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Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat

2002; Elsevier BV; Volume: 43; Issue: 1 Linguagem: Inglês

10.1016/s0022-2275(20)30187-5

1539-7262

Thad A. Rosenberger, Jun Oki, A. David Purdon, Stanley I. Rapoport, Eric J. Murphy,

Metabolism and Genetic Disorders

The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-3H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol·g−1·min−1, respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter. The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function. —Rosenberger, T. A., J. Oki, A. D. Purdon, S. I. Rapoport, and E. J. Murphy. Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat. J. Lipid Res. 2002. 43: 59–68. The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-3H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol·g−1·min−1, respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter. The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function. —Rosenberger, T. A., J. Oki, A. D. Purdon, S. I. Rapoport, and E. J. Murphy. Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat. J. Lipid Res. 2002. 43: 59–68. Ether phospholipids are naturally occurring lipids having either a 1-O-alkyl (plasmanyl) or a 1-O-alk-1′-enyl (plasmenyl) linkage at the sn-1 position of the glycerol backbone. In mammalian cells, these lipids belong almost exclusively to the choline and ethanolamine glycerophospholipid classes (1Ansell G.B. Phospholipids and the nervous system.in: Ansell G.B. Hawthorne J.N. Dawson R.M.C. Form and Function of Phospholipids. Elsevier, New York1973: 377-422Google Scholar) and are especially abundant in the heart, kidney, and central nervous system (CNS) (2Panganamala R.V. Horrocks L.A. Geer J.C. Cornwell D.G. Positions of double bonds in the monounsaturated alk-1-enyl groups form the plasmalogens of human heart and brain.Chem. Phys. Lipids. 1971; 6: 97-102Google Scholar, 3Druilhet R.E. Overturf M.L. Kirkendall W.M. Structure of neutral glycerides and phosphoglycerides of human kidney.Int. J. Biochem. 1975; 6: 893-901Google Scholar, 4Blank M.L. Snyder F. Byers L.W. Brooks B. Muirhead E.E. Antihypertensive activity of an alkyl ether analog of phosphatidylcholine.Biochem. Biophys. Res. Commum. 1979; 90: 1194-1200Google Scholar). In the human brain, plasmenyl-type ether phospholipids (plasmalogens) compose approximately 23% of total brain phospholipids (2Panganamala R.V. Horrocks L.A. Geer J.C. Cornwell D.G. Positions of double bonds in the monounsaturated alk-1-enyl groups form the plasmalogens of human heart and brain.Chem. Phys. Lipids. 1971; 6: 97-102Google Scholar) while in the rat brain and monkey spinal cord, 33% to 35% of myelin phospholipids are plasmalogens (5Horrocks L.A. Composition of myelin from peripheral and central nervous system of the squirrel monkey.J. Lipid Res. 1967; 8: 569-576Google Scholar, 6Eng L.F. Noble E.P. The maturation of rat brain myelin.Lipids. 1968; 3: 157-162Google Scholar). Although the large proportion of ether phospholipids within the central nervous system implies a structural role, evidence exists that suggests important physiological functions. For example, the mammalian brain contains anabolic acyltransferases, phosphotransferases (7Choy P.C. Skrzypczak M. Lee D. Jay F.T. Acyl-GPC and alkenyl/alkyl-GPC:acyl-CoA acyltransferases.Biochim. Biophys. Acta. 1997; 1348: 124-133Google Scholar, 8Yamashita A. Sugiura T. Waku K. Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells.J. Biochem. 1997; 122: 1-16Google Scholar), and catabolic lipases (9Gunawan J. Vierbuchen M. Debuch H. Studies on the hydrolysis of 1-alk-1′-enyl-sn-glycero-3-phosphoethanolamine by microsomes from myelinating rat brain.Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 971-978Google Scholar, 10Gunawan J. Debuch H. Alkenylhydrolase: a microsomal enzyme activity in rat brain.J. Neurochem. 