The activities of the proposed triacylglycerol synthetase complex, acyl-CoA ligase, acyl-CoA acyltransferase (AAT), monoacylglycerol acyltransferase (MGAT), and diacylglycerol acyltransferase (DGAT), coeluted upon Cibacron blue 3GA-agarose affinity chromatography of detergent-solubilized rat intestinal microsomes. The AAT activity is associated with a 54-kDa protein, that binds covalently an acyl group from acyl-CoA via a thiol ester linkage (Lehner, R. and Kuksis, A. (1993) J. Biol. Chem. 268, 24726-24733). Reagents that prevent the acyl-AAT formation inhibit triacylglycerol synthesis as does the removal of AAT from the complex by immunoprecipitation. In the absence of mono- and diacylglycerol acceptors, the acyl group is transferred to water. It is proposed that triacylglycerol synthesis proceeds via a sequential transfer of acyl groups from acyl-CoA ligase to the AAT, from which they are passed to the mono- and diacylglycerol acyltransferases for incorporation into the di- and triacylglycerols depending on the availability of the acyl acceptors.
Glycosylation is a major pathway for posttranslational modification of tissue protein and begins with nonenzymatic addition of carbohydrate to the primary amino groups. Excessive glycation of tissue protein has been implicated in the pathogenesis of diabetes and ageing. While glycation of aminophospholipids has also been postulated, glycated aminophospholipids have not been isolated. Using normal phase HPLC with on-line electrospray mass spectrometry we found glycated ethanolamine phospholipids to make up 10-16% of the total phosphatidylethanolamine (PE) of the red blood cells and plasma of the diabetic subjects. The corresponding values for glycated PE of control subjects were 1-2%.Key words: Glucosylated aminophospholipid; Glucose; Phosphatidylethanolamine; Phosphatidylserine; Electrospray; Thin-layer chromatography; Liquid chromatography, mass spectrometry; Normal phase HPLC bovine brain phosphatidylinositol (PI) and sphingomyelin (SPH) were obtained from Sigma Chemical Co., St. Louis, MO. All chemicals were of reagent grade quality, while the solvents were of chromatographic purity and were obtained from local suppliers. The purity of the reference compounds was ascertained by thin-layer chromatography (TLC) [3,7].
Isolation of phospholipids from bloodBlood was obtained from six diabetic patients and six non-diabetic donors. The diabetics were selected for elevated blood glucose levels indicated by their content of glycosylated hemoglobin (9-15%). EDTA blood was centrifuged (2300xg for 10 min) in a swinging bucket rotor to separate the plasma from the red cells. The cells were washed three times with five volumes of phosphate buffered saline (150 mM NaC1, 50 mM sodium phosphate, pH 8.0) and centrifuged (2300xg for 10 min). The red blood cell phospholipids were extracted according to Rose and Oaklander [8]. The plasma phospholipids were extracted with chloroform-methanol 2:1 modified from Folch et al. [9]. Glucosylated PE could be stored at -20°C in neutral chloroform methanol for several days without decomposition. The Schiff base dissociated in dilute acetic acid.
