The very low density lipoproteins (VLDL (MF)) from the granules of hen's egg yolk were isolated by a combination of preparative ultracentrifugation and agarose gel filtration. Ultracentrifugal, electrophoretic and chromatographic analyses were performed to characterize and assess the purity of the VLDL (MF) and apoVLDL. The Beckman Prep UV Scanner accessory to the ultracentrifuge was used in conjunction with an on-line data acquisition system for the determination of flotation/sedimentation coefficients. The software that was developed to permit rapid data analysis using this system is described. An agarose disc gel electrophoretic system was developed which, in combination with the use of sudan black B prestained lipoproteins, allowed for rapid electrophoretic analysis of large VLDL (MF) particles. Flotation velocity analysis of VLDL (MF) indicated a heterogeneous preparation with two major floating boundaries (Sf 75 and 207S). Alkylation of VLDL (MF) with iodoacetamide to prevent aggregation due to oxidation of sulfhydryl groups decreased the flotation rates of the macromolecules to Sf 41 and 108S. It is possible that the VLDL (MF) particles exist in a partially aggregated state in fresh egg yolk. Concanavalin A affinity chromatography of VLDL (MF) produced a retained and an unretained fraction. The retained fraction contained twice as much total carbohydrate as the unfractionated VLDL (MF), while the unretained VLDL (MF) contained half as much carbohydrate. All fractions were glycoproteins containing hexose, hexosamine and sialic acid sugar residues. VLDL (MF) was partially delipidated with n-heptane which preferentially extracted the neutral lipids and preserved the solubility of the phospholipid-protein residues. Two fractions were isolated by gel filtration and characterized by analytical ultracentrifugation and by their phospholipid/protein ratios. VLDL (MF) was delipidated using sodium deoxycholate (NaDOC) and ethanol-ether procedures. Removal of lipids by both methods produced aggregated apoprotein, but the NaDOC apoprotein remained soluble. That obtained from ethanol-ether was precipitated, but could be solubilized in sodium dodecylsulfate (SDS) or urea. Tetramethylurea (TMU) was used to delipidate VLDL (MF) and dissociate the apoprotein. Electrophoretic analysis indicated the presence of three apoprotein subunits, one of which was an aggregate of the other two. SDS electrophoresis of the proteins extracted from the TMU gels demonstrated that each band was heterogeneous, containing many polypeptides. ApoVLDL was dissociated into 22 polypeptides (MW ranging from 9,600-136,000 daltons) in the presence of urea, SDS and 2-mercapto-ethanol (BME). Fourteen of these bands were glycoprotein in nature, staining positively with Schiff reagent. In the absence of BME, only 18 bands were resolved, most of which contained higher molecular weight aggregates not present in the reduced samples. The apoVLDL was fractionated on 6% agarose columns containing 8M urea or 2mM SDS. Two apoprotein fractions were eluted from the SDS column, and three from the-urea column. The protein from the void volume of the urea column was an aggregate of the other two proteins. A correlation was found between the fractions eluted from the SDS column and the proteins extracted from the TMU gels. The molecular weights of the two apoproteins from the SDS column were analyzed by sedimentation equilibrium techniques. As plots of 1n c vs. r² were nonlinear, weight average molecular weights were calculated for each measured radial distance from the meniscus to the bottom of the cell. The high molecular weight component ranged from 33,000 to 166,000 daltons and the low molecular weight component ranged from 6,000 to 28,000. These values corresponded well with the molecular weight ranges of the polypeptides characterized by SDS gel electrophoresis. / Land and Food Systems, Faculty of / Unknown

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Includes bibliographical references (leaves 172-217). Examines the effects of estrogens and phytoestrogens on plasma lipoprotein levels and other risk factors for cardiovascular disease, including the oxidisability of low density lipoprotein

Includes bibliographical references (leaves 172-217). / viii, 217 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the effects of estrogens and phytoestrogens on plasma lipoprotein levels and other risk factors for cardiovascular disease, including the oxidisability of low density lipoprotein / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1999

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 3 uppsatser.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

This study was designed to investigate the mechanism(s) underlying the hypercatabolism of high density lipoprotein in Tangier disease (TD). Initially, the metabolism of normal HDL incubated in Tangier plasma in vitro was examined. Sufficient normal human HDL was added to TD plasma to raise the concentration of HDL-cholesterol to within normal levels. During incubation the concentration of HDL-cholesterol in the TD plasma fell by up to 50% in a time dependent manner. This was not seen in control samples treated in a similar manner. The loss of HDL-cholesterol in the TD could be completely accounted for by the loss of HDL-cholesteryl ester and was accompanied by a 2.3-fold increase in the concentration of HDL-triglyceride. These observations could not be accounted for by lecithin: cholesterol acytransferase activity, cholesteryl ester hydrolysis, or the triglyceride level in the TD plasma. However, preliminary evidence suggested that the activity of cholesteryl ester transfer protein in TD plasma is responsible for the changes in HDL-lipid composition. The resulting triglyceride-rich, cholesteryl-poor HDL was shown to have a normal affinity for the human skin fibroblast HDL receptor. However, this finding does not exclude other pathways of HDL catabolism that may contribute to the rapid turnover of modified HDL in TD plasma. The metabolism of normal HDL by TD fibroblasts and monocytes in vitro was also studied in an attempt to identify a cellular defect of HDL metabolism in TD. However, both TD fibroblasts and monocytes were normal with respect to their ability to bind/internalize and degrade normal HDL invitro. It is concluded that the hypercatabolism of normal HDL in TD involves alterations of HDL-lipid and protein composition prior to removal from the plasma component. Thus, these studies support the hypothesis that the defect in TD resides in the plasma and not in the cells of these patients. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy