Pros and cons of phospholipid asymmetry in erythrocytes.

Phospholipids of erythrocyte are found as bilayer with choline containing phospholipid like phosphatidyl choline and sphingomylein in the outer layer and amine containing phospholipid like phosphatidyl ethanolamine and phosphatidyl serine in the inner layer. This arrangement is known as phospholipid asymmetry. Lipid asymmetry is maintained throughout the entire life span of red blood cell and is disturbed when cells enter into the stage of apoptosis. We here discuss some of the conditions in which phospholipid asymmetry of erythrocyte is maintained and disturbed and the various detection methods to check the distortion phospholipid asymmetry of it.

Cell is the structural and functional unit of all living organism. The membrane that bound the cell and cell organelle is made up of phospholipids. Accordingly we can say our body as a puddle of phospholipids. Most components of cells are water soluble; however phospholipids are water insoluble, which provide integrity to the cell. Apart from phospholipids other lipids such as glycolipids and cholesterol also play an important role in providing integrity to the cell. Phospholipids are found as bilayer with choline containing phospholipid like phosphatidyl choline (PC) and sphingomylein in the outer layer and amine containing phospholipid like phosphatidyl ethanolamine and phosphatidyl serine in the inner layer. These are non-randomly arranged in bilayer in normal cells hence the name lipid asymmetry. The phospholipid bilayer act as semi permeable membrane, thus protecting cells from the external environment, while transport the essential component to the cell for its metabolic activity. Phospholipid contains one hydrophilic head and two hydrophobic tails. There is one more type of asymmetry with respect to lipid head group. The fatty acyl chains of the exterior phospholipids are highly saturated than the acyl chains of the interior phospholipids. This type of lipid asymmetry is known as transverse phospholipid asymmetry. This makes the cells interior more lipophilic. Once, this transverse phospholipid asymmetry is established, it is maintained by the combination of slow transbilayer movement of lipids and the presence of selective lipid transporters. Lipid asymmetry is maintained by the co-operative activities of three transporters namely flippase, floppase and scramblase.[12]

Flippase

It is an ATP dependent amino phospholipid – specific translocase, which transports phosphatidl Serine and Phosphatidyl ethanaol amine from the cells outer to the inner leaflet. Flippases are transmembrane lipid transporter enzymes located in the membrane.

Floppase

It is ATP dependent nonspecific translocase, which slowly transport lipid from the cell's inner to the outer leaflet.

Scramblase

Ca2+ dependent non-specific lipid transporter, which allows lipid to move randomly between the leaflets; that means they can transport the negatively charged phospholipids from the inner-leaflet to the outer-leaflet and vice versa. In humans, phospholipid scramblases constitute a family of five homologous proteins, which are hPLSCR1, hPLSCR2, hPLSCR3, hPLSCR4 and hPLSCR5.[23]

Role of Spectrin in Phospholipid Asymmetry

Spectrin is a major protein component of human red blood cells (RBC) located in intracellular region. Spectrin forms pentagonal or hexagonal arrangements, forming scaffolding and plays an important role in maintenance of phospholipid asymmetry by interacting with the lipid component. When oxidation takes place in membrane the sulfhydryl group converts to disulfide bond and the cross linking of membrane skeletal protein and aggregation of membrane proteins.[45]

Role of Calcium in Lipid Asymmetry

Increased cytoplasmic Ca2+ concentration disrupt lipid asymmetry by activating the scramblase and inhibiting the Mg2+ ATPase amino phospholipid translocase. Elevation of Ca2+ leads to calpalin induced degradation of variety of membrane protein.[6]

Condition in Which Phospholipid Asymmetry is Maintained and or Disturbed

Phospholipid asymmetry is maintained throughout the life span of the cell. Once the cell enters into apoptosis stage, this phospholipid asymmetry gets distorted. When lipid asymmetry is distorted, amine containing phosphatidy Serine will exposed, phosphatidyl Serine act as a self-marker formacrophages and removed from the body by engulfing them[7] [Figure 1].

Figure 1

Lipid asymmetry in normal cells and exposure of phosphatidyl serine in apoptotic cell

We here described some of the physiological condition and pathologic conditions in which phospholipid asymmetry is maintained and or distorted [Figures 2 and 3] and various methods to detect the phospholipid asymmetry.

Figure 2

Conditions in which phospholipid asymmetry maintained

Figure 3

Conditions in which lipid asymmetry distorted

In uremic patients

In patients with the chronic kidney disease, anemia occurs due to interaction of various factors such as uremic toxins, free radical and elevated Ca2+ concentration with erythrocyte membrane accompanied by loss of asymmetry.[89]

Erythrocytes as drug carrier

The relationship between phospholipid asymmetry and erythrocyte as drug carrier can be studied under two heading

As slow release depot formulation.

For targeting mononuclear phagocyte system.

Slow release depot formulation

Erythrocytes can be used as drug carrier for slow release depot formulation provided that there should not any exposure of phosphatidyl Serine during the loading procedure. Exposure of phosphatidyl Serine will leads to rapid recognition of erythrocytes by mononuclear phagocytic system.

