Contribution of de novo fatty acid synthesis to very low density lipoprotein triacylglycerols: evidence from mass isotopomer distribution analysis of fatty acids synthesized from [2H6]ethanol.

A detailed comparison of the structures of plasma very low density lipoprotein (VLDL) and liver triacylglycerols (TG) (Yang et al. 1995. J. Lipid Res. 36: 125-136) has demonstrated that a minimum of 60% of the secreted TG could have been derived from partial lipolysis and reesterification of stored TG and a maximum of 40% could have been derived from direct secretion of newly made TG. To investigate the processes involved in the transfer of TG to VLDL in vivo, we have determined the distribution of deuterium among the molecular species of the liver-TG and VLDL-TG during the infusion of perdeuterated ethanol along with fructose or glucose and during the provision of either glucose or fructose in the drinking water for 2 weeks. The deuterium labeling (percent excess and percent replacement) of the total fatty acids was determined by GC/MS of the methyl esters while the labeling of the glycerol and the glycerol plus fatty acids of the enantiomeric diacylglycerol moieties of TG was determined by LC/MS with on-line mass spectrometry. Supplementation of the diet for 2 weeks with either glucose and fructose stimulated the synthesis of TG containing new fatty acids and glycerol. The proportion of the newly made to preexisting TG differed in VLDL from that in the liver. The 2H % replacement in glycerol and in total fatty acids was greater in VLDL-TG than in the liver-TG. On the basis of the mass isotopomer distribution analysis it was estimated that a maximum of 30% of the VLDL-TG could have been derived directly from TG that was made de novo and did not equilibrate with the liver-TG stores. The transfer of the stored TG to VLDL was best accounted for by a degradation to 2-monoacylglycerols and resynthesis via the 2-monoacylglycerol pathway with addition of an excess of newly synthesized fatty acids to the resynthesized TG.

Complementary chromatographic analysis of free diacylglycerols and potential glycerophospholipid precursors in human SH-SY5Y neuroblastoma cells following incubation with lithium chloride.

We performed detailed chromatographic analyses on the molecular species of the major glycerophospholipids (GPLs) and free sn-1,2-diacylglycerols (DAGs) from SH-SY5Y human neuroblastoma cells following incubation with or without LiCl. For this comparison the inositol, choline, ethanolamine and serine GPLs were dephosphorylated with phospholipase C and the released sn-1,2-diacylglycerols along with the DAGs were subjected to high-temperature GLC on polar and non-polar capillary columns as their trimethylsilyl and tert.-butyl-dimethylsilyl ethers. A 30-min incubation with 10 mM LiCl increased the total amount of human neuroblastoma DAGs by 32-58% (P < 0.05) to 2.6 pmol/micrograms cell protein. This was accompanied by a limited qualitative shift in the molecular species pattern, the most obvious of which was the increase (13%) in the major saturated-polyunsaturated molecular species and the ca. 46% increase in the minor 18:1-18:1 species over control levels. The DAGs originated mainly from the inositol GPLs (IGPLs), as indicated by the high levels of the characteristic 18:0-20:4n6 (18:0-20:3n9) species in both IGPLs and DAGs, and to a lesser extent from the choline GPLs (CGPLs), as indicated by the high proportion in CGPLs of the oligoenoic species, which were largely absent from IGPLs. Alkenylacylglycerols were not detected in DAGs, although they made up some 60% of the total ethanolamine GPLs (EGPLs). No significant changes in the molecular species composition of the cellular GPLs, including IGPLs, were detected after exposure to LiCl.

Surface components of chylomicrons from rats fed glyceryl or alkyl esters of fatty acids: minor components.

