Measurement of plasma lipoproteins by electrophoresis on polyacrylamide gel.

1977 ◽  
Vol 23 (10) ◽  
pp. 1826-1833 ◽  
Author(s):  
N Muñiz
Keyword(s):  
Clinical Application ◽  
Classical Method ◽  
Correlation Coefficients ◽  
Low Density Lipoproteins ◽  
Polyacrylamide Gel ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Densitometric Analysis ◽  
Routine Clinical Application

Abstract Results of densitometric analysis of lipoprotein peaks after electrophoresis on polyacrylamide gel and values obtained by the classical method of ultracentrifugation/precipitation correlate well. The correlation coefficients for high-density, low-density, and very-low-density lipoproteins are 0.85, 0.99, and 0.99, respectively. Thus the densitometric method for lipoprotein analysis appears to be suitable for routine clinical application.

New micromethod for measuring cholesterol in plasma lipoprotein fractions.

1977 ◽  
Vol 23 (11) ◽  
pp. 2089-2098 ◽  
Author(s):  
T J Bronzert ◽  
H B Brewer
Keyword(s):  
Plasma Density ◽  
Correlation Coefficients ◽  
Cholesterol Content ◽  
Oxygen Electrode ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Centrifuge Tube ◽  
Solvent Density

Abstract A method is described for the reliable, fast, and relatively inexpensive fractionation of plasma lipoproteins and quantitation of their cholesterol content. This procedure requires 350 microliter of plasma and can be completed within 3 h. Plasma lipoproteins (175 microliter of plasma) were prestained with Fat Red 7B and centrifuged (Beckman Airfuge) at plasma density (d = 1.006 kg/liter) and at a solvent density of 1.060 kg/liter, adjusted by adding solid KBr. Prestained centrifuged samples demonstrated the characteristic elevation of chylomicrons in phenotypes I and V, low-density lipoproteins of phenotype II, very-low-density lipoproteins in phenotype IV and V, and continuum of pink color throughout the centrifuge tube, diagnostic of the floating beta lipoprotein of type III. Centrifuged samples were separated into top and bottom fractions by aspiration. Cholesterol was quantitated with an enzymic oxygen-electrode analyzer (Beckman Cholesterol Analyzer). Correlation coefficients between cholesterol values for plasma from normal hyperlipidemic individuals obtained with the Beckman Analyzer vs. the Technicon AutoAnalyzer II and SMAC systems were 0.977 and 0.973, respectively.

The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver

1976 ◽  
Vol 54 (7) ◽  
pp. 617-628 ◽  
Author(s):  
A. Christine Nestruck ◽  
David Rubinstein
Keyword(s):  
Golgi Apparatus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Density Lipoproteins ◽  
Polyacrylamide Gel ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Relationship Analysis ◽  
Discontinuous Sucrose Gradient

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor–product relationship. Analysis of the apoproteins of the Golgi VLDL by polyacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF3, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1 showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent.The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.

Subcellular Localization of “B” Apoprotein of Plasma Lipoproteins in Rat Liver

1974 ◽  
Vol 32 ◽  
pp. 238-239
Author(s):  
Cheryl Ann Alexander ◽  
Robert L. Hamilton ◽  
Richard J. Havel
Keyword(s):  
Horseradish Peroxidase ◽  
Sodium Phosphate ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Step Method ◽  
Very Low Density Lipoproteins ◽  
Sodium Phosphate Buffer ◽  
Mammalian Liver ◽  
Long Evans

The B apoprotein is thought to be essential for the synthesis and secretion of triglyceriderich lipoproteins from mammalian liver. Antibodies were prepared against rat low density lipoproteins (LDL, density 1.025-1.045) and very low density lipoproteins (VLDL). By Ouchterlony immunodiffusion and Immunoelectrophoresis, the major determinant shared by these antisera had the characteristics of the B apoprotein. Fab fragments were prepared by the method of Porter and conjugated to horseradish peroxidase (HRP) by the two step method of Avrameas and Ternynck. Male Long-Evans rats (250-300 gm) were fasted overnight, anesthetized with Brevital (Lilly), and perfused through the portal vein with fresh 4% formaldehyde in 0.135 M sodium phosphate buffer.

scholarly journals Characterization of plasma lipoproteins separated and purified by agarose-column chromatography

1974 ◽  
Vol 139 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Lawrence L. Rudel ◽  
Jason A. Lee ◽  
Manford D. Morris ◽  
James M. Felts
Keyword(s):  
Human Plasma ◽  
Column Chromatography ◽  
Low Density Lipoproteins ◽  
High Density ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Simple Method ◽  
Lipoprotein Class

1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.

