Temperature effects on plasmalogen profile and quality characteristics in Pacific oyster (Crassostrea gigas) during depuration.

The quality of Pacific oyster (Crassostrea gigas) can be affected by many factors during depuration, in which temperature is the major element. In this study, we aim to determine the quality and plasmalogen changes in C. gigas depurated at different temperatures. The quality was significantly affected by temperature, represented by varying survival rate, glycogen content, total antioxidant capacity, alkaline phosphatase activity between control and stressed groups. Targeted MS analysis demonstrated that plasmalogen profile was significantly changed during depuration with PUFA-containing plasmalogen species being most affected by temperature. Proteomics analysis and gene expression assay further verified that plasmalogen metabolism is regulated by temperature, specifically, the plasmalogen synthesis enzyme EPT1 was significantly downregulated by high temperature and four plasmalogen-related genes (GPDH, PEDS, Pex11, and PLD1) were transcriptionally regulated. The positive correlations between the plasmalogen level and quality characteristics suggested plasmalogen could be regarded as a quality indicator of oysters during depuration.

Distinctive Lipid Characteristics of Colorectal Cancer Revealed through Non-targeted Lipidomics Analysis of Tongue Coating.

The metabolites and microbiota in tongue coating display distinct characteristics in certain digestive disorders, yet their relationship with colorectal cancer (CRC) remains unexplored. Here, we employed liquid chromatography coupled with tandem mass spectrometry to analyze the lipid composition of tongue coating using a nontargeted approach in 30 individuals with colorectal adenomas (CRA), 32 with CRC, and 30 healthy controls (HC). We identified 21 tongue coating lipids that effectively distinguished CRC from HC (AUC = 0.89), and 9 lipids that differentiated CRC from CRA (AUC = 0.9). Furthermore, we observed significant alterations in the tongue coating lipid composition in the CRC group compared to HC/CRA groups. As the adenoma-cancer sequence progressed, there was an increase in long-chain unsaturated triglycerides (TG) levels and a decrease in phosphatidylethanolamine plasmalogen (PE-P) levels. Furthermore, we noted a positive correlation between N-acyl ornithine (NAOrn), sphingomyelin (SM), and ceramide phosphoethanolamine (PE-Cer), potentially produced by members of the Bacteroidetes phylum. The levels of inflammatory lipid metabolite 12-HETE showed a decreasing trend with colorectal tumor progression, indicating the potential involvement of tongue coating microbiota and tumor immune regulation in early CRC development. Our findings highlight the potential utility of tongue coating lipid analysis as a noninvasive tool for CRC diagnosis.

Optimized 13C-TrEnDi Enhances the Sensitivity of Plasmenyl Ether Glycerophospholipids and Demonstrates Compatibility with Other Derivatization Strategies.

The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses.

Rapid Analysis of Plasmalogen Individual Species by High-Resolution Mass Spectrometry.

Analysis and quantification of ether-lipid phospholipid species-also known as plasmalogens-is a crucial step in the study of the biological functions played by these lipids. Application of analytical separation methods and high-resolution mass spectrometry has gained much attention in this regard, while resolution issues and time-consuming sequences interfered with these advances. Herein, we describe a simple and rapid method for the analysis of plasmalogen (Pl) species by HILIC-HRMS. This method is able to identify and quantify relative levels of ethanolamine-plasmalogens (PlsEtn) and choline-plasmalogens (PlsCho) in biological matrices such as whole blood, plasma, erythrocytes, and also retina. Moreover, we provide a detailed and modified lipid extraction method that is applicable to almost all biological matrices.

Metabolomic and exposomic biomarkers of risk of future neurodevelopmental delay in human milk.

