Effect of portacaval anastomosis on rat lipoproteins

The distribution of cholesterol (C), triglycerides (TG), phospholipids (PL) and protein in the different lipoproteins was studied in male Wistar rats under 2 conditions: control and 2 months after portacaval anastomosis (PCA). PCA decreased the levels of cholesterol and the other components in chylomicrons (-90%), very low density lipoproteins (-65 to -78%), LDL2 (1.040 less than d less than 1.063 g/ml; -51 to -61%) and HDL (1.063 less than d less than 1.21 g/ml), whereas no change was observed in LDL1 (1.006 less than d less than 1.040 g/ml). Apoprotein C contents were decreased in all lipoproteins. The relative proportions of C, TG, PL and proteins in lipoproteins were essentially unchanged by the shunt, suggesting a reduced number of lipoprotein particles in plasma after PCA. It was concluded that PCA reduced the levels of all lipoproteins secreted by liver and/or the intestine without modifying those of intraplasmatic origin (LDL1).     

Changes in plasma very low density and low density lipoprotein content, composition, and size after a fatty meal in normo- and hypertriglyceridemic man.

Four subfractions of plasma VLDL characterized by decreasing Sf value and LDL were isolated by density gradient preparative ultracentrifugation from normotriglyceridemic (NTG) and hypertriglyceridemic (HTG) (type IV) subjects in the fasting state and after a fatty meal. Chemical analysis and computation of numbers of particles in each fraction showed that the hyperlipidemia of type IV subjects was accounted for by an increase in total numbers of VLDL and a shift in the distribution of VLDL towards particles of larger diameter. Postprandial hyperlipidemia was due to the presence of chylomicron remnants rather than intact chylomicrons, and was accounted for by an increase in particle diameter of the largest VLDL subfraction rather than by an increase in particle numbers. Postprandial hyperlipedemia was accompanied by a shift in the distribution of VLDL towards particles of larger diameter in both NTG and HTG subjects, probably because of competition for the triglyceride-depletion process between chylomicrons and hepatic VLDL. Most chylomicron remnants were removed from the circulation without degradation to smaller VLDL or to LDL, but some remnants were sufficienty small to contribute to smaller VLDL subfractions. The LDL of type IV subjects contained more apoprotein B than those from NTG subjects, and this difference was associated with increases in diameter, molecular weight, density, and the ratio of protein: phospholipid in LDL from type IV subjects. Defective degradation of large VLDL to small VLDL, and of VLDL to LDL may be related to this alteration in apoprotein B content of the lipoproteins in type IV subjects.     

On the polydispersity of mitochondria in tissue homogenates and the determination of the average sedimentation coefficients of mixed populations of mitochondria

The sedimentation profile (sediterm) of subcellular particles in homogenous media depends on the average sedimentation coefficient ( value) and the size distribution. The present study has focused on the two common types of polydispersity, i.e., (i) a variable standard deviation in a normal (Gaussian) size distribution, and (ii) two populations of partieles with defined values and size distributions. Theoretical considerations and experimental data indicate that rat liver mitochondria have a normal size distribution, ( ) with much smaller standard deviation than previously assumed (σ = 0.118 μm) based on isokinetic gradient centrifugation and electron microscopy. Sedimentation of a mixture of rat liver and guinea pig ileal mitochondria having the values 17,040 S and 5640 S, respectively, gave the expected profile (sediterm) of two populations of particles. Their values were estimated to be identical to those obtained when the individual mitochondrial populations were sedimented. The ratio between the populations (based on the assay of marker enzyme) was found to be identical to the expected value.     

Development of a novel method to determine very low density lipoprotein kinetics

Isotopic tracer methods of determining triglyceride-rich lipoprotein (TRL) kinetics are costly, time-consuming, and labor-intensive. This study aimed to develop a simpler and cost-effective method of obtaining TRL kinetic data, based on the fact that chylomicrons compete with large VLDL (VLDL(1); S(f) = 60-400) for the same catalytic pathway. Ten healthy subjects [seven men; fasting triglyceride (TG), 44.3-407.6 mg/dl; body mass index, 21-35 kg/m(2)] were given an intravenous infusion of a chylomicron-like TG emulsion (Intralipid; 0.1 g/kg bolus followed by 0.1 g/kg/h infusion) for 75-120 min to prevent the clearance of VLDL(1) by lipoprotein lipase. Multiple blood samples were taken during and after infusion for separation of Intralipid, VLDL(1), and VLDL(2) by ultracentrifugation. VLDL(1)-apolipoprotein B (apoB) and TG production rates were calculated from their linear increases in the VLDL(1) fraction during the infusion. Intralipid-TG clearance rate was determined from its exponential decay after infusion. The production rates of VLDL(1)-apoB and VLDL(1)-TG were (mean +/- SEM) 25.4 +/- 3.9 and 1,076.7 +/- 224.7 mg/h, respectively, and the Intralipid-TG clearance rate was 66.9 +/- 11.7 pools/day. Kinetic data obtained from this method agree with values obtained from stable isotope methods and show the expected relationships with indices of body fatness and insulin resistance (all P < 0.05). The protocol is relatively quick, inexpensive, and transferable to nonspecialist laboratories.     

