Loss Of Phospholipids Research Articles

Phospholipase-mediated phosphate recycling during plant leaf senescence

BackgroundPhosphorus is a macronutrient necessary for plant growth and development and its availability and efficient use affect crop yields. Leaves are the largest tissue that uses phosphorus in plants, and membrane phospholipids are the main source of cellular phosphorus usage.ResultsHere we identify a key process for plant cellular phosphorus recycling mediated by membrane phospholipid hydrolysis during leaf senescence. Our results indicate that over 90% of lipid phosphorus, accounting for more than one-third of total cellular phosphorus, is recycled from senescent leaves before falling off the plants. Nonspecific phospholipase C4 (NPC4) and phospholipase Dζ2 (PLDζ2) are highly induced during leaf senescence, and knockouts of PLDζ2 and NPC4 decrease the loss of membrane phospholipids and delay leaf senescence. Conversely, overexpression of PLDζ2 and NPC4 accelerates the loss of phospholipids and leaf senescence, promoting phosphorus remobilization from senescent leaves to young tissues and plant growth. We also show that this phosphorus recycling process in senescent leaves mediated by membrane phospholipid hydrolysis is conserved in plants.ConclusionsThese results indicate that PLDζ2- and NPC4-mediated membrane phospholipid hydrolysis promotes phosphorus remobilization from senescent leaves to growing tissues and that the phospholipid hydrolysis-mediated phosphorus recycling improves phosphorus use efficiency in plants.

Differences in membrane lipid homeostasis confer contrast tolerance to low phosphorus in two wheat (Triticum aestivum L.) cultivars

Although lipids are involved in plant adaptation to phosphorus (P) deficiency, the underlying relationships are not fully understood, especially in wheat. Thus, a comparative study of changes in leaf lipid content, composition and implicated gene expression during low-P was conducted hydroponically using two wheat cultivars differing in low-P tolerance. Low-P reduced phospholipid content but increased digalactosyldoacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) content in older fully unfolded leaves (OL), with ND2419 (tolerant) showing greater changes than ZM366 (sensitive), suggesting a greater capacity to recycle internal P in ND2419. Interestingly, a novel lipid remodeling pattern was detected in the youngest unfolded leaf (YL) of ND2419, which reduced less in phospholipid content but accumulated more DGDG and SQDG with higher unsaturation levels than ZM366. These changes favored better chloroplast development, functionality and membrane integrity of ND2419, which together improved low-P tolerance. The expression of genes encoding glycolipid synthases and enzymes involved in phospholipid biosynthesis and degradation explained the lipid changes. Furthermore, our findings indicate that phospholipid loss in OL is primarily caused by enhanced degradation, whereas that in YL is driven by decreased biosynthesis and increased degradation. The detailed description of the changes in lipid profiles and gene expression under low P provides a basis for improving wheat low-P tolerance.

Membrane phospholipids and nucleotide derivatives in “big six” Escherichia coli are targets of chlorine-based sanitisers during lettuce decontamination

Sodium chlorite (NaClO), the most commonly used sanitiser for fresh produce decontamination, is associated with the problems of pungency and corrosiveness. In search for a more environmentally- and user-friendly chlorine-based sanitiser, acidic electrolysed water (AEW), came into view in recent years. To determine whether AEW is an ideal alternative to NaClO, using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), this study thoroughly investigated the effectiveness of AEW and NaClO, both containing 4 mg/L of free available chlorine (FAC), in inhibiting the “big six” Escherichia coli pathogens (serotypes O26, O45, O103, O111, O121 and O145) on lettuce as well as the underlying antimicrobial mechanisms.The two sanitisers led to generally comparable E. coli cell reductions after a 10-min treatment, which indicated the decisive role of FAC in their antimicrobial effects. Among the strains, mortalities varied from approximately 0.68 log CFU/g in O111 under both treatments to 1.48 ± 0.04 log CFU/g in AEW-treated O26 and 1.23 ± 0.04 log CFU/g in NaClO-treated O45. O111's high resistance was confirmed by its almost undisturbed metabolome whereas the high lethality of O26 and O45 could be attributed to the loss of membrane phospholipids. Besides, retarded cell growth and energy deficiency as implied by a vast depletion of nucleotide derivatives further challenged O45's survival. Overall, the comparable sanitising efficacy and the similarity in antimicrobial mechanisms between the two sanitisers provided a scientific basis for replacing NaClO with the “greener” AEW for fresh produce sanitisation in the future.

