Core modification of human low-density lipoprotein by artificial triacylglycerol emulsion

To determine whether an apolipoprotein-free artificial triacylglycerol emulsion can substitute for VLDL in studying cholesterol ester-triacylglycerol exchange processes between triacylglycerol-rich lipoproteins and cholesterol ester-rich lipoproteins, we used Intralipid to modify human plasma LDL. Intralipid was incubated with LDL in the presence of lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. Intralipid served as an acceptor for cholesterol ester and as a donor of triacylglycerol, modifying the low-density lipoproteins so that triacylglycerol became the major core lipid in the particle - the contribution of cholesterol ester to LDL mass decreased from 38% to 18%, while that of triacylglycerol increased from 4.9% to 26%. On lipolysis most added LDL triacylglycerol (59-72%) was hydrolyzed, resulting in a smaller particle than the "native' LDL particle with net loss of cholesterol ester. Incubation of LDL with the original Intralipid emulsion resulted in modified LDL with a high relative weight of phospholipid (27.7%). On removal of excess phospholipid from Intralipid and incubation of the resultant "washed' Intralipid with LDL, the relative weight of phospholipid in modified LDL decreased to 20%, which was similar to that observed after incubation of LDL with VLDL. We demonstrate that artificial triacylglycerol emulsion can indeed substitute for VLDL in neutral lipid exchange processes, and further confirm that transfer of core cholesterol ester and triacylglycerol occurs independently of the apolipoproteins present in triacylglycerol-rich lipoproteins and LDL.     

Alteration by Diacylglycerols of the Transport and Fatty Acid Composition of Lymph Chylomicrons in Rats

Lymph fistula rats were continuously infused with emulsions containing diacylglycerol consisting of 1,3-species (65.6%), 1(or 3),2-species (32.6%), and triacylglycerol (rapeseed oil) at the rate of 3ml/h for 1 h through a cannula inserted into the stomach. The lymph fluids were collected every hour for 5 h after starting the infusion of the lipid emulsions, and the lymph chylomicrons were isolated, purified and analyzed. Test emulsions were prepared to provide the same amount of fatty acids (144mg/h) as that in these acylglycerols. The rates for triacylglycerol transport at 2–3 h and for cholesterol transport by chylomicrons at 2–3 h and 3–4 h of the experimental period in rats infused with the diacylglycerol emulsion were significantly lower than the corresponding values in the rats infused with the triacylglycerol emulsion. As a consequence, the cumulative value for triacylglycerol transport at the end of the experimental period in rats infused with the diacylglycerol emulsion was significantly lower than the corresponding value in the rats infused with the triacylglycerol emulsion. In addition, cumulative values for cholesterol transport from 3h to the end of the experimental period were significantly lower in the former than in the latter. There was no difference in the total fatty acid compositon of chylomicron-triacylglycerol between the rats receiving the triacylglycerol and diacylglycerol emulsions. However, a considerable difference existed in the fatty acid composition at the 2-position of triacylglycerol between the two groups of rats. Thus, intragastric infusion of diacylglycerol mainly consisting of the 1,3-species compared to triacylglycerol not only altered the rate of lipid transport by lymph chylomicrons but also altered the structure of the triacylglycerol moiety in the rats.     

Physico-chemical characterization of Intralipid emulsions.

Fat emulsions containing soy triacylglycerols (100-300 g/l) and egg-yolk phospholipids (12 g/l) are often used for intravenous feeding. Previous studies have shown that these emulsions contain chylomicron-like emulsion particles of diameters of 300-400 nm and excess phospholipids aggregated as vesicles (liposomes), which remain in the infranatant upon floatation of the emulsion particles by ultracentrifugation. This work is devoted to the characterization of the commercial lipid emulsions commonly denoted Intralipids, with special emphasis on the presently ill-defined liposomes. The lipid particles composing commercial lipid emulsions (10%, 20% and 30% Intralipids, Kabivitrum Nutrition) were characterized by the combined use of physical and chemical methods. Each of the emulsions was fractionated by ultracentrifugation in saline into a 'cream' layer which floats to the top of the dispersion upon ultracentrifugation and a relatively transparent infranatant. The cream layer contains large emulsion particles of diameters ranging from 300 to 400 nm, in agreement with theoretical considerations based on their chemical composition as determined by chemical analysis. The infranatants contain about 1 g/l triacylglycerols in addition to phospholipids (from 7.2 g/l in 10% Intralipid to 2.4 g/l in 30% Intralipid) in the form of smaller particles of 70-100 nm diameter. Cryo-transmission electron microscopy shows that the infranatants contain vesicles (mostly unilamellar) at the side of residual small emulsion particles. This conclusion is also consistent with the distribution of phospholipids between outer and inner lamellae, as determined by 31P-NMR.     