Intestinal apoB synthesis, lipids, and lipoproteins in chylomicron retention disease

Chylomicron retention disease is characterized by fat malabsorption, hypocholesterolemia, normal fasting triglycerides, and marked intestinal steatosis despite the presence of both plasma and intestinal apoprotein B. The defect remains unknown but presumably involves the synthesis or secretion of chylomicrons. The present investigation examines this hypothesis by studying the biosynthesis of chylomicrons in cultured jejunal explants and by defining the quantitative and qualitative abnormalities of plasma lipids and of circulating lipoproteins. Following 2-3 years of a low fat diet supplemented with medium chain triglycerides, six patients with chylomicron retention disease had significantly higher triglyceride (TG) levels coupled with a decrease in both free (FC) and esterified cholesterol (EC) as well as in essential fatty acids and phospholipids (PL) when compared to healthy controls. The low total plasma cholesterol was largely accounted for by low levels of both low density (LDL) and high density lipoprotein (HDL) cholesterol. VLDL and LDL were characterized by a diminished percentage of CE with an increase of TG while HDL contained relatively more FC as well as PL and less CE. The diameter of VLDL was larger whereas those of LDL and HDL were smaller than in normal controls. Jejunal explants, when incubated with [14C]palmitate, were capable of normal biosynthesis of TG, diglycerides, PL, and CE. These lipids, however, except for PL, were retained in the tissue and could not be secreted into the culture medium. Incubation of intestinal biopsies with [3H]leucine and [14C]mannose resulted in normal protein synthesis and reduced glycosylation. The presence of intestinal apoB-48 was confirmed by immunoblot using 2D8 antibodies. These data suggest that the intestinal defect in this disease results from a disorder of the final assembly of chylomicrons or in the mechanism of their exocytosis.     

Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.

To develop a cell culture model for chyclomicron (CM) assembly, the apical media of differentiated Caco-2 cells were supplemented with oleic acid (OA) together with either albumin or taurocholate (TC). The basolateral media were subjected to sequential density gradient ultracentrifugations to obtain large CM, small CM, and very low density lipoproteins (VLDL), and the distribution of apoB in these fractions was quantified. In the absence of OA, apoB was secreted as VLDL/LDL size particles. Addition of OA (>/=0.8 mM) with TC, but not with albumin, resulted in the secretion of one-third of apoB as CM. Lipid analysis revealed that half of the secreted phospholipids (PL) and triglycerides (TG) were associated with CM. In CM, TG were 7-11-fold higher than PL indicating that CM were TG-rich particles. Secreted CM contained apoB100, apoB48, and other apolipoproteins. Secretion of large CM was specifically inhibited by Pluronic L81, a detergent known to inhibit CM secretion in animals. These studies demonstrate that differentiated Caco-2 cells assemble and secrete CM in a manner similar to enterocytes in vivo. Next, experiments were performed to identify the sources of lipids used for lipoprotein assembly. Cells were labeled with [3H]glycerol for 12 h, washed, and supplemented with OA, TC, and [14C] glycerol for various times to induce CM assembly and to radiolabel nascent lipids. TG and PL were extracted from cells and media and the association of preformed and nascent lipids with lipoproteins was determined. All the lipoproteins contained higher amounts of preformed PL compared with nascent PL. VLDL contained equal amounts of nascent and preformed TG, whereas CM contained higher amounts of nascent TG even when nascent TG constituted a small fraction of the total cellular pool. These studies indicate that nascent TG and preformed PL are preferentially used for CM assembly and provide a molecular explanation for the in vivo observations that the fatty acid composition of TG, but not PL, of secreted CM reflects the composition of dietary fat. It is proposed that in the intestinal cells the preformed PL from the endoplasmic reticulum bud off with apoB as primordial particles and the assembly of larger lipoproteins is dependent on the synthesis and delivery of nascent TG to these particles.     

