Which phospholipids are the most abundant in the body

While the phospholipid asymmetry of the RBC plasma membrane has been known for more than 40 years, methods to analyze the outer leaflet lipid composition still depend on enzymatic degradation of the outer leaflet lipids. In this study, we have developed a method, based on cyclodextrin-mediated lipid exchange to characterize the outer leaflet phospholipids of RBCs. This method, which builds up on previous studies to determine outer membrane leaflet composition in cancer cells 4 , does not perturb membrane integrity and provides results that are compatible with the enzymatic degradation method.

Importantly, the delivery of lipids to the outer leaflet of RBCs facilitated by this method can be useful in ex vivo preservation of RBCs, by reconstituting their membrane and preventing their uptake by macrophages. The method in the current study is based on cyclodextrin-mediated lipid exchange to extract the outer leaflet of RBCs.

The pioneering study of Li et al. Here, this process was optimized to cause the lowest level of damage to the membrane integrity of RBCs during lipid extraction. Similar to the methods of Li et al.

A better understanding of the mechanisms of complex formation and lipid exchange is needed to tease out why certain lipids and lipid concentrations are more efficient at maintaining RBC membrane integrity. Lorent et al. Thus, the moderate exchange efficiency for SM, especially compared to PC which shows near complete removal is somewhat surprising. One potential explanation for this lower extraction efficiency is the favorable interaction of SM with cholesterol 50 , Thus, it is possible that the high concentration of cholesterol in the outer leaflet of RBCs hinders cyclodextrin-mediated extraction of SM as well.

Experiments with cholesterol oxidase, to reduce the amount of membrane cholesterol, followed by membrane lipid extraction using cyclodextrin, or experiments with asymmetric vesicles, with varying degrees of cholesterol and SM in the outer leaflet could help shed light on this issue.

Here, a note on cholesterol asymmetry in the RBC membrane is also warranted. While there are reports of cholesterol preferentially residing in the outer leaflet due to its affinity to saturated chains of SM and PC, there are also studies suggesting that PE and PS can draw cholesterol to the inner leaflet The rapid flip-flop rate of cholesterol 55 , 56 , along with the differences in its distribution in different cells have led to conflicting reports on its distribution 54 , Regardless, evidence on cholesterol affinity for certain phospholipids 52 and toward more ordered membranes 58 exist.

Thus, it is possible that the cholesterol in the outer leaflet interacts more with the SM, due to its long and saturated acyl chains, which further inhibits SM extraction from the membrane.

The abundance values for the species of loaded lipids were replaced by averaging the abundance values of the species of that lipid from samples exchanged using the other two lipids. This allowed for removal of the values for the excess loaded lipid that would confound the mass spectrometry analysis otherwise. Here, the drawbacks of using cyclodextrins to probe plasma membrane lipids should also be pointed out.

Extensive extraction of lipids from the plasma membrane by cyclodextrins can lead to cell lysis While this can be prevented by loading cyclodextrins with exogenous lipids 4 , 38 , this process will likely need to be optimized individually for each cell, increasing the burden for such analyses. In addition, the possibility that cyclodextrin might be biased toward certain lipid structures cannot be ruled out. For example, while strong bias toward certain lipid headgroups, except for phosphatidylinositol, are not generally observed 35 , the chain length and saturation, as noted above, are known to affect cyclodextrin-lipid complex formation 36 , 48 , The composition of the individual leaflets of the plasma membrane of RBCs was first characterized over 40 years ago by Verkleij et al.

It is worth mentioning that the reported bar graphs of Verkleij et al. The methods used in that study were not capable of reporting the composition of other lipids, now known to reside in the membrane e.

Those methods were also not capable of providing a detailed breakdown of phospholipid acyl chain structure in each leaflet. Recently, Lorent et al. Contradictory to the results by Verkleij et al. Importantly, while the methods are clearly distinct, there are remarkable similarities in the breakdown of outer leaflet PC and SM between the current study and the study of Lorent et al.

Interestingly, the abundance of SM species in the outer leaflet is also completely consistent between the two studies. Despite striking similarities between results from the two methods, differences are also observed. For example, our results show PC as the most and PC as the second most abundant PC species in the outer leaflet, while the reverse is reported by Lorent et al.

These minor differences could be due to the fact that cyclodextrin favors more saturated lipids over polyunsaturated ones However, as the abundance of the SM species shows, the lipid exchange was capable of extracting the SM species from the outer leaflet, in an unbiased manner. While the characterization of the membrane outer leaflet lipid composition can be important in the context of membrane biology and RBC function in health and disease, an important application of this method could be to deliver lipids of choice to the outer leaflet of the RBC membrane.

