Rat Brain Phosphatidyl-N,N-Dimethylethanolamine Is Rich in Polyunsaturated Fatty Acids
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Journal oj Neurochemistry Raven Press. New York 10 1985 International Society for Neurochemistry Rat Brain Phosphatidyl-N,N-Dimethylethanolamine Is Rich in Polyunsaturated Fatty Acids Mariateresa Tacconi and Richard J. Wurtman Laboratory of Neuroendocrine Regulation, Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. Abstract: Phosphatidyl - N,N - dimethylethanolamine analysis of its FAs (36.9 :t 1.8 g/g). The FAs in the PE (PDME), an intermediate in the formation of phosphati- and PC of rat brain synaptosomes were also analyzed; dylcholine (PC) by the sequential methylation of phos- too little PDME was present in synaptosomes to permit phatidylethanolamine (PE), was purified from rat brain similar analysis. The percentage of unsaturated FAs in- and its fatty acid (FA) composition compared with those synaptosomal PE was even higher (43.4 vs. 27.7) than that of brain PC and PE. The proportion of polyunsaturated in PE prepared from whole brain. Since synaptosomes fatty acids (PUFAs) in the PDME (29.8%) was similar to have a very high activity of phosphatidyl-N-methyltrans- that of PE (27.7%) and much greater than in PC (2.8%). ferase, the enzyme complex that methylates PE to form Like the PUFAs of PE, the major PUFAs found in PDME PC, this enzyme may serve, in nerve endings, to produce were arachidonic acid (20:4) and docosahexaenoic acid a particular pool of PC, rich in PUFAs, which may have (22:6). An isotopic method was developed to quantify the a distinct physiological function. Key Words: Fatty PDME purified from brain; a tritiated methyl group from acid composition - Phos phatid ylethanolamine-N -meth- CHJI was transferred to the PDME in the presence of yltransferase - Phosphatidyl - N,N - dimethylethanol- cyclohexylamine to form pH]PC, and the radioactivity of amine-Rat brain. Tacconi M. and Wurtman R. J. the PC was then counted. The concentration of rat brain Rat brain phosphatidyl-N,N-dimethylethanolamine is PDME obtained using this method (33.0 :t 1.8 g/g brain) rich in polyunsaturated fatty acids. J. Neurochem. 45, was very similar to that obtained using quantitative GLC 805-809 (1985). Phosphatidylcholine (PC) in cells can be synthe- via all three pathways, has a much lower average sized by three pathways: by combining CDP-cho- PUFA content (Skrbic and Cumings, 1970; Albert line and diacylglycerol; by exchanging free choline and Anderson, 1977; Crawford and Wells, 1979). It for a serine in phosphatidylserine (PS) or an etha- therefore seemed possible that the brain PC formed nolamine in phosphatidylethanolamine (PE) (base- via PEMT might contain more PUFAs than the PC exchange); or by sequentially methylating PE. Of provided by CDP-choline or base-exchange path- these pathways only the latter actually forms new ways, and might fulfill different physiological func- choline molecules. PE-methylation activity, cata- tions. (For example, a PC rich in PUFAs might lyzed by the enzymes phosphatidylethanolamine- modify its microenvironment, either by changing N-methyltransferase (PEMT), has been demon- the viscosity of the biomembrane or by being a strated in many tissues (Bremer and Greenberg, better substrate for phospholipases, thereby making 1961; Hirata and Axelrod, 1978, 1980) including PUFAs or other degradation products, such as cho- brain (Blusztajn et aI., 1979; Crews et aI., 1980). line, more available for further metabolic pro- The PE in brain is very rich in polyunsaturated fatty cesses.) In attempting to characterize the pool of acids (PUFAs), whereas brain PC, probably formed PC formed via PE methylation, we examined the Re"eived October 15. 1984; accepted February 28, 1985. Abbreviations used: BHT, butylated hydroxy toluene; FA, Address correspondence and reprint requests to Dr. R. J. fatty acid; PC, phosphatidylcholine; PDME, phosphatidyl-N,N- Wurtman at Laboratory of Neuroendocrine Regulation, M.LT., dimethylethanolamine; PE, phosphatidylethanolamine; PEMT, E25-604, Cambridge, MA 02139, U.S.A. phosphatidylethanolamine-N-methyltransferase; PL, phospho- The present address of Dr. M. Tacconi is Istituto di Ricerche lipid; PMME, phosphatidyl-N-monomethylethanolamine; PS, Farmacologiche Mario Negri, Via Eritrea 62,20157 Milano. phosphatidylserine; PUFA, polyunsaturated fatty acid. Fatty Italy. acids are abbreviated as number of carbon atoms. 805 - - - .- .. .._ _. _.n. ......._.._.._._.. 