The transport of DDT from chylomicrons to adipocytes does not mimic triacylglycerol transport

Despite being banned in the U.S., organochlorine toxins such as DDT are frequently detected in human adipose tissue. The main route of exposure is through the consumption of contaminated foods and subsequent intestinal packaging of DDT into chylomicrons. These chylomicrons, which also contain dietary triacylglycerol (TG), are delivered directly to peripheral tissues without first being metabolized by the liver. The physiological process by which these compounds are delivered from chylomicrons to adipose is not well understood, but is clinically relevant since it bypasses first-pass metabolism. Based on its highly lipophilic nature, it has been assumed that DDT is transferred to peripheral tissues similar to TG; however, this has not been measured. Here, we use the lymph fistula rat to isolate chylomicrons containing both DDT and TG. These chylomicrons are the in vivo DDT delivery vehicle. Using 3T3-L1 adipocytes, we investigated the rate at which DDT transfers from chylomicrons to adipocytes, and mediators of this process. This novel approach closely approximates the in vivo DDT exposure route. We show that: 1) DDT repartitions from chylomicrons to adipocytes, 2) this transport does not require hydrolysis of TG within the chylomicron, and is stimulated by the inhibition of LPL, 3) albumin does not inhibit DDT uptake, 4) DDT dissolved in DMSO does not appropriately mimic in vivo DDT transport; and most importantly, 5) DDT uptake from chylomicrons does not mimic the uptake of TG from the same particles. Understanding these factors is important for designing interventions for human populations exposed to DDT.     

Lymphatic chylomicron size is inversely related to biliary phospholipid secretion in mice

Biliary phospholipids (PL) stimulate dietary fat absorption by facilitating intraluminal lipid solubilization and by providing surface components for chylomicron (CM) assembly. Impaired hepatic PL availability induces secretion of large very-low-density lipoproteins, but it is unclear whether CM size depends on biliary PL availability. Biliary PL secretion is absent in multidrug resistance protein 2-deficient (Mdr2(-/-)) mice, whereas it is strongly increased in essential fatty acid (EFA)-deficient mice. We investigated lymphatic CM size and composition in mice with absent (Mdr2(-/-)) or enhanced (EFA deficient) biliary PL secretion and in their respective controls under basal conditions and during enteral lipid administration. EFA deficiency was induced by feeding mice a high-fat, EFA-deficient diet for 8 wk. Lymph was collected by mesenteric lymph duct cannulation with or without intraduodenal lipid administration. Lymph was collected in 30-min fractions for up to 4 h, and lymphatic lipoprotein size was determined by dynamic light-scattering techniques. Lymph lipoprotein subfractions were isolated by ultracentrifugation, and lipid composition was measured. Lymphatic CMs were significantly larger in Mdr2(-/-) mice than in Mdr2(+/+) controls either without (+50%) or with (+25%) enteral lipid administration, and molar core-surface ratios were increased [triglyceride (TG)-to-PL ratio: 4.4 +/- 1.4 in Mdr2(-/-) mice vs. 2.7 +/- 0.8 in Mdr2(+/+) mice, P < 0.001]. In contrast, EFA-deficient mice secreted lipoproteins into lymph that were significantly smaller than in EFA-sufficient controls (173 +/- 32 vs. 236 +/- 47 nm), with correspondingly decreased core-surface ratios (TG-to-PL ratio: 3.0 +/- 1.0 in EFA-deficient mice vs. 6.0 +/- 1.9 in EFA-sufficient mice, P < 0.001). CM size increased during fat absorption in both EFA-deficient and EFA-sufficient mice, but the difference between the groups persisted. In conclusion, the present results strongly suggest that the availability of biliary PL is a major determinant of the size of intestinally produced lipoproteins both under basal conditions and during lipid absorption. Altered CM size may have physiological consequences for postprandial CM processing.     

