for cellular energy metabolism mediated through mitochondrial oxidative phosphorylation. Recent studies indicate that it plays a diverse role in cellular metabolism. Eukaryotic cardiolipin is synthesized de novo from phosphatidic acid via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway and is deacylated to monolysocardiolipin in order for it to be remodelled into the form that is observed in mitochondrial membranes. This resynthesis of deacylated cardiolipin from monolysocardiolipin occurs via the Barth Syndrome gene product tafazzin and acyllysocardiolipin acyltransferase-1, monolysocardiolipin acyltransferase-1 and the alpha subunit of trifunctional protein. Heart failure is a disease condition in which the amount and type of cardiolipin is altered. Several animal models have been generated to study the role of altered cardiolipin in heart failure. In many of these models loss of the tetralinoleoyl-cardiolipin species is observed during the development of the heart failure. In the doxycycline inducible short hairpin RNA tafazzin knock down mouse, loss of tetralinoleoyl-cardiolipin is associated with a mitochondrial bioenergetic disruption. Reduction in mitochondrial supercomplex formation and NADH dehydrogenase activity within these supercomplexes is observed. Modulation of CL fatty acyl composition may serve as a therapeutic strategy for the treatment of several pathologies including cardiac dysfunction.We propose that increasing cardiolipin may improve mitochondrial function and potentially serve as a therapy for diseases which exhibit mitochondrial dysfunction involving reduced cardiolipin.

Lipid profile remodeling in response to nitrogen deprivation in the microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae).

Many species of microalgae produce greatly enhanced amounts of triacylglycerides (TAGs), the key product for biodiesel production, in response to specific environmental stresses. Improvement of TAG production by microalgae through optimization of growth regimes is of great interest. This relies on understanding microalgal lipid metabolism in relation to stress response in particular the deprivation of nutrients that can induce enhanced TAG synthesis. In this study, a detailed investigation of changes in lipid composition in Chlorella sp. and Nannochloropsis sp. in response to nitrogen deprivation (N-deprivation) was performed to provide novel mechanistic insights into the lipidome during stress. As expected, an increase in TAGs and an overall decrease in polar lipids were observed. However, while most membrane lipid classes (phosphoglycerolipids and glycolipids) were found to decrease, the non-nitrogen containing phosphatidylglycerol levels increased considerably in both algae from initially low levels. Of particular significance, it was observed that the acyl composition of TAGs in Nannochloropsis sp. remain relatively constant, whereas Chlorella sp. showed greater variability following N-deprivation. In both algae the overall fatty acid profiles of the polar lipid classes were largely unaffected by N-deprivation, suggesting a specific FA profile for each compartment is maintained to enable continued function despite considerable reductions in the amount of these lipids. The changes observed in the overall fatty acid profile were due primarily to the decrease in proportion of polar lipids to TAGs. This study provides the most detailed lipidomic information on two different microalgae with utility in biodiesel production and nutraceutical industries and proposes the mechanisms for this rearrangement. This research also highlights the usefulness of the latest MS-based approaches for microalgae lipid research.

Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.

Impact of high dietary lipid intake and related metabolic disorders on the abundance and acyl composition of the unique mitochondrial phospholipid, cardiolipin

Excessive dietary lipid intake, coupled with lack of exercise, are the major causes of the development and progression of metabolic syndrome features e. g. obesity, hepatic steatosis, insulin resistance, type 2 diabetes and cardiovascular diseases. These metabolic diseases are associated with both structural and functional alterations of mitochondria. Cardiolipin (CL) is a unique phospholipid that is almost exclusively localized in the mitochondrial inner membrane. Cardiolipin is at the heart of mitochondrial metabolism playing a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics, and in many of the mitochondrial-dependent steps of apoptosis. Indeed, alterations to CL content and acyl chain profile have been associated with mitochondrial dysfunction in multiple tissues in Barth syndrome and in many other physio-pathological conditions. After a brief overview of the biological roles of CL, we highlight the consequences of lipid overload-related nutritional manipulations as well as related metabolic disorders on both CL content and its fatty acid composition in the major metabolic tissues, the heart, muscle and liver. The goal of this review is to fill a void in the CL literature concerning the effects of CL abundance and form that arise following high lipid supplementation and the related metabolic disorders.

Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells

Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained.

Engineering the biosynthesis of novel rhamnolipids inEscherichia colifor enhanced oil recovery

The interfacial tension of rhamnolipids and their applications in enhanced oil recovery are dependent on their chemical structures and compositions. To improve their performances of interfacial tension and enhanced oil recovery, the engineered strategies were applied to produce novel rhamnolipids with different chemical structures and compositions. By introducing different key genes for rhamnolipid biosynthesis, Escherichia coli was firstly constructed to produce rhamnolipids that showed different performances in interfacial tension from those from Pseudomonas aeruginosa due to the different fatty acyl compositions. Then, the mutant RhlBs were created by directed evolution and subsequent site-directed mutagenesis and resulted in the production of the novel rhamnolipids with the different performances in interfacial tension as well as enhanced oil recovery. Lastly, computational modelling elucidates that the single amino acid mutation at the position 168 in RhlB would change the volume of binding pocket for substrate and thus affect the selectivity of rhamnolipid formation in E. coli. The novel rhamnolipids that showed the improved performances of interfacial tension and the potential different applications in enhanced oil recovery were successfully produced by engineered E. coli. This study proved that the combination of metabolic engineering and protein engineering is an important engineered strategy to produce many novel metabolites in micro-organisms.

