Protocols

Radiochemical and fluorometric assays for plasmalogen-selective phospholipase A2

Posted on Friday, November 17, 2006

Description
Among glycerophospholipids, plasmalogens represent a special class that is characterized by the presence of a long-chain enol ether (vinyl ether) bond at the sn-1 position of the glycerol moiety. In plasmalogen sn-2 position of glycerol moiety is enriched in arachidonic acid, AA, 20:4 n-6 or decosahexaenoic acid, DHA, 22:6 n-3. Plasmalogens are found in all mammalian and non-mammalian cells. In mammals, these phospholipids are especially abundant in brain and heart. Choline plasmalogen is enriched in heart tissue whereas ethanolamine plasmalogen is rich in brain tissue. Plasmalogens are involved in membrane fusion, ion transport, cholesterol efflux, and act as a reservoir for second messengers. The vinyl ether linkage of plasmalogens may also play a role in protection of cellular membranes against oxidative stress. Plasmalogen-selective phospholipase A2 (PlsEtn-PLA2) hydolyzes AA from the sn-2 position of the plasmalogen. The levels of ethanolamine plasmalogen in brain tissue rapidly increase during myelination. Neural membranes contain a considerably higher proportion of plasmalogens. Plasmalogen levels are decreased in several neurological disorders including Alzheimer disease, ischemia, and spinal cord trauma. This is due to stimulation of PlsEtn-PLA2. A deficiency of plasmalogens has also been reported in peroxisomal disorders. This is due to decreased activities of plasmalogens synthesizing enzymes resulting in impaired membrane trafficking. PlsEtn-PLA2 has been purified from different sources (bovine brain, canine myocardium, renal proximal tubule). BrainPlsEtn-PLA2 differs from kidney and heart enzyme in kinetic properties, substrate specificities and response to detergents and inhibitors.
Immunocytochemical studies have indicated that this enzyme is localizes in astrocytes. The purpose of this protocol is to introduce Science Advisory Board members an updated version of determining PlsEtn-PLA2 activity by fluorometric and radiochemical methods (Farooqui et al., 1998).


Procedure
***Radiochemical Assay

This procedure is fast. The disadvantage of this procedure is that radiolabled substrate preparation is laborious, low yield and costly.

1. Lysoplasmenylcholine was prepared from bovine heart phosphatidylcholine as described earlier (Farooqui et al., 1998).

2. Mix 34 mg of lysoplasmenylcholine (71 mmol) with 70 mmol of free arachidonate and 50 mCi of [3H] AA together in a 2 ml reaction vial and evaporate with nitrogen.

3. Recrystallize DMAP in a vacuum desicator in the presence of phosphorous pentoxide and evacuate at less than 50 mTorr overnight. Dissolve lipids in 1.7 ml chloroform. Add 10 mg of dried recrystallized DMAP (80 mmol) to the lipid mixture.

4. Add 4 mg of DCC (20 mmol) to start the reaction. It is a light sensitive reaction, therefore avoid keeping in light. Add DCC every 4 hr (10 additions of 0.1-0.15 mol equivalents) with continuous stirring.

5. Terminate the reaction by adding methanol (lipid phase dried under nitrogen). Extract the residue in 200 ml chloroform/methanol (1:1). Shake for 1 min and let the mixture settle down into two layers. Collect the upper chloroform layer and dry it in nitrogen.

6. Purify labeled plasmalogen by HPLC (using linear gradient of hexane/2-propanol/water from 48.5/48.5/3 to 46/46/8 at a flow rate of 10 ml/min and 4 hr duration.

7. Use tandem Dynamax Macro-HPLC silica column (21.4 mm x 25 cm) in combination with a linear gradient (100% chloroform/0% methanol- 0%chloroform/100% methanolat a flow rate of 10 ml/min) in case of large amount of DMAP present in the reaction extract.

8. In assay buffer containing 100 mM Tris-HCl, 4 mM EGTA, 5% glycerol, pH 7.0, add 5 to 50 ml of enzyme solution and 10 ml of 2 mM [3H] plasmenylcholine and incubate at 37 degree C for 5 min.

9. Stop the reaction with 100 ml of butanol. Vortex and centrifuge at 1000g for 10 min.

10. Isolate released [3H] fatty acid by 25 ml of butanol to Silica Gel G plates. Develop these plates in petroleum ether/diethylether/acetic acid (70/30/1, v/v).

11. After scraping [3H] fatty acid band from plate, use liquid scintillation counting for
quantification.

*** Fluorometric Assay

This procedure has several advantages over radiochemical assay. It does not require radioactivity. Furthermore, it is more sensitive, inexpensive and provides a better yield of fluorescent substrate.

1. Purify ethanolamine plasmalogen from bovine brain mixed ethanolamine glycerophospholipids (For details see Farooqui et al., 1998).

2. Load the lipids on Unicil silicic acid column (2.5 x 30 cm), previously equilibrated with 100% ethanol. Elute free fatty acids and other neutral lipids by 300 ml of chloroform.

3. Elute ethanolamine plasmalogen by applying 500 ml of chloroform/methanol and lysophospholipids by 500 ml of methanol.

4. Determine the purity of ethanolamine plasmalogen by 2D TLC using a silica G plate under the solvent system of chloroform/methanol/ammonium hydroxide (65/25/4; v/v). Hydrolyze TLC plate with concentrated HCl fumes before developing second dimension.

