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The influence of lipid transfer protein I on the binding and transfer of cyclosporine A between lipoproteins Kwong, Mona 2002

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The Influence of Lipid Transfer Protein I on the Binding and Transfer of Cyclosporine A Between Lipoproteins by Mona Kwong BSc(Pharm), University of British Columbia, 1997 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Faculty of Pharmaceutical Sciences (Division of Pharmaceutics and Biopharmaceutics) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A September 2002 © Mona Kwong, 2002 UBC Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her re p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of (Faculty of fiharma^jxhccJI^CA*V\CJ?<!) The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date ID£khr (*> />J ZtytiX . http://www.library.ubc.ca/spcoll/thesauth.html 10/10/2002 A B S T R A C T 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, [l4C]=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 (1.2pg/mL) in the absence and presence of a monoclonal antibody, TP1 (30pg 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 n 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 [ HJCSA-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. T A B L E OF CONTENTS A B S T R A C T ii T A B L E O F C O N T E N T S iv L I S T O F T A B L E S vii L I S T O F F I G U R E S viii L I S T O F A B B R E V I A T I O N S x A C K N O W L E D G E M E N T S xii D E D I C A T I O N xiii C H A P T E R 1 I N T R O D U C T I O N 1 1.1 CYCLOSPORINE A 2 1.2 PLASMA LIPOPROTEINS 8 1.3 CSA ASSOCIATION WITH PLASMA LIPOPROTEINS 14 1.4 LIPID TRANSFER PROTEIN I. 15 1.5 LTP I MEDIATED TRANSPORT OF LIPOPHILIC DRUGS 20 1.5.1 LTP I and Halofantrine 20 1.5.2 LTP I and Amphotericin B & Amphotericin B Lipid Complex 22 1.5.3 LTP I and Cyclosporine 24 1.6 OBJECTIVE 31 1.7 SIGNIFICANCE OF RESEARCH 31 1.8 HYPOTHESIS 31 C H A P T E R 2 A I M S 32 2.1 SPECIFIC AIMS 33 2.2 RATIONALE 33 C H A P T E R 3 M A T E R I A L S A N D M E T H O D S 36 3.1 MATERIALS AND REAGENTS 37 3.2 LIPOPROTEIN PREPARATION 38 3.2.1 Radiolabelling of Plasma Lipoproteins 38 iv 3.2.2 Lipoprotein separation 38 3.3 LIPID TRANSFER PROTEIN I TRANSFER ACTIVITY 41 3.3.1 Determination of amount of acceptor and donor lipoproteins to add 41 3.4 LTP I TRANSFER OF CYCLOSPORINE AND PHOSPHOLIPID 44 3.5 BINDING OF CE, CSA, AND EGG PC LIPOSOMAL VESICLES TO LTP 1 48 3.5.1 Binding of CE to LTP 1 48 3.5.1.1 Protein mass analysis 49 3.5.1.2 Radioactive profile 52 3.5.1.3 LTP I ELISA 52 3.5.1.3.1 Plate Coating 53 3.5.1.3.2 ELISA Procedure 53 3.5.2 Binding of CSA to LTP 1 57 3.6 STATISTICAL ANALYSIS 57 C H A P T E R 4 R E S U L T S 58 4.1 PC AND CSA TRANSFER BETWEEN HDL AND LDL 59 4.2 LTP I AND PROTEIN ELUTION PROFILES 63 4.2.1 LTP I Alone 63 4.2.2 LTP I and CE 64 4.2.3 LTP I and CSA 64 4.3 CE ELUTION PROFILES 67 4.4 CSA ELUTION PROFILES 69 C H A P T E R 5 D I S C U S S I O N 71 5.1 LTP TRANSFER OF PC AND CSA 72 5.2 LTP I BINDING 73 5.3 CONCLUSION 77 5.4 SIGNIFICANCE 77 5.5 PROBLEMS ENCOUNTERED AND LIMITATIONS 80 5.6 FUTURE STUDIES 81 R E F E R E N C E S 84 A P P E N D I X A P R E P A R A T I O N A N D C O M P O S I T I O N O F D E N S I T Y S O L U T I O N S F O R G R A D I E N T U L T R A C E N T R I F U G A T I O N 93 A P P E N D I X B L T P I T R A N S F E R A S S A Y S U M M A R Y S H E E T 94 A P P E N D I X C P A C K I N G A S E P H A C R Y L H R 100 X K C O L U M N 96 v A P P E N D I X D V O I D V O L U M E D E T E R M I N A T I O N O F C O L U M N 97 A P P E N D I X E M A K I N G P C E N C A P S U L A T E D L I P O S O M A L V E S I C L E S 98 A P P E N D I X F E X T R U D E D V S S O N I C A T E D [3H]-CE L I P O S O M A L V E S I C L E S 100 A P P E N D I X G E L I S A S O L U T I O N C A L C U L A T I O N S 102 A P P E N D I X H L T P I A N D P R O T E I N D E T E R M I N A T I O N O F E L U T I O N F R A C T I O N S F R O M A P L A S M A I N J E C T I O N 103 vi LIST OF T A B L E S Table 1 Physical chemical characteristics of major lipoprotein subclasses 11 Table 2 Dissociation Constants of antibody binding to immobilized LTP I 17 Table 3 %kt [3H]-CSA or [3H]-TG transfer from HDL to LDL lipoproteins with plasma source of LTP I or purified LTP I corrected for TG 30 Table 4 Net %kt [3H]-CSA transfer between HDL and LDL lipoproteins with plasma source of LTP I or purified LTP I in the absence and presence of TP1 (30 pg total protein) corrected for PC 61 vii LIST OF FIGURES Figure 1 Molecular structure of cyclosporine 6 Figure 2 Mechanism of action of cyclosporine 7 Figure 3 General structure of a lipoprotein 10 Figure 4 Reverse cholesterol transport 13 Figure 5 (A) An epitope map of LTP I based on the pattern of reactivities of mAbs with expressed LTP I fragments (B) Model of the carboxyl-terminal of LTP I based on results of epitope mapping and antibody competition experiments 18 Figure 6 Percent recovery of cyclosporine in the HDL/LPDF and VLDL/LDL fractions in human plasma supplemented with increasing amounts of exogenous LTP 1 25 Figure 7 Cholesteryl ester (CE) and cyclosporine (CSA) percent transfer from LDL to HDL, in the presence or absence of a monoclonal antibody (TP2) directed against LTP I 28 Figure 8 Cholesteryl ester (CE) and cyclosporine (CSA) percent transfer from HDL to LDL, in the presence or absence of a monoclonal antibody (TP2) directed against LTP I 29 Figure 9 Triglyceride (TG) and cyclosporine (CSA) percent transfer from LDL to HDL, in the presence or absence of a monoclonal antibody (TP1) directed against LTP 1 30 Figure 10 A representation of the separation of plasma lipoprotein fractions in an ultracentrifuge tube following ultracentrifugation in a Beckman SW41-Ti swinging bucket rotor 40 Figure 11 Representative standard curve for determination of PL 43 Figure 12 Diagram of LTP I lipoprotein transfer experiments with radiolabeled PC or CSA 47 Figure 13 Sample protein concentration curve 51 Figure 14 Basic description of the steps in the LTP I ELISA procedure 55 Figure 15 Effect of LTP I concentration on measured optical density in the LTP I ELISA using TP2 monoclonal antibody (sample standard curve) 56 Figure 16 [14C] -PC percent kt transfer from HDL to LDL in the presence and absence of a monoclonal antibody (TP1 30pg) directed against LTP 1 60 viii Figure 17 [14C]-PC and [3H]-CSA percent kt transfer from LDL to HDL in the presence and absence of a monoclonal antibody (TP1) directed against LTP 1 62 Figure 18 LTP I concentration (ng/mL) and protein concentration (pg/mL) in eluted fractions from injections onto a FPLC system with a Sephacryl HR 100 XK column (18mmx20cm) a) 9 pg LTP I alone b) LTP I 9pg + [3H]-CE liposomal vesicles (egg PC 590nmol, CE 9.06nmol) b) LTP I + [3H]-CSA liposomal vesicles (egg PC 510nmol, CSA 8.10nmol) 66 Figure 19 [3H]-CE radioactivity (CPM/mL) in eluted fractions from injections onto a FPLC system with a Sephacryl HR 100 XK column (18mmx20cm) a) [3H]-CE liposomal vesicles (egg PC 590nmol, CE 9.06nmol) b) LTP I 9pg + [3H]-CE liposomal vesicles (egg PC 590nmol, CE 9.06nmol) 68 Figure 20 [3H]-CSA radioactivity (CPM/mL) in eluted fractions from injections onto a FPLC system with a Sephacryl HR 100 XK column (18mmx20cm) a) [3H]-CSA liposomal vesicles (egg PC 510nmol, CSA 8.10nmol) b) LTP I 9pg + [3H]-CSA liposomal vesicles (egg PC 510nmol, CSA 8.10nmol) 70 Figure 21 Model of LTP I interactions with CE, TG, PC, and CSA 79 Figure F 1 Particle size distribution of sonicated [3H]-CE liposomal vesicles 100 Figure F 2 Particle size distribution of extruded [ H]-CE liposomal vesicles 100 Figure F 3 [3H]-CE Elution of sonicated versus extruded [3H]-CE liposomal vesicles injected through a Sephacryl HR 100 XK column 101 ix LIST OF A B B R E V I A T I O N S AmpB amphotericin B ABLC amphotericin B lipid complex CE cholesteryl ester CETP cholesteryl ester transfer protein CPM counts per minute CSA cyclosporine A EDTA ethylenediaminetetraacetate FPLC fast protein liquid chromatography HDL high density lipoprotein IDL intermediate density lipoprotein IL-2 interleukin-2 LCAT lecithin cholesterol acyl transferase LDL low density lipoprotein LPDF lipoprotein deficient fraction LTP I lipid transfer protein I LTP II lipid transfer protein II mab monoclonal antibody OD optical density PC phosphatidylcholine PL phospholipid PLTP phospholipid transfer protein RCT reverse cholesterol transport TCR T cell receptor TG triglyceride TP 1 monoclonal antibody directed against lipid transfer protein I TP 2 monoclonal antibody directed against lipid transfer protein I VLDL very low density lipoprotein XI A C K N O W L E D G E M E N T S I would like to thank my supervisor, Dr. Kishor Wasan, for his words of encouragement, guidance, and his never ending enthusiasm during the course of my studies. Thank you for providing an environment where I could constantly learn and challenge myself. I would also like to thank the members of my research committee, Dr. Frank Abbott, Dr. Thomas Chang, Dr. John Hill, and Dr. Marc Levine for their input and feedback. Thank you also to Dr. Wayne Riggs for his suggestions and feedback. Thank you also to Dr. Kathleen MacLeod. I would like to thank to Dr. Richard Morton for his generous donation of LTP I for the completion of this project. To my coworkers in the lab, Agatha, Alison, Manisha, Nancy, Olena, Verica, and Swamy, and to my fellow graduate students thank you for the support, for listening, for all the discussions, and most of all the friendship. To Marguerite Yee - thank you for listening - 1 have really enjoyed the time here. I would like to acknowledge the Michael Smith Foundation for Health Research for their financial support. D E D I C A T I O N This work is dedicated to my parents, Cindy Mee Sin and Simon M Wah Kwong, and to my brothers, David, Vincent and Evan. Thank you for your faith. Chapter 1 INTRODUCTION 1.1 Cyclosporine A Transplantation success and the treatment of autoimmune diseases have benefited from the introduction of immunosuppressant agents. The main aim in transplantation is to increase graft survival by preventing the development of acute and chronic rejections, with the result being an increase in patient survival. In the 1960s, steroids provided part of the basis of immunosuppressive therapy, particularly for acute rejection. For the next two decades, steroids provided the backbone of maintenance therapy [1]. However, steroids are non-selective immunosuppressants and are associated with many long-term side effects. The most clinically useful immunosuppressive agents are ones that target specific parts of the immune system. One such example is Cyclosporine A (CSA), which acts by specifically inhibiting the T cell response. Cyclosporine A is an immunosuppressant commonly used in organ transplantation and in the treatment of several autoimmune disorders [2]. Sandoz originally discovered it in the drug screening process for antifungal drugs. CSA was originally obtained from the fermentation products of the fungal species, Tolypocladium inflatum Gams [3]. The structure of CSA (Figure 1) was elucidated by X-ray crystallography as well as chemical degradation [4, 5]. It is a cyclic undecapeptide (C62H111N11O12) with a molecular weight of 1202.63. The structure consists of eleven amino acids cyclically linked with seven N-methylated and one unique 9-carbon amino acid, now named (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (MeBmf) [6, 7]. CSA is a highly lipophilic with the aliphatic amino acids responsible for this property. It has a low solubility of 0.04 mg/mL in water [8]. It has an experimental log P o c t value of 2.92 and a calculated log P o c t value of 14 [9]. The partition co-efficient, log Po ct, is a physicochemical 2 property describing a partitioning equilibrium of solute molecules between a water and an organic solvent, the organic solvent was octanol in this case. Several analogues exhibit in vitro biologic activities, but only CSA and some analogues cause selective inhibition of the immune response in vivo [2]. CSA was the most potent immunosuppressant and was purified from these analogues in 1973. The introduction of CSA in the 1980's revolutionized transplantation by not only increasing graft survival, but also by reducing the use of steroids and their associated side effects [2]. In addition to transplantation use, CSA is indicated for and has successfully treated autoimmune disorders including such conditions as psoriasis [8, 10], uveitis [11], and rheumatoid arthritis [2, 8, 12]. T cells contribute to immune response by the recognition of foreign antigens. The binding of foreign antigens results in the transcription of cytokines and creates an effective immune response by activating other components of the immune response such as T cells and B cells. T cell activation begins with the binding of an antigen to the T cell receptor (TCR) which is coupled to a cell surface protein [2]. CSA functions as an immunosuppressant by the selective inhibition of T lymphocyte signal transduction (Figure 2) [2]. It targets calcineurin, an enzyme that phosphorylates other proteins as part of the signal transduction pathways and is recognized as one of the rate limiting steps in lymphocyte activation [2]. CSA alters the activity of calcineurin to its substrates in T cells by binding to several proteins in the cell of which the cyclophilins are most important. The CSA/cyclophilin complex blocks the enzymatic function of the calcium-activated calcineurin, which in turn prevents dephosphorylation and activation of the interleukin-2 (IL-2) enhancer, the antigen-inducible transcription factors NFAT1 (nuclear factor of activated T cells), and the DNA binding proteins [2]. As a result, translocation to 3 the nucleus is prevented, IL-2 synthesis is down regulated, and an effective