BACKGROUND: The chemical composition of human milk has long-lasting effects on brain development. We examined the prognostic value of the human milk metabolome and exposome in children with the risk of neurodevelopmental delay (NDD). METHODS: This retrospective cohort study included 82 mother-infant pairs (40 male and 42 female infants). A total of 59 milk samples were from mothers with typically developing children and 23 samples were from mothers of children at risk. Milk samples were collected before 9 months of age (4.6 ± 2.5 months, mean ± SD). Neurocognitive development was assessed by maternal report at 14.2 ± 3.1 months using the Ages and Stages Questionnaires-2. RESULTS: Metabolome and exposome profiling identified 453 metabolites and 61 environmental chemicals in milk. Machine learning tools identified changes in deoxysphingolipids, phospholipids, glycosphingolipids, plasmalogens, and acylcarnitines in the milk of mothers with children at risk for future delay. A predictive classifier had a diagnostic accuracy of 0.81 (95% CI: 0.66-0.96) for females and 0.79 (95% CI: 0.62-0.94) for males. CONCLUSIONS: Once validated in larger studies, the chemical analysis of human milk might be added as an option in well-baby checks to help identify children at risk of NDD before the first symptoms appear. IMPACT: Maternal milk for infants sampled before 9 months of age contained sex-specific differences in deoxysphingolipids, sphingomyelins, plasmalogens, phospholipids, and acylcarnitines that predicted the risk of neurodevelopmental delay at 14.2 months of age. Once validated, this early biosignature in human milk might be incorporated into well-baby checks and help to identify infants at risk so early interventions might be instituted before the first symptoms appear.

Action of alkyl glycerol ethers and n-3 polyunsaturated fatty acids diet on hematological parameters of blood and liver plasmalogen level in aged rats.

One of the ways to reduce age-related changes can be a diet correction by adding biologically active substances. We studied the effect of a diet including alkyl glycerol ethers (AGs) and n-3 polyunsaturated fatty acid (PUFA) concentrate isolated from the hepatopancreas of Berrytheuthis magister squid on hematological parameters and plasmalogens level in the liver of elderly rats. The senile animals showed decrease in hemoglobin, a three-fold decrease in leukocytes, a three-fold increase in platelet count, and a double decrease of blood coagulation time in the peripheral blood. Age-related changes in rats were characterized by the development of anemia, hypercoagulation, and a decrease in the number of immunocompetent cells. AGs, both separately and in combination with n-3 PUFAs, induced an increase in the number of red blood cells and hemoglobin, a decrease in the number of platelets, and an immunostimulating activity. Under the action of AGs and n-3 PUFAs, the concentration of plasmalogens and docosahexaenoic acid in the rat liver increased 2- and 1.5 folds, respectively. PRACTICAL APPLICATION: This study showed that the combined use of AGs and n-3 PUFAs improves the rheological properties of the blood and the state of the immune system during aging. The enrichment of diet with dietary supplements, whose structure contains AGs and n-3 PUFAs can increase the content of plasmalogens in the body.

Chemical Derivatization-Aided High Resolution Mass Spectrometry for Shotgun Lipidome Analysis.

Chemical derivatization coupled with nano-electrospray ionization (nESI) and ultra-high resolution accurate mass spectrometry (UHRAMS) is an established approach to overcome isobaric and isomeric mass interference limitations, and improve the analytical performance, of direct-infusion (i.e., "shotgun") lipidome analysis strategies for "sum composition" level identification and quantification of individual lipid species from within complex mixtures. Here, we describe a protocol for sequential functional group selective derivatization of aminophospholipids and O-alk-1'-enyl (i.e., plasmalogen) lipids, that when integrated into a shotgun lipidomics workflow involving deuterium-labeled internal lipid standard addition, monophasic lipid extraction, and nESI-UHRAMS analysis, enables the routine identification and quantification of >500 individual lipid species at the "sum composition" level, across four lipid categories and from >30 lipid classes and subclasses.

Lipidomic Analysis of Postmortem Prefrontal Cortex Phospholipids Reveals Changes in Choline Plasmalogen Containing Docosahexaenoic Acid and Stearic Acid Between Cases With and Without Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive and incurable brain disorder that has been associated with structural changes in brain phospholipids (PLs), including diacyl species and ether-linked PLs known as plasmalogens. Most studies have characterized total changes in brain PL pools (e.g., choline plasmalogens), particularly in prefrontal cortex, but detailed and quantitative information on the molecular PL species impacted by the disease is limited. In this study, we used a comprehensive mass-spectrometry method to quantify diacyl and plasmalogen species, alkyl synthetic precursors of plasmalogens, and lysophospholipid degradation products of diacyl and plasmalogen PLs, in postmortem samples of prefrontal cortex from 21 AD patients and 20 age-matched controls. Total PLs were also quantified with gas-chromatography analysis of bound fatty acids following thin layer chromatography isolation. There was a significant 27% reduction in the concentration (nmol/g wet weight) of choline plasmalogen containing stearic acid (alkenyl group) and docosahexaenoic acid in AD compared to controls. Stearic acid concentration in total PLs was reduced by 26%. Our findings suggest specific changes in PLs containing stearic acid and docosahexaenoic acid in AD prefrontal cortex, highlighting structural and turnover PL pathways that could be targeted.