Effects of moderate exercise on VLDL₁ and Intralipid kinetics in overweight/obese middle-aged men

Prior moderate exercise reduces plasma triglyceride (TG)-rich lipoprotein concentrations, mainly in the large very low-density lipoprotein (VLDL?) fraction, but the mechanism responsible is unclear. We investigated the effects of brisk walking on TG-rich lipoprotein kinetics using a novel method. Twelve overweight/obese middle-aged men underwent two kinetic studies, involving infusion of Intralipid to block VLDL? catabolism, in random order. On the afternoon prior to infusion, subjects either walked on a treadmill for 2 h at ～50% maximal oxygen uptake or performed no exercise. Multiple blood samples were taken during and after infusion for separation of Intralipid (S(f) 400) and VLDL? (S(f) 60-400). VLDL?-TG and -apoB production rates were calculated from their linear rises during infusion; fractional catabolic rates (FCR) were calculated by dividing linear rises by fasting concentrations. Intralipid-TG FCR was determined from the postinfusion exponential decay. Exercise reduced fasting VLDL?-TG concentration by 30% (P = 0.007) and increased TG enrichment of VLDL? particles [30% decrease in cholesteryl ester (CE)/TG ratio (P = 0.007); 26% increase in TG/apoB ratio (P = 0.059)]. Exercise also increased VLDL?-TG, VLDL?-apoB, and Intralipid-TG FCRs by 82, 146, and 43%, respectively (all P < 0.05), but had no significant effect on VLDL?-TG or -apoB production rates. The exercise-induced increase in VLDL?-apoB FCR correlated strongly with the exercise-induced changes in VLDL? CE/TG (r = -0.659, r = 0.020) and TG/apoB (r = 0.785, P = 0.002) ratios. Thus, exercise-induced reductions in VLDL? concentrations are mediated by increased catabolism, rather than reduced production, which may be facilitated by compositional changes to VLDL? particles that increase their affinity for clearance from the circulation.     

Effect of saturated and unsaturated lipid on the composition of mesenteric triglyceride-rich lipoproteins in the rat

The effect of saturated and unsaturated lipids on the composition of mesenteric lymph triglyceride-rich lipoproteins was studied in rats. A short-term steady-state infusion model was developed in mesenteric lymph fistula rats. Micellar solutions of linoleate, oleate, or palmitate were infused intraduodenally. Steady-state conditions of lymph flow, triglyceride, and apoA-I and apoB secretion rates were achieved in hours 3-5 after the start of the infusion. During this steady-state period, triglyceride-rich lipoproteins were prepared and characterized. With lipid infusion there were the expected increases in secretion rates of triglyceride, apoB, and apoA-I both in whole lymph and in the d less than 1.006 g/ml lipoproteins. Compositional analysis of d less than 1.006 g/ml lipoproteins revealed no difference in the ratios of phospholipid or apoA-I (surface) to triglyceride (core) constituents between saturated and unsaturated lipids, suggesting a similar particle size. This was directly verified by agarose gel filtration and electron microscopy carried out at 27 degrees C, which showed no difference in particle size between linoleate and palmitate chylomicrons. When these lipoproteins were prepared at 4 degrees C, palmitate lipoproteins exhibited dramatically changed gel filtration elution profiles, suggesting a shift to smaller or at least distorted particles and questioning earlier results suggesting a smaller size for saturated fat d less than 1.006 g/ml lipoproteins. Despite the similarity of size between saturated and unsaturated chylomicrons, the apoB content of unsaturated linoleate chylomicrons was significantly lower than that of palmitate chylomicrons. This difference was present whether chylomicrons were prepared by centrifugation or by gel filtration. The clearance of palmitate chylomicrons from the circulation of recipient rats was slightly more rapid than that of linoleate chylomicrons. The mechanism for this apparently selective increase in the apoB content of saturated fat chylomicrons is unknown but the present studies suggest that these changes may be of physiologic significance, perhaps relating to the potential atherogenicity of saturated lipids.     

Lipoproteins and apolipoproteins in intestinal lymph of the preruminant calf, Bos spp., at peak lipid absorption

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluidity changes and chemical composition of lipoproteins in type IIa hyperlipoproteinemia

