Experimentation with Phospholipids for HPLC Analysis

1. Monique Honeyghan HCS Class of 2008 Professor Mark Borden Columbia University Chemical Engineering Experimentation with Phospholipids for HPLC Analysis

2. Overview • Phospholipid Structures • Motivation • Procedure • Data • Conclusion • References • Acknowledgements

3. Phospholipid Structures Monomer SUV Bilayer MLV

4. Motivation • Prepare a protocol for a lab assignment designated for Columbia undergrad seniors majoring in Chemical Engineering • Create a solution of phospholipids and lysophospholipids • Run the lipid solution through the HPLC to identify and quantify both types of lipids for analysis • Future component of research involving phospholipids • Provide further understanding of lipids in relation to enzymatic digestion

5. Procedure • Preparation of lipids solution • DSPC 1,2-Distearoyl-sn-Glycero-3-phosphocholine C44H88NO8P • stored in chloroform • Evaporation using nitrogen gas • Vacuum overnight • 10mg/ml Concentration vs. 15 mg/ml Concentration • PBS – Pure Buffer Solution

6. Procedure • Sonication : sonic energy breaks up large vesicles into SUVs. • Bath vs. Probe • Clear, yet translucent (hazy blue) • Heating Block above 60o C

7. Procedure • Extrusion – mechanical energy in the form of force to push larger vesicles though small pores in order to help form uniformed vesicles. • 11 passes • 0.2 micron polycarbonate membrane • Heat block above 60o C • * Bath sonicate stock sample to be extruded • * Heat extruded samples • Syringe filter (0.2 micron) vs. (0.45 micron) vs. no filtering • Variable: hot bath vs. quenching with ice bath

8. Procedure • Testing on DLS machine • Dynamic Light Scanner • Size of liposomes • Size distribution by volume

9. Procedure SUV • Enzyme Reaction • Phospholipase A2 from bovine pancreas • 100 µL to 1mL lipid solution • 45 minutes on rotator* • Reactant: liposome (DSPC) • Products: lysophospholipid + fatty acid MLV Phospholipase A2

10. Procedure • Extraction (Method by Folch et al) • Solvents - Chloroform: methanol in a 2:1 ratio • 0.6 mL per 1 ml reacted lipid solution • Agitation to homogenize (3 minutes) • Centrifugation (2000 rpm x 3 minutes) • Separates phases distinctly • Upper: methanol, water, enzyme • Lower: chloroform, PC and Lyso PC • Remove upper phase • Add 0.4 ml Filtered H2O • Agitation to homogenize (1 minute) • Centrifugation (2000 rpm x 3 minutes) • Filtered using a 0.2 μm pore syringe filter • Test upper phase on DLS* • Test lipid phase (lower) on HPLC for efficiency of the Phospholipase A2 enzyme

11. Procedure • HPLC - High Performance Liquid Chromatography – identify and quantify the phospholipids and lysophospholipids via an isocratic elution that is run through a silica column • Buffer Components: Hexane: 2-Propanol: 25 mM Potassium Acetate: Acetonitrile: Glacial Acetic Acid 442: 490: 62: 25: 0.6 by volume • Run on buffer to establish a baseline • Run on PC and LysoPC to get readings (Control) • Inject samples of prepared lipid solution to see if the results match the standards

12. Procedure (HPLC Diagram)

13. Procedure (HPLC) Detector Injection site PUMPS Column Chamber r Buffer

14. Procedure (HPLC) Pump B Injection site Column Pump A

15. Data • Graph showing size distribution by volume of 10 mg/mL DSPC

16. Data • Graph showing size distribution by volume of 10 mg/mL DSPC

17. Data • Graph showing size distribution by volume of 15 mg/mL DSPC

18. Data • Graph showing size distribution by volume in 10 mg/mL enzyme extract (upper phase)

19. Data • Graph showing size distribution by volume of 10 mg/mL enzyme extract

20. Data • Graph showing size distribution by volume of 10 mg/mL enzyme extract

21. Results • Probe sonication is faster, but there is a risk to contaminating the sample with particles from the metal probe. • Hot bath is more efficient than quenching with cold water, however, if samples need to be cooled, it is better to do it rapidly. • Virtually no difference between syringe filtering and not doing so • Making 10 mg/ml samples is easier and more efficient than making 15 mg/ml samples. • HPLC has no final results. • Progress with the reacted liposome samples shown by 2 peaks, which might correspond to the PC and Lyso-PC standards

22. Conclusion • Able to produce unilamellar vesicles averaging around 100 nm in diameter • Developed protocol so the undergrads can reproduce the experiment • Safety concerns established • Working under a fume hood with chloroform and the HPLC buffer components • Wear nitrile gloves when working with chloroform • Wear heat resistant gloves when using Avanti Mini extruder on heat block

23. References • “Interfacial Enzyme Kinetcs at the Phospholipid/Water Interface: Practical Considerations “by Raymond A. Deems. www.ideallibrary.com • “Preparation of liposomes” and extrusion technique by Avanti Polar Lipids Inc. www. avantilipids.com • Phospholipase A2 at the Bilayer Interface” by Faustoramirez and Mahendra Kumar Jain • “Separation and Quantitation of Phospholipids and Lysophospholipids by High Performance Lipid Chromatography” by Edward Lesnefsky, Maria Stroll, Paul Minker and Charles Hoppel • “HPLC – High Performance Liquid Chromatography” and HPLC diagram by Resource Library of www.waters.com • “Folch (et al) method of lipid extraction “ www.cyberlipid.org • Cuvette image www.krackeler.com/products/1092-Cuvettes/1071... • Image of DLS machine (Malvern Zetasizer) www.azon.com • Image of bath sonicator by all-spec.com • Multilamellarvessicle image by encapsula.com

24. Acknowledgements • Professor Borden • Cherry Chen • Melissa Moy • Borden Lab group • Dr. Sat Bhattacharya • Harlem Children Society • Dr. Zarou • Bronx Health Sciences High School • Family and Friends