Renovating a double fence with or without notifying the next door and across the street neighbors: why the biogenic cytoplasmic membrane of Gram-negative bacteria display asymmetry?

The complex two-membrane organization of the envelope of Gram-negative bacteria imposes an unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on the cytoplasm facing leaflet of the cytoplasmic (inner) membrane (IM), across the IM and between the IM and outer membrane (OM). Balanced growth of two membranes and continuous loss of phospholipids in the periplasmic leaflet of the IM as metabolic precursors for envelope components and for translocation to the OM requires a constant supply of phospholipids in the IM cytosolic leaflet. At present we have no explanation as to why the biogenic E. coli IM displays asymmetry. Lipid asymmetry is largely related to highly entropically disfavored, unequal headgroup and acyl group asymmetries which are usually actively maintained by active mechanisms. However, these mechanisms are largely unknown for bacteria. Alternatively, lipid asymmetry in biogenic IM could be metabolically controlled in order to maintain uniform bilayer growth and asymmetric transmembrane arrangement by balancing temporally the net rates of synthesis and flip-flop, inter IM and OM bidirectional flows and bilayer chemical and physical properties as spontaneous response. Does such flippase-less or 'lipid only", 'passive' mechanism of generation and maintenance of lipid asymmetry exists in the IM? The driving force for IM asymmetry can arise from the packing requirements imposed upon the bilayer system during cell division through disproportional distribution of two negatively curved phospholipids, phosphatidylethanolamine and cardiolipin, with consistent reciprocal tendency to increase and decrease lipid order in each membrane leaflet respectively.

IPLA2β-Null Mice Show HCC Protection by an Induction of Cell-Cycle Arrest after Diethylnitrosamine Treatment.

Group VIA phospholipase A2 (iPLA2β) play diverse biological functions in epithelial cells and macrophages. Global deletion in iPLA2β-null (KO) mice leads to protection against hepatic steatosis in non-alcoholic fatty liver disease, in part, due to the replenishment of the loss of hepatocellular phospholipids. As the loss of phospholipids also occurs in hepatocellular carcinoma (HCC), we hypothesized that global deletion in KO mice may lead to protection against HCC. Here, HCC induced by diethylnitrosamine (DEN) was chosen because DEN causes direct injury to the hepatocytes. Male wild-type (WT) and KO mice at 3-5 weeks of age (12-13 mice/group) were subjected to a single intraperitoneal treatment with 10 mg/kg DEN, and mice were killed 12 months later. Analyses of histology, plasma cytokines, and gene expression were performed. Due to the low-dose DEN used, we observed a liver nodule in 3 of 13 WT and 2 of 12 KO mice. Only one DEN-treated WT mouse was confirmed to have HCC. DEN-treated KO mice did not show any HCC but showed suppressed hepatic expression of cell-cycle cyclinD2 and BCL2 as well as inflammatory markers IL-1β, IL-10, and VCAM-1. Notably, DEN-treated KO mice showed increased hepatic necrosis and elevated levels of plasma lactate dehydrogenase suggesting an exacerbation of liver injury. Thus, global iPLA2β deficiency in DEN-treated mice rendered HCC protection by an induction of cell-cycle arrest. Our results suggest the role of iPLA2β inhibition in HCC treatment.

Free Radical Chain Reactions and Polyunsaturated Fatty Acids in Brain Lipids.

