A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

The clinical and laboratory investigation of dysbetalipoproteinemia.

Familial dysbetalipoproteinemia (type III hyperlipoproteinemia) is a potentially underdiagnosed inherited dyslipidemia associated with greatly increased risk of coronary and peripheral vascular disease. The mixed hyperlipidemia observed in this disorder usually responds well to appropriate medical therapy and lifestyle modification. Although there are characteristic clinical features such as palmar and tuberous xanthomata, associated with dysbetalipoproteinemia, they are not always present, and their absence cannot be used to exclude the disorder. The routine lipid profile cannot distinguish dysbetalipoproteinemia from other causes of mixed hyperlipidemia and so additional investigations are required for confident diagnosis or exclusion. A range of investigations that have been proposed as potential diagnostic tests are discussed in this review, but the definitive biochemical test for dysbetalipoproteinemia is widely considered to be beta quantification. Beta quantification can determine the presence of "ß-VLDL" in the supernatant following ultracentrifugation and whether the VLDL cholesterol to triglyceride ratio is elevated. Both features are considered hallmarks of the disease. However, beta quantification and other specialist tests are not widely available and are not high-throughput tests that can practically be applied to all patients with mixed hyperlipidemia. Using apolipoprotein B (as a ratio either to total or non-HDL cholesterol or as part of a multi-step algorithm) as an initial test to select patients for further investigation is a promising approach. Several studies have demonstrated a high degree of diagnostic sensitivity and specificity using these approaches and apolipoprotein B is a relatively low-cost test that is widely available on high-throughput platforms. Genetic testing is also important in the diagnosis, but it should be noted that most individuals with an E2/2 genotype do not suffer from remnant hyperlipidemia and around 10% of familial dysbetalipoproteinemia cases are caused by rarer, autosomal dominant mutations in APOE that will only be detected if the gene is fully sequenced. Wider implementation of diagnostic pathways utilizing apo B could lead to more rational use of specialist investigations and more consistent detection of patients with dysbetalipoproteinemia. Without the application of a consistent evidence-based approach to identifying dysbetalipoproteinemia, many cases are likely to remain undiagnosed.

1H NMR spectroscopy quantifies visibility of lipoproteins, subclasses, and lipids at varied temperatures and pressures.

NMR-based quantification of human lipoprotein (sub)classes is a powerful high-throughput method for medical diagnostics. We evaluated select proton NMR signals of serum lipoproteins for elucidating the physicochemical features and the absolute NMR visibility of their lipids. We separated human lipoproteins of different subclasses by ultracentrifugation and analyzed them by 1H NMR spectroscopy at different temperatures (283-323 K) and pressures (0.1-200 MPa). In parallel, we determined the total lipid content by extraction with chloroform/methanol. The visibility of different lipids in the 1H NMR spectra strongly depends on temperature and pressure: it increases with increasing temperatures but decreases with increasing pressures. Even at 313 K, only part of the lipoprotein is detected quantitatively. In LDL and in HDL subclasses HDL2 and HDL3, only 39%, 62%, and 90% of the total cholesterol and only 73%, 70%, and 87% of the FAs are detected, respectively. The choline head groups show visibilities of 43%, 75%, and 87% for LDL, HDL2, and HDL3, respectively. The description of the NMR visibility of lipid signals requires a minimum model of three different compartments, A, B, and C. The thermodynamic analysis of compartment B leads to melting temperatures between 282 K and 308 K and to enthalpy differences that vary for the different lipoproteins as well as for the reporter groups selected. In summary, we describe differences in NMR visibility of lipoproteins and variations in biophysical responses of functional groups that are crucial for the accuracy of absolute NMR quantification.

Resolution of Lipoprotein Subclasses by Charge Detection Mass Spectrometry.

Lipoproteins are micelle-like assemblies that are key players in the pathogenesis of atherosclerosis. High-density lipoprotein (HDL), low-density lipoproteins (LDL), and very low density lipoprotein (VLDL) are the three major classes present in fasting plasma. Within each class, there is a broad size distribution with wide variations in protein and lipid content. The development of better metrics for cardiovascular risk is thought to depend on better characterization of lipoprotein subclasses. Using charge detection mass spectrometry (CDMS), the mass distributions of HDL, LDL, and VLDL have been directly measured for the first time. In the case of HDL, seven distinct subpopulations were resolved using a two-dimensional correlation of charge and mass. The resolved components are assigned to HDL particles containing different numbers of the key structural proteins apolipoprotein A-I and apolipoprotein A-II.

A power set-based statistical selection procedure to locate susceptible rare variants associated with complex traits with sequencing data.

