Characterization of oxysterol transport & metabolism in human serum
Authors:   , Lin Chen Yi , Morel Diane W. 
Published: July 2024

The studies summarized in this thesis were designed to explore the transport and metabolism of oxysterols in human serum in detail. When added in vitro to human serum via an ethanol vehicle, 25OHC associated with both lipoproteins and LPDS over a wide range of 25OHC concentrations, unlike cholesterol which associated exclusively with serum lipoproteins. Gel filtration of LPDS demonstrated that most 25OHC was protein-associated. Subsequent purification (ion exchange chromatography and non-denaturing polyacrylamide gel electrophoresis) revealed that 25OHC in human LPDS associated with a single protein with molecular weight of 67 kDa. Immunoprecipitation with an anti-albumin antibody demonstrated that albumin was a carrier protein for 25OHC. In competition studies, 19-hydroxycholesterol, 7[beta]-hydroxycholesterol and 7-ketocholesterol were shown to effectively compete with radiolabeled 25OHC for association with BSA, suggesting that in serum other oxysterols may also associate with albumin. When putative albumin-ligands, such as long-chain fatty acids, bilirubin, and bile salts were tested for their abilities to compete for oxysterol-binding, only bilirubin competed with 25OHC for binding to BSA, suggesting that oxysterols likely bind to albumin at or close to the bilirubin-binding region. Subsequent Scatchard analysis revealed that the binding of 25OHC to BSA is of low affinity (Kd = 5 x 10-4 M and Bmax = 0.11 mole 25OHC/mole albumin). When radiolabeled 25OHC was added in vitro to rabbit serum, more 25OHC was lipoprotein-associated in hypercholesterolemic rabbit serum than in normocholesterolemic rabbit serum, suggesting that an increase in serum lipoprotein concentrations leads to an increase in the association of oxysterols to the lipoproteins. A similar shift of 25OHC from LPDS to the lipoproteins was seen when an increasing amount of lipoproteins was mixed with a constant amount of 25OHC-labeled LPDS. As further demonstrated in Chapter 4, using phosphatidylcholine (PC) and free cholesterol (FC) liposomes as a model for serum lipoproteins, an increase in liposomal PC/FC ratio in liposomes from 0.31 to 1.93 increased the amount of liposome-associated 25OHC obtained from the 25OHC-labeled LPDS. 25OHC added to human serum was shown to be readily esterified at 37[degrees]C. This esterification was effectively suppressed by the inhibitors of serum lecithin cholesterol acyltransferase (LCAT), suggesting that LCAT is likely to be the enzyme responsible for the esterification of oxysterols in serum. The 25OHC-esters formed are monoesters, as determined by comparison of their elution on HPLC and TLC with that of chemically synthesized 25OHC mono- and diesters. Esterification led to redistribution of 25OHC in serum, substantially increasing the percentage of 25OHC associated with lipoproteins and decreasing that associated with albumin. Esterification of oxysterols in serum also modified the association of oxysterols with the lipoproteins. Esterified 25OHC remained immobile unless human LPDS was present. Suppression of this lipid-transferring effect of human LPDS by a monoclonal antibody demonstrated the role of cholesterol ester transfer protein (CETP). Esterification of 25OHC in serum substantially limited the access of serum 25OHC to both human erythrocytes as well as a human fibroblast cell line (GM 3468 A cells). Up-regulation of the LDL-receptor in fibroblasts by preincubation in serum-free medium enhanced cellular uptake of 25OHC, but did not offset the limitation in 25OHC uptake imposed by esterification. (Abstract shortened by UMI.).