Triacylglycerol-rich Particles Research Articles

Alpha-tocotrienol is the most abundant tocotrienol isomer circulated in plasma and lipoproteins after postprandial tocotrienol-rich vitamin E supplementation

BackgroundTocotrienols (T3) and tocopherols (T), both members of the natural vitamin E family have unique biological functions in humans. T3 are detected in circulating human plasma and lipoproteins, although at concentrations significantly lower than α-tocopherol (α-T). T3, especially α-T3 is known to be neuropotective at nanomolar concentrations and this study evaluated the postprandial fate of T3 and α-T in plasma and lipoproteins.MethodsTen healthy volunteers (5 males and 5 females) were administered a single dose of vitamin E [526 mg palm tocotrienol-rich fraction (TRF) or 537 mg α-T] after 7-d pre-conditioning on a T3-free diet. Blood was sampled at baseline (fasted) and 2, 4, 5, 6, 8, and 24 h after supplementation. Concentrations of T and T3 isomers in plasma, triacylglycerol-rich particles (TRP), LDL, and HDL were measured at each postprandial interval.ResultsAfter TRF supplementation, plasma α-T3 and γ-T3 peaked at 5 h (α-T3: 4.74 ± 1.69 μM; γ-T3: 2.73 ± 1.27 μM). δ-T3 peaked earlier at 4 h (0.53 ± 0.25 μM). In contrast, α-T peaked at 6 h (30.13 ± 2.91 μM) and 8 h (37.80 ± 3.59 μM) following supplementation with TRF and α-T, respectively. α-T was the major vitamin E isomer detected in plasma, TRP, LDL, and HDL even after supplementation with TRF (composed of 70% T3). No T3 were detected during fasted states. T3 are detected postprandially only after TRF supplementation and concentrations were significantly lower than α-T.ConclusionsBio-discrimination between vitamin E isomers in humans reduces the rate of T3 absorption and affects their incorporation into lipoproteins. Although low absorption of T3 into circulation may impact some of their physiological functions in humans, T3 have biological functions well below concentration noted in this study.

Reversible flow of cholesteryl ester between high-density lipoproteins and triacylglycerol-rich particles is modulated by the fatty acid composition and concentration of triacylglycerols

We determined the influence of fasting (FAST) and feeding (FED) on cholesteryl ester (CE) flow between high-density lipoproteins (HDL) and plasma apoB-lipoprotein and triacylglycerol (TG)-rich emulsions (EM) prepared with TG-fatty acids (FAs). TG-FAs of varying chain lengths and degrees of unsaturation were tested in the presence of a plasma fraction at d > 1.21 g/mL as the source of CE transfer protein. The transfer of CE from HDL to FED was greater than to FAST TG-rich acceptor lipoproteins, 18% and 14%, respectively. However, percent CE transfer from HDL to apoB-containing lipoproteins was similar for FED and FAST HDL. The CE transfer from HDL to EM depended on the EM TG-FA chain length. Furthermore, the chain length of the monounsaturated TG-containing EM showed a significant positive correlation of the CE transfer from HDL to EM (r = 0.81, P < 0.0001) and a negative correlation from EM to HDL (r = -041, P = 0.0088). Regarding the degree of EM TG-FAs unsaturation, among EMs containing C18, the CE transfer was lower from HDL to C18:2 compared to C18:1 and C18:3, 17.7%, 20.7%, and 20%, respectively. However, the CE transfer from EMs to HDL was higher to C18:2 than to C18:1 and C18:3, 83.7%, 51.2%, and 46.3%, respectively. Thus, the EM FA composition was found to be the rate-limiting factor regulating the transfer of CE from HDL. Consequently, the net transfer of CE between HDL and TG-rich particles depends on the specific arrangement of the TG acyl chains in the lipoprotein particle core.

