The CDP-ethanolamine Pathway and Phosphatidylserine Decarboxylation Generate Different Phosphatidylethanolamine Molecular Species

In mammalian cells, phosphatidylethanolamine (PtdEtn) is mainly synthesized via the CDP-ethanolamine (Kennedy) pathway and by decarboxylation of phosphatidylserine (PtdSer). However, the extent to which these two pathways contribute to overall PtdEtn synthesis both quantitatively and qualitatively is still not clear. To assess their contributions, PtdEtn species synthesized by the two routes were labeled with pathway-specific stable isotope precursors, d3-serine and d4-ethanolamine, and analyzed by high performance liquid chromatography-mass spectrometry. The major conclusions from this study are that (i) in both McA-RH7777 and Chinese hamster ovary K1 cells, the CDP-ethanolamine pathway was favored over PtdSer decarboxylation, and (ii) both pathways for PtdEtn synthesis are able to produce all diacyl-PtdEtn species, but most of these species were preferentially made by one pathway. For example, the CDP-ethanolamine pathway preferentially synthesized phospholipids with mono- or di-unsaturated fatty acids on the sn-2 position (e.g. (16:0-18:2)PtdEtn and (18:1-18:2)PtdEtn), whereas PtdSer decarboxylation generated species with mainly polyunsaturated fatty acids on the sn-2 position (e.g. (18:0-20:4)PtdEtn and (18:0-20:5)PtdEtn in McArdle and (18: 0-20:4)PtdEtn and (18:0-22:6)PtdEtn in Chinese hamster ovary K1 cells). (iii) The main PtdEtn species newly synthesized from the Kennedy pathway in the microsomal fraction appeared to equilibrate rapidly between the endoplasmic reticulum and mitochondria. (iv) Newly synthesized PtdEtn species preferably formed in the mitochondria, which is at least in part due to the substrate specificity of the phosphatidylserine decarboxylase, seemed to be retained in this organelle. Our data suggest a potentially essential role of the PtdSer decarboxylation pathway in mitochondrial functioning. In mammalian cells, phosphatidylethanolamine (PtdEtn) is mainly synthesized via the CDP-ethanolamine (Kennedy) pathway and by decarboxylation of phosphatidylserine (PtdSer). However, the extent to which these two pathways contribute to overall PtdEtn synthesis both quantitatively and qualitatively is still not clear. To assess their contributions, PtdEtn species synthesized by the two routes were labeled with pathway-specific stable isotope precursors, d3-serine and d4-ethanolamine, and analyzed by high performance liquid chromatography-mass spectrometry. The major conclusions from this study are that (i) in both McA-RH7777 and Chinese hamster ovary K1 cells, the CDP-ethanolamine pathway was favored over PtdSer decarboxylation, and (ii) both pathways for PtdEtn synthesis are able to produce all diacyl-PtdEtn species, but most of these species were preferentially made by one pathway. For example, the CDP-ethanolamine pathway preferentially synthesized phospholipids with mono- or di-unsaturated fatty acids on the sn-2 position (e.g. (16:0-18:2)PtdEtn and (18:1-18:2)PtdEtn), whereas PtdSer decarboxylation generated species with mainly polyunsaturated fatty acids on the sn-2 position (e.g. (18:0-20:4)PtdEtn and (18:0-20:5)PtdEtn in McArdle and (18: 0-20:4)PtdEtn and (18:0-22:6)PtdEtn in Chinese hamster ovary K1 cells). (iii) The main PtdEtn species newly synthesized from the Kennedy pathway in the microsomal fraction appeared to equilibrate rapidly between the endoplasmic reticulum and mitochondria. (iv) Newly synthesized PtdEtn species preferably formed in the mitochondria, which is at least in part due to the substrate specificity of the phosphatidylserine decarboxylase, seemed to be