In addition to diacyl glycerophosphocholine and sphingomyelin, human plasma also contains small amounts of other glycerophospholipids, which may have special metabolic function. The structure and origin of these minor plasma lipids has not been determined. Knowledge of the detailed composition of the phospholipids of red blood cells (Myher et al., Lipids 24, 1989) permits evaluation of one of the possible sources. This study reports the detailed analyses of plasma glycerophospholipids made in parallel to those of the erythrocyte lipids obtained from the same blood using HPLC and GLC methods. The proportions of the major phospholipid classes in the plasma and erythrocytes were similar to published values, including the essential absence of diradyl glycerophosphoserine from plasma. Plasma diradyl glycerophosphocholine contained 93.0% diacyl, 3.4% alkylkacyl and 3.6% alkenylacyl, whereas the diradyl glycerophosphoethanolamine consisted of 71.8% alkenylacyl, 19.9% diacyl and 8.3% alkylacyl subclasses. The diradyl glycerophosphoinositol was 100% diacyl. The content of the minor subclasses of plasma diradyl glycerophosphocholine is similar to that of the red cells, but the ether content of the diradyl glycerophosphoethanolamine is higher in plasma than in cells. The lipid ether subclasses of plasma glycerophospholipids also contained a higher proportion of the C20, C22 and C24 alkyl and alkenyl chains than those of the cells. Furthermore, the C16 and C18-containing species in diradyl glycerophosphoethanolamine subclasses varied with the nature of the polyunsaturated acid, whereas in diradyl glycerophosphocholine subclasses the polyunsaturated acids were combined with the C16 and C18 acids in equal proportions. The significant differences in the molecular species of glycerophospholipids and sphingomyelin between plasma and red cells would appear to limit any direct transfer or equilibration of their lipid components.
High density lipoprotein (HDL) is rich in polyunsaturated phospholipids that are sensitive to oxidation. However, the effect of apolipoprotein A-I and paraoxonase-1 (PON-1) on phosphatidylcholine oxidation products has not been identified. We subjected native HDL, trypsinized HDL, and HDL lipid suspensions to oxidation by the peroxynitrite donor, 3-morpholinosydnonimine. HDL had a basal level of phosphatidylcholine mono-and di-hydroperoxides that increased to a greater extent in HDL, compared with either trypsinized HDL or HDL lipid alone. Phosphatidylcholine core aldehydes, which were present in small amounts, increased 10-fold during oxidation of native HDL, compared with trypsinized HDL (p ؍ 0.004), and 4-fold compared with HDL lipid suspensions (p ؍ 0.0021). In addition, the content of lysophosphatidylcholine increased 300% during oxidation of native HDL, but only 80 and 25%, respectively, during oxidation of trypsinized HDL and HDL lipid suspensions. Phosphatidylcholine isoprostanes accumulated in comparable amounts during the oxidation of all three preparations. Incubation of apolipoprotein A-I with 1-palmitoyl-2-linoleoyl glycerophosphocholine proteoliposomes in the presence of 3-morpholinosydnonimine or apoAI with phosphatidylcholine hydroperoxides resulted in a significant increase in phosphatidylcholine core aldehydes with no formation of lysophosphatidylcholine. We propose that apolipoprotein A-I catalyzes a one-electron oxidation of alkoxyl radicals. Purified PON-1 hydrolyzed phosphatidylcholine core aldehydes to lysophosphatidylcholine. We conclude that, upon HDL oxidation with peroxynitrite, apolipoprotein AI increases the formation of phosphatidylcholine core aldehydes that are subsequently hydrolyzed by PON1.
Selected elution factors were determined for model oxotriacylglycerols as an aid in identification of the peroxidation products of natural triacylglycerols by reverse-phase high-performance liquid chromatography (HPLC) with electrospray mass spectrometry (LC/ES/MS). For this purpose synthetic triacylglycerols of known structure were converted to hydroperoxides, hydroxides, epoxides, and core aldehydes and their dinitrophenylhydrazones by published procedures. The oxotriacylglycerols were resolved by normal-phase thin-layer chromatography and reverse-phase HPLC, and the identities of the oxotriacylglycerols confirmed by LC/ES/MS. Elution factors of oxotriacylglycerols were determined in relation to a homologous series of saturated triacylglycerols, ranging from 24 to 54 acyl carbons, and analyzed by reverse-phase HPLC, using a gradient of 20-80% isopropanol in methanol as eluting solvent and an evaporative light-scattering detector. It was shown that the elution times varied with the nature of the functional group and its regiolocation in the triacylglycerol molecule. A total of 31 incremental elution factors were calculated from chromatography of 33 oxygenated and nonoxygenated triacylglycerol species, ranging in carbon number from 36 to 54 and in double-bond number from 0 to 6.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.