Interfacial Properties of a Complex Multi-Domain 490 Amino Acid Peptide Derived from Apolipoprotein B (Residues 292−782)

ApolipoproteinB (ApoB) is a lipid binding protein that is a nonexchangeable component of chylomicrons, VLDL, and LDL. In the liver and intestinal cells ApoB recruits lipid to form nascent triacylglycerol rich particles cotranslationally in the endoplasmic reticulum membrane which are then processed and secreted to form plasma lipoproteins. The N-terminal domain, which comprises the first 22% of apoB, recruits lipid in a controlled manner. The first 6% (residues 1-291) of the N-terminus does not bind lipid. The first lipid binding domain, including residues 292-782 (B6-17), forms a lipid binding pocket which is predicted to consist of 17 alpha-helices and 6 beta-strands. A structural model based on the X-ray structure of the homologues protein lipovitellin suggests that the N-terminal 6-8 helices and the beta-sheet interact with lipid while the C-terminal helices form a structural unit stabilizing the beta-sheet. Using isothermal drop tensiometry we showed that ApoB6.4-17 is surface active and binds to a triolein/water interface and exerts 16-19 mN/m of pressure (Pi) on that surface. The protein initially adsorbs slowly from aqueous solution to the surface but following compression and re-expansion it reaches equilibrium much faster. When Pi exceeds 16.9 mN/m part of the protein is ejected from the surface, but when compressed to high Pi the protein is never completely ejected indicating that part of the peptide is irreversibly anchored to the interface. The surface dilation modulus (epsilon) varies between 25-38 mN/m, and is predominantly elastic with a small viscous component. When compressed at an air/water interface ApoB6.4-17 has a limiting area of approximately 11 A2 per amino acid at lift off and only approximately 7 A2 per amino acid at the collapse Pi (28 mN/m). These values are about half the anticipated values if all the residues are at the surface. This suggests that ApoB6.4-17 retains some globular structure at an interface and does not completely denature at the surface, as many other globular proteins do. We suggest that while bound to the surface ApoB6.4-17 exhibits properties of both alpha and beta structure giving it unique and versatile characteristics at a hydrophobic interface.

Postprandial metabolic fate of tocotrienol-rich vitamin E differs significantly from that of α-tocopherol

The detection of tocotrienols in human plasma has proven elusive, and it is hypothesized that they are rapidly assimilated and redistributed in various mammalian tissues. The primary study objective was to evaluate the postprandial fate of tocotrienols and alpha-tocopherol in human plasma and lipoproteins. Seven healthy volunteers (4 males, 3 females) were administered a single dose of vitamin E [1011 mg palm tocotrienol-rich fraction (TRF) or 1074 mg alpha-tocopherol] after a 7-d conditioning period with a tocotrienol-free diet. Blood was sampled at baseline (fasted) and 2, 4, 5, 6, 8, and 24 h after supplementation. Concentrations of tocopherol and tocotrienol isomers in plasma, triacylglycerol-rich particles (TRPs), LDLs, and HDLs were measured at each interval. After intervention with TRF, plasma tocotrienols peaked at 4 h (4.79 +/- 1.2 microg/mL), whereas alpha-tocopherol peaked at 6 h (13.46 +/- 1.68 microg/mL). Although tocotrienols were similarly detected in TRPs, LDLs, and HDLs, tocotrienol concentrations were significantly lower than alpha-tocopherol concentrations. In comparison, plasma alpha-tocopherol peaked at 8 h (24.3 +/- 5.22 microg/mL) during the alpha-tocopherol treatment and emerged as the major vitamin E isomer detected in plasma and lipoproteins during both the TRF and the alpha-tocopherol treatments. Tocotrienols are detected in postprandial plasma, albeit in significantly lower concentrations than is alpha-tocopherol. This finding confirms previous observations that, in the fasted state, tocotrienols are not detected in plasma. Tocotrienol transport in lipoproteins appears to follow complex biochemically mediated pathways within the lipoprotein cascade.

