PREPARATION AND CHARACTERIZATION OF MICROEMULSIONS

CONTAINING SPHINGOMYELIN AND CHOLESTERYL OLEATE: A 3lP NMR STUDY

Lisa Yajie Zhao

B. Sc., Hunan University, 1984

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

in the department

of

chemistry

O Lisa Yajie Zhao 1993

SIMON FRASER UNIVERSITY

June 1993

All rights reserved. This work may not be

reproduced in whole or in part, by photocopy

or other means, without permission of the author

Approval

Name: Lisa Yajie Zhao

Degree: Master of Science

Title of Thesis: PREPARATION AND CHARACTERIZATION OF MICROEMULSIONS CONTAINING SPHINGOMYELIN AND

CHOLESTERYL OLEATE: A 3lP NMR STUDY

Examining Committee:

Chairman: Dr.

Dr. R. J. Cushley Senior Supervisor

Dr. Y. L. Chow Supervisory committee

Dr. W. R. Richards Supervi~qry committee,

Dr. N. Haunerland Internal examiner

Date approved: '$$, 1993

PARTIAL COPYRIGHT LICENSE --

I hereby grant to Simon Fraser University the right to lend my

thesis, project or extended essay (the title of which is shown below) to

users of the Simon Fraser University Library, and to make partial or

single copies only for such users or in response to a request from the

library of any other university, or other educational institution, on its own

behalf or for one of its users. I further agree that permission for multiple

copying of this work for scholarly purposes may be granted by me or the

Dean of Graduate Studies. It is understood that copying or publication

of this work for financial gain shall not be allowed without my written

permission.

Title of Thesis/Project/Extended Essay:

Author: (signature)

" (date)

ABSTRACT

As a model for the lipid organization of lipoproteins, sphingomyelinl

cholesteryl oleate (SPMICO) microemulsions about 23-26 nm in diameter were

prepared, which is in the size range of LDL. A temperature study using

phosphorus-31 NMR showed that the linewidth for SPMICO microemulsions is

slightly larger than that of SPM vesicles. This suggests that the neutral core

(CO) of the microemulsions may modify the motion of the phospholipid (SPM)

monolayer.

In order to study the lipid-protein interaction, microemulsions in the

presence of protein (apo HDL3) were prepared. A temperature dependence

study indicated that the 31P NMR spectra could not be simulated using a single

Lorentzian lineshape function; instead, a superposition of two Lorentzians was

needed to get a reasonably good fit at low temperatures (below 25•‹C).

Lineshape analysis suggested the presence of two magnetically inequivalent

domains within the reconstituted lipoproteins.

The lateral diffusion coefficient (D) of sphingomyelin in SPM/CO

microemulsions was determined from the viscosity-dependence of 3lP NMR

linewidths. At 25 "C, D was 1 + 0.3 x 10-9 cm2 s-1 for SPM / CO

microemulsions. The value of D in SPM / CO microemulsions is approximately

1.4 times smaller than in native LDL. A possible explanation is that the core of

the microemulsions may play a role in slowing SPM diffusion, or the relatively

rigid sphingomyelin phospholipid monolayer may be responsible for the slower

diffusion.

The residual chemical shift anisotropy of sphingomyelin in SPh

microemulsions at 25•‹C was 33 ppm, measured by field-dependence of the 31 P

NMR linewidth. This value is similar to that found for egg PC vesicles and egg-

PCitriolein microemulsions, indicating that the three systems may have similar

headgroup orientations.

The lateral diffusion constant of sphingomyelin in SPMICOlapo HDL3

reconstituted particles was determined from the viscosity-dependence of 3lP

NMR linewidths. NMR spectra were fitted by a superposition of two Lorentzians.

The lateral diffusion constants for the two environmentally different domains

were determined to be 1 + 0.4 x 10-9 cm2 s-1 (broad component ) and 2.1 + 0.8

x 10-8 cm2 s-1 (narrow component), respectively. A possible explanation for two

quite different diffusion coefficients is that the diffusion constant of the broad

component is due to the "protein-deficient" phospholipids (or phospholipids not

close to the protein), whereas the diffusion constant of the narrow component is

due to the "protein-rich" phospholipids (or phospholipids adjacent to protein).

DEDICATION

To Daiqing

and

Jennifer

ACKNOWLEDGMENTS

I would like to thank my supervisor Dr. R. J. Cushley for his guidance,

encouragement and support throughout the course of this research project. I

also wish to extend my thanks to Dr. Richards and Dr. Chow for their time as

members of my supervisory committee.

To Dr. W. D. Treleaven, for his advice, expertise, encouragement,

friendship and warmth for the past two years, I am grateful.

To the members of the research group for their assistance.

To Fred Chin, for his computer assistance.

To Jennifer, for making my life much brighter, and bringing me all the

happiness I needed. Thank you, my dear baby.

To my parents and my sister for their love, support and the source of my

strength.

Finally, I would like to thank my husband, Daiqing Liao for everything. His

knowledge and full understanding make this degree possible.

TABLE OF CONTENTS

Title ...................................................................................................

Approval. ...........................................................................................

~ b s t ract .............................................................................................

Dedication .........................................................................................

............................................................................. Acknowledgments

.............................................................................. Table of Contents

..................................................................................... List of Tables

................................................................................... List of Figures

.......................................................................... List of Abbreviations

1. INTRODUCTION

1.1 Biological Membranes ........................................................

1.2 Plasma Lipoproteins ..........................................................

............................................. 1.2.1 Lipoprotein Metabolism

............................... 1 .3 Structure of Low Density Lipoproteins

................. 1.4 Microemulsions and Reconstituted lipoproteins

1.5 Research Objectives.. ........................................................

2. THEORY

2.1 Quasi-Elastic Light Scattering ............................................

2.2 Phosphorus-31 NMR Theory .............................................

2.3 Lateral Diffusion Studies of Phopholipids in Model Particles

3. MATERIALS AND METHODS

3.1 Materials ............................................................................

3.2 Methods .............................................................................

i

II

iii

v

vi

vi i

ix

X

xiii

1

3

3

9

9

16

17

19

29

32

33

vii

3.2.1 Isolation of High Density Lipoproteins .....................

3.2.2 Isolation of HDL Apolipoproteins .............................

3.2.3 Preparation of Protein-free Microemulsions ............

3.2.4 Sphingomyelin Vesicles ..........................................

3.2.5 Quasi-Elastic Light Scattering .................................

3.2.6 Preparation of Reconstituted Lipoproteins ..............

3.2.7 NMR Measurements ...............................................

........................................... 3.2.8 Diffusion Measurement

3.2.9 NMR Lineshape Analysis ........................................

4 . RESULTS AND DISCUSSION

4.1 Unilamellar Vesicles ...........................................................

4.2. Microemulsions: Model for Smaller Cholesterol

Rich Lipoproteins ..............................................................

4.3. SPMICOlapo-HDL3 Reconstituted Lipoprotein

System (LDL size) .............................................................

4.4 A Lateral Diffusion Study of SPMICO Microemulsion ........

4.5 A Fileld Dependence Study of SPMICO Microemulsion ....

4.6. A Lateral Diffusion Study of SPMICOlapo-HDL3

Reconstituted Lipoproteins ...............................................

5 . CONCLUSION .............................................................................

6 . REFERENCES .............................................................................

LIST OF TABLES

Table

Composition (weight %) of human plasma lipoproteins .........

Properties of the apoproteins of the major human lipoprotein classes ..................................................................

The linewidth of sphingomyelin vesicles at selective temperature ............................................................................

The linewidth of SPMICO microemulsion at selected temperature ............................................................................

3' P NMR linewidths of SPMICOlapo-HDL3 recombinant lipoproteins at selective temperature .....................................

The viscosity dependence of linewidths of SPMICO microemulsions .....................................................................

Comparison of lateral diffusion coefficient for serum

lipoproteins, microemulsions, reconstituted lipiprotein

........................ particles and phospholipid bilayers (vesicles)

Variation of linewidths of 31P NMR spectra of SPMICO .................... microemulsions at different NMR field strengths

Page

4

5

44

53

69

75

80

82

Comparison of chemical shift anisotropy for serum lipoproteins, microemulsions, and phospholipid bilayers ............................ 83

The Viscosity dependence of Linewidths of SPMICO

Microemulsions in the Presence of apo-HDL at 25 "C ........... 97

LIST OF FIGURES

Figure

Singer and Nicolson's model for biomembrane ......................

The schematic structure of low density lipoproteins ...............

Molecular structures of SPM and PC .....................................

Schematic diagram of the NICOMP 370 dynamic light

scattering apparatus ...............................................................

The three different orientations of the chemical shielding tensors in the molecular frame of the phosphate ...................

3lP NMR (1 18 MHz) proton-decoupled spectrum of

anhydrous dipalmitoyl phosphatidylcholine powder at 15 "C .

Proton decoupled 31 P NMR spectra of polymorphic phases available to liquid crystalline phospholipids ...........................