The chemical composition and the physical properties of lipoproteins (VLDL, LDL and HDL) were studied in two groups of patients: 14 healthy normolipidemic subjects and 15 type IIa familial hypercholesterolemic patients. The steady-state fluorescence anisotropy rs was estimated in lipoproteins by the fluorescence depolarization of two fluorescent probes: the DPH (1,6-diphenyl-1,3,5-hexatriene) and the TMA-DPH (1,4-trimethylammonium phenyl-6-1,3,5-hexatriene). A structured order parameter S was calculated from the DPH fluorescence anisotropy. The flow activation energies were calculated for LDL and HDL from both groups from the Arrhenius plots (log r DPH versus 1/T). By using TNBS (trinitrobenzene sulfonic acid) as a distance control quencher, the two probes were located in the outer shell of LDL. In HDL, TMA-DPH remained at the surface of the particles, while DPH was more deeply embedded in the lipid core. There was no difference in the physico-chemical properties of VLDL between the two groups studied. DPH fluorescence anisotropies were significantly increased in LDL and HDL from the hypercholesterolemic group compared to the control particles (P less than 0.05 and P less than 0.01, respectively). In LDL this modification of the fluorescence anisotropy can be related to a change in the lipid composition of particles. LDL from hypercholesterolemic patients contained significantly less triacylglycerol (P less than 0.01) and more cholesteryl ester (N.S.). Their cholesteryl ester to triacylglycerol ratio was significantly higher. In HDL, there was no difference in chemical composition between the two groups. The increase in DPH fluorescence anisotropy can be related to the presence of smaller particles in HDL from HC group. No difference was noted in the TMA-DPH fluorescence anisotropy at 37 degrees C in the LDL from the two groups. In contrast, TMA-DPH fluorescence anisotropy in HDL from hypercholesterolemic group was significantly higher than in control HDL. The flow activation energy of DPH was also significantly higher in both LDL and HDL from the hypercholesterolemic group than in control group particles. In both LDL and HDL from the control group, DPH fluorescence anisotropy was negatively correlated with TG/protein and TG/PL ratios and positively correlated with the CE/TG ratio. No correlation was observed between lipid composition and DPH fluorescence anisotropy values in hypercholesterolemic particles. The modification in fluidity parameters, especially the increase in the flow activation energies in LDL and HDL from hypercholesterolemic patients, could lead to a restriction of cholesterol movements in these particles. From a physiological point of view, this could represent a loss of functional capacity.     

Low-density lipoprotein density determination by electric conductivity

The predominance of small dense low-density lipoprotein (LDL) particles is associated with an increased risk of coronary heart disease. A simple but precise method has been developed, based on electrical conductivity of an isopycnic gradient of KBr, to obtain density values of human LDL fraction. The results obtained can distinguish LDL density populations and their subfractions from different patients. These data were corroborated by Fourier transform infrared spectroscopy (FTIR) (structure) and light-scattering analyses (size).     

Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis

The proportion of the electronegative low density lipoprotein [LDL(-)] subfraction, which is atherogenic, is increased in type 2 diabetes but is not reduced by glycemic control. Therefore, we evaluated the ability of a new technique, capillary isotachophoresis (cITP), to quantify charge-based LDL subfractions and examined the relation between insulin resistance and the cITP fast-migrating (f) LDL levels. Seventy-five 10-year-old boys were included. The two cITP LDL subfractions, fLDL and major LDL subfractions, were proportional to the LDL protein content within the range of 0.1-0.8 mg/ml LDL protein. Levels of cITP fLDL were positively correlated with triglyceride (TG) levels and negatively correlated with LDL size. Insulin resistance as assessed by the homeostasis model assessment (HOMA-IR) was positively correlated (P < 0.01) with cITP fLDL levels (r = 0.41). The relation between HOMA-IR and cITP fLDL levels depended on TG levels but was independent of body mass index and LDL size. cITP lipoprotein analysis is an accurate and sensitive method for quantifying charge-based LDL subfractions in human plasma, and insulin resistance is related to cITP fLDL independent of LDL size.     

Regulation of triacylglycerol and phospholipid trafficking by fatty acids in newborn swine enterocytes

We (Wang H, Berschneider HM, Du J, and Black DD. Am J Physiol Gastrointest Liver Physiol 272: G935-G942, 1997; Wang H, Lu S, Du J, Yao Y, Berschneider HM, and Black DD. Am J Physiol Gastrointest Liver Physiol 280: G1137-G1144, 2001) previously showed that different fatty acids influence synthesis and secretion of triacylglycerol (TG) and phospholipid (PL) in a newborn swine enterocyte cell line (IPEC-1). The most striking effects were produced by stearic acid (SA; 18:0), which modestly affected TG and PL synthesis but reduced TG and PL secretion, and by eicosapentaenoic acid (EPA; 20:5), which reduced TG and PL synthesis and TG secretion relative to oleic acid (OA; 18:1). To define the mechanism of these effects, differentiated IPEC-1 cells were incubated for 24 h with OA, SA, or EPA and [(3)H]glycerol. Endoplasmic reticulum (ER) and Golgi (G) content of labeled lipids and apolipoprotein (apo) B and apoAI protein were measured. Relative to OA, SA did not impair ER TG synthesis, but reduced movement of labeled TG from ER to G. EPA impaired both ER TG synthesis and movement of labeled TG from ER to G. PL followed the same pattern, except ER synthesis of PL was relatively unaffected by EPA. Carbonate treatment demonstrated decreased partitioning of labeled lipid from ER membrane to lumen in EPA-treated cells. Organelle apoB and apoAI content demonstrated opposite patterns after SA and EPA incubation. We conclude that SA and EPA adversely influence immature enterocyte ER to G lipid trafficking, compared with OA. Furthermore, EPA inhibits ER lipid synthesis and transfer of membrane lipid to luminal particles. Regulation of apoAI ER to G trafficking is independent of that of apoB.     