Effect of orlistat on postprandial lipemia, NMR lipoprotein subclass profiles and particle size

Evidence suggests that metabolic phenomena during postprandial lipemia may be important in the pathogenesis of atherosclerosis. Both lipid concentrations and lipoprotein subclass patterns may be important cardiovascular risk modifiers. The pancreatic lipase inhibitor orlistat reduces fat absorption by 30% and is used for the treatment of overweight and obesity. We evaluated the effect of orlistat on postprandial lipemia and lipoprotein particle distribution after moderate-and high-fat meals in healthy volunteers. In this double-blind, randomized, cross-over study, 10 healthy young men received orlistat 120 mg plus a high-fat meal (HFO), orlistat plus a moderate-fat meal (MFO) or placebo plus a high-fat meal (HFP). Plasma triacylglycerol, glucose, insulin, and free fatty acids were measured at baseline (fasting) and postprandially for 8 h. Lipoprotein subclass profile was assessed by nuclear magnetic resonance spectroscopy. The 8 h postprandial mean triacylglycerol area under the curve (AUC) was significantly lower with MFO and HFO (0.79 versus 1.33 mmol/l h) versus HFP (4.33 mmol/l h; p = 0.02). Mean change in large VLDL subclass concentration during the 4–8 h and mean VLDL size after 8 h was significantly lower with HFO and MFO versus HFP ( p < 0.001). Small HDL particle concentration decreased significantly with HFP versus MFO or HFO ( p < 0.001). There was no significant difference in postprandial concentrations of glucose, insulin or free fatty acids on the different regimens. The lowering of postprandial triacylglycerol AUC, shorter postprandial lipemia, lower concentration of large triacylglycerol-rich particles and decrease of VLDL particle size supports the hypothesis of a less atherogenic postprandial lipoprotein profile following orlistat ingestion.

Postprandial lipaemia in familial combined hyperlipidaemia.

FCHL (familial combined hyperlipidaemia) is the most frequent inherited disorder of lipid metabolism leading to premature atherosclerosis. The usual phenotype in FCHL is elevated fasting plasma triacylglycerols, low HDL (high-density lipoprotein)-cholesterol concentrations and elevated plasma apolipoprotein B concentrations. The metabolic basis for this phenotype is hepatic overproduction of VLDL (very-low-density lipoprotein), which is only partly linked to the insulin resistance associated with FCHL. At this stage the molecular basis for this VLDL overproduction is not known, but emerging evidence points to a disturbed trapping of peripheral fatty acids, resulting in enhanced hepatic flux of NEFA [non-esterified ('free') fatty acids]. Postprandial hyperlipidaemia with accumulation of lipoprotein remnants and NEFA have been implicated in the development of atherosclerosis in this disorder. It has been proposed that, by VLDL overproduction, fasting hypertriglyceridaemia may lead to 'overflow' of the catabolic cascade for triacylglycerol-rich particles, thereby explaining the delayed catabolism of remnants in FCHL. Delayed clearance of remnants of VLDL and chylomicrons leads to enhanced interaction of these highly atherogenic particles with the endothelium, and enhanced trans-endothelial migration of the particles, resulting in a chronic inflammatory response that is the initiation of the atherosclerotic lesion. In this process, activated leucocytes (either directly by the remnants or indirectly by released NEFA) play an important role by adherence to the endothelium and migration into the subendothelial space, where the uptake of atherogenic remnants results in a vicious cycle of activation of endothelium, leucocytes and production of cytokines.

Bile salt-stimulated carboxyl ester lipase influences lipoprotein assembly and secretion in intestine: a process mediated via ceramide hydrolysis.

