Characterization and metabolic fate of two very-low-density lipoprotein subfractions separated by heparin-sepharose chromatography

Human very-low-density lipoproteins (VLDL) have been separated into two discrete subfractions by heparin-Sepharose chromatography. The retained fraction relative to the unretained fraction is characterized by an increased cholesterol ester/triacylglycerol ratio and an increased ratio of apolipoprotein E relative to apolipoprotein C. We have subfractionated VLDL from type IV hyperlipoproteinemic subjects and characterized these subfractions with respect to (i) composition and (ii) the metabolic fate of apolipoprotein B of each subfraction. The unretained fraction accounted for an average of 42% of total VLDL in type IV subjects. A similar distribution was obtained with VLDL from Type III subjects; however, only 25% of normal VLDL is in the unretained fraction. The apolipoprotein E/apolipoprotein C ratio was 2-8-fold higher in the retained fraction. The distribution of apolipoprotein E isomorphs and the individual C apolipoproteins were similar in each fraction. Retained and unretained fractions were labelled with 125I and/or 131I and injected simultaneously into miniature pigs. Apolipoprotein B of retained fractions was catabolized at a greater rate (fractional catabolic rate = 0.98 h-1 vs. 0.54 h-1, n = 7, P less than 0.05) compared to unretained fractions. These results are consistent with the concept that reduced content of C apolipoproteins in VLDL is correlated with enhanced uptake by perfused rat livers. Apolipoprotein B from retained fractions was converted to intermediate-density lipoproteins (IDL) at a greater rate, and apolipoprotein B from both fractions were converted to low-density lipoproteins (LDL). Although the unretained fraction may be the precursor of the retained fraction, the possibility exists that each fraction is largely synthesized and catabolized independently.     

Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006-1.019 g/mL) and LDL (d 1.019-1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.     

A novel explanation for the reduced LDL cholesterol in severe hypertriglyceridemia

When [3H]cholesteryl ester-labeled low density (LDL) and intermediate density lipoproteins (IDL) from a normotriglyceridemic, hypercholesterolemic rabbit were injected into severely hypertriglyceridemic, hypercholesterolemic rabbits, 60% of the label appeared in very low density lipoproteins (VLDL) at 3 hr. A similar experiment showed that 40% of injected 131I-protein-labeled LDL appeared in the IDL fraction at 4 hr. Taken together, these data suggest that the exchange of LDL cholesteryl ester for VLDL triglyceride results in a density shift of injected LDL to the IDL density range. Furthermore, the percent of injected 131I-labeled LDL from normotriglyceridemic rabbits that appeared in the IDL fraction increased in rabbits with increasing levels of plasma triglyceride. This LDL density shift was reproduced in vitro by incubating iodinated LDL from normotriglyceridemic, hypercholesterolemic rabbits with concentrations of VLDL from hypertriglyceridemic, hypercholesterolemic rabbits similar to those in plasma. With such a system, it was shown that the percentage of LDL that appeared in the IDL fraction increased with time, was enhanced fourfold by the addition of plasma lipid transfer protein, increased with increasing molar ratio of triglyceride to cholesteryl ester in VLDL, but apparently did not increase with increasing VLDL particle number. These studies suggest that a pronounced decrease in density of lipoproteins that would normally appear in the LDL density range, resulting from loss of cholesteryl ester in exchange for VLDL triglyceride, may explain, at least in part, the reduced LDL levels in severe hypertriglyceridemia.     

Intestinal lipoprotein synthesis in control and hypercholesterolemic rats

Previous studies in our laboratory have shown that very-low-density lipoproteins (VLDL) synthesized by the intestine of the diet-induced hypercholesterolemic rat are enriched in cholesteryl esters and unesterified cholesterol compared with intestinal VLDL from control rats. In these studies, we isolated and characterized nascent intestinal Golgi intermediate-density lipoproteins (IDL, d 1.006-1.040 g/ml) and studied isotope incorporation into apoliproteins of Golgi VLDL from control and hypercholesterolemic rats. IDL were triacylglycerol-rich lipoproteins but contained more cholesteryl ester and protein than the corresponding Golgi VLDL fractions. IDL from hypercholesterolemic rats were enriched in cholesteryl esters to a greater extent than IDL from control rats. The apolipoprotein patterns of IDL fractions were the same as those of intestinal Golgi VLDL, consisting of apolipoproteins (apo) B-48, A-I and A-IV. Time-course isotope incorporation curves for apo A-I and A-IV in Golgi VLDL were similar, but they differed from curves for apo B-48. None of these curves was markedly altered in the hypercholesterolemic rat. We conclude that the major effect of increased dietary cholesterol on intestinal lipoprotein biosynthesis is to increase the percentage of cholesteryl esters in Golgi lipoproteins. Dietary cholesterol does not alter the apolipoprotein composition of Golgi lipoproteins, nor does it have a significant effect on the pattern of isotope incorporation into apolipoproteins of Golgi VLDL. The effect of cholesteryl ester enrichment on the subsequent metabolism of these particles in the circulation and the effect of these particles on hepatic lipoprotein production remain to be determined.     

