Purification of very high density lipoproteins by differential density gradient ultracentrifugation

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.     

Isolation of crustacean egg yolk lipoproteins by differential density gradient ultracentrifugation

• 1.1. Egg yolk lipoproteins from four species of Crustacea were isolated by differential density gradient ultracentrifugation.
• 2.2. Egg yolk proteins from freshwater prawn, striped stone crab and mitten crab consissted of high-density lipoprotein (HDL) and lipid-free protein, while low-density lipoprotein (LDL) was present in the egg yolk protein of sand crayfish as well as HDL and lipid-free protein.
• 3.3. HDL was a major component in the egg yolk proteins from four species of Crustacea. HDL was identical to egg yolk lipovitellin.
• 4.4. Both HDL and LDL possessed phospholipid as a major lipid.
• 5.5. HDL, but not LDL, contained carotenoids. The color of HDL from mitten crab showed a reddish purple and was distinct from other Crustacea whose color was orange. The reddish purple color was characterized by an absorption flexion at 600–650 nm.

     

A potential artifact generated by pelleting viral particles during preparative ultracentrifugation

Pelleting in an ultracentrifuge produces fundamental changes in the structural and functional characteristics of some types of subviral particles of reovirus. Attention is drawn to this phenomenon as a possible source of artifact in experiments where pelleting of bioparticles is employed in the preparative procedure, as is common practice in a wide variety of studies.     

A micro-enzymatic method to measure cholesterol and triglyceride in lipoprotein subfractions separated by density gradient ultracentrifugation from 200 microliters of plasma or serum

A micro-enzymatic method was developed to measure total cholesterol (CHOL) and triglyceride (TG) in lipoproteins and their subfractions separated by density gradient ultracentrifugation. This method had a detection limit and sensitivity below 2 mg/dl and accuracy (bias to reference sera) and imprecision (coefficient of variation) of less than 3% between 2 and 30 mg/dl for both CHOL and TG. In addition, the method was in good agreement with standardized Abell-Kendall CHOL (r = 0.98) and enzymatic TG (r = 0.99) methods. Lipoproteins from 200 microliters of plasma or serum were separated by either equilibrium (EQ)- or rate zonal (RZ)-density gradient ultracentrifugation and the resulting fractions were analyzed for CHOL and TG by the micro-enzymatic method. Lipoprotein measurements by these micro-enzymatic/density gradient methods were highly correlated with standardized Lipid Research Clinic (LRC) procedures and preparative ultracentrifugation. The EQ-density gradient procedure also allowed determination of CHOL and TG in LDL and HDL subfractions within any desired density interval. These methods will facilitate the measurements and study of lipoproteins and their subfractions especially in infants, children, the elderly, and small animals. In addition, the micro-enzymatic method may be adapted to other modes of lipoprotein separation such as liquid chromatography, electrophoresis, and precipitation. CHOL or TG determinations could be made on approximately 500 density gradient fractions per hour.     

The selective isolation ofMicromonospora from soil by cesium chloride density gradient ultracentrifugation

Summary A novel method is described for the selective isolation ofMicromonospora from mixed microbial populations in soil. Microorganisms were released from soil by sonication, and the bulk of the soil debris was discarded by low-speed centrifugation. The supernatant microbial suspension was concentrated and applied to a continuous, linear (1.1–1.6 g/ml) gradient of CsCl which was then centrifuged at high speed. The gradient was fractionated, and each fraction was diluted and plated on a medium devoid of antimicrobial agents.Micromonospora were found in the 1.35–1.42 g/ml density band. Occasionally,Bacillus species were also obtained in this density range, but other nonfilamentous bacteria or actinomycetes were usually not observed. This technique allowed the isolation of portions of the soilMicromonospora population which were suppressed by conventional isolation techniques employing heat and antibiotics.     

Separation of subclasses of human serum high density lipoproteins by zonal ultracentrifugation.

An improved one-step method for the preparative separation of three subfractions of high-density lipoproteins from normal human serum has been developed. It employs the method of rate zonal ultracentrifugation in a z-60 rotor using a discontinuous NaBr gradient in the density range of 1.0-1.4. The density gradients were monitored directly by a flow-through density meter allowing the direct read-out of the actual densities in the process of filling and emptying the rotor. The separation of the three density fractions from 5 to 15 ml serum was achieved during a single 12 hours run at 59.000 rpm. The three fractions showed characteristically different patterns on polyacrylamide gel electrophoresis and differences in their lipid and protein composition.     

