Differences in phosphatidylcholine profiles and identification of characteristic phosphatidylcholine molecules in meat animal species and meat cut locations.

Phosphatidylcholine (PC) is an essential component of the plasma membrane. Its profile varies with species and tissues. However, the PC profiles in meat have not been explored in depth. This study aimed to investigate the differences in PC profiles between various meat animal species and meat cut sites, along with the identification of characteristic PC molecules. The results demonstrated that the PC profiles of chicken meat differed from those of other species. Significant differences were also observed between the PC profiles of pork meat and the meat obtained from other species. The amount of PCs containing ether bonds was high in pork meat. PCs containing an odd number of carbon atoms were characteristic of beef and lamb meats. Furthermore, PC profiles differed based on the muscle location in chicken and pork. These results suggest that the PC profiles of skeletal muscles are indicators of animal species and muscle location.

Simultaneous quantification of the lipids phosphatidylcholine, 3-sn-phosphatidylethanolamine, sphingomyelin, and L-α-lysophosphatidylcholine extracted from the tissues of the invasive golden apple snail (Pomacea canaliculata) using UHPLC-ESI-MS/MS.

Lipids such as phosphatidylcholine (PC), 3-sn-phosphatidylethanolamine (PE), sphingomyelin (SM) and L-α-lysophosphatidylcholine (LPC) are the major components of biological membranes and play important roles in physiological functions. Here, PC, PE, SM, and LPC were extracted from golden apple snails (GAS, Pomacea canaliculata) and GAS flesh (GASF) using an ethanol/hexane sequential scheme and quantified simultaneously using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) to evaluate whether the GAS could be the source of the four lipids. Our results suggest that ethanol extracts contained the most crude lipids, and the yield of dry (evaporated) lipids were 3.45 g per 100 g fresh GASF and 1.82 g per 100 g of fresh GAS. Quantification of the lipids using UHPLC-ESI-MS/MS suggested that GAS contained PE, PC, SM and LPC, with SM being the most abundant lipid (after purification: 1.71 and 1.42 mg g-1 dry weight from 100 g of GASF and GAS, respectively). The method we used is cost-effective, and the recovery rates of ethanol and hexane ranged from 80-91% and 87-91% respectively. Overall, GAS and GASF are potential raw materials for lipids such as SM and PC extraction using the ethanol/hexane method. Comparatively, lipids extraction from the GAS is more effective and timesaving. Our finding would provide a way to utilize GAS and potentially control its invasion.

Exploration of natural phosphatidylcholine sources from six beans by UHPLC-Q-HRMS.

Beans are a rich source of phosphatidylcholine (PC). This study aims to explore natural PC sources rich in polyunsaturated fatty acid (PUFA) with nutritional interest. PCs from six beans were purified (purity > 98.2%) by thin layer chromatography (TLC), and subsequently identified by ultra-high performance liquid chromatography-Quadrupole (Q)-high-resolution mass spectrometry (UHPLC-Q-HRMS). Results showed that the PC content of chickpea (Cicer arietinum) and soybean (Glycine max) was 50.0 and 34.0 mg/g, respectively, which was significantly higher than that of other beans (P < 0.05). Gas chromatographic analysis showed that soybean contained high proportion of PUFA (58.78%), and chickpea contained high proportion of docosahexaenoic acid (DHA) (2.73%). A total of 49 molecular species were identified by UHPLC-Q-HRMS. (18:2-18:2)PC was predominant in soybean, adzuki bean, runner bean, and common bean. (16:0-18:1)PC was the major species of chickpea PC, and many ether PC species and DHA-PC were identified. Discriminatory analysis by principal component analysis (PCA) indicated that the molecular profiles of chickpea PC were significantly different from other beans studied. The findings suggest that chickpea appears to be an interesting plant source of DHA and ether lipids for dietary supplement. PRACTICAL APPLICATION: In this study, we reported an UHPLC-Q-HRMS technique to identify PC molecular species of six beans. The diversity of PC molecular species in the different beans was classified using chemometrics. This analytical method not only provides comprehensive information to nutritionists about the PC distribution in different beans, but also can identify biomarkers for bean flour fraud identification in food supplementation. Furthermore, the approach gives fragmentation patterns of several PC species and could be further applied to determine the chemical structure of PC molecular species from many natural resources.

