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The influence of lipid transfer protein I on the binding and transfer of cyclosporine A between lipoproteins Kwong, Mona

Abstract

Lipid transfer protein I (LTP I) is responsible for all facilitated transfer of the core lipoprotein neutral lipids, cholesteryl ester and triglycerides, and approximately one-third of the coat lipoprotein lipid, phosphatidylcholine (PC) between different plasma lipoproteins. LTP I has a binding site for neutral lipid and monoclonal antibodies against this site has been developed. It has been found LTP I can facilitate the transfer of lipophilic drugs between different lipoprotein subclasses. Our hypothesis is the human body may appear to recognize exogenous lipophilic drugs as lipid-like particles, resulting in these compounds interacting with lipoproteins just like endogenous plasma lipids. Thus their transfer between lipoproteins may be facilitated by plasma LTP I. Previous work has demonstrated LTP I facilitates the co-transfer of CSA with the neutral lipids, CE and TG, from low density lipoproteins (LDL) to high density lipoproteins (HDL) at the neutral lipid binding site. However the role of LTP I in the facilitated co-transfer of PC and CSA between lipoproteins has not been studied. Purpose: The purpose of this work was to further understand the role of LTP I in the transfer of lipophilic drugs using CSA as a model lipophilic drug. There were two working hypotheses in this work: 1) LTP I is involved in the facilitated co-transfer of the CSA and PC between lipoproteins and 2) LTP I directly binds CSA to its neutral lipid binding site Methods: To assess if LTP I facilitated PC transfer activity regulates the plasma lipoprotein distribution of CSA, [¹⁴C]=PC- or [³H]-CSA-enriched HDL or LDL were incubated in T150 buffer containing a radiolabeled free counterpart with purified LTP I or in delipidated human plasma (1.2μg/mL) in the absence and presence of a monoclonal antibody, TP1 (30μg protein/mL), directed against LTP I at the neutral lipid binding site at 37°C. The transfer was determined by radioactive scintillation counting. To assess if CSA binds to LTP I at the neutral lipid binding site, a series of samples (LTP I alone, [³H]-CE liposomal vesicles alone, [³H]-CSA liposomal vesicles alone, [³H]-CE liposomal vesicles with LTP I, or [³H]-CSA liposomal vesicles with LTP I) were injected onto a FPLC Sephacryl XK (18mm x 20cm) column set at a sample load of 0.25mL/min for 3 minutes followed by the collection of lmL fractions from 3 to 35 at a flow rate of 0.5mL/min. CE binding to LTP I at the neutral lipid binding site was evaluated as a positive control. The LTP I, protein, and radioactivity concentrations of the eluted FPLC fractions were determined accordingly via an LTP I ELISA, protein enzymatic assay, and scintillation counting, respectively. Results: From the transfer studies in this thesis, it appears the distribution of CSA is not influenced by LTP I-mediated PC transfer activity between the lipoproteins HDL and LDL but is partially dependent on the LTP I-mediated CE and TG transfer activities as demonstrated in previous experiments. The LTP I in fractions eluted from [³H]-CE liposomal vesicles/LTP I injections and [³H]CSA-liposomal vesicles/LTP I injections were similar but the radioactive elution profile was not similar. The FPLC studies suggest CSA may bind with LTP I at the neutral lipid binding site, at other regions, to albumin, or non-specifically to the FPLC column. In conclusion, LTP I mediated transfer of CSA between lipoproteins may be a result of the direct binding of CSA to LTP I at the neutral lipid binding site. The distribution/redistribution of drugs among plasma lipoproteins by the facilitated transfer of LTP I may serve as a possible mechanism for determining the ultimate fate of drug compounds. The work presented represents a prerequisite for further studies both, in vitro and in vivo testing, of the complex between various drugs and LTP I. A challenge in the future could be the development of LTP-mediated transport and controlled release of low molecular weight drugs.

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