Improved Techniques for Assessment of Serum Lipoprotein Patterns. II. Rapid Method for Diagnosis of Type III Hyperlipoproteinemia without Ultracentrifugation

1973 ◽  
Vol 19 (10) ◽  
pp. 1139-1141 ◽  
Author(s):  
Heinrich Wieland ◽  
Dietrich Seidel
Keyword(s):  
Rapid Method ◽  
Serum Lipoprotein ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Agarose Gel ◽  
Low Density ◽  
Electrophoretic Separation ◽  
Very Low Density Lipoproteins ◽  
Type Iii ◽  
Type Iii Hyperlipoproteinemia

Abstract Based on the previously described technique [Clin. Chem.19, 737 (1973)] of precipitating plasma lipoproteins with polyanions after their electrophoretic separation in gels, a new method is presented for diagnosing type III hyperlipoproteinemia without need for ultracentrifugation or immunologic techniques. Used in the procedure are 0.1 mol/liter MgCl2, 1.5 g/liter heparin, and 10 g/liter NaCl to visualize selectively the very-low-density lipoproteins in agarose gel after electrophoresis. The technique is simple, inexpensive, accessible to every laboratory, and provides the answer in less than 2 h after electrophoresis of the patient’s whole serum. The results obtained are the same as those obtained by ultracentrifugation followed by lipoprotein electrophoresis of the isolated fractions.

scholarly journals Membrane receptors for very low density lipoprotein (VLDL) inhibitor of lymphocyte proliferation

Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1055-1064 ◽  
Author(s):  
PI Yi ◽  
G Beck ◽  
S Zucker
Keyword(s):  
Dna Synthesis ◽  
Lymphocyte Proliferation ◽  
Human Lymphocytes ◽  
Low Density Lipoproteins ◽  
Membrane Receptors ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Scatchard Analysis ◽  
Low Density ◽  
Very Low Density Lipoproteins

Abstract Physiologic concentrations of human plasma very low density lipoproteins inhibit the DNA synthesis of lymphocytes stimulated by allogeneic cells or lectins. In this report we have compared the effects of isolated lipoproteins [very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)] and lipoprotein-depleted plasma (LDP) on DNA synthesis by phytohemagglutinin-stimulated human lymphocytes. The relative potency for the inhibition of lymphocyte proliferation was VLDL greater than LDL greater than HDL greater than LDP. Fifty percent inhibition of DNA synthesis was observed at a VLDL protein concentration of 1.5--2.0 microgram/ml. We have further demonstrated the presence of specific receptors for VLDL on human lymphocytes. Native VLDL was more effective than LDL in competing for 125I-VLDL binding sites. Subsequent to binding to lymphocytes, 125I-VLDL was internalized and degraded to acid- soluble products. Based on a Scatchard analysis of VLDL binding at 4 degrees C, the number of VLDL receptors per lymphocyte was estimated at 28,000 +/- 1300. Based on an estimated mean binding affinity for the VLDL receptor complex at half saturation of approximately 8.8 X 10(7) liter/mole, it is estimated that 91% of lymphocyte VLDL receptors are occupied at physiologic VLDL concentrations in blood. Although the immune regulatory role of plasma lipoproteins is uncertain, we suggest tha VLDL and LDL-In may maintain circulating blood lymphocytes in a nonproliferative state via their respective cell receptor mechanisms.

scholarly journals Inhibition of lymphocyte proliferation stimulated by lectins and allogeneic cells by normal plasma lipoproteins.