Here, this method was used to reconstitute the membrane of senescent RBCs to alter their interactions with macrophages.

As RBCs age, PS translocates to the membrane outer leaflet, this is a signal for initiation of erythrophagocytosis i. While in the current study PS removal has been used to show one application of cyclodextrin-mediated lipid exchange in RBCs, we anticipate such lipid manipulations to be more widely used in studies of RBC membrane biology and to find applications in altering the RBC membrane to affect cell—cell interactions. The mixture was then vortex-mixed at 3, rpm for 10 min to form lipid vesicles.

The resulting turbid solution was centrifuged at 14, g for 5 min to obtain a clear solution without any precipitates.

All hematocrit contents are on a volume basis. The mixture was incubated at room temperature without shaking for different times as mentioned in Supplementary Table S1. This protocol was exactly followed anytime the exchange was performed.

Presence of hemoglobin in the exchange solution was used as a measure of RBC membrane disruption hemolysis. RBCs suspended in DPBS and water for the same incubation period, at the same hematocrit concentrations as the ones used for exchange experiments, were used as negative and positive controls for hemolysis, respectively.

As phospholipid exchange proceeds, exogenous phospholipids are delivered to the membrane, replacing the endogenous phospholipids of the outer leaflet of RBCs. According to the specific volumes provided by this method, solutions of methanol:chloroform, chloroform and water were added to the samples, with vigorous vortex-mixing after each addition.

The final milky suspension was centrifuged at g for 5 min and the bottom phase was collected using a Pasteur pipette, with positive pressure bubbling through the top phase to avoid contamination. The plate was then placed in a TLC tank, containing a mobile phase of acetic acid:water:methanol:chloroform.

When the solvent front reached the top of the plate, the plate was taken out of the tank and air dried. To identify the phospholipids delivered to the RBCs, the supernatant of the exchange was discarded after centrifugation and the RBCs were lysed with water.

Untreated RBCs from the same donors as the ones for the exchange was collected to the same hematocrit content and were lysed in water. The lipid extraction and TLC experiments were performed exactly as described earlier, with the exception of the mobile phase which was ammonium hydroxide:methanol:chloroform for this plate.

The solvent was changed to obtain a better separation between all the phospholipids as well as cholesterol. The plate was also air dried, sprayed, and charred as described earlier. The bands in each lane of this plate were quantified and normalized with respect to cholesterol. Delivery of phospholipids to the membrane was studied using fluorescence. A quenching agent was used to diminish the fluorescence and evaluate whether the lipids are delivered to the membrane outer leaflet.

The other steps prior to the exchange experiment were performed as mentioned before. Sodium dithionite solution was made freshly and added to an aliquot of the exchanged RBCs to achieve a final concentration of 50 mM just prior to loading RBCs onto glass slides and imaging. To assess the effects of sodium dithionite treatment on the RBCs, triple washed expired and fresh RBCs were incubated with 50 mM of sodium dithionite for 2 min, were then centrifuged at g for 5 min, and washed with DPBS.

The RBCs were then washed twice with centrifugation and the fluorescence of the samples was recorded. Quantification of the phospholipids and their respective species were performed using mass spectrometry.

After exchanging the outer leaflet of RBCs with POPS, the cells were collected, lysed using water to remove the hemoglobin, and a total lipid extraction was performed on the cells using the methods of Bligh and Dyer The lipids were then dried, weighed, and used for mass spectrometry. Samples contained 0. Phospholipid internal standards were mixed with the samples, and a solvent mixture chloroform:methanol mM ammonium acetate in water, Samples were analyzed by a series of precursor and neutral loss scans, as described by Narayanan et al.

Data were processed as described by Narayanan et al. Pooled quality control samples were included in the analysis to determine analytical precision, but they were not used to correct the sample data.

Lipidomics data were used as obtained. The only changes made in the data were removing the entries that were below the detection limit of the instrument. After that, the species of each phospholipid headgroup were summed together to obtain the total abundance of each headgroup. Presence of PS in the outer leaflet of the mammalian cells is attributed to the apoptosis of the cells.

Milk fat globule epidermal growth factor 8 protein a. A flow cytometer was used to detect the FITC intensity for , events. Macrophages were derived from THP-1 monocytes as described by Starr et al. The macrophages were washed twice with warm media, then incubated with exchanged and untreated senescent RBCs separately at a ratio of macrophages:RBCs for two hours.