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques.

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 µm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

Two-step modification of silica for efficient adsorption of phosphatidylcholine in water and application in phospholipase D-catalyzed transphosphatidylation.

An efficient and green aqueous-solid system was introduced for phospholipase D-mediated transphosphatidylation. γ-(2,3-epoxypropoxy) propytrimethoxysilane was covalently bound to silica and esterified by acetic acid, which acted as an anchor molecule to facilitate the adsorption of phosphatidylcholine (PC) in aqueous solutions. Obtained silica-adsorbed PC was successfully used for transphosphatidylation to produce phosphatidylserine (PS). The PC loading and PS yield reached 98.8 and 98.3%, respectively. A new model was proposed to illustrate the adsorption and enzymatic processes. Moreover, this aqueous-solid system provides a promising way for the continuous production. Four kinds of phospholipids were biosynthesized in the pack-bed reactor. The stability of the aqueous-solid system was excellent, as demonstrated by its use 30 times without any loss of the productivity. The product was eluted by coconut oil and manufactured into microcapsules. Toxic agents were completely avoided in the whole production process.

Novel Cubosome System Resistant to Lipid Removal by Serum Albumin.

Cubosomes are lipidic nanoparticles containing bicontinuous cubic structures. Their unique architecture and potential as drug delivery vehicles have attracted researchers' attention. However, cubosome systems that are more robust in the presence of plasma components are being sought after for applications in intravenous administration. In this study, we prepared cubosomes consisting of 1,2-dioleoyl-sn-glycero-3-hexylphosphocholine (hexyl-DOPC) and compared their interaction with bovine serum albumin (BSA), the most abundant protein in plasma, with that of conventional cubosome systems consisting of several bicontinuous cubic phase-forming lipids, including 1-monoolein (MO), 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCO C22), or 1-O-(5,9,13,17-tetramethyloctadecyl)-ß-D-xylopyranoside (ß-XP). The average number of lipids bound to each BSA molecule was between 1.2-4.0 for MO, EROCO C22, and ß-XP. On the other hand, hexyl-DOPC exhibited negligible binding to BSA. This result suggests that hexyl-DOPC, which was shown to resist removal from particles by BSA, can be used as a new lipid component of cubosomes, and has higher plasma stability than the other cubic phase-forming lipids.

Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS.

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature.

Removal of Serum Lipids and Lipid-Derived Metabolites to Investigate Breast Cancer Cell Biology.

The use of cultured cells has been instrumental in studying biochemical, molecular, and cellular processes. The composition of serum that cells are maintained in can have a profound impact on important cellular checkpoints. Cell growth and apoptosis are analyzed in an estrogen receptor positive breast cancer cell line in the presence of serum that have been treated to remove steroids or lipids, as well-described in the literature. It is shown that maintaining cells in the presence of charcoal-dextran-treated serum causes reduced growth rate, which can be reversed by the addition of estradiol. Silica-treated-serum also slows down cell growth and induces apoptosis. In order to investigate the role of lipids in these phenotypes, the levels of a wide range of lipids in different sera are investigated. It is shown that silica-treatment significantly depletes phosphatidylcholines and cholesterol. It is also shown that lipogenesis is stimulated when cells are cultured with silica-treated-serum and this is reversed by the addition of exogenous lipids, which also restores growth rate and apoptosis. The results show that cultured cells are sensitive to different serum, most likely due to the differences in levels of structural and signaling metabolites present in their growth environment.