Quantitative and comparative study of plasmalogen molecular species in six edible shellfishes by hydrophilic interaction chromatography mass spectrometry.

Shellfishes contain plasmalogens correlating to the functions of brain, heart, etc. Herein, a mild acid hydrolysis and hydrophilic interaction chromatography (HILIC) tandem mass spectrometry method was developed for analyzing plasmalogens in six shellfish species. A total of 19 plasmalogen molecular species were successfully identified, including nine phosphatidylcholine plasmalogen (plasPC), seven phosphatidylethanolamine plasmalogen (plasPE), and three phosphatidylserine plasmalogen (plasPS). The quantitative results indicated that mussel (32 µg·mg-1) possessed the highest content of plasmalogens, followed by oyster (21 µg·mg-1) and razor clam (15 µg·mg-1). The statistic models showed that the plasPE P-18:0/20:5 (m/z 748), plasPE P-16:0/22:2 & P-18:0/20:2 (m/z 754) and plasPS were the most contributing difference between shellfishes. The results indicated that this method was sensitive and precise to determine plasmalogens in shellfish, and mussel was demonstrated to be a good choice for the large-scale preparation of plasmalogens.

Unequivocal Mapping of Molecular Ether Lipid Species by LC-MS/MS in Plasmalogen-Deficient Mice.

Deficient ether lipid biosynthesis in rhizomelic chondrodysplasia punctata and other disorders is associated with a wide range of severe symptoms including small stature with proximal shortening of the limbs, contractures, facial dysmorphism, congenital cataracts, ichthyosis, spasticity, microcephaly, and mental disability. Mouse models are available but show less severe symptoms. In both humans and mice, it has remained elusive which of the symptoms can be attributed to lack of plasmanyl or plasmenyl ether lipids. The latter compounds, better known as plasmalogens, harbor a vinyl ether double bond conferring special chemical and physical properties. Discrimination between plasmanyl and plasmenyl ether lipids is a major analytical challenge, especially in complex lipid extracts with many isobaric species. Consequently, these lipids are often neglected also in recent lipidomic studies. Here, we present a comprehensive LC-MS/MS based approach that allows unequivocal distinction of these two lipid subclasses based on their chromatographic properties. The method was validated using a novel plasmalogen-deficient mouse model, which lacks plasmanylethanolamine desaturase and therefore cannot form plasmenyl ether lipids. We demonstrate that plasmanylethanolamine desaturase deficiency causes an accumulation of plasmanyl species, a too little studied but biologically important substance class.

Recovery of brain DHA-containing phosphatidylserine and ethanolamine plasmalogen after dietary DHA-enriched phosphatidylcholine and phosphatidylserine in SAMP8 mice fed with high-fat diet.

BACKGROUND: Glycerophospholipids were the main components of cerebral cortex lipids, and there was a close association between lipid homeostasis and human health. It has been reported that dietary DHA-enriched phosphatidylcholine (DHA-PC) and phosphatidylserine (DHA-PS) could improve brain function. However, it was unclear that whether supplementation of DHA-PC and DHA-PS could change lipid profiles in the brain of dementia animals. METHODS: SAMP8 mice was fed with different diet patterns for 2 months, including high-fat diet and low-fat diet. After intervention with DHA-PC and DHA-PS for another 2 months, the lipid profile in cerebral cortex was determined by lipidomics in dementia mice. RESULTS: High-fat diet could significantly decrease the levels of DHA-containing PS/pPE, DPA-containing PS, and AA-containing PE, which might exhibit the potential of lipid biomarkers for the prevention and diagnosis of AD. Notably, DHA-PC and DHA-PS remarkably recovered the lipid homeostasis in dementia mice. These might provide a potential novel therapy strategy and direction of dietary intervention for patients with cognitive decline. CONCLUSIONS: DHA-PC and DHA-PS could recover the content of brain DHA-containing PS and pPE in SAMP8 mice fed with high-fat diet.