The uptake of RBCs is measured using a calorimetric method based on specific oxidation of 2,7-diaminofluorene DAF by hemoglobin The macrophages were washed two times with media to remove the excess RBCs and then the surface bound RBCs are lysed using a hypotonic solution 0. The wells were washed twice again using media to remove any RBCs or free hemoglobin in the well.

The remaining cells were then lysed using a solution of 0. Op den Kamp, J. Lipid asymmetry in membranes. Verkleij, A. The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Acta Biomembr. CAS Google Scholar. Vance, D. Google Scholar. Li, G. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids. Lorent, J. Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape.

A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24 , — PubMed Google Scholar. Fadeel, B. The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease.

Bevers, E. Changes in membrane phospholipid distribution during platelet activation. Vermes, I. Flow cytometry of apoptotic cell death. Methods , — Moras, M.

From erythroblasts to mature red blood cells: organelle clearance in mammals. Kumar, A. Red cell membrane abnormalities in chronic myeloid leukaemia. Nature , Zwaal, R. Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance. Yasin, Z. Phosphatidylserine externalization in sickle red blood cells: associations with cell age, density, and hemoglobin F. Blood , — Yashar, V.

Wahid, S. Increased platelet and erythrocyte external cell membrane phosphatidylserine in type 1 diabetes and microalbuminuria. Diabetes Care 24 Surface exposure of phosphatidylserine in pathological cells. Life Sci. Connor, J. Exposure of phosphatidylserine in the outer leaflet of human red blood cells.

Relationship to cell density, cell age, and clearance by mononuclear cells. Lang, K. Mechanisms of suicidal erythrocyte death. Of macrophages and red blood cells; a complex love story. Klei, T. From the cradle to the grave: the role of macrophages in erythropoiesis and erythrophagocytosis. Phosphatidic acid can be used in the synthesis of several phospholipids by two different mechanisms. The first mechanism involves the hydrolysis of the phosphate group from phosphatidic acid to yield diacylglycerol.

This is achieved through the association of the cytosolic phosphatidic acid phosphatase also known as lipin with phosphatidic acid in the endoplasmic reticulum membrane. Diacylglycerol is used in the subsequent biosynthetic pathways for phosphatidylcholine and phosphatidylethanolamine, which will be discussed in following sections.

Diacylglycerol is also the precursor to the main storage form of energy, triacylglycerol. The second method whereby phosphatidic acid is used to synthesize additional phospholipids utilizes cytidine triphosphate CTP as an energy source and creates a CDP-diacylglycerol molecule.

Overall, this mechanism allows for the replacement of the phosphate group of phosphatidic acid by other phosphate functional groups to form phosphatidylinositol, phosphatidylglycerol or cardiolipin also known as diphosphatidylglycerol. The synthesis of these glycerophospholipids will not be covered in this overview. Most of the PC in the plasma membrane is found within the outer leaflet. PC is cylindrical in shape; as such, it is an important structural component that contributes to the integrity and function of membranes.

PC is essential for the formation and secretion of very-low-density lipoproteins by the liver, which is responsible for the delivery of hydrophobic cargo cholesterol and energy in the form of fat to other organs. This phospholipid also plays a role in bile salt-mediated micelle formation in the intestinal lumen, which facilitates the absorption of lipid-soluble nutrients from the diet.

Choline entering the cell is rapidly phosphorylated by choline kinase, converting choline to phosphocholine. Although these isoforms perform the same enzymatic reaction, they differ structurally in two significant ways. The two isoforms also differ in the length of the phosphorylation domain found at the carboxyl terminus.

The addition of the phosphocholine moiety to diacylglycerol completes the synthesis of PC. This reaction is catalyzed by CDP-choline:1,2-diacylgylcerol cholinephosphotransferase, or CPT, and occurs at the surface of the endoplasmic reticulum.

PC production via the PEMT pathway occurs primarily in the liver, where the demand for PC is high due to the production and secretion of very-low-density lipoproteins and PC secretion in bile, in addition to the normal cellular requirement for the synthesis of membranes.

PEMT is an intrinsic membrane protein, containing four membrane-spanning domains. PEMT is active in the endoplasmic reticulum, where it performs three repeated methylation reactions converting phosphatidylethanolamine PE to PC.

The methyl donor S -adenosylmethionine is required for each step of the reaction, generating three molecules of S -adenosylhomocysteine for each PC molecule produced. However, when choline is limiting in the diet, the PEMT pathway is critical for maintaining the supply of PC in the liver.

