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Platelets used for general NMR analysis were obtained from the North London Blood Transfusion Centre, Brentwood, Essex. Blood used in the investigation of platelets and disease studies was obtained from outpatients in the department of cardiology. King’s College Hospital as well as from healthy volunteers at the School of Pharmacy, University of London.

Standard phospholipids were obtained from the Sigma chemical company, UK. Deuterated chloroform (99.8%) and perdeuteromethanol (99.8%) were obtained from the Aldrich chemical company.

All other reagents were of the highest grade available and were obtained from BDH Ltd.

3.2.2 Methods

3.2.2.1 Isolation of platelets from whole blood

20 ml of blood was obtained from each subject and placed in a 50 ml falcon tube containing 2 ml of 3.18% sodium citrate solution.

The blood sample was centrifuged at 200g at 20°C for 10 min to precipitate the red cells. This produced a platelet rich plasma (prp) which was decanted into another centrifuge tube, the pellet of red blood cells being discarded. The prp was centrifuged at 900g at 20°C for 20 min to precipitate the platelets and to produce a platelet free

Chapter 3_________________________________________________ NMR lipid profiles of platelets

plasma.

The platelet pellet was resuspended in 1 ml of washing buffer (140 mM NaCl, 5 mM glucose, 1 mM EDTA and 15 mM Tris-HCl, pH 7.4) and centrifuged for 20 min at 900g at 20°C. This procedure was repeated a second time and the final pellet obtained was immediately frozen in liquid nitrogen or dry ice before being stored at -80°C.

3.2.2.2 Total lipid extraction

Lipid extraction was carried out using a modified form of the Bligh and Dyer method [132]. All the lipid extraction procedures were performed in glassware. All solvents were “Analar” grade. To avoid oxidation of the highly unsaturated lipids dissolved oxygen was removed by bubbling nitrogen through the solvent.

A small volume of cold methanol (<0°C) was added to the pellet to aid transfer to a glass tube for lipid extraction. Chloroform and methanol were added to the glass tube to give a final composition of chloroform/methanol 2:1 (v/v). The organic solvent was 5 times the pellet volume.

The suspension was vortexed and sonicated for 20 mins at 0°C, with occasional vortexing. The mixture was then centrifuged at 1500 rpm to separate the aqueous from the organic phase. The upper aqueous layer was carefully removed and discarded. The bottom organic layer was transferred to another clean glass tube. The interface between the two layers contained denatured proteins. They were re-extracted using the same volume of chloroform/methanol (2:1, v/v) as before. The mixture was vortexed, left to stand for 10 min on ice with occasional vortexing, before centrifuging at 1500 rpm for 5 min at room temperature. The upper aqueous layer was removed and discarded. The lower organic layer was combined with the organic layer from the previous extraction step.

Chapter 3_________________________________________________ NMR lipid profiles of platelets

The extract was washed twice using a volume of 50 % methanol/ 0.5M KCl equal to the extraction volume added previously. The mixture was vortexed, centrifuged at 1500 rpm for 5 mins at room temperature and the upper aqueous layer removed by Pasteur pipette. This procedure was repeated with the lower layer extract.

The washed extract was then dried using a small quantity of dry Na2S0 4. Any remaining particles were then removed by filtering through tightly packed glass whool. The drying agent was washed twice with small volumes of chloroform which were filtered and mixed with the extract.

The solvents were evaporated under a stream of nitrogen. The extract was redissolved in a small volume of chloroform and stored in a sealed glass container, under nitrogen, at -80°C.

3.2.2.3 Proton NMR spectra analysis of lipids

Spectra were recorded on a Bruker AM500 NMR spectrometer. The proton spectra were recorded at 298K in the Fourier transform mode with 16K data points, using a 45° detection pulse and a 2.0s acquisition time, with water presaturation during relaxation to remove excess HOD signal. The 2D COSY experiment was performed on extracted lipids in a non-phase sensitive mode, with solvent presaturation. The 2D spectrum consisted of 2K data points obtained from 512 FIDs of 48 scans, with zero filling in the FI dimension. The data was multiplied with a square sine bell function in both directions prior to transformation. Chemical shifts were referenced in both cases to the residual methanol resonance at 3.31 ppm.

3.2.2.4 Calculation of lipid and water soluble metabolite proportions Chemical shifts were identified as described elsewhere [24-33] and from the 2D

Chapter 3_________________________________________________ NMR lipid profiles of platelets

COSY spectra of the extracts. After baseline correction, characteristic peaks in the ID NMR spectra were integrated. The integrals directly related to the amounts of each lipid present, correcting for any signal overlap. The sum of each individual lipid characteristic integral was taken as 100 %. The number of protons giving rise to the signal was considered in the calculations. In estimating the fatty acid composition of phospholipids, the integral at ca. 2.3 ppm was taken as a measure of total fatty chains. The integrals of individual fatty acid were compared to this value.

3.3 RESULTS