MOTIVATION: Existing association methods for rare variants from sequencing data have focused on aggregating variants in a gene or a genetic region because of the fact that analysing individual rare variants is underpowered. However, these existing rare variant detection methods are not able to identify which rare variants in a gene or a genetic region of all variants are associated with the complex diseases or traits. Once phenotypic associations of a gene or a genetic region are identified, the natural next step in the association study with sequencing data is to locate the susceptible rare variants within the gene or the genetic region. RESULTS: In this article, we propose a power set-based statistical selection procedure that is able to identify the locations of the potentially susceptible rare variants within a disease-related gene or a genetic region. The selection performance of the proposed selection procedure was evaluated through simulation studies, where we demonstrated the feasibility and superior power over several comparable existing methods. In particular, the proposed method is able to handle the mixed effects when both risk and protective variants are present in a gene or a genetic region. The proposed selection procedure was also applied to the sequence data on the ANGPTL gene family from the Dallas Heart Study to identify potentially susceptible rare variants within the trait-related genes. AVAILABILITY AND IMPLEMENTATION: An R package 'rvsel' can be downloaded from http://www.columbia.edu/∼sw2206/ and http://statsun.pusan.ac.kr.

Parameter trajectory analysis to identify treatment effects of pharmacological interventions.

The field of medical systems biology aims to advance understanding of molecular mechanisms that drive disease progression and to translate this knowledge into therapies to effectively treat diseases. A challenging task is the investigation of long-term effects of a (pharmacological) treatment, to establish its applicability and to identify potential side effects. We present a new modeling approach, called Analysis of Dynamic Adaptations in Parameter Trajectories (ADAPT), to analyze the long-term effects of a pharmacological intervention. A concept of time-dependent evolution of model parameters is introduced to study the dynamics of molecular adaptations. The progression of these adaptations is predicted by identifying necessary dynamic changes in the model parameters to describe the transition between experimental data obtained during different stages of the treatment. The trajectories provide insight in the affected underlying biological systems and identify the molecular events that should be studied in more detail to unravel the mechanistic basis of treatment outcome. Modulating effects caused by interactions with the proteome and transcriptome levels, which are often less well understood, can be captured by the time-dependent descriptions of the parameters. ADAPT was employed to identify metabolic adaptations induced upon pharmacological activation of the liver X receptor (LXR), a potential drug target to treat or prevent atherosclerosis. The trajectories were investigated to study the cascade of adaptations. This provided a counter-intuitive insight concerning the function of scavenger receptor class B1 (SR-B1), a receptor that facilitates the hepatic uptake of cholesterol. Although activation of LXR promotes cholesterol efflux and -excretion, our computational analysis showed that the hepatic capacity to clear cholesterol was reduced upon prolonged treatment. This prediction was confirmed experimentally by immunoblotting measurements of SR-B1 in hepatic membranes. Next to the identification of potential unwanted side effects, we demonstrate how ADAPT can be used to design new target interventions to prevent these.

Does a diet high or low in fat influence the oxidation potential of VLDL, LDL and HDL subfractions?

BACKGROUND AND AIMS: High-fat diets have become increasingly popular for weight-loss, but their effect on the oxidation potential of lipoprotein subfractions has not been studied. Therefore, this study compared the effects of high-fat vs. low-fat weight reduction diets on this parameter. METHODS AND RESULTS: Very-low, low- and high-density lipoprotein (VLDL, LDL & HDL) subfractions were isolated by rapid ultracentrifugation from 24-overweight/obese subjects randomised to a high- or low-fat diet. The lipoprotein subfractions were assessed for oxidation potential by measuring conjugated diene (CD) production and time at half maximum. We found a significant between-group difference in oxidation potential. Specifically, a high-fat diet led to increased CD production in VLDL(A-D) and HDL(2&3), and a prolongation of time at half maximum. Within-group differences found that CDs increased in VLDL(A&D), LDL(I-III) and HDL(2&3) in the high-fat group and fell in VLDL(A-C) and HDL(2&3) and increased in LDL(I&II), in the low-fat group. Furthermore, following both diets all lipoprotein subfractions, except LDL(II) in the low-fat group, were protected against oxidation. CONCLUSION: These results demonstrate that at first glance, a high-fat diet may be indicative of having heart-protective properties. However, this may be erroneous, as although the time for oxidation to occur was prolonged, once this occurred these lipoproteins had the potential to produce significantly more oxidised substrate. Conversely, a low-fat diet may be considered anti-atherogenic, as these subfractions were protected against oxidation and mainly contained fewer oxidised substrate. Thus, increased fat intake may, by increasing the oxidation product within lipoprotein subfractions, increase cardiovascular disease.

Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity.

Visceral adipose tissue (VAT) is an important risk factor for obesity-related metabolic disorders. Therefore, a reduction in VAT has become a key goal in obesity management. However, VAT is correlated with intrahepatic triglyceride (IHTG) content, so it is possible that IHTG, not VAT, is a better marker of metabolic disease. We determined the independent association of IHTG and VAT to metabolic function, by evaluating groups of obese subjects, who differed in IHTG content (high or normal) but matched on VAT volume or differed in VAT volume (high or low) but matched on IHTG content. Stable isotope tracer techniques and the euglycemic-hyperinsulinemic clamp procedure were used to assess insulin sensitivity and very-low-density lipoprotein-triglyceride (VLDL-TG) secretion rate. Tissue biopsies were obtained to evaluate cellular factors involved in ectopic triglyceride accumulation. Hepatic, adipose tissue and muscle insulin sensitivity were 41, 13, and 36% lower (P < 0.01), whereas VLDL-triglyceride secretion rate was almost double (P < 0.001), in subjects with higher than normal IHTG content, matched on VAT. No differences in insulin sensitivity or VLDL-TG secretion were observed between subjects with different VAT volumes, matched on IHTG content. Adipose tissue CD36 expression was lower (P < 0.05), whereas skeletal muscle CD36 expression was higher (P < 0.05), in subjects with higher than normal IHTG. These data demonstrate that IHTG, not VAT, is a better marker of the metabolic derangements associated with obesity. Furthermore, alterations in tissue fatty acid transport could be involved in the pathogenesis of ectopic triglyceride accumulation by redirecting plasma fatty acid uptake from adipose tissue toward other tissues.

Intravascular transfer contributes to postprandial increase in numbers of very-low-density hepatitis C virus particles.

BACKGROUND & AIMS: The physical association of hepatitis C virus (HCV) particles with lipoproteins in plasma results in distribution of HCV in a broad range of buoyant densities. This association is thought to increase virion infectivity by mediating cell entry via lipoprotein receptors. We sought to determine if factors that affect triglyceride-rich lipoprotein (TRL) metabolism alter the density and dynamics of HCV particles in the plasma of patients with chronic HCV infection. METHODS: Fasting patients (n = 10) consumed a high-fat milkshake; plasma was collected and fractionated by density gradients. HCV- RNA was measured in the very-low-density fraction (VLDF, d < 1.025 g/mL) before and at 7 serial time points postprandially. RESULTS: The amount of HCV RNA in the VLDF (HCV(VLDF)) increased a mean of 26-fold, peaking 180 minutes after the meal (P < .01). Quantification of HCV RNA throughout the density gradient fractions revealed that HCV(VLDF) rapidly disappeared, rather than migrating into the adjacent density fraction. Immuno-affinity separation of the VLDF, using antibodies that recognize apolipoprotein B-100 and not apolipoprotein B-48, showed that HCV(VLDF) is composed of chylomicron- and VLDL-associated HCV particles; peaking 120 and 180 minutes after the meal, respectively. Plasma from fasting HCV-infected patients mixed with uninfected plasma increased the quantity of HCV(VLDF), compared with that mixed with phosphate-buffered saline, showing extracellular assembly of HCV(VLDF). CONCLUSIONS: Dietary triglyceride alters the density and dynamics of HCV in plasma. The rapid clearance rate of HCV(VLDF) indicates that association with TRL is important for HCV infectivity. HCV particles, such as exchangeable apolipoproteins, appear to reassociate with TRLs in the vascular compartment.

Effects of palmitic acid on lipid metabolism homeostasis and apoptosis in goose primary hepatocytes.

Studies have shown that not only does palmitic acid promote triglyceride (TG) accumulation, but it also affects cell viability in in vitro steatosis models. However, to what degree these effects are mediated by steatosis in goose primary hepatocytes is unknown. In this study, the effects of palmitic acid on the lipid metabolism homeostasis pathway and on apoptosis were determined. The authors measured the mRNA levels of genes involved in TG synthesis, lipid deposition, fatty acid oxidation and the assembly and secretion of VLDL-TG in goose primary hepatocytes. The results indicated that palmitic acid can significantly reduce the activity of goose hepatocytes, and that palmitic acid had a significant effect on TG accumulation; however, with increasing palmitic acid concentrations, the extracellular TG and extracellular VLDL concentration gradually decreased. With increasing palmitic acid concentrations, the gene expression levels of DGAT1, DGAT2, PPARα, CPT-1, FoxO1 and MTTP (which regulate hepatic TG synthesis, fatty acid oxidation and the assembly and secretion of VLDL-TGs) first increased and then decreased; the change in PLIN gene expression was palmitic acid dose-dependent, similar to the regulatory mode of intracellular TG accumulation. In conclusion, this study clearly shows that palmitic acid can promote TG accumulation and induce apoptosis in goose primary hepatocytes, and this effect may be related to the lipid metabolism pathway.