retained in this organelle. Our data suggest a potentially essential role of the PtdSer decarboxylation pathway in mitochondrial functioning. Phosphatidylethanolamine (PtdEtn) 2The abbreviations used are: PtdEtnphosphatidylethanolamineCDP-EtnCDP-ethanolamineCDP-ChoCDP-cholineChocholineERendoplasmic reticulumEtnethanolamineMAMmitochondria-associated membranePSDphosphatidylserine decarboxylasePtdChophosphatidylcholinePtdSerphosphatidylserineSerserineCHOChinese hamster ovaryHPLChigh performance liquid chromatographyMSmass spectroscopy. is the second most abundant phospholipid subclass in mammalian cells, comprising 15–25% of total phospholipids (1Vance D.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B.V., Amsterdam, The Netherlands1996: 1-33Google Scholar). Three pathways are present for PtdEtn biosynthesis. The majority of PtdEtn is synthesized via the CDP-ethanolamine pathway and PtdSer decarboxylation, whereas the third route, calcium stimulated base-exchange, is of little significance (2Vance J.E. Vance D.E. Biochem. Cell Biol. 2004; 82: 113-128Crossref PubMed Scopus (267) Google Scholar). In the CDP-ethanolamine (CDP-Etn) pathway, ethanolamine is converted to PtdEtn by the sequential actions of ethanolamine kinase, CTP: phosphoethanolamine cytidylyltransferase and finally choline/ethanolaminephosphotransferase. Choline/ethanolaminephosphotransferase has a dual specificity, as it can use both CDP-choline (CDP-Cho) and CDP-Etn as substrates for the biosynthesis of phosphatidylcholine (PtdCho) and PtdEtn, respectively (3Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (91) Google Scholar). In addition to choline/ethanolaminephosphotransferase, a CDP-choline-specific cholinephosphotransferase is available for PtdCho biosynthesis (4Henneberry A.L. Wistow G. McMaster C.R. J. Biol. Chem. 2000; 275: 29808-29815Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). phosphatidylethanolamine CDP-ethanolamine CDP-choline choline endoplasmic reticulum ethanolamine mitochondria-associated membrane phosphatidylserine decarboxylase phosphatidylcholine phosphatidylserine serine Chinese hamster ovary high performance liquid chromatography mass spectroscopy. The PtdSer decarboxylation pathway for PtdEtn biosynthesis was first described by Borkenhagen et al. (5Borkenhagen L.F. Kennedy E.P. Fielding L. J. Biol. Chem. 1961; 236: PC28-PC30Abstract Full Text PDF Google Scholar). In this pathway PtdSer synthesized from PtdCho or PtdEtn by phosphatidylserine synthase-1 and -2, respectively (2Vance J.E. Vance D.E. Biochem. Cell Biol. 2004; 82: 113-128Crossref PubMed Scopus (267) Google Scholar, 6Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 24184-24189Abstract Full Text PDF PubMed Google Scholar, 7Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), is decarboxylated by the enzyme phosphatidylserine decarboxylase (PSD) to generate PtdEtn. To date, only one mammalian PSD has been cloned (8Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 6370-6376Abstract Full Text PDF PubMed Google Scholar), and the enzyme was shown to be located on the external aspect of the inner mitochondrial membrane (9Zborowski J. Dygas A. Wojtczak L. FEBS Lett. 1983; 157: 179-182Crossref PubMed Scopus (92) Google Scholar, 10van Golde L.M. Raben J. Batenburg J.J. Fleischer B. Zambrano F. Fleischer S. Biochim. Biophys. Acta. 1974; 360: 179-192Crossref PubMed Scopus (115) Google Scholar). Because PtdSer synthesis occurs in the ER and especially in ER-related membranes termed mitochondria-associated membranes (MAM) (2Vance J.E. Vance D.E. Biochem. Cell Biol. 2004; 82: 113-128Crossref PubMed Scopus (267) Google Scholar, 11Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar), PtdSer