The lipid class, fatty acid and molecular species composition of the minor polar surface components of rat lymph chylomicrons were determined during absorption of menhaden oil and corn oil or of the corresponding fatty acid ethyl esters. In addition to the previously reported minor polar lipids (sphingomyelin, phosphatidylserine, phosphatidylinositol, phosphatidic acid and lysophosphatidylcholine), we identified phosphatidylglycerol, dimethylphosphatidylethanolamine, ceramide and cholesteryl sulfate in the chylomicrons from both oil and ester feeding. The dietary fatty acids were found to be incorporated to a variable extent into the different phospholipid classes, the proportions of which remained the same during both types of feeding. No evidence was obtained for the presence of the minor glycerophospholipids characteristic of the lysosomal membranes (e.g., bis-phosphatidic, lysobisphosphatidic and semilysobis-phosphatidic acids), although special efforts were made to identify them. These results indicate that the chylomicrons arising from the monoacylglycerol and phosphatidic acid pathways of triacylglycerol biosynthesis become enveloped in closely similar monolayers of phospholipids. Hence, all triacylglycerols may be secreted from the villus cells via a common mechanism as suggested by the previously demonstrated convergence (at the 2-monoacylglycerol stage) of the monoacylglycerol and the phosphatidic acid pathways of mucosal triacylglycerol formation [Yang, Y.L., and Kuksis, A. (1991) J. Lipid Res. 32, 1173-1186].

Similarities in surface lipids of chylomicrons from glyceryl and alkyl ester feeding: major components.

This study tests the hypothesis that the rat chylomicrons are assembled and released into lymph similarly regardless of the site (rough or smooth endoplasmic reticulum) or pathway (phosphatidic acid or monoacylglycerol) of triacylglycerol biosynthesis. For this purpose we determined the lipid class, fatty acid and molecular species composition of the choline, ethanolamine, inositol and serine phospholipids of lymph chylomicrons during absorption of menhaden, mustard-seed and corn oil (monoacylglycerol pathway) or the corresponding fatty acid methyl or ethyl esters (phosphatidic acid pathway). The dietary fatty acids were found to be incorporated to various extents into different phospholipid classes, the proportions of which were not affected by the nature of the dietary fat. The chylomicron phospholipids contained 80-82% choline, 8% ethanolamine and 2.5% inositol glycerophospholipids, and much smaller amounts of serine and other minor phospholipids. Administration of a meal of each dietary fat resulted in a retention of approximately 50% endogenous fatty acids in the major glycerophospholipids of the chylomicrons. A minimum of 50% of the molecular species of the choline and ethanolamine glycerophospholipids contained at least one exogenous fatty acid. No significant discrepancies were found in the fatty acid and molecular species composition of the glycerophospholipids between chylomicrons from the oil and corresponding ester feeding. It is concluded that the chylomicrons arising from the monoacylglycerol (oil feeding) and the phosphatidic acid (ester feeding) pathways of triacylglycerol biosynthesis become enveloped in surfactant monolayers containing qualitatively and quantitatively identical classes and molecular species of phospholipids.

Intestinal absorption of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat.

The relative cellular uptake and incorporation into prechylomicrons and chylomicrons was investigated for the menhaden and rapeseed oil fatty acids, when given by stomach tube as the original oils or the corresponding methyl and ethyl esters. The intermediates and final products of cellular acylation were determined by chromatographic methods at various times over a period of 1-24 h. There was little selectivity in the uptake among the oligo- and poly-unsaturated fatty acids of menhaden oil, when either oil or esters were fed. In contrast, the long-chain saturated and monounsaturated fatty acids of rapeseed oil were discriminated against during both cellular uptake and reacylation (60% overall reduction in utilization). Also, there was detectable discrimination against the long-chain polyunsaturated monoacylglycerols of menhaden oil and against the long-chain saturated and monounsatured monoacylglycerols of rapeseed oil during both cellular uptake and reacylation (30% overall reduction in utilization). Evidence was obtained for an indiscriminate cellular uptake of variable amounts (4-22%) of intact dietary methyl and ethyl esters of fatty acids, which, however, appeared in the chylomicrons only to a very limited extent (0.1-1.0% of total lipid). During peak absorption the cellular and lymphatic appearance of fatty acids from the digestion and absorption of the alkyl esters was nearly 50% lower than that from the corresponding triacylglycerols. The slower absorption of the fatty acids from the alkyl ester feeding is hypothetically attributed to a lower efficiency of the phosphatidic acid pathway, which is required in the absence of dietary 2-monoacylglycerols, but other mechanisms cannot be excluded.

