Chylomicrons isolation

For the convenience of the reader, we have outlined the method of sequential flotation employed in our laboratory for separating chylomicrons VLDL, LDL, HDLa, HDLs, VHDL, and d> 1.25 bottom (Table 1). This method, the result of years of experience, has been highly reproducible in terms of the normal human population examined in this laboratory. Such a method may not necessarily apply to dyslipoproteinemic states, where modifications may be necessary, depending on the type of abnormality under consideration. It should also be stressed that any lipoprotein isolated is in need of purification this may be achieved by ultracentrifugation based on the assumption that contaminants are in loose association with the main complex. Whenever this purification is not achieved, other methods may be used as outlined below. For a discussion of the application of density gradient ultracentrifugation to the study of plasma lipoproteins, the reader is referred to a recent review (L3). 

Gershkovich, P., and A. Hoffman. 2005. Uptake of lipophilic drugs by plasma derived isolated chylomicrons Linear correlation with intestinal lymphatic bioavailability. Fur J Pharm Sci 26 394. 

Apolipoprotein C-II can also be isolated from VLDL or HDL (H20, L5, N3). It contains 78 residues (J3) and has been shown by Chou-Fasman analysis to bind phospholipids (M26, M40), with three predicted helical sequences (M26). ApoC-II has attracted a great deal of attention because it activates one of the most important enzymes in plasma lipid metabolism, lipoprotein lipase, responsible for the hydrolysis of triglyceride in chylomicrons and VLDL. Sparrow and Gotto have summarized a number of studies on structure-function relationships (S52). These, taken together, indicate that there are separate functional domains in apoC-II, in that lipoprotein lipase activation is mediated by residues 55-78 and phospholipid binding by... 

Blood plasma contains a number of soluble lipoproteins, which are classified, according to their densities, into four major types. These lipid-protein complexes function as a lipid transport system. Isolated lipids are insoluble in blood, but they are rendered soluble, and therefore transportable, by combination with specific proteins, the so-called lipoproteins. There are four basic types in human blood (1) chylomicrons, (2) very low density lipoproteins (VLDL), (3) low-density lipoproteins (LDL). and (4) high-density lipoproteins (HDL). Their properties are summarized in Table 6.2. 

Cholesterol and triacylglycerols are transported in body fluids in the form of lipoprotein particles. Each particle consists of a core of hydrophobic lipids surrounded by a shell of more polar lipids and apoproteins. The protein components of these macromolecular aggregates have two roles they solubilize hydrophobic lipids and contain cell-targeting signals. Lipoprotein particles are classified according to increasing density (Table 26.1) chylomicrons, chylomicron remnants, very low density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Ten principal apoproteins have been isolated and characterized. They are synthesized and secreted by the liver and the intestine. 

Tsujita and Okuda demonstrated in vitro that lipoprotein lipase is capable of catalyzing FAEE synthesis (Tsujita. 1994a). Chang and Borensztajn supported this observation by demonstrating that FAEE synthesis in an isolated rat heart perfused with chylomicrons and ethanol is mediated by lipoprotein lipase (Chang, 1997). The basic characteristics of enzymes involved in FAEE synthesis is presented in Table 1. 

Several different classes of lipoproteins exist whose structure and function arc closely related. Apart from the largest species, the chylomicron, the.se are named according to their density, as they are most commonly isolated by ullracen-irifugaiion. The four main lipoproteins and their functions are shown in Table 2. 

Chylomicrons (CM) were isolated during absorption of fat meals from plasma or lymph. 

Lipoproteins are classified into four major types in human blood, according to their densities chylomicrons, very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Isolated lipids are generally insoluble in blood, but lipoproteins allow lipids to be transported in the blood. 

In our studies, we have administered tritium-labeled vitamin A in one of its two physiological plasma transport vehicles (associated with either retinol-binding protein or chylomicrons) so that tracer data can be extrapolated to the vitamin A compounds of interest (retinol, retinyl esters, and metabolites). To prepare pH]retinol in its plasma transport complex (Green and Green, 1990b), vitamin A-depleted rats are used as donors to maximize hepatic secretion of the labeled vitamin on acciunulated liver apoRBP. pH]Retinol or pH]retinyl acetate in an emulsion with Tween 40 is administered intravenously to donor rats and blood is harvested 100 min later when plasma radioactivity is maximal. Plasma is isolated and stored under a nitrogen atmosphere at 4°C plasma is used for in vivo studies within 23 days. 

To prepare pHJvitamin A-labeled chylomicrons (Green et ai, 1993), [ Hjretinol or retinyl acetate is administered intraduodenally to thoracic lymph duct-cannulated donor rats and lymph is collected at 4°C. Chylomicrons containing mainly pHJretinyl esters can be isolated from lymph by preparative ultracentrifugation for administration to recipient rats. Alternatively, aliquots of whole lymph can be injected to minimize handling of the dose (see below). Then the proportions of total lymph radioactivity and vitamin A mass in chylomicrons (typically >8590%) can be determined analytically. In either case, lymph preparations should be used for in vivo studies within 12 days of collection. 