1985; 44: 370-375Google Scholar, 11Van Iderstine S.C. Byers D.M. Ridgway N.D. Cook H.W. Phospholipase D hydrolysis of plasmalogen and diacyl ethanolamine phosphoglycerides by protein kinase C dependent and independent mechanisms.J. Lipid Med. Cell Signal. 1996; 15: 175-192Google Scholar, 12Farooqui A.A. Yang H.C. Rosenberger T.A. Horrocks L.A. Phospholipase A2 and its role in brain tissue.J. Neurochem. 1997; 69: 889-901Google Scholar) that are selective for plasmanyl and plasmenyltype phospholipids. A 39 kDa phospholipase A2 (PLA2) has been found in the brain that is highly selective for plasmenylethanolamine (PlsEtn) and may be involved in both plasmalogen acyl chain remodeling and arachidonate release (13Hirashima Y. Farooqui A.A. Mills J.S. Horrocks L.A. Identification and purification of calcium-independent phospholipase A2 from bovine brain cytosol.J. Neurochem. 1992; 59: 708-714Google Scholar). The brain also contains microsomal enzymes that metabolize 2-lysoplasmenylethanolamine produced by the 39 kDa PLA2, forming ethanolamine or phosphoethanolamine (9Gunawan J. Vierbuchen M. Debuch H. Studies on the hydrolysis of 1-alk-1′-enyl-sn-glycero-3-phosphoethanolamine by microsomes from myelinating rat brain.Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 971-978Google Scholar). These enzymes degrade 2-lysoplasmanylethanolamine in a similar fashion (14Vierbuchen M. Gunawan J. Debuch H. Studies on the hydrolysis of 1-alkyl-sn-glycero-3-phosphoethanolamine in subcellular fractions of rat brain.Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 1091-1097Google Scholar). Platelet activating factor, an ether phospholipid, is also synthesized in the brain and is involved in CNS function (15Feuerstein G.Z. Platelet-activating factor: a case for its role in CNS function and brain injury.J. Lipid Med. Cell Signal. 1996; 14: 109-114Google Scholar) and signal transduction (16Izumi T. Shimizu T. Platelet-activating factor receptor: gene expression and signal transduction.Biochim. Biophys. Acta. 1995; 1259: 317-333Google Scholar). Collectively, the presence of these enzymes in the brain suggests that the substrate phospholipids and their metabolites have an important biological function. In this regard PlsEtn promotes membrane fusion and vesicle formation (17Glaser P.E. Gross R.W. Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adopt an HII phase with its ability to promote membrane fusion.Biochemistry. 1994; 33: 5805-5812Google Scholar) suggesting a potential role in membrane trafficking (18Thai T.P. Rodemer C. Jauch A. Hunziker A. Moser A. Gorgas K. Just W.W. Impaired membrane traffic in defective ether lipid biosynthesis.Hum. Mol. Genet. 2001; 10: 127-136Google Scholar). Ether phospholipids are also a reservoir of arachidonate, (19Farooqui A.A. Yang H-C. Horrocks L.A. Plasmalogens, phospholipases A2 and signal transduction.Brain Res. Rev. 1995; 21: 152-161Google Scholar) and the high reactivity of the vinyl ether linkage with singlet oxygen and reactive oxygen species suggests that plasmalogens may serve as potential membrane localized antioxidants (20Zoeller R.A. Lake A.C. Nagan N. Gaposchkin D.P. Legner M.A. Lieberthal W. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether.Biochem. J. 1999; 338: 769-776Google Scholar). Ether phospholipids in the brain are synthesized in peroxisomes and microsomes (21Paltauf F. Ether lipids in biomembranes.Chem. Phys. Lipids. 1994; 74: 101-139Google Scholar, 22Lee T.C. Biosynthesis and possible biological functions of plasmalogens.Biochim. Biophys. Acta. 1998; 1394: 129-145Google Scholar) and are then transferred to synaptic membranes or to myelin (18Thai T.P. Rodemer C. Jauch A. Hunziker A. Moser A. Gorgas K. Just W.W. Impaired membrane traffic in defective ether lipid biosynthesis.Hum. Mol. Genet. 