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat.

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.

Facile gas-liquid chromatographic resolution of saturated and unsaturated sterols using a polar capillary column.

The 5 alpha-stanol-3 beta-ol analogs of cholesterol, campesterol and beta-sitosterol along with many other sterol types were resolved using a Supelcowax 10 (Bonded Carbowax PEG 20M) fused silica capillary column (15 m x 0.25 mm). Unlike non-polar capillary columns, the semi-polar Carbowax liquid phase retains the unsaturated sterols longer than the corresponding saturated sterols, allowing an orderly elution and quantitation. The unnatural epicholestanol (5 alpha-cholestan-3 alpha-ol) was used as internal standard. By this method the stanol content of human plasma and of the unsaponifiable matter of dietary fats and oils is readily determined. Because of low column bleed the Supelcowax 10 liquid phase can be used for combined GC/MS analysis.

Stereochemical course of intestinal absorption and transport of mustard-seed oil triacylglycerols in the rat.

Male rats with thoracic duct cannulae were intubated with mustard-seed oil or the corresponding fatty acid methyl esters and the lymph was collected over 0-24 h. The chylomicron and very low density lipoprotein fractions were obtained by conventional ultracentrifugation. The triacylglycerols and glycerophospholipids were isolated and the positional distribution and molecular association of fatty acids were determined by stereospecific and chromatographic methods. The oleic, linoleic, and linolenic acids were recovered in the lymph in the proportion in which they occurred in the fat fed, while eicosenoic, erucic, and lignoceric acids were rejected to about the same extent by the two pathways of intestinal triacylglycerol biosynthesis. It is shown that the lymph triacylglycerols arising via the monoacylglycerol or the phosphatidic acid pathway possess structures that are closely similar to each other and to that of the original mustard-seed oil. It is proposed that this is a result of comparable fatty acid and positional specificity of the acyltransferases associated with the acylglycerol synthesis in the animal and plant tissues and the wide range of fatty acid chain lengths in the mustard-seed oil.

Effect of saturated and unsaturated fat diets on molecular species of phosphatidylcholine and sphingomyelin of human plasma lipoproteins.

Four healthy 21-23-year-old males with normal lipoprotein patterns and plasma lipid concentrations were subjected voluntarily to two diets of 5 weeks duration each: I, highly saturated fat diet; II, highly polyunsaturated fat diet. The VLDL, LDL and HDL3 fractions were isolated by conventional ultracentrifugation from each subject on the high fat diets and the molecular species of the component phosphatidylcholines and sphingomyelins were identified and quantitated by GC-MS of the t-butyldimethylsilyl ethers of the corresponding diacylglycerols and ceramides. It was shown that the diet markedly and rather evenly affected the molecular species of the phosphatidylcholines of all lipoprotein classes. However, the changes in the corresponding major molecular species were reciprocal in nature and were consistent with a demonstrated relative resistance to alterations in surface fluidity. In contrast, the dietary fat had only a minor effect on the composition of the sphingomyelins, and did not alter the characteristic differential distribution of the molecular species among the low and high density lipoprotein classes. These results, which were free of the uncertainties introduced by analyses of derived fatty acid and which were obtained on samples isolated from the same subjects, clearly demonstrate that a complete equilibration of the molecular species of the phospholipids is not attained amont the plasma lipoprotein classes even in the fasting state. The possible physico-chemical and metabolic basis of these observations is briefly discussed.

Comparative determination of plasma phospholipids by automated gas--liquid chromatographic and manual colorimetric phosphorus methods.