Bile salt-stimulated carboxyl ester lipase (CEL), also called cholesterol esterase, is one of the major proteins secreted by the pancreas. The physiological role of CEL was originally thought to be its mediation of dietary cholesterol absorption. However, recent studies showed no difference between wild type and CEL knockout mice in the total amount of cholesterol absorbed in a single meal. The current study tests the hypothesis that CEL in the intestinal lumen may influence the type of lipoproteins produced. A lipid emulsion containing 4 mm phospholipid, 13.33 mm [(3)H]triolein, and 2.6 mm [(14)C]cholesterol in 19 mm taurocholate was infused into the duodenum of lymph fistula CEL(+/+) and CEL(-/-) mice at a rate of 0.3 ml/h. Results showed no difference between CEL(+/+) and CEL(-/-) mice in the rate of cholesterol and triglyceride transport from the intestinal lumen to the lymph. However, CEL(-/-) mice produced predominantly smaller lipoproteins, whereas the CEL(+/+) mice produced primarily large chylomicrons and very low density lipoprotein. The proximal intestine of CEL(-/-) mice was also found to possess significantly less ceramide hydrolytic activity than that present in CEL(+/+) mice. By using Caco2 cells grown on Transwell membranes as a model, sphingomyelinase treatment inhibited the secretion of larger chylomicron-like lipoproteins without affecting total cholesterol secretion. In contrast, the addition of CEL to the apical medium increased the amount of large lipoproteins produced and alleviated the inhibition induced by sphingomyelinase. Taken together, this study identified a novel and physiologically significant role for CEL, namely the promotion of large chylomicron production in the intestine. The mechanism appears to be mediated through CEL hydrolysis of ceramide generated during the lipid absorption process.

Interaction of physical activity and diet: implications for lipoprotein metabolism.

To consider how physical activity interacts with diet to modify lipoprotein metabolism and comment on implications for human health. An overview of lipoprotein metabolism is followed by a summary of the main effects of physical activity on lipoprotein metabolism. Interactions with dietary practice and the disposition of dietary lipid are reviewed, with comment on links with body fatness. Literature is reviewed in relation to the risk of atherosclerotic disease. Although some data are presented on athletic groups, evidence relating to individuals with normal physical activity habits is mainly discussed. Physical inactivity is a risk factor for cardiovascular disease and one mechanism may involve changes to lipoprotein metabolism. The consensus is that aerobic activity involving an expenditure of > or = 8 MJ week(-1) results in an increase in HDL cholesterol and probably decreases in fasting triacylglycerol. These changes Occur despite the spontaneous increase in the proportion of dietary energy from carbohydrate which accompanies increased exercise. For this reason, exercise may be a means of reducing the hypertriglyceridaemic and HDL-lowering effects of low fat (high carbohydrate) diets. Decreases in total and low density lipoprotein cholesterol are sometimes, but not always, reported in sedentary individuals beginning exercise. One mechanism linking all these changes may be alterations to the dynamics of triacylglycerol-rich particles, particularly in the fed state. The expenditure of considerable amounts of energy through regular, frequent physical activity increases the turnover of lipid substrates, with effects on their transport and disposition which may reduce the progression of atherosclerosis.

Estrogen Induction of VLDLy Assembly in Egg-Laying Hens

The yolk of a 60-g chicken egg contains 6 g of triacylglycerols transported to the oocyte from the liver of the laying hen in apolipoprotein (apo) B–containing particles. With the onset of egg production, estrogen shifts hepatocytic lipoprotein production from generic VLDL to VLDLy (yolk targeted). These VLDLy are triacylglycerol-rich particles; they are reduced in size by one half, are resistant to lipoprotein lipase and are taken up intact by oocyte receptors. The VLDLy pathway for apoB provides sufficient energy for the caloric requirements of chick development. VLDLy size reduction occurs in spite of surplus liver triacylglycerols and is necessary for VLDL particles to pass through the granulosa basal lamina and reach the receptors located on the oocyte surface. New ultrastructural data show that some proximal tubule cells of bird kidney secrete generic VLDL, perhaps providing energy and other VLDL-associated nutrients to tissues bypassed by VLDLy. Birds are an apoB100-only species, providing a natural in vivo model with which to investigate mechanisms of apoB100 VLDL assembly. Preliminary studies of liver lipoprotein assembly intermediates isolated from the biosynthetic membranes (endoplasmic reticulum) of the laying hen are consistent with the presence of both putative first- and second-step precursor particles of VLDLy. These findings suggest that the two-step mechanism of apoB core lipidation is an ancient development in apoB biology, handed down to mammals from oviparous ancestors.