Polyunsaturated fatty acyl chains (PUFAs) concentrate in the brain and give rise to numerous oxidative chemical degradation products. It is widely assumed that these products are the result of free radical chain reactions, and reactions of this type have been demonstrated in preparations where a single PUFA substrate species predominates. However, it is unclear whether such reactions can occur in the biologically complex milieu of lipid membranes where PUFA substrates are a minority species, and where diverse free radical scavengers or other quenching mechanisms are present. It is of particular interest to know whether they occur in brain, where PUFAs are concentrated and where PUFA oxidation products have been implicated in the pathogenesis of neurodegenerative disorders. To ascertain whether free radical chain reactions can occur in a complex brain lipid mixture, mouse brain lipids were extracted, formed into vesicles, and treated with a fixed number of hydroxyl radicals under conditions wherein the concentrations and types of PUFA-containing phospholipids were varied. Specific phospholipid species in the mixture were assayed by tandem mass spectrometry to quantify the oxidative losses of endogenous PUFA-containing phospholipids. Results reveal crosstalk between the oxidative degradation of ω3 and ω6 PUFAs that can only be explained by the occurrence of free radical chain reactions. These results demonstrate that PUFAs in a complex brain lipid mixture can participate in free radical chain reactions wherein the extent of oxidative degradation is not limited by the number of reactive oxygen species available to initiate such reactions. These reactions may help explain otherwise puzzling in vivo interactions between ω3 and ω6 PUFAs in mouse brain.

Monogalactosyl diacylglycerol synthase 3 affects phosphate utilization and acquisition in rice.

Galactolipids are essential to compensate for the loss of phospholipids by 'membrane lipid remodelling' in plants under phosphorus (P) deficiency conditions. Monogalactosyl diacylglycerol (MGDG) synthases catalyse the synthesis of MGDG which is further converted into digalactosyl diacylglycerol (DGDG), later replacing phospholipids in the extraplastidial membranes. However, the roles of these enzymes are not well explored in rice. In this study, the rice MGDG synthase 3 gene (OsMGD3) was identified and functionally characterized. We showed that the plant phosphate (Pi) status and the transcription factor PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout and overexpression lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared with the wild type, OsMGD3 knockout lines displayed a reduced Pi acquisition and utilization while overexpression lines showed an enhancement of the same. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed a role of OsMGD3 in membrane lipid remodelling, in addition to a role in regulating diacylglycerol and phosphatidic acid contents that affected the expression of Pi transporters. Our study highlights the role of OsMGD3 in affecting both internal P utilization and P acquisition in rice.

Porcine liver decomposition product-derived lysophospholipids promote microglial activation in vitro

Cognitive impairments such as dementia are common in later life, and have been suggested to occur via a range of mechanisms, including oxidative stress, age-related changes to cellular metabolism, and a loss of phospholipids (PLs) from neuronal membranes. PLs are a class of amphipathic lipids that form plasma membrane lipid bilayers, and that occur at high concentrations in neuronal membranes. Our previous study suggested that a porcine liver decomposition product (PLDP) produced via protease treatment may improve cognitive function at older ages, by acting as a rich source of PLs and lysophospholipids (LPLs); however, its specific composition remains unclear. Thus, the present study used a novel liquid chromatography electrospray ionization tandem mass spectrometric (LC-MS/MS) protocol to identify the major PLs and LPLs in PLDP. Furthermore, it assessed the effect of identified LPLs on microglial activation in vitro, including cell shape, proliferation, and cell morphology. The results of the conducted analyses showed that PLDP and PLDP-derived LPLs concentration-dependently modulate microglial activation in vitro. In particular, lysophosphatidylcholine (LPC) concentration-dependently promotes cell morphology, likely via effects mediated by the enzyme autotaxin (ATX), since inhibiting ATX also promoted cell morphology, while conversely, increasing ATX production (via treatment with high levels of LPC) abolished this effect. These findings suggest that LPC is likely neuroprotective, and thus, support the importance of further research to assess its use as a therapeutic target to treat age-related cognitive impairments, including dementia.

IPla2β deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes

Group VIA calcium-independent phospholipase A2 (iPla2β) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2β deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2β-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2β-null mice. However, iPla2β deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptorγ, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2β deficiency under MCD did not have any effects on inflammasomes and pro-inflammatory markers but exacerbated hepatic expression of myofibroblast α-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2β inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2β mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types.

Phospholipolysis Caused by Different Types of Bacterial Phospholipases During Cold Storage of Bovine Raw Milk Is Prevented by N2 Gas Flushing.