1977 ◽  
Vol 146 (6) ◽  
pp. 1791-1803 ◽  
Author(s):  
J H Morse ◽  
L D Witte ◽  
D S Goodman
Keyword(s):  
Human Plasma ◽  
Rank Order ◽  
Human Lymphocytes ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Normal Plasma

Lipoproteins, isolated by sequential flotation at densities 1.006, 1.019, 1.063, and 1.21, were examined for their ability to inhibit human lymphocytes stimulated by allogeneic cells and by lectins (phytohemagglutinin-P and concanavalin A). All the classes of normal plasma lipoproteins inhibited lymphoproliferation when peripheral blood lymphocytes were cultured in autologous, heterologous, or lipoprotein-deficient plasma (d greater than 1.21). The rank order of inhibitory potency was intermediate density lipoprotein (IDL) greater than very low density lipoproteins (VLDL) greater than low density lipoproteins (LDL) greater than high density lipoproteins (HDL), regardless of the mode of stimulation. The concentrations of IDL, VLDL, and LDL required for complete inhibition of stimulated lymphoproliferation were considerably below the levels of each of these lipoproteins normally found in human plasma. In addition, the concentration of HDL required for 50-90% inhibition was in the range of HDL levels normally found in human plasma. Moreover, at relatively higher concentrations, lipoproteins suppressed the incorporation of [3H]thymidine into DNA below the levels seen with reseting, unstimulated lymphocytes. The results suggest that circulating lymphocytes may normally be highly suppressed by the combined effects of all the endogenous lipoproteins and that the lipoproteins may play important roles in vivo in modulating lymphocyte functions and responses.

Diagnosis of Type III Hyperlipoproteinemia by Chromatography of Plasma Lipoproteins on Columns Containing Agarose

1975 ◽  
Vol 21 (13) ◽  
pp. 1887-1891 ◽  
Author(s):  
James Shepherd ◽  
Christopher J Packard ◽  
Frances J Dryburgh ◽  
Jane L H C Third
Keyword(s):  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Type Iii ◽  
Elution Pattern ◽  
Type Iii Hyperlipoproteinemia ◽  
Primary Type ◽  
Relative Deficiency

Abstract Agarose column chromatography has been used to separate plasma lipoproteins into very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Applied to the diagnosis of primary type III hyperlipoproteinemia, the procedure is capable of demonstrating three characteristic and specific changes from normality in the elution pattern of lipoproteins from patients with this condition. In the type III profile there is (a) incomplete separation of VLDL from putative LDL material, (b) early elution of the type III LDL with respect to a normal LDL marker, and (c) relative deficiency of type III LDL with elution characteristics of normal LDL. We advocate the use of this method in the diagnosis of type III hyperlipoproteinemia.

Comparison of four procedures for separating apolipoprotein A- and apolipoprotein B-containing lipoproteins in plasma.

1987 ◽  
Vol 33 (9) ◽  
pp. 1597-1602 ◽  
Author(s):  
P Puchois ◽  
C Luley ◽  
P Alaupovic
Keyword(s):  
Risk Factors ◽  
Coronary Heart Disease ◽  
Heart Disease ◽  
Apolipoprotein B ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Low Density ◽  
Total Plasma ◽  
Very Low Density Lipoproteins ◽  
Apolipoprotein A

Abstract Because lipoproteins containing apolipoprotein A (ApoA-I + ApoA-II) or apolipoprotein B (ApoB) seem to exert opposite effects as risk factors for coronary heart disease, we decided to determine the separability of these two major plasma lipoproteins by procedures originally designed to separate high-density from low- and very-low-density lipoproteins. The presumably ApoB-free lipoproteins isolated from normal plasma by (a) ultracentrifugation at d = 1.063; precipitation with (b) heparin-Mn2+ or (c) phosphotungstate-Mg2+; or (d) immunoprecipitation with antibodies to ApoB were characterized by quantifying cholesterol and apolipoproteins A-I, A-II, B, C-II, C-III, D, E, F, and Lp(a). ApoA- and ApoB-containing lipoproteins were completely separated only by immunoprecipitation with antibodies to ApoB. The ApoB-containing lipoproteins isolated by other procedures always contained 4% to 20% of total plasma ApoA-I and differed substantially from one another with respect to the content of some of the minor apolipoproteins. Measuring apolipoproteins was more reliable than measuring cholesterol for monitoring this separation and for expressing the concentrations of ApoA- and ApoB-containing lipoproteins.

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