Abstract
In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g. 16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g. 18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver.
Highlights
Substrate and is catalyzed by three enzymes: choline kinase, CTP:phosphocholine cytidylyltransferase (CT), and cholinephosphate transferase, with CT as the rate-limiting enzyme [1]
Hepatocytes are unique because they possess a high activity of phosphatidylethanolamine methyltransferase (PEMT) that converts PE to PC via three sequential steps of methylation [4] in addition to a high level of CDP-choline pathway activity
The PC synthesized via this methylation pathway does not substitute for the role of PC synthesized from the CDP-choline pathway [12]
Summary
Materials—D4-ethanolamine and D9-choline chloride were from Isotec, Inc. [3H]ethanolamine was from American Radiolabeled Chemicals, Inc. [3H]ethanolamine was from American Radiolabeled Chemicals, Inc. Dulbecco’s modified essential medium (MEM) and fetal bovine serum were from Life Technologies, Inc. Silica gel H plates were from Analtech, Inc. Phospholipid standards were from Avanti Polar Lipids. Incorporation of Tritium-labeled Precursors into PC—3.5 ϫ 105 cells were labeled with 1 Ci/ml each, [3H]choline chloride, or [3H]ethanolamine HCl for 24 h in low choline medium (choline-free MEM ϩ 2% fetal bovine serum). The lipid extracts were separated on silica gel H plates in a solvent system of 65/35/8 chloroform/methanol/ammonium hydroxide. Cells were scraped into methanol and lipids were extracted. Lipid extract equaling 500 pmol total lipid phosphorus in 100 l of 2:1 methylene chloride:methanol was analyzed on a Micromass Quattro II triple quadruple mass spectrometer (Micromass, United Kingdom) in a solvent system of 45/ 45/10 methylene chloride/methanol/H2O. PEMT protein was displayed by a reaction with Supersignal Chemiluminescent Substrate (Pierce) and exposure to x-ray films
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