immune response is not generated. Due to its lipophilicity, CSA must be solubilized for administration. CSA can be administered intravenously as a 50 mg/mL solution made of an ethanol-polyoxyethylated castor oil mixture [7]. It can also be administered orally as a soft gelatin capsule (Sandimmune®) or as the newer oral microemulsion formulation (Sandimmune Neoral®). The original soft gelatin capsule formulation has a wide range in bioavailability ranging from 20% to 50% [7]. This was shown to be due more to a variability in absorption rather than a first pass effect [13]]. The new microemulsion formulation was developed to improve the absorption characteristics of CSA. It is a mixture of CSA with DL-a-tocopherol, castor oil, propylene glycol, corn oil and ethanol which forms a microemulsion upon contact with gastrointestinal fluids. The bioavailability of CSA in this microemulsion formulation is improved by 40-60% compared to the original oral formulation and there has been demonstrated reduction in the inter- and intrapatient variability of pharmacokinetic parameters [14, 15]. Food does not affect the absorption of CSA in the microemulsion formulation but a fatty meal does delay the absorption of CSA in the original oral formulation[15, 16]. Peak levels of CSA occur from 1.3 to 4 hours after oral administration [17]. CSA has a high volume of distribution with values of 3-5 L/kg reported in solid transplant patients [8]. CSA is extensively metabolized in the liver by the cytochrome P450 3A enzymes and to a lesser extent in the gastrointestinal system and the kidney [8]. More than 25 metabolites have been reported from patients taking CSA [6, 7, 18]. The majority of metabolites produced are N-demethylated and/or hydroxylated with the cyclic backbone remaining intact 4 [6, 19]. Some of these metabolites exhibit immunosuppressive activity and may contribute to the varied CSA pharmacokinetics, therapeutic outcomes and severity of side effects found in patients taking this therapy [18]. Only 0.1% of CSA is excreted unchanged in urine and elimination.is primarily via the biliary route [8]. 5 6 7 Despite its effectiveness, CSA is associated with a number of side effects. Its clinical use is limited by its nephrotoxicity which is indicated by increased serum creatinine levels and changes in creatine clearance [2]. A complicating factor accompanying CSA nephrotoxicity is distinguishing this adverse effect from chronic rejection. Nephrotoxicity can be minimized by drug combinations or by dose reduction during chronic maintenance therapy [20]. Other adverse effects of CSA include hypertension, hepatotoxicity, neurotoxicity, hirsutism, gingival hyperplasia, and gastrointestinal toxicity [8]. Many disease states have abnormal metabolic or physiological conditions that can affect drug characteristics. In the diseased state, lipid metabolism can be affected. This may result in dyslipidemic plasmas such as hypocholesterolemia and hypertriglyceridemia, which may alter the concentration of the different lipoprotein classes [21]. A number of lipophilic drugs bind to lipoproteins including halofantrine (Hf) [22, 23], amphotericin B [24, 25], probucol [26], nystatin [27], and cyclosporine [28]. Hence it is possible that changes in lipoprotein profiles may affect the association of a compound with lipoproteins as well as its distribution amongst the lipoprotein subclasses. 1.2 Plasma lipoproteins Lipoproteins are complexes carrying lipids and proteins in plasma that primarily transport lipids throughout the vascular and extravascular body fluids to cells requiring energy [21]. Lipoproteins contain a hydrophobic core of cholesteryl ester (CE) and triglyceride (TG) surrounded by a monolayer of phospholipid (PL) embedded with both unesterified 8 cholesterol and apolipoproteins (Figure 3). The types of PL include phosphatidylcholine (PC), phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin with PC being the most abundant. The lipids found in lipoproteins are delivered from the liver and intestine to other tissues in the body for either storage or in the production of energy. 9 Lipoproteins are traditionally classified according to density, with those having a high protein to lipid ratio, being of higher density [21]. There are four main categories of lipoproteins: triglyceride rich lipoproteins composed of chylomicrons and very low density lipoprotein (VLDL), low density lipoprotein (LDL), intermediate density lipoprotein (IDL), and high density lipoprotein (HDL). Table 1 Physical chemical characteristics of major lipoprotein subclasses [30] Chvlomicrons V L D L IDL L D L H D L Diameter (nm) 75-1200 30-80 25-35 18-25 . 5-12 Density (g/mL) 0.95 0.95-1.006 1.006-1.019 1.019-1.063 1.063-1.21 Composition (% dry weight) Cholesterol 5 19 38 50 19 Trielvcerides 86 55 23 6 4 Phospholipids 7 18 20 22 30 Protein 1-2 8 19 22 47 Apoproteins A l . A2 A l . A2 B-48 B-100 B-100 B-100 C1.C2.C3 C1.C2. C3 C1.C2. C3 C1.C2. C3 E E E E Chylomicrons are the largest of the lipoprotein particles and are most abundant after a meal. They range in diameter from 75 to 1200 nm with the largest component comprised of TG. VLDL has a diameter range of 30 to 80 nm, and is the major transport vehicles for endogenous TG in the plasma. It is synthesized in the liver [31] and to a lesser degree in the intestine. Excess fatty acids and carbohydrates in the body are converted into TG and are stored in VLDL. The TG in both chylomicrons and VLDL are used as a cellular energy source or stored in adipose tissue after hydrolysis by lipoprotein lipase to release free fatty acids. 11 Intermediate-density lipoproteins (IDL) remain after the TG in the VLDL has been hydrolysed. IDL undergoes further hydrolysis of core TG and form LDL which is rich in CE. LDL has a diameter of 18 to 25 nm and is the major transport vehicle for cholesterol. LDL is removed from the circulation primarily via the LDL-receptor pathway. Once endocytosed, its contents are hydrolysed with free cholesterol being stored or incorporated into membranes. HDL, the smallest lipoproteins with a diameter of 5 to 12 nm, has the highest protein content, and are involved in a process called reverse cholesterol transport (RCT) (Figure 4). RCT is the process of transporting cholesterol from peripheral tissues back to the liver [32-34]. Disc shaped nascent HDL particles are precursors to HDL. The esterification of the free cholesterol in the nascent HDL by lecithin cholesterol acyl transferase (LCAT) and its accumulation in the core result in a spherical shaped particle known as HDL 3. The further exchange of CE in HDL3 for TG from triglyceride rich lipoproteins is facilitated by lipid transfer protein I (LTP I), also known as cholesteryl ester transfer protein (CETP). The end result of this process is the mature HDL 3 particle, called HDL 2. The liver then takes the triglyceride rich lipoproteins back and the cholesterol obtained from HDL 3 particles is removed, hence the term RCT. The HDL 2 particle regenerates into HDL 3 particles by the hepatic lipase mediated hydrolysis of TG and the cycle of cholesterol removal can begin again. 12 Lipoproteins appear to have a wider biological role than simply the movement of water-soluble lipids from the systemic circulation to tissues. They are also involved in the binding and subsequent transport of a number of lipophilic compounds [22-28]. Lipophilic compounds associate with different lipoproteins with varying effects on their efficacy and toxicity and this will be discussed in the thesis. 1.3 CSA Association wi th plasma lipoproteins The lipophilic properties of CSA facilitate its association with lipoproteins in blood, serum, and plasma [24, 37]. In blood, CSA is mainly found in erythrocytes (40-50%) and plasma (30-40%) with the remainder located in granulocytes and lymphocytes [28]. In plasma, CSA is mainly associated with lipoproteins of which approximately 33-46% is associated with HDL, 21%-31% with LDL, and 8%-19% with VLDL [37]. LDL and HDL appear to be the lipoproteins mostly associated with CSA. The interaction of CSA with lipoproteins is poorly understood. Researchers have shown the activity of CSA may be dependent to which lipoprotein it is bound. One proposed consequence of CSA association with lipoproteins is the decrease in the pharmacological effect of the drug. It has been suggested that the pharmacological or toxic effects of CSA may depend to which lipoprotein CSA is bound [28]. We have further shown changes in the total and plasma lipoprotein lipid concentration and composition influence the lipoprotein binding of CSA [24]. Other studies have reported decreases in CSA activity in patients with elevated plasma triglyceride levels [38, 39], while an increase in CSA toxicity has been associated in patients with hypolipidemia [40], in heart transplant patients with high total 14 plasma cholesterol levels [41]. It appears CSA association with lipoproteins may result in alterations in its distribution and toxicity profile in different patient populations. Transplantation patients who are administered CSA often exhibit or will exhibit plasma dyslipidemias [41-44], including hypocholesterolemia and hypertriglyceridemia. Plasma LTP I activity has been shown to be influenced by a number of plasma factors including neutral lipids and free cholesterol [45]. Dyslipidemias may also have an elevation in LTP I levels [46-51]. Other work has proposed CSA to be transferred by a facilitated process between lipoproteins rather than through simple diffusion [52]. Since the human body may recognize lipophilic compounds as lipid-like particles, LTP I is hypothesized to be involved in the facilitated transfer of the lipophilic drug, CSA, similar to how it facilitates the transfer of lipids. 1.4 Lipid transfer protein I LTP I is a 476 amino acid glycoprotein with a molecular weight of 74,000 daltons [53]. LTP I and phospholipid transfer protein (PLTP or LTP II), are believed to have evolved from a common ancestor belonging to the family of lipopolysaccharide binding proteins. These two proteins share a 20% homology to each other and have homologous regions to lipopolysaccharide binding proteins [32, 54, 55]. LTP I expression between mammalian species is variable, with undetectable levels in rats and mice but moderate levels in humans and rabbits [45, 56]. In humans, LTP I is mainly synthesized in the liver [57], with lower levels synthesized in the adipose, kidney, heart and spleen [58]. It has been shown that LTP I is responsible for all facilitated transfer of the core lipoprotein lipids (CE and TG) and 15 approximately one-third of the coat lipoprotein lipid (PC) [53, 59-62]. It is involved in the removal of CE generated by the LCAT reaction through transferring from HDL3 to triglyceride rich lipoproteins [21] with a reciprocal transfer of TG [53, 63, 64]. LTP I, along with LTP II, are involved in the metabolism and remodeling of plasma lipoproteins [32, 65]. LTP I may also play a role in certain disease processes, such as atherosclerosis, by redistributing cholesterol from the anti-atherogenic HDL particles to the pro-atherogenic LDL particles. Conversely, LTP I as mentioned previously, it is also involved in the process of RCT, which removes cholesterol from peripheral tissues and is thus viewed as anti-atherogenic (Figure 4). The exact role of LTP I in the development atherosclerosis remains uncertain [58]. LTP I is regulated by cholesterol levels, with an increase in activity and expression seen in response to cholesterol [66]. In studies using human-LTP I transgenic mice, the increased activity is attributed to an increase in transcription of the LTP I gene [67]. Conversely, LTP I activity has been reduced in response to corticosteroids and lipopolysaccharides [68, 69]. The LTP I binding domain for neutral lipids (CE and TG) is located in the 26 amino acids comprising the carboxyl-terminus of LTP I [53, 59-62]. A LTP I variant lacking the amino acid residues 470-475 cannot bind CE or TG [60, 70]. Binding to LTP I is also blocked by anti-LTP I monoclonal antibodies (mAb), such as TP1 and TP2, specific to the neutral lipid binding site of LTP I [59, 61, 62, 71]. TP2 has a higher affinity than TP1. This is illustrated by Table 3 [71]. Table 3 summarizes the differences in affinity to immobilized LTP I of different mAbs raised against LTP I [71]. 16 Table 2 Dissociation Constants of antibody binding to immobilized LTP I [71] MAb Kd ( M ^ TP1 2.4 x 1(T TP2 1.9 x 10"8 TP4 2.2 x 10"8 TP 5 5.4 x 10"8 TP7 1.3 x IO-8 TP 8 2.3 x 10"7 TP9 3.9 x IO-7 TP10 5.5 x 10"8 TP11 1.2 x IO-8 TP12 5.3 x 10"7 TP13 6.2 x 1(T8 TPH 4.8 x IO"9 TP15 7.9 xlO- 9 TP16 4.8 x IO"7 TP17 4.9 x IO"8 TP18 9.7 x 10"9 TP19 4.1 x IO"9 TP20 1.6 x 10"9 Another group [71] has prepared other anti-human LTP I mAbs with a proposed LTP I epitope map (Figure 5). The domain is comprised of an amphipathic helix, consisting of a charged/polar residue face and a hydrophobic residue face to which binding of neutral lipid occurs [72]. The binding of neutral lipids may induce a conformational change which in turn enhances binding to lipoproteins [62]. 