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.     

Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation?

Chylomicrons produced by the human gut contain apolipoprotein (apo) B48, whereas very-low-density lipoproteins made by the liver contain apo B100. To study how these molecules function during lipid absorption, we examined the process as it occurs in apobec-1 knockout mice (able to produce only apo B100; KO) and in wild-type mice (of which the normally functioning intestine makes apo B48, WT). Using the lymph fistula model, we studied the process of lipid absorption when animals were intraduodenally infused with a lipid emulsion (4 or 6 micromol/h of triolein). KO mice transported triacylglycerol (TG) as efficiently as WT mice when infused with the lower lipid dose; when infused with 6 micromol/h of triolein, however, KO mice transported significantly less TG to lymph than WT mice, leading to the accumulation of mucosal TG. Interestingly, the size of lipoprotein particles from both KO and WT mice were enlarged to chylomicron-size particles during absorption of the higher dose. These increased-size particles produced by KO mice were not associated with increased apo AIV secretion. However, we found that the gut of the KO mice secreted fewer apo B molecules to lymph (compared with WT), during both fasting and lipid infusion, leading us to conclude that the KO gut produced fewer numbers of TG-rich lipoproteins (including chylomicron) than the wild-type animals. The reduced apo B secretion in KO mice was not related to reduced microsomal triglyceride transfer protein lipid transfer activity. We propose that apo B48 is the preferred protein for the gut to coat chylomicrons to ensure efficient chylomicron formation and lipid absorption.     

Interactions between model triacylglycerol-rich lipoproteins and high-density lipoproteins in rat, rabbit and man

There are inverse relationships between HDL cholesterol and plasma triacylglycerol concentrations in normal and in hypertriglyceridemic individuals. To investigate the interactions between triacylglycerol-rich lipid particles and HDL, a lipid emulsion model of the triacylglycerol-rich lipoproteins was prepared. When emulsion particles were incubated with rat high-density lipoproteins (HDL) in the presence of lipid transfer activity (d greater than 1.21 g/ml fractions) from rabbit or human plasma there was a rapid bi-directional exchange of cholesteryl oleate (CO) and phospholipid (PL) labels between lighter and heavier fractions of HDL and emulsion particles. The transfers of CO and PL labels between both light and heavy fractions of HDL and the emulsion particles were increased with increasing amounts of emulsion added to the incubations. Incubation with the d greater than 1.21 g/ml fraction from rat plasma resulted in only a small exchange of CO whereas PL exchange was similar to rabbit and human plasma. Retinyl palmitate label was not transferred from emulsion particles to the HDL fractions even in the presence of lipid transfer activity from rabbit or human plasma. The present study shows that the transfer protein-mediated exchanges of surface and core lipids between HDL and the triacylglycerol-rich lipoproteins are affected by the quantity of triacylglycerol-rich particles in the system. This mechanism may contribute to the inverse relationships between plasma triacylglycerol concentrations and HDL concentrations in normal and hypertriglyceridemic individuals.     

Further studies on the mechanism of inhibition of intestinal chylomicron transport by Pluronic L-81

This study explored further the hypothesis that intestinal cells have two pathways for producing large triacylglycerol-rich lipoprotein particles. The hydrophobic surfactant Pluronic L-81 (L-81) inhibits formation of chylomicrons (containing triacylglycerol synthesized from dietary fatty acids and monoacylglycerol, through the monoacylglycerol pathway), but not formation of very-low-density lipoproteins. L-81 does not inhibit lymphatic lipid transport during infusion of egg phosphatidylcholine, whose fatty acid is processed through the alpha-glycerol phosphate pathway and is transported in lymph in very-low-density lipoproteins. Thus, the first part of this study tested whether L-81 cannot inhibit the alpha-glycerol phosphate pathway, and thus L-81 can only affect chylomicron lipid secretion. Intestinal lymph fistula rats were infused with a lipid emulsion containing [1-14C]oleic acid, but no monoacylglycerol, to ensure that the oleic acid will be channeled to the alpha-glycerol phosphate pathway. Experimental rats received 1 mg/h of L-81 in their emulsion whereas control rats lacked L-81. Lymphatic triacylglycerol output, measured both chemically and radioactively, was markedly suppressed in the experimental rats as compared to the controls. Thus, these data indicate that the reason why lipid transport was unaffected by L-81 when egg phosphatidylcholine was infused was not because of the pathway used for the resynthesis of triacylglycerol from phosphatidylcholine. In the second part of this study, we measured the appearance time for chylomicron (in control rats) and for very-low-density lipoprotein (in L-81-treated rats). The appearance time is defined as the time between placement of radioactive fatty acid into the intestinal lumen and the appearance of radioactive lipid in the central lacteal. The average appearance time for the control rats was 10.8 min, which was significantly shorter than the 16.2 min in the L-81-treated experimental rats. This difference in appearance time further supports the hypothesis that chylomicron and very-low-density lipoprotein are packaged separately in the enterocytes and only the formation of chylomicron is inhibited by L-81.     

Effects of chronic glucagon administration on rat lipoprotein composition

Male adult rats of the Wistar strain received daily at 9 a.m. and 5 p.m. 10 micrograms of Zn-protamine glucagon (Novo) for 21 days by subcutaneous injections. Plasma levels of cholesterol, triacylglycerol and phospholipids were decreased by 47, 40 and 21%, respectively. Lipoproteins were separated by sequential ultracentrifugation. Concentrations of cholesterol, phospholipids and proteins were decreased in chylomicrons, VLDL, LDL2 (1.040-1.063 g/ml) and HDL, LDL2 being the most affected by glucagon treatment (-70%). Triacylglycerol levels were decreased only in chylomicrons and VLDL. The relative proportions of cholesterol, triacylglycerol, phospholipids and proteins in lipoproteins were virtually unchanged by glucagon, suggesting a reduced number of some lipoprotein particles in plasma. However, lipoproteins of glucagon-treated rats were depleted in cholesteryl esters, while the proportion of triacylglycerol increased in LDL and HDL. Apo E contents were decreased in plasma, LDL1 (1.006-1.040 g/ml), LDL2 and HDL, whereas apo B100 proportions increased in VLDL and LDL1 in glucagon-treated rats. Glucagon appeared to be a potent hypolipidemic agent affecting mainly the apo-E-rich lipoproteins.     

Identification of factors regulating lipoprotein lipase catalyzed hydrolysis in rats with the aid of monoacid-rich lipoprotein preparations(1)

To identify the substrate specificity and regulatory factors in lipoprotein lipase (LPL) catalyzed hydrolysis of triacylglycerol-rich lipoprotein, monoacid-rich lipoproteins were used to study the kinetic parameters of LPL. Feeding growing rats with diets rich in palmitic acid (16:0), oleic acid (18:1) or linoleic acid (18:2) for 10 days increased the corresponding acid content in the triacylglycerols of the lipoproteins. Force-feeding the monoacid-rich triacylglycerols, particularly 16:0 or 18:1, increased the respective fatty acid content in both chylomicrons and VLDLs. Major apolipoproteins and lipid compositions were essentially similar among all lipoproteins differing in monoacid species, except for apo A-IV. The Vmax of LPL for 16:0-rich chylomicrons and VLDLs were higher than for 18:1- or 18:2-rich lipoproteins. Order parameter (S), an indicator of the surface fluidity of lipoproteins, decreased with the chain length and unsaturation of monoacid in similar manner as the Vmax. The Vmax of LPL increased linearly (P < 0.05) with an increase in either the palmitic acid content of the lipoprotein triacylglycerols or order parameter (S) of the lipoproteins. The order parameter (S) and Vmax of LPL were higher in 16:0 triacylglycerol emulsions with apo B than with 18:1 or 18:2 triacylglycerols. The apo A-IV in triacylglycerol emulsions stimulated Vmax of LPLs in the presence of apo B and apo C-II. The binding of apo A-IV to 16:0 triacylglycerol emulsions was higher than to other triacylglycerol emulsions. These findings suggest that lipoprotein catalysis by LPL is modulated by the 16:0 level in the lipoprotein triacylglycerol, which affects the surface fluidity and apo A-IV content of lipoproteins.     

Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters

Male rats with thoracic duct cannulae were intubated with corn oil or fatty acid methyl esters and the lymph was collected over the next 2-72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate - polyacrylamide - glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas--liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phosphatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B-100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from corn oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.     

The effect of added monoacylglycerols on the removal from plasma of chylomicron-like emulsions injected intravenously in rats

Lipid emulsions were prepared with compositions similar to the triacylglycerol-rich plasma lipoproteins, but also incorporating added small amounts of monoacylglycerols. Control emulsions without monoacylglycerol were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. The emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the bloodstream, with the removal rates of triacylglycerols faster than those of cholesteryl esters. Much of the removed cholesteryl ester was found in the liver, but only a small fraction of the triacylglycerol, consistent with hepatic uptake of the triacylglycerol-depleted remnants of the injected emulsion. Emulsions incorporating added monooleoylglycerol or stearic acid were metabolized similarly. Added 1- or 2-monostearoylglycerol had no effect on triacylglycerol removal from plasma, but the removal rate of cholesteryl esters was decreased and less cholesteryl ester was found in the liver. These effects are similar to those recently described when emulsions and chylomicrons contained triacylglycerols with a saturated acyl chain at the glycerol 2-position, suggesting that saturated monoacylglycerol produced by the action of lipoprotein lipase may cause triacylglycerol-depleted remnant particles to remain in the plasma instead of being rapidly taken up by the liver.     

Enteral infusion of phosphatidylcholine increases the lymphatic absorption of fat, but lowers alpha-tocopherol absorption in rats fed a low zinc diet*

Our previous study has shown that the lymphatic absorption of both fat and alpha-tocopherol (alphaTP) is lowered markedly in rats fed a low zinc diet, with a parallel decrease in lymphatic phospholipid (PL) output. This study was conducted to determine if enteral infusion of phosphatidylcholine (PC) could restore lymphatic absorption of fat and alphaTP in zinc-deficient rats. One group of rats was fed an AIN-93G diet containing 3 mg Zn/kg (low zinc; LZ) and the other was fed the same diet but containing 30 mg Zn/kg (adequate zinc; AZ). Rats were trained to consume two meals daily of equal amounts of food. At 6 wk, each rat with lymph fistula was infused at 3 mL/h with a lipid emulsion containing 3.6 &mgr;mol alphaTP and 565 &mgr;mol [carboxyl-14C]-triolein (14C-OA), with or without 40 &mgr;mol 1,2-dilinoleoyl-PC in 24 mL PBS at pH 6.4. The lymphatic absorptions of fat and alphaTP were determined by measuring 14C-radioactivity and alphaTP appearing in the mesenteric lymph collected hourly for 8 h. When the emulsion devoid of PC was infused, the absorptions of both 14C-OA (41 +/- 4% dose) and alphaTP (431 +/- 55 nmol) in LZ rats were significantly lower than in AZ rats (48 +/- 2% 14C-OA dose and 581 +/- 70 nmol alphaTP). When the emulsion containing PC was infused, the absorption of 14C-OA was restored rapidly to normal in LZ rats, along with a parallel increase in lymphatic PL output. However, PC infusion further lowered the absorption of alphaTP to 311 +/- 20 nmol/8 h in LZ rats and also lowered the absorption of alphaTP in AZ rats (347 +/- 48 nmol/8 h). The results demonstrate that low zinc intake results in impaired intestinal absorption of both alphaTP and fat. The findings also indicate that PC significantly improves the intestinal absorption of fat, but inhibits alphaTP absorption, suggesting that PC affects the intestinal absorption of alphaTP and fat via distinctly different mechanisms.     

Release of lipoprotein lipase to plasma by triacylglycerol emulsions. Comparison to the effect of heparin.

It was previously known that lipoprotein lipase (LPL) activity in plasma rises after infusion of a fat emulsion. To explore the mechanism we have compared the release of LPL by emulsion to that by heparin. After bolus injections of a fat emulsion (Intralipid) to rats, plasma LPL activity gradually rose 5-fold to a maximum at 6-8 min. During the same time the concentration of injected triacylglycerols (TG) decreased by about half. Hence, the time-course for plasma LPL activity was quite different from that for plasma TG. The disappearance of injected 125I-labelled bovine LPL from circulation was retarded by emulsion. This effect was more marked 30 min than 3 min after injection of the emulsion. The data indicate that the release of LPL into plasma is not solely due to binding of the lipase to the emulsion particles as such, but involves metabolism of the particles. Emulsion increased the fraction of labelled LPL found in adipose tissue, heart and the red muscle studied, but had no significant effect on the fraction found in liver. The effects of emulsion were quite different from those of heparin, which caused an immediate release of the lipase to plasma, decreased uptake of LPL in most extrahepatic tissues by 60-95%, and increased the fraction taken up in the liver.     