Plasmalogen fingerprint alteration and content reduction in beef during boiling, roasting, and frying.

Plasmalogens are dietary phospholipids with beneficial health effects. In this work, plasmalogen characteristics and changes in beef during boiling, frying, and roasting were comprehensively investigated by liquid-chromatography-mass spectrometry. The alteration of plasmalogen fingerprint during cooking processes was found by untargeted omics approach, in which time of boiling, temperature of roasting, and meat core/surface of frying were responsible for the observed variations. Moreover, the targeted determination of representative plasmalogen species showed significant loss with a temperature- and time-dependent manner in roasting and frying. And frying even showed an extra loss in meat surface compared with core. Furthermore, an artificial neural network-based predictive model elucidated the dynamics of plasmalogen species during cooking. Finally, batter-coating pretreatment was performed to show its protection against plasmalogens loss during frying. These results might provide a potential strategy to better control and improve the quality of functional foodstuffs during cooking processes.

Eicosapentaenoic Acid-Enriched Phosphoethanolamine Plasmalogens Alleviated Atherosclerosis by Remodeling Gut Microbiota to Regulate Bile Acid Metabolism in LDLR-/- Mice.

Eicosapentaenoic acid (EPA)-enriched phosphoethanolamine plasmalogens (EPA-PlsEtns) might be retained in the intestine rich in gut microbiota for a long time after treatment. It reminded us that EPA-PlsEtns might affect intestinal microbiota composition and its metabolites, which have been identified as a contributing factor in the development of cardiovascular diseases. In the present study, EPA-PlsEtn administration for 8 weeks significantly reduced the atherosclerotic lesion area in low-density lipoprotein receptor deficient (LDLR-/-) mice. Notably, the serum total cholesterol and low-density lipoprotein cholesterol levels were significantly reduced by 33.6 and 38.2%, respectively, by EPA-PlsEtns instead of EPA in the form of ethyl ester (EPA-EE) treatment compared with the model group. EPA-PlsEtn administration also increased total neutral sterol and bile acids in feces by 92 and 39%, respectively, rather than EPA-EE. Mechanistically, EPA-PlsEtns might affect the abundance of gut microbiota contributing to the alteration of bile acid profiles, which might further accelerate bile acid synthesis via increasing cholesterol 7 α-hydroxylase expression induced by the inhibition of farnesoid X receptor activation.

Metabolomics analysis of human acute graft-versus-host disease reveals changes in host and microbiota-derived metabolites.

Despite improvement in clinical management, allogeneic hematopoietic stem cell transplantation (HSCT) is still hampered by high morbidity and mortality rates, mainly due to graft versus host disease (GvHD). Recently, it has been demonstrated that the allogeneic immune response might be influenced by external factors such as tissues microenvironment or host microbiota. Here we used high throughput metabolomics to analyze two cohorts of genotypically HLA-identical related recipient and donor pairs. Metabolomic profiles markedly differ between recipients and donors. At the onset of acute GvHD, in addition to host-derived metabolites, we identify significant variation in microbiota-derived metabolites, especially in aryl hydrocarbon receptor (AhR) ligands, bile acids and plasmalogens. Altogether, our findings support that the allogeneic immune response during acute GvHD might be influenced by bile acids and by the decreased production of AhR ligands by microbiota that could limit indoleamine 2,3-dioxygenase induction and influence allogeneic T cell reactivity.

[Oral cavity microbiocenosis assessment on the basis of bacterial endotoxin and plasmalogens in a saliva by method GAS-liquid chromatography-mass spectrometry.]