17 f A.Z 183 261 331 366 410 TP14 T P 6 TP12 T P 2 T P 9 TP1S TP11 TP10 T P 5 T P 1 3 TP18 TP7 TP17 TP20 T P 8 T P 1 8 (A) TP19 TP14 Figure 5 (A) An epitope map of LTP I based on the pattern of reactivities of mAbs with expressed LTP I fragments (B) Model of the carboxyl-terminal of LTP I based on results of epitope mapping and antibody competition experiments. Antibodies that are capable of greater than 20% inhibition of LTP I-mediated CE transfer are underlined (from [71]) 18 LTP I facilitated lipid transfer has been proposed to occur by one of two methods: 1) a carrier mediated process [73], in which LTP I acts a shuttle between the donor and acceptor lipoprotein and 2) a ternary mechanism [74], in which a donor-LTP I complex collides with an acceptor forming a ternary complex. In the carrier-mediated process LTP I binds to the donor lipoprotein (HDL), which could induce a conformational change in the protein, exposing a neutral lipid binding site. A molecule of CE is then relocated to the this binding site and LTP I dissociates from the HDL particle. This LTP I/CE complex then diffuses and will eventually bind to an acceptor lipoprotein (VLDL or LDL) where the CE is exchanged for a molecule of TG. The LTP I then dissociates from the acceptor lipoprotein and the process repeats. In the ternary mechanism of lipid exchange, the LTP I/HDL complex would not dissociate from each other, rather this complex would collides and binds to the acceptor lipoprotein to form a ternary complex. The exchange of a CE molecule for a TG molecule is facilitated by LTP I. Once this exchange is completed, the three components dissociate into their separate entities and the process repeats again. LTP I is believed to transfer these lipids via a carrier mediated mechanism rather than by the formation of a ternary complex comprised of LTP I, the donor, and the acceptor lipoproteins [75]. Since the human body may recognize lipophilic compounds as lipid-like particles, LTP I was originally hypothesized to be involved in the facilitated transfer of lipophilic drugs amongst lipoproteins at the same neutral binding lipid site as where it facilitates the transfer of lipids. 19 Evidence to support this hypothesis is presented in the subsequent sections with the discussion of the role of LTP I in the lipoprotein distribution of Hf, AmpB, and CSA. The understanding of the mechanism by which these drugs distribute into lipoprotein fractions may be important in understanding their efficacy and toxicity. The aims presented in this thesis further explores the role of LTP I in the distribution of CSA between lipoproteins. 1.5 L T P I mediated transport of lipophilic drugs 1.5.1 LTP I and Halofantrine Hf is a lipophilic agent indicated for the treatment of malaria, particularly against Plasmodium falciparum and other multi-drug resistant strains [76]. One study has suggested HDL may be a source of phospholipids for Plasmodium falciparum to support its growth in vitro [77]. There has been observed decreases in plasma HDL-cholesterol levels and increases in plasma VLDL-TG levels in patients infected with malaria compared to non-infected patients [78, 79]. Hf has been shown to bind to lipoproteins upon incubation in human blood [23] and in plasma [22]. It was observed that Hf interacts and binds to LDL and HDL upon incubation in human blood from both malaria infected individuals and noninfected individuals[23]. Another study has observed the relative pre-prandial and post-prandial lipoprotein profiles may influence the lipoprotein distribution of Hf within fasted and fed beagle plasma [80]. This group has also demonstrated the IC50 of Hf significantly increases when incubated in the presence of 10% post prandial serum with an elevation in triglyceride rich lipoproteins compared with normolipidemic serum in an in vitro culture of Plasmodium falciparum [81]. These differences may be 20 regulated by core neutral lipoprotein lipids [22]. As stated before, LTP I is involved in the facilitated transfer of neutral lipids between lipoproteins. Hence, from these studies, experiments were done to determine the role of lipoprotein lipid and protein concentration and the role of LTP I in the plasma distribution of Hf [82]. In these studies, Hf (1000 ng/mL) was incubated in plasma from three different patients (hypo-, normo-, or hyper-lipidemic human plasma) for 60 minutes at 37°C. Following incubation, the plasma was cooled to 4°C to prevent drug redistribution, then separated into its lipoprotein and lipoprotein deficient fractions by density gradient ultracentrifugation. Each fraction was subsequently quantified for Hf by high pressure liquid chromatography [82]. The activity of LTP I in these dyslipidemic plasma samples was determined in terms of its ability to transfer CE between LDL and HDL. The fraction of lipids and drug transferred (kt) was calculated as described by Pattnaik and Zilversmit [83]. kt = -In (1-A/Do) where D 0 and At are the radioactivities of the donor at time 0 and the acceptor at time t, respectively. The constant k is the fraction of the radio-labeled compound transferred per unit time (t). Acceptor radioactivity in the absence of LTP I (usually < 2-3%) is subtracted before calculating kt values. Calculations assume steady-state conditions in which all lipid and drug transfer are in an exchange process. From these experiments, it was observed, that as the total plasma and lipoprotein lipid concentrations increased from the hypolipidemic plasma to the hyperlipidemic plasma, LTP I 21 activity increased from 2.2 ± 0.8 %kt to 14.6 ± 2.5 %kt. As the LTP I mediated transfer of CE increased, the amount of Hf recovered in the HDL fraction decreased with an increased amount recovered in the triglyceride rich lipoprotein fractions. In this work [82], fasted normolipidemic plasma was further supplemented with an exogenous supply of LTP I (0.67pg protein/mL) to determine if these differences in Hf lipoprotein distribution are a function of LTP I. A significantly greater percentage of Hf was recovered in the TRL fraction with a significantly lower percentage of Hf recovered in the LDL, HDL, and lipoprotein deficient plasma fractions compared to normolipidemic plasma [82]. Hence, these studies demonstrated as LTP I activity increases, the proportion of Hf associated with HDL decreases and increases with the TRL fraction. Since LTP I catalyses the transfer exchange of neutral lipids between lipoproteins, the studies suggest Hf plasma distribution is related to its lipoprotein neutral lipid content or to LTP I. 1.5.2 LTP I and Amphotericin B & Amphotericin B Lipid Complex Despite the development of a number of new lipid-based anti-fungal formulations [84], AmpB, formulated as a colloidal suspension, remains one of the most effective and affordable agents in the treatment of systemic fungal infections [84]. However, the clinical use of AmpB has been limited by dose-dependent renal toxicity. AmpB's association with serum LDL is involved in the development of AmpB-induced kidney toxicity. This association is regulated by an increase in LTP I [63, 64, 85-87]. However, when AmpB is incorporated into non-toxic phospholipids to form AmpB-lipid complex (ABLC), 22 AmpB binding to serum LDL decreases and AmpB-induced kidney toxicity is significantly reduced [88, 89]. When AmpB was incubated in human serum for 1 hour at 37°C, over 70% of the drug was bound with serum HDL [90]. However, the preliminary findings of the research suggest that an increase in LTP I concentration increased the binding of AmpB with serum LDL [90]. This implies that changes in LTP I concentration may regulate the distribution of AmpB between the HDL and LDL fractions of human serum. This observation was recently supported by other research [91] which reported AmpB forms a complex with plant lipid transfer protein I, even though no binding affinity could be determined. An increase in LTP I concentration, however, did not increase the binding of AmpB with serum LDL when ABLC was incubated in human serum [90]. Furthermore, the presence of ABLC decreased the ability of LTP I to transfer CE from HDL to LDL [90]. These observations suggest that the presence of lipid complexes in serum result in the reduction of LTP I-mediated transfer of CE from HDL to LDL. Since AmpB binds to unesterified and esterified cholesterol in serum [92], this finding may partially explain the decreased association of AmpB with serum LDL when formulated into these lipid complexes. Taken together these, studies suggest that AmpB is related to its lipoprotein neutral lipid content. 23 1.5.3 LTP I and Cyclosporine As mentioned in previous sections, LTP I has a role in the distribution of the lipophilic drugs, Hf and AmpB. In the research of this thesis, CSA was used as a model of a lipophilic drug to further explore the relationship with LTP I. Because CSA's activity and toxicity may be dependent upon which lipoprotein it is bound to and differences in activity and toxicity are noted in patients with different lipoprotein lipid concentration and composition [38-41], understanding the mechanism of how this drug is transferred from one lipoprotein to another may be important in understanding the efficacy and toxicity of this drug. Exploring the relationship of CSA with LTP I may also be important because of its relationship with dyslipidemic plasmas. Transplantation patients who are administered CSA often exhibit plasma dyslipidemias [41-44], and dyslipidemias may have an elevation in LTP I levels [46-51]. Previous data suggest LTP I has a direct role in lipoprotein distribution of CSA [93]. CSA incubated in plasma results in a predominant association with HDL and LDL [24]. When this plasma was supplemented with exogenous LTP I, CSA redistributes from LDL to HDL, as shown in Figure 6 [93]. 24 Amount of LTP I added (|ig protein) Figure 6 Percent recovery of cyclosporine in the H D L / L P D F and V L D L / L D L fractions in human plasma supplemented with increasing amounts of exogenous LTP I. Cyclosporine was incubated at a concentration of lOOOng/mL for 60 min at 37°C. After incubation, plasma was separated by affinity chromatography and the percentage of cyclosporine recovered in each fraction was determined by radioactivity. Data expressed as mean SD (n=6). (From [93]). LTP I has been shown to facilitate the transfer of mainly the neutral lipids contained in the core of lipoproteins at the neutral lipid binding site located in the 26 amino acids comprising the carboxyl-terminus of LTP I. We had originally hypothesized LTP I may transfer lipophilic compounds in the same way as lipids. Thus, we investigated the role of the neutral core lipids, CE and TG, with the transfer of CSA at the neutral lipid binding site. We have demonstrated the transfer of CSA between LDL and HDL is partially dependent on the LTP I facilitated transfer of CE and TG [93, 94]. Briefly, in these experiments there was an incubation of radio-labeled enriched CE or CSA HDL or LDL with a corresponding non-labeled counterpart in the presence of a purified source of LTP I in T150 buffer or in the presence of a delipidated source of plasma containing LTP I and other endogenous transfer proteins. In a further experiment, a mAb against the neutral lipid binding site of LTP I was also added. Transfer of the radio-25 labeled lipoprotein compound of interest to its non-radiolabeled counterpart was quantitated for each of these experiments. The experiments were repeated for radio-labeled enriched TG or CSA lipoproteins. To account for diffusional transfer, radio-labeled donor lipoproteins and its corresponding non-labeled counterpart were added in the volume equivalent tolOpg cholesterol or triglyceride. The fraction of lipids and drug transferred (kt) was calculated as described by Pattnaik and Zilversmit, as previously described [83]. These studies showed LTP I is partially involved in the transfer of CSA from LDL to HDL. Transfer rates were increased when the experiment was performed in delipidated plasma compared to the experiment in T150 buffer system with CE (Figure 7). The percent transfer of CSA from only LDL to HDL was greater in T150 buffer than in human plasma with TG [94]. When the transfer of CE and CSA (Figure 7) and TG and CSA (Figure 9) were determined in the presence of a mAb against the neutral lipid binding site, the percent transfer from LDL to HDL were significantly decreased compared to the controls. This suggests the neutral lipid binding site of LTP I has a role in the facilitated co-transfer of either CE or TG with CSA. The transfer of CSA from HDL to LDL does not appear to be LTP I facilitated. When a mAb against the neutral lipid binding site of LTP I was added in amounts to significantly reduce the transfer of CE (Figure 8) from HDL to LDL there was no significant change in the transfer of CSA. An undetectable transfer of CSA from HDL to LDL was observed in T150 buffer containing purified LTP I when TG transfer was observed (Table 3). These findings suggest that along with LTP I, there may also be other factors involved in the transfer of CSA, such as other endogenous plasma transfer proteins. Our data also suggest LTP I may partially regulate 26 CSA distribution amongst different lipoprotein classes in human plasma. This transfer could be explained by CSA: • binding to lipoprotein core neutral lipids, CE or TG, or to the coat phospholipid, PC, which then binds to LTP I at the neutral lipid binding site • binding to the neutral lipid binding site of LTP I, independently of the lipids • binding to an alternative site(s) on LTP I Since LTP I is also involved in the transfer of one-third of the coat lipoprotein lipid, the role of PC in the transfer of CSA was further investigated in this thesis to determine if only the neutral lipids in the core of the lipoproteins play a role in the transfer of CSA at the neutral lipid binding site. The research in this thesis also investigated if CSA independently binds to LTP I at the neutral lipid binding site. 27 WlOnwl Tl'2 VMlfiTPl Without W i W I K T150 Buffer 'Human Plasma Figure 7 Cholesteryl ester (CE) and cyclosporine (CSA) percent transfer from LDL to HDL, in the presence or absence of a monoclonal antibody (TP2) directed against LTP I. Experiment involved incubation of radiolabeled CE- and CSA-enriched LDL with unlabeled HDL (at 10 pg lipoprotein cholesterol) for 60 min at 37°C in T150 buffer supplemented with LTP I (1.0 pg protein/ml) or delipidated human plasma which contained 1.0 pg protein/ml of LTP I. Data expressed as mean ± S.D. (n = 6). *P < 0.05 vs. CE or CSA percent transfer without TP2. (From [931). 