Phosphatidylcholine metabolism after transfer from lipid emulsions injected intravenously in rats. Implications for high-density lipoprotein metabolism

When injected intravenously in rats, emulsion models of triacylglycerol-rich lipoproteins were metabolized like natural lipoproteins and during the hydrolysis of emulsion triacylglycerols, a large fraction of the emulsion phosphatidylcholine was transferred to the plasma high-density lipoproteins. The removal from plasma of emulsion phosphatidylcholine was followed for 2 h in unanaesthetized rats. The half-lives for removal of phospholipid after injection of emulsions stabilized with dioleoylphosphatidylcholine or 1-palmitoyl-2-oleolyphosphatidylcholine were 58-63 min when traced with isologous label. In comparison, the published half-lives of HDL mixed phospholipids in rats are approx. 40 min, indicating that much of the clearance of the emulsion phospholipid could be accounted for by HDL catabolism. Measured LCAT activity was sufficient to account for not more than 2% of the catabolism of the HDL phospholipids labelled by this physiological procedure. Removal from plasma of label was more rapid when the same emulsions were labelled with tracer amounts of the heterologous dipalmitoylphosphatidylcholine, showing that individual phosphatidylcholine species were handled distinctly even when present only in tracer amounts in a bulk of another phosphatidylcholine differing in acyl chains.     

Size of lipoproteins in intestinal lympho of sheep and suckling lambs.

The relative importance of chylomicrons (Sf greater than 400) and very low density lipoproteins (Sf 20--400) in transporting lipids in lymph was investigated in surgically prepared adult sheep and pre-ruminant lambs fed low fat diets or infused intraduodenally with corn oil. The concentration of triacylglycerol in the intestinal lymph of sheep and lambs was increased from 520 and 925 mg/100 ml to 2326 and 2367 mg/100 ml respectively when corn oil was infused into the duodenum and the ratio of triacylglycerol to phospholipid changed from 3.7 and 5.5 to 9.5 and 9.7 respectively. The flow of lymph also increased. Electron microscopy and analytical and preparative ultracentrifugation showed that lymph lipoproteins from sheep and lambs fed low fat diets consisted mainly of lipoproteins 50 nm in diameter and that very low density lipoproteins (Sf 20--400) contirbuted up to 75% of the Sf greater than 20 lipoproteins. There were no lipoproteins with diameters above 150 nm. Infusion of corn oil into the duodenum of sheep and lambs increased the diameters of lymph lipoproteins. Most were 80--100 nm in diameter but substantial numbers above 150 and up to 400 mn were observed. The maximum contribution of very low density lipoproteins (Sf 20--400) to lipoproteins of Sf greater than 20 was 27--30%. The above findings demonstrate that the size of intestinal lymph lipoprotein particles increases with the amount of lipid absorbed from the small intestines and that the transport of lymph lipids, in ruminants, is similar to that previously found in rats, rabbits and man.     

Cholesterol is necessary for triacylglycerol-phospholipid emulsions to mimic the metabolism of lipoproteins

Emulsions with lipid compositions similar to the triacylglycerol-rich lipoproteins were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. Radioactive labels tracing the emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the blood stream, but the removal rate of triacylglycerols was faster than that of cholesteryl ester. Most of the removed cholesteryl ester label was found in the liver, but only a small fraction of the triacylglycerol label was found in this organ, consistent with hepatic uptake of the remnants of the injected emulsion. Emulsions otherwise identical but excluding unesterified cholesterol were metabolized differently. The plasma removal of triacylglycerols remained fast, but the cholesteryl esters were removed very slowly. Heparin stimulated lipolysis, but failed to increase the rate of removal of cholesteryl esters from emulsions lacking cholesterol. Evidently, emulsions lacking cholesterol were acted on by the enzyme lipoprotein lipase, but the resultant triacylglycerol-depleted remnant particle remained in the plasma instead of being rapidly taken up by the liver. Therefore, the presence of emulsion cholesterol is a critical determinant of early metabolic events, and the findings point to a similar role for cholesterol in the natural triacylglycerol-rich lipoproteins.     