decarboxylation requires transport of PtdSer from its site of synthesis to the inner mitochondrial membrane, where PSD is located (12Shiao Y.J. Lupo G. Vance J.E. J. Biol. Chem. 1995; 270: 11190-11198Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 13Vance J.E. J. Biol. Chem. 1991; 266: 89-97Abstract Full Text PDF PubMed Google Scholar, 14Voelker D.R. J. Biol. Chem. 1990; 265: 14340-14346Abstract Full Text PDF PubMed Google Scholar). The relative importance of the CDP-Etn and PtdSer decarboxylation pathways to overall PtdEtn biosynthesis appears to vary depending on cell type and the availability of the substrates ethanolamine and serine, respectively. From studies in Chinese hamster ovary (15Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5790-5794Abstract Full Text PDF PubMed Google Scholar, 16Miller M.A. Kent C. J. Biol. Chem. 1986; 261: 9753-9761Abstract Full Text PDF PubMed Google Scholar) and baby hamster kidney (17Voelker D.R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2669-2673Crossref PubMed Scopus (146) Google Scholar) cells that were cultured in medium with fetal bovine serum being the sole source of ethanolamine, it was concluded that PtdSer decarboxylation was the major pathway for PtdEtn synthesis. However, observations in hamster heart (18Zelinski T.A. Choy P.C. Can. J. Biochem. 1982; 60: 817-823Crossref PubMed Scopus (46) Google Scholar) and in rat liver, hepatocytes, heart, and kidney (19Arthur G. Page L. Biochem. J. 1991; 273: 121-125Crossref PubMed Scopus (49) Google Scholar, 20Tijburg L.B. Geelen M.J. van Golde L.M. Biochem. Biophys. Res. Commun. 1989; 160: 1275-1280Crossref PubMed Scopus (44) Google Scholar) illustrated that the vast majority of PtdEtn is synthesized via the CDP-Etn pathway. A possible explanation for these opposite results is the availability of exogenous ethanolamine. All studies mentioned above employed incorporation of radioactive serine and ethanolamine into PtdEtn. Because of the presence of endogenous pools of unlabeled ethanolamine and serine, the quantitative contributions of both pathways to overall PtdEtn synthesis are still unclear. An intriguing question is why two pathways for PtdEtn biosynthesis exist, whereas only one pathway is available for the de novo synthesis of PtdCho in most mammalian cells. First, a second biosynthetic pathway could serve as a backup pathway under conditions where one of the two pathways is not able to function properly. Second, the two pathways could serve mainly to "locally" supply certain organelles with PtdEtn for maintaining specific molecular species profiles within these organelles. Finally, it is possible that the two pathways yield different molecular species profiles as was shown for the synthesis of PtdCho (CDP-Cho pathway versus PtdEtn methylation) in rat hepatocytes (21DeLong C.J. Shen Y.J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The large molecular diversity of PtdEtn and other phospholipid subclasses is dictated by the combination of different lengths, number of unsaturations, and types of linkages of the hydrocarbon chains. An ester linkage at the sn-1 position defines a diacyl molecular subspecies, whereas an ether linkage at this position defines a plasmanyl subspecies, and a plasmalogen subspecies is defined by a vinyl ether bond at the sn-1 position, as in all three subspecies the hydrocarbon chain at the sn-2 position is known to be linked to the glycerol backbone via an ester bond (22Hsu F.F. Turk J. Thukkani A.K. Messner M.C. Wildsmith K.R. Ford D.A. J. Mass Spectrom. 