28 115$ Buffer Bximw Plasma Figure 8 Cholesteryl ester (CE) and cyclosporine (CSA) percent transfer from HDL to LDL, in the presence or absence of a monoclonal antibody (TP2) directed against LTP I. Experiment involved incubation of radiolabeled CE- and CSA-enrichedHDL with unlabeled LDL (at 10 pg lipoprotein cholesterol) for 60 min at 37°C in T150 buffer supplemented with LTP I (1.0 pg protein/ml) or delipidated human plasma which contained 1.0 pg protein/ml of LTP I. Data expressed as mean ± S.D. (n = 6). *P < 0.05 ro. CE or CSA percent transfer without TP2. (From [93]) 29 • TG Transfer 30 25 ^ 20 >*-to c ra c a> u i_ CD a. 15 a x O 10 5 0 • CSA Transfer Wi thou t TP1 With TP1 ** * T 1 5 0 Withou t TP1 Plasma With TP1 Figure 9 Triglyceride (TG) and cyclosporine (CSA) percent transfer from L D L to HDL, in the presence or absence of a monoclonal antibody (TP1) directed against LTP I. Experiment involved incubation of radiolabeled TG- and CSA-enrichedLDL with unlabeled HDL (at 10 ug lipoprotein triglyceride) for 60 min at 37°C in T150 buffer supplemented with LTP I (1.0 ug protein/ml) or delipidated human plasma which contained 1.0 ug protein/ml of LTP I. Data expressed as mean ± S.D. (n = 6). *P < 0.05 vs. TG or CSA percent transfer without TP1. (From [94]) "1 O Table 3 %kt [ H]-CSA or [ H]-TG transfer from HDL to LDL lipoproteins with plasma source of LTP I or purified source of LTP I corrected for TG Treatment Groups Net TG transfer (kt%) Net CSA transfer (kt%) Plasma LTP I 18.6 ± 2.1 16.8 ± 3 . 2 Purified LTP I 32.4 ± 1.7* ND Data expressed as mean ± standard deviation (n=4); *p < 0.05 vs. TG percent transfer with plasma LTP I; ND = nondetectable Experiment involved incubation of radiolabeled T G - and CSA-enrichedLDL with unlabeled H D L (at 10 ug lipoprotein triglyceride) for 60 min at 37°C in T150 buffer supplemented with LTP I (1.0 ug protein/ml) or delipidated human plasma which contained 1.0 ug protein/ml of LTP I 30 Objective The overall aims of this proposal was to understand the role of LTP I in the facilitated co-transfer of CSA with PC between lipoproteins and to investigate if CSA binds to LTP I at its neutral lipid binding site. 1.6 Significance of research Dyslipidemias may result in a different pharmacological or toxic profile of a lipophilic drug by differences seen in its lipoprotein distribution. Many transplantation patients exhibit lipid disturbances [41-44] and dyslipidemias may also have an elevation in LTP I levels [47-51, 95]. Since LTP I facilitates the transfer of mainly lipids between lipoproteins in the body then it may also facilitate the transfer of lipophilic drugs. Understanding the role of LTP I in the transfer of lipophilic drugs between lipoproteins may help provide a mechanism by which these drugs distribute into lipoprotein fractions. This may be important in understanding the efficacy and toxicity of lipophilic drugs when distributed into lipoproteins of patients with different lipid profiles. 1.7 Hypothesis There are two hypotheses in this thesis: 1. LTP I is involved in the facilitated co-transfer of the CSA and PC between lipoproteins. 2. LTP I directly binds CSA to its neutral lipid binding site. 31 Chapter 2 AIMS 2.1 Specific Aims Aim 1: To determine if LTP I there is facilitated co-transfer of PC, the major coat lipid of lipoproteins, with CSA between lipoproteins. Aim 2: To determine if CSA directly binds to LTP I at the neutral lipid binding site by using a modified fast protein liquid chromatography (FPLC) method. 2.2 Rationale Aim 1: In a preliminary experiment, we have demonstrated LTP I can influence the lipoprotein distribution of CSA [93]. In this experiment, the CSA incubation in plasma resulted in a predominant association with HDL and LDL [93]. When the plasma was supplemented with exogenous LTP I, CSA redistributed from LDL to HDL [93]. This result suggested LTP I may have a role in facilitating the transfer of CSA. Hence, we started to study the role of LTP I in the transfer of CSA in in vitro systems utilizing isolated [ Hj-CSA lipoprotein complexes. Because LTP I primarily is involved in the transfer of the neutral lipids, CE and TG, between lipoproteins at the neutral lipid binding site, we 33 started to investigate the role of LTP I in the co-transfer of CSA along with CE or TG at the neutral lipid binding site as previously discussed. We reported LTP I facilitated transfer of CSA between HDL and LDL is only partially dependent on LTP I's CE and TG transfer activities from LDL to HDL [93, 94]. This was shown by evaluating CE, TG, and CSA co-transfer between lipoproteins, using radioactivity, mAbs directed against the neutral lipid binding site of LTP I, and two different sources of LTP I; lipoprotein-deficient plasma or a purified source. The lipoprotein-deficient plasma source of LTP I also contained other endogenous transfer proteins. Since LTP I is also involved in the transfer of one-third of the lipoprotein coat lipid, the role of the coat lipid, PC, in the transfer of CSA at the neutral lipid binding site was further investigated in this thesis. This was done to determine if only the neutral lipids in the core of the lipoproteins play a role in the transfer of CSA. To date, the role of the main coat lipoprotein lipid, PC, on the transfer of CSA between lipoproteins with LTP I has not been investigated. The purpose of this aim was to establish if LTP I facilitated a co-transfer of PC with CSA between lipoproteins. An assay to measure LTP I facilitated transfer of PC and CSA, utilizing a mAb against the neutral lipid binding site of LTP I was developed to answer this aim. A i m 2: From the results of the experiments completed in Aim 1 and in previously published experiments [93, 94], it appears CSA is partially dependent on the LTP I facilitated transfer of the core neutral lipids, CE and TG, between lipoproteins but not of PC at the neutral lipid 34 binding site. LTP I was only involved in the some of the facilitated co-transfer of CE and TG with CSA between lipoproteins from LDL to HDL. This suggests a number of possibilities: • Spontaneous transfer of CSA and/or • Facilitated transfer of CSA by other endogenous transfer proteins and/or • Facilitated transfer of CSA by LTP I at the neutral lipid binding site and/or at other binding site(s) either independently or co-transferred along with the core neutral lipids, CE or TG To start understanding if CSA is independently transferred at the neutral lipid binding site of LTP I, the purpose of the second aim was to investigate if CSA binds directly to the neutral lipid binding site of LTP I. This was accomplished by the determination of LTP I, protein, and radioactive concentrations of eluted FPLC fractions, when appropriate, following an injection onto the column (LTP I alone, [ H]-CE liposomal vesicles alone, [ Hj-CSA liposomal vesicles alone, [3H]-CE liposomal vesicles with LTP I, or [3H]-CSA liposomal vesicles with LTP I). 35 Chapter 3 MATERIALS AND METHODS 36 3.1 Materials and Reagents Radio-labeled CSA ([mebmt-$- H] Cyclosporine A dissolved in 100% ethanol solution; Specific Activity, 5.82 mCi/mg), radio-labeled PC (L-3-Phosphatidylcholine,l-palmitoyl-2-[l-14C]oleoyl dissolved in a 50:50 toluene:ethanol solution; Specific Activity,72.2pCi/mg), and radio-labeled cholesteryl ester ([la,2oc(n)- H]-cholesteryl oleate dissolved in 100% ethanol solution; Specific Activity, 73.4 mCi/mg) were purchased from Amersham Pharmacia Biotech (Oakville, Ontario, Canada). Tris-HCl, sodium azide, ethylenediaminetetraacetate (EDTA), sodium bromide, sodium chloride, sodium carbonate, sodium bicarbonate, sodium phosphate (anhydrous), potassium phosphate monobasic, potassium chloride, bovine serum albumin, cholesteryl oleate, sodium hydroxide, p-nitrophenyl phosphate, triton x-100, and cyclosporine A were purchased from Sigma Chemical Company (St. Louis, MO, USA). LTP I (1690 pg protein/mL, 30pg LTP I/mL), stabilized with albumin in a buffer was donated by Dr. Richard E. Morton (Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA) and was purified from human lipoprotein-deficient plasma as previously described [63]. Scintillation fluid was purchased from ICN Ltd (Montreal, QC, Canada). Phospholipids were bought from Avanti Lipids (Alabaster, AL, USA). Affinity purified goat anti-mouse phosphatase conjugate was purchased from Promega (Madison, Wl, USA). Normolipidemic fasted pooled human plasmas (total plasma cholesterol concentrations ranging 135-150 mg/dL were obtained from Canadian Blood Services (Vancouver, BC, Canada) and Bioreclamation (Hicksville, NY, USA). TP 1 and TP 2 were obtained from the Ottawa Heart Institute (Ottawa, ON, Canada). Total plasma and lipoprotein triglycerides, cholesterol (free and cholesteryl ester), phospholipids and protein 37 concentrations were determined by enzymatic assays purchased from Sigma Diagnostics (St. Louis, MO, USA), Hoffmann-La Roche Diagnostics (Laval, QC, Canada), and Biorad Laboratories (Mississauga, ON, Canada). BSA protein standards were purchased from Sigma Diagnostics (St. Louis, MO, USA). Sterile 0.2 um syringe filters were purchased from Millipore (Bedford, MA, USA). A XK column (18mm x 20cm) and Sephacryl HR 100 packing were obtained from Amersham Pharmacia Biotech (Oakville, ON, Canada). Chloroform and Nunc Maxisorp brand 96 well plates were obtained from Fisher Scientific (Nepean, ON, Canada). A Lipex ® extruder and lOOnm filters were obtained from Northern Lipids (Vancouver, BC, Canada). 3.2 Lipoprotein Preparation 3.2.1 Radiolabeling of Plasma Lipoproteins ['4C]-PC (2300 ng/ml) or [3H]-CSA (1000 ng/ml) was incubated in plasma for 20-24 hours in plasma at 37°C. The plasma was then separated into its HDL and LDL fractions by the ultracentrifugation procedure described below. 3.2.2 Lipoprotein separation The lipoprotein subfractions, HDL and LDL, were obtained by separation of plasma by gradient ultracentrifugation [96, 97]. An additional sequential ultracentrifugation for concentrated HDL 14 3 was done. Labeling of the donor lipoproteins with [ C]-PC or [ H]-CSA was done as described 38 above, prior to separation. All plasma and sodium bromide solutions were kept at 4°C prior to layering. Human plasma samples (3.0 mL) were placed in centrifuge tubes and its solvent densities were adjusted to 1.25mg/mL by the addition of 1.02 g of sodium bromide. This was followed by layering with 2.8mL of the appropriate density solutions on top (1.21mg/mL, 1.063mg/mL, and 1.006mg/dL) to form a gradient. Following gradient ultracentrifugation on a SW 41 Ti swinging bucket rotor (Beckman, Palo Alto, CA, USA) at 40,000 rpm for 18 hours at 15°C in a Beckman L8-80M Ultracentrifuge (Beckman, Palo Alto, CA, USA), only the LDL-rich and HDL-rich sub-fractions were recovered from each tube. Figure 10 shows a sample tube with four visibly distinct regions represented by the VLDL, LDL, HDL, and lipoprotein deficient plasma fractions. To further concentrate the HDL-rich fraction, 0.9g sodium bromide was added to each lOmL of HDL-rich fraction, and the resulting solution was spun at 40,000 rpm for an additional 7 hours at 15°C. The HDL-rich concentrated fraction was then recovered. Fractions were further purified by dialyzing against sodium chloride 0.9% solution (500mL) for 16-20 hours at 4°C using dialysis tubing with a molecular weight cut-off of 500 or 1000. Following dialysis, lipoprotein fractions were filtered through a 0.2-micron filter. The dialysis and filtration steps were performed to remove PC or CSA not incorporated into either the coat or core of HDL and LDL. 39 Very low density lipoproteins Low density lipoproteins High density lipoproteins Lipoprotein-deficient plasma Figure 10 A representation of the separation of plasma lipoprotein fractions in an ultracentrifuge tube following ultracentrifugation in a Beckman SW41-Ti swinging bucket rotor at 40,000 rpm and 15°C for 18 hours in a Beckman L8-80M ultracentrifuge. Separation is based on the density of the plasma components; therefore the constituents of low density will rise to the top and those of heavier density will remain at the bottom. Only the LDL and HDL fractions were collected and used. 40 3.3 L ip id Transfer Protein I Transfer Activi ty 3.3.1 Determination of amount of acceptor and donor lipoproteins to add For all transfer studies, radio-labeled HDL and LDL and their non-radio-labeled acceptor counterparts were added in equal amounts by using a volume equivalent to 20pg of PL, as determined by an enzymatic assay and calculated from a standard curve. This was done to account for diffusional transfer. The following enzymatic procedure was used to determine phospholipids concentration. Phospholipase D is used to catalyse the conversion of PL to produce choline and phosphatidic acids. The choline is then oxidized by choline oxidase to yield betaine and hydrogen peroxide (H2O2) which then reacts with 4-aminophenazone and phenol in the presence of peroxidase to produce 4-(p-benzo-quinone-monoimino)-phenazone (the chromagen) and water [98]. The chromagen is a coloured dye with an absorbance maximum at 500nm. In summary, the series of reactions are as follows: phospholipids + H20 phospholipase D choline + phosphatidic acids choline+ 202 + H20 choline oxidase' > betaine + 2H202 2H202 + 4-aminophenazone + phenol peroxidase 4-(p-benzoquinone-monoimino) phenazone + 4H20 41 Phospholipid reagent was prepared by dissolving the contents of one enzyme reagent bottle in 40mL of the provided buffer solution. This working reagent solution contained phenol 20mmol/L, 4-aminophenazone 8 mmol/L, phospholipase D > 1000 U/L, choline oxidase > 1400 U/L, and peroxidase > 800U/L. This solution was stable for up to two weeks when stored at 2 to 8°C. A standard curve was developed by serial dilution of the standardized stock solution of 300 mg phospholipids/dL equivalent to 54.1 mg/dL choline chloride rather than using a one point standard as suggested by the kit. This standard curve was preformed each time new batches of lipoproteins were made. 10 pL of the sample or standard was added to 1.5 mL of the prepared phospholipid reagent solution, mixed, and incubated at 37°C for 10 minutes and read against the reagent blank at 500nm using a Hewlett Packard 8452A Diode Array Spectrophotometer interfaced with a Hewlett Packard Vectra N2 Personal computer with UV-Vis software. The absorbance values of the standards were used to generate a standard curve (Figure 11) and the volume equivalent to 20pg of PL of radio-labeled HDL and LDL and their non-radio-labeled acceptor counterparts were calculated from the fitted linear equation. 