The size distribution of sphingomyelin vesicles by

Quasi-Elastic Light Scattering ................................................

P NMR spectra of sphingomyelin (SPM) vesicles at a). 15 "C, b). 20 "C, c). 25"C, d). 50 "C ..................................

The schematic representation of ultracentrifugation separation of microemulsion ..................................................

The size distribution of sphingomyelin/cholesteryI

microemulsions measured by Nicomp 370 particle sizer .......

Page

2

10

14

20

24

25

3 1

39

4 1

46

48

3lP NMR spectra of SPMJCO microemulsions at

a). 15 "C, b). 25 "C, c). 35 OC, d). 45 OC ................ .................

Light scattering results from the SPMICO microemulsion

samples before and after addition of apo-HDL3 ....................

31 P NMR spectra of SPM/CO/apo-HDL3 particles at 1 5 OC ...

31P NMR spectra of SPMJCOIapo-HDL3 particles at 20 OC ...

31 P NMR spectra of SPM/CO/apo-HDL3 particles at 25 OC ...

3' P NMR spectra of SPM/CO/apo-HDL3 particles at 30•‹C ....

P NMR spectra of SPM/CO/apo-HDL3 particles at 45 OC ...

3lP NMR spectra of SPM / CO microemulsions at several

different viscosities at 25 OC ...................................................

The linewidths of 31 P NMR spectra of SPMJCO microemulsions as a function of viscosity ..............................

Plot of ( Av - C) versus q of SPM / CO

microemulsions ..... . . . . ..... .. . . . . . . ... . .. . . .. . . . . . .. ... . . . . . . .... . . . . .. .. ... ... . . . .

(AvlI2 - C) as a function of vO2 for SPMICO

microemulsion at 25 "C ..........................................................

P NMR spectra of SPM/CO/apo-HDLyeconstituted lipoprotein particles at 25 OC (viscosity = 0.99 centipoise) ....................... 85

P NMR spectra of SPM/CO/apo HDLgreconstituted lipoprotein

particles at 25 OC (viscosity = 1.53 centipoise) ....................... 87

3lP NMR spectra of SPM/CO/apo HDL3 reconstituted lipoprotein ....................... particles at 25 "C (viscosity = 2.71 centipoise) 89

3' P NMR spectra of SPM/CO/apo HDL3 reconstituted lipoprotein ....................... particles at 25 "C (viscosity = 5.30 centipoise) 9 1

3lP NMR spectra of SPM/CO/apo HDL3 reconstituted lipoprotein ...................... particles at 25 "C (viscosity = 9.89 centipoise) 93

3 l P NMR spectra of SPM/CO/apo HDL3 reconstituted lipoprotein ..................... particles at 25 "C (viscosity = 17.24 centipoise) 95

Plot of linewidth of broad component of SPM/CO/apoHDLg reconstituted lipoprotein versus viscosity ............................... 100

The plot of broad component ( Avl/* - C) -'XI 0 versus q -' ...................... of SPM/CO/apoHDL3 reconstituted lipoprotein 101

Plot of linewidth of narrow component of SPM/CO/apoHDL3 reconstituted lipoprotein versus viscosity ............................... 102

The plot of narrow component ( A V ~ , ~ - C) -'XI 0 versus q ..................... of SPM/CO/apoHDLg reconstituted lipoprotein 103

xii

LIST OF ABBREVIATIONS

CO:

CM:

CSA:

DMPC:

DPPC:

HDL:

QELS:

LB:

LDL:

PC:

SPM:

TO:

VLDL:

cholesteryl oleate

chylomicron

chemical shift anisotropy

dimyristoyl phosphatidylcholine

dipalmitoyl phosphatidylcholine

high density lipoprotein

quasi-elastic light scattering

line-broadening

low density lipoprotein

phosphatidylcholine

sphingomyelin

trioleoylg lycerol

very low density lipoprotein

31P NMR: phosphorus-31 nuclear magnetic resonance

CHAPTER 1

INTRODUCTION

1.1 Biological Membranes

Biological membranes are central elements of cellular structure and

function. Biomembranes are selectively permeable barriers rather than

impervious. They control the molecular flow of information both within the cell as

well as between the cells and the environment (Stryer, 1988). An understanding

of biomembrane structure and dynamics is, therefore, an attractive area to study.

Biomembranes differ from each other on the basis of both structure and

function. However, biomembranes do share a number of important features:

1. The composition of membranes includes primarily lipids, proteins and some

carbohydrate, and the membrane lipids are relatively small molecules with

both a hydrophobic and a hydrophilic moiety.

2. Membranes are organized into sheetlike assemblies (bilayers). The

thicknesses of membranes are dependent on the size of phospholipid acyl

chains as well as the hydration layer.

3. Specific proteins mediate distinctive functions of the membrane.

4. Membranes are structurally and functionally asymmetric.

The amphipathic nature of membrane components drives the elements

together in a noncovalent macromolecular array in the presence of an aqueous

environment (Stryer, 1988; Ginsberg et al., 1982; Barenholz, 1984; Seelig and

Seelig, 1980; Lipowsky, 1991 ). Lipids and proteins, the two major components

of membranes, organize themselves according to the distribution of hydrophobic

and hydrophilic regions on the molecular surfaces. The lipids, consisting of a

hydrophilic headgroup and long hydrophobic hydrocarbon tails, form a bilayer

spontaneously with the polar headgroup pointed out towards the aqueous

surrounding (An interaction between the hydrophobic tails of the phospholipid

and the aqueous phase is thermodynamically unfavorable. In contrast, the

hydrophilic headgroups of the lipids are usually able to interact with the aqueous

phase very well) (Ginsberg et al., 1982; Barenholz, 1984; Seelig and Seelig,

1980; Yeagle, 1990).

Singer and Nicolson described the macroscopic organization of the

biomembranes as a " fluid mosaic model " (Singer and Nicolson, 1972). This

model has helped focus attention on the mobility of membrane components by

conceptualizing the membrane as a sea of lipid in which embedded proteins are

freely floating. Figure 1.1 shows the Singer and Nicolson's fluid mosaic model of

Figure 1.1 : Singer and Nicolson's model for biomembrane (Singer and

Nicolson, 1972).

1.2 Plasma Lipoproteins

Plasma lipoproteins are spherical macromolecular complexes of specific

proteins and lipids. Their primary function is to transport hydrophobic lipids of

dietary or endogenous origin to the tissues where they may be utilized for

oxidative metabolism, storage, synthesis of triglyceride, steroid hormone and bile

acidslbile salts or maintenance of cellular membrane and function (Dolphin,

1 985).

Lipoproteins are classified according to their buoyant densities and can be

isolated by sequential ultracentrifugation. The four major groups of lipoproteins

are chylomicra (CM, ~ 0 . 9 4 glml), very low density lipoprotein (VLDL, 4 .006

glml), low density lipoprotein (LDL, 1 .019-1.063 glml), and high density

lipoprotein (HDL2, 1.063-1.1 25 glml; HDL3, 1.1 25-1.21 glml) (Gotto, 1987;

Atkinson and Small, 1986). The characteristic properties of the plasma

lipoproteins are the electrophoretic mobility, molecular weight, size, lipid and

protein composition. Table 1.1 lists the chemical composition and physical

properties of human lipoproteins. Table 1.2 lists the function and properties of

apoproteins in human plasma lipoproteins.

1.2.1 Lipoprotein Metabolism

Chylomicra are the largest and the lightest particles, with diameters

ranging from 100 to 500 nm (Gotto, 1987; Atkinson and Small, 1986). They are

triglyceride-rich particles (with extremely low free cholesterol) formed in the

intestine during the absorption of fat and contain apolipoproteins A-I, A-11, A-IV,

Table 1.1 : Composition (weight O/O) of human plasma lipoproteins.

Component

Protein

Trig lyceride

Cholesterol

Cholesteryl ester

Phospholipids

Diameter (nm)

MW X 106

Density (glml)

Apoprotein compositions

VLDL LDL

46

3

4

19

28

11

0.22

1.063 - 1.125

A-I, A-Ill

c-I, c-11, C-Ill, D, E

6 1

2

2

14

20

8.8

0.16

1.125 - 1.21

A-I, A-Ill

c-ll c-Il l C-Ill, D, E

Adopted from Dolphin (1 985), Zubay (1 988) and Scanu (1 979).

- --

Table 1.2: .Properties of the apolipoproteins of the major human lipoprotein classes.