Presence of two forms of apolipoprotein B in patients with dyslipoproteinemia

Current information suggests that the major forms of the human B apolipoproteins, B-100 and B-48, are under separate genetic control and are synthesized by the liver and intestine, respectively. The apolipoprotein B composition of plasma lipoproteins has been determined in order to gain insight into the metabolic defects in patients with dyslipoproteinemias. Patients with type I and type V hyperlipoproteinemia can be separated into two groups based on apolipoprotein composition and triglyceride concentration. The first group had markedly elevated plasma triglycerides with B-48 in the 1.006 g/ml density fraction and only B-100 within IDL and LDL. The second group had plasma triglycerides less than 1200 mg % and only B-100 in all density fractions. Patients with type III hyperlipoproteinemia had B-48 in only the density less than 1.006 g/ml with B-100 in IDL and LDL; the type III hyperlipoproteinemic patient with apolipoprotein E deficiency, however, had B-48 in density less than 1.006 g/ml fraction, IDL, and LDL. Patients with type IIa, IIb, and IV hyperlipoproteinemia had only B-100 in all density fractions. These combined results are interpreted as indicating that B-48 is associated with triglyceride-rich lipoproteins synthesized by the intestine and that patients with phenotypes I, III, and V have defects in chylomicron remnant metabolism. In addition, in patients with types I and V hyperlipoproteinemia, mild hypertriglyceridemia appears to be associated with lipoprotein particles of liver origin.     

Apolipoprotein B metabolism in homozygous familial hypercholesterolemia

This report describes the metabolism of apolipoprotein B-containing lipoproteins in seven familial hypercholesterolemic (FH) homozygotes and compares the results to the values obtained from five healthy control subjects. The concentration, composition, and metabolism of large, triglyceride-rich very low density lipoproteins (VLDL1, Sf 60-400) were the same in the control and FH groups, indicating that this component of the VLDL delipidation cascade ws unaffected by the absence of receptors. In contrast, familial hypercholesterolemic small VLDL2 (Sf 20-60) was enriched with cholesterol and depleted in triglyceride. Moreover, its plasma concentration was elevated as a result of an increase in its synthesis and a defect in the removal of a remnant population within this density interval. The latter accounted for up to 50% of the total mass of the fraction. Onward transfer of apolipoprotein B (apoB) from small VLDL through intermediate density lipoprotein (IDL) to low density lipoprotein (LDL) was retarded, suggesting that receptors were involved in this supposedly lipase-mediated event. IDL and LDL concentrations increased up to fourfold above normal in the plasma of the FH patients due partly to the delay in maturation and partly to defective direct catabolism. We conclude that the LDL receptor plays multiple and important roles in the metabolism and transformation of apoB-containing particles in the Sf 0-400 flotation interval.     

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.     

Metabolsim of apoB and apoC lipoproteins in man: kinetic studies in normal and hyperlipoproteininemic subjects.

The kinetics of apolipoproteins B and C were studied in 14 normal and hyperlipoproteinemic subjects after injection of exogenously (125)I-labeled very low density lipoprotein (VLDL) particles. Plasma radioactivities of apoB and apoC were determined over a period of 4 days in VLDL (d < 1.006) and total radioactivity in intermediate (IDL) (1.006 < d < 1.019), low (LDL) (1.019 < d < 1.063), and high (HDL) (1.063 < d < 1.21) density lipoproteins. The data were analyzed by the use of a model, developed mostly from these data, with the following results. The VLDL particle undergoes a series of incremental density changes, most likely due to a number of delipidation steps, during which apoB stays with the particle until the density reaches the IDL range. There is, however, a loss of apoC associated with these delipidation steps. In our normal subjects, all IDL apoB eventually becomes LDL. In our hyperlipemic subjects some of the apoB on IDL is also degraded directly. The apoC lost by VLDL and IDL recycles to HDL, and most of it is picked up again by newly synthesized VLDL. There is a slowdown of the stepwise delipidation process in all hyperlipemic individuals studied. Three additional features became apparent in the type III subjects. First, there is a significant increase (a factor of 2 compared to normal) in the apoB synthesis rate by way of VLDL; second, there is an induced direct apoB synthesis pathway by way of IDL (and/or LDL); third, a bypass of the regular stepwise VLDL delipidation pathway is induced by which VLDL particles lose apoC but none of their apoB, thereby forming a new particle with metabolic properties similar to LDL, but with a density still in the VLDL density range. Two type III patients treated with nicotinic acid and clofibrate showed a sharp decrease in their VLDL apoB synthesis rates. This was somewhat compensated by an increased IDL apoB synthesis rate. A type I patient on a medium chain triglyceride diet also showed a number of metabolic changes, including reduced VLDL apoB synthesis and the induction of considerable IDL and/or LDL apoB synthesis.     