42 3.4 L T P I transfer of cyclosporine and phospholipid To investigate the hypothesis that the transfer of CSA between lipoproteins is regulated by LTP I facilitated PC transfer activity, two strategies involving the supplementation and inhibition of LTP I were used. LTP I transfer activity was measured following a modified protocol of Pattnaik and Morton [63, 64, 83, 99]. The first strategy involved incubation of [3H]-CSA-enriched HDL or LDL with a corresponding drug-free lipoprotein counterpart in a T150 buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% sodium azide, 0.01% disodium EDTA, pH 7.4) in the presence of a purified source of LTP I (1.2 pg protein). Endogenous LTP I concentration within normolipidemic human plasma is usually 1-2 pg protein/ml [63]. In a further experiment LTP I was co-incubated with TP1 (30 pg protein per system), a monoclonal antibody (mAb) directed against LTP I at the neutral lipid binding site, before transfer studies were done [59]. The second strategy was to incubate delipidated human plasma as a source ofLTPIwith[3H]-CSA-enriched HDL or LDL with a corresponding drug-free lipoprotein counterpart. This human plasma source was chosen from bags of pooled normolipidemic plasma to obtain a transfer activity of at least 15%. To confirm the transfer of CSA is due to LTP I and not other endogenous plasma factors, mAb TP1 (30 pg protein per system) was co-incubated with the delipidated plasma before transfer studies were started. The above 2 strategies were designed to determine if the movement of CSA between lipoprotein particles was partially facilitated by 44 LTP I and/or was a result of non-facilitated drug transfer rather than from the influence of other plasma components. 14 [ C]-PC transfer between HDL and LDL in the presence and absence of TP1 was also determined for both sources of LTP I to observe if TP1 plays a role in inhibiting LTP I facilitated PC transfer activity. For all the aforementioned experiments, incubations were carried out for 90 minutes at 37°C. For experiments with TP1, an additional 90-minute incubation time with the LTP I source was carried out at 37°C before transfer studies were started. Following the final incubation, samples were separated out into their individual lipoprotein constituents by a manganese and phosphate precipitation step (0.1M manganese chloride solution and 0.45M sodium phosphate, 0.02% sodium azide, pH 7.4 solution). For all experiments, spontaneous transfer of the component of interest was accounted for by the performance of the transfer assay devoid of a LTP I source and this was subtracted from the total transferred. Lipid and drug transfer between donor and acceptor lipoprotein was quantitated by scintillation counting using a Beckman LS6000TA scintillation counter. Figure 12 shows a diagram of the steps preformed in these transfer experiments. 45 As discussed earlier in the thesis, the fraction of lipids and drug transferred (kt) was calculated as described by Pattnaik and Zilversmit [83]. kt = -In (1-At/D0) where D 0 and At are the radioactivities of the donor at time 0 and the acceptor at time t, respectively. The constant k is the fraction of the radiolabeled compound transferred per unit time (t). Acceptor radioactivity in the absence of LTP I (usually < 2-3%) is subtracted before calculating kt values. Calculations assume steady-state conditions in which all lipid and drug transfer are in an exchange process. 46 •«-» *** o S | ^ -h ^ "a -3 « S ^  •is "Q Q -a 3 HH HH B HH rt o .y C/3 "53 V r2 ca "3 S M rt ? rt ^ H ^ -1 •s ^ *s rt 00 <u • • H 47 3.5 Binding of CE, CSA, and Egg PC liposomal vesicles to L T P I 3.5.1 Binding ofCE to LTP I Binding of CE to LTP I was evaluated as a positive control since CE has been shown to bind to LTP I by other investigators [61, 70]. [3H]-CE liposomal vesicles were firstly made. 21.6pmol of egg PC, 0.328pmol of cholesteryl oleate, 80pL of [3H]-cholesteryl oleate (73.4mCi/mg, Amersham) were mixed in chloroform, dried to a thin layer in a glass tube under a stream of nitrogen over a hot water bath (> 25°C), and then placed in a vacuum for 2 hours. The lipids were then resuspended in 7mL of buffer (lOmM Tris-HCl and ImM EDTA, pH 7.4) and vortexed. The solution was aliquoted in lmL aliquots into cyrotubes and subjected to 5 freeze thaw cycles using liquid nitrogen and a hot water bath. The samples were put away in a -70°C freezer until extrusion. Samples were extruded through a lOOnm filter in a Lipex® extruder 10 times then sized by a Malvern Zetasizer and used for experiments. The total recovery after extrusion was 96.2%. The FPLC column was equilibrated for 2 hours prior to any injections with a mobile phase buffer consisting of lOmM Tris Cl and ImM EDTA buffer, pH 7.4. Egg PC (590 nmol) vesicles containing cholesteryl oleate and [3H]-cholesteryl oleate (9.06nmol total) were incubated with LTP I (9pg) for 1 hour in a total volume of 0.5mL. As stated before, the LTP I solution used is stabilized with albumin. The mixture was loaded onto a Sephacryl HR 100 XK column (18mm x 20cm) controlled by a FPLC apparatus and mobile phase was run through to separate the components. The flow rate was set at 0.25mL/min for a 3-minute load and adjusted 48 to 0.5mL/min for collection. The sample loop used was 0.5mL. Fractions of 1 mL were collected and elution fractions were assayed for [3H]-CE profile, protein mass (Biorad protein DC assay), LTP I mass (ELISA), and PC profile (enzymatic assay as previously described). LTP I injected alone on the column and [3H]-CE vesicles injected alone on the column served as controls. The column was washed with 300mL of buffer after each run. As an added step, a 0.5mL plasma sample was first injected onto the column to determine how dilute the eluted fractions were in order to minimize the usage of the pure LTP I source. A LTP I assay and a protein assay were conducted on the collected plasma fractions (Appendix G). 3.5.1.1 Protein mass analysis All eluted fractions from the column were analyzed for protein content to determine the amount of LTP I and other proteins in the fractions. As stated before, the LTP I solution used is stabilized with albumin. This assay was done to further confirm the presence of this protein in the eluted fractions. The LTP I ELISA described later is more specific for LTP I. The method for measuring protein concentration levels utilized a modification of the Lowry assay [100, 101]. Protein standard solution equivalent to BSA 400pg/mL was prepared by addition of distilled water to the vial as indicated by the labeled instructions. The appropriate amount of standard or 5uL of sample from each of the eluted fractions were added, either in duplicate or triplicate, in wells of a microtitre plate. 25pL of Reagent A (alkaline copper tartrate) were added to each well followed by 200pL of Reagent B (Folin reagent). Alkaline copper tartrate reacts with proteins and forms a blue colour when the Folin reagent is added. The resulting blue colour has a maximum absorbance of 750nm. The plate was gently mixed in 49 a plate shaker for 15 minutes after which the absorbance was read at 620nm with a plate reader (Labsystems Multiskan Ascent®). A standard curve (Figure 13) was constructed from the absorbance readings for the standards and the protein concentration of each sample was determined. 50 51 3.5.1.2 Radioactive profile 20uL of the collected eluted [ H]-CE lmL fractions from the experiments incubated with and without LTP I was aliquoted into scintillation vials with 5.4mL scintillation fluid and read in a scintillation counter (Beckman LS 6000TA) to detect tritium for a time period of 3 min per sample. CPM per mL was reported. 3.5.1.3 LTP I ELISA A widely used assay for measurement of LTP I protein in samples is a radioimmunoassay (RIA) that utilizes a monoclonal antibody to LTP I [102]. Glenn and Melton (1996) [103] have modified this type of method to eliminate the need for radiolabeled mAb. They have developed an ELISA involving competitive immunoreaction of unlabelled TP2, a mAb against the neutral lipid binding site of LTP I, between the LTP I absorbed to 96 well polystyrene plates and the samples of LTP I. The amount of the primary mAb, TP2, bound to the immobilized LTP I is detected with a secondary mAb consisting of an affinity-purified goat anti-mouse immunoglobulin G (IgG) linked to alkaline phosphatase. P-nitrophenyl phosphate is added as a substrate for the reaction to occur, and the end product, p-nitrophenol, is read at an absorbance of 405nm with a reference wavelength of 620nm. The measured level of TP2 bound to immobilized LTP I on the plates is indirectly proportional to the amount of soluble LTP I competing for immunoreaction. Because TP2 binds to the neutral lipid binding site of LTP I and our results from previous experiments have shown a partial transfer of CSA by this site [93, 94], this procedure should have been modified using a mAb not directed at this neutral lipid binding site [71]. However, due to material constraints, only TP2 was used to duplicate the published procedure to ensure 52 replication [103]. If a mAb not against the neutral lipid binding site of LTP I was used, then the LTP I ELISA procedure would have to optimized which would require the use of more materials. The procedure was followed from Glenn and Melton (1996) [103]: 3.5.1.3.1 Plate Coating Partially purified LTPI was diluted in coating buffer (15mM Na 2C0 3, 35mM NaHC03, 0.02% (w/v) NaN3, pH 9.8) to 0.4pg/mL and dispensed at lOOuL/well, leaving 3 wells on the top left as controls in a 96-well Nunc Maxisorp plate. Following an overnight incubation at room temperature, the plates was rinsed twice with washing buffer (138mM NaCl, 8. ImM NaiHPO^ 1.2mM KH 2P0 4 , 2.7mM KC1, pH 7.2). 250pl/well saturation buffer (washing buffer containing 1% (w/v) BSA, ImM EDTA) was added per well prior to incubation at room temperature for 4 hours. Plates were stored at -20°C or used immediately. 3.5.1.3.2 ELISA Procedure The saturation buffer treated plates were rinsed 4 times with washing buffer. Dilutions of LTP I standards were done first. Standards were dispensed as lOOpL aliquots into triplicate wells of 10, 20, 40, 80, 160, and 320 ng/well. lOOuL of triton buffer (saturation buffer containing 0.2% (w/v) Triton X-100) was added to the 3 uncoated LTP I wells and to 3 additional coated LTP I wells to serve as controls. Test samples were dispensed in lOOuL aliquots/well in duplicates. lOmL at a concentration of lOOng/mL in TB, was prepared from an aliquot of stock TP2. lOOpL/well of this solution was dispensed in all 96 wells of each plate and the plates were incubated at 37°C for 4 hours. 53 Each plate was rinsed four times with washing buffer prior to addition of lOOpL/well of the goat anti-mouse alkaline phosphatase conjugate (a 1:7000 dilution of stock using saturation buffer). The plate was incubated at 37°C for one hour and rinsed four times with washing buffer. lOOpL alkaline phosphatase substrate was added to each well and the plates were incubated at 37°C for 30 minutes. Non-LTP I coated wells were used as a blank. Plates were read at 405nm with a reference wavelength of 620nm. Figure 14 shows a simplified diagram of the LTP I ELISA procedure. Each LTP I ELISA assay was done independently for each injection containing LTP I. A LTP I standard curve was run each time. The LTP I standard curve was plotted and a linear regression analysis of LTP I concentration relative to the mean optical density (OD) was used to calculate LTP I levels in test samples (Figure 15). The standard curve was best fitted linearly. However, validation of this assay in our laboratory should be done further with more standard curves to evaluate if a semi-log fit is more appropriate. Validation should also be done with different operators over a longer period of time. 54 JZ. CD ' c i_ CD > CD _co C L c o c CD _CT> C ll - J CD X I CO 3 o o O .E "D CD 03 O O o CD CD CO J5 C L CD 2 CD C/D •4—» co "co o to ~ - O CD T 3 - Q i CD CO -2 JS to CO C L ^ ~ - O co t - CD CO CO C L Z5 — 2 C L O c o x : . £ I— c CO c o o CO CD CD X I to c 'co E CD s_ >> T 3 O JO "•4—« C CO to to CD SCNJ — CD i i " O CD C CD x: CD -*—1 CD 0- ~ Ti CD 4—» co Q . ® C C L O E -co Q. to f± CD _ J 2 £ - c -c C L co _ CD ^ CL a fZ CD n CD i >< & CD C -C O JO O CD to -S CD CD C L - Q E = CO $ . CD CD J Z JS o C L - » -X " ° to <= > ° > xi co g o CM CO PQ CD L . PH 55 56 3.5.2 Binding of CSA to LTP I Egg PC liposomal vesicles containing CSA and [ H]-CSA were prepared in a manner similar to the preparation of [3H]-CE labeled liposomal vesicles. Cold and radiolabeled CSA were added at the beginning of the procedure in place of cold CE and radio-labeled CE to the egg PC in chloroform. Total recovery after extrusion was 82.74%. The same procedure as that used for CE was used in evaluating the binding assay for CSA to LTP I. Purified LTP I (9pg) was incubated with egg PC liposomal vesicles (510 nmol) containing CSA and [3H]-CSA (8.10nmol total) for 1 hour in a total volume of 0.5mL. The sample was analyzed the same way as described for [ H]-CE binding to LTP I using a Sephacryl HR 100 XK column (18mm x 20cm), FPLC system, scintillation counting, LTP I ELISA, and PC analysis. [ H]-CSA liposomal vesicles injected alone on the column served as a control. 