-

Apolipo- protein

Plasma concentration (mgI100 ml)

Miscellaneous

Major protein in HDL (64%): activate LCAT

Mainly in HDL: two identical chains joined by

disulfide bond

Major protein in LDL: very difficult to solubilize in

detergents

A protein found in chylomicra

Activate LCAT*

Activate lipoprotein lipase

Contain 79 a.a and Gal, GalNAc, and NeuNAc

Cholesterol ester transfer protein and associated with

HDL and LCAT

Arginine-rich lipoprotein

Adapted from Zubay (1 988). ( *LCAT=Lecithin-cholesterol acyltransferase)

and apo 8-48. (Apo B-48 appears to be essential for the release of the

chylomicron from the intestinal cell). Chylomicra can be rapidly degraded and

removed within minutes from the circulation. In the plasma, the triglyceride core

is modified by lipoprotein lipase, and as a result, small particles (diameter of the

particles is 40-1 00 nm) are derived, which have reduced t riglyceride content.

During production of small particles, the surface components become redundant,

buckle to form bilayers, and dissociate from the chylomicron as surface

remnants. The final products are core and surface remnants, of which the

former are enriched in cholesterol and are rapidly removed by the apo-B, E-

receptor and chylomicron-receptor of the liver, and the latter enter into the HDL

pool (Brown and Goldstein, 1983; Mahley and Innerarity, 1983).

Very low density lipoproteins (VLDL) are nascent particles originating in

the liver and some of their plasma remnants. VLDLs were so termed originally

because of the flotation characteristics of these lipoproteins. VLDLs are

triglyceride-rich particles, and contain apo B-100, apo C, and apo E, and are

synthesized in the endoplasmic reticulum and in the golgi apparatus of the liver

(Gotto, 1987; Atkinson and Small, 1986; Alexander et al., 1976; Higgins and

Hutson, 1984; Higgins, 1988). VLDLs are involved in transporting endogenous

triglyceride and cholesterol. VLDLs are hydrolyzed by lipoprotein lipase,

producing diacylglycerol, monoacylglycerol and fatty acids (lipoprotein lipase

may also hydrolyze the phospholipids at the surface) (Cryer, 1981 ; Hamosh and

Hamosh, 1 983; Breckenridge, 1985; Fielding, 1978; Gotto, 1987). During the

lipolytic process, the structure of VLDL undergoes a series of changes which

result in a depletion of the triglyceride core, a reduction of particle size, an

increase in the ratio of cholesteryl esterltriglyceride and increase in particle

density. The resulting VLDL remnant, also called intermediate density

lipoprotein (IDL), can have different metabolic fates. IDL may either be

removed (recognized) rapidly from circulation by the liver or further metabolized

and remodeled, giving rise to low density lipoproteins (LDL) in the plasma (Gotto,

1987; Atkinson and Small, 1986; Havel, 1984). During the conversion to LDL

(cholesteryl ester-rich microemulsions), most of the triglycerides (98%) are

transferred, and most of the surface layer phospholipids (75%), free cholesterol

(85%), and all of the proteins (with the exception of apo 8-100) are depleted

from the lipoprotein surface (Eisenberg, 1980; Gotto, 1 987; Atkinson and Small,

1 986).

Low density lipoproteins are isolated in a density of 1 .019 - 1.063 glml,

with a diameter of 22 - 24 nm (Gotto, 1987; Atkinson and Small, 1986). LDL

contains a single copy of apo B-100, and a core which is predominantly

cholesteryl ester (Gotto, 1987; Reisinger and Atkinson, 1990). A high level of

LDL in blood has been positively correlated with the development of

atherosclerosis, an arterial disease characterized by the lipid-containing lesions,

which is the chief cause of death in North America and Europe (Ginsburg, et al.,

1982; Scanu, 1979; Benade et al., 1988; Darfler, 1990; Yla-Herttuala et al.,

1989). LDL is the major carrier of cholesterol to the peripheral tissues and is

involved in the regulation of cholesterol biosynthesis by the LDL pathway

(Stryer, 1988). The apo B-100 in LDL is recognized by apo 8, E-receptor, a

glycoprotein located in clathrin-coated pits on the cell membrane of many

tissues. Binding of LDL to the LDL receptor leads to internalization of the

receptor-LDL complexes by endocytosis, which results in the hydrolytic

degradation of both proteins and cholesteryl esters. The liberated free

cholesterol plays an important role in cell cholesterol regulation; it suppresses

the synthesis of cholesterol and LDL receptors, resulting in a decrease of both

cholesterol synthesis and uptake from LDL (Stryer, 1988).

High density lipoproteins (HDL) are essential to the regulation of

lipoprotein metabolism. H DL is involved in the major intravascular metabolic

process of the triglyceride-rich lipoprotein and controls the extracellular

transport of cholesterol (Redgrave et al., 1992; Gotto, 1987). Cholesterol efflux

is a process by which the cholesterol is transported back to where it is ultimately

excreted from the body (for example, the transport of cholesterol from peripheral

tissues to the liver, the main organ of cholesterol utilization) (Stein et al., 1988,

1989(a), 1989(b); Schmitz et al., 1990; Gotto, 1987). It was demonstrated that

increased binding of cholesterol to HDL, correlates with increased cholesterol

efflux (Hara and Yokoyama, 1992). Furthermore, a high level of HDL in plasma

may prevent the development of atherosclerosis (Fenske et a1.,1988; Miller and

Miller, 1975; Gotto, 1987). HDLs are synthesized by both liver and intestine in

two different forms: (i) discoidal HDLs, and (ii) small spherical HDL3 particles

(Atkinson and Small, 1986, Gotto, 1987). It is believed that the nascent discoidal

HDL particles containing apo A-l are converted into small HDLg-like particles by

lecithin-cholesterol-acyl-transferase (LCAT) (as shown in Table 1.2 , apo A-I is

the activator of LCAT) and then LCAT converts some cholesterol and

phospholipids to cholesteryl ester (Atkinson and Small, 1986, Gotto, 1987). This

transformation may act as the basis for the process of cholesterol efflux (Fenske

et al., 1988).

1.3 Structure of Low Density Lipoproteins

Low density lipoproteins (LDL) are biological microemulsions of particular

interest because of their important role (as a carrier) in cholesterol transport and

metabolism as discussed previously. As a result, LDLs have been the subject of

considerable study (Ginsberg et al., 1982). LDLs are quasi-spherical particles,

20-26 nm in diameter (Stryer, 1988; Zubay, 1988; Atkinson and Small, 1986;

Gotto, 1987; Ginsburg et al., 1984). The predominant lipids of LDL are

phosphatidylcholine, sphingomyelin and cholesteryl esters (cholesteryl linoleate

and cholesteryl oleate), which comprise more than 80% of total LDL lipids (for

details see Table 1 .I). The polar phospholipids form an outer monolayer to

provide surface stability to a nonpolar neutral core comprised of cholesteryl

esters and triacylglycerol (TG). It was demonstrated that all the lipoproteins

share the common structural features as determined by various physical studies

(Hamilton and Morrissett, 1986; Parmar et al., 1983; 1985; Treleaven et al.,

1986; Forte et al., 1968; Deckelbaum et al., 1977; Atkinson et al., 1977, 1980;

Laggner and Muller, 1978; Baumstark et al., 1983; Laggner et al., 1981 ;

Vauhkonen and Somerharju, 1990). Figure 1.2 shows the schematic molecular

structure of low density lipoprotein.

1.4 Microemulsions and Reconstituted Lipoproteins

The reconstitution of plasma lipoproteins represents an important aspect

of our overall understanding of the relationships between lipoprotein structure

and function. Recently, reconstituted lipoproteins were shown to offer several

advantages over native lipoproteins in the study of lipoprotein structure and

PROTEIN v CHOLESTEROL f-

CHOLESTERYL ESTER w

PHOSPHOLIPID c

TRIGLY CERIDE d

Figure 1.2: The schematic structure of low density lipoproteins

(modified after Laggner and Muller, 1978).

function (Fenske et al., 1988, 1990; Cushley et a1.,1987; Chana et al., 1990;

Yang et al., 1992; Mims et al., 1986; Reisinger and Atkinson, 1990; Ginsburg et

al., 1982). Such advantages include the following:

. Reconstituted lipoprotein system, in which the molecular complexity of the

native particle is simplified by the use of a single species of lipid and a

single species of apoprotein, provides well defined model systems for

studying lipoprotein structure and function. Therefore, we can investigate

the contribution of individual lipids or proteins to lipoprotein properties.

The composition of the reconstituted lipoprotein can be precisely controlled;

thus, we can design particles of given size and density.

Lipid-protein reconstituted complexes, with rigidly controlled composition,

also provide means to study lipid-protein interactions. However, the majority of

studies reported to date employed synthetic phosphatidylcholines with very few

studies employing sphingomyelin (SPM) as a lipid (Barenholz, 1984; Fenske et

al., 1988, 1990; Cushley et al., 1987; Chana et al., 1990; Mims et al., 1986;

Reisinger and Atkinson, 1990; Ginsburg et al., 1982). Yet several pieces of

evidence indicate that the study of sphingomyelin is of particular interest

(Bentejac et al., 1988, 1990). Firstly, sphingomyelin is one of the major

phospholipid components of serum lipoproteins, and is second in abundance

only to phosphatidylcholine (Barenholz, 1984; Gotto, 1987; Fenskeet a1.,1988).