For targeting disease affecting reticuloendothelial system/mononuclear phagocytic system

Exposure of phosphatidyl Serine is preferred if we are supposed to target RES system because phosphatidyl Serine is a self-marker for macrophages. Thus we can able to target diseases like liver tumor, certain parasitic diseases in liver, disease affecting spleen etc.[101112]

Microvascular occlusion

The loss of phospholipid asymmetry of erythrocytes activate coagulation factors like platelet activating factor (PAF) so that blood cell coagulate and this will adhere to endothelial cells, micro vascular occlusions happen and ultimately lead to cardiovascular complications such as shock, myocardial infarction etc.[13]

In sickle cell anemia

In sickle cell anemia, erythrocyte loss their lipid asymmetry as a consequence of rapid transbilayer diffusion of PC so that phospholipids will exposed. It will lead to the activating of factors responsible for clotting (like PAF) leading to micro vascular occlusion, clinically manifested most often as an acute painful episode or “crisis,” but with many other acute and chronic complications. Hence microvascular occlusion can be considered as secondary complications of loss of phospholipid asymmetry in much pathological condition.[131415]

In blood coagulation

Activation of procoagulant cascade is not very significant in erythrocyte phosphatidyl Serine exposure. But is significant when comes in the case of platelet's phosphatidyl Serine exposure. However the exposure of phosphatidyl Serine s of erythrocyte in diabetic mellitus, sickle cell anemia and the resulting blood coagulation and microvasular occlusion is a matter to be considered.[161718]

During inflammation or tissue remodeling

During tissue remodeling or inflammation it is important to remove apoptotic cells from body in order to restore normal tissue structure and function. Loss of phospholipid asymmetry makes the inflamed cell recognized by the macrophages. So that rapid clearance from the body will happen.[19]

Chronic myeloid leukemia

Chronic myeloid leukemia is a clonal disorder common to granulocyte.

Platelet and erythrocyte precursors due to phosphatidyl Serine exposure the spectrin cross linking occur because of the formation of disulfide between its two subunits.[2021]

Hyperbilirubinema

High secretion or low excretion of bilirubin results in hyperbilirubinema in infants. When excess of bilirubin accumulate in body it binds with erythrocyte membrane and dissolves majority of glycerophospholipids like PC and cholesterol and expose the phosphatidyl Serines on the outer layer.[22]

During chemotherapy

Mature RBC the lipid asymmetry is disturbed during chemotherapy resulting in anemia; this condition may sometimes fatal.[23]

β thalassemia

Thalassemia is an inherited disease, clinically manifested as chronic anemia, which is partially due to ineffective erythropoiesis and decreased survival of erythrocyte in peripheral blood because of loss of lipid asymmetry.[24]

Detection of Phospholipid Asymmetry

By binding with fluorescently labeled Annexin V

Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for phosphatidyl Serine and binds to cells with exposed phosphatidyl Serine. The binding assay result will be provided by flow cytometry in a four quadrant table [Figure 4].

Figure 4

Annexin V binding assay test four quadrant table

Quadrant 3 (Q3): This quadrant shows the no. of cells that do not have affinity for annexin, which means all cells are live and no exposure of phosphatidyl Serine.

Quadrant 4 (Q4): Annexin V positive cell is quantified in this table. That means it gives information about the apoptotic cell or phosphatidyl Serine exposed erythrocytes.

Q1 and Q2 have no role in determining the phospholipid asymmetry of RBC as these quadrants are meant for nucleus containing cells. This quadrants give positive results for propidium iodide dye which stains the nucleus.[25]

In vitro macrophages uptake studies

Damaged/senescent cell are removed from the circulation by macrophages. Macrophages cellular uptake studies give valuable information about the lipid asymmetry. Phosphatidyl Serine s exposure will make erythrocytes easily recognizable by these macrophagal cell lines.

By confocal microscopy

Confocal microscopy is used for quantifying cellular uptake of erythrocyte. Phosphatidyl Serine exposed erythrocytes are internally localized in live macrophages cells.

Limitation

One of the most important limitations of confocal microscopy is the cost of instrument. Statistical problem also arise because only small number of cells can be monitored by it. Moreover, expertise needed to perform the experiment.

Procedure

Cells were grown to about 80% confluence in a 35 mm gloss bottom dishes. Fluorescently labeled erythrocytes is added to culture medium and incubated at 37°C. The cells are washed with phosphate-buffered saline after incubating and visualized under confocal microscope.[26]

Fluorescent assisted cell sorting

Erythrocytes are coupled with a flourophore and then added to cultured macrophages cell line and the fluorescent intensity of cells is taken by FACS.[2728]

Limitation

It gives false positive result if the erythrocytes bind to the surface of macrophages cells.

Even though the detection of phospholipid asymmetry uses the sophisticated techniques, the uses of these techniques are limited to biomedical research.

Detection of phospholipid asymmetry is not only a diagnosis test for majority of diseases discussed above but also an important stability test parameter of storing RBC for transfusion. Erythrocytes which maintain lipid asymmetry throughout the entire storage period and storage condition is used for transfusion. Increase in storage period will lead to lipid asymmetry distortion. Proper storage is also a criteria for maintain phospholipid asymmetry.[29] Detection of phospholipid asymmetry can be considered an important recently updated evaluation parameter of resealed erythrocytes used as carrier for parenteral controlled release of drugs.

Conclusion

Maintenance of phospholipid asymmetry of erythrocyte membrane is essential for retaining the normal lifespan of the cell. Whenever distortion in lipid asymmetry occurs, the cells undergo apoptosis and are then removed from circulation by the mono nuclear phagocytic system. Many patho physiological conditions are attributed due to distortion in lipid asymmetry. Even though the lipid asymmetry seems to a minor phenomenon, a balance in maintenance and disturbance in lipid asymmetry is essential for a better life processes. This nature's own phenomenon of this lipid asymmetry of erythrocyte is used by man to make drug carrier for slow release depot formulation and also for targeting reticulo endothelial system.