Nano electrospray differential mobility analysis based size-selection of liposomes and very-low density lipoprotein particles for offline hyphenation to MALDI mass spectrometry.

Gas-phase electrophoresis of single-charged analytes (nanoparticles) enables their separation according to the surface-dry particle size (Electrophoretic Mobility Diameter, EMD), which corresponds to the diameter of spherical shaped particles. Employing a nano Electrospray Differential Mobility Analyzer (nES DMA), also known as nES Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA), allows sizing/size-separation and determination of particle-number concentrations. Separations are based on a constant high laminar sheath flow and a tunable, orthogonal electric field enabling scanning of EMDs in the nanometer size range. Additionally, keeping the voltage constant, only nanoparticles of a given EMD pass the instrument and can be collected on corresponding supporting materials for subsequent nanoparticle analyses applying e.g. microscopic, immunologic or spectroscopic techniques. In our proof-of-concept study we now focus for the first time on mass spectrometric (MS) characterization of DMA size-selected material. We carried out size-selection of liposomes, vesicles consisting of a lipid bilayer and an aqueous lumen employed as carriers in e.g. pharmaceutic, cosmetic or nutritional applications. Particles of 85 nm EMD were collected on gold-coated silicon wafers. Subsequently, matrix was applied and Matrix-Assisted Laser Desorption / Ionization (MALDI) MS carried out. However, we not only focused on plain liposomes but also demonstrated the applicability of our approach for very heterogeneous low density lipoprotein (VLDL) particles, a transporter of lipid metabolism. Our novel offline hyphenation of gas-phase electrophoresis (termed nES DMA or nES GEMMA) and MALDI-MS opens the avenue to the molecular characterization of size-select nanoparticles of complex nature.

Proteomic analysis of human low-density lipoprotein reveals the presence of prenylcysteine lyase, a hydrogen peroxide-generating enzyme.

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid-phase IEF has been used to resolve LDL proteins into well-defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL-associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol-binding protein, and paraoxonase-1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time-dependent generation of H(2)O(2) which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H(2)O(2) generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.

Sites of lipoprotein particles in normal rat hepatocytes.

Very low density lipoprotein (VLDL) particles are packaged by the Golgi apparatus into vacuoles which move to the plasma membrane and empty the particles into the space of Disse, via exocytosis. Traditionally, all lipoprotein-containing cisternae and vacuoles are thought to be parts of this pathway. Observations reported here demonstrate that there is a second population of lipoprotein-containing cisternae and vacuoles. This population is part of GERL, an organelle we consider to be a specialized hydrolase-rich region of the endoplasmic reticulum (ER). To our knowledge, this is the first systematic study of GERL in normal rat hepatocytes.

Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes. Evidence obtained by immunoadsorption method.

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 mum) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.

Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.

Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.

Heterogeneity of lipoprotein particles in hepatic Golgi fractions.

Newly synthesized phospholipids, labeled with either [14C]choline, [3H]myo-inositol, or [33P]phosphate, partioned preferentially (greater than 80% of total incorporated radioactivity) in a Golgi membrane subfraction, although the cognate content subfraction contained a relatively large amount of secretory lipoproteins. The labeling pattern was the same for all phospholipids tested in the two subfractions. An active exchange process of polar lipids between Golgi membranes and Golgi secretory lipoproteins is postulated as a plausible explanation for these findings. Less than half of all Golgi lipoprotein particles have the density of serum VLDLs and a similar, but not identical, biochemical composition. The remaining lipoprotein particles are characterized by a continuous spectrum of sizes, and (to the extent tested) by a lipid and protein composition different from that of serum VLDLs and HDLs. Results obtained in control experiments rule out the possibility that the heterogeneous population of Golgi lipoprotein particles is an artefact caused by our preparation procedures. It is assumed that these heterogeneous particles are immature precursors of both VLDLs and HDLs.

Cytochemistry of Golgi fractions prepared from rat liver.

