Effect of protein depletion on the VLDL triacylglycerol secretion and apoprotein synthesis by the perfused liver from pregnant rats

The effect of protein depletion in the pregnant rat on the polyunsaturated fatty acid incorporation into very low density lipoproteins (VLDL) has been investigated. The apoprotein pattern of these particles was determined. In in vivo experiments the amounts of serum and liver triacylglycerol were determined. VLDL were isolated and their apo C concentration calculated. In in vitro experiments the radioactivity of [³H] leucine incorporated into VLDL apoproteins was measured. The results show that protein depletion during pregnancy promotes a drastic increase in serum and liver triacylglycerol. The VLDL isolated from these animals show an increase in the triacylglycerol/protein ratio and a decrease in their content of apo C. Meanwhile, a significant reduction in the [³H]leucine incorporation into apo C peptides by the perfused liver of protein depleted rats was detected. On the other hand, protein deprivation did not affect labeled linoleic and arachidonic acid incorporation into triacylglycerol of the newly secreted VLDL. Taking these results together, let us deduce that a defective VLDL is secreted by the liver of the protein depleted pregnant rats. The abnormal composition of these particles may influence its normal metabolism through their effects on lipoprotein lipase and this fact could affect the normal supply of polyunsaturated fatty acids to the fetus.     

p-Chlorophenoxyisobutyrate enhanced retention of homologous lipoproteins by human aortic smooth muscle cells

Human aortic smooth muscle cells (SMC) specifically bind and take up indiscriminately both the lipid and protein moietics of homologous²⁵I-very low density lipoproteins (VLDL) and¹²⁵I-low density lipoproteins LDL). Sixty-five to 80% of absorbed lipids are incorporated into the cell lipids, preferentially into the phospholipid fraction. Twenty to 35% of the lipid bound and the protein moiety are eliminated from the cells. Half of the eliminated protein label is recovered as TCA soluble products. Five mM of p-chlorophenoxyisobutyrate (CPIB) raise the level of intracellular radioactivity derived from the lipid moieties of VLDL and LDL by about 40% via a reduced elimination. The processing of the protein moiety and lipoprotein binding to the cell surface are not affected by 5.0 mM of CPIB. CPIB lowers the incorporation of¹⁴C-acetate,¹⁴C-pyruvate, and³²phosphate radioactivity into fatty acids and phospholipids of aortic SMC. Five mM of CPIB reduce the overall palmitic acid synthesis by shifting from de novo synthesis to the mechanism of chain elongation, although the further elongation to saturated C₁₈–C₂₄ fatty acids is also depressed. The CPIB-enhanced retention of the lipid-derived lipoprotein radio-activity is interpreted as a compensatory mechanism providing cellular fatty acids which are deficient as a result of the CPIB inhibited synthetic processes.     

Diurnal variation of plasma methyl sterols and cholesterol in the rat: Relation to hepatic cholesterol synthesis

Free cholesterol of plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) of the rat was high and that of plasma very low density lipoproteins (VLDL) was low during the dark period of the diurnal cycle. Variations in the esterified plasma sterols were inconsistent. Free methyl sterols were high in all lipoproteins during the dark phase. Simultaneously, the incorporation of¹⁴C-acetate into nonsaponifiable sterols and the concentrations of free methyl sterols and cholesterol in the liver were elevated.     

Eicosenoic and docosenoic acid incorporation in serum lipoproteins in rats fed rapeseed oil

Rats were fed rapeseed oil rich in eicosenoic (20∶1) and docosenoic (22∶1) acids for 7 days, and the fatty acid composition of the lipid classes of serum and serum lipoproteins was determined. Concentrations of 20∶1 and 22∶1 acids in the lipid classes were variable, especially among lipoproteins, and were a direct function of the alimentary state of the animal. The results suggest differences in the incorporation of the above acids among the major lipoprotein types and various lipid classes within a given lipoprotein type. The quick partial disappearance of very low density lipoproteins (VLDL) and of low density lipoproteins (LDL) containing 20∶1 and 22∶1 acids upon starvation and the preferential incorporation of these acids in the triacylglycerols of high density lipoproteins (HDL) are discussed.     

