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The main focus of the project is on study of the composition of membrane fatty acids of red blood cell membrane in both the genders. The techniques for the determination of the fatty acids were studied on the mock blood samples before using the actual blood samples.
The mock blood samples were subjected to all the procedures as per the protocol for the determination of membrane fatty acids. This step helped in understanding the techniques in much details and it also helped in understanding the risks and caution while dealing with the blood samples and chemicals involved in the process.
The fatty acids and their long chain derivatives are very much essential for the development of the human body and also for optimal health.(3) For optimal cardiovascular health, the international society for the study of fatty acids and lipids.(ISSFAL) recommends a minimum combined intake of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) as 500 mg/day(5). Previous studies have suggested that woman synthesise DHA more efficiently than men, the study carried out by Institute of Brain Chemistry and Human Nutrition, London Metropolitan University, investigated the relationship between diet, platelet phospholipids fatty acid and gender. The study was carried out on the Caucasian men and women. They found that the absolute and % energy intakes of arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA, and the ratios of total n-6/n-3 was faound to be same in both the sexes, however women have higher DHA level in phosphatidyl-choline (PC) and PE than men also EPA was higher in womenââ‚¬â„¢s phosphatidylethanolamine (PE). On the contrary it was also revealed that men had high level of AA and total n-6 fatty acids in PC (1).
All the research was carried out on the Platelets, but the composition of fatty acids in the platelets and the RBC are very different so the study of variation of composition of fatty acid in Red blood cell membrane on gender basis is important our project deals with the same, the blood is collected from the healthy volunteers of both the sexes male -15 and female -15. The ethical approval for the collection of blood sample was granted by the Camden and Islington Community research Ethics committee.
The fatty acid from RBC will be first extracted by chloroform methanol + BHT mixture BHT is added to prevent oxidation of fatty acids. The extracted fatty acids are then run on TLC to separate it into its individual components. Then the fatty acids are converted into methyl ester form i.e. FAME (fatty acid methyl ester) then the Fame is run in GLC to analyse it quantitatively. The data obtained is interpreted by use of EZChrom which will analyse the data and test the hypothesis that weather gender compromises the fatty acid composition of red blood cell membrane in healthy volunteers.
Materials and methods:
All the chemicals used were ordered from Sigma Aldrich by the dispensary. Following are the chemicals and apparatus used for the experimental purpose.
Apparatus : P1000 automatic pipette & blue tips,Pasteur pipettes,Measuring cylinders (100 mL, 250 mL) (2 each),Glass beaker (100 mL ; 500 mL) (2 each),separating funnel,round-bottomed flask (rotavap) & stoppers,Thin layer chromatography plates,Thin layer chromatography tank,Marker,vials holding stands 2.
Chemicals: HPLC grade Methanol-1650ml,HPLC grade chloroform-2450ml,Whatmann round filter paper-40,Silica gel 20 x 20 plates-14,Methylamine-190ml,Anhydrous methanol-650ml,Acetyl chloride-100ml,HPLC grade petrol spirit-570ml,3.5ml glass vial-150,Sodium sulphate-130g, HPLC grade Heptane.
The determination of fatty acids from the mock blood samples was done by following methods. Extraction of 1ml RBC was done by using 15ml of methanol+ BHT in a100 ml extraction tube the RBC was suspended in methanol to prevent clumping(1). Then to the resultant solution 30 ml chloroform +BHT was added and the tube was shaken and was flushed with nitrogen gas for 1 min and the cap was placed. The tube was stored at 40 C for 24 hrs. After 24 hrs the sample was filtered into separating funnel and the extraction tube was rinsed with first 10 ml Chloroform methanol + BHT (C/M) and then with 5 ml of C/M + BHT and each rinse was used to wash the filter paper. Then to the separating funnel 15 ml 0.85% saline solution was added. The liquids in the separating funnel were flushed with nitrogen and cap was put on the funnel it was checked for leaks and was stored at 4o C overnight.