2001; 10: 127-136Google Scholar, 23Radominska-Pyrek A. Horrocks L.A. Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines by the CDP-ethanolamine: 1-radyl-2-acyl-sn-glycerol ethanolaminephosphotransferase from microsomal fraction of rat brain.J. Lipid Res. 1972; 13: 580-587Google Scholar, 24Sun G.Y. Horrocks L.A. Metabolism of palmitic acid in the subcellular fractions of mouse brain.J. Lipid Res. 1973; 14: 206-214Google Scholar, 25Radominska-Pyrek A. Strosznajder J. Dabrowiecki Z. Goracci G. Chojnacki T. Horrocks L.A. Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.J. Lipid Res. 1977; 18: 53-58Google Scholar). Added to the evidence that ether phospholipids participate in a large number of metabolic processes, such transfer suggests that they likely play an active role in signal transduction and synaptic activity. Nevertheless, measurements of decay half-lives of ether phospholipids, following their labeling by intracerebral injection of [14C]ethanolamine, [3H]glycerol, [14C]choline, and [32P]phosphate, indicate half-lives of 11 to 58 days in myelin and microsomal lipids, 30 days in whole brain, and 6 and 9.5 days in neurons and glia, respectively (26Freysz L. Bieth R. Mandel P. Kinetics of the biosynthesis of phospholipids in neurons and glial cells isolated from rat brain cortex.J. Neurochem. 1969; 16: 1417-1424Google Scholar, 27Miller S.L. Benjamins J.A. Morell P. Metabolism of glycerophospholipids of myelin and microsomes in rat brain. Reutilization of precursors.J. Biol. Chem. 1977; 252: 4025-4037Google Scholar). On the other hand, consistent with a more dynamic role, is the rapid accumulation of radioactivity into brain ether phospholipids within 1 to 4 h after intracerebral injection of these precursors. Thus, using standard tracer incorporation equations (28Zilversmit D.B. Entenman C. Fishler M.C. On the calculation of "turnover time" and "turnover rate" from experiments involving the use of labeling agents.J. Gen. Physiol. 1942; 26: 325-331Google Scholar), Masuzawa et al. (29Masuzawa Y. Sugiura T. Ishima Y. Waku K. Turnover rates of the molecular species of ethanolamine plasmalogen of rat brain.J. Neurochem. 1984; 42: 961-968Google Scholar) showed that intracerebrally injected [1-3H]-glycerol in myelinating rats gave a rate of synthesis of PlsEtn equal to 32 nmol·g−1·min−1, equivalent to a turnover rate of 5.3% per h and a half-life of 13 h. This suggests that the biosynthetic reactions involved in the incorporation of the tracer into the phospholipid fractions occur at a more rapid rate than efflux calculations demonstrate. However, the long half-lives calculated from classic efflux experiments appear excessive and likely reflect the brain's ability to recycle the tracer as well as reflecting the dilution of labeled phospholipids into metabolically inactive membranes (27Miller S.L. Benjamins J.A. Morell P. Metabolism of glycerophospholipids of myelin and microsomes in rat brain. Reutilization of precursors.J. Biol. Chem. 1977; 252: 4025-4037Google Scholar, 30Hennacy D.M. Horrocks L.A. Recent developments in the turnover of proteins and lipids in myelin and other plasma membranes in the central nervous system.Bull. Mol. Biol. Med. 1978; 3: 207-221Google Scholar, 31Rapoport S.I. Purdon D. Shetty H.U. Grange E. Smith Q. Jones C. Chang M.C. In vivo imaging of fatty acid incorporation into brain to examine signal transduction and neuroplasticity involving phospholipids.Ann. N.Y. Acad. Sci. 1997; 820: 56-74Google Scholar). Furthermore, if synthesis takes place in only a small, rapidly turning over brain phospholipid pool (21Paltauf F. Ether lipids in biomembranes.Chem. Phys. Lipids. 