Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation.

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.

Preparation and investigation of 99m technetium-labeled low-density lipoproteins in rabbits with experimentally induced hypercholesterolemia.

Low-density lipoproteins (LDL) were radiolabeled in atherosclerosis studies. The aim was to investigate the biodistribution and uptake of 99mTc-labeled LDL by atherosclerotic plaques in experimentally induced hyperlipidemia. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate human LDL aliquots, to prepare radioactive-labeled lipoproteins and in rabbits with induced hyperlipidemia. Preparative density gradient centrifugation was applied for the simultaneous isolation of the major lipoprotein density classes, which form discrete bands of lipoproteins in the preparative tubes. The cholesterol and protein levels in the lipoprotein fractions were determined. LDL was subsequently dialysed against physiological solution and sterilized and apolipoprotein fragments and aggregates were eliminated by passage through a 0.22-micron filter. LDL was radiolabeled with 99mTc by using sodium dithionite as a reducing agent. Radiochemical purity and in vitro stability were controlled by paper chromatography in acetone. The labelling efficiency was 85-90% for human LDL. Two months after the start of cholesterol feeding, the total cholesterol in the blood serum had increased approximately 33-fold in comparison with the basal cholesterol content of hypercholesterolemic rabbits. Investigation of LDL was performed by Schlieren analysis after adjustment of the density of serum and underlayering by salt solution in a spinning ultracentrifugation capillary band-forming cell. Quantitative results were obtained by measuring the Schlieren areas between the sample curves and the reference baseline curve by means of computerized numerical and graphic techniques. In this manner we measured the concentrations of human LDL and analyzed rabbit LDL levels in induced hyperlipidemia. Gamma scintillation camera scanning of the rabbits was performed. Overnight fasted rabbits were injected in the marginal ear vein with 99mTc-labeled human LDL (4-10 mCi, 0.5-1.5 mg protein). The initial scintigram showing a typical blood-pool scan, gradually changing with time to an image of specific organ uptake of radioactivity by the liver, kidneys and brain and in the bladder. Gamma camera in vivo scintigraphy on rabbits revealed visible signals corresponding to atherosclerotic plaques in the aorta and carotid arteries. Our results show that 99mTc-LDL can be used to assess the organ distribution pattern of LDL in the rabbit, and to detect and localize areas of arterial atherosclerotic lesions.

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry.

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic 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 composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.