Tetradecylthioacetic acid (a 3-thia fatty acid) decreases triacylglycerol secretion in CaCo-2 cells.

The effects of the hypolipidemic fatty acid analogue tetradecylthioacetic acid (TTA) on synthesis and secretion of lipoproteins in CaCo-2 cells were studied. Radiolabeled tetradecylthioacetic acid was absorbed and metabolized as efficiently as oleic acid, although a discrepancy in the metabolic fate was evident. Whereas tetradecylthioacetic acid was incorporated into cell-associated triacylglycerol to the same extent as normal fatty acids (e.g., oleic acid and palmitic acid), the amount of triacylglycerol secreted from cells incubated with tetradecylthioacetic acid was 8 to 10 times lower than the amount secreted from cells incubated with palmitic acid and oleic acid, respectively. On the other hand, there was an enhanced incorporation of tetradecylthioacetic acid into cell-associated and secreted phospholipids. Despite incorporation of tetradecylthioacetic acid into cellular triacylglycerol, unlike oleic acid, tetradecylthioacetic acid did not stimulate production of triacylglycerol-rich particles. Ultracentrifugation of basolateral media from cells incubated with tetradecylthioacetic acid revealed low amounts of triacylglycerol in the triacylglycerol-rich fraction (p < 1.006 g/ml), suggesting secretion of lipoproteins with a higher density than chylomicrons. However, the present study shows that the stimulated triacylglycerol secretion caused by oleic acid was inhibited in the presence of TTA. The decreased rate of triacylglycerol secretion from these cells was not accompanied by a stimulation of fatty acid oxidation. Based on these findings, we therefore suggest that tetradecylthioacetic acid mainly affects secretion of lipoproteins in CaCo-2 cells.

The influence of cholesteryl ester content on hepatocyte triolein emulsion uptake

When triolein emulsions are enriched with cholesteryl oleate they are more readily removed by primary rat hepatocytes and HepG2 cells via an apolipoprotein E-responsive pathway. The increment in the cholesteryl ester does not appreciably change the size of the emulsion or its affinity for the apo E protein. The cholesteryl ester enriched-emulsion demonstrates increased apo E-mediated HepG2-binding as well as endocytosis. The higher the content of cholesteryl ester of the particle, the greater was both the binding and the endocytosis. The increased endocytosis was associated with increased degradation of the apo E. Cholesteryl linoleate and palmitate produced the same effects as the oleate. Lecithin-cholesterol acyltransferase additions were also able to increase the HepG2 uptake of the emulsion. The data indicates that the increment in cholesterol ester occurring during maturation of plasma triacylglycerol-rich particles facilitates hepatic remnant assimilation by an apo E-dependent pathway.

Sequence analysis of the 5′ flanking sequence of ovine Lipoprotein Lipase (LPL)