Secondly, it was reported recently that lipoproteins present in the peripheral

lymph of humans are enriched in sphingomyelin (Reichl and Sterchi, 1992).

Third, it was shown that sphingomyelin is positively correlated with the

development of atherosclerosis (Gotto, 1987). Furthermore, the physiological

importance of sphingomyelin is demonstrated by the high degree of regulation of

the chain length of the fatty acid in the ceramide molecules in different

lipoprotein classes. In general, the weight percent of sphingomyelin molecules

increases with lipoprotein density (Myher, et al., 1981).

It was shown that sphingomyelin interacts with apolipoprotein A-I, forming

discoidal particles, in a manner similar to dimyristoylphosphatidylcholine

(DMPC). However, discs formed with sphingomyelin are more resistant to

guanidine-HCI denaturation than those formed with DMPC (Swaney 1983). For

example, in the case of sphingomyelin-containing discs, apolipoprotein A-I

continued to display significant helical character up to 7 molar guanidine-HCI.

Since both lipids possess phosphoryl choline headgroups, the increased particle

stability can not be a consequence of the headgroup. Interaction between

sphingomyelin and apolipoprotein A-I appears to take place at or near the region

of the acyl chains, since it was reported that the protein-lipid interaction

diminished as the length of the acyl chain increased (Swaney and Chang 1980).

Moreover, it appears that sphingomyelin significantly enhances the stability of

reconstituted LDL (DMPCISPMICO) (Treleaven et al., 1991 ; W.D. Treleaven,

unpublished results).

Both SPM and PC (phosphatidylcholine) are classified in the same lipid

subclass, and have very similar packing parameters, because of their similar

headgroup (phosphorylcholine) and hydrophobic region (composed of two

hydrocarbon chains). These gross similarities enable them to replace each

other in membranes (Barenholz, 1984). However, there are marked differences

of structure in the fine details of these molecules. PC has two hydrocarbon

chains of about equal length, while SPM has one hydrocarbon chain of constant

chain length, contributed by sphingosine, and a second of varying length,

contributed by the N-acyl group. The latter can be up to 10 carbons longer than

the sphingosyl residue. This asymmetric nature is responsible in part for

interdigitation between the opposing monolayers of the lipid bilayer. The higher

degree of saturation of SPM relative to PC is another factor which contributes to

the increased rigidity in the SPM bilayer. The differences of hydrogen bond-

forming capability in the interface region between SPM and PC are even more

striking. The amide bond and hydroxyl group of SPM afford an important

hydrogen bond donor capability not found in PC (SPM can act as hydrogen

bond donors and acceptors), whereas in PC, the carboxyl oxygen can act only

as hydrogen acceptors (Barenholz, 1984). Figure 1.3 shows the structures of

sphingomyelin and phosphatidylcholine.

Thus, we have chosen SPM as a monolayer phospholipid and cholesteryl

ester as a neutral core to prepare microemulsions with the particle size of 23 -

26 nm which are in the size range of LDL. Cholesteryl oleate (CO) was selected

as the hydrophobic core because it is one of the cholesteryl esters which

predominate in the core of native low density lipoprotein. To date, the

preparation of homogeneous SPMICO microemulsions in large quantity has not

been reported.

For rnicroemulsions containing proteins, we were interested in studying

the protein-lipid interactions in LDL-sized microemulsions containing

/OH SPM -CH

Figure 1.3: Molecular structures of sphi ngomyelin and phosphatidylcholine.

sphingomyelin and apo-HDL3. We wish to shed some light on general aspects

of lipid-protein interaction using this simple reconstituted particle based on a

number of considerations:

(1). Natural LDL contains apo 8-1 00, phospholipids, and cholesteryl ester. The

size of apo 8-100 (540,000 kD) and its propensity to aggregate in the lipid-free

state and in the absence of very high concentration of detergents make

experiments very difficult (Atkinson and Small, 1986; Gotto; 1987; Chen et al.,

1 989).

(2). The reconstituted particle containing sphingomyelin is very stable, and

amenable to analysis by NMR techniques. We have initiated this study, and

previous data in this laboratory indicated that reconstituted LDL spectra differ

from those of native LDL (Treleaven et al, 1991).

(3). Apo-HDL3 was selected for reconstitution of protein-containing, LDL-sized

particles because it is water-soluble and readily available.

Moreover, such a study also has biological relevance. In vitro studies

have shown that reverse cholesterol transport from cells takes place in the

presence of suitable acceptors. Plasma HDL as well as reconstituted HDL

(apolipoproteins with phospholipids) appear to be the most efficient acceptors

(Bentejac et al., 1990; Hara and Yokoyama, 1991, 1992; lbdah et al., 1989).

Stein and coworkers showed that complexes of HDL apolipoproteins with

sphingomyelin appear to be better acceptors than complexes with

phosphatidylcholine (Stein et al., 1988, 1989 a,b).

31 P NMR is a powerful, nonperturbing mean of investigating the

structure and dynamics of membrane headgroups. When proteins associate

with phospholipids, they must interact with the phospholipid headgroup region

(Yeagle, 1990; Seelig, 1978). 3' P NMR primarily senses the behavior and the

environment of the phosphorous, thus is an ideal method of characterization of

lipid-lipid interaction and lipid-protein interaction near the headgroup. The 31 P

nucleus is 100•‹/o naturally abundant. 31 P NMR can be acquired from

membranes without addition of any probes to the membrane under study.

Therefore, we used the 31P NMR technique to study lipid-protein interactions in

reconstituted model particles (SPMICOlapo-HDL3) and protein-free

microemulsions(SPM/CO).

1.5 Research Objectives

The first objective of the present research was to develop an optimized

method to reproducibly prepare microemulsions of sphingomyelin (SPM) and

cholesteryl oleate (CO) in good yield and defined size.

The second objective was the characterization of reconstituted lipoprotein

particles of SPMICOlapo-HDL3 by 3' P NMR.

The third objective was to study the diffusion behavior of the monolayer

of SPMICO microemulsions and SPMICOlapo-HDL3 reconstituted lipoproteins.

CHAPTER 2

THEORY

2.1 Quasi-Elastic Light Scattering

Quasi-Elastic Light Scattering was used to characterize the size of the

submicron particles for this study. Figure 2.1. shows the schematic diagram of

the Nicomp 370 DLS (dynamic light scattering) apparatus. The principle of DLS

is as follows: A laser beam is focused into a cell containing a solution of

suspended particles. A small fraction of the incident light is scattered by the

particles and collected at an angle (usually 90") by a sensitive PMT detector.

The suspended particles are not stationary; rather, they randomly move about or

diffuse by a process known as Brownian motion. Each particle illuminated by the

laser beam produces a scattered light wave whose phase at the detector

depends on the position of the particle in solution. As a result, the phase of each

of the scattered waves arriving at the PMT detector fluctuates randomly in time.

The reason that the intensity of scattered light fluctuates in time is that the

different scattered waves interfere with each other. Each individual scattered

wave arriving at the detector bears a phase relationship with respect to the

incident laser wave which depends on the precise position of the suspended

particle from which it originates. Although individual fluctuations occur randomly,

there is a well defined lifetime z for their buildup and decay, roughly equal to the

average time required for a pair of particles to change their separation by one-

half the laser wavelength h. The lifetime z is inversely related to the diffusivity D.

In order to get a reliable quantitative measure of t (where t is time), one should

The autocorrela ition function, C(t), is used to study the correlation or similari

between the value of I(t) at given time t' and a later time (t'-t). From this

equation, we can easily see that the largest value of C(t) occurs for a very small

value of t, where the sampled intensities are nearly identical, because the

particles have moved only slightly. As a result, the value of C(t) fort + 0 is

<12(t)>. Conversely, the smallest value of C(t) occurs in the limit of large t, where

the sampled intensities are no longer correlated with each other due to the

Brownian motion. Hence, at t + -, C(t) reduces to the square of the average

scattering intensity <I (t)> 2.

A digital autocorrelator is used to obtain C(t) simultaneously for many

values of t, resulting in a smooth quasi continuous function. For the simplest

case of particles of uniform size, C(t) consists of a single decaying exponential

after subtraction of the baseline < I (t) > :

C(t)= A exp (- 2Dk't) ( 2-2 )

Here k' = (4xn / h)sin 812 is scattering wavevector, n is the index of refraction of

the solvent, 8 is the angle at which scattered light is intercepted by the PMT

detector. In the case of Nicomp 370, h = 632.8 nm, 8=90•‹, k' equals 1.868~1 o5

cm-1. One can compute the autocorrelation function of the fluctuating intensity,

obtain a decaying exponential curve in time. From the decay time constant, one

can get the particle diffusivity (D). Finally the radius R of the particles can be

obtained from the well known Stokes-Einstein relation:

Where, T is the absolute temperature, R is the radius, q is the viscosity of the

solution and k is the Boltzman constant. In the case of a polydisperse mixture of

M different particle sizes, each of which has radius R and diffusivity D, a more

complicated expression is needed:

M ~ ( t ) = A (zfi exp ( - 2 ~ ~ k ~ t ) ) 2

The correlation function consists of the square of a weighted sum of decaying

exponentials, each of which decays at a rate corresponding to a particular

particle radius. The weighting coefficient is proportional to the concentration of

particles, a given radius, the square of their volume, and the square of the

scattering intensity " form factor," which is due to intraparticle interference. The

purpose of distribution analysis is to invert C(t) successfully so as to obtain the

weighting coefficient.