Serum lipoprotein and apolipoprotein profiles of the genetically obese ob/ob mouse

The lipid transport system of 3-month-old male C57BL/6J obese (ob/ob) mice was investigated. Serum lipoproteins were separated by density gradient ultracentrifugation and characterized by their chemical and electrophoretic properties as well as their relative apolipoprotein contents, defined according to molecular weight and charge. Obese, ob/ob mice exhibited a marked hyperlipoproteinemia resulting from large increases in low-density lipoproteins (LDL, d 1.021-1.058 g/ml) and high-density lipoproteins (HDL, d 1.058-1.137 g/ml), particularly, the HDL2 subclass (d 1.058-1.109 g/ml). This increase in lipoproteins was entirely responsible for their hypercholesterolemia and hyperphospholipidemia. By contrast, these obese mice had a net decrease in very-low-density lipoproteins (VLDL, d less than 1.016 g/ml) and intermediate-density lipoproteins (IDL, d 1.016-1.021 g/ml), which accounted for their moderate hypotriglyceridemia. The chemical composition of heterogeneous light LDL (d 1.021-1.040 g/ml and dense LDL (d 1.040-1.058 g/ml) overlapped by HDL-like particles was highly modified. These modifications consisted of increases in the percentages of cholesteryl ester and phospholipid and decreases in that of triacylglycerol. There were also marked changes in the relative values of the apolipoproteins of VLDL, but principally, IDL and LDL. IDL and light LDL were poorer in apolipoproteins BH (Mr 340,000-320,000) and eventually in apolipoprotein BL (Mr 220,000-200,000) and enriched in apolipoproteins E (Mr 37,000-35,000) and C-A-II (Mr approximately equal to 12,000). A similar and very significant change occurred in VLDL for both the apolipoproteins BL and C-A-II. Dense LDL, mainly poorer in apolipoprotein BH and enriched in apolipoprotein A-I (Mr 28,000-27,000), closely resembled HDL2 in all the groups, and were enriched in apolipoproteins C-A-II in only the obese mice. We suggest that ob/ob mice are probably protected against atheromata because of the low VLDL and IDL levels, and the increase in HDL2.     

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.     

A new micromethod for routine measurement of serum LDL-apolipoprotein B

A new method for low density lipoprotein (LDL) (d 1.019-1.063 g/ml)-apolipoprotein B (apoB) determination has been developed, based on the fact that very low density and intermediate density lipoproteins (VLDL and IDL) contain apolipoprotein C-I (apoC-I), whereas this apolipoprotein is apparently absent in LDL. VLDL and IDL were quantitatively precipitated with a monospecific anti-apoC-I antibody whereafter LDL-apoB in the supernatant was quantitated by Laurell rocket electrophoresis. Over a wide range of cholesterol and triglyceride values there was a linear correlation with LDL-apoB values measured after ultracentrifugation. The method would be useful for routine measurements, especially in children, since only 25 microliter of serum is required, and for making the diagnosis of hyperapobetalipoproteinemia, which at present is complicated.     

Apolipoprotein B overproduction by the perfused liver of the St. Thomas' mixed hyperlipidemic (SMHL) rabbit

The St. Thomas' mixed hyperlipidemic (SMHL) rabbit (previously St. Thomas' Hospital rabbit) is a putative model of familial combined hyperlipidemia (FCH). When fed a low (0.08%) cholesterol diet, it exhibits elevations in both plasma cholesterol and triglyceride compared to New Zealand White (NZW) controls. To determine the mechanism for this hyperlipidemia we studied the secretion of apolipoprotein B (apoB)-containing lipoproteins from perfused livers of both young and mature rabbits. During a 3-h perfusion we measured the total cholesterol and triglyceride content of the medium and the cholesterol, triglyceride, and apoB content of very low density lipoprotein (VLDL)(1) (S(f) 60;-400), VLDL(2) (S(f) 20;-60), intermediate (S(f) 12;-20), and low (S(f) 0;-12) density lipoproteins (IDL, LDL). Lipoprotein concentrations increased linearly throughout the perfusion period. The rate of cholesterol output was 3-fold higher (459 vs. 137 ng/g liver/min, P = 0.003) in SMHL versus NZW rabbits whilst that of triglyceride was similar (841 vs. 662 ng/g liver/min, NS). VLDL(1) cholesterol output was elevated 2-fold (232 vs. 123 ng/g liver/min, P < 0.05) and VLDL(2) + IDL + LDL cholesterol output, 4.5-fold (106 vs. 23 ng/g liver/min, P < 0. 005) in SMHL versus NZW rabbits. ApoB output in VLDL1 was 38 ng/g liver per min in SMHL and 14 ng/g liver per min in NZW (NS). In SMHL VLDL(2) + IDL + LDL apoB was increased 9-fold at 53 versus 6 ng/g liver per min in NZW (P < 0.001). We conclude that the SMHL rabbit overproduces apoB-containing lipoproteins particularly in the VLDL(2) + IDL + LDL fraction, a characteristic consistent with its use as a model of FCH.     