2003; 38: 752-763Crossref PubMed Scopus (83) Google Scholar). The development and refinement of mass spectrometry in combination with the availability of deuterated pathway-specific precursors has opened the possibility of specifically displaying the PtdEtn species synthesized via the CDP-Etn or PtdSer decarboxylation pathways. We report here that McA-RH7777 cells, when cultured at equimolar concentrations of ethanolamine and serine, prefer the CDP-Etn pathway over PtdSer decarboxylation in a ratio of ∼2:1, with the decarboxylation route having a preference for the synthesis of long chain, polyunsaturated species. Materials—Dulbecco's modified Eagle's medium, fetal bovine serum, and horse serum were from Invitrogen. d9-choline (HO(CH2)2N+ (CD3)3), l-(2,3,3-d3)-serine, and d4-ethanolamine (HOCD2CD2NH2) were from Cambridge Isotope Laboratories, Andover, MA. Tissue culture flasks were from Corning Inc., Acton, MA. Cell Culture—McArdle (McA-RH7777, ATCC CRL-1601) and Chinese hamster ovary cells (CHO-K1, ATCC CRL-9618) were cultured in Dulbecco's modified Eagle's medium supplemented with 6% fetal bovine serum and 6% horse serum and Ham's F-12 containing 10% fetal bovine serum, respectively. The cells were maintained in 80-cm2 culture flasks at 37 °C, 5% CO2, and 90% humidity. Incorporation of Deuterium-labeled Precursors into PtdEtn, PtdCho, and PtdSer—Cells were grown in "full" Dulbecco's modified Eagle's medium to 60–80% confluency in 175-cm2 culture flasks. For deuterium-label studies, cells were washed twice with phosphate-buffered saline and incubated in serine- and choline-free Dulbecco's modified Eagle's medium (supplemented with serum as mentioned earlier) for 6, 24, or 72 h in the presence of 400 μm d9-choline (d9-Cho) and either 400 μm d4-ethanolamine (d4-Etn) or d3-serine (d3-Ser). To maintain similar substrate concentrations during incubation, 400 μm unlabeled serine was supplemented to the culture medium of the d4-ethanolamine incubations and vice versa. Incubations were stopped by washing the cells three times with ice-cold phosphate-buffered saline, and cells were scraped into methanol. Total lipids were extracted, and phospholipid classes were isolated by normal-phase high performance liquid chromatography (HPLC) and analyzed as described in one of the sections below. Subcellular Fractionation of McA-RH7777 Cells—Mitochondrial and microsomal fractions were isolated from labeled McA-RH7777 cells essentially as described for rat liver by Shiao et al. (12Shiao Y.J. Lupo G. Vance J.E. J. Biol. Chem. 1995; 270: 11190-11198Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Briefly, cells were scraped into phosphate-buffered saline, pelleted, and homogenized in ice-cold isolation medium (250 mm mannitol, 5 mm HEPES (pH 7.4), 0.5 mm EGTA, and 0.1% bovine serum albumin). After removal of nuclei and cell debris, the supernatant was centrifuged at 10,000 × g for 10 min to pellet crude mitochondria. The resulting supernatant was centrifuged at 100,000 × g for 1 h to pellet microsomes. The mitochondrial pellet was further purified from MAM by hand homogenization in isolation medium and layering of the homogenate on top of Percoll medium (225 mm mannitol, 25 mm HEPES (pH 7.4), 1 mm EGTA, and 0.1% bovine serum albumin and 30% (v/v) Percoll) followed by centrifugation for 30 min at 95,000 × g. Purity of mitochondria and characterization of microsomes was assessed by assaying for succinate dehydrogenase (mitochondrial marker enzyme) and aryl esterase (ER marker) and by Western blotting for cytochrome c (mitochondrial marker) and calnexin (ER marker). Analysis of Phosphatidylethanolamine Molecular Species—Total lipids were extracted from cells and mitochondrial and microsomal fractions according to the method of Bligh and Dyer (23Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). The obtained total lipid extract was dissolved in hexane/isopropanol/acetone (82:17:1, v/v/v). Lipid classes were separated on a normal-phase HPLC column as described (24Brouwers J.F. Gadella B.M. van Golde L.M. Tielens A.G. J. Lipid Res. 1998; 39: 344-353Abstract Full Text Full Text PDF PubMed Google Scholar), and the PtdEtn fraction was collected manually from the column effluent using a flow splitter, dried under nitrogen, and stored at -20 °C till analysis. The fraction was dissolved in chloroform/methanol (1:1, v/v), and PtdEtn species were separated on two Synergi 4 μm MAX-RP 18A columns (250 × 3 mm) (Phenomenex, CA) in series as described (25Brouwers J.F. Vernooij E.A. Tielens A.G. van Golde L.M. J. Lipid Res. 1999; 40: 164-169Abstract Full Text Full Text PDF PubMed Google Scholar) with a slightly modified mobile phase of acetonitrile/methanol (2:3, v/v). Identification of molecular species was performed by on-line tandem mass spectrometry in the negative-ion mode on an API 4000 Q Trap mass spectrometer fitted with an Atmospheric Pressure Chemical Ionization source (Sciex, Ontario, Canada). Analysis of PtdEtn molecular species compositions and deuterium labeling was performed by on-line single quadrupole mass spectrometry in the negative-ion mode on an API 3000 triple stage quadrupole mass spectrometer fitted with an Atmospheric Pressure Chemical Ionization source (Sciex). Nitrogen was used as nebulizer gas and curtain gas. PtdEtn molecular species compositions were determined by extracting the (labeled) molecular ions and isotope peaks of the various species from the negative Q1 chromatogram representing total PtdEtn, which was verified by detection with a Varex MKIII evaporative light scattering detector (Alltech, Deerfield, IL) operated at 100 °C at a gas flow of 1.8 liters/min. Determination of the position of the ester linkage of fatty acids to glycerophosphoethanolamine was performed according to Brouwers et al. (25Brouwers J.F. Vernooij E.A. Tielens A.G. van Golde L.M. J. Lipid Res. 1999; 40: 164-169Abstract Full Text Full Text PDF PubMed Google Scholar). Evaporative light scattering detector data were analyzed using EZChrom software (Scientific Software, San Ramon, Canada), and mass spectrometric data were analyzed using Analyst1.4 software (Sciex). Analysis of Phosphatidylcholine Molecular Species—The PtdCho fraction of cells and mitochondrial and microsomal fractions was isolated from total lipid extracts by normal-phase HPLC as described in the previous section. The fraction was dissolved in chloroform/methanol (1:1, v/v), and PtdCho molecular species were separated on two LiChrospher 100 RP18-e columns (5 μm, 250 × 4.6 mm; Merck) in series as previously (24Brouwers J.F. Gadella B.M. van Golde L.M. Tielens A.G. J. Lipid Res. 1998; 39: 344-353Abstract Full Text Full Text PDF PubMed Google Scholar), with a slightly modified mobile phase of acetonitrile/methanol/triethylamine (25:24:1, v/v/v). Identification of molecular species was performed by on-line tandem mass spectrometry in the positive-ion mode on an API 4000 Q Trap mass spectrometer fitted with an electrospray ionization source (Sciex). Deuterium labeling of the various molecular species was determined by on-line mass spectrometry in the positive-ion mode on an API 2000 Q Trap mass spectrometer operated in enhanced (trapping) mode fitted with an electrospray ionization source (Sciex). PtdCho molecular species compositions were determined by evaporative light scattering detector detection as described above. Determination of the position of the ester linkage of fatty acids to glycerophosphocholine was performed