Simplified method for the diameter sizing of serum low-density lipoprotein using polyacrylamide gradient gel electrophoresis

2004 ◽  
Vol 42 (9) ◽  
Author(s):  
Hideko Tsukamoto ◽  
Izumi Takei ◽  
Keiko Ishii ◽  
Kiyoaki Watanabe
Keyword(s):  
Fatty Acids ◽  
Gel Electrophoresis ◽  
Low Density Lipoprotein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Internal Standard ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Increased Risk ◽  
Gradient Gel Electrophoresis

AbstractThe appearance of small, dense, low-density lipoprotein in serum has been demonstrated to be associated with increased risk of coronary artery disease. The molecular diameter of low-density lipoprotein is usually measured on the basis of mobility differences on polyacrylamide gel electrophoresis. However, since mobility assessed by this method is seriously affected by the increased levels of serum free fatty acids associated with hypertriglyceridemia, we used polyacrylamide gradient gel electrophoresis to eliminate the interference by fatty acids and devised a simple, precise method of polyacrylamide gradient gel electrophoresis to measure the diameter of small, dense, low-density lipoproteins in serum. We used apoferritin and thyroglobulin, which have a molecular diameter of 12.2 nm and 17.0 nm, respectively, and standard low-density lipoprotein particles having a diameter of 25.7 and 27.0 nm as calibrators, estimated by measurement of negative staining of electron microscopy. We also included apoferritin as an internal standard for polyacrylamide gradient gel electrophoresis. The only stain used was Coomassie brilliant blue, and it was used for lipoprotein staining. When we used low-density lipoprotein of 25.73 nm in diameter as a quality control specimen, the coefficient of variation of the size measurements obtained by our method was less than 1.2%. The new method markedly improved the laboratory procedure for measuring the diameter of low-density lipoproteins.

Single-spin density-gradient ultracentrifugation vs gradient gel electrophoresis: two methods for detecting low-density-lipoprotein heterogeneity compared

1991 ◽  
Vol 37 (6) ◽  
pp. 853-858 ◽  
Author(s):  
Tom P J Dormans ◽  
Dorine W Swinkels ◽  
Jacqueline de Graaf ◽  
Jan C M Hendriks ◽  
Anton F H Stalenhoef ◽  
...  
Keyword(s):  
Density Gradient ◽  
Spin Density ◽  
Gel Electrophoresis ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Density Gradient Ultracentrifugation ◽  
Low Density ◽  
Single Spin ◽  
Screening Programs ◽  
Gradient Gel Electrophoresis

Abstract Single-spin density-gradient ultracentrifugation (DUC) has proven to be a reproducible method for detection of low-density-lipoprotein (LDL) heterogeneity. Recently another method has been described for this: gradient gel electrophoresis (GGE) of serum, a method that might be more suitable for screening. To gain insight into the relationship of GGE to DUC and into their reproducibility, we determined LDL heterogeneity by DUC and GGE in 41 healthy individuals. In 90.2% (n = 37) of the subjects, the number of LDL subfractions found by both methods agreed. In addition, the density and the relative migration distance of the predominant LDL subfraction observed with the respective methods showed a strong correlation (Pearson correlation, r = 0.85, P less than 0.0001). Although it was not possible to compare for all aspects of LDL heterogeneity, these data suggest that GGE is a valid method of analysis for LDL heterogeneity. In screening programs, it may be necessary to store samples. Therefore, we studied in 24 sera the influence of storage at -80 degrees C for one, four, and 12 weeks on the LDL subfraction distribution detected by each method. LDL heterogeneity was maintained during storage under these conditions.

scholarly journals Stimulation of triacylglycerol synthesis in rat adipocytes by plasma very-low-density lipoproteins

1978 ◽  
Vol 176 (1) ◽  
pp. 169-174 ◽  
Author(s):  
P Thomopoulos ◽  
M Berthelier ◽  
D Lagrange ◽  
M J Chapman ◽  
M H Laudat
Keyword(s):  
Fatty Acids ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Rat Adipocytes ◽  
Triacylglycerol Synthesis