Cold storage aims to preserve the quality and safety of raw milk from farms to dairies; unfortunately, low temperatures also promote the growth of psychrotrophic bacteria, some of which produce heat-stable enzymes that cause spoilage of milk or dairy products. Previously, N2 gas flushing of raw milk has demonstrated significant potential as a method to hinder bacterial growth at both laboratory and pilot plant scales. Using a mass spectrometry-based lipidomics approach, we examined the impact of cold storage [at 6°C for up to 7 days, the control condition (C)], on the relative amounts of major phospholipids (phosphatidylethanolamine/PE, phosphatidylcholine/PC, phosphatidylserine/PS, phosphatidylinositol/PI, and sphingomyelin/SM) in three bovine raw milk samples, and compared it to the condition that received additional N2 gas flushing (N). As expected, bacterial growth was hindered by the N2-based treatment (over 4 log-units lower at day 7) compared to the non-treated control condition. At the end of the cold storage period, the control condition (C7) revealed higher hydrolysis of PC, SM, PE, and PS (the major species reached 27.2, 26.7, 34.6, and 9.9 μM, respectively), compared to the N2-flushed samples (N7) (the major species reached 55.6, 35.9, 54.0, and 18.8 μM, respectively). C7 samples also exhibited a three-fold higher phosphatidic acid (PA) content (6.8 μM) and a five-fold higher content (17.3 μM) of lysophospholipids (LPE, LPC, LPS, and LPI) whereas both lysophospholipids and PA remained at their initial levels for 7 days in N7 samples. Taking into consideration the significant phospholipid losses in the controls, the lipid profiling results together with the microbiological data suggest a major role of phospholipase (PLase) C (PLC) in phospholipolysis during cold storage. However, the experimental data also indicate that bacterial sphingomyelinase C, together with PLases PLD and PLA contributed to the degradation of phospholipids present in raw milk as well, and potential contributions from PLB activity cannot be excluded. Altogether, this lipidomics study highlights the beneficial effects of N2 flushing treatment on the quality and safety of raw milk through its ability to effectively hinder phospholipolysis during cold storage.

Palmitic and Stearic Free Fatty Acids Are Consistently Found in Materials used for Dried Blood Spot Collection

BackgroundDried blood spotting has been used to collect samples for fatty acid analysis, particularly when access to analytical laboratories or ultra‐cold storage is limited. However, the materials used to collect dried blood spots are often contaminated with lipids and/or fatty acids.ObjectiveTo measure background/containment fatty acid and lipids on materials used to collect dried blood spots.MethodsThree materials used for dried blood spot collection that included 903 protein saver cards (Fischer Scientific, St. Louis, USA), chromatography paper cut into strips (Analtech Inc., Newark, DE), and novel Mitra Microsamplers (Neoteryx, California, USA). Blank collection materials as purchased were handled with nitrile gloves and preexisting lipids were extracted using 2:1 chloroform:methanol (v/v) solution. Lipid extracts were divided into two, with one portion for gas chromatography (GC) analysis of fatty acids and the other portion for lipidomic analyses using ultra‐high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC‐MS/MS) in a quadrupole‐orbitrap system (Q‐Exactive, Thermo Scientific, New York, USA). Fatty acid methyl esters were prepared for GC analysis by direct transesterification of the lipid extracts using boron trifluoride. Lipid extracts were dried and re‐suspended in 65:35:5 acetonitrile:isopropanol:water + 0.1% formic acid and analyzed using a 47‐minute reversed‐phase UHPLC multi‐step binary protocol with top‐5 data dependent MS/MS acquisition. Samples were run twice, once under positive ESI‐MS/MS and once under negative ESI‐MS/MS to enable the identification of compounds that preferentially ionize with different polarities.ResultsGC analysis identified 0.64±0.05 μg of palmitic acid (C16:0) and 0.88±0.04 μg stearic acid (C18:0) per 6mm punch from 903 protein saver cards, 0.97±0.08 μg 16:0 and 1.43±0.16 μg 18:0 per 6mm punch of Whatman chromatography paper and 0.64±0.08 μg 16:0 and 0.88±0.10 μg 18:0 per Mitra tip. In the positive ESI‐MS/MS analyses, scanning for fragment losses of phospholipids (choline, ethanolamine, serine head groups) did not result in extracted ion chromatograms that would indicate that these compounds were present. Neutral‐loss scanning of fatty acids like palmitate and stearate in positive ESI also showed no evidence of other complex lipids such as triacylglycerols or cholesteryl esters in the blank samples. However, negative ESI‐MS/MS experiments confirmed that the palmitate and stearate that were identified using GC are found as free fatty acids and not as part of complex lipids.ConclusionsMaterials used to collect dried blood spots appear consistently contaminated with palmitic and stearic free fatty acids. GC determinations of fatty acids from dried blood spots samples need to consider this contamination and/or take steps to prewash sample collecting materials. For lipidomic analysis, these contaminants can be ignored if complex lipids and not free fatty acids are being examined.Support or Funding InformationThis work was supported by a Natural Sciences and Engineering Research Council (NSERC) Discovery grant (327149, to K.D.S), and an NSERC doctoral scholarship to J.J.A.H. K.D.S. is also supported by a Canada Research Chair in Nutritional Lipidomics.