3.6 Statistical Analysis Differences in LTP I mediated CE and CSA transfer activity in the presence of different treatment groups were determined by paired sample t-test (Jumpln, SAS Institute Inc.) with critical differences set at p<0.05, data expressed as mean +/- standard deviation. Data in other experiments were expressed as mean +/- standard deviation. 57 Chapter 4 RESULTS 4.1 PC and CSA transfer between HDL and LDL These experiments were done to assess the role of the lipoprotein coat lipid, PC on the transfer of CSA by LTP I. To date, only the role of the lipoprotein neutral core lipids, TG and CE, was assessed in the transfer of CSA between lipoproteins [93, 94]. To assess the ability of LTP I to promote the co-transfer of PC and CSA from HDL to LDL, radiolabeled PC or CSA enriched HDL was incubated in T150 buffer containing purified LTP I (1.2pg/mL) or in lipoprotein deficient plasma with endogenous LTP I (1.2pg/mL). Detectable PC transfer occurred from HDL to LDL with both sources of LTP I, with no statistical difference when TP1 was added (Figure 16). This percent transfer was greater with the delipidated human plasma as compared to the purified source (Figure 16). Undetectable [3H]-CSA transfer occurred from HDL to LDL in T150 buffer containing purified LTP I and in the delipidated human plasma containing LTP I in the presence and absence of TP1 (Table 4). The percent transfer of PC and CSA from LDL to HDL was greater in delipidated human plasma containing LTP I than in T150 buffer containing purified LTP I (Figure 17). The percent transfer of PC determined in the presence of TP1 from LDL to HDL was significantly decreased in T150 buffer with purified LTP I as well as with the delipidated human plasma compared to controls (Figure 17). CSA transfer from LDL to HDL in T150 buffer containing purified LTP I was non-detectable (Figure 17). 59 CD ^ ll n c c C L C L CD 03 E CO 03 Q_ • • 3 O CO LO CN O CN L O L O jajsue.il W% 73 cu o U l-l ^ J 'bO a . o m r—1 OH H 73 O - O QL 'H—> c r - cj c~ C H — • o ocl a o s CCS ^ — <+H Q . O cu 1 o I C t i CU Z3 cn . 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O 73 £ a a * S H CD cn * a 73 CD £ cn C3 CD 43 73 CD 43 CD O H CD - H i—l Q Q i—l £ o l - l <+H cn a CCJ ti Y> PH U iC PH - C 73 J D 13 4 3 ccj a CM | T 3 — CD cn , 1 OH . « CD cn ^ a <3 .a -*-» CD ccj o Q - ~ £ 2 c/i <D w g £ ^ -3 oo a J CD ,<D « .a « cn o 2 S -2 "a3 S ^ a -C O « CD O • 2 V CD CD )H a o cn CD sO -T3 O Q H CD ccj C+H CD 43 a CD CD l -CD O H < C O U O H a cn l - l C+H ccj - O a o a . a 00 4 3 .£ o H .S 73 a ccj CD _a -a 73 ^ ccj 43 O H cn O 43 O H PH H +H a o 43 P i h J g *-> a cn fo a a U ' 5 PH SP TO CD CD kH CD a ° o o ^ 'C g U oj ^ u PH I u 2 ^ O rH cn CD 1— C CD *a CCJ - H 8P.I O O O S CD , IH IH ua ,o 73 C+H 5 S o -fc O H d -B Co ^ CD ' o +2 in » 2 !L a o ^ o .tJ.'S cn 4H cn 62 4.2 L T P I and protein elution profiles LTP I interactions with the lipids CE, TG, and PC in defined assays have been evaluated using FPLC by other groups [61, 70]. Our previous work [93, 94] has shown CSA to be partially dependent on the facilitated co-transfer of the neutral lipids, CE and TG, from LDL to HDL but not of PC. Since the neutral lipid binding site of LTP I is responsible for the transfer of the neutral lipids between lipoproteins, there is a possibility that the neutral lipid binding site also binds CSA. Previous experiments have demonstrated CE, TG, and PC binding the neutral lipid binding site of LTP I using FPLC [61, 70]. As a positive control, CE binding to LTP I at the neutral lipid binding site was assessed using a modification of this FPLC method. PC binding to LTP I at this site was also assessed. This FPLC method was also used to assess CSA independent binding to LTP I at the neutral lipid binding site [61, 70]. 4.2.1 LTP I Alone First, LTP I (9pg) was separated by FPLC (Figure 18a). The LTP I elution profile was determined with a LTP I ELISA and the protein elution profile was determined by a protein assay [103]. LTP I injected alone on the column eluted out into three main LTP I areas, at fraction numbers 10, 14, and 30, and there were minor peaks at fractions 18 and 27. There was a major protein peak at fraction 14 and also minor peaks at fractions 19, 27, and 30, corresponding to the some of the LTP I peaks. 63 4.2.2 LTP I and CE As a positive control, CE binding to LTP I was first assessed. Unilamellar egg PC liposomal vesicles containing cold CE and [3H]-CE were incubated with purified LTP I (9pg) then separated by FPLC. The elution profile of LTP I and protein were determined by both a LTP I ELISA and a protein assay (Figure 18b). The LTP I peak at fraction 10 decreased and fraction 30 decreased towards baseline. The LTP I peaks obtained from the combination LTP I and [ H]-CE liposomal vesicle injection at fractions 11, 18, and 26 increased by 2x, 1.6x, and 4.4x compared to the control LTP I fractions at 14, 18, and 26. There was an additional peak appearing at fraction 34. The protein assay resulted in only a peak at fraction 12 (Figure 18b) that was 1.6x the magnitude of the protein peak found in the control at fraction 14 (Figure 18a). 4.2.3 LTP I and CSA Unilamellar egg PC liposomal vesicles containing cold CSA and [3H]-CSA incubated with purified LTP I (9pg) was separated by FPLC. The elution profile of LTP I and protein were determined (Figure 18c) by a LTP I ELISA and a protein assay as previously described. A pattern similar to similar to the [ H]-CE and LTP I injection was seen. There was a decrease in fraction 10 and fraction 30 decreased towards the baseline. The LTP I peak obtained from the combination LTP I and [ ]H-CSA liposomal vesicle injection at fractions 11, 18, and 25 increased by 2x, 1.8x, and 4.4x compared to the control LTP I fractions at peaks 14, 18, and 26. There was also an additional peak seen at fraction 34. The protein pattern seen with the [3H]-CSA/LTP I injection was different than the [3H]-CE/LTP I injection. There were more peaks seen with the [3H]-CSA/LTP I injection (Figure 18c) which followed some of the 64 minor peaks seen with the control LTP I run (Figure 18a). The protein peaks at 12, 19, 28, and 33 were 2.1x, 1.3x, 1.9x, and 1.8x greater than the control LTP I fractions at peaks 14, 19, 27, and 32 respectively (Figures 18c and 18a). 65 a) Fraction number > protein Fraction number Figure 18 LTP I concentration (ng/mL) and protein concentration (pg/mL) in eluted fractions from injections onto a FPLC system with a Sephacryl HR 100 XK column (18mmx20cm) a) 9 pg LTP I alone b) LTP I 9pg + [3H]-CE liposomal vesicles (egg PC 590nmol, CE 9.06nmol) b) LTP I + [3H]-CSA liposomal vesicles (egg PC 5 lOnmol, CSA 8. lOnmol) Flow rate was set at 0.25mL/min for a 3 min load and adjusted to 0.5mL/min for collection. The sample loop used was 0.5mL. Fractions of lmL were collected. n=l for injections; eluted sample concentrations were determined n=l to 2 for each assay 66 4.3 CE elution profiles The elution profile [ H]-CE is reported in Figure 19a following the injection of [ H]-CE liposomal vesicles on the column. The radioactivity peak at fraction 12 was the [3H]-CE eluting out in the void volume. When [3H]-CE liposomal vesicles were preincubated with LTP I (9pg), the amount of [3H]-CE eluting out at the void volume increased by 1.9x (Figure 19b) compared to injection of [ H]-CE liposomal vesicles alone (Figure 19a). There was also [3H]-CE eluting past fraction 18 but the magnitudes of these radioactive peaks closely match that of the control (Figures 19a and 19b insets). The total radioactivity recovery was 29% from the control [3H]-CE liposomal vesicles as compared to 44% from the [3H]-CE liposomal vesicles injected with LTP I. 67 a) 600000 500000 400000 E S 300000 Q. ° 200000 100000 i o 14000 12000 t 10000 ^ 8000 n 6000 4000 ^ 2000 0 15 17 19 21 23 25 27 29 31 33 35 Fraction number 11 13 15 17 19 21 23 25 27 29 31 33 35 Fraction number Figure 19 [ H]-CE radioactivity (CPM/mL) in eluted fractions from injections onto a FPLC system with a Sephacryl HR 100 XK column (18mmx20cm) a) [3H]-CE liposomal vesicles (egg PC 590nmol, CE 9.06nmol) b) LTP I 9pg + [3H]-CE liposomal vesicles (egg PC 590nmol, CE 9.06nmol) Insets show fractions 15 to 35 with an additional y axis showing amount of LTP I in the eluted fractions. Flow rate was set at 0.25mL/min for a 3 min load and adjusted to 0.5mL/min for collection. The sample loop used was 0.5mL. Fractions of lmL were collected. n=T for injections; eluted sample concentrations were determined n=l to 2 for each assay 68 4.4 C S A elution profiles The distribution of [ H]-CSA is shown in Figure 20a for [ HJ-CSA liposomal vesicles injected onto the column. The radioactivity peaking at fraction 12 is the CSA eluting out in the void volume. When [3H]-CSA liposomal vesicles were preincubated with LTP I (9pg), the amount of [ H]-CSA eluting out at the void volume decreased by 0.4x as compared to control counts. There was also [3H]-CSA eluting out at later fractions (Figure 20b). The [3H]-CSA eluting out at fractions 18, 26, and 36 corresponded to the LTP I peaks at these fractions (Figure 20b inset). The magnitude of the radioactive peak at 18 closely matches to that of the control but the magnitude was 3x and 1.4x higher at fractions 26 and 34 . The total radioactivity recovery was 14% from the control [3H]-CSA liposomal vesicles compared to 8% from the [3H]-CSA liposomal vesicles injected with LTP I. 69 a) 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Fraction number b) 140000 x 120000 100000 g 80000 I £ 60000 40000 20000 15 17 19 21 23 25 27 29 31 33 35 Fraction number 13 15 17 19 21 23 25 27 29 31 33 35 Fraction number Figure 20 [3H]-CSA radioactivity (CPM/mL) in eluted fractions from injections onto a FPLC system with a Sephacryl HR 100 XK column (18mmx20cm) a) [3H]-CSA liposomal vesicles (egg PC 5lOnmol, CSA 8.lOnmol) b) LTP 19pg + [3H]-CSA liposomal vesicles (egg PC 510nmol, CSA 8. lOnmol) Insets show fractions 15 to 35 with an additional y axis showing amount of LTP I in the eluted fractions. Flow rate was set at 0.25mL/min for a 3 min load and adjusted to 0.5mL/min for collection. The sample loop used was 0.5mL. Fractions of lmL were collected. n=l for injections; eluted sample concentrations were determined n=l to 2 for each assay 70 Chapter 5 DISCUSSION 71 5.1 L T P Transfer of PC and CSA LTP I is responsible for all facilitated transfer of the core lipoprotein neutral lipids, CE and TG, and approximately one-third of the coat lipoprotein lipid, PC [53, 59-62]. In previous studies, the LTP-I facilitated transfer of CSA was found to be partially dependent on its CE and TG transfer activity [93, 94] from LDL to HDL. In this thesis, since LTP I also facilitates the transfer of PC, we chose to assess if there was LTP I facilitated co-transfer of PC with CSA between lipoproteins. In the experiments that were designed to directly measure the potential role of LTP I to facilitate CSA transfer based on HDL and LDL PC content, LTP I-mediated percent transfer of PC from LDL to HDL in delipidated human plasma containing other endogenous transfer proteins and in T150 buffer containing purified LTP I was different from that of CSA. The percent transfer of CSA from LDL to HDL is undetectable in T150 buffer containing purified LTP I, while PC transfer was observed and decreased in the presence of TP1 (Figure 17) [104]. In delipidated plasma, the percent transfer of CSA from LDL to HDL was not significantly different than with TP1, while PC transfer was observed and decreased in the presence of TP1 (Figure 17) [104]. The percent transfer of CSA from HDL to LDL is undetectable in T150 buffer containing purified LTP I and in delipidated plasma (Table 4). PC transfer is noted and not decreased in the presence of TP1 with both purified LTP I and delipidated plasma LTP I (Figure 16) [104]. Taken together, these findings found that the distribution of CSA is not influenced by LTP I-mediated PC transfer activity between HDL and LDL. These findings further support the notion that the PC transfer activity may be due 72 to facilitated transfer by other endogenous plasma factors (such as LTP II) and/or facilitated by LTP I at another site. Sgoutas and coworkers have proposed the nature of CSA's association with HDL and LDL particles appears to be non-specific and with low affinity and high capacity, suggesting CSA is physically dissolved within the lipoprotein-lipid component [37]. From previous studies, assessing LTP I facilitated co-transfer of CE or TG with CSA, it appears CSA is only partially recognized by LTP I as an endogenous lipid compound [93, 94]. The distribution of CSA between lipoproteins does not seem to be influenced by LTP I-mediated PC transfer activity between HDL and LDL but is partially dependent on the LTP I-mediated CE and TG transfer activities [93, 94, 104]. Since the neutral lipids, CE and TG, are found in the lipid core of lipoproteins and are transferred between lipoproteins by binding to the neutral lipid binding site of LTP I this lead us to hypothesize that LTP I may facilitate CSA's transfer by directly binding CSA to its neutral lipid binding site. The next experiments in this thesis addressed whether there is independent binding of CSA to the neutral lipid binding site of LTP I. 5.2 L T P I Binding The binding of LTP I with CE, TG, and PC in defined assays have been assessed by other groups [61, 70]. We had originally hypothesized