Lipid metabolism and tissue composition in Atlantic salmon (Salmo salar L.)—Effects of capelin oil, palm oil, and oleic acid-enriched sunflower oil as dietary lipid sources

Triplicate groups of Atlantic salmon (Salmo salar L.) were fed four diets containing different oils as the sole lipid source, i.e., capelin oil, oleic acid-enriched sunflower oil, a 1∶1 (w/w) mixture of capelin oil and oleic acid-enriched sunflower oil, and palm oil (PO). The β-oxidation capacity, protein utilization, digestibility of dietary fatty acids and fatty acid composition of lipoproteins, plasma, liver, belly flap, red and white muscle were measured. Further, the lipid class and protein levels in the lipoproteins were analyzed. The different dietary fatty acid compositions did not significantly affect protein utilization or β-oxidation capacity in red muscle. The levels of total cholesterol, triacylglycerols, and protein in very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and plasma were not significantly affected by the dietary fatty acids. VLDL, LDL, and HDL fatty acid compositions were decreasingly affected by dietary fatty acid composition. Dietary fatty acid composition significantly affected both the relative fatty acid composition and the amount of fatty acids (mg fatty acid per g tissue, wet weight) in belly flap, liver, red and white muscle. Apparent digestibility of the fatty acids measured by adding yttrium oxide as inert marker, was significantly lower in fish fed the PO diet compared to the other three diets.     

Preparation of fatty acid methyl esters from lipoprotein and macrophage lipid subclasses on thin-layer plates

A simple, accurate, and fast procedure for quantitative analysis of fatty acids (FA) in simple lipid subclasses from different biological specimens is presented. Lipid extracts of isolated plasma lipoproteins (very low, low, and high density lipoproteins; VLDL, LDL, and HDL, respectively) and permanent J774 mouse macrophages were fractionated into lipid subclasses by thin-layer chromatography (TLC) on silica gel 60 plates. Bands comigrating with authentic lipid standards were scraped off under argon and subjected to direct,in situ transesterification with BF₃/MeOH in the presence of the TLC adsorbent. Fatty acid methyl esters were subsequently quantitated by capillary gas chromatography. A comparison of the FA content present in total lipid extracts and in lipid subclasses separated by TLC revealed recoveries ranging from 93 (J774 cell extracts) to 99.7% (LDL). The method described is applicable for the measurement of FA in individual lipid subclasses and was successfully applied to quantitatively analyze the FA composition of the phospholipid, triacylglycerol, and cholesteryl ester fraction derived from VLDL, LDL, and HDL. In J774 lipid extracts, the FA composition of the phospholipid-, monoacylglycerol-, diacylglycerol-, free fatty acid-, triacylglycerol-, and cholesteryl ester fraction was quantitated. In addition we have analyzed the time-dependent loss of the major HDL polyunsaturated fatty acids (18:2, 20:4) in the phospholipid and cholesteryl ester fraction during copper-dependent peroxidation of HDL. We have not encountered analytical problems concerning low FA recoveries from CE-rich lipid extracts as indicated by almost quantitative recoveries of FA in LDL, HDL, and J774 extracts.     

Stearic acid modifies very low density lipoprotein lipid composition and particle size differently from shorter-chain saturated fatty acids in cultured rat hepatocytes

Stearic acid as compared to myristate, palmitate, or oleate is poorly incorporated into triacylglycerol, a major lipid component of very low density lipoprotein (VLDL). The present study investigated the effects of these fatty acids on VLDL metabolism in cultured rat hepatocytes. All fatty acids stimulated [2-³H] glycerol incorporation into VLDL lipids and secretion of [³H]-labeled VLDL by hepatocytes. However, the rate of [³H]-labeled VLDL secretion in the presence of nonlabeled stearate (12.8±0.7 pmol/mg protein/4h) was 46, 59, and 22% of that observed for those treated with myristate, palmitate, and oleate, respectively. [1-¹⁴C]Stearate as a substrate was also less effective than other labeled fatty acids to be incorporated into VLDL lipids. Of total VLDL lipids synthesized from [1-¹⁴C] stearate, triacylglycerol accounted for 78% as compared to 88–97% of that derived from palmitate, myristate, and oleate. The amounts of apoB100 and apoB48 were the same in hepatocytes treated with or without exogenous fatty acids. Similarly, the rate of apoB synthesis from [³⁵S] methionine was not affected by exogenous fatty acids. The treatment of cells with various saturated fatty acids increased the particle size of VLDL to different extents. The largest particles of VLDL, with a mean diameter of 79.3±11.9 nm, were seen in the cells treated with stearate, followed by those treated with palmitate and myristate (45.5±9.8 and 38.6±6.8 nm, diameter, respectively). Clearly, hepatocytes treated with stearate secrete less VLDL and produce larger VLDL particles than those treated with shorter-chain saturated fatty acids.     

Acetate and mevalonate labeling studies with developingCuphea lutea seeds

Cuphea seeds contain large amounts of medium chain (C₈ to C₁₄) fatty acids, mainly as triacylglycerols. The biosynthesis of these lipids was studied in vivo by incubating developingCuphea lutea seeds with labeled acetate. Incorporation of label into triacylglycerols and into medium chain fatty acids occurred principally during the period of endogenous lipid deposition, but some label was encountered in these products even during seed dehydration. At this later stage palmitate and oleate were the dominant labeled fatty acids. During the period of rapid endogenous lipid deposition acyl lipids other than triacylglycerols were minor labeled components. The labeling patterns were consistent with the Kennedy pathway for triacylglycerol biosynthesis. The fatty acid composition of the acyl-CoA pool was similar to the total lipid fatty acid composition, but the acyl-ACP pool contained relatively more short chain acyl groups. Squalene was labeled from acetate throughout the period of seed development, but labeled sterols were not detected. Using [2-¹⁴C]mevalonic acid lactone as substrate, squalene was the principal labeled product. Small amounts of label were found in free sterols. However, in terms of mass, free sterol dominated over squalene. The possibility of two independent sites of isoprenoid biosynthesis in the developing embryo is discussed.     

The effect of elaidic acid incorporation upon the lipid composition of ehrlich ascites tumor cells and of the host’s liver

The incorporation of elaidic acid into Ehrlich ascites tumor cells (EATC) upon feeding the host an elaidic acid-rich diet has been investigated in the present study. The EATC lipids contained only one-half the concentration of elaidic acid found in the lipids of either the host livers or of livers from normal mice. On the other hand, elaidic acid incorporation into tumor cells was close to that of ascites fluid. This incorporation was mainly into phospholipids; the highest into choline phospholipids and ethanolamine phospholipids. Some changes in the EATC fatty acid composition were noted due to this incorporation. EATC phospholipids had reduced polyunsaturated fatty acids as compared with oleic acid-grown cells. The same was true with respect to ascites fluid phospholipids, but neutral lipids were not altered. Tumor development was accompanied by an increase in elaidic acid of the host’s liver. Elaidic acid incorporation into tumor cells resulted in a reduction in the amount of all major lipids in the tumor. In contrast, elaidic acid had no effect on lipid composition of livers from normal mice and-tumor bearing mice, and also had no effect upon the lipids of the ascites fluid that bathes the tumor cells. The incorporation of elaidic acid into the lipids of EATC, normal liver and host liver did not affect the relative composition of phospholipids in these tissues. The development of the tumor did result in decreases in triacylglycerols and esterified cholesterol, and increases in phospholipids and free cholesterol in the livers of host animals.     

Cholesteryl ester and triacylglycerol fatty acids in type V hyperlipidemia

The fatty acid compositions of plasma cholesteryl esters (CE) and triacylglycerols (TG) from seven healthy individuals and five patients with type V hyperlipoproteinemia were determined. Very low density (VLDL), low density (LDL) and high density lipoproteins (HDL) were isolated. The lipids were extracted from the lipoproteins and the triacylglycerols and cholesteryl esters separated for analysis. The fatty acid compositions of triacylglycerols from healthy and type V individuals were very similar. The cholesteryl esters from type V patients had increased contents of palmitic and decreased amounts of linoleic and arachidonic acids as compared to the normal individuals. The fatty acid composition of the cholesteryl esters from the high density lipoproteins had the greatest deviation. The fatty acid compositions of the triacylglycerols from the two groups were similar. However, the triacylglycerols in all lipoprotein fractions contained more palmitic and oleic and less linoleic and arachidonic acids than the cholesteryl esters.     