The concentration of plasmalogen bacterial and endotoxin levels in the saliva of patients with different severity of periodontal disease, injury prosthetic bed and with various degrees of the oral cavity microbiocenosis violations was studied. Determination of the presence of the pathological process was carried out clinically, according to the condition of periodontal tissues. The degree of microbiological disorders was assessed by the quantitative ratio of the types of microorganisms isolated from the smear taken from the gingival groove. It was found that the concentration of plasmalogen for normal microbiocenosis is not less than 0.7 µg/g. For the intermediate type of microbiocenosis, the concentration of 1.82 µg/g was determined; for dysbiosis - 5.64 µg/g, and for the expressed violation of the microbial composition accompanied by inflammatory processes - 6.54 µg/g. An increase in the concentration of bacterial endotoxin (be) more than 6.25 nanomole/g indicates the pronounced inflammatory process, regardless of the determined intensity of contamination of opportunistic gram-negative microflora.

Influence of Myo-inositol Plus Ethanolamine on Plasmalogens and Cell Viability during Oxidative Stress.

Previously, we demonstrated that treatment of rats with myo-inositol plus ethanolamine (ME) elevated brain ethanolamine plasmalogens (PE-Pls) and protected against phosphine-induced oxidative stress. Here we tested the hypothesis that ME treatment elevates PE-Pls in a neuro-2A (N2A) cell culture system and protects against hydrogen peroxide (H2O2)-induced oxidative stress, and we assessed the effects of treatments using myo-inositol with or without (+/-) ethanolamine on ethanolamine phospholipids (PLs) and cell viability following H2O2 exposure. Cells were treated with equimolar amounts (500 µM) of myo-inositol, ethanolamine (Etn), or their combination (ME) for 24 h, followed by an additional 24 h exposure to 650 µM H2O2. NMR analyses evaluated the treatment effects on Etn PLs, while LC-MS/MS analyses assessed the molecular species of Etn PLs preferentially affected by ME and H2O2 treatments, especially PE-Pls and their degradation byproducts-lysophosphatidylethanolamine (LPE) and glycerophosphoethanolamine (GPE). Only ME influenced the cellular levels of PLs. ME yielded a 3-fold increase in PE-Pls and phosphatidylethanolamine (PE) ( p < 0.001) and a preferential 60% increase in PE-Pls containing saturated and monounsaturated fatty acids (SFA+MUFA), while polyunsaturated fatty acid (PUFA) species increased by only 10%. Exposing cells to 650 µM H2O2 caused a significant cell death (56% viability), a 27% decrease in PE-Pls, a 201% increase in PUFA-rich LPE, and a ca. 3-fold increase in GPE. H2O2 had no impact on PE, suggesting that LPE and GPE were primarily the byproducts of PE-Pls (not PE) degradation. Surprisingly, ME pretreatment ameliorated H2O2 effects and significantly increased cell survival to 80% ( p < 0.05). Cellular PE-Pls levels prior to H2O2 treatment were highly correlated ( R2 = 0.95) with cell survival, suggesting a relationship between PE-Pls and cell protection. Data suggest that a preferential increase in PE-Pls containing SFA+MUFA species may protect cells from oxidative stress. Such studies aid in our understanding of the neuroprotective mechanisms that may be associated with plasmalogens and the relevance of these phospholipids to neurodegenerative diseases/disorders.

Tissue-selective alteration of ethanolamine plasmalogen metabolism in dedifferentiated colon mucosa.

Human colon lipid analysis by imaging mass spectrometry (IMS) demonstrates that the lipid fingerprint is highly sensitive to a cell's pathophysiological state. Along the colon crypt axis, and concomitant to the differentiation process, certain lipid species tightly linked to signaling (phosphatidylinositols and arachidonic acid (AA)-containing diacylglycerophospholipids), change following a rather simple mathematical expression. We extend here our observations to ethanolamine plasmalogens (PlsEtn), a unique type of glycerophospholipid presenting a vinyl ether linkage at sn-1 position. PlsEtn distribution was studied in healthy, adenomatous, and carcinomatous colon mucosa sections by IMS. In epithelium, 75% of PlsEtn changed in a highly regular manner along the crypt axis, in clear contrast with diacyl species (67% of which remained constant). Consistently, AA-containing PlsEtn species were more abundant at the base, where stem cells reside, and decreased while ascending the crypt. In turn, mono-/diunsaturated species experienced the opposite change. These gradients were accompanied by a gradual expression of ether lipid synthesis enzymes. In lamina propria, 90% of stromal PlsEtn remained unchanged despite the high content of AA and the gradient in AA-containing diacylglycerophospholipids. Finally, both lipid and protein gradients were severely affected in polyps and carcinoma. These results link PlsEtn species regulation to cell differentiation for the first time and confirm that diacyl and ether species are differently regulated. Furthermore, they reaffirm the observations on cell lipid fingerprint image sensitivity to predict cell pathophysiological status, reinforcing the translational impact both lipidome and IMS might have in clinical research.