IPLA2beta deficiency protects mice from diet-induced obesity and steatosis by replenishing the loss of hepatic phospholipids containing unsaturated fatty acids

iPLA2beta deficiency protects mice from diet-induced obesity and steatosis by replenishing the loss of hepatic phospholipids containing unsaturated fatty acids

Abstract 13218: Remodeling of High Density Lipoprotein Induced by CSL112: Structure and Function of the Remodeled Lipoproteins

Introduction: CSL112 is a novel formulation of human apolipoprotein A-I (apoA-I) suitable for intravenous infusion, currently in development for reducing the high rate of early recurrent events after acute myocardial infarction. Infusion of CSL112 into healthy volunteers causes HDL remodeling, with an immediate rise in lipid-poor apoA-I and a strong and immediate increase in the ABCA1-dependent cholesterol efflux capacity of plasma. Interaction of CSL112 with whole plasma or purified HDL gives rise to three new remodeled HDL species: large (L-HDL rem ) and small (S-HDL rem ) species, and lipid-poor apoA-I. Here we characterized the composition and morphology of remodeled HDL species and compared their ability to efflux cholesterol via different transporters. Methods and Results: CSL112 and HDL3, fluorescently labeled on protein or lipid, were incubated together for varying periods of time at 37°C. Resultant HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescence imaging. Morphology of the individual purified products of HDL remodeling was examined by negative stain electron microscopy. We show that L-HDL rem is a large, spherical species composed of apoA-I and phospholipid from both native HDL and CSL112. S-HDL rem is a small, disc-shaped species composed of apoA-I from CSL112 that is smaller due to the loss of phospholipids. The smallest species, lipid-poor apoA-I, is composed of apoA-I from HDL and CSL112. Lipid-poor apoA-I and S-HDL rem were found to have the greatest ability to efflux cholesterol via ABCA1. While S-HDL rem and L-HDL rem showed comparable ability to efflux cholesterol via ABCG1, L-HDL rem mediated additional efflux via SR-B1 and by an aqueous diffusion process. Conclusion: The generation of these remodeled HDL species can account for the strong and immediate increase in cholesterol efflux capacity observed upon infusion of CSL112, and make CSL112 a promising approach for rapidly removing cholesterol from atherosclerotic plaque.

Enhanced HDL Functionality in Small HDL Species Produced Upon Remodeling of HDL by Reconstituted HDL, CSL112