2.2 3 1 ~ NMR Theory

For a spin-1/2 phosphorus nucleus, NMR absorption is a result of

transitions between two energy levels (-1/2, 1/2) which are stimulated by the

-- S L I T

DIGITAL AUTOCORRLUTOR

Y i D t O M I C I O C O V V T U D I S P U Y

Figure 2.1: Schematic diagram of the Nicomp 370 DLS apparatus.

applied r.f. field. The nucleus will rotate at the Larmor frequency around the

applied field. The Larmor frequency is determined by the strength of the applied

field and the gyromagnetic ratio (y) as shown in equation 2.5:

The mathematical technique called Fourier transformation allows one to

transform data from the representation in the time domain into an equivalent

representation in the frequency domain and vice versa as shown in equation 2.6:

where f(t) represents the time domain data, and f(o) represents the frequency

domain data. Relaxation is the process by which the magnetization approaches

equilibrium. There are two relaxation times: longitudinal relaxation time or spin-

lattice relaxation time (Ti) and transverse relaxation time or spin-spin relaxation

time (T2). T i measures the efficiency of attaining thermal equilibrium between

the spin and its surrounding after applying a radio frequency pulse. According to

the assumption of exponential growth of the induced field, the magnetization in

the Z direction can be deduced as shown in equation 2.7 (Derome, 1987). T2 is

the time constant for the decay of the precessing x-y component of the

magnetization following a perturbation. When molecular motion is very fast as in

the case of non-viscous liquids, TI = T2. However, in the case of solids, TI

>>T2.

Because of the field inhomogeneities, the linewidth at half-height (Av,,,) usually

is not determined by ideal relaxation time T2 as deduced by classical Block

equations, but instead by effective relaxation time T2' as shown in equation 2.8

(Harris, 1983; Seelig and Seelig, 1980; Ames, 1966; Abragam, 1961 ):

The 3 1 ~ NMR spectra of the biomembrane reflect contribution from -

P dipolar interactions and chemical shift anisotropy (Ao) of the phosphorus

nucleus. The dipolar interactions can be easily removed by proton-decoupling.

The chemical shift anisotropy arises from the fact that electronegative oxygen

L G nuclei shield the electron from the external magnetic field. The interaction of the

electron cloud surrounding the phosphorus nucleus with the external applied

magnetic field gives rise to a local magnetic field. The induced field further

shields the nucleus. Because chemical shift anisotropy is from the anisotropic

shielding of the nucleus by the bonding electrons, this is the informative

parameter on the headgroup of the phospholipids.

Asymmetry of the charge distribution of the phospholipid suggests that

31P chemical shifts vary as a function of the orientation of the molecule relative

to the external magnetic field. The shielding is smallest along the axis with the

lowest electron density and largest along the molecular axis with the highest

electron density (Seelig, 1982). The chemical shift anisotropy can be defined by

three principal components oil, (322, (333 of the chemical shielding tensor. The

static chemical shielding tensor op is given by 2.9 (Seelig, 1978):

where the subscript refers to the molecule-based principal coordinate system.

The orientation of the chemical shielding tensor about the phosphate segment

was determined for single crystals of phosphoethanolamine (Kohler and Klein,

1976) and barium diethylphosphate (Herzfeld et al., 1978). The magnitude and

the orientation of the principal elements were determined by monitoring the

variation of the peaks as a function of crystal rotations with respect to the

magnetic field. Figure 2.2 illustrates the principal orientations of chemical

shielding tensors 011, 022, 033 for the phosphate segment of a phosphodiester,

barium diethylphosphate (Herzfeld et al., 1978). The ol axis is approximately

perpendicular to the O(3) - P - O(4) plane, 022 bisects the O(3) - P - O(4) angle

and the 033 is orthogonal to these two directions. The orientation of the principal

axes in phospholipid molecules has proven to be very difficult to measure

because the difficulties encountered with growing large enough crystals for the

NMR investigation. Recently, Hauser et al. (1 988) have determined the principal

axis orientations in a phospholipid analogue, 1 -hexadecyl-2-deoxyglycero-3-

phosphoric acid monohydrate. The values are very close to those reported for

the phosphomonoesters, phosphoethanolamine and 2-deoxyribose- 5'-

monophosphate, and the orientations only tend to deviate 7 - 13" from the

barium diethylphosphate. The magnitude of principal elements in phospholipids

can be measured with less certitude from the P NMR spectra of crystalline

powders (Seelig, 1978). The principal components o;; are obtained from the

spectra of phospholipid powders, where the phosphate group is completely

immobilized. The 3 lP NMR spectrum of anhydrous dipalmitoyl

phosphatidylcholine (DPPC) is shown in Figure 2.3. The principal components

are as follows: o 11 = -98 ppm, o 22 = -34 ppm, o 33 = 134 ppm. For the

monohydrate of DPPC, the principal values of the chemical shift anisotropy are

reduced, suggesting that the binding of one molecule of water increases

phosphate symmetry. With the increasing concentration of water molecules, the

spectrum of dipalmitoyl phosphatidylcholine (DPPC) is converted from an

asymmetric lineshape to an axially symmetric one. This change is due to the

molecular motions, in particular the phospholipids undergo rapid rotation around

the director axis (perpendicular to membrane surface). This averages the

components of the shielding tensor in the plane of the membrane, giving rise to

Figure 2.2: The three different orientations of the chemical shielding tensors in

the molecular frame of the phosphate. O(1) and O(2) are the

esterified oxygen bonded to the headgroup residue and glycerol backbone respectively. While O(3) and O(4) are the nonesterified

oxygens (Herzfeld et al., 1978).

1 I I I I I v- 2 0 0 100 0 -100 - 200

CHEMICAL SHIFT (PPM) RELATIVE TO EXT. H,PO,

Figure 2.3: 31P NMR (1 18 MHz) proton-decoupled spectrum of

anhydrous dipalmitoylphosphatidylcholine power at 15 "C.

an effective tensor o,ff which is axially symmetric about the director axis (Seelig,

1978) as shown by the relation:

Where o,, and o I are the time averaged components parallel and perpendicular

to the director axis. For planar oriented bilayers, a single resonance is

observed. The resonance is dependent on the angle between the director axis

and the applied magnetic field (Seelig, 1978). The residual chemical shift

anisotropy Ao is defined as:

AG = 0, - Oil

The quantitative relationship betwe e static component oii and time

averaged components o,, (where the magnetic field is parallel to the bilayer

normal) and o, (where the magnetic field is perpendicular to the bilayer normal)

is given by (Seelig, 1978):

Where @ and 8 are the Eular angles relating to the orientation of the rotation axis

with respect to the principal axis of the shielding tensor as defined in Figure 2.2.

For a membane bilayer, the chemical shift anisotropy is between 40 - 50

ppm. A quantitative measure of phosphate headgroup motion has been

developed by Seelig, where relationship between do and the static chemical

shift anisotropy value is represented by order parameters (order parameters Sii

reflect the amplitude of the motion of the principal coordinate axes of the

chemical shielding tensor about the director axis) (Seelig, 1978):

the phosphate group and S33 refers to the order parameter of the axis I i

connecting to the nonesterified oxygens.

Ao is a measure of the orientation and the average fluctuation of the

phosphate group. Unfortunately, Ao is determined by two independent order

parameters, and it is impossible to determine the two independent order

parameters at the same time only by one measurement. This makes the

analysis of 31P NMR spectra much more complicated.

When dispersed in water, not all lipids adopt a bilayer organization which

is the generally accepted molecular model for biomernbrane. Some lipids, such

as phosphatidylethanolamine, instead tend to form hexagonal (11) structures.

The hexagonal (11) phase is characterized by tubular structures. The polar

headgroups of these molecules face toward the inside of the cylinder while the

hydrocarbon chains face the hydrophobic phase (Cullis and DeKruijff, 1979).