Lipoprotein lipase is found in many species and Ussue types 11) and is the pivotal enzyme in controlling lipid flux between plasma triacylglycerol rich lipoprotein particles and the tissues. Because tissue LPL activities alter with the physiological state of an organism, it is of great commercial and economic importance, and an understanding of the way factors interact to regulate its expression may, eventually, allow the remote manipulation of deposition of tissue lipids in v i m The 5 flanking sequence of Human I2.31. Mouse 141 and Chicken [5] LPL have already been determined and although chicken, in general, shares a lower level of sequence homology with human and mouse there is a remarkable level of conservation of cisacting elements across the species. Therefore this study was devised to investigate the 5 flanking sequence of ovine LPL to investigate whether these control elements were also conserved in sheep, a ruminant, with a very different metabolism to the species already investigated. High molecular weight ovine genomic DNA was isolated, from liver, and used to construct a sheep genomic library. This library consisted of 1.2 million independent clones with an average insert size of 14Kbk4Kb. A 1.7% ovine LPL cDNA (R1660) had already been isolated and sequenced previously[6] and was used to screen approx. 200.000 plaques of the sheep genomic library. From this screen one putative plaque was isolated, purified to homogeneity and designated LPLl. During the sequencing of R1660 a fragment corresponding to 225bp of the 5 end of R1660 had been generated using Pst I. This fragment was cloned into pGEM 3Zf and was designated LPL225c. LPLl was characterised by single and double digests with a range of restriction enzymes (HindIII. Sacl. Kpnl. BamHI, EcoFU and PstI). I t was determined from these digests that LPLl contained an insert of approx. 18Kb. The above digests were transferred to a nylon membrane and probed with 32P-labelled LPL225c. A single low molecular weight band of approx. 700bp was observed in the Pst I digest. This fragment was then subcloned into a vector suitable for sequencing. The vector chosen was pGEM 3Zf. Ligations were performed, transformed into JM109 and recombinants selected by blue/white screening. Plasmids were extracted from recombinant colonies and digested with Pst I. Digests were separated on an agarose gel, the DNA was transferred to a nylon membrane and probed again with 32P labelled LPL225c. A positive clone was identified and designated LPL7OOg. LPL7OOg was used as the template for sequencing reactions using the M 1 3 universal forward and reverse sequencing primers. Using the sequence already obtained, oligonucleotides were synthesised to internal sites in LPL7OOg in an attempt to walk through the clone. The sequence obtained from this Pst I sub-clone of LPLl included part of exon 1 and in addition 430bp of the 5 flanking sequence of LPL. This sequence was searched for known cis-elements using FINDPA'ITERNS from the Genetics Computer Group (GCG) suite of programs using the transcription factors database as the dictionary of ciselements and additionally by REGULON the authors own programme. The results of this analysis is shown in Table 1.

Intravascular metabolism of different fatty acids during lipid infusion in man

The differential intravascular metabolism of individual fatty acids contained in triacylglycerol-rich particles was studied by infusing 6 normal subjects for 5h with a conventional soy-based emulsion and an experimental olive oil-based emulsion. Both emulsions contained similar amounts of palmitate (11%) and stearate (3–4%) but the former was quite rich in linoleate (54%) and alpha-linolenate (7%), while the latter was rich in oleate (69%). During hydrolysis of circulating triacylglycerols by endothelial lipases, the associated rise of non-esterified fatty acids (FFA) in plasma represents the balance between fatty acid release and tissue uptake. Plasma levels of triacylglycerols and FFA increased about 3 fold and total body fat oxidation was raised to similar values with both emulsions. Fatty acid pattern quickly changed in plasma triacylglycerols to resemble the composition of emulsion particles, with an exception for palmitate which increased markedly more, suggesting a high level of hepatic re-esterification and re-appearance in nascent very low density lipoprotein triglycerides (VLDL-TG) secreted into the circulation. In plasma FFA, stearate and palmitate increased more and alpha-linolenate much less than expected from their content in the emulsion, indicating probably low tissue uptake for the former ones but avid removal for the latter.

Apolipoprotein A-II modulates HDL remodeling in plasma

Native and reduced-carboxamidomethylated (RCM) HDL 3 are incubated with a lipoprotein-depleted plasma fraction in the presence of triacylglycerol-rich particles isolated from Intralipid. Unmodified HDL 3 are mainly converted into large HDL 2b particles (diameter: 9.84 ± 0.15 nm); RCM-HDL 3 are transformed into both large HDL 2b (9.76 ± 0.10 nm) and small HDL 3c (7.68 ± 0.07 nm). These findings indicate that the apo A-II dimers participate in the remodeling of HDL, by inhibiting the formation of small HDL particles.

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.

