Action of liproprotein lipase on apoprotein-depleted chylomicrons.

1. Rat lymph chylomicrons were exposed to soluble and to immobilized trypsin. This treatment caused no detectable changes in the chylomicron structure or lipid composition, but did result in virtually total depletion of all their tetramethylurea-soluble apoproteins. 2. The capacity of these apoprotein-depleted chylomicrons to act as substrate for lipoprotein lipase in vitro and in situ (i.e. isolated perfused rat heart) was decreased by about 90 and 75% respectively, compared with intact chylomicrons. 3. On incubation with rat plasma high-density lipoproteins, trypsin-treated chylomicrons readily acquired a full apoprotein complement. This resulted in the complete restoration of their capacity to act as substrate for lipoprotein lipase both in vitro and in situ. 4. It is suggested that with the use of try,sin-treated chylomicrons it is now possible for the first time to investigate the physiological role that individual apoproteins play in the catabolism of triacylglycerol-rich lipoproteins by lipoprotein lipase.     

Uptake of phospholipid-depleted chylomicrons by the perfused rat liver.

1. Rat lymph chylomicrons were depleted of their surface phospholipids by treatment with pure phospholipase A2 from Crotalus adamanteus venom. 2. About 80% of the phospholipids could be removed from the chylomicrons without any apparent effect on their size, neutral lipid composition or qualitative profile of their tetramethylurea-soluble apoproteins. 3. Phospholipid-depleted chylomicrons were rapidly taken up whole by liver cells when perfused through isolated rat liver preparations. The rate of uptake was dependent on the extent of phospholipid depletion and reached a maximum (4-6.5-fold greater than control chylomicrons) when 80% of the phospholipids had been removed. 4. It is speculated that the hepatic uptake of phospholipid-depleted chylomicrons occurs by a mechanism to that of chylomicron-remnants uptake.     

Fractionation of chylomicrons by heparin-sepharose chromatography. Characterization of two heparin-binding proteins

Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed.     

Competition between chylomicrons and their remnants for plasma removal: a study with artificial emulsion models of chylomicrons

In previous studies, protein-free emulsions of defined lipid composition were shown capable of simulating either the metabolism of chylomicrons (chylomicron-like emulsion) or their remnants (remnant-like emulsion), depending on the content of free, unesterified cholesterol. To validate further the assumption that remnant-like and chylomicron-like emulsion have metabolic pathways in common with their natural counterparts, studies of competition for plasma removal were undertaken: the remnant-like emulsion labeled with [3H]triolein was injected sequentially twice in the carotid arteries of rats to compare the clearance of remnant-like emulsion of the second injection with the first (control). Prior to the second injection, a large bolus of the chylomicron-like emulsion or rat lymph chylomicron was injected, to check the hypothesis that remnant generated from chylomicron-like emulsion or natural chylomicrons could compete with and displace remnant-like emulsion particles from their tissue receptor sites. Experiments were also performed in rats treated with Triton WR-1339, to block the generation of remnants. Results showed that remnants derived from either natural chylomicrons or chylomicron-like emulsion both strongly competed with the remnant-like emulsion. In contrast, when transformation of remnants was prevented by Triton, the undegraded particles of chylomicron-like emulsion or natural chylomicron were unable to compete with or displace remnant-like emulsion from its sites of removal from the plasma. In agreement with plasma clearance data, the hepatic uptake of the remnant-like emulsion was inhibited by the surplus dose of natural chylomicrons. In contrast, the spleen uptake was unaffected by it.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.     