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF(1), GF(2), GF(3)) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF(1) and GF(2), and along the outside of the cisternal membranes in GF(3). In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF(1) and within many VLDL-filled vacuoles in GF(1) and GF(2), indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF(3) and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF(1) and GF(2), and was not found in the cisternae in GF(3). The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF(1) and GF(3), representing primarily trans-Golgi elements from the secretory Golgi face, and GF(3) consisting largely of cis-Golgi components from the opposite face.

Thermal transitions in human plasma low density lipoproteins.

Thermal analysis of human plasma low density lipoproteins reveals a broad reversible transition encompassing body temperature. The calorimetric and x-ray scattering data identify this transition as a cooperation, liquid-crystalline to liquid phase change involving the cholesterol esters in the lipoprotein. This behavior requires the presence of a region rich in cholesterol ester within the lipoprotein.

Radioimmunoassay studies of human apolipoprotein E.

This report describes the development and first applications of a sensitive and specific double antibody radioimmunoassay for human apoplipoprotein E (apoE). ApoE was purified from the very low density lipoproteins of hypertriglyceridemic patients by heparin-agarose affinity chromatography, DEAE-cellulose chromatography, and preparative polyacrylamide gel electrophoresis. The purified apoprotein had an amino acid composition characteristic of apoE and resulted in the production of monospecific antisera when injected into rabbits. The radioimmunoassay, which was carried out in the presence of 5 mM sodium decyl sulfate, had a working range of 0.8-12 ng. The withinassay coefficient of variation was 9% and the coefficient of variation for systematic between-assay variability was 3%. Prior delipidation of samples with organic solvents did not alter their immunoreactivity. In 26 normal volunteers, the mean plasma apoE concentration was 36 +/- 13 microgram/ml. Hyperlipidemic patients (n = 68) had higher mean apoE levels. A single patient with type III hyperlipoproteinemia had a plasma apoE level of 664 microgram/ml. The plasma apoE level was independently related to plasma cholesterol and triglyceride levels in a population of 108 normal and nonchylomicronemic hyperlipidemic patients. The multiple correlation coefficient for this relationship was 0.73. Thus, variation in plasma cholesterol and triglyceride concentrations described 53% of the variation in apoE concentrations in this population. The lipoprotein distribution of apoE was investigated by agarose column chromatography and ultracentrifugation of plasma. Agarose column chromatography demonstrated that all or nearly all plasma apoE is associated with lipoproteins. In plasma from normal volunteers and hypercholesterolemic patients, apoE was found in two discrete lipoprotein classes: very low density lipoproteins and a set of lipoprotein particles with size and density characteristics similar to HDL2. In hypertriglyceridemic patients, nearly all apoE was associated with the triglyceride-rich lipoproteins.

Apoprotein composition of very low density lipoproteins of human serum.

Methods for quantitation of the major apoproteins of human serum very low density lipoprotein have been developed employing tetramethylurea, which delipidates the lipoprotein and selectively precipitates apolipoprotein B. Six soluble apoproteins are separated by electrophoresis in polyacrylamide gel. One of these is a previously unrecognized species of R-alanine (R4-alanine), more anionic than the R3-alanine polypeptide. Conditions of staining have been found which yield reproducibly linear chromogenic response with native lipoprotein and with each purified apoprotein. Recovery of protein in the seven species measured accounts for over 97% of the total in the very low density lipoprotein of normolipidemic individuals and in most samples from individuals with endogenous hyperlipemia. The mean content of apolipoprotein B in 43 samples from normolipidemic subjects was 36.9(+/-1.2 SEM)% of total protein, The distribution of the major soluble apoproteins as mean (+/-SEM) percentage of the soluble fraction was : R-serine, 5.3+/-o.5; arginine-rich, 20.6+/-1.0; R-glutamic, 10.6+/-0.4; R2-alanine, 28.3+/-0.7; R3-alanine, 26.9+/-0.5; and R4-alanine, 8.0+/-0.5. Distribution of the apoproteins was a function of particle diameter of very low density lipoprotein in fractions separated by gel permeation chromatography and by density gradient ultracentrifugation. In fractions below 700-800 A, apolipoprotein B comprised an increasing percentage of the total protein with decreasing particle diameter. Among the soluble proteins the percentage of the arginine-rich and R-serine polypeptides increased and that of the R-glutamic polypeptide declined progressively with decreasing particle size. Apoprotein distribution was similar in fractions of similar particle size from normolipidemic and hyperlipemic subjects with the exception that all fractions from the hyperlipemic subjects contained more R-serine and some, more arginine rich polypeptide. Even in the absence of chylomicrons, the distribution of soluble apoproteins in particles of diameters greater than 700-800 A was usually similar to that of the smallest particles. This suggests that the largest particles may include products of the partial catabolism of chylomicrons.