Effects of intravenous infusions of commercial fat emulsions (Intralipid 10 or 20%) on rat plasma lipoproteins: Phospholipids in excess are the main precursors of lipoprotein-X-like particles

Like most commercial parenteral emulsions, Intralipid ® contains the same amount of phospholipids (12 mg/ml) to stabilize 100 or 200 mg of soybean oil (10 or 20% formula, respectively). By centrifugation, 10 or 20% Intralipid was separated into a supernatant, fat particles containing the bulk of triacylglycerols stabilized by a fraction of phospholipids and an infranatant — called mesophase — consisting mainly of phospholipids used in excess as emulsifier. We observed that the initial triacylglycerol phospholipid ratio of the emulsion ( 100 12 and 200 12 , respectively) determines the size of the triacylglycerol-rich particles (260 and 350 nm) as well as the phospholipid content of the mesophase (6.02 and 4.67 mg/ml). To understand the mechanism of the lipoprotein-X (LPX) accumulation generally reported after intravenous fat infusions, plasma lipid levels and lipoprotein profiles were first compared in the rats after infusion (at a constant rate of 0.5 or 1 ml/h for 43 h) of Intralipid 10 or 20%. For the same intravenous triacylglycerol load (100 mg/h), rats infused with Intralipid 10% at 1 ml/h displayed higher triacylglycerol levels than rats infused with the 20% emulsion at 0.5 ml/h, suggesting that the size of exogenous fat particles modulated the catabolic rate of their triacylglycerols. The plasma levels of LPX varied according to the infusion rate of phospholipids not associated with triacylglycerol-rich particles of the emulsion. Moreover, an apo E and apo B enrichment of plasma and an elevation of the apo B48/apo B100 ratio was always observed after Intralipid infusions. In order to confirm that phospholipids of the mesophase are the main LPX precursors, lipoprotein profiles were then compared in the rats after intravenous infusion, at a constant rate of 1 ml/h, of either the mesophase or a suspension of triacylglycerol-rich particles isolated from Intralipid 20%. As expected, significant LPX amounts were only detected in rats infused with the pure mesophase of the emulsion. It was concluded that products of the lipolysis of exogenous fat particles play only a minor role in the formation of LPX. In fact these abnormal lipoproteins are generated by phospholipids of the mesophase which, like infused liposomes, actively mobilize endogenous free cholesterol. Consequently, in order to be considered as true chylomicron models for safe fat delivery in parenteral nutrition and in order to prevent some detrimental effects on cholesterol metabolism, commercial emulsions should be cleared of phospholipid excess.

Experimental nephrotic syndrome: removal and tissue distribution of chylomicrons and very-low-density lipoproteins of normal and nephrotic origin

Lymph chylomicrons and plasma VLDL, 14C-labelled in vivo, were isolated from normal and nephrotic rats and injected into normal or nephrotic recipients. In normal recipients, the half-life of chylomicrons of nephrotic vs. normal origin was significantly longer (5.2 ± 0.5 vs. 3.5 ± 0.4 min −1). The nephrotic chylomicrons were larger in size, deficient in apo-E and apo A-I, rich in triacylglycerol and cholesterol, but poor in phospholipids, indicating that factors related to composition affected their removal. The half-life of nephrotic vs. normal VLDL, given to normal recipients, was unexpectedly shorter, (4.5 ± 0.2 vs. 5.8 ± 0.2 min −1). The nephrotic VLDL were also triacylglycerol- and cholesterol-rich and phospholipid-poor, but had a large diameter spread and contained a dense fraction according to the zonal ultracentrifugation pattern, suggesting the presence of faster removable IDL-like particles. When nephrotic rats received normal particles, a pronounced removal delay was seen, paralleling the extent of plasma triacylglycerol elevation. The half-life of chylomicrons was 8.3 ± 1.4 and 15.2 ± 2.5 min −1 in moderately and severely nephrotic rats, respectively, that of VLDL was 11.72 ± 2.1 and 37.8 ± 7.1 min −1 correspondingly. The chylomicron-triacylglycerol uptake was reduced both by adipose tissues and muscles of normal or nephrotic recipients, with some increase in entry into lungs, kidneys and spleen. Tissue distribution patterns of VLDL-triacylglycerol was similar to that of chylomicrons, except that the liver took up approx. 90% of the label. The low share of triacylglycerol uptake by tissues rich in lipoprotein lipase indicates that the activity of this enzyme was unlikely to limit the rate of removal. Lipoprotein lipase activity in adipose tissue and heart was slightly decreased in moderately nephrotic rats and declined only by approx. 35% in severely nephrotic ones. These results indicate that the removal defect in nephrosis seems to be due, in part, to changes in the composition of triacylglycerol-rich particles, compromising their accessibility to lipolysis and, in part, to their abundance, saturating the lipolytic capacity.