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.     

Particle distribution of human serum high density lipoproteins.

Density gradient ultracentrifugation of human serum high density lipoproteins (HDL) from both normolipemic males and females results in a distribution of HDL concentration versus subfraction hydrated density which has three maxima. Gradient gel electrophoresis of total HDL is characterized by three banding maxima, the positions of which suggest the presence of three particle size ranges: I. 10.8-12.0 nm, II. 9.7-10.7 nm, and III. 8.5-9.6 nm. Gradient gel electrophoresis of density gradient subfractions established an inverse relationship between particle size and particle hydrated density which was corroborated by electron microscopy and analytic ultracentrifugation. Comparison of male HDL from size ranges I, II, and III with female HDL from the same size ranges showed only small differences in the mean value of the peak F degrees 1.20 rate, size, molecular weight, protein weight percent, and weight protein/weight phospholipid. Major differences between males and females were seen in the relative amounts of HDL in density gradient subfractions 1-3 (size range I material) and 11-12 (size range III material); the percent total HDL in the group of subfractions 1-3 was greatly increased in female HDL while that of the group of subfractions 11-12 was increased in the male HDL. These studies indicate the presence of at least three major components in HDL instead of two (HDL2 and HDL3) and that peak F degrees 1.20 rate differences in HDL schlieren patterns between males and females are a function of the relative levels of these three components.     

Size distributions of chylomicrons from human lymph from dynamic light scattering measurements

Chylomicrons, the vehicles for the transport of exogeneous triglycerides and cholesterol in the lymph and the blood, were characterized by their size from dynamic light scattering measurements. To achieve an appropriate resolution, correlation data were collected over several hours. Analysis was performed with an extended version of the regularization method CONTIN, and special attention was given to errors in the experimental baseline and to randomness of the residuals. The solutions selected by means of Fisher's F-test by CONTIN agreed with those obtained with the stability plot of Schnablegger and Glatter, when in the case of data of lower statistical accuracy the solution was taken from the lower part of the confidence interval of the F-test. The intensity-weighted size distributions indicated two classes of particle, their mean diameters being 100–140 nm and 330–350 nm. The ability to resolve two peaks of such a size ratio is demonstrated. The numbers of particles associated with the two peaks were estimated by means of the scattering properties of the particles, which showed that the overwhelming majority were small ones. This estimation also suggested that the mean size of the first peak of the number distribution is significantly smaller than the typical size of chylomicrons. This was consistent with the finding that the sample contained not only apolipoprotein B-48 but also a similar amount of apolipoprotein B-100, which is associated with lipoproteins of smaller size. The larger particles of the second peak are probably dietary triglyceride-rich chylomicrons. Received: 12 October 1997 / Revised version: 26 June 1998 / Accepted: 7 August 1998     

Serum sphingomyelin levels are related to the clearance of postprandial remnant-like particles

It is known that sphingomyelin (SM) content is higher in apolipoprotein B-containing particles (BLps) than in high density lipoproteins and that BLp levels, including chylomicrons and their remnant particles, are positively related to atherosclerosis. To evaluate the relationship between serum SM and postprandial remnant particle levels, we determined SM, triglyceride (TG), and cholesterol levels in serum and in remnant-like particles (RLPs) before and 3, 5, 7, and 10 h after a high-fat meal in 31 healthy subjects. We found that serum SM, like serum TG, was increased to its maximum 3 h after fat loading and then gradually decreased to basal levels after 10 h. More important, we determined that SM and TG levels in RLPs were parallel. Serum SM was positively correlated with serum TG (P <0.001), RLP SM (P <0.001), RLP TG (P <0.001), and RLP cholesterol (P <0.001) levels. It is our conclusion that serum SM is a marker for the clearance of RLPs.     

Direct evidence for a two-step assembly of ApoB48-containing lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes.

The aim of this study was to investigate the types and characteristics of chylomicron precursors in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low fat diet, newly synthesized and immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized, and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.