The differential effect of eicosapentaenoic acid and oleic acid on lipid synthesis and VLDL secretion in rabbit hepatocytes

The suppression of plasma very low density lipoprotein (VLDL) triglyceride levels by dietary fish oils rich in polyunsaturated n−3 fatty acids has been attributed to decreased hepatic VLDL secretion. To investigate the effect of n−3 fatty acids on lipid metabolism and VLDL secretion in a tissue culture system, we incubated rabbit hepatocytes with oleic acid and eicosapentaenoic acid (EPA) and examined [³H]glycerol and [¹⁴C]fatty acid incorporation into hepatocyte triglyceride and phospholipid and into media VLDL. Glycerol incorporation studies showed that EPA failed to stimulate VLDL triglyceride secretion from hepatocytes as occurred with oleic acid (P<0.05). Oleic acid preferentially enhanced hepatocyte triglyceride synthesis while EPA stimulated significantly phospholipid synthesis (P<0.01). Varying the relative concentrations of oleic acid and EPA at a constant total fatty acid concentration corroborated preferential triglyceride synthesis from oleic acid. Synthesis shifted predominantly to phospholipids with increasing concentrations of EPA and lower levels of oleic acid. Incorporation of the [¹⁴C]fatty acids (800 μM) followed similar patterns: 87% of [¹⁴C]oleic acid was incorporated into hepatocyte triglyceride and 44% of [¹⁴C]EPA was assimilated in hepatocyte phospholipid (p<0.001). Fatty acids at trace concentrations (53 nM) showed a more divergent pattern of lipid incorporation: 60% of [¹⁴C]oleic acid was incorporated into triglyceride while 91% of [¹⁴CEPA was incorporated into phospholipid (p<0.001). We conclude that in primary rabbit hepatocyte culture, which appears to be a useful model to study lipid metabolism and VLDL secretion, EPA is avidly incorporated into phospholipid while oleic acid predominantly becomes esterified in triglyceride. In addition, EPA, unlike oleic acid, fails to stimulate hepatocyte VLDL secretion. These divergent effects on hepatocyte lipid metabolism are, at least in part, likely to be responsible for fish oil induced suppression of plasma triglycerides.     

The biosynthesis of n-[2H7] fatty acids byArthrobacter globiformis from [U-2H15] octanoic acid

The principal fatty acids present whenArthrobacter globiformis is grown on a glycine medium free of normal fatty acids were found to be the C15 and C16 anteiso fatty acids; only a small amount of the normal fatty acids (C14 and C16) were present. Cells grown on the same medium but supplemented mented with 0.1 mg/ml [U-²H₁₅]octanoic acid were found to contain an increased amount of the normal fatty acids and these fatty acids were found to be labeled with 7 deuteriums. I concluded that the octanoic acid is degraded by β-oxidation in these cells to [U-²H₇] butyryl-CoA, which then competes with 2-methylbutyryl-CoA for the initiation of fatty acid biosynthesis.     

Dual-labeled technique for human lipid metabolism studies using deuterated fatty acid isomers

Two deuterated fatty acids, elaidate-d ₂ and oleate-d ₄, were fed simultaneously to a human subject as a mixture of trielaidin-d ₆ and triolein-d ₁₂. Periodically, blood samples were drawn, and red blood cells were separated from the plasma. Red blood cells and plasma lipids were fractionated and analyzed by combined gas chromatography—multiple ion mass spectroscopy. Dual deuterium-labeling allows rate and extent of fatty acid incorporation to be followed in various plasma and red cell neutral and phospholipid fractions. Maximum amount of deuterated fat varied from 4% in cholesterol ester to 64% in phosphatidyl ethanolamine. The highest levels of deuterated fat occurred in either 6-, 8-, or 12-hr samples; generally, <1% labeled fatty acids could be detected in 72-hr samples. Because the method is based on dual-labeling, differences in the relative incorporation of both fatty acid isomers can be compared directly. Differences in rates of incorporation, rates of removal, and extent of incorporation of labeled fatty acids into blood plasma can also be determined reliably. Our experimental labeling of fats with deuterium permits for the first time the metabolism of two fatty acid isomers to be compared simultaneously in human subjects. This new method should be applicable to a variety of other lipid metabolic studies. Presented at the AOCS meeting Dallas, Texas, April 27–30, 1975.     

Net lipid transfer between lipoproteins in fish-eye disease plasma supplemented with normal high density lipoproteins

Native fish-eye disease plasma, which is deficient of both high density lipoproteins (HDL) and lecithin-cholesterol acyltransferase activity (α-LCAT), processing the free cholesterol of these lipoproteins, has been supplemented with normal isolated HDL₂ or HDL₃ and incubated in vitro at 37 C. After incubation for 0,7.5 and 24 hr the very low density (VLDL) and low density (LDL) lipoproteins as well as HDL were isolated, and their contents of triglycerides, phospholipids and free, esterified and total cholesterol were quantified. The resulting net mass transfer of the different lipids revealed a functioning transfer of cholesteryl esters and all other analyzed lipids between the lipoproteins, although no de novo esterification of the HDL cholesterol by LCAT in this plasma occurred. In accordance with previous findings there was a functioning esterification process of the free cholesterol of the combined VLDL and LDL of fish-eye disease plasma. The present results make it reasonable to conclude that the lack of HDL cholesterol esterification in this disease is not a result of a deficiency of cholesteryl ester transfer or lipid transfer activities.     

Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis: II. Perfused rat liver

Livers from rats with experimental hypoproteinemia induced by aminonucleoside-nephrosis or plasmapheresis were perfused with a [¹⁴C]-labeled amino acid mixture at physiological concentration. Compared to control rats, a significantly increased incorporation of the amino acid label was found in the apolipoproteins of the ultracentrifugally separated very low and high density lipoproteins (VLDL, HDL), and into albumin secreted into the perfusate. However, no increase in the amino acid-derived label was detected in VLDL- or HDL-borne lipids in nephrosis or plasmapheresis. Perfusion with U-[¹⁴C] leucine as a lipogenesis precursor at >10 times higher than physiological concentration resulted in 5-fold increase in the label incorporation into perfusate proteins in nephrosis but only in a slightly significant increase in perfusate lipids. In contrast, the incorporation of a preformed fatty acid, 9,10-[³H] oleate into VLDL and HDL lipids increased 3- to 4-fold in nephrosis. Both with leucine and oleate as precursors, the increments in the label appearing in perfusate proteins or lipids, respectively, were markedly greater than the increases in hepatic tissue proteins or lipids. The results indicate that amino acids are preferentially directed by the liver into the synthesis of circulating apolipoproteins and albumin in hypoproteinemia and do not seem to constitute an important precursor of the lipoprotein lipids. The increased production of apolipoproteins is associated with an increased incorporation of preformed fatty acids into lipoprotein lipids in addition to the previously reported stimulation of hepatic de novo lipid synthesis from precursors other than amino acids.     

Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside: Hepatic synthesis of lipoproteins and apolipoproteins

Hepatic synthesis of lipoproteins and apolipoproteins was investigated in male Wistar rats with severe nephrotic syndrome induced by puromycin aminonucleoside by incubating liver slices with a mixture of¹⁴C-amino acids. Labeled lipoproteins were separated by preparative ultracentrifugation from the incubation medium after the addition of carrier plasma. The incorporation of¹⁴C-amino acids into very low density lipoproteins (VLDL) (1.006 g/ml), low density lipoproteins (LDL) (1.006–1.063 g/ml) and high density lipoproteins (HDL) (1.063–1.210 g/ml) was increased in nephrotic liver 6.1-, 5.7- and 5.0-fold, respectively. The measurement of radioactivity associated to apolipoproteins isolated by SDS-PAGE documented an increased incorporation into apolipoprotein E (apoE) of nephrotic VLDL (33.1% vs 20% of the total radioactivity incorporated into VLDL apoproteins) and a markedly increased incorporation into apolipoprotein A-I (apoA-I) of nephrotic HDL (44.3% vs 16.3% of the total radioactivity incorporated into HDL apoproteins). In nephrotic liver, the total incorporation of amino acids into apolipoproteins (apoVLDL+apoLDL+apoHDL) was increased 12.6 times for apoA-I, 6.4 times for apoB, 5.0 times for apoE, 4.2 times for apoC+apoA-II and 2.5 times for apoA-IV. We suggest that, in nephrotic liver: (a) the synthesis of VLDL, LDL and HDL is increased, and (b) the total synthesis of apoA-I is selectively increased when compared to that of the other apolipoproteins. Preliminary reports of this work were presented at the Annual Meeting of the European Society for the Study of the Liver (Düsseldorf, September 13–15, 1979); at the 5th International Symposium on Atherosclerosis (Houston, November 6–9, 1979) and at the Annual Meeting of the Italian Society for the Study of the Liver (Rome, December 14–15, 1979).     

Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis. I: Studies in rat in vivo

The incorporation of L-4,5-[³H]leucine into the ultracentrifugally separated apolipoproteins of very low, low, and high density lipoproteins (VLDL, LDL, HDL) and into serum albumin was found three-to four-fold higher in nephrotic than in normal rats one hour after intravenous injection. Incorporation of leucine into the circulating lipids was negligible. Increases of similar magnitude were obtained in the incorporation of simultaneously injected 1,5[¹⁴C] citrate into the lipids of VLDL, LDL, and HDL of nephrotic rats. Of the citrate carbons incorporated into serum and liver lipids, the proportion in cholesterol was higher in nephrotic rats when compared to normal rats. The incorporation of both precursors into total proteins and lipids of the liver vs. the incorporation into the lipoproteins was relatively lower in nephrotic than in control rats, indicating a preferential channeling into secretable products. The occurrence of enhanced new lipid synthesis in nephrosis was corroborated by the finding of markedly enhanced synthesis of lipoprotein-borne fatty acids and cholesterol from³H₂O. These results point out that while leucine is not an efficient in vivo precursor of lipoprotein lipids in nephrosis, de novo lipogenesis proceeds from other precursors. Similar trend of changes, though of smaller magnitude, was elicited in rats after double plasmapheresis, 18 hr apart, when measured 3 hr after the second plasma withdrawal. This indicates that the loss of circulating proteins either by direct removal or through kidney lesion stimulates the compensatory hepatic response involving excessive lipoprotein synthesis. Time-course studies showed that peak incorporation of leucine and citrate into the protein and lipid components of lipoproteins, respectively, as well as into serum albumin, occurred coincidentally 3 hr after the second plasmapheresis, suggesting an interdependence of the enhanced protein and lipid synthesis.     

Effect of nicotine on lipoprotein metabolism in rats

Nicotine, a major component of cigarette smoke, plays an important role in the development of cardiovascular disease and lung cancer in smokers. The effect of nicotine on lipoprotein metabolism was studied using rats as the experimental animal. There was a significant increase in the total cholesterol, phospholipids, and triglycerides as well as the amount of lipids associated with very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in sera of nicotine-treated rats. The incorporation of ³H labeled leucine into the apo B was found to be increased both in the medium and associated cells in the hepatocytes isolated from nicotine-treated rats indicating an increased synthesis and secretion of the apo B containing lipoproteins. This was further confirmed by the higher incorporation of ¹⁴C acetate into total and individual lipids of LDL and VLDL secreted into the medium as well as that associated with different lipids in the cell layer. The activity of lipoprotein lipase in extrahepatic tissues and plasma lecithin cholesterol acyltransferase activity were significantly lower in nicotine-treated rats. These results indicate that nicotine exerts hyperlipidemic effects particularly by increasing the synthesis and secretion of triglyceride-rich lipoproteins. Since nicotine is one of the major hazardous components present in cigarette smoke and tobacco, one can extrapolate that the deleterious effect exerted by nicotine on rats extends to cigarette smokers and those who use other forms of tobacco.     

Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

Different metabolism of saturated and unsaturated long chain plasma free fatty acids by intestinal mucosa of rats

During fat absorption, unsaturated long chain fatty acids are esterified at a higher rate than saturated fatty acids of similar chain length. This phenomenon has been attributed to differences in the binding affinity of fatty acids to a cytosolic fatty acid-binding protein. As intestinal mucosa utilizes plasma free fatty acids as well, we investigated whether long chainplasma free fatty acids of different degree of saturation are metabolized also at different rates.³H-Palmitic and¹⁴C-linoleic acid complexed to rat serum were injected rapidly into a tail vein of fasting rats. One, 2 and 4 min later there was no difference between³H and¹⁴C-radioactivity in intestinal mucosa, suggesting equal initial uptake of the two labeled fatty acids from plasma. Despite their equal uptake, the incorporation of the isotopes into ester lipids was significantly different, however: at 2 min, 53.1±3.9% of³H and 73.8±4.6% of¹⁴C were recovered in ester lipids. Phospholipids and triglycerides accounted for most of the mucosal³H and¹⁴C. At 4 min, a similar distribution of isotopes in intestinal mucosal metabolites was found. These data show that despite equal initial uptake by intestinal mucosa unsaturated long chain fatty acids taken up from plasma are esterified to a higher and oxidized to a lower extent than saturated plasma free fatty acids. Unsaturated plasma free fatty acids, therefore, may provide a more important source of fatty acids for endogenous intestinal lipoprotein lipids than saturated plasma free fatty acids. It is speculated that the fatty acid binding protein might be operative not only in the intracellular transport and metabolism of luminal fatty acids but of plasma free fatty acids as well.