Gram-Negative Bacteria Sepsis in the Rat and Tissue Lipolytic Activity on LCT and MCT/LCT-Based Commercial Parenteral Emulsions

The aim of this study was to evaluate the effect of a gram-negative bacteria sepsis on the activity of the enzymes lipoprotein lipase (LPL) and hepatic lipase (HL), involved in the clearance of circulating triacylglycerol-rich fat particles. Fasting rats were intravenously injected with NaCl9 g.l-1, live or heat-killed Pseudomonas aeruginosa bacteria. After 18 h the animals were killed. When compared to controls, the 2 treated groups showed an increase in body temperature, cholesterolemia, triglyceridemia and a decrease in ketonemia, proteinemia, albuminemia and in the in vitro activity of diaphragm, heart and adipose tissue LPL and of HL. The decrease in the enzyme activities occurred independent of the type of emulsion used as in vitro substrate, whether it was based on long-chain triglycerides or on medium- and long-chain triglycerides, but in any case the activity was lower with the first than with the second type of fat emulsion.

Electron microscopic visualization of fatty acids in tissues.

Long-chain fatty acids are amphipathic molecules with important structural and metabolic functions in tissues. Fatty acids are derived from triacylglycerol-rich particles in capillaries (chylomicrons and very-low-density lipoproteins) and from triacylglycerol stored in cells (lipid droplets) by the hydrolytic activity of tissue lipases. The identification and localization of fatty acids in tissues has been considered difficult to obtain by using conventional ultrastructural techniques. However, structural findings from our studies on fatty acid transport in tissue became interpretable due to the use of many overlapping techniques. We present here these ultrastructural techniques developed to study fatty acids in tissues and review data which demonstrate lipase activity and fatty acid production from triacylglycerol in aldehyde-fixed tissue. Accumulations of fatty acid in tissue are present as lamellar structures with periodicity of 40-50 A in sections of resin-embedded tissue and as hydrated myelin figures in freeze fracture replicas of unfixed and fixed tissue. Finally, a new method, using the ionization properties of fatty acids combined with freeze fracture, locates these amphipathic molecules to leaflets of membrane bilayers.

Immunological heterogeneity of rat apolipoprotein B epitope expression. A study using monoclonal antibodies to rat apolipoprotein B

Epitope expression of rat apolipoprotein B on lipoproteins was investigated with the help of six monoclonal antibodies produced from mice. Through a variety of techniques, which include cotitrations, ELISAs and quantitative immunoadsorption precipitation, we concluded that the six monoclonal antibodies recognize five different epitopes. LRB 110 and LRB 260 recognize epitopes that may be overlapping. LRB 240 and LRB 250 recognize epitopes that are preferentially expressed in triacylglycerol-rich particles. LRB 220 recognizes an epitope that is expressed by all apolipoprotein-B-containing lipoproteins. We have also determined that apolipoprotein B epitope expression in rat lipoproteins is very similar to its human counterpart. Both rat and human apolipoprotein B epitope expression on lipoproteins showed heterogeneities even in homologous lipoprotein preparations. We concluded that a variety of techniques are necessary to fully characterize monoclonal antibodies to apolipoproteins. The possible implications of epitope expression in pathophysiology are also discussed.