Assessment of RBCs membrane protective activity of citicoline and eicosapentanoic- decosahexanoic acid in osmotic fragility model

Different organic compounds possess a good membranes stabilizing effects that protect cells like erythrocytes from strains exerted by change in medium osmolarity . These changes in tonicity could predispose RBCs for hemolysis . This disorder could be assessed by osmotic fragility test . From those compounds that widen osmotic fragility test values are citicoline (membrane precursor) and polyunsaturated fatty acids EPA-DHA. Assessment of these compounds on osmotic fragility test revealed that both of citicoline and EPA-DHA will protect RBCs from hemolysis for a wider range than values of the control blank. Although these effects were statistically not significant on considering statistical correlation test, however, the differences were important on considering t test for the range of saline concentration between 0.4-0.5 at P<0.05 . From the overall results, both citicoline and pufa induced a significant change in statbility of RBCs membrane upon exposure of osmotic effects in the saline concentration between 0.4-0.5. Further evaluation of citicoline and EPA- DHA effects may be necessary before recommending the use these compounds in protection against hemolytic diseases.


Abstract
Different organic compounds possess a good membranes stabilizing effects that protect cells like erythrocytes from strains exerted by change in medium osmolarity . These changes in tonicity could predispose RBCs for hemolysis . This disorder could be assessed by osmotic fragility test . From those compounds that widen osmotic fragility test values are citicoline (membrane phospholipids precursor) and polyunsaturated fatty acids EPA-DHA.
Assessment of these compounds on osmotic fragility test revealed that both of citicoline and EPA-DHA will protect RBCs from hemolysis for a wider range than values of the control blank. Although these effects were statistically not significant on considering statistical correlation test, however, the differences were important on considering t test for the range of saline concentration between 0.4-0.5 at P<0.05 .

Introduction
Many disorders of erythrocytes membrane can attenuate ability of RBCs in overcoming osmotic stress like hereditary spherocytosis, hemolytic anemias, sickle cell anemia, thalassemia major and conditions that exert oxidative stresses on cells lipid bilayer can induce rupture of erythrocytes and cause hemolytic sequalae (1). Human red blood cells RBCs have the ability to minimize strain exerted on their cell membranes by their concave shape and specific lipid constituents of their membranes that enable RBCs resistant to rupture under higher osmotic stress like hypotonic conditions. RBCs will swell under hypotonic blood conditions to make intracellular osmolarity as similar as to that of extracellular (2) . However , under genetic or oxidative disorders above or under abnormal hypotonicity this ability of RBCs is lacking giving rise to fragile RBCs that eventually will hemolyse when for example traversing splenic network .
As many disorders can cause RBCs to lack one or more of their plasma membrane constituents, supplement of some essential lipid bilayer supporting agents like unsaturated fatty acids ( eicosopentanoic EPA and decosohexanoic DHA acids ) and adenosine analoque citicoline are expected to increase RBCs resistance for strains exerted by hypotonic or stressful conditions since these compounds could optimize structural integrity of plasma membrane of many cell types (3) . Citicoline is a phosholipid precursor that stabilizes cell membrane (Chemical IUPAC Name 2-[[[5-(4-amino-2-oxopyrimidin-l-yl)-3,4-dihydroxy -oxolan -2-yl] methoxy -hydroxy-phosphory l]oxy-hydroxy -phosphory l ] oxyethy l -trimethy l -azanium ). It has the structure: Figure (1) citicoline structure, a membrane phospholipids precursor (4) In regard to polyunsaturated fatty acids (pufa) and omega-3 pufa they have the following properties: Osmotic fragility is a test to measures red blood cell (RBC) resistance to hemolysis when exposed to a series of increasingly dilute saline solutions. It has a normal range: Hemolysis begins 0.45% and complete 0.35%. The sooner hemolysis occurs, the greater the osmotic fragility of the cells .

Materials and Methods
The research was carried out at Therapeutic Research Unit College of Medicine Kufa University. Collection Medium: EDTA blood collection tubes Minimum: 5 ml whole blood. Specimen: whole blood Rejection Criteria: Hemolyzed specimen. Methodology: Spectrophotometer .At 540 nm .

EPA-DHA
There was an obvious left graphic shift (increased RBCs resistance for more hypotonic solution) as compared with blank.However this effect is significant not along the whole dilution range (correlative assessment) other than saline concentration between 0.4-0.5 at P<0.05.

Discussion
It was noticeable from the result graph that both citicoline and EPA-DHA induced a protective effect for RBCs against hypotonic swelling exerted on their plasma membranes due to the aggressive hypotonic medium.
However that change in RBCs osmotic fragility was only statistically significant for citicoline and EPA-DHA at P<0.05 on considering NaCl concentration between 0.4-0.5. This minor changes in osmotic fragility, although limited but they could safe a large proportion of blood cells from hemolysis in critical conditions like hereditary spherocytosis .
It is expected that both citicoline and those unsaturated fatty acids will incorporate with RBCs plasma membrances and give them more flexibility and strength that prevent them from rapture. Omega -3 fatty acids like EPA-DHA expand the spectrum of osmotic fragility test for human RBCs similar studies agreed with this attribution (6), (7) . Another studies showed that citicoline as a part of phospholipids will stabilize cell membranes and protect against stress as well (8). These RBCs stabilizing effects may be beneficial in treatment of patients with hemolytic conditions and heridetary and aquired spherocytosis.

Conclusion
Citicoline and EPA-DHA had a beneficial erythrocyte protective effect against hemolytic stimuli like hypotonicity. This property could make them a good candidate for use in thalassemia, sickle cell anemis and spherocytosis after being further evaluated.