5. Spray TLC plate with TNS solution or expose it to iodine vapor.

6. Visualize UV bands under UV light.

7. Identify plasmenylethanolamine, phosphatidylethanolamine, and lysophospholipid bands, scrape and quantify by phosphorus assay.

8. Label plasmenylethanolamine with pyrenesulfonylchloride in dark as described earlier (Farooqui et al., 1998).

9. For each substrate, dry 74 mmol of pyrene-labeled plasmenylethanolamine under a stream of nitrogen. Add 345 ul of H20 to the dried lipid and subject to sonication on ice.

10. Add 74 ul of 5% BSA in 20 mM MOPS, pH 7.4; 74 ul of Triton X-100 in 20 mM MOPS and 4 mM EGTA. Start the reaction by adding 100 all of plasmalogen-specific PLA2 enzyme source to the substrate, and substrate blank.

11. Incubate the reaction for 2 hr with gentle agitation at 37 degree centigrade. Stop the reaction by adding 4 ml of chloroform/methanol (2/1, v/v), vortex, add 159 ul of water and vortex.

12. Centrifuge the mixture at 1000g for 10 min. Discard the upper phase, transfer 1.2 ml of the lower phase to each of two clean test tubes and dry under a stream of nitrogen. Redissolve the two dried lower phase samples with 30 ul of chloroform each. Spot each sample on a separate Silica G plate (10 x 10 cm).

13. Subject one of the plates to acid hydrolysis by putting it in a tank containing fumes of concentrated hydrochloric acid for ~ 5 min.

14. Develop both plates under the solvent system of chloroform/methanol/ammonium hydroxide.

15. Identify the pyrene-labeled phosphoglyceroethanolamine band under UV light. Scrape off the band and extract with 2 ml methanol. Read the fluorescence intensity at excitation and emission of 340 and 376 nm respectively.



Recipes


Supplies
1. Supplies for radiochemical assay
Bovine heart lecithin (phosphatidylcoline, AvantiPolar Lipids, Inc., Albaster, AL); chloroform used in this procedure should be redistilled by fluxing over phosphorous pentoxide in a distillation flask; glacial acetic acid; N, N'-dimethyl-4-aminopyridine (DMAP, Aldrich); AA and [3H] AA (Nu Chek Prep., Elysian, MN and Dupont-NEN, Boston, MA); Econosil octadecyl silica column (10 mm x 25 cm), 10 mm particle size (Altech Association, Deerfield, IL); Dynamax Macro-HPLC Silica column (21.4 mm x 25 cm) (Rainin instrument, Woburn, MA); Silica gel G plates (20 x20 cm) 50 ?m (Analtech, Newarc, DE); methanol/water/acetonitrile (57/23/20, v/v); hexane/2-propanol/water (48.5/48.5/3, v/v); and hexane/2-propanol/water (46/46/8, v/v); N,N'-Dicyclohexylcarbodiimide (DCC, Aldrich Chem, Co, Milwaukee, WI).


2. Supplies for fluorometric assay
Bovine brain ethanolamine glycerophospholipid, 100 mg (Doosan Serdary Laboratory, Englewood Cliffs, NJ); sodium deoxycholate (Sigma); bovine serum albumin (Sigma); chloroform and methanol (Fisher Scientific Co.); silica gel TLC plates (250 micron) 20 x 20 cm (Anatech, Newark, DE) ; Unisil activated silicic acid (200-325 mesh) (Clarkson Chem., Williamsport, PA); Rhizopus arrhizus (R.A.) lipase, 50,000 U/ml (Boehringer Mannheim, Indianapolis, IN); pyrene sulfonylchloride (Molecular Probes. Eugene, OR), stored at -20 degree centigrade; triethanolamine, store at room temperature; 0.02 M 3-[N-morpholino] propane sulfonic acid (MOPS buffer) pH 7.4; Branson Sonifier cell disruptor 185; Perkin-Elmer L55 Spectrofluorometer.


Tips
1. Always store plasmalogen under nitrogen at -80 degree centigrade.
2. Check for the purity of plasmalogen. If it is less than 95% pure, then purify it using lipase digestion.
3. Radio-labeling PLA2 assay is the most sensitive method for determining PlsEtn-PLA2 activity. The disadvantages of this assay are its cost, discontinuity, and having safety issues because of using radioactive compound in the procedure. Preparation of radioactive plasmalogen has been reported earlier (Robertson and Lands, 1962).
4. Fluorometric PLA2 assay is less sensitive and inexpensive than radiochemical assay but it is rapid, reliable and reproducible.
5. Other assay procedures for PLA2 including radiochemical and fluorometric have been described in detail by Farooqui and Horrocks (2007).
6. This protocol is an updated version of Farooqui et al (1998). I thought it is a good idea to share this protocol with a broader audience.


Referenses:

Robertson, A.F., Lands, W.E. (1962) Positional specificities in phospholipids hydrolysis. Biochemistry: 1, 804-810.

Farooqui, A.A., Yang, H-C., Hirashima, Y., Horrocks, L.A. (1998) Determination of plasmalogen-selective phospholipase A2 activity by radiochemical and fluorometric assay procedures. In: Methods in Molecular Biology. Lipase and phospholipase protocols (Ed. Mark Doolittle and Karen Reue). Volume 109, pp 39-47. Humana Press, Tatowa, New Jersey.

Farooqqui, AA., Horrocks, L.A. (2007) Assay methods for phospholipase A2 activities in brain. In: Glycerophospholipids in brain pp. 321-340. Springer, New York.

Submitted by: buckeyetf