The effect of human plasma lipoproteins on lipogenesis from glucose has been studied in isolated rat adipocytes. The very-low-density lipoproteins increased lipogenesis specifically, whereas low-density lipoproteins and high-density lipoproteins were without effect. Such stimulation could be reproduced with partially delipidated very-low-density lipoproteins. Nod-esterified fatty acids and glycerol were also without effect. Pretreatment of the adipocytes with trypsin did not alter the effect of very-low-density lipoprotein. The presence of Ca2+ was required for the full activation of lipogenesis. The synthesis of acylglycerol fatty acids and of acylglycerol glycerol were equally increased. The effect of very-low-density lipoprotein was not additive to that of insulin. It is suggested that very-low-density lipoprotein may directly stimulate lipogenesis in fat-cells, particularly in states when the lipoproteins are present at high concentration in the circulation.

scholarly journals Comparison of Low-Density Lipoprotein Size by Polyacrylamide Tube Gel Electrophoresis and Polyacrylamide Gradient Gel Electrophoresis

2003 ◽  
Vol 119 (3) ◽  
pp. 439-445 ◽  
Author(s):  
Shaina V. Hirany ◽  
Yusra Othman ◽  
Patricia Kutscher ◽  
David L. Rainwater ◽  
Ishwarlal Jialal ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Gradient Gel Electrophoresis ◽  
Lipoprotein Size

Insufficient accuracy and specificity of polyanion precipitation methods for quantifying low-density lipoproteins

1990 ◽  
Vol 36 (12) ◽  
pp. 2109-2113 ◽  
Author(s):  
R Siekmeier ◽  
W März ◽  
W Gross
Keyword(s):  
Fatty Acids ◽  
Dextran Sulfate ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Nonesterified Fatty Acids ◽  
High Concentrations

Abstract Recently, polyanion precipitation assays for low-density lipoprotein (LDL)-cholesterol have been found to underestimate their analyte in normolipidemic samples (Siekmeier et al., Clin Chim Acta 1988;177:221-30). Therefore, accuracy, specificity, and interference by nonesterified fatty acids have been studied for three precipitants (obtained by heparin, dextran sulfate, or polyvinyl sulfate precipitation). At normal concentrations of LDL, precipitation is incomplete, whereas it is nearly quantitative at high concentrations of LDL. The polyvinyl sulfate reagent markedly responds to variations in the amount of non-LDL protein present in the precipitation mixture. In the dextran sulfate and the polyvinyl sulfate method, but not in the heparin method, the percentages of LDL precipitated notably increase as the concentration of the polyanion compound is decreased. In either assay, very-low-density lipoproteins, but not high-density lipoproteins, are significantly coprecipitated (dextran sulfate 28%, polyvinyl sulfate and heparin 66%) in a concentration-independent fashion. Increased concentrations of nonesterified fatty acids markedly interfere with the dextran sulfate and polyvinyl sulfate assay, but do not much affect results with the heparin reagent.

scholarly journals The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 186-192 ◽  
Author(s):  
Edward V. Quadros ◽  
Yasumi Nakayama ◽  
Jeffrey M. Sequeira
Keyword(s):  
Plasma Membrane ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Low Density Lipoprotein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Density Lipoprotein Receptor

Abstract The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC- cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. TCblR belongs to the low-density lipoprotein receptor family, with 2 low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. The 282-amino acid sequence includes a signal peptide of 31 residues, extracellular domain of 198 residues, a transmembrane region of 21 residues, and a cytoplasmic domain of 32 residues. The binding of TC-Cbl does not require the cytoplasmic domain or its orientation in the plasma membrane because the recombinant extracellular domain binds TC-Cbl with high affinity and specificity. The protein is heavily glycosylated and accounts for the 58-kDa size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human gene first identified as 8D6A and more recently as CD 320 encoding TCblR is located at p13.2 on the short arm of chromosome 19, spans a length of 6.224 kb, and is composed of 5 exons and 4 introns.

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