1994; 74: 101-139Google Scholar, 22Lee T.C. Biosynthesis and possible biological functions of plasmalogens.Biochim. Biophys. Acta. 1998; 1394: 129-145Google Scholar), this fact should be taken into account when estimating turnover and half-lives. This has not been the case in prior in vivo studies. Therefore, to assess the possibility that ether phospholipids are rapidly metabolized in the brain, rates of synthesis, turnover, and half-lives of microsomal brain ether phospholipids were measured based on known pathways of synthesis. In these experiments, [1,1-3H]hexadecanol was infused intravenously in the awake rat, and its incorporation into different brain ether phospholipid compartments was quantified as a function of time by both analytical and quantitative autoradiographic techniques. Since the enzyme catalyzing the first committed step in ether phospholipid synthesis, alkyl-DHAP synthase, is not selective toward fatty alcohols of chain lengths between C10 and C22 (32Hajra A.K. Glycerolipid biosynthesis in peroxisomes (microbodies).Prog. Lipid Res. 1995; 34: 343-364Google Scholar), [1,1-3H]hexadecanol was used as a representative substrate for this enzyme and for the subsequent reactions outlined in Fig. 1. Also, the use of [1,1-3H]hexadecanol permits measurement of only ether phospholipid synthesis because the tritium atoms are lost during the oxidation of [1,1-3H]hexadecanol to palmitate. Furthermore,there is stoichiometric retention of the label following the desaturation step involved in plasmalogen formation allowing for the quantitative measure of plasmalogen biosynthesis. This combined approach allows us to determine the distribution of the tracer following infusion, and estimate the rate at which these phospholipids turnover by calculating the flux of the tracer through the microsomal ether phospholipid pool. We report here, for the first time, synthesis rates, turnover times, and half-lives of brain microsomal ethanolamine and choline ether phospholipids in vivo, and show that in the adult rat the tracer is preferentially incorporated into gray matter regions. We also present an animal model in which ether phospholipid function in the post-myelinating rat can be assessed. [1,1-3H]hexadecanol (55 Ci ·mmol−1 ≥97% pure) was purchased from Moravek Biochemicals (Brea, CA). Phospholipid and neutral lipid standards were from Nu-Chek-Prep (Elysian, MN) and fatty alcohol standards and "essentially fatty acid free" bovine serum albumin was from Sigma Chemical Co. (St. Louis, MO). Acetic anhydride, anhydrous pyridine, and thin-layer chromotography plates were from Analtech (Deer field, IL). High performance liquid chromatography grade n-hexane and 2-propanol were from EM Science (Gibbstown, NJ). Reagent grade chloroform, methanol, and other chemicals were from Mallinckrodt (Paris, KY) unless noted otherwise. Scintillation cocktail (Beckman Ready-Safe, Fullerton, CA) containing 1% glacial acetic acid was used to determine radioactivity in all samples. Extracts were stored in n-hexane: 2-propanol (3:2, v/v) + 5.5% H2O under N2 at −20°C unless otherwise noted. Surgery was performed, as previously described (33Washizaki K. Smith Q.R. Rapoport S.I. Purdon A.D. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat.J. Neurochem. 1994; 63: 727-736Google Scholar), in accordance with NIH guidelines (NIH Publication no. 80-23). Briefly, a 3-month-old male Sprague-Dawley rat, weighing 150–280 g (Charles River, Wilmington, MA), was anesthetized with 2% to 3% halothane (Halocarbon, River Edge, NJ). Polyethylene catheters (PE 50, Becton Dickinson, Sparks, MD) filled with 100 IU sodium heparin were tied into the right femoral artery and vein. The skin was closed with clips and 1% lidocaine was applied as a local anesthetic. The animal was wrapped loosely in a fast-setting plaster body cast, taped to a wooden block, and allowed to recover from anesthesia for at least 3 h. Body temperature was