Simultaneous quantification of serum monounsaturated and polyunsaturated phosphatidylcholines as potential biomarkers for diagnosing non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide. In this study, we investigated Ultrahigh Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry and Gas Chromatography Time-of-Flight/Mass Spectrometry-based non-targeted metabolomic profiles of serum samples obtained from early-stage NSCLC patients and healthy controls (HC). Metabolic pathways and the biological relevance of potential biomarkers were extensively studied to gain insights into dysregulated metabolism in NSCLC. The identified biomarker candidates were further externally validated via a targeted metabolomics analysis. The global metabolomics profiles could clearly distinguish NSCLC patients from HC. Phosphatidylcholine (PC) levels were found to be dysregulated in glycerophospholipid (GPL) metabolism, which was the top altered pathway in early-stage NSCLC. Compared with those in HC, significant increases in the levels of saturated and monounsaturated PCs such as PC (15:0/18:1), PC (18:0/16:0) and PC (18:0/20:1) were observed in NSCLC. Additionally, relative to those in HC, the levels of 9 polyunsaturated PCs, namely, PC (17:2/2:0), PC (18:4/3:0), and PC (15:0/18:2), and so on were significantly decreased in NSCLC patients. A panel of 12 altered PCs had good diagnostic performance in differentiating early-stage NSCLC patients from HC, and these PCs may thus be used as serum biomarkers for the early diagnosis of NSCLC.

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy.

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature (Tm) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above-as well as below-the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10-2-105 Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm. The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures.

Endometriosis foci differentiation by rapid lipid profiling using tissue spray ionization and high resolution mass spectrometry.

Obtaining fast screening information on molecular composition of a tissue sample is of great importance for a disease biomarkers search and for online surgery control. In this study, high resolution mass spectrometry analysis of eutopic and ectopic endometrium tissues (90 samples) is done using direct tissue spray mass spectrometry in both positive and negative ion modes. The most abundant peaks in the both ion modes are those corresponding to lipids. Species of three lipid classes are observed, phosphatidylcholines (PC), sphingomyelins (SM) and phosphoethanolamines (PE). Direct tissue analysis gives mainly information on PC and SM lipids (29 species) in positive ion mode and PC, SM and PE lipids (50 species) in negative ion mode which gives complementary data for endometriosis foci differentiation. The biggest differences were found for phospholipids with polyunsaturated acyls and alkils. Although, tissue spray shows itself as appropriate tool for tissue investigation, caution should be paid to the interpretation of mass spectra because of their higher complexity with more possible adducts formation and multiple interferences must be taken into account. The present work extends the application of direct tissue analysis for the rapid differentiation between endometriotic tissues of different foci.

Regional changes in CNS and retinal glycerophospholipid profiles with age: a molecular blueprint.

We present here a quantitative molecular blueprint of the three major glycerophospholipid (GPL) classes, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), in retina and six regions of the brain in C57Bl6 mice at 2, 10, and 26 months of age. We found an age-related increase in molecular species containing saturated and monoenoic FAs and an overall decrease in the longer-chain PUFA molecular species across brain regions, with loss of DHA-containing molecular species as the most consistent and dramatic finding. Although we found very-long-chain PUFAs (VLC-PUFAs) (C28) in PC in the retina, no detectable levels were found in any brain region at any of the ages examined. All brain regions (except hippocampus and retina) showed a significant increase with age in PE plasmalogens. All three retina GPLs had di-PUFA molecular species (predominantly 44:12), which were most abundant in PS (∼30%). In contrast, low levels of di-PUFA GPL (1-2%) were found in all regions of the brain. This study provides a regional and age-related assessment of the brain's lipidome with a level of detail, inclusion, and quantification that has not heretofore been published.

Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic approach.