according to Brouwers et al. (25Brouwers J.F. Vernooij E.A. Tielens A.G. van Golde L.M. J. Lipid Res. 1999; 40: 164-169Abstract Full Text Full Text PDF PubMed Google Scholar). Evaporative light scattering detector and mass spectrometric data were analyzed using software as described above. Analysis of Phosphatidylserine Molecular Species—The Ptd-Ser fraction of cells and mitochondrial and microsomal fractions was isolated from total lipid extracts by normal-phase HPLC as described above. The fraction was dissolved in chloroform/methanol (1:1, v/v), and PtdSer molecular species were separated on a Synergi 4 μm MAX-RP 18A column (250 × 3 mm) (Phenomenex, CA) with a mobile phase of acetonitrile/methanol/H2O (15:22.5:12.5, v/v/v) containing 1 μm serine and 2.5 mm ammonium acetate. Analysis and identification of molecular species was performed by on-line (tandem) mass spectrometry in the negative enhanced mass spectrometry ion mode on an API 4000 Q Trap mass spectrometer fitted with an electrospray ionization source. Ptd-Ser species compositions were determined in PtdSer fractions obtained from unlabeled cells by extracting the molecular ions and isotope peaks of various species from the negative neutral loss 87-chromatogram representing total PtdSer. Interpretation of Mass Spectra—The incorporation of d4-Etn into PtdEtn and d9-Cho into PtdCho was calculated for all molecular species by determining the intensities of the unlabeled and labeled molecular ion peak in the negative or positive Q1 mass spectrum and expressing the intensity of the labeled molecular ion as the percentage of the sum of the unlabeled and labeled molecular ion. d3-Ser incorporation into PtdEtn and PtdSer was calculated similarly from (enhanced) negative Q1 spectra. Because d3-Ser labeling also, unexpectedly, yielded significant d2-Ser labeling (see "Discussion"), this d2-Ser incorporation had to be taken into account for determination of the total d3-Ser incorporation into the various PtdEtn and PtdSer species. All PtdCho, PtdEtn, and PtdSer molecular ions display isotope peaks in Q1 mass spectra (Fig. 1, C and D), originating from the natural presence of 13C in these large biomolecules. To determine total d3-Ser incorporation into PtdEtn and PtdSer molecular species, the natural contribution of 13C to the intensities of the isotope peaks of each molecular ion was subtracted from the actual, measured intensities of the isotope peaks in the Q1 mass spectra obtained, the remaining "isotope peak" intensities (exemplified for PtdEtn in Fig. 1D) representing total d3-Ser incorporation into the various PtdEtn and PtdSer species. Measuring the Intracellular Deuterated to "Cold" Serine and Ethanolamine Ratio—Cells were labeled with 400 μm d3-Ser or d4-Etn for various times up to 6 h and washed twice with ice-cold phosphate-buffered saline before extracting the water-soluble components (23Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar). The water-methanol phase was collected and evaporated to dryness, and the primary amines Ser and Etn were subsequently derivatized with fluorescamine exactly as described (26De Bernardo S. Weigele M. Toome V. Manhart K. Leimgruber W. Bohlen P. Stein S. Udenfriend S. Arch. Biochem. Biophys. 