The separating funnel was then allowed to stand at room temperature for 30 min to stop condensation. The lower organic layer of the separating funnel was removed into 100 ml round bottom flask and the flask was put on rotary evaporator (Rotavap) and the solvent was removed under reduced pressure in a water bath at 370 C. When dry 1-2 ml of methanol was added rinsing the inside of the flask then it was rotavap again this was done twice to remove the residual water in the sample. The dried whole lipid extract was removed in 10 ml vials using 2ml washes of C/M + BHT thrice. The sample was reduced to a small volume on a heating block and was stored at 4oC.
The TLC was performed on the sample obtained from the previous process. The solvent used for elution was chloroform / methanol / methylamine + BHT (65:35:15)
The tank was set up lined with filter paper and was filled with solvent and was allowed to equilibrate for 30 min before use. 20 cm square plated were used with 0.25mm thickness
since the blood sample is small in size the plates were activated by keeping it at 1000 C in a desiccator for 1 hr. The sample was applied to the plate using a micro pipette. After application of the sample the plates were kept in the tank and the lid was put on and was kept in tank for 1 hr time. After the required time the plates were removed out of the tank and was dried in cold air and was sprayed with visualizing agent i.e. 0.1% solution of 2, 7- dichlorofluroscein in methanol. The plate was dried in cold air and was observed under UV light and the bands were marked by pencil. The bands were scrapped and transferred to a methylating tube.
The methylation was carried out using 15 % acetyl chloride solution in methanol. To the bands scrapped from the TLC plates 4 ml of the methylating reagent was added the tube was flushed with nitrogen and the tube was capped and kept in oven at 700C for three hours. The tubes were removed from the oven and cooled to each tube 4ml, 5% saline was added and 2 ml petrol spirit+ BHT the tube was capped and shaked well. The upper petrol layer was removed to a test-tube containing 2ml 2% potassium bicarbonate solution to neutralise the sample. Then again the upper layer was separated and transferred to a tube containing granular sodium sulphate to remove the residual water. Finally the solution of fatty acids in petrol was transferred to a 3ml vial the vials were flushed and stored at 4oC until used for GLC.
Results and Disscussions:
No significant results have been obtained so far as the Gas Chromatography is yet to be performed. So, far the fatty acids were only extracted and converted to methyl ester form for its Gas chromatographic analysis. Fatty acids cannot be directly analysed using GC since they have high boiling points so they are converted into fatty acid methyl esters (FAME) which are their volatile counter parts. So far 5 mock blood samples were worked on and all the experimental except GC was performed on it, then 5 real blood samples were used to carry out the same procedure. In total 30 samples need to be worked on i.e. 15 male and 15 female blood samples. The samples will be extracted and methylated and the extract will be stored until all the samples done with the same process. The GC will be done on the entire sample at once.
The following problems encountered during the experimental using mock samples were later rectified by adopting the correct techniques when actual samples were used. When extraction of RBC was carried out addition of chloroform methanol +BHT produced clumping for that reason first methanol was added then the tube was shaken and eventually chloroform was added this prevented clumping. In the extraction of fatty acid methyl esters addition of petrol spirit produces turbid emulsion it is broken by addition of small volume of methanol by a forceful pipette flow.
So far only five samples have been processed for the Gas chromatography. First the remaining 25 samples will be extracted methylated and again extracted for the GC analysis. These 25 samples will be processed in a batch of first 10 and then 15 (10+15=25).
Once the extraction and processing is done with all the samples the GC analysis will be performed on the samples this is the main step of the fatty acid determination since GC will determine the fatty acid present in the samples quantitatively. The samples will be loaded by the auto sampler. After the run in GC is complete the data output obtained will be analysed by EZChrom and the results will be reported. EZChrom interpretates the results obtained from the GC analysis and helps in understanding the variation of composition in each samples.