Even when care is taken to handle the doses carefully, we have found that 215% of the tracer in the case of pHjretinol-labeled plasma, and up to 40% in the case of isolated pHJretinyl ester-labeled chylomicrons, acts nonphysiologically when preparations are injected into recipient rats. That is, a variable fraction of the dose (the nonphysiological component) is cleared from plasma within a few minutes. Presumably nonphysiological... 

Although apoprotein A-IV exhibits the properties of an apolipoprotein [2], and recent data on its sequence [11, 12, 21] have shown that it contains 14.5 tandemly repeated docosapeptides that possess the potential to form amphipathic a-helices [11], it is mainly found unassociated with lipoproteins in human plasma [4,18,19,36]. The Apo A-IV fraction in the lipoprotein-free plasma compartment is still able to bind lipids, as shown by Weinberg and Scanu [37], who were able to reassociate Apo A-IV from the d = 1.21 g/ml infranate to a phospholipid-triglyceride emulsion. After reassociation Apo A-IV could be isolated by flotation in chylomicron-like particles upon ultracentrifugation. 

The possible mechanism of Apo A-IV displacement from the surface of chylomicrons upon entry into the plasma was studied in an in vitro model by Weinberg and Spector [41]. They used Apo A-IV associated with a phospholipid-triglyceride emulsion for displacement studies. When these chylomicron-like particles were incubated with HDL, they found a displacement of Apo A-IV from these particles, mainly by Apo C-III. Thus, one of the mechanisms responsible for the dissociation of Apo A-IV frbm chylomicrons upon entry into the plasma compartment could be a displacement by other circulating apoproteins that have a higher affinity for chylomicrons (or their remnants after attack by hpoprotein lipase). In contrast to Apo A-IV, which decays and is later mostly found in the lipoprotein-free plasma compartment, the other major chylomicron apoprotein, A-I, reassociates with HDL, as shown by tracer kinetic studies [20, 29]. It thus remains puzzling why the bulk of Apo A-IV, despite its apoprotein structure, does not reassociate with lipoproteins. In addition, lymph and plasma Apo A-IV have similar a-helical contents and properties in solution [10]. This seems to be an unexplained phenomenon since we were able to show that apoprotein A-IV, isolated either from chylomicrons or from lipoprotein-free plasma, recombines with hpids to form stable complexes [34]. 

Utermann G, Beisiegel U (1979) Apohpoprotein A-FV a protein occuring in human mesenteric lymph chylomicrons and free in plasma. Isolation and quantification. Eur J Biochem 99 333-343... 

The LDL density class of cholestatic patients often shows a very heterogeneous picture in the electron microscope normal LP-B particles, the stacked discs of LP-X, and larger spherical particles. These are remnants of triglyceride-rich lipoproteins. LP-B and remnants can be separated from LP-X by Cohn-fractionation and the isolation of remnants can be achieved by immunoaffinity chromatography on an anti-Apo-C column. The remnant contains Apo-B and Apo-C, as demonstrated by Immunoelectrophoresis. These particles most probably originate from chylomicrons, since their concentration can be diminished by a fat-free diet. 

The anatomic sites (subcellularly) and the details of the enzymatic processes involved in the hydrolysis of chylomicron cholesteryl esters newly taken up by the liver have not been fully defined. It is clear that one of the major processes consists of receptor-mediated endocytosis of chylomicron remnants, followed by hydrolysis of cholesteryl esters and other remnant components within lysosomes. In rare genetic diseases characterized by lysosomal acid lipase deficiency (Wol-man s disease and cholesteryl ester storage disease), cholesteryl esters accumulate in liver cells and in other tissues as well [see Assmann and Frederickson (1983) for review and references]. An acid cholesteryl ester hydrolase from rat liver lysosomes has been partially purified and characterized (Brown and Sgoutas, 1980 Van Berkel etal., 1980). Enzymatic activity was found in preparations of both parenchymal and nonparenchymal liver cells (Van Berkel et al., 1980). Hydrolysis of chylomicron cholesteryl esters taken up by isolated rat hepatocytes was inhibited by chloroquine (Florin and Nilsson, 1977), an agent which inhibits the action of acid hydrolases in lysosomes. Finally, there is also evidence that the rate of cholesteryl ester hydrolysis may be limited by the rate at which internalized remnant particles are moved to the presumably lysosomal site of hydrolysis (Nilsson, 1977 Florin and Nilsson, 1977 Cooper and Yu, 1978). 

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