טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentEtzioni Shulamit
SubjectLipidic Polyols from Plants as a Natural Source for
Phospholipase A2 Inhibitors
DepartmentDepartment of Biotechnology
Supervisor Mr. Ishak Neeman (Deceased)


Abstract

This thesis is about inhibition of phospholipase A2 (PLA2) by natural lipidic polyols, isolated from avocado seeds. The enzyme PLA2 hydrolyses phospholipids at the sn-2 position of the glycerol moiety. The products of the hydrolysis are unsaturated fatty acid, and a lysophospholipid. The hydrolysis of phospholipids in the cell membrane by PLA2 leads to the synthesis of many lipidic mediators in different pathological conditions. These mediators include lysophospholipids, arachidonic acid and their respective derivatives (eicosanoids). The enzyme phospholipase A2 is very ubiquitous and is found in all kinds of cells, in venom of reptiles and insects, and in pancreatic secretions. Ten different kinds of PLA2 were characterized until now. It's very hard to specify which of them participates in certain pathological conditions, as in inflammation.

In this work lipidic polyols (lipids with some hydroxyl groups) were purified from the avocado seed. The four compounds isolated and identified are: 1-acetoxy-2,4-dihydroxy-n-heptadeca-16-en (olefin A), 1,2,4-trihydroxy-n-heptadeca-16-en (olefin B), 1-acetoxy-2,4-dihydroxy-n-heptadeca-16-yn (acetylene A) and 1,2,4-trihydroxy-n-heptadeca-16-yn (acetylene B). These compounds were found to be not toxic to cell cultures. These compounds are also stable at room temperature for many years. It was found that those compounds influence the activity of PLA2. The influence of these compounds has been tested on several PLA2 enzymes, in several different systems and in three different ways.

Three types of secreted PLA2 enzymes were used: (a)a bee venom PLA2, (b) a porcine pancreatic PLA2, and (c) a Crotalus Atrox (a rattle snake) venom PLA2. The three different systems, in which the activity of the PLA2 enzymes under the influence of the lipidic polyols was tested were: (a) The phospholipid was as a monomer because of the high concentration of n-propanol, (b) The phospholipids were arranged as unilamellar vesicles, and (c) The phospholipids were arranged as inverted micelles. Three different methods for measuring the activity of PLA2 were used: (a) measuring the pH of the reaction, (b) using a radioactive substrate, which is labeled in the sn-2 fatty acid, and (c) separation of the phospholipid (substrate) from the lysophospholipid (product) on a thin-layer chromatography plate. Then a method for phosphate analysis is needed. Initially we tried to use the wet ashing method, which is known and accepted. However, this method was found to be laborious and very complicated to be implemented. Therefore, we developed a method, which is based on a computer software, and gives rather accurate analytical results. In this work there is a comparison between the two methods on the basis of the calibration curves.

The results have shown that the tested compounds influenced the tested enzymes. In the system in which the substrate is not in an aggregated form, and the enzyme originated from bee venom, the tested compounds inhibited the enzyme. In the system in which the substrate was in unilamellar vesicles form, and the enzyme was originated from Crotalus Atrox' venom, acetylene B inhibited the activity of the enzyme only in high concentrations. In low concentration, however, acetylene B accelerated the activity of the enzyme. In the last system, in which the substrate was in inverted micelles form, the acetylene B accelerated the activity of the enzyme, in a dose-dependent manner. When the compounds were tested on cell cultures, it was found that they were not toxic to the cells but slowed down the proliferation rate.