1974; 163: 390-399Crossref PubMed Scopus (261) Google Scholar). The deuterated to cold ratio was determined using mass spectrometry. Determination of the Substrate Specificity of Phosphatidyl-serine Decarboxylase—The substrate (d3-labeled egg-PtdSer and various PtdSer molecular species) required to determine the substrate specificity of PSD by using a mass spectrometry approach was synthesized from their respective PtdCho species. Briefly, 2.5 mg of PtdCho was dissolved in 1 ml of chloroform, after which 25 mg of silica (kieselgel 60 for column chromatography) was added. The mixture was stirred for 30 min and carefully dried under a gentle stream of nitrogen. Subsequently, 250 μl of 100 mm acetate buffer (pH 5.6) containing 100 mm CaCl2, 50 mg/ml d3-serine, and 10 units of phospholipase D (Streptomycin species) was added to the silica, and the suspension was incubated for 36–48 h at 30 °C while shaking continuously. The reaction was stopped by adding 470 μl of H2O and 80 μl of 6 m HCl, and phospholipids were extracted (23Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42878) Google Scholar, 27Yamane T. Iwasaki Y. Mizumoto Y. Kasai M. Okada T. 2003Google Scholar). The amount of d3-PtdEtn formed was quantified using the phosphorus assay (28Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1319) Google Scholar). PSD activity was measured in mitochondria, prepared as described in the subcellular fractionation section, as the formation of d3-PtdEtn from d3-PtdSer (29Voelker D.R. J. Biol. Chem. 1985; 260: 14671-14676Abstract Full Text PDF PubMed Google Scholar). The reaction mixture (final volume 0.4 ml) consisted of 100 mm KH2PO4 (pH 6.8), 10 mm EDTA, 0.5 mg of Triton X-100/ml of assay mixture, 50 μm PtdSer (d3-egg PtdSer or a mixture of equal amounts (10 μm) of d3-(16:0-16:0) PtdSer, d3-(16:0-20:4) PtdSer, d3-(18:0-18:1) PtdSer, d3-(18:0-18:2) PtdSer, and d3-(18:0-20:4) PtdSer) and enzyme (∼250 μg of mitochondrial protein). Assays were carried out for 45 min at 37 °C and were terminated by adding 3.4 ml of chloroform/methanol/H2O (15:29:6, by volume) followed by lipid extraction. After removing the Triton X-100 using small silica (kieselgel 60) columns, the amount of d3-PtdSer (substrate) and d3-PtdEtn (product) was quantified as described above. Pathway-specific Monitoring of Phosphatidylethanolamine Biosynthesis in McA-RH7777 Cells—To get insight into the qualitative and quantitative contributions of the CDP-Etn pathway and PtdSer decarboxylation to overall PtdEtn synthesis in mammalian cells, McA-RH7777 (McArdle) cells were incubated in the presence of deuterated, pathway-specific precursors. After various times, total lipids were extracted and subfractionated into phospholipid subclasses, and the PtdEtn fraction was analyzed by HPLC-mass spectrometry (MS). A typical chromatogram of the HPLC separation of McArdle PtdEtn molecular species is shown in Fig. 1A, with the peak identification given in Table 1. The major advantage of on-line HPLC separation before MS is that isobaric molecular species, i.e. species having the same mass but different radyl groups in their diacylglycerol backbones, are largely separated, thus allowing individual analysis.TABLE 1Phosphatidylethanolamine molecular species composition in McA-RH7777 cells, mitochondria and microsomes The PtdEtn fraction was collected from total lipid extracts of McA-RH7777 cells, mitochondria and microsomes, and the molecular species compositions were determined as described under "Experimental Procedures." Shown in this table is the PtdEtn molecular species composition (first row) of the total cell homogenate (third row), the mitochondrial fraction (fourth row), and the microsomal fraction (fifth row). The second row indicates the chromatogram peak in which the various molecular species elute in the HPLC-MS chromatogram displayed in Fig. 1A. Data are expressed as the mean ± S.D. of three experiments, performed in triplicate.PtdEtn speciesPeak (Fig. 1A)Whole cellMitochondriaMicrosomes% of total PtdEtn (average ± S.D.)16:0-18:1718.2 ± 0.114.4 ± 1.019.5 ± 3.716:0-18:256.1 ± 0.45.7 ± 0.66.2 ± 1.616:0-20:431.7 ± 