Cesium chloride density distributions in preparative equilibrium ultracentrifugation

A computer program has been developed for calculating equilibrium centrifugation density distributions by direct integration of tabulated data, which eliminates curve fitting. Error calculations showed that a high accuracy is required for the measurement of the data needed a determine a density distribution either by computation or by direct measurements. In particular, the variation in drop size during the fractionation of the contents of a centrifuge tube have been investigated by computer simulation. A direct measurement of the drop size indicated that it may increase somewhat during collection. In addition, plots are presented of the dependence upon seven parameters of the centrifugation required to approach equilibrium to a desired degree (based on the formula of Van Holde and Baldwin).     

Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation.

Very low density lipoproteins (VLDL) have been isolated and subfractionated on the basis of their differing flotation rates. The procedure consists of a single 45-min zonal ultracentrifugation step using a linear density gradient of d = 1.00 to 1.15 g/ml. Appropriate fractions of the zonal rotor effluent containing the entire VLDL spectrum were characterized by analytical ultracentrifugation, gel filtration chromatography, and complete chemical analysis. Flotation rates of VLDL subspecies from hypertriglyceridemic and normolipemic plasmas correlated directly with their Stokes radii and triglyceride content and inversely with their proportion of cholesterol, cholesteryl esters, phospholipids, and total protein. There was also an inverse correlation of flotation rate with the fraction of tetramethylurea-insoluble protein. This procedure provides a reliable methodology for a rapid isolation of VLDL subfractions and the accurate determination of their flotation rates.     

Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation

The major lipoprotein density classes (chylomicrons-VLDL, LDL, HDL(2) and HDL(3)) were isolated from human plasma in a two-step ultracentrifugal procedure using the Ti-14 zonal rotor. The isolation of the two major high density lipoprotein subclasses (HDL(2) and HDL(3)) was achieved in a 24-hr run using a nonlinear NaBr gradient in the density range of 1.00-1.40. The lipoproteins with a density < 1.063 found in the rotor's center were isolated in a second run of 140 min duration using a continuous linear NaBr gradient in the density range of 1.00-1.30. The isolated lipoproteins were analyzed for chemical composition and for electrophoretic mobility; purity of isolated fractions was checked by immunochemistry. The lipoproteins exhibited flotation rates, chemical compositions, and molecular weights similar to those found with the common sequential procedures in angle-head rotors. The amount of lipoprotein lipids in the bottom fraction of the zonal rotor was comparable to that of the angle-head rotor. The described method yields the main lipoprotein density classes free from albumin in a very short running time; compared with the rate-zonal techniques already in use, this method allows the quantitative separation of an additional lipoprotein density class (HDL(2)) without increasing the running time. Furthermore, this procedure proved to be suitable for isolation of plasma lipoproteins from subjects with various types and varying degrees of hyperlipoproteinemia.     

Nano-sized and micro-sized polystyrene particles affect phagocyte function

Adverse effect of nanoparticles may include impairment of phagocyte function. To identify the effect of nanoparticle size on uptake, cytotoxicity, chemotaxis, cytokine secretion, phagocytosis, oxidative burst, nitric oxide production and myeloperoxidase release, leukocytes isolated from human peripheral blood, monocytes and macrophages were studied. Carboxyl polystyrene (CPS) particles in sizes between 20 and 1,000 nm served as model particles. Twenty nanometers CPS particles were taken up passively, while larger CPS particles entered cells actively and passively. Twenty nanometers CPS were cytotoxic to all phagocytes, ≥500 nm CPS particles only to macrophages. Twenty nanometers CPS particles stimulated IL-8 secretion in human monocytes and induced oxidative burst in monocytes. Five hundred nanometers and 1,000 nm CPS particles stimulated IL-6 and IL-8 secretion in monocytes and macrophages, chemotaxis towards a chemotactic stimulus of monocytes and phagocytosis of bacteria by macrophages and provoked an oxidative burst of granulocytes. At very high concentrations, CPS particles of 20 and 500 nm stimulated myeloperoxidase release of granulocytes and nitric oxide generation in macrophages. Cytotoxic effect could contribute to some of the observed effects. In the absence of cytotoxicity, 500 and 1,000 nm CPS particles appear to influence phagocyte function to a greater extent than particles in other sizes.