806 M. TACCONJ AND R. J. WURTMAN fatty acid (FA) composition of brain phosphatidyl- to visualize the TLC region containing PDME. A I-cm N,N-dimethylethanolamine (PDME), an interme- band, corresponding to the Rr of a true PDME standard, diate in the formation of the PC. was scraped, extracted three times with methanol (2 + I + I mt) containing 0.01% BHT, evaporated to dryness, spotted on high-performance TLC plates (HETLC-HL MATERIALS AND METHODS Uniplates with preadsorbent, 10 x 10 cm, Analtech, Male Sprague-Dawley rats (200-300 g, Charles River Newark, DE, U.S.A.), and chromatographed in chloro- Laboratories) were housed under a 12-h light-dark form-methanol-water (70:30:4). A brain sample to which schedule (VITA-LITE; Duro-Test, North Bergen, NJ, PDME standard had been added was always run in par- U.S.A.) and given free access to food (Charles River Lab- allel. After visualization as described above, the band oratories, RMH 3000) and water. The animals were de- corresponding to the PDME standard was scraped and capitated and their brains quickly removed, weighed, and then transferred in glass tubes for further analysis. In one immediately processed. To prepare synaptosomes, brains experiment, uniformly labeled [14C]PDME (0.05 fJ.Ci/mg) were homogenized in 0.32 M sucrose and centrifuged was extracted and chromatographed following the pro- under a sucrose gradient as described by Dodd et al. cedure described above; the radioactivity recovered (1981). The protein contents of synaptosomes were de- was 55%. termined according to Lowry et al. (1951). Analysis of FA composition Extraction of phospholipids and separation Silica gels containing PDME (purified by the above into classes procedure), PE, and PC (separated by TLC using chlo- Brain tissues (0.5-1 g) were homogenized in chloro- roform-methanol-ammonia, 70:30:4) were methylated di- form-methanol (2: I) containing 0.01% butylated hydroxy- rectly by alkaline methanolysis (Glass, 1971). Dihepta- toluene (BHT) as antioxidant and lipids were extracted decanoyl-PC (20 fJ.g)was added to each sample as internal by the method of Folch et al. (1957) with minor modifi- standard. Each sample was mixed thoroughly with I ml cations; they were first homogenized in 10 volumes of saturated NaOH in chloroform-methanol (2: I); 10 min methanol, then the chloroform (20 volumes) was added. later, I ml I M HCI in saline was added and the sample Extracts were stirred for 5 min, after which they were was mixed and centrifuged. Approximately 300 fJ.Iof the filtered over filter paper and the filters were washed with lower phase was then aspirated, evaporated to dryness, additional chloroform-methanol (2: I); the mixture was and resuspended in 20 fJ.Ichloroform, 1-2 fJ.Iof which then layered with 0.75% wt/vol aqueous KCI (0.2 mUmt), were injected in a gas chromatography apparatus Packard nitrogen was layered over it, and it was allowed to stand 430, equipped with a FID detector and an electronic in- overnight. The upper phase was discarded and the lower tegrator. The chromatographic column (6 feet long and 2 phase, containing phospholipids (PLs), was evaporated mm internal diameter) contained SP 2330 as liquid phase; to dryness under vacuum. When the lipids in synapto- the carrier gas was nitrogen (22 mUmin). The initial tem- somes were to be extracted, aliquots corresponding to perature was kept at 160°C for 4 min, and then was in- 200-300 fJ.gprotein were resuspended in 200 fJ.1of 0.32 creased to 220°C at a rate of 2°C/min; the injector and M sucrose and extracted with chloroform-methanol (2: I), detector temperatures were both 275°C. The FA meth- as described above. To separate PLs from neutral lipids, ylesters were identified by comparing their retention fac- the extracts were redissolved in chloroform and layered tors with those of standard solutions run under identical onto a I x 25 cm column of activated silicic acid (10 g, conditions. Measurements of peak areas were made with Biosil A, 100-200 mesh, Bio-Rad) (Rouser et aI., 1976). the automatic integrator attached to the gas chromato- Chloroform, acetone, and methanol (10 column volumes graph. FA contents 'were expressed as percentages of each) were used to elute lipids; the first two ~Iuates-the total FAs. Total FAs in PDME were quantitated by com- neutral lipids and free FAs in chloroform, and the cere- parison with the internal standard (diheptadecanoyl-PC) brosides and sulfatides in acetone-were discarded. PLs and corrected for recoveries. eluted with methanol were evaporated to dryness under Methylation of PDME to PC vacuum and redissolved in 2 ml chloroform; aliquots were To affirm the authenticity of the FAs obtained through then spotted onto silica gel plates (Silica gel G, Adsor- the above procedure, an additional assay was developed bosil Plus I, Alltech) and chromatographed using the involving the transfer of a tritiated methyl group from solvent system chloroform-methanol-ammonia, 70:30:4 by vol. CH3I to the PDME, yielding tritiated PC (Stoffel, 1975). Aliquots of 200-300 mg of brain were extracted and chro- Purification of PDME matographed as described above. Silica gel containing PDME has a Rr of 0.62 in the solvent system previously PDME was scraped and then extracted three times with described; it thus separates well from the major PLs [PE, methanol; the extracts were evaporated to dryness. To Rf = 0.47; PC, Rr = 0.33; PS, Rr = 0.07; phosphatidyl- the dry extracts 2 fJ.Iof cyclohexylamine (50 fJ.mol),24 fJ.I N-monomethylethanolamine (PMME), Rr = 0.40] but not [3H]CH3I (25 fJ.mol,0.1 fJ.Ci/fJ.mol),and then methanol to from others such as cardiolipin and, possibly, the cere- a final volume of 500 fJ.1were added.