Comprehensive study of rodent olfactory tissue lipid composition.

The peripheral olfactory tissue (OT) plays a primordial role in the detection and transduction of olfactory information. Recent proteomic and transcriptomic studies have provided valuable insight into proteins and RNAs expressed in this tissue. Paradoxically, there is little information regarding the lipid composition of mammalian OT. To delve further into this issue, using a set of complementary state-of-the-art techniques, we carried out a comprehensive analysis of OT lipid composition in rats and mice fed with standard diets. The results showed that phospholipids are largely predominant, the major classes being phosphatidylcholine and phosphatidylethanolamine. Two types of plasmalogens, plasmenyl-choline and plasmenyl-ethanolamine, as well as gangliosides were also detected. With the exception of sphingomyelin, substantial levels of n-3 polyunsaturated fatty acids, mainly docosahexaenoic acid (22:6n-3; DHA), were found in the different phospholipid classes. These findings demonstrate that the rodent OT shares several features in common with other neural tissues, such as the brain and retina.

Analysis of phosphatidylethanolamine, phosphatidylcholine, and plasmalogen molecular species in food lipids using an improved 2D high-performance liquid chromatography system.

Phospholipids are an important class of lipids in cell membranes and food. Several high-performance liquid chromatography (HPLC) methods have been developed to analyze phospholipids at the molecular species level. We developed a two-dimensional HPLC system with a charged aerosol detector and mass spectrometry (MS) to analyze phosphatidylethanolamine (PE), phosphatidylcholine (PC), and their plasmalogens (pls) extracted from food materials. Accordingly, the phospholipid molecular species can be analyzed in a single step despite using smaller samples. We confirmed that chromatogram peaks from soybean lecithin are mostly baseline separated, assigned, and quantified (24 molecular species for PE and 27 for PC). In addition, it was confirmed that chromatograms of lipids extracted from chicken breast meat include plasmalogen peaks. The PE fraction in lipids extracted from chicken breast meat contained 17 types of ethanolamine plasmalogens, corresponding to approximately 57% of the total by weight. The PC fraction contained only four choline plasmalogens, corresponding to approximately 11% of the total weight. The composition of the pls-PC molecular species differed from that of pls-PEs. The polyunsaturated fatty acids connected at the sn-2 positions of the pls-PEs consisted of 20.5% 20:4 fatty acid and were independent of the carbon chain at the sn-1 position. However, the 18:1 fatty acid at the sn-2 position was dependent on the carbon chain at the sn-1 position.

The Phospholipid Composition of Kangaroo Spermatozoa Verified by Mass Spectrometric Lipid Analysis.

Cryopreservation of kangaroo sperm has not been successful so far, and yet there is no promising cryopreservation protocol for these cells available. However, conservation of gametes is extremely important, particularly in the context of preserving endangered species. As spermatozoa are comprised of different membrane systems, the composition of these membranes might account for difficulties in cryopreservation. Lipids, as the main components, affect the physical properties of biological membranes and play a major role in sperm maturation. Therefore, knowledge of the lipid composition is crucial for any further step toward the preservation of the species. We used MALDI-TOF, ESI-IT, tandem mass spectrometry, and thin layer chromatography to investigate the lipid composition of epididymal spermatozoa of four different kangaroo species. Spectra of these species were very similar with respect to the identified lipid species. Tremendous changes in the lipid composition during the transit of sperm from caput to cauda epididymis could be seen, specifically an increase in poly-unsaturated fatty acids, ether lipids, and plasmalogens, as well as a reduction in mono- and di-unsaturated fatty acids. Additionally, phosphatidylcholines containing docosatrienoic acid (22:3), a heretofore unknown fatty acid for sperm membranes, showed the highest abundance in kangaroo sperm.