that the human body may recognize lipophilic compounds as lipid-like particles, thus we modified these assays to assess the interaction of LTP I with CSA. CE binding to LTP I was evaluated as a positive control. PC binding to LTP I was also evaluated because the [3H]-CE liposomal vesicles and [3H]-CSA liposomal vesicles were made of PC. However, the PC in the diluted buffer fractions was 73 below the detection limit of the enzymatic assay employed. A further experiment should be done to evaluate LTP I binding with PC using radioactivity. In experiments that were designed to evaluate CE and CSA binding to the neutral lipid binding site of LTP I, [3H]-CE liposomal vesicles or [3H]-CSA liposomal vesicles were co-incubated with LTP I (9pg) and injected onto a FPLC. The resulting LTP I elution profiles were found to be similar for both these experiments but the protein elution profiles (Figure 18) and radioactive elution profiles were different (Figure 19 and Figure 20). First LTP I (9pg) was injected onto the column. The LTP I elution profile showed peaks coinciding with the protein elution profile (Figure 18) except there was one additional peak at fraction 10. The magnitude of the protein peaks were also much larger than the LTP I peaks. This suggests that there are unbound and protein bound LTP I available. Since the LTP I preparation contains both LTP I and albumin in a buffer, the higher magnitude of the protein peaks corresponding to the LTP I peaks suggest that albumin is binding to LTP I. Because the LTP I ELISA detects LTP I using a primary mAb against the neutral lipid binding site of LTP I, the LTP I detected in the elution fractions is not occupied at the neutral lipid binding site of LTP I. If this site was occupied then the LTP I would not be detected. In the next experiments, [ H]-CE liposomal vesicles or [ FfJ-CSA liposomal vesicles were co-incubated with LTP I (9pg) and injected onto a FPLC. A similar LTP I elution pattern was seen with both these experiments. There was a disappearance of the unbound LTP I detected at fraction 10 in the control experiment, an increase of some elution peaks by the same magnitude (peaks located at fractions 11, 18, 25 or 26), a disappearance of the peak at fraction 30, and an appearance of a peak at fraction 34. The disappearance of the unbound 74 LTP I at fraction 10 suggest both CE and CSA bind to the neutral lipid binding site of LTP I. The similar pattern seen with the LTP I detected in the other eluted fractions could be explained by PC associated LTP I since PC is the common component to both the radioactive liposomal vesicles and it can be transferred by LTP I [32]. The two protein peaks at the void volume from the [3H]-CE vesicles or [3H]-CSA vesicles co-injection with LTP I were also higher than that of control (Figure 18). This could be due in part to the nonspecific interaction of the PC or CE or CSA with albumin which then could interact with LTP I at a non-neutral lipid binding site. The disappearance of the unbound LTP I peak at fraction 10 does suggest CE and CSA binding to the neutral lipid binding site of LTP I as discussed before in Figure 18. However, further evaluation of the radioactive data suggest CSA binds differently than CE. Co-administration of [3H]-CE liposomal vesicles with LTP I resulted in an increase in [3H]-CE radioactivity recovered at the fraction number where unbound LTP I eluted versus the [ H]-CE radioactivity following [ H]-CE liposomal vesicles injected alone onto the column (Figure 19). This confirms CE binding to the neutral lipid binding site of LTP I. Co-administration of [ H]-CSA liposomal vesicles with LTP I resulted in the decrease of the [ H]-CSA radioactivity recovered at the fraction number where unbound LTP I eluted compared to control [3H]-CSA vesicles injected alone on the column, however there was still a significant amount of [ H]-CSA recovered (Figure 20). [ H]-CSA radioactivity was also recovered at other fraction numbers, some of which corresponded to where the protein-bound LTP I eluted (Figure 20 inset). Taken together, this data suggest CSA does bind to the neutral lipid binding site of LTP I, but it may also be binding to albumin, other regions on LTP I, or non-specifically to the FPLC column. 75 In these experiments, both CE and CSA were found to have non-specific interaction with the column by the determination of radioactive recovery rates. There was a low radioactive recovery from the column for both radioactive samples, demonstrating non-specific interactions: only 29% for the [3H]-CE liposomal vesicle injection, 44% for the [3H]-CE liposomal vesicle/LTP I injection, 14% for the [3H]-CSA liposomal vesicle injection, and 8% for the [3H]-CSA liposomal vesicle/LTP I injection were recovered calculated to fraction 35. [JH]CSA was also chaotically distributed passed fraction 26 at levels higher than control with the [ Hj-CSA liposomal vesicle/LTP I injection (Figure 20) which could suggest that there are more non-specific interactions occurring with CSA compared to CE. CSA may have more non-specific interactions due to its size since it is almost double in molecular weight compared to CE. Some of this non-specific binding not solely be due to the column, but could be due to non-specific interactions with the tubing, or components used to transfer the sample. Analysis to fraction 82 showed 15% recovery in the [3H]-CSA liposomal vesicle injection and 11% in the [3H]-CSA liposomal vesicle/LTP I injection. Non-specific binding could be occurring with the resin of the column and the compound could be washing out at later fractions with the mobile phase as it flows through the column. In a previous experiment done by our laboratory, CSA was injected onto a Sepharose column without incorporation into a vesicle. This resulted in the CSA being stuck on the column indefinitely. In this set of experiments, we have determined both CE and CSA bind to LTP I at the neutral lipid binding site. CE binding to LTP I was a positive control. These binding experiments demonstrate CSA may also be binding to albumin, other regions on LTP I, or non-specifically to the FPLC column. 76 5.3 Conclusion In the current studies, LTP I has been studied to see if it plays a role in the transfer of CSA between lipoproteins by binding at the neutral lipid binding site. In the first set of experiments in this thesis, it was found LTP I does not facilitate the transfer of CSA with PC amongst lipoproteins while our previous studies have shown that LTP I does facilitate the transfer of CSA with the core neutral lipids, CE and TG [82, 93], suggesting CSA may be residing in the core of the lipoproteins. In the second set of experiments in this thesis, the results suggested LTP I binds to CE and CSA at the neutral lipid binding site. The data also suggest CSA may bind with LTP I at other regions, bind 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. Figure 21 describes a model of the interaction of LTP I with the lipoprotein lipids and CSA. 5.4 Significance The distribution/redistribution of drugs among plasma lipoproteins facilitated by LTP I may serve as a possible mechanism for determining the ultimate fate of drug compounds. LTP I may have potential application in the field of drug delivery since it can facilitate the transfer of lipophilic drugs such as Hf, AmpB, and CSA between different lipoprotein subclasses. 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. When drugs have to penetrate lipid membranes, the use of LTP I could be evaluated since it exhibits a transfer activity. A recent 77 paper [91] suggests a potential application of plant lipid transfer proteins for drug delivery. This group reports that skin lipids such as sphingosine, sphingomyelin and cerebroside (an azole derivative with antitumoral and/or antileishmania properties), and amphotericin B were shown to bind to plant lipid transfer protein I. These binding studies suggest a challenge for the future could be the development of LTP-mediated transport and controlled release of low molecular weight drugs. 78 • a) Interaction with Cholesteryl Ester b) Interaction with Triglyceride c) Interaction with Phospholipid d) Interaction with Cyclosporine A Figure. 2.1 rAODEu O F WTP \ INTERACTIONS WITK C E ^ T C , , pct M^J> es* 5.5 Problems encountered and limitations The major limitation in all these studies was the lack of available purified LTP I, resulting in the performance of only one experiment each with the purified source. In the transfer studies, two transfer studies were done on different days with a delipidated source of LTP I before using a delipidated source of LTP I along with the purified source of LTP I in the third transfer study. In the binding studies, each experiment was only done once. Purified LTP I was used to coat enough ELISA plates to almost run duplicates of each fraction from each experiment for the determination of LTP I concentration. Considerable time was spent to determine if a commercial source of LTP I could be used in these experiments. However, this source was not purified enough for our studies. Transfer studies did not perform with this source of LTP I and ELISA plates could not be coated with this source, nor could it be quantitated with ELISA plates coated with the old source of LTP I. Another limitation of the study was the LTP I ELISA used the primary mAb TP2 against the neutral lipid binding site of LTP I. We had expected if there was binding at the neutral lipid binding site of LTP I, then LTP I would not be detected with this particular ELISA. A more ideal experiment would be to use a primary mAb that is not against this site. This way, the LTP I would still be detected if the component of interest was bound to the neutral lipid binding site of LTP I. This particular LTP I ELISA was not developed first due to the lack of purified LTP I. Control radio-labeled liposomal vesicles eluted out at the same location as some of the control LTP I. A longer column may have allowed enough column interactions to occur, enabling the control radio-labeled liposomal vesicles and the control LTP I to elute at 80 different fractions. If binding of CE and CSA with LTP I were to occur, then there may be a more distinctive visual decrease in the location of the control peaks. If the compound of interest was to bind with LTP I then there would also be an additional peak appearing. Since the lipoprotein coat lipid, PC, is known to interact with LTP I with one-third of its transfer between lipoproteins being mediated with LTP I [32], it should be further evaluated with regards to its binding with LTP I. PC and LTP I binding was evaluated as part of this thesis, however an enzymatic reaction was used to detect the PC content in each fraction. The samples fell below the limit of detection to be of use. A more ideal experiment would be to use a radioactive isotope of PC to increase the sensitivity of detection. Dual-labeled liposomal vesicles of radioactive PC and radioactive lipid or CSA could also be incubated with LTP I and eluted out on the FPLC. 5.6 Future studies The data suggest CSA does bind to the neutral lipid binding site of LTP I but the role of the neutral core lipids of lipoproteins in this interaction has not been evaluated. CSA could be binding to this neutral lipid binding site independently or with the neutral lipids as well. The relationship between the neutral core lipids, CE and TG, and CSA could be explored further. A simple experiment to do would be to repeat the transfer experiments from one lipoprotein component to another, utilizing purified LTP I and an increasing amount of CSA with the same concentration of a neutral lipid (CE or TG) and vice versa. This would determine if neutral lipid influences the transfer of CSA. Further experiments could utilize dual labeling of CSA and the neutral lipid (CE or TG) to follow both components in both transfer studies and binding studies. 81 The data also suggest the control LTP I injected onto the FPLC column is free at the neutral lipid binding site. A transfer study of neutral lipids (CE or TG) between lipoproteins using the control LTP I elution fractions from the FPLC with and without a mAb against the neutral binding site could be done to confirm this. This experiment can also confirm whether the LTP I is still active. As mentioned, in our experiments, role of PC binding to LTP I could not be determined due the low sensitivity of the PC assay. This experiment should be repeated with radioactive PC liposomal vesicles. To determine if other binding sites play a role in CSA transfer between lipoproteins, transfer experiments from one lipoprotein component to another, utilizing LTP I and other mAb against LTP I at other sites or utilizing mutant LTP I could be done. A FPLC procedure could then be developed further to determine the non-neutral lipid binding site if warranted. Since the human body may recognize lipophilic compounds as lipid-like particles, we had originally hypothesized LTP I may also be involved in the facilitated transfer of the lipophilic drug, CSA. CSA was used as a model lipophilic compound. Other lipophilic drugs should also be tested. One limitation of this would be the lack of commercial readily available radio-labeled lipophilic drug compounds. 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Biochem Pharmacol, 2001. 62(5): p. 555-60. 91 92. Kennedy, A.L. and Wasan, K.M., Preferential distribution of amphotericin B lipid complex into human HDL3 is a consequence of high density lipoprotein coat lipid content. J Pharm Sci, 1999. 88(11): p. 1149-55. 93. Wasan, K.M., et al., Lipid transfer protein I facilitated transfer of cyclosporine from low- to high-density lipoproteins is only partially dependent on its cholesteryl ester transfer activity. J Pharmacol Exp Ther, 1998. 