Plasma samples obtained during a prevalence study of hyperlipemia in a free-living urban population were analyzed for phosphatidylcholine, sphingomyelin and lysophosphatidylcholine content by automated high-temperature gas--liquid chromatographic (GLC) and manual colorimetric phosphorus (thin-layer chromatographic, TLC) methods. The GLC estimates were obtained from a quantitative analysis of the diacylglycerol, ceramide and monoacylglycerol moieties released from the parent phospholipids by digestion with phospholipase C, while the TLC estimates were derived by manual colorimetric phosphorus analyses of the individual phospholipid classes resolved by TLC. On samples analyzed over a two-year period the methods gave excellent correlation for the total phospholipids (r = 0.98), phosphatidylcholine (r = 0.98) and sphingomyelin (r = 0.90), but resulted in a poor agreement for lysophosphatidylcholine (r = 0.69). Comparable results were obtained for estimates of these phospholipids in plasma very low density, low density and high density lipoproteins. The between-method coefficient of variation ranged from 3 to 5% for phosphatidylcholine and from 5 to 10% for sphingomyelin. The relative error for the estimates of lysophosphatidylcholine ranged from 10 to 25%, and was due to the inclusion in the GLC estimates of a variable proportion of plasma free monoacylglycerols. Other differences between the two methods are due to various analytical errors and biases inherent in the two techniques. The within-day, within GLC, relative error averaged 1% for phosphatidylcholine, 3% for sphingomyelin and 5% for lysophosphatidylcholine. The apparent high precision and accuracy of the GLC method recommend it as an alternative to conventional direct methods of phospholipid analyses based on TLC isolation of lipid classes and colorimetric measurements of their phosphorus content. The GLC analyses of the plasma phospholipids are particularly convenient in conjunction with GLC measurements of plasma cholesterol and triacylglycerols, where a smaller throughput of samples is not a limitation and where both total amount and relative proportion of the lipids are of interest.

Acylglycerol structure of mustard seed oil and of cardiac lipids of rats during dietary lipidosis.

Stereospecific degradation and combined gas chromatographic--mass spectrometric (gc/ms) analysis were employed in a detailed investigation of the triacylglycerol structure of mustard seed oil and of the triacylglycerols transiently accumulating in the hearts of young rats receiving the oil in their diet. It was shown that feeding of mustard seed oil at 40% of the daily caloric requirement resulted in a deposition of cardiac triacylglycerols containing a high proportion of enantiomers of a positional distribution and molecular association of fatty acids which were closely similar to those found in the dietary oil. Complete structures were derived for a total of 88 species representing 75 to 85% of the triacylglycerols. About 90% of the accumulated triacylglycerol contained at least one long-chain (C20--C22) monounsaturated fatty acid per molecule. The long-chain acids were confined mainly to the primary positions and preferentially to the sn-3-position of the glycerol molecule. The dietary lipidosis, is, therefore, accompanied by little or no accumulation of the normal rat tissue triacylglycerols containing C16 and C18 fatty acids. It is suggested that the deposition and eventual clearance of the enantiomeric long-chain triacylglycerols in the rat heart during mustard seed oil feeding may be largely a result of a gradual change in specificity of the cardiac lipases.

Improved resolution of natural diacylglycerols by gas-liquid chromatography on polar siloxanes.

A new cyanoalkylphenylsiloxane (SILAR 5CP) liquid phase is shown to possess sufficient polarity to permit improved GC separations of natural diacylglycerols based on unsaturation and positional placement of fatty acids as well as on molecular weight, which was previously possible only on ethylene glycol succinate polyesters. Unlike the polyesters, the polar siloxane polymer has moderate thermal stability and provides GC columns which can be used for several months without replacing the packing. The GC analyses were made with conventional columns containing 3% SILAR 5CP on Gas Chrom Q at 270 degrees C isothermally. The diacylglycerols were chromatographed as the TMS ethers. Excellent seperations were obtained for the 1,2(2,3)- and 1,3-diacyglycerols derived from corn, linseed, peanut and cod liver oils and for the 1,2-diacyl-sn-glycerols from hepatic glycerophospholipids.