maintained at 36.5°C using a rectal probe and a feedback-heating device (Yellow Springs Laboratories, Yellow Springs, OH). Using an infusion pump (Harvard Apparatus, South Natick, MA) an awake rat was infused intravenously for 5 min at a rate of 0.4 ml·min−1 with isotonic saline containing 1.75 mCi ·kg body wt−1 [1,1-3H]hexadecanol suspended in 0.06 mg bovine serum albumin. Arterial samples were collected during and following the infusion to determine the radioactivity and levels of unlabeled lipids in whole blood and plasma. At fixed times between 5 and 240 min following the start of infusion, the rat was killed by sodium pentobarbital (100 mg ·kg body wt−1, i.v.). Animals used to analyze whole brain lipids were subjected to head-focused microwave irradiation (5.5 kw, 3.4 s, Cober Electronics, Stamford, CT). All brains were immediately excised, frozen on dry ice, and stored at −80°C. To isolate different brain fractions, frozen brain was dispersed using a glass Tenbroeck homogenizer in 50 mM Tris buffer, pH 7.5, containing 0.32 M sucrose. Frozen brain has been studied previously in this manner (34Farooqui A.A. Rapoport S.I. Horrocks L.A. Membrane phospholipid alterations in Alzheimer's disease: deficiency of ethanolamine plasmalogens.Neurochem. Res. 1997; 22: 523-527Scopus (146) Google Scholar). Myelin and gray matter membranes (microsomes and synaptosomes) were separated using continuous sucrose density centrifugation as described previously (24Sun G.Y. Horrocks L.A. Metabolism of palmitic acid in the subcellular fractions of mouse brain.J. Lipid Res. 1973; 14: 206-214Google Scholar). Myelin fractions were purified by osmotic shock, resuspension, and washing with water to remove the sucrose. Fractions obtained in this way have been characterized by electron microscopy, enzymic assay, and lipid analysis (5Horrocks L.A. Composition of myelin from peripheral and central nervous system of the squirrel monkey.J. Lipid Res. 1967; 8: 569-576Google Scholar, 35Whittaker V.P. The morphology of fractions of rat forebrain synaptosomes separated on continuous sucrose density gradients.Biochem. J. 1968; 106: 412-417Google Scholar, 36Horrocks L.A. Metabolism of the ethanolamine phosphoglycerides of mouse brain myelin and microsomes.J. Neurochem. 1969; 16: 13-18Google Scholar, 37Sun G.Y. Horrocks L.A. The acyl and alk-1-enyl groups of the major phosphoglycerides from ox brain myelin and mouse brain microsomal, mitochondrial and myelin fractions.Lipids. 1970; 5: 1006-1012Google Scholar, 38Sun A.Y. Samorajski T. Effects of ethanol on the activity of adenosine triphosphatase and acetylcholinesterase in synaptosomes isolated from guinea-pig brain.J. Neurochem. 1970; 17: 1365-1372Google Scholar, 39Sun A.Y. Sun G.Y. Samorajski T. The effect of phospholipase C on the activity of adenosine triphosphatase and acetylcholinesterase in synaptic membranes isolated from the cerebral cortex of squirrel monkey.J. Neurochem. 1971; 18: 1711-1718Google Scholar). With regard to lipid analysis, the phospholipid content of each of our fractions was comparable to published values (1Ansell G.B. Phospholipids and the nervous system.in: Ansell G.B. Hawthorne J.N. Dawson R.M.C. Form and Function of Phospholipids. Elsevier, New York1973: 377-422Google Scholar, 24Sun G.Y. Horrocks L.A. Metabolism of palmitic acid in the subcellular fractions of mouse brain.J. Lipid Res. 1973; 14: 206-214Google Scholar). Regional brain radioactivity was determined at 30, 60, and 240 min following the start of infusion by quantitative autoradiography (40Noronha J.G. Bell J.M. Rapoport S.I. Quantitative brain autoradiography of [9,10-3H]palmitic acid incorporation into brain lipids.J. Neurosci. Res. 1990; 26: 196-208Google Scholar). Frozen brains were secured to mounts with embedding media and cut into 20-μm coronal sections