Effect of fat pre-feeding on bile flow and composition in the rat

Studies were conducted in rats to determine if the increase in lymph triacylglycerol output on pre-feeding a 20% glyceryltrioleate diet (Mansbach, C.M., II and Arnold, A. (1986) Am. J. Physiol. 251, G263-269) was due to an increase in phosphatidylcholine output into bile. Rats who were fed chow or pre-fed the 20% fat diet were equipped with biliary and duodenal cannulas and infused with glucose-saline while bile was collected hourly. The next day a taurocholate-glyceryltrioleate infusion was given and bile collected for 5 h. Bile flow, bile acid, phosphatidylcholine and cholesterol output were greater in the chow fed group than controls during the 6 h of the glucose saline period. Outputs were low overnight. During the taurocholate-glyceryltrioleate infusion, bile flow, bile acid, phosphatidylcholine and cholesterol output were all greater in the fat pre-fed group than the chow fed controls. We conclude that fat pre-feeding profoundly influences biliary composition and flow. The 2-fold increase in biliary phosphatidylcholine output during duodenal lipid infusion offers a potential explanation for the increased delivery of triacylglycerol into the lymph in rats on a similar fat pre-feeding program.     

Metabolomics reveals increased isoleukotoxin diol (12,13-DHOME) in human plasma after acute Intralipid infusion

Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipid and saline into 10 healthy men in a double-blind, placebo-controlled experiment and used GC/MS, LC/MS, and NMR to profile the small-molecule composition of their plasma before and after infusion. Multivariate statistical analysis of the 40 resulting plasma samples revealed that after Intralipid infusion, a less-well-characterized pathway of linoleic acid metabolism had resulted in the appearance of (9Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DHOME, P < 10(-3)), a leukotoxin that has powerful physiological effects and is known to inhibit the neutrophil respiratory burst. Intralipid infusion caused increased plasma 12,13-DHOME. Given that 12,13-DHOME is known to directly affect neutrophil function, we conclude that untargeted metabolomics may have revealed a hitherto-unknown mechanism of intralipid-induced immunosuppression.     

Effect of intralipid infusion on lecithin:cholesterol acyltransferase and lipoprotein lipase in young rats

We compared the effects of Intralipid and dextrose infusion on plasma lecithin:cholesterol acyltransferase (LCAT), plasma lipid profiles and lipolytic activity. We used 5-week-old male Sprague-Dawley rats which were given total parenteral nutrition (TPN) with either Intralipid (3 g/kg body weight) or an equicaloric amount of 25% dextrose in the presence or absence of heparin (1 or 10 IU/ml of TPN). 40 min after the end of 4 h of infusion, plasma LCAT activity was significantly decreased (P less than 0.001), while total cholesterol and free fatty acid levels were significantly (P less than 0.05) increased in rats given Intralipid as compared to those given dextrose. We found associations (P less than 0.005) between LCAT activity and total cholesterol and between LCAT and free fatty acid levels; the coefficients of negative correlation were 0.543 and 0.607, respectively. Concomitantly to the increment in plasma total cholesterol levels, there was a decrease in the high-density lipoprotein (HDL) cholesterol fraction; the latter, which was 40% of the total plasma cholesterol in control and dextrose-infused rats, declined to 9% in rats given Intralipid. Administration of heparin during Intralipid infusion, even up to 10 IU/ml of TPN, did not affect any of these changes. After dextrose infusion, the values of all three parameters were similar to those of the control group. Plasma lipolytic activity was not significantly different between rats given infusion (Intralipid or dextrose) and controls. However, in the presence of heparin, plasma lipolytic activity increased similarly in both infused groups. These data indicate that in young rats, Intralipid infusion leads to an increase in plasma total cholesterol and free fatty acid levels, which correlates with a decrease in LCAT activity; the concurrent decrease in HDL cholesterol levels might account, in part, for the loss of LCAT activity. The administration of heparin results in an elevation of plasma lipolytic activity; however, it does not prevent the hypercholesterolemia, nor the decline in LCAT activity associated with Intralipid infusion.