31P NMR is sensitive to the phase changes between different phase

structures (Gennis, 1989). The proton decoupled 31P NMR spectra of soy bean

phosphatidylethanolamine yield powder patterns of reverse asymmetry, with

about half of the chemical shift anisotropy of that can be found in bilayer

membranes. Because the tube-like structure found in hexagonal (11) phase,

additional motional averaging (over what is found in phospholipid bilayer) results

from rotation of the long cylinder and lateral diffusion of the phospholipid

molecule around the aqueous channel of the cylinders. This causes a reduced

value of Ao to 112 of its original value (in the phospholipids bilayer) and changes

the sign. Figure 2.4 shows the 31 P NMR spectra of polymorphic phase of

phospholipids. In small unilamellar vesicles, microemulsions, recombinant

lipoproteins and native lipoproteins, the chemical shift anisotropy cannot be

observed directly. Rapid, isotropic tumbling of the small particle averages the

chemical shift anisotropy, and as a result, a single Lorentzian lineshape is

observed. The 31 P NMR linewidth A v l ~ can be constituted in terms of the

effective correlation time for phospholipid reorientation ze and the residual

second moment M2 (Abragam, 1961 ):

The effective correlation time for isotropic motion is determined by particle

tumbling (z t) and phospholipid lateral diffusion (z d).

where the rotational correlation time (q) is determined by Stokes-Einstein

relationship

~t =4xR% / 3kT

and the diffusion correlation time (zd) is determined by

Where T = absolute temperature in Kelvin

28

R = particle radius

q = viscosity

D = coefficient of lateral diffusion

k = Boltzmann constant

The second moment M2 is given by (McLaughlin et al., 1975)

M2 = ( 4 I 45 ) ( ~ K V ~ ) ~ A O ~

where A o is the chemical shift anisotropy.

2.3 The Theory of Lateral Diffusion of Phospholipids in Model particles

The lateral diffusion coefficients of the reconstituted particles

(microemulsion and reconstituted particles) were measured according to the

method of Cullis (1 976). A gradual increase in glycerol concentration was used

to broaden the 3IP NMR linewidths. It is well known that the line-narrowing was

chiefly due to two mechanisms: particle tumbling and lateral diffusion. With the

increasing glycerol concentration, particle tumbling is slowed down and lateral

diffusion will become the dominant mechanism contributing to the linewidth. By

combining equations (2.15) to (2.18), the linewidth can be represented by:

In order to analyze the linewidth variation as a function of viscosity, the constant

"C" is required. C is a constant containing the contribution from z, independent

processes, such as magnetic field inhomogenity and spin-lattice relaxation. C is

the natural linewidth of the phospholipid in chloroform, which is the isotropic

value. The value of C was measured to be 15 + 3 Hz ( Parmar et al., 1986).

A plot of ( Av 112 - C) -' versus q -' is a straight line, and from the ratio of

intercept to slope we can obtain the lateral diffusion coefficient D:

D= ((intercept 1 slope)kT) 1 ( 8nR ) . ( 2.21 )

By combining equations 2.15 and 2.19, chemical shift anisotropy can also be

obtained from a plot of ( nAv l12 - C) versus the vO2 by equation 2.22.

Where v, = frequency

A o = chemical shift anisotropy.

Corresponding Corresponding Phospholipid phases 31P-NMR spectra Fracture-faces

Bilayer

B

Phases where isotropic motion occurs

I 1. Vesicles 2. Inverted micellar 3. Micellar 4. Cubic 5. Rhombic - 40 ppm- H--

Figure 2.4: proton decoupled 3lP NMR spectra of polymorphic phases

available to liquid crystalline phospholipids (Gennis, 1989).

A. Bilayer: egg phosphatidylcholine

B. Hexagonal (H It): Soya bean phosphatidylethanolamine C. Isotropic Motions: Small Unilamellar Vesicles,

Microemulsions

CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

Bovine brain sphingomyelin and cholesteryl oleate were purchased from

Sigma Chemical Company, St. Louis, Missouri. No impurities were detected by

thin layer chromatography in chloroform/methanol/water (65:25:4). Lipids were

used without further purification. Aquacide I I (sodium salt of

carboxymethylcellulose, MW = 500,000) was purchased from Calbiochem

Corporation. Glycerol was purchased from Fisher Scientific Company. The lipid

analysis kits for determining total cholesterol were purchased from Boehringer

Mannheim.

Sonication buffer

The sonication buffer contains 100 mM KCI, 10 mM Tris-HCI. Na2C03

was used to adjust the pH of the buffer to 8.6. Solid KBr was used to raise the

buffer density to 1.21 g/ml.

Dialysis buffer

The dialysis buffer contains 100 mM KCI, 10 mM Tris-HCI. Na2C03 was

used to adjust the pH of the buffer to 8.6.

3.2 Methods

3.2.1 Isolation of High Density Lipoproteins (HDL)

HDL3 was isolated by sequential isopycnic floatation between the

densities p=1.125 and p=1.210 glml, at 4 "C using a Beckman L5-75

ultracentrifuge, and a Ti 50.2 rotor. Potassium bromide was used to raise the

density. Samples were centrifuged at 45,000 rpm for 24 hours at p = 1.1 25 glml,

and 48 hours at p=1.210 glml.

3.2.2 Isolation of HDL3 Apolipoproteins

HDL3 ( 0.45 ml, 10 to 15 mg protein per ml ) was added to one of the 30

ml glass centrifuge tubes. Approximately 25 ml of cold (-20 "C)

chloroform/methanol (CHC13:MeOH 2:1 vlv) was added to each tube. The

solution was incubated at -20 "C for 1 hour. The tubes were centrifuged for

approximately 5 minutes using a desktop centrifuge, and the solvent was

decanted from the precipitate. Apolipoproteins were incubated four times with

cold anhydrous diethyl ether (15 minutes each at -20•‹C ). Following the final

wash, the apolipoproteins were air dried, and stored at -20 "C. Analysis of the

apolipoproteins by SDS polyacrylamide gel electrophoresis revealed that no

human serum albumin was present.

3.2.3 Preparation of Microemulsions

Homogeneous microemulsions were prepared by the following

procedures (Darfler, 1990; Ginsburg et al., 1982). About 150 mg sphingomyelin

and 60 mg cholesteryl oleate were codissolved in chloroform / methanol (2: l)

mixture. The solvent was evaporated under a stream of nitrogen, and the lipid

mixture was further dried under high vacuum overnight. A lipid-buffer emulsion

was formed by mixing lipid with approximately 10 ml of sonication buffer. The

mixture was sonicated for 5.5 hours (9O0lO duty cycle) at 60 "C under nitrogen

using a Heat Systems W-375 sonicator operating at 30% output power. The

circulation temperature was monitored by a thermocouple system around the

sonication mixture. The sonication mixture was centrifuged for 15 minutes to

remove titanium particles, then was dialyzed against hypotonic saline buffer with

3 - 4 changes of 1 -liter each, over 14 hours. The sample was centrifuged at

50,000 rpm (1 65,0009) for 30 minutes(l5"C). The very top layer was removed

and saved for the light scattering measurement. The second top layers were

collected and recentrifuged again at 50,000 rpm from 15-30 hours. The bottom

vesicle-containing layer was discarded. The microemulsions were removed from

the top of the second spin. The microemulsions can be prepared reproducibly

with about 23-27 nm in diameter as shown by the light scattering measurement,

and these samples are stable for several weeks. The microemulsions were

further concentrated using either ultracentrifugation or aquacide at room

temperature. The microemulsions were then transferred to an NMR tube.

Temperature dependent 3 1 ~ NMR experiments were conducted immediately.

3.2.4 Sphingomyelin Vesicles

Sphingomyelin vesicles were prepared according to the procedure of

Barenholz (Barenholz et al., 1976). In short, sphingomyelin was suspended in

50 mM KCI; the lipid suspensions were sonicated using a Heat Systems

Sonicator (W-375) under nitrogen for 30 minutes. The sonication temperature

(0•‹C) was controlled by an ice-water mixture. Following sonication, the vesicles

were separated by ultracentrifugation. The vesicles were prepared reproducibly

with approximately 25 - 27 nm in diameter. Vesicles were stable for several

days to two weeks. The temperature dependence of 31 P NMR spectra was

recorded.

3.2.5 Quasi-Elastic Light Scattering

Quasi-elastic light scattering measurements were performed using a

Nicomp model 270 or 370 submicron particle sizer.

3.2.6 Preparation of Reconstituted Lipoproteins

The appropriate apoproteins were dialysed against buffer (1 00 mM KCI,

10 mM Tris HCI, Na2C03, density = 1.005 glml, pH = 8.6) for several hours. The

apoprotein solution was transferred to a vial, and the microemulsion solutions

were added to the vial very gently. The apolipoprotein-microemulsion mixture

was stirred and incubated at 50 + 1 "C for 18 hours. 31 P NMR experiments at

various temperatures were conducted using these samples. After NMR

measurements, protein concentration of the reconstituted lipoprotein was

measured by the Lowry method (Lowry et al., 1951). The phospholipid

concentration in recombinant lipoprotein was measured as described (Ames,

1966). Cholesteryl oleate concentration in recombinant lipoprotein was

determined with the specific assay kits from Boehringer Mannheim.

