Combination of preparative and analytical ultracentrifugation methods for the investigation of low-density lipoprotein aliquots to prepare radioactive-labelled lipoproteins
Low-density lipoprotein (LDL) can be radiolabelled with different techniques and various tracers (99mTc, 123I, 125I, 131I, 11 1In, 67 Ga) and can be used as a tool for the noninvasive exploration of a variety of disorders of lipoprotein metabolism. Density-gradient centrifugation allows the simultaneous isolation of the major lipoprotein density classes, i.e. very low density lipoproteins, LDLs, high-density lipoproteins, forming discrete bands of lipoproteins in preparative tubes. The analysis of the principal lipid constituents was done in isolated fractions. The cholesterol and triglyceride values in the fractions were determined by enzymatic methods. The fraction limits of the bands were defined by means of prestaining the serum lipoproteins. Aliquots of the fractions were taken for Schlieren analysis after adjusting the density and underlayering with a salt solution, in a spinning ultracentrifugation capillary band-forming cell. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by computerised numerical and graphical techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the con stituent Gaussian subclasses. With the LDL particle concentration technique, we measured the concentration of LDL and analysed the polydispersity of the aliquots for the preparing of the radioactive-labelled lipoproteins. LDL is the major transport protein for endogenous cholesterol in human plasma. Radioiodination of LDL was performed using the iodogen method and LDL was radiolabelled with 99mTc using sodium dithionite as a reducing agent. The development of intracellularly trapped ligands permitted the sites of in vivo degradation of LDL to be identified. The methods described proved to be useful for a clear and immediate visual presentation of the concentration values of lipoproteins in the whole spectra and for the identification of the heterogeneity of lipoproteins subfractions. Combination of preparative and analytical ultracentrifugation methods allow lipoprotein aliquots to be investigated before and after radioactive labelling with isotopes to identify labelled materials.
Key wordsSingle-spin density gradient ultracentrifugation Preparative isolation of lipoproteins Investigation of lipoproteins by Schlieren curves Radioactive-labelled lipoproteins
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