Metabolism of chylomicrons by the isolated rat liver

Isolated livers perfused with washed corn oil chylomicrons labeled in vivo with palmitic acid-1-(14)C removed a large proportion of the chylomicrons. Slices from these livers oxidized chylomicron fatty acid esters to both carbon dioxide and acetoacetate. The liver slices also generated free fatty acids from chylomicron lipids and converted chylomicron triglycerides to phospholipids. Similar activities were observed in rat liver slices prepared shortly after the intravenous administration of chylomicrons to intact rats. The observed chylomicron uptake and lipid conversions were similar in livers from both fed and fasted rats. Fasting increased the oxidation of chylomicron fatty acid esters by livers labeled in vivo and by perfusion. In livers removed from intact rats given labeled chylomicrons, the triglyceride-(14)C to phospholipid-(14)C ratio was high, a finding unexpected if the liver had acquired this (14)C by removal of circulating fatty acids formed by extrahepatic lipolysis. These results demonstrate the ability of the liver to remove and utilize chylomicrons directly and suggest that direct removal accounts for a significant portion of the chylomicron fatty acids utilized by the liver of intact rats.     

The surface coat of chylomicrons: lipid chemistry

Chylomicrons from the thoracic duct lymph of dogs fed corn oil were isolated by centrifugation and disrupted by either freezing and thawing or rotary evaporation and rehydration. A pellet, representing the surface coat, was isolated by centrifugation. Pellets isolated by freezing and thawing contained a higher percentage of saturated triglycerides than pellets isolated by rotary evaporation; the presence of saturated triglyceride in the pellet was probably an artifact of the preparation of the surface coat material at low temperature. Exchange of free cholesterol between surface and core lipid of chylomicrons was complete within 1 hr. The percentage of cholesterol in pellets of surface material isolated by freezing and thawing was about twice that found for pellets after rotary evaporation at 25-40 degrees C. Cholesteryl ester was not present in the surface lipid and that present in the core lipid did not exchange with serum lipoprotein cholesteryl ester. For phosphatidyl choline, the percentage of linoleic acid in lymph chylomicrons was markedly higher than that in clear lymph or plasma, while the percentage of arachidonic acid was lower. Sphingomyelin of lymph chylomicrons was characterized by very high levels of 16:0 and relatively small percentages of very long-chain fatty acids as compared with clear lymph or plasma. The data are consistent with the view that in lymph chylomicrons: (a) cholesteryl esters are dissolved in a core of triglycerides which contain fatty acids derived primarily from dietary fatty acids, (b) free cholesterol is partitioned between core and surface and is freely exchangeable between the two, (c) the phospholipid fractions are present on the surface and are intracellular in origin.     

Hepatic uptake of phospholipid-depleted chylomicrons in vivo. Comparison with the uptake of chylomicron remnants.

1. Rats pretreated with Triton WR-1339 to prevent the formation of remnants were injected with [3H]cholesterol-labelled remnants, intact chylomicrons or chylomicrons depleted of most of their surface phospholipids by treatment with phospholipase A2. Within 5 min about 80% of the injected label of remnants and phospholipid-depleted chylomicrons was incorporated into the livers compared with less than 10% of the injected radioactivity of intact chylomicrons. A similar rapid hepatic uptake of radioactivity occurred when rats not pretreated with Triton were injected with [3H]cholesterol-labelled phospholipid-depleted chylomicrons. This rapid hepatic uptake of phospholipid-depleted chylomicrons occurred apparently without any alteration in the apoprotein composition of the particles. 2. The participation of hepatocytes in the uptake of remnants and phospholipid-depleted chylomicrons was examined. Both types of particles were taken up by the hepatocytes. However, small chylomicrons (Sf less than 400) were taken up more efficiently than were large chylomicrons (Sf greater than 400), but neither was taken up as efficiently as the remnants. 3. The results of this study lend support to the hypothesis that phospholipid-depleted chylomicrons and chylomicron remnants are taken up by the liver by a similar mechanism, which depends on the loss of surface phospholipids.     

A rapid method for staining large chylomicrons

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.     

The surface coat of chylomicrons: electron microscopy

Electron microscope studies were performed on thoracic duct lymph and on washed chylomicrons from dogs fed corn oil. High-resolution electron micrographs showed the presence of a surface coat that differed from the core material and did not resemble a plasma membrane. This was true for both chylomicrons in whole lymph and those that had been subjected to repeated washing. Apparently, the chylomicrons, while passing from the intracellular to the extracellular space, do not acquire their surface coat from pinched off cellular membrane.