3.2.7 NMR Measurements

The 3lP NMR experiments were performed at 102.2 MHz without proton

decoupling using a home-built spectrometer and a 5.9 T Nalorac

superconducting magnet. Data collection and the Fourier transformation were

performed on a Vax station I. Temperature was controlled by a solid state

temperature controller built by the Simon Fraser University electronics shop with

an accuracy of + 0.25 "C. The signal to noise ratio was enhanced by using

exponential multiplication. The samples were allowed to equilibrate for 30 min.

at a given temperature before data were acquired. The spectral parameters are

given in the figure legends.

For the field dependence studies, 3lP NMR spectra were collected at 40

MHz (SY-100),102.2 MHz (home-built NMR), 160.2 MHz (Bruker AMX-400), and

202.46 MHz (Bruker AMX-500) .

3.2.8 Diffusion Measurements

Glycerol was added to the freshly prepared samples (SPMICO

microemulsions and SPMICOlapoHDL3 reconstituted particles) to increase the

viscosity. The viscosity of SPMICO microemulsions and reconstituted

measured using an Ostwald viscometer. 31 P NMR spectra were collected at

each viscosity at 25 "C.

3.2.9 NMR Lineshape Analysis

Phosphorus NMR spectra of SPMICO microemulsions, sphingomyelin

vesicles and reconstituted lipoproteins were analyzed using a four parameter

(root-mean-square base line, signal amplitude, and estimated linewidth for each

signal plus the chemical shift) iterative least-squares fit of the resonance to a

single Lorentzian function. The 3IP spectra of reconstituted lipoprotein at 15 "C

and 25 "C were analyzed using a seven parameter (baseline, signal amplitude of

the broad component, chemical shift of broad component, linewidth at half-height

of broad component, signal amplitude of narrow component, chemical shift of

narrow component, linewidth at half-height of the narrow component) iterative

least-squares fit of the phosphorus resonance to a superposition of two

Lorentzian functions.

P

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Unilamellar Vesicles

The unilamellar vesicles were made successfully by using the method of

Barenholz (Barenholz et al., 1976). As described in Chapter 3, sphingomyelin

was dissolved in chloroform and dried under vacuum for about 2 hours, and then

allowed to interact with buffer at pH 8.6 and specific ionic strength (100 mM KCI).

They then swelled to form multilamellar liposomes. Multilamellar liposomes were

subsequently subjected to ultrasonic irradiation to produce small unilamellar

vesicles. The vesicles were 25 nm in diameter and contained a single bilayer.

Figure 4.1 shows the size distribution of sphingomyelin vesicles determined by

light scattering (the particles in the range of 60-100 nm are large unilamellar

vesicles). The inner and outer leaflet of the bilayers were shown not to be

equivalent as demonstrated by 3lP nuclear magnetic resonance studies using

the chemical shift reagent Pr 3+ to demonstrate the difference between the inner

and outer layer (Yeagle, 1990). The homogeneous vesicles are also thought to

provide a surface that has curvature similar to low density lipoprotein (Diameter

= - 25 nm). Therefore, we may extrapolate from the vesicle system to the

biomembrane or lipoprotein system. But caution should be exercised when one

attempts to make some extrapolation to the native lipoprotein because of the

simplicity of the small unilamellar vesicles.

Figure 4.1 : The size distribution of sphingomyelin vesicles by Quasi-Elastic

Light Scattering.

i representative 3' P NMR spectra of sphingomyelin vesicles at selected

temperatures. The solid line represents an iterative least-square fit of single

Lorentzian function to the data (crosses). The linewidths of sphingomyelin

vesicles at different temperatures over the range 15 "C to 50 "C are shown in

Table 4.1. The trend of decreasing linewidth as temperature was raised is

reversible. From Table 4.1, we can see that below 45 "C, the linewidth of 31 P

NMR spectra shows great temperature dependence. Above 45 "C, the linewidth

of 31P NMR spectra shows slight temperature dependence.

4.2 Microemulsions: Models for Smaller Cholesterol-Rich Lipoproteins

As models to study the lipid organization in lipoproteins, protein free

homogeneous microemulsions have been prepared using specific phospholipid

and cholesteryl esters, which constitute the important components of

lipoproteins. The microemulsion system was formed with a single species of

phospholipid, therefore, fundamental details of the lipid-lipid and lipid-protein

interaction could be studied without the complexity of heterogeneous molecular

compositions found in the native lipoproteins.

Typically microemulsions are formed by extensive sonication of both

phospholipid and cholesteryl ester in an aqueous solution at a temperature

above the main order-disorder transition temperature of both lipid components.

Fractionation was carried out by ultracentrifugation. Ultracentrifugation is the

most widely used technique for lipoprotein separation because particles can be

separated from each other on the basis of difference in the buoyant densities or

Figure 4.2: 31P NMR spectra of sphingomyelin vesicles a). 15 "C, b). 20 "C, c). 25 "C, d). 50 "C The spectra were simulated by an iterative least-squared fit to a Lorentzian lineshape function (solid line) to the spectral data

points (crosses). Spectral parameters for all cases are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 10,000 Hz,

delay between pulses = 2.5 s, dataset = 2K zero filled to 4K, dwell time = 50 ps, line broadening = 5 Hz, Number of scans =

10,000.

Figure 4.2 (a): The P NMR spectrum of sphingomyelin vesicles at 15 "C

Figure 4.2 (b): The P NMR spectrum of sphingomyelin vesicles at 20 "C

42

Figure 4.2 (c): The 31P NMR spectrum of sphingomyelin vesicles at 25 "C

Figure 4.2 (d): The 31P NMR spectrum of sphingomyelin vesicles at 50 "C

Table 4.1: The linewidth of sphingomyelin vesicles at selective

temperature

I Temperature ( "C ) Linewidth ( Av 1/, ) (Hz)*

I * Numbers in parentheses are about 10O/0 of the linewidths

on the basis of their sedimentation rate. The density of the sonication buffer was

adjusted to 1.21 glml before sonication. Following sonication, the sample was

dialyzed against the dialysis buffer (1 00 mM KCI, 10 mM Tris-HCI, density=

1.005 glml, pH = 8.6). Na2C03 was used to adjust the pH of the dialysis buffer

to a desired value. The reason for doing this is as follows: After sonication for

five and half hours, the products of the sphingomyelin and cholesteryl oleate

mixture were small-sized microemulsions, large-sized microemulsions,

multilamellar liposomes and small unilamellar vesicles. When the density of the

sonication buffer was adjusted to 1.21 glml, the multilamellar liposomes and

small unilamellar vesicles that are produced during the process of sonication are

much heavier than microemulsions. Following sonication, the background

density was adjusted to 1.005 glml. Under the untracentrifugation condition, all

the particles can be separated from each other according to buoyant density,

with microemulsions floating at the top of the centrifuge tube, the background

buffer in the middle layer, and multilamellar liposomes and vesicles sinking to the

bottom of the centrifuge tube. The schematic representation of the separation is

shown in Figure 4.3. Therefore, adjusting the sonication buffer density to 1.21

glml is the critical step to have a good separation of homogeneous

microemulsions.

Preparation of LDL-sized microemulsions also requires other critical

steps. Lipids in the appropriate ratio were thoroughly mixed in solvent, and then

the solvent was completely removed prior to sonication. The temperature of the

mixture was maintained at 60•‹C throughout the sonication procedure. Failure to

maintain this temperature would result in the formation of undesired particles.

The volume of sonication buffer added to the sonication vessel should be in the

+-)Bigger-sized microemulsion

Desired-sized microemulsion

+-I Background buffer

-Vesicles or multilamellar liposomes

Figure 4.3 : Schematic representation of ultracentrifugation separation of microemulsion.

sonication. The distance between the microtip and the bottom of the sonication

vessel should be in the range of 0.8 - 1 cm. The sonication would be inefficient if

the microtip is too far away from the bottom of the vessel, while the sonication

vessel would break easily if the microtip is too close to the bottom of the vessel.

Sonication for 5.5 hours gave optimum yields of the desired particles.

In order to make sure that the sonication process did not damage

sphingomyelin or cholesteryl oleate, thin layer chromatography was used to

check the chemical compositions of lipids after the sonication. The experimental

results showed that no degradation products of sphingomyelin or cholesteryl

oleate could be detected. That the SPMICO microemulsion can be successfully

prepared by the above-mentioned protocol demonstrates that sonication is an

efficient method to prepare stable, homogeneous SPMICO microemulsions of

LDL size. The homogeneous size distribution was confirmed by quasi-elastic

light scattering.