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.     

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.     

A density gradient study of the lipoprotein and apolipoprotein distribution in the chicken, Gallus domesticus

Plasma lipoproteins from 5-week old male chickens were separated over the density range 1.006-1.172 g/ml into 22 subfractions by isopycnic density gradient ultracentrifugation, in order to establish the distribution of these particles and their constituent apolipoproteins as a function of density. Lipoprotein subfractions were characterized by electrophorectic, chemical and morphological analyses, and their protein moieties were defined according to net charge at alkaline pH, molecular weight and isoelectric point. These analyses have permitted us to reevaluate the density limits of the major chicken lipoprotein classes and to determine their main characteristics, which are as follows: (1) very-low-density lipoproteins (VLDL), isolated at d less than 1.016 g/ml, were present at low concentrations (less than 0.1 mg/ml) in fasted birds; their mean diameter determined by gradient gel electrophoresis and by electron microscopy was 20.5 and 31.4 nm respectively; (2) as the the density increased from VLDL to intermediate density lipoproteins (IDL), d 1.016-l.020 g/ml) and low-density lipoproteins (LDL, d 1.020-1.046 g/ml), the lipoprotein particles contained progressively less triacylglycerol and more protein, and their Stokes diameter decreased to 20.0 nm; (3) apolipoprotein B-100 was the major apolipoprotein in lipoproteins of d less than 1.046 g/ml, with an Mr of 350000; small amounts of apolipoprotein B-100 were detectable in HDL subfractions of d less than 1.076 g/ml; urea-soluble apolipoproteins were present in this density range as minor components of Mr 38000-39000, 27000-28000 (corresponding to apolipoprotein A-1) and Mr 11000-12000; (4) high density lipoprotein (HDL, d 1.052-1.130 g/ml) was isolated as a single band, whose protein content increased progressively with increase in density; the chemical composition of HDL resembled that of human HDL2, with apolipoprotein A-1 (M 27000-28000) as the major protein component, and a protein of Mr 11000-12000 as a minor component; (5) heterogeneity was observed in the particle size and apolipoprotein distribution of HDL subfractions: two lipoprotein bands which additional apolipoproteins of Mr 13000 and 15000 were detected. These studies illustrate the inadequacy in the chicken of the density limits applied to fractionate the lipoprotein spectrum, and particularly the inappropriateness of the 1.063 g/ml density limit as the cutoff for LDL and HDL particle populations in the species.     

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Dietary restriction amplifies the metabolic disturbances of very-low-density lipoproteins in cholesterol-fed rabbits

The effect of dietary restriction (half of the control ration) on VLDL turnover was investigated in cholesterol-fed rabbits. Rabbits on standard, cholesterol and restricted cholesterol diets were injected with homologous 125I-labelled VLDL. Accompanying the amplification of hypercholesterolemia, additional disturbances of VLDL turnover were observed when cholesterol feeding was associated with dietary restriction. Cholesterol-fed rabbits with normal caloric ration exhibited delayed clearance of 125I-labelled apolipoprotein B component of VLDL compared to control rabbits. This was markedly accentuated in underfed rabbits, indicating further down-regulation of apolipoprotein B,E receptors in these animals. Furthermore, a reduced proportion of radiolabelled apolipoprotein B was converted from VLDL to intermediate-density lipoprotein (IDL) and LDL in both groups receiving cholesterol-rich diets. Thus, the combination of further impairment in plasma clearance of VLDL and the poor conversion into IDL and LDL could account for the massive increase of beta-VLDL in underfed animals on cholesterol-rich diets.     

Synthesis,secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac

Summary Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d<1.006 g/ml, d=1.006–1.020 g/ml and d=1.020–1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with preformed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.Abbreviations apo apolipoprotein - ER endoplasmic reticulum - IDL intermediate density-lipoprotein - LDL low density-lipoprotein - VLDL very low density-lipoprotein - PBS phosphate-buffered salt solution - RER rough endoplasmic reticulum - TEM transmission electron microscopy     

Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.

Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.     

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized.The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.