The freshwater alga Chroothece richteriana (Rhodophyta) as a potential source of lipids

During an ecological study of Chroothece (Rhodophyta) in a small river in a semi-arid region of south-east Spain it became clear that most of these cells had a high lipid content. This suggested potential uses in biotechnology, which has been investigated further. The colonies, which occur in full sunlight, are typically orange-brown. Most, perhaps all, the yellow-orange colour is associated with their high carotenoid content, with the carotenoid to chlorophyll ratio up to 2.7. The polyunsaturated fatty acyl composition of the glycerides was 35.3% of the dry weight. This consisted mainly of omega-3 (5.9%) and omega-6 (29.4%) fats. The relatively high proportion of docosahexaenoyl (1.78%), eicosapentaenoyl (14.15%), arachidonoyl (0.92%) and γ-linolenoyl (0.78%) suggests use for medical and dietary purposes. All cells have a high phycocyanin content whilst phycoerythrin is absent. The alga has a wide distribution globally and hence provides scope for selecting strains with optimum properties.

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2cisΔ9,12; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.

Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids

Normal phase thin-layer chromatography (NP TLC) is an established method of (phospho)lipid analysis. The determination of the fatty acyl composition is, however, a more challenging task by NP TLC. The direct coupling of TLC separation with mass spectrometric detection (e.g., matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS), however, enables a detailed characterization of complex lipid mixtures. Here we show that the thickness of the silica gel layer has a considerable effect on the quality of the mass spectra recorded directly from the TLC plate. In particular, the intensity of the matrix background signals can be reduced if “thinner” TLC layers are used.

193 nm Ultraviolet Photodissociation Mass Spectrometry for the Structural Elucidation of Lipid A Compounds in Complex Mixtures

Here we implement ultraviolet photodissociation (UVPD) in an online liquid chromatographic tandem mass spectrometry (MS/MS) strategy to support analysis of complex mixtures of lipid A combinatorially modified during development of vaccine adjuvants. UVPD mass spectrometry at 193 nm was utilized to characterize the structures and fragment ion types of lipid A from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa using an Orbitrap mass spectrometer. The fragment ions generated by UVPD were compared to those from collision induced dissociation (CID) and higher energy collision dissociation (HCD) with respect to the precursor charge state. UVPD afforded the widest array of fragment ion types including acyl chain C–O, C–N, and C–C bond cleavages and glycosidic C–O and cross ring cleavages, thus providing the most comprehensive structural analysis of the lipid A. UVPD exhibited virtually no dependence on precursor ion charge state and was best at determining lipid A structure including acyl chain length and composition, giving it an advantage over collision based methods. UVPD was incorporated into an LC–MS/MS methodology for the analysis of a number of structural variants in a complex mixture of combinatorially engineered Escherichia coli lipid A.

Head‐group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl‐galactose acyl composition varies with the plant species and applied stress

Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.

A MALDI MS Investigation of the Lysophosphatidylcholine/Phosphatidylcholine Ratio in Human Spermatozoa and Erythrocytes as a Useful Fertility Marker

The human spermatozoa membrane is characterized by a unique fatty acyl composition with significant amounts of highly unsaturated fatty acids, particularly docosahexaenoic acid (22:6), whereby phosphatidylcholine (PtdCho) (16:0/22:6) is the most abundant glycerophospholipid. The large amount of highly unsaturated fatty acyl residues is crucial for the fluidity of the membrane and, therefore, the successful fertilization process. Consequently, however, the spermatozoa are very sensitive to reactive oxygen species (ROS) that are generated under conditions of "oxidative stress" and key players in many pathological conditions. Lipid oxidation of the sperm membrane is accompanied by the loss of the oxidatively modified unsaturated residue (normally in the sn-2 position) and the generation of saturated lysophosphatidylcholine (LysoPtdCho). Although other lysolipids are also generated, LysoPtdCho is the "marker" lipid of choice due to the high abundance of PtdCho. In particular, obesity (body mass index >30kg/m(2)) is characterized by increased ROS generation and negatively affects the reproductive potential. We will show here that the LysoPtdCho/PtdCho ratio can be easily determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The data found do correlate with clinical markers of sperm quality. A very interesting aspect is that the LysoPtdCho/PtdCho ratios determined in the spermatozoa extracts correlate with the LysoPtdCho/PtdCho values determined in the organic extracts of erythrocytes. Thus, there is no absolute need for a sperm investigation, but an estimation of the fertilizing ability of the corresponding male could be also made directly from the blood which is more readily available than the spermatozoa.