284(2): p. 599-605. 94. Wasan, K.M., et al., Cyclosporine transfer from low- and high-density lipoproteins is partially influenced by lipid transfer protein I triglyceride transfer activity. Pharm Res, 1999. 16(7): p. 1067-73. 95. Moulin, P., et al., Gender effects on the distribution of the cholesteryl ester transfer protein in apolipoprotein A-I-defined lipoprotein subpopulations. J Lipid Res, 1994. 35(5): p. 793-802. 96. Ramaswamy, M., et al., Human plasma distribution of free paclitaxel andpaclitaxel associated with diblock copolymers. J Pharm Sci, 1997. 86(4): p. 460-4. 97. Havel, R., Eder, H., and Bragdon, J., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest, 1955. 34: p. 1345-1353. 98. Takayama, M., et al., A new enzymatic method for determination of serum choline-containingphospholipids. Clin Chim Acta, 1977. 79(1): p. 93-8. 99. Pattnaik, N.M., et al., Cholesteryl ester exchange protein in human plasma isolation and characterization. Biochim Biophys Acta, 1978. 530(3): p. 428-38. 100. Lowry, O.H., Protein measurement with the folin phenol reagent. J Biol Chem, 1951. 193: p. 265-275. 101. Peterson, G.L., A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem, 1977. 83(2): p. 346-56. 102. Marcel, Y.L., et al., Distribution and concentration of cholesteryl ester transfer protein in plasma of normolipemic subjects. J Clin Invest, 1990. 85(1): p. 10-7. 103. Glenn, K.C. and Melton, M.A., Quantification of cholesteryl ester transfer protein: activity and immunochemical assay. Methods Enzymol, 1996. 263: p. 339-51. 104. Kwong, M., et al., Cyclosporine A transfer between high- and low-density lipoproteins: independent from lipid transfer protein I-facilitated transfer of lipoprotein-coated phospholipids because of high affinity of cyclosporine a for the protein component of lipoproteins. J Pharm Sci, 2001. 90(9): p. 1308-17. 105. Connolly, D.T., et al., Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein. Biochem J, 1996. 320(Pt 1): p. 39-47. 92 A P P E N D I X A P R E P A R A T I O N A N D C O M P O S I T I O N O F D E N S I T Y S O L U T I O N S E M P L O Y E D F O R S E P A R A T I O N O F P L A S M A C O M P O N E N T S I N T O L I P O P R O T E I N A N D L I P O P R O T E I N - D E F I C I E N T F R A C T I O N S B Y G R A D I E N T U L T R A C E N T R I F U G A T I O N Solution Density Composition Amount 1.006 g/mL Sodium chloride 11.4g IM Sodium hydroxide 1 mL Water qslOOOmL 1.478 g/mL Sodium bromide 195.8 g 1.006 g/mL density solution 250 mL 1.063 g/mL 1.006 density solution 400 mL 1.478 density solution 55 mL 1.210 g/mL 1.006 density solution 200 mL 1.478 density solution 152.2 mL 93 A P P E N D I X B S U M M A R Y SHEET OF T H E L I P I D T R A N S F E R P R O T E I N ASSAY Materials • 3.5% bovine serum albumin in 50mM Tris-HCl, 0.02% NaN 3 , pH 7.4 (37°C) • 50mM Tris-HCl, 150 mM NaCl, 0.02% NaN 3 , 0.01% disodium E D T A , pH 7.4 (T150) • unlabeled H D L or L D L • radiolabeled L D L or H D L • 0.1 M M n C l 2 i n H 2 0 • 0.45 M sodium P0 4 , 0.02% NaN 3 , pH 7.4, in H 2 0 • LTP source (Purified LTP I or Delipidated Plasma) Procedure 1. In 1.5 ml microfuge tubes, combine the following amounts of reagents for assay blanks, assay samples, and assay totals: (For LTP I human source, in a separate microfuge tube, add 200uL BaCl 2 and 250uL 0.4% P T A in NaOH solution, leave it for 15 minutes, spin at 6740 x g (9000 rpm) for 3 minutes in a Beckman Microfuge 12, or equivalent and remove the supernatant). Reagent 3 Assay Blanks (6x ) Assay Samples*** (2x ) Assay Totals 3.5% BSA 200 u l 200 ul 200 ul LDL L + U l L u l L u l HDL These three should total 200 nl. H* ul Hul Hul H 2 0 (200-H-L) u l (200-H-L) ul (200-H-L) ul LTP source** 200 ul 200 ul T150 300 ul 100 ul 100 ul Mix well - Incubate all tubes at 37°C for 90 minutes (typical) PO4 reagent 100 ul 100 ul H2O 7 i0 u l Mix well MnCh reagent 150ul 150 ul Mix well - Allow to stand at room temperature for 15 minutes * where x = number of samples + where L = volume equivalent to give 20 p.g of phospholipid, as determined by BM Phospholipid Assay or * where H = volume equivalent to give 20 ng of phospholipid, as determined by BM Phospholipid Assay or ** for pure source of LTP I, this may be less; the volume will be added to the Tl 50 buffer in this case ***6x assay samples with a mAb can be added; a preincubation of 90 min at 37°C with LTP I is done first; the volume will be deducted from the Tl 50 buffer in this case 2. Centrifuge blank and sample tubes at 6740 x g (9000 rpm) for 3 minutes in a Beckman Microfuge 12, or equivalent, to pellet the L D L . 3. For blanks and samples, remove all supernatant into scintillation vials. For totals, remove entire volume into scintillation vials. Determine radioactivity by scintillation counting. (Use 5.4 mL of scintillation fluid.) (For HDL to LDL transfer blanks and samples, discard all supernatants. Pipette lmL of normal saline to each sample to suspend the precipitated LDL, and pipette into scintillation vials.) 94 After appropriate corrections of raw counting data for background radioactivity, calculate the extent of lipid transfer as follows. For LDL to HDL Transfer: kt = -ln (1 - ((sample HDL, cpm - blank HDL, cpm) / total LDL 0 cpm)) where HDL, is the supernatant radioactivity at time t for the assay sample and assay blank, and LDL 0 is the total LDL radioactivity added at time 0. Transfer values are reported as percent lipid transfer; i.e., 100 x kt or %kt. For HDL to LDL Transfer: kt = -ln (1 - ((sample LDL, cpm - blank LDL, cpm) / total HDL 0 cpm)) 95 APPENDIX C PACKING A SEPHACRYL HR 100 X K COLUMN 1. Slurry the gel in a total volume of 1.5x that of the final packed column (use buffer). 2. Dilute to 2x volume of the final packed column volume for the slurry to be poured into the column. 3. Level the column with a Teveler' before clamping it. 4. Prepare the adapter by removing the air with 20% EtOH so no bubbles are in the net. Close the tube once all air is removed and leave in a beaker until needed. Repeat this process with the bottom piece and insert this piece on the column. 5. Initially pour about 5cm of buffer in the column. 6. Attach the reservoir. Pour in the homogeneous slurry. Make sure the reservoir is filled to the top with buffer and open the tubing on the bottom of the column to allow outflow of buffer. 7. Once the gel is poured into the column, pump the buffer at the specified rate (l.OmL/min for 2 hours then 1.8mL/min for 1 hour for Sephacryl HR 100). Afterwards, stopper the tubing that is coming out of the bottom of the column. 8. Stop the pump, detach tubing from the top of the reservoir then detach the column from the post. 9. Remove the reservoir over a beaker or the sink and pour out the remaining buffer. 10. Remove excess slurry so the gel bed is visible underneath the black top piece (mark level), and fill to top with buffer. Insert the 'wetted' adapter top piece on top of gel bed at 45 degrees, making sure not to trap air. 11. Tighten nut and remove excess liquid around the top of the adaptor. 12. Remove the cap from the adapter tubing. Screw on red top of the adapter and push gently down to remove the excess buffer from the top of the column. "2 clicks on the video" 13. Start pump at low flow and flush all bubbles from the VALVE before connecting the tubing. 14. Connect the adapter tubing to the valve ad open the bottom tubing. 15. Once the bed is stabilized, mark level, sop the pump and close the outlet from the bottom of the column. 16. Switch the valve to connect the column and the sample port. 17. Adjust the adapter to the new bed height slightly below the bed height (5mm), allowing excess buffer to run out through the open tube leading out from the adapter. 18. Start pump and connect the column and pump. 19. Equilibrate column with 2 volumes of buffer. 20. Test column with a small volume of acetone to measure peak symmetry: Leading - too high a packing rate will cause small cracks in the gel bed or poor contact between the gel bed and column wall. Tailing - caused by too low a packing rate or large dead volume or air under the top net (filter). The valve may be the source of bubbles, so make sure the same channel for pump-to-column and port-to-column is used. 96 APPENDIX D VOID VOLUME DETERMINATION OF X K 18MMX20CM SEPHACRYL HR 100 COLUMN 1 i -0.2 J Fraction number (mL) 97 APPENDIX E MAKING PC ENCAPSULATED LIPOSOMAL VESICLES 1 Litre Buffer - adjust to pH 7.4 and filter Tris-HCl EDTA 157.6 372.2 MW MW lOmM = 0.01 M ImM =0.001 M Tris-HCl EDTA -> need 1.576g -> need 0.3722g Add Tris-HCl and EDTA to distilled water to make up to 1L, mix, then filter, and store in the fridge. 1. Take out the PC and cholesterol oleate (cold) from the freezer. In a capped test tube, quickly weigh out enough lipid to make 2mL of a 20mg/mL in chloroform for each. Back calculate to add the appropriate amount of chloroform. Vortex well. You now know that you have x mg lipid in x mL of chloroform. Take 2 x 20uL samples of this for scintillation counting. Dry the chloroform off the samples (chloroform quenches counts). 2. Set up a vacuum system in the fume hood for step 4. 3. Dry down in the fume hood with N2 gas. Put the adapter on the gas cylinder and put a glass pipette on the end of the rubber hose. Get the N2 gas to blow very gently - check the gas flow on your hand - it should be very light. Get a beaker 3A full of hot tap water. Dip test tube inside and blow down the liquid with the glass pipette - GO SLOW and blow down until you get a thin layer with lots of bubbles. 4. Once you've dried your lipid to a thin layer, keep it WARM. The warmer the sample is the easier it is to boil away the excess chloroform. Cover the top of the test tube with kimwipe taped or rubber band bound it to the top of the test tube. This will absorb some of the liposome if it shoots up into the vacuum. Put the test tube into the erlenmyer vacuum flask and start the vacuum slowly and watch as the chloroform boils off. You may get a thick film building up. Just swirl the test tube until the film breaks. Be careful - if you have the vacuum on too high and the film breaks, the sample will shoot right out of the test tube. The lipid should foam and bubble as the chloroform boils off and you should be left with a fluffy lipid film (depending on the lipid). Keep the vacuum on for 2 hours. 5. At this point you may wish to stop. You can use a cap and parafilm seal the test tube and store it in the freezer. This is handy so that if you want fresh liposomes, you just have to reconstitute from this step rather than starting at step 1. 6. Set up a hot water bath. Heat your buffer and the lipid. Add 7mL of buffer to the film of lipid. Vortex very thoroughly. Procedure 98 7. Fill a dewar with liquid N2. Transfer the lipid mixture into plastic cyro tubes (I put lmL in the 1.8mL cyro tubes), (glass test tubes will shatter when you freeze thaw and the cyro tubes cannot be used with chloroform at the beginning step because chloroform dissolves plastic). Attach cyro tubes to the lower portions of the metal crane. Freeze the sample in liquid N2 and then thaw in the hot water bath at the transition state temperature. Repeat this 5 times. 8. You can stop here if you run out of time at the freezing part and put away in a -70 C freezer. 9. Assemble the extruder and put sample through the extruder 10 times. 10. Size the sample with a particle sizer. Take about a drop or two and dilute with your filtered buffer. You should run the sizer again using a 2x dilution of the sample you just ran. If the two samples agree with +/- 5nm then it is fine. If not, keep on diluting until you have this error. (The particle sizer works by measuring the movement of the molecules. If the solution is too concentrated then the molecules will be bigger than they actually are). 11. Take 2 x 20uL samples of the final solution to determine the final concentration. For interest sake the filters can be counted to see how much lipid is stuck to the filters. Cap the sample and store in the fridge. 12. Wash the extruder with detergent then rinse very well (detergent will lyse any liposomes that are extruded next time if you don't rinse well). Wash well with radiowash as well if radioactivity is used. Rinse with 70% isopropranol then with distilled water and leave to air dry. 99 Appendix F EXTRUDED VS SONICATED [3H]-CE LIPOSOMAL VESICLES Size distribution(s) 10 • 510 50 500 Diameter (nm) Figure F 1 Particle size distribution of sonicated [3H]-CE liposomal vesicles (mean 318nm after 24min) Size distribution(s) 5 i t i : * « • ' s :' » — - — f i i .it ¥ 1 \ I Diameter (nm) Figure F 2 Particle size distribution of extruded [3H]-CE liposomal vesicles (mean 48.7nm) 100 900000 800000 700000 600000 £ 500000 2 400000 O 300000 200000 100000 0 -100000 - • — sonicated vesicles • extruded vesicles 5 7 9 11 13 1 5 1 7 1 9 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 S3 65 67 69 71 73 75 77 79 81 Fraction number Figure F 3 [ H]-CE Elution of sonicated versus extruded [ H]-CE liposomal vesicles injected through a Sephacryl HR 100 X K column 101 Z o H H H < U - J u z o H -o H H h-l W 0 X H H Q Z W PH < -J I o CD c -S o •a « C E fi CO « C •** fc -J CM X. fi CO CD . c CD O O 05 LO CN ID CO CD T f T f d CN CD CM O 1^ Tf CD O O o> LO oo uo o x— CO 00 iri T f o p CD O TO LO OO o E IO CO oo z CO 00 I CU CO "55 £ 3 CQ O) w o o jCD jCN •CO oo CM IO CD CD T f T f TT O CO 00 05 CO •S: tod. CO ,00 LO r-O CD co T f O CL X CN CO Z s DL CM X E CM i i a CQ 0) . c •£ w Ii x> CN Q) s : I CO a x> JZ > CO cr CN CN 00 o CM d CD CO CM CN CO T f T f T f CN oo ol -CO -5 ° < cy) m 3 -co 3 3 CQ C ,o 2 3 •** ro CO CN d lg , 3 CQ C o 102 APPENDIX H LTP I AND PROTEIN DETERMINATION OF ELUTION FRACTIONS FROM A PLASMA INJECTION (0 .5ML) THROUGH A SEPHACRYL HR 100 X K COLUMN 103 

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