Rationale: CSL112, human apolipoprotein A-I (apoA-I) reconstituted with phosphatidylcholine, is known to cause a dramatic rise in small high-density lipoprotein (HDL). Objective: To explore the mechanisms by which the formation of small HDL particles is induced by CSL112. Methods and Results: Infusion of CSL112 into humans caused elevation of 2 small diameter HDL fractions and 1 large diameter fraction. Ex vivo studies showed that this remodeling does not depend on lipid transfer proteins or lipases. Rather, interaction of CSL112 with purified HDL spontaneously gave rise to 3 HDL species: a large, spherical species composed of apoA-I from native HDL and CSL112; a small, disc-shaped species composed of apoA-I from CSL112, but smaller because of the loss of phospholipids; and the smallest species, lipid-poor apoA-I composed of apoA-I from HDL and CSL112. Time-course studies suggest that remodeling occurs by an initial fusion of CSL112 with HDL and subsequent fission leading to the smaller forms. Functional studies showed that ATP-binding cassette transporter 1–dependent cholesterol efflux and anti-inflammatory effects in whole blood were carried by the 2 small species with little activity in the large species. In contrast, the ability to inactivate lipid hydroperoxides in oxidized low-density lipoprotein was carried predominantly by the 2 largest species and was low in lipid-poor apoA-I. Conclusions: We have described a mechanism for the formation of small, highly functional HDL species involving spontaneous fusion of discoidal HDL with spherical HDL and subsequent fission. Similar remodeling is likely to occur during the life cycle of apoA-I in vivo.

Impairment of myocardial mitochondria in viral myocardial disease and its reflective window in peripheral cells.

BackgroundViral myocardial disease (VMD) is a common disease inducing heart failure. It has not been clear the roles of mitochondrial damage in the pathological changes of cardiomyocytes in VMD.MethodsMyocardial tissues and lymphocytes were collected from 83 VMD patients. Control groups included 12 cases of healthy accidental death with myocardial autopsy and 23 healthy blood donors. The mouse model of viral myocarditis (VMC) was established by Coxsackie virus B3 infection and myocardial tissues and skeletal muscle were collected. Mitochondrial DNA (mtDNA) deletion rate was quantitatively determined using polymerase chain reaction.ResultsThere was significantly difference of myocardial mitochondrial DNA deletion rate between VMD or VMC group and control group (P<0.05). Moreover, the loss of mitochondrial membrane phospholipids was significantly different between VMD or VMC group and control group. In VMC mice, there were negative correlations between myocardial mtDNA3867 deletion rate and left ventricular peak systolic pressure (LVPSP) (r = −0.66, P<0.05), and between myocardial mtDNA3867 deletion rate and +dp/dtmax (r = −0.79, P<0.05), while there was positive correlation between myocardial mtDNA3867 deletion rate and −dp/dtmax (r = 0.80, P<0.05).ConclusionMitochondrial damage is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction. The mitochondrial damage in the skeletal muscle and lymphocytes reflect a “window” of myocardial mitochondrial damage.

Erythrocyte phospholipid and polyunsaturated fatty acid composition in diabetic retinopathy.

BackgroundLong chain polyunsaturated fatty acids (LCPUFAs) including docosahexaenoic acid and arachidonic acid are suspected to play a key role in the pathogenesis of diabetes. LCPUFAs are known to be preferentially concentrated in specific phospholipids termed as plasmalogens. This study was aimed to highlight potential changes in the metabolism of phospholipids, and particularly plasmalogens, and LCPUFAs at various stages of diabetic retinopathy in humans.Methodology and Principal FindingsWe performed lipidomic analyses on red blood cell membranes from controls and mainly type 2 diabetes mellitus patients with or without retinopathy. The fatty acid composition of erythrocytes was determined by gas chromatography and the phospholipid structure was determined by liquid chromatography equipped with an electrospray ionisation source and coupled with a tandem mass spectrometer (LC-ESI-MS/MS). A significant decrease in levels of docosahexaenoic acid and arachidonic acid in erythrocytes of diabetic patients with or without retinopathy was observed. The origin of this decrease was a loss of phosphatidyl-ethanolamine phospholipids esterified with these LCPUFAs. In diabetic patients without retinopathy, this change was balanced by an increase in the levels of several phosphatidyl-choline species. No influence of diabetes nor of diabetic retinopathy was observed on the concentrations of plasmalogen-type phospholipids.Conclusions and SignificanceDiabetes and diabetic retinopathy were associated with a reduction of erythrocyte LCPUFAs in phosphatidyl-ethanolamines. The increase of the amounts of phosphatidyl-choline species in erythrocytes of diabetic patients without diabetic retinopathy might be a compensatory mechanism for the loss of LC-PUFA-rich phosphatidyl-ethanolamines.