Under nitroxidative stress conditions, lipids are prone to be modified by reaction with reactive nitrogen species (RNS) and different modifications were reported to occur in fatty acids. However, in the case of phospholipids (PL) studied under nitroxidative stress conditions, only nitroalkene derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were reported when using both in vitro biomimetic conditions and in vivo model system of type 1 diabetes mellitus. Therefore, in order to further explore other nitroxidative modifications of PL, a biomimetic model of nitroxidation combined with liquid chromatography mass spectrometry (MS) and MS/MS approaches were used to characterize the nitrated and nitroxidized derivatives of PCs and PEs. Single and multiple nitrated derivatives of phospholipids (PLs) such as nitroso and dinitroso, nitro, dinitro, and nitronitroso derivatives, together with nitroxidized derivatives were identified. Further, the specific MS/MS fragmentation pathways of these products were studied. Product ions arising from loss of HNO and HNO2, from the combined loss of HNO (or HNO2) and polar head groups, [NOn-FA+On+H]+ and [NOn-FA+On-H]- (n=1-2) product ions corresponding to the modified fatty acyl chains were observed, depending on each modification. The knowledge obtained from the study of the MS/MS fragmentation pattern has allowed us to identify nitrated PCs, including NO2-PC, (NO2)2-PCs, (NO2)(NO)-PC, NO-PC; nitrated PEs, NO2-PEs; and nitroxidized PCs, (NO2)(2O)-PC in H9c2 cells under starvation, but not under ischemia or control conditions. The physiological relevance of this nitrated and nitroxidized PCs and PEs species observed exclusively in cardiomyoblast cells (H9c2) under starvation is still unknown but deserves to be explored.

MALDI-TOF/TOF Mass Spectrometric Determination and Antioxidative Activity of Purified Phosphatidylcholine Fractions from Shrimp Species.

Purification, characterization, and antioxidative activity in vitro of shrimp phosphatidylcholines (PCs) were investigated. The molecular structures of shrimp PCs were determined by MALDI-TOF/TOF MS. The MS2 fragments produced from protonated PC precursors and sodiated PC precursors were identified. The specific fragments including [M + Na - trimethylamine]+, [M + Na - 205]+, [M + Na - RCOOH - trimethylamine]+, and [M + H - RCOOH - trimethylamine]+ could distinguish the precursor type to confirm PC molecular structures. The antioxidative activities of purified shrimp PC fractions were evaluated by assay of DPPH free radical scavenging activity, and their effects on the oxidative stability of camellia oil were measured by monitoring changes in the peroxide value assay during oxidation. The PC fractions from Penaeus chinesis and Macrobranchium nipponense showed stronger antioxidative activities than those of other species. All of the shrimp PCs at 0.2% (w/w) improved the oxidative stability of camellia oil significantly (P < 0.05) compared to controls. The experimental findings suggest that shrimp PCs might be a valuable source of natural antioxidants for edible oils or other food dispersions.

Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion-trap mass spectrometer.

Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PCs. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PCs. This approach is comprised of two MS methods: a precursor ion scanning (PIS) of mass m/z 184 in positive mode (PIS m/z +184) and MS3 fragmentation in negative mode, both performed on the same instrument, a hybrid triple quadrupole ion-trap mass spectrometer. The MS3 experiment identified the FA composition and the relative abundance of isobaric and sn-1, sn-2 positional isomeric PC species, which were subsequently combined with absolute quantitative data obtained by PIS m/z +184 scan. This approach was applied to the analysis of a National Institute of Standards and Technology human blood plasma standard reference material (SRM 1950). We quantified more than 70 PCs and confirmed that a majority are present in isobaric and isomeric mixtures. The FA content determined by this method was comparable to that obtained using GC with flame ionization detection, supporting the quantitative nature of this MS method. This methodology will provide more in-depth biomarker information for clinical and mechanistic studies.

A novel chiral stationary phase HPLC-MS/MS method to discriminate between enzymatic oxidation and auto-oxidation of phosphatidylcholine.

