A Novel Assay for Phospholipase A Activity


I am in the process of revising the Handbook of Biochemistry and Molecular Biology.  While this can be frustrating when I work in areas outside of my own parochial interests; however, this activity  can be rewarding when one finds a clever paper as described below.  I am currently  working though lipidomics with the current emphasis on fatty acids.  Thanks to the efforts of the late Don Hanahan at the University of Washington, I had a rudimentary understanding of lipids . Somewhat later I found that while saturated fatty acids had useful IR absorbance, there was no useful UV-Vis absorbance other than carboxyl group absorbance at 210 nm (1-3).  Polyunsaturated fatty acids (PUFA) contain double bonds but most are separated by methylene group.  There are PUFA which contain conjugated double bonds where absorbance at longer wavelength (bathochromic effect) with greater intensity (hyperchromic effect) occurs with increasing conjugation length (1,4).

   
I was surprised (and pleased) to find a paper where 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine was developed as a substrate for phospholipase A (5). α-Eleostearoic acid [(9Z,11E,13E)-octadecatrienoic acid] contains three conjugated double bonds and has high absorbance at 270 nm (6). 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine was coated on a microplate where it is not removed by the solvents used in phospholipase A assay.   α-Eleostearoic acid is released into solution (solubilized by β-cyclodextrin) by the action of phospholipase A with an increase in absorbance at 272 nm.  Another group (7), also from France, has taken this a step further by using pomegranate oil, which contains punicic acid, a diastereoisomer of α-eleostearoic acid, as a substrate for phospholipase.  It is refreshing to see that there are still clever people doing science. Now, if we could just get nomenclature under control


References


1.  Kates, M., Techniques of Lipidology  Isolation, Analysis and Identification of Lipids, Volume 3 in Laboratory Techniques in Biochemistry and Molecular Biology, pps. 168-175, Elsevier, Amsterdam, Netherlands, 1968
2.  Chapman, D. and Goni, F.M., Physical properties: Optical and spectral characteristics, in The Lipid Handbook, 2nd edn., Chapman and Hall, London, United Kingdom, 1994
3.   Shahadi, F. and Wanasundam, P.D., Extraction and analysis of lipid, in Food Lipids Chemistry, Nutition, and Biotechnology, ed. C.C Akoh and D.B. Min, Chapter 5, pps. 115-138, Marcel Dekker, New York, New York, USA, 1998
4.  Meier, H., Stalmach, U., and Kolshorn, H., Effective conjugation length and UV/vis spectra of oligomers, Acta Polymer. 48, 379-384,
5.  El Alaoui, M., Noiriel, A., Soulère, L., et al., Development of a high-throughput assay for measuring phospholipase A activity using synthetic 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine coated on microtiter plates, Anal.Chem. 86, 10576-10583, 2014
6. Hoffman, J.S., O’Connor, R.T., Heinzelman, D.C., and Bickford, W.G., A simplified method for the preparation of α- and β-eleostearoic acids and a revised spectrophotometric procedure of their determination, J.Am.Oil Chem.Soc. 34, 338-342, 1957
7.  Ülker, S, Placidi, C., Point, V., et al., New lipase assay using Pomegranate oil coating in microtiter plates, Biochimie 120, 110-118, 2016