at −20°C in a cryostat (Model OTF, Bright Instruments, Huntington, England). The sections were placed on glass cover slips and dried at 60°C to 70°C for 45 min. Dry slides were attached to boards, inserted into film cassettes beside radioactive tissue standards (Amersham, Corp.), covered with RPN-12 Hyper film™ (Amersham Corp, Arlington Heights, IL), and exposed for 20 weeks. The films were developed, fixed, and analyzed. Regional brain radioactivity was measured in sextuplet by digital quantitative densitometry using NIH Image (Version 1.55, created by Wayne Rasband, NIH). The whole brain was weighed and extracted as previously described (33Washizaki K. Smith Q.R. Rapoport S.I. Purdon A.D. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat.J. Neurochem. 1994; 63: 727-736Google Scholar). Samples of plasma, blood, and membrane fractionations were extracted using the Folch method (41Folch J. Lees M. Sloane-Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissue.J. Biol. Chem. 1957; 226: 497-509Google Scholar). The extracts were concentrated in vacuo (Savant, Hickville, NY), dissolved in n-hexane [2-propanol (3:2, v/v) + 5.5% H2O] and filtered using a 0.2 μm nylon filter. Standards and samples were applied to 20 cm × 20 cm Whatman silica gel 60A LK6 TLC plates and separated as previously described (42Jolly C.A. Hubbell T. Behnke W.D. Schroeder F. Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation.Arch. Biochem. Biophys. 1997; 341: 112-121Google Scholar). Bands corresponding to ethanolamine (EtnGpl) and choline glycerophospholipid (ChoGpl), diradyl-sn-glycerol-3-phosphate (HOGpl), and neutral lipids were scraped from TLC plates stored. EtnGpl and ChoGpl fractions were extracted from the silica gel using n-hexane [2-propanol (3:2, v/v) + 5.5% H2O] and concentrated with N2 at 40°C. Samples were incubated in 2 ml methanol containing 0.1 M of [KOH]hydroxide of potassium hydrate at 30°C for 15 min to cleave the acyl chains. At 15 min, 1 ml methyl formate (Sigma Chemical Co., St. Louis, MO) was added followed by 4 ml chloroform [n-butanol (4:2, v/v)]. Adding 2 ml 0.1 M KCl formed two phases and their separation was promoted by centrifugation at 1000 × g (43Ansell G.B. Spanner S. The alkaline hydrolysis of the ethanolamine plasmalogen of brain tissue.J. Neurochem. 1963; 10: 941-945Google Scholar). The lower solvent phase, containing the lyso-ether phospholipid analogs 1-O-alkyl-2-lyso-sn-glycero-3-phosphoethanolamine (2-lysoPakEtn) + 1-O-alk-1′-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (2-lysoPlsEtn) or 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (2-lysoPakCho) + 1-O-alk-1′-enyl-2-lyso-sn-glycero-3-phosphocholine (2-lysoPlsCho), were retained. The extracts were concentrated with N2 at 40°C and dissolved in chloroform. Aliquots of each sample were spotted onto two 10 cm × 10 cm silica gel G TLC plates (Analtech, Deerfield, IL), and the lipid classes were separated using chloroform-methanol-ammonium hydroxide (65:25:4, v/v/v). The second TLC plates were exposed to HCl fumes for 15 min before separation using the solvent system described above. Bands corresponding to 2-lysoPakEtn + 2-lysoPlsEtn, and 2-lysoPakCho + 2-lysoPlsCho (plate #1) or 2-lysoPakEtn, and 2-lysoPakCho (plate #2) were scraped from the TLC plates and transferred to acid-washed 16 mm × 120 mm test tubes. A portion of the sample was used to quantitate radioactivity, while the remainder was assayed for lipid phosphorus (44Rouser G. Siakotos A.N. Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.Lipids. 