Low density lipoprotein for oxidation and metabolic studies. Isolation from small volumes of plasma using a tabletop ultracentrifuge.
A rapid method is described for the isolation of small volumes of plasma low density lipoprotein (LDL) free of plasma protein contaminants using the TL-100 Tabletop Ultracentrifuge (Beckman). The isolation of LDL was achieved by a 25 min discontinuous gradient density centrifugation between the density range of 1.006 and 1.21 g/ml, recovery of LDL by tube slicing followed by a 90 min flotation step (d = 1.12 g/ml). The purity of LDL and apolipoprotein B100 (apo B100) were monitored by agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), radial immunodiffusion and micropreparative fast protein liquid chromatography (FPLC). The ability of LDL oxidation was assessed by following absorbance at 234 nm after addition of copper ions. The functional integrity of the isolated LDL was checked by clearance kinetics after injection of [125I]-labelled LDL in estrogen-treated rats. The additional purification step led to LDL fractions free of protein contamination and left apo B100, alpha-tocopherol and beta-carotene intact. The LDL prepared in this way was free of albumin, as evident from analytic tests and from its enhanced oxidative modification by copper ions. Used for analytical purposes, this method allows LDL preparations from plasma volumes up to 570 microliters. This method is also convenient for metabolic studies in small animals, especially those relating to the determination of kinetic parameters of LDL in which LDL-apo B100 has to be specifically radiolabelled.
Himber
J
J
Pharma Division, F. Hoffmann-La Roche AG, Basel, Switzerland.
Bühler
E
E
Moll
D
D
Moser
U K
UK
D000818:Animals; D001055:Apolipoproteins B / Q000097:blood; D002338:Carotenoids / Q000097:blood; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid / Q000379:methods; D003300:Copper; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D005779:Immunodiffusion; D008077:Lipoproteins, LDL / Q000097:blood* / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D014461:Ultracentrifugation / Q000379:methods*; D014810:Vitamin E / Q000097:blood; D019207:beta Carotene
D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D019207:beta Carotene; D014810:Vitamin E; D002338:Carotenoids; D003300:Copper