The antiproliferative properties of the milk fat globule membrane are affected by extensive heating

The milk fat globule membrane (MFGM), the material surrounding milk fat globules, is not only interesting from a technological standpoint but it also shows great potential as a health ingredient, as it exerts cytotoxic and apoptotic effects against colon cancer cells. Although the effects of milk processing on the MFGM composition and functionality are well documented, less is understood on how processing may affect its bioactivity. This study aimed to determine if heating can affect the antiproliferative capacity of the MFGM. To do so, MFGM was extracted from milk heated at 80 °C for 10 min, as this temperature/time regime is known to cause extensive protein-protein interactions with changes in the processing functionality of milk. Two cell lines, whose morphological features are representative of two different stages of colon carcinogenesis (HT-29 and Caco-2), were used to test the antiproliferative capacity of MFGM isolates obtained either from untreated or heated milk. Cell proliferation analysis showed a similar dose-dependent decrease of DNA synthesis in both cell lines exposed to 6.25–200 μg of MFGM protein.mL−1, isolated from unheated milk. The heat treatment diminished the efficacy of the MFGM isolates, as only the highest concentrations of MFGM tested following heating showed a significant effect on cell proliferation. The decreased ability of MFGM isolates to affect the carcinoma cell proliferation was attributed to changes in composition, mainly phospholipid losses. Changes to the supramolecular structure of the MFGM caused by heating may also have played a role. This work demonstrates the importance of processing history in assessing the biological functionality of MFGM.

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10°C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1week at low temperature, followed by a further decrease after 2weeks. The enzyme activity was not changed during 25°C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10°C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25°C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.

112 Membrane changes in liposome-treated rat red blood cells upon hypothermic storage

Liposomes, which are microscopic synthetic lipid vesicles, have been shown to improve red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42 day storage. Considering that the use of animal models is an essential step in preclinical studies for evaluating transfusion efficacy with novel manipulations of blood transfusion products, the aim of this study was to evaluate the effect of four liposome formulations on rat RBC membrane-related quality parameters during hypothermic storage. Unilamellar liposomes were synthesized using an extrusion method to contain lipid bilayer of saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):cholesterol, 7:3 mol%), unsaturated (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):cholesterol, 7:3 mol%), saturated charged (DPPC:cholesterol: phosphatidylserine (PS), 6:3:1 mol%) and unsaturated charged (DOPC:cholesterol:PS, 6:3:1 mol%) phospholipids. The leukoreduced, packed RBCs from Sprague-Dawley rats ( n = 3) were incubated for 1 h at 37 °C in a mixture of AS-3 solution with either HEPES-NaCl solution (control) or 2 mM lipid from four different liposome compositions. RBC membrane quality was assessed after treatment, one and five weeks of hypothermic storage by % hemolysis (Drabkins), deformability (ektacytometry), hematological indices (Coulter), morphology (microscopy), cholesterol and phospholipid concentration (colorimetry). All four liposome treatments resulted in significant decrease in percent hemolysis, with the effect most prominent with DOPC-treated RBCs (1.6 ± 0.1% vs. 3.1 ± 0.2 %, p = 0.05). No alterations in rat RBC membrane deformability, morphology, or hematological indices were detected in liposome treated RBCs immediately after the treatment. While DOPC liposome treatment resulted in significant increase in rat RBC phospholipid concentration, DPPC liposome treatment seemed to induce loss of phospholipids from the rat RBC membranes (0.30 ± 0.03 mg vs 0.15 ± 0.01 mg, p = 0.05). The cholesterol: phospholipid ratio in DOPC-treated rat RBCs was comparable to control (0.49 ± 0.06 vs 0.51 ± 0.08, p = 0.80). Upon the first week of storage DOPC-treated RBCs demonstrated significantly lower hemolysis (1.4 ± 0.1% vs. 2.5 ± 0.1%, p = 0.05) with values that are comparable to the ones of fresh RBC samples ( p = 0.25). All liposome treated RBCs continued to exhibit decreased hemolysis after 5 weeks of storage compared to the control ( p = 0.05), with uncharged formulations resulting in lower hemolysis than RBCs treated with charged liposomes (3.4 ± 0.2% vs 3.9 ± 0.4%, p = 0.01). The cell rigidity was significantly improved in DOPC-treated RBCs as demonstrated by the decreased KEI values compared to non-treated RBCs (1.01 ± 0.13 vs 1.36 ± 0.16, p = 0.05). The liposome treatment resulted in an improvement in rat RBC in vitro quality upon storage. The stabilizing effect was related to the chemical characteristics of each liposome formulation. The storage induced detrimental changes in the rat RBC membrane were minimized to a greater extent with the unsaturated than saturated liposome treatment. Also, negatively charged liposomes seemed to increase hemolysis upon storage. Therefore, unsaturated-uncharged liposomes will be used in the upcoming in vivo rat studies to further evaluate the pre-clinical effect of transfusing liposome-treated RBCs. Source of funding: Canadian Blood Services. Conflict of interest: None declared. jelena.holovati@ualberta.ca