In addition, a source of diacylglycerol is required. This enzyme exists in a soluble form, which acts as an inactive reservoir. When the enzyme forms an association with lipids within the membrane, it acquires a greater affinity for its substrate, CTP. Lipids that stimulate CT activity include phosphatidylglycerol, oleic acid, and diacylglyerol. Translocation is regulated by phosphorylation of the enzyme, promoting the soluble inactive configuration, and dephosphorylation, favouring the active-membrane associated form.

An accumulation of S -adenosylhomocysteine inhibits the methylation of PE. This was the first evidence that PC biosynthesis is required for survival of macrophages following a cholesterol load, a process characteristic of the development of atherosclerotic lesions.

The cause of this disease is unclear, but appears to be linked to a combination of lower hepatic PC and higher hepatic ceramide and diacylglyerol levels. This liver failure is prevented by adding choline back to the diet or by preventing PC secretion into the bile by deleting the multiple drug resistance-2 protein.

A link between PEMT and whole body energy expenditure has recently been established. Interestingly, this phenotype was reversed by supplementing the high-fat diet with extra choline.

PE has a relatively small head group, which can accommodate the insertion of proteins within the membrane while still maintaining the integrity of the membrane. Another characteristic of PE arising from the small head group size is the propensity to form non-bilayer structures. This is important in the formation of new membranes and vesicles, as well as membrane fusion and budding processes.

PE is also enriched in the membranes of mitochondria and is essential for the growth and stability of these energy-producing organelles. PE is used in the production of glycosylphosphatidylinositol, which facilitates the anchoring of proteins to the membrane.

PE is synthesized in the cell through the phosphatidylserine decarboxylation pathway and the CDP-ethanolamine pathway. Phosphatidylserine decarboxylase PSD is produced as a 42 kDa protein that undergoes proteolytic cleavage to yield the active enzyme. A mitochondrial targeting sequence is located at the amino terminus of the full-length peptide. The adjacent functional domain is an inner mitochondrial membrane signal. The decarboxylation of phosphatidylserine is the main route for the production of PE found in mitochondrial membranes.

PE produced in the mitochondria is also efficiently transported to other membranes within the cell. Ethanolamine is phosphorylated to form the head group of PE by ethanolamine kinase EK in the cytosol. Alternative splicing of the ET transcript yields two isoforms of this protein with different levels of enzymatic activity.

Similar to CT, the ET protein forms a homo- or heterodimer. In the final step, phosphoethanolamine is attached to the sn -3 position of diacylglycerol. This reaction is catalyzed by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransfease EPT in the endoplasmic reticulum and yields the end product PE.

The synthesis of PE through the decarboxylation of PS occurs rapidly in the inner mitochondrial membrane. However, PS is made in the endoplasmic reticulum. PC, as reported in European trials, has the capacity to attenuate arterial plaque. As an emulsifier, PC breaks down fats. Besides forming bilayers, all phospholipids maintain a gradient of chemical and electrical processes that ensure cell survival.

PC enhances cognition, response time, learning, memory, and executive function. Phosphatidylcholine is a major lipid in the protective mucus of the gastrointestinal tract, has been shown to exert an anti-inflammatory effect. Studies have shown that PC can be helpful to those with gastrointestinal issues as well as by providing protection against NSAIDs , which can be detrimental to the GI system.

The best documented clinical use of PC is its significant amelioration of liver damage, primarily because damage demands substantial restoration of cell membrane mass. This eventuates to the improvement of enzyme function and related biochemical indicators of liver health, applicable in hepatitis, drug-induced insults, alcoholic steatosis, and NAFLD.

As we age, cells change in size and function and become less able to divide and multiply. Derogating fats increase inside the cell, and waste accumulates. Connective tissue stiffens, organs become more rigid, and membranes have more difficulty passing oxygen and nutrients in and out of the cell.

Illness, environmental insults, medications, increase in physical demands, and diet influence the manner of a cell's aging and the time it takes to do so. Replacing lost lipids with mixtures of cell membrane phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid restores membrane, cellular and mitochondrial functions while enhancing the removal of waste materials and toxins, thereby reducing symptoms of degeneration.

These products are not intended to diagnose, treat, cure, or prevent any disease. Home Supplements View All Supplements. Learn Blog. Video Library. Health Terms. About Us. Where are phospholipids found? What does the bilayer do? Phosphatidylcholine PC Of the tens of thousands of molecules that make up the life of a cell, PC is one of the most important.

Phosphatidylethanolamine PE Phosphatidylethanolamine PE is the second most abundant phospholipid, after PC, and is located in the inner leaflet of the cell membrane and the inner membrane of mitochondria. Phosphatidylserine PS Phosphatidylserine is important to cognitive function and has many biological functions in the cell.