To elucidate the role of enzymatic lipid peroxidation in disease pathogenesis and in food deterioration, we recently achieved stereoselective analysis of phosphatidylcholine hydroperoxide (PCOOH) possessing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE) using HPLC-MS/MS with a CHIRALPAK OP (+) column. Because enzymatic oxidation progresses concurrently with auto-oxidation, we need to distinguish them further. Here, we attempted such an analysis. First, we used lipoxygenase, linoleic acid, and lysophosphatidylcholine (LPC) to synthesize the enzymatic oxidation product 13(S)-9Z,11E-HPODE PC, and the auto-oxidation products 13(RS)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC, which were used as standards to test the ability of various columns to separate the enzymatic oxidation product from auto-oxidation products. Separation was achieved by connecting in series two columns with different properties: CHIRALPAK OP (+) and CHIRALPAK IB-3. The CHIRALPAK OP (+) column separated 13(R)-9Z,11E-HPODE PC and 13(S)-9Z,11E-HPODE PC, whereas CHIRALPAK IB-3 enabled separation of 13(S)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC. The results for the analysis of both enzymatically oxidized and auto-oxidized lecithin (an important phospholipid mixture in vivo and in food) indicate that our method would be useful for distinguishing enzymatic oxidation and auto-oxidation reactions. Such information will be invaluable for elucidating the involvement of PCOOH in disease pathogenesis and in food deterioration.

Improved specificity of serum phosphatidylcholine detection based on side-chain losses during negative electrospray ionization tandem mass spectrometry.

Many current tandem mass spectrometry (MS) methods for measuring phosphatidylcholines (PtdChos) rely only on precursor ion scanning of the common 184 m/z phosphocholine fragment with positive electrospray ionization (+ESI), and thus measure pools of PtdChos rather than specific isoforms. In this paper, we developed and compared an isotope dilution, tandem MS method capable of quantifying PtdChos based on specific fatty acid side-chains to the traditional 184 m/z method. The method is based on the detection of PtdCho ammonium formate (AmF) adduct as parent ions and fatty acid fragment daughter ions under negative electrospray ionization (-ESI). Accuracy, imprecision, and recovery were below 15 %, with acceptable linearity (R 2 > 0.99) up to 5 µg/mL. We used the method to analyze the distributions of PtdChos with common side-chain combinations among 60 subjects and showed that it was possible for two individuals to have the same PtdCho pool concentration based on detection of the 184 m/z fragment, but up to a fourfold difference in the levels of specific isoforms comprising the pool based on our method. We then compared the results of both methods across 572 patients with mild cognitive impairment (MCI), Alzheimer's disease (AD), or no impairment (NI), which showed that statistically significant associations between specific PtdCho isoforms and AD were masked with the 184 m/z method. Our findings demonstrate the importance of isoform specificity for quantifying PtdChos, and suggest caution when interpreting analytical data based on pools of biomarkers.

An integrated strategy for the rapid extraction and screening of phosphatidylcholines and lysophosphatidylcholines using semi-automatic solid phase extraction and data processing technology.

This study attempts to establish a comprehensive strategy for the rapid extraction and screening of phosphatidylcholines (PCs) and lysophosphatidylcholines (LysoPCs) in biological samples using semi-automatic solid phase extraction (SPE) and data processing technology based on ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). First, the Ostro sample preparation method (i.e., semi-automatic SPE) was compared with the Bligh-Dyer method in terms of substance coverage, reproducibility and sample preparation time. Meanwhile, the screening method for PCs and LysoPCs was built through mass range screening, mass defect filtering and diagnostic fragments filtering. Then, the Ostro sample preparation method and the aforementioned screening method were combined under optimal conditions to establish a rapid extraction and screening platform. Finally, this developed method was validated and applied to the preparation and data analysis of tissue samples. Through a systematic evaluation, this developed method was shown to provide reliable and high-throughput experimental results and was suitable for the preparation and analysis of tissue samples. Our method provides a novel strategy for the rapid extraction and analysis of functional phospholipids. In addition, this study will promote further study of phospholipids in disease research.

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

OBJECTIVE: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. DESIGN: Experimental study. SETTING: University-affiliated tertiary hospital. PATIENT(S): A total of 20 normospermic fertile men. INTERVENTION(S): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). MAIN OUTCOME MEASURE(S): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). RESULT(S): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. CONCLUSION(S): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.