J. Biochem, 1997, Vol. 121, No. 6 1096-1101
© 1997 Japanese Biochemical Society
research-article |
A Simple and Rapid Purification Procedure Minimizes Spontaneous Oxidative Modifications of Low Density Lipoprotein and Lipoprotein (a)
*Dipartimento di Medicina Clinica e Sperimentale
Dipartimento di Biochimica e Biotecnologie Mediche
Servizio di Spettrometria di Massa CNR
§Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" e CEOS, CNR, Facolta' di Medicina e Chirurgia, Universita' di Napoli "Federico II," Via Sergio Pansini, 5, 80131 Naples, Italy
1To whom correspondence should be addressed at: Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Via Sergio Pansini, 5, 80131 Naples, Italy. Phone: +39 81 7463249; Fax: +39 81 7701016 E-mail: Palumbo{at}.unina.it
Usual purification procedures of LDL and Lp(a) require numerous, extensive and prolonged sample handlings: this greatly increases the possibility of spontaneous oxidation. We have developed a method which, making use of two short-run ultracentrifugations in vertical rotors alternated by two rapid column-chromatography steps (SRUC), significantly shortens the preparation time to 3.5 h (LDL) and does not demand additional instrumentation or particular accuracy. Purification of Lp(a) requires a further wheat germ agglutinin chromatographic step, which can be accomplished within 30 min. More importantly, the method significantly reduces spontaneous oxidation as compared with classical isolation procedures. LDL isolated by the standard sequential method exhibits more extensive apolipoprotein B100 degradation, lipid peroxidation, and endogenous antioxidant (vitamin E) loss than the same lipoproteins obtained by means of the SRUC. This procedure may have be particularly valuable in experiments evaluating the effects of oxygen radical-induced modifications, especially in vitro.
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