0.82.6 ± 1.00.6 ± 0.416:0-20:510.4 ± 11.4 ± 0.70.3 ± 0.316:0-22:620.8 ± 0.21.2 ± 0.21.3 ± 0.518:0-18:196.1 ± 2.05.5 ± 2.67.8 ± 1.418:0-18:2/18:1-18:1720.9 ± 1.415.4 ± 0.927.5 ± 3.618:0-20:380.5 ± 0.10.5 ± 0.20.2 ± 0.118:0-20:465.1 ± 0.512.3 ± 3.30.9 ± 0.818:0-20:548.7 ± 1.315.3 ± 3.51.6 ± 1.318:0-22:5/20:1-20:460.8 ± 0.21.3 ± 0.10.4 ± 0.318:0-22:652.1 ± 0.12.3 ± 0.11.3 ± 0.418:1-18:255.8 ± 0.34.6 ± 0.25.7 ± 1.618:1-18:320.2 ± 0.10.3 ± 0.20.2 ± 0.118:1-20:1/18:0-20:291.0 ± 0.30.5 ± 0.21.5 ± 0.218:1-20:431.7 ± 0.61.4 ± 0.51.0 ± 0.618:1-20:510.3 ± 0.11.1 ± 0.50.3 ± 0.218:1-22:620.4 ± 0.10.6 ± 0.10.5 ± 0.1Plas(16:0-18:1)80.5 ± 0.20.2 ± 0.10.8 ± 0.3Plas(16:0-22:6)31.8 ± 0.511.3 ± 0.11.9 ± 0.9 Open table in a new tab Labeling of cells with d4-Etn or d3-Ser in combination with mass spectrometric phospholipid analysis allowed us to distinguish between PtdEtn species synthesized via the CDP-Etn pathway and species formed by Ptd-Ser decarboxylation. d4-Etn-labeled PtdEtn species de novo synthesized via the CDP-Etn pathway could be easily discriminated from unlabeled PtdEtn species because of a 4-Da mass difference (Fig. 1C) between the molecular ions in the (negative) Q1 spectrum (Fig. 1B). In d3-Ser, three carbon-bound protons of serine are replaced by deuterium atoms. Once the d3-Ser label is incorporated into the head group of PtdSer species, the 3-Da mass difference between labeled PtdSer species and their non-labeled counterparts is retained upon decarboxylation to PtdEtn (Fig. 1D; the unexpected appearance of d2-PtdSer will be explained under "Discussion" and is corrected for, see "Experimental Procedures"). In addition to PtdSer decarboxylation, (labeled) serine can be incorporated into PtdEtn via two other routes; (i) it can enter the CDP-Etn pathway as (labeled) phosphoethanolamine, generated as an intermediate of sphingomyelin metabolism (30Merrill A.H. Jones Jr., D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (395) Google Scholar), and (ii) it can be incorporated into the diacylglycerol moiety (31Steenbergen R. Nanowski T.S. Nelson R. Young S.G. Vance J.E. Biochim. Biophys. Acta. 2006; 1761: 313-323Crossref PubMed Scopus (34) Google Scholar). However, experiments with β-chloro-l-alanine, a potent inhibitor of sphingomyelin synthesis (32Medlock K.A. Merrill Jr., A.H. Biochemistry. 1988; 27: 7079-7084Crossref PubMed Scopus (69) Google Scholar), revealed that phosphoethanolamine liberated from sphingomyelin breakdown only marginally (<2%) contributed to serine labeling of PtdEtn in our experimental system (data not shown). Furthermore, only a slight amount of deuterium label was detected in the diacylglycerol moiety of PtdEtn and PtdCho in McArdle cells labeled with d3-Ser for 24 h, which was clear from the fact that less than 4% of the PtdCho molecules were detected with a mass up to 3 mass units heavier than the parental molecular species. Therefore, it was concluded that in McArdle cells deuterated Ser-labeled PtdEtn species were derived from PtdSer decarboxylation (>95%). Qualitative and Quantitative Contributions of the CDP-ethanolamine Pathway and PtdSer Decarboxylation to Overall Phosphatidylethanolamine Synthesis—We first determined a suitable labeling time to study the contribution of the various pathways to phospholipid biosynthesis by labeling McArdle cells with 400 μm d9-Cho and 400 μm d4-Etn for 6, 24, and 72 h. Because McArdle cells have only one route for de novo PtdCho synthesis, namely the CDP-Cho pathway, it was expected that all PtdCho species would be labeled to the same extent when remodeling was completed. Fig. 2A shows that already after 6 h 24% of PtdCho mass in the cells is de novo-synthesized, but n