028 Successful cryopreservation of avian germplasm: Why a multifaceted approach is required

The value of the ability to cryopreserve and store germplasm has long been recognized for indefinite preservation of genetic material, especially for “at-risk” populations. For domestic livestock species, options for preserving genetic material in frozen form range from sperm to oocytes to embryos. More than sixty years ago, the discovery of glycerol’s cryoprotective properties for rooster sperm pioneered the success of modern cryobiology and led to the development of semen cryopreservation for a wide range of species. Despite decades of research, the overall fertility rates of frozen/thawed poultry sperm are highly variable and not reliable enough for preservation of genetic stocks. The relatively low fertilizing ability most likely results from physiological sensitivity to the cryogenic process coupled with the requirement for prolonged sperm functionality in the hen reproductive tract. We have conducted systematic experiments to understand how and why poultry sperm lose functional competence after cryopreservation, and have shown that the glycocalyx, the carbohydrate-rich zone on the sperm surface, is altered by the cryogenic cycle. The amount of sialic acid, a terminal sugar critical for sperm transport in the female reproductive tract, was reduced after cryopreservation; however, we have shown that sperm cells can incorporate exogenous sialic acid, suggesting a strategy for sperm freezing protocols. Second, sperm lipids such as phosphotidylcholine also are altered during cold storage. The ratio of lipids in the sperm membrane determine the overall fluidity of the membrane and impact the ability of sperm to remain viable during the wide temperature fluctuations that are part of the cryogenic cycle. We have shown that supplemental lipids can circumvent the loss of phospholipids during cryopreservation. Finally, we have conducted the first proteomic analysis of poultry sperm and have identified three proteins not previously found in sperm from any species to date (dihydropyrimidinase, mitofilin and mitochondrial tri-functional protein). We currently are evaluating the proteomic profile of frozen/thawed sperm to determine the impact of cryopreservation on sperm proteins. Taken together, these data suggests innovative strategies for developing reliable poultry semen cryopreservation methods. For most species, sperm cryopreservation effectively captures the entire genome; however, in birds, the female is the heterogametic sex. Cryopreservation of avian oocytes and embryos is problematic because of the macrolecithal characteristics of the egg, where the surface-to-volume ratio poses a significant barrier to the movement of solutes/cryoprotectants and impedes effective protection of the cells from freeze/thaw damage. Recent advances for manipulation of poultry primordial germ cells and culture of embryonic stem cells suggests that cryopreservation of these cell types, followed by re-implantation in recipient embryos, could provide a mechanism to preserve the entire genome; albeit the efficiency rates of reconstitution associated with the current technologies are rather low. A novel innovation of cryopreservation and transplantation of ovarian tissue offers an alternative for whole genome preservation in birds (Song and Silversides, 2008); although chemical treatment to reduce the growth of host germ cells does not completely prevent the production of host-derived offspring. Reconstitution of poultry lines will most likely rely on more than one type of cryopreserved material. Source of funding: USDA ARS Project Number: 1245-31000-105-00. Conflict of interest: None declared. [email protected]