1969; 1: 85-86Google Scholar). Neutral lipids and brain HOGpl from the phospholipid separation were extracted from the silica gel using n-hexane [2-propanol (3:2, v/v) + 5.5% H2O]. Brain neutral lipids, plasma, and whole blood lipid extracts were concentrated with N2 at 40°C and dissolved in chloroform. Samples and standards were applied to 20 cm × 20 cm Analtech silica gel G TLC plates and the neutral lipids separated as previously described (45Breckenridge W.C. Kuksis A. Specific distribution of short-chain fatty acids in molecular distillates of bovine milk fat.J. Lipid Res. 1968; 9: 388-393Google Scholar). Bands corresponding to fatty alcohol (brain or plasma) or to HOGpl (brain) were transferred to separate acid washed tubes and dissolved in n-hexane [2-propanol (3:2, v/v) + 5.5% H2O]. Liquid scintillation counting quantitated radioactivity and the specific activity was determined by assaying for lipid phosphorus (44Rouser G. Siakotos A.N. Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.Lipids. 1969; 1: 85-86Google Scholar) following methanolic KOH hydrolysis of the HOGpl and separation via TLC, as described above. The fatty alcohol was quantitated following acetate derivatization by gas liquid chromatography (GLC). GLC of acetate derivatives from brain, whole blood, and plasma fatty alcohols were separated using a gas chromatograph (Model 5890, series II, Hewlett-Packard, King of Prussia, PA) equipped with a capillary column (SP 2330; 30 m × 0.32 mm i.d., Supelco, Bellefonte, PA) and a flame ionization detector. The equations that we applied to our data are based on known pathways of brain ether phospholipid biosynthesis (Fig. 1) and on evidence that ether phospholipid synthesis occurs in brain peroxisomes and microsomes (microsomal fraction) (21Paltauf F. Ether lipids in biomembranes.Chem. Phys. Lipids. 1994; 74: 101-139Google Scholar, 22Lee T.C. Biosynthesis and possible biological functions of plasmalogens.Biochim. Biophys. Acta. 1998; 1394: 129-145Google Scholar). The radioactivity (dpm ·gram−1) of hexadecanol and of the different ether phospholipids was measured in plasma and whole brain extracts of rats killed at 5, 10, 20, 30, 60, or 120 min following the start of infusion. Parenchymal brain radioactivity in each pool i, cbr,i∗ (dpm·ml−1) was then calculated by subtracting the intravascular contribution for that compound from its net brain radioactivity. Intravascular radioactivity was taken as the compound's radioactivity (dpm ·ml−1) in whole blood at the time of brain sampling and multiplied by the brain blood volume (0.023 ml·g brain−1) (46Grange E. Deutsch J. Smith Q.R. Chang M. Rapoport S.I. Purdon A.D. Specific activity of brain palmitoyl-CoA pool provides rates of incorporation of palmitate in brain phospholipids in awake rats.J. Neurochem. 1995; 65: 2290-2298Google Scholar). The radioactivity of plasma hexadecanol ( cpl,hex∗), brain hexadecanol, and ether phospholipids [ cbr,i∗(T2)] was used to calculate the integrated compartmental radioactivity from the beginning of infusion at time 0 to the time of brain sampling T2(∫0T2cbr,i∗dt)using the trapezoidal rule (SigmaPlot Windows, Version 5.0, SPSS, Chicago, IL). The unidirectional transfer coefficient ( ki−1→i∗, equation 1) for transfer of labeled hexadecanol from a substrate compartment (i – 1) to product compartment (i) was calculated as the ratio of the brain radioactivity cbr,i∗(T2) in i to the integrated radioactivity of i – 1 between T1 and T2. dcbr,i∗∕dt=ki−1→i∗cbr,i−1∗(Eq. 1) T1 and T2 were chosen to cover the period of the linear rate of change of radioactivity in i, when radioactivity essentially reflected synthesis de novo. Specific equations of the form in equation 1 can be written on the basis of the pathways outlined in Fig. 1. For PakOH, PakCho, PakEtn, PlsEtn, and PlsCho these equations are: dcbr,PakOH∗∕dt=khex→PakOH∗cbr,hex∗(Eq. 1a) dcbr,PakCho∗∕dt=kPakOH→PakCho∗cbr,PakOH∗(Eq. 1b) dcbr,PakEtn∗∕dt=kPakOH→PakEtn∗cbr,PakOH∗(Eq. 1c) dcbr,PlsEtn∗∕dt=kPakEtn→PlsEtn∗2(cbr,PakEtn∗)(Eq. 1d) dcbr,PlsCho∗∕dt=kPlsEtn→PlsCho∗cbr,PlsEtn∗(Eq. 1e) Therefore, T1 an