Figure 4.4 shows the size distribution of SPMICO microemulsions

determined by the light scattering technique. Greater than 95% of the

microemulsions fell within the measured size range (23-27 nm). Occasionally

trace amounts of large microemulsions (about 80 nm) contaminated the sample

if ultracentrifugal separation was incomplete. It can easily be demonstrated that

these particles make no contribution to the NMR spectra within the spectral

window in these experiments. The big particles (80 nm) are about 3 times

bigger than the small ones (about 25 nm in diameter). If the linewidth

contribution from diffusion is neglected, and we consider only the isotropic

Mean Diameter of Major Particles (98 O/O) = 25 nm

Figure 4.4: The size distribution of SPMICO microemulsions measured by Nicomp 370 particle sizer.

rotation of the particle, from the Stokes-Einstein equation (equation 2.1 7 in

chapter 2), zt of the larger particles is calculated to be 33 times larger than that

of the small ones. Since linewidth varies directly with correlation time, the

linewidth of the big particle will be about 33 times larger than the small particles,

therefore, the linewidth of the analyzed big particles can only contribute to the

baseline of the NMR spectra.

The sphingomyelin/cholesteryl oleate microemulsions were studied by 31 P

NMR. Figure 4.5 shows representatives of the 3lP NMR spectra of SPMICO

microemulsions at different temperatures. The spectra at all the temperatures

can be represented by a single Lorentzian function as demonstrated by Figure

4.5. This suggests that all of the phospholipids are magnetically equivalent.

The temperature dependence of 31 P linewidth of SPMICO

microemulsions is shown in Table 4.2. Linewidths are calculated by the

computer simulation to the s1 P spectra to a single Lorentzian lineshape function.

By comparing Table 4.1 with Table 4.2, it is clear that the linewidth of the spectra

of sphingomyelin vesicles and SPM /CO microemulsions are similar within the

experimental error at temperatures below 40 "C. This indicates that the motions

of the headgroup at temperature below 40•‹C are similar. The gel to liquid

crystalline phase transition temperature of around 37 "C is in good agreement

with the data presented here. At temperatures above 40 "C, the SPMICO

microemulsions have slightly broader linewidths compared to SPM vesicles.

This suggests that the neutral core may alter the motion or orientation of the

headgroup in the microemulsion monolayer.

Figure 4.5: 3' P NMR spectra of sphingomyelin/cholesteryl oleate microemulsions at a). 15 "C, b). 25 "C, c). 35 "C, d). 45 "C The spectra were simulated by an iterative least-square fit of a Lorentzian lineshape function (solid line) to the spectral data

points (crosses). Spectral parameters for those cases are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 25,000 Hz,

delay between pulses = 2.5 s, dataset = 2k zero filled to 4K, dwell time = 20 1s. line broadening = 5 Hz at 15 "C and 25 "C, line

broadening = 0 Hz at 35 "C and 45 "C.

Figure 4.5 (a): 31P NMR spectra of SPMICO microemulsion at 15 OC

Figure 4.5 (b): 31P NMR spectra of SPMICO microemulsion at 25 OC

5 1

Figure 4.5 (c): 3lP NMR spectra of SPM/CO microemulsion at 35 'C

Figure 4.5 (d): 3lP NMR spectra of SPM/CO microemulsion at 45 "C

Linewidth (Av 1/, ) (Hz) *

4.3 SPM/CO/apo-HDL3 Reconstituted Lipoprotein System (LDL size)

Recombinant lipoproteins were prepared by incubating the SPMICO

microemulsion with the purified apo-HDL3 above the order-disorder transition

temperature. Two steps were found to be critical for the successful preparation

of recombinant lipoprotein:

Exhaustive dialysis of the apo-HDL3 against dialysis buffer (1 00 mM KCI, 10

mM Tris-HCI, Na2C03, density= 1.005 glml, pH = 8.6) shoud be carried out in

order to get the same background buffer as microemulsions. After the

dialysis, the appearance of apo-HDL solution should be clear (no precipitate

appears in the solution).

The mixing procedure should be performed very gently.

In addition, the size distribution of the SPMICO microemulsion should be

very uniform and in the range of 23-26 nm. To date, we have reproducibly

prepared the recombinant lipoprotein (SPMICOlapo-HDL3 ) in low density

lipoprotein size. In order to study the effect of temperature on the structure of

reconstituted particles, the 3lP NMR spectra were collected over the

temperature range 10-50 "C. It is interesting to note that at lower temperature

when apo-HDL3 was added to the protein-free SPMICO microemulsions, the 31 P

linewidth of the reconstituted lipoprotein increased by approximately 20-40 Hz

compared to SPM vesicles and SPMICO microemulsions.

After the reconstituted lipoprotein was made, the size of the reconstituted

lipoprotein was checked immediately by quasi-elastic light scattering as shown in

Figure 4.6. The size was the same as that before addition within experimental

error. From this observation, we can conclud e that the linewidth increase after

apo-HDL, was incorporated was not due to any increase of particle size. One

possible explanation for this observation (the linewidth increase at lower

temperatures was not due to increase in particle size) is that bulky apo-HDL,

may influence the motion of sphingomyelin headgroup after the incorporation.

Another possible explanation is that the bulky apo-HDL3 changes the orientation

of phospholipid headgroups, thus resulting in the change in chemical shift

anisotropy of the phospholipid headgroup. From equations 2.1 5 and 2.19 in

Chapter 2, we can see that this could lead to a change in linewidth. The spectra

of reconstituted lipoproteins were presented in Figure 4.7 to Figure 4.9. Each

spectrum can be simulated by a superposition of two Lorentzian functions whose

linewidth at half-height (AVt/2) differ significantly.

However, we were able to represent the data by a single Lorentzian when

the temperature reaches above 35•‹C. Indeed, as demonstrated in Figure 4.10,

the spectrum recorded at 45 "C could be fitted to a single Lorentzian with very

good accuracy. It is possible that a superposition of Lorentzians also exists

above 35"C, but the linewidth of the respective spectral components is not

sufficiently different so that we can resolve them. Table 4.3 shows the linewidths

of 3' P NMR spectra SPMICOlapo-HDL3 reconstituted particles as a function of

the temperature. The linewidths (both broad and narrow components) were

obtained from simulation of the superposition of two Lorentzian functions fitting

to the experimental data points.

The ability to resolve two superimposed spectral components

unambiguously depends on the magnitude of the difference in linewidth between

Mean Diameter of Major Particles (97 %) = 24.0 nm

Figure 4.6 (a) Light scattering results from the SPMICO microemulsion samples before addition of apo-HDL3.

Mean Diameter of Major Particles (98 'lo) = 24.0 nm

Size (nrn)

Figure 4.6 (b) Light scattering results from the SPMICO microemulsion

samples after addition of apo-HDL3.

r

the broad and the narrow component. When the two Lorentzian functions differ

in width by a factor of three or more, they can be easily resolved. On the other

hand, if the difference in linewidth between the broad and narrow component is a

factor of two or less, they may be very difficult to resolve.

The possible explanation for this phenomenon (spectra were comprised of

two components) is that protein alters the motion or the orientation of the

headgroup of phospholipids differently in its different domains. It is possible that

the broad component of the spectra is due to the influence of the protein. We

can not rule out the possibility that the broad component is not influenced by the

protein, but that the narrow component is influenced. But no matter what is the

case, we did observe two different domains in the particle. As the temperature

increased, the rate of the exchange between the "protein - rich" domain and

"protein - deficient" domain increases, thus averaging the observed linewidth.

For instance, at temperatures above 35 "C, the motion of the molecule was so

fast that we were unable to detect a spectral difference between the two

components. A similar phenomenon was also observed for the spectrum at

45•‹C.

Phospholipid - protein interaction is one of the most important topics in the

study of the structure of lipoproteins and biological membranes. The interaction

between the two major components of the membrane (protein and lipid) is very

complex, and the influence of one component on the other cannot be expected

to be the same because the structures of the two components are greatly

different from each other. Therefore, an elucidation of the effects of each

component on the other is crucial to an understanding of the relationship

Figure 4.7: 31 P NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3

particles at 1 5 "C. The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points (crosses). The solid line represents the calculated fit.

Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 50,000 Hz,

delay between pulses = 2.5 s, dataset= 2k zero filled to 4K, dwell time = 10 ps, number of scans = 20,000, line broadening = 5 Hz.

Figure 4.7 (a): 3'P NMR spectrum of SPMICOlapo-HDL3 reconstituted particles at 15 "C (single Lorentzian).

Figure 4.7 (b): 31P NMR spectrum of SPMICOlapo-HDL3 reconstituted

particles at 15 "C (sum of two Lorentzians).

Figure 4.8: 31 P NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3 reconstituted lipoprotein particles at 20 OC. The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters)

of the spectral data points (crosses). The solid line represents the calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 50,000 Hz,

delay between pulses = 2.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans = 20,000, line broadening = 5 Hz.

Figure 4.8 (a): 31 P NMR spectrum of SPMICOlapo-HDL3 reconstituted

particles at 20 "C (single Lorentzian).

Figure 4.8 (b): 31P NMR spectrum of SPMICOlapo-HDL3 reconstituted particles at 20 "C (sum of two Lorentzians).

Figure 4.9: 3lP NMR spectra of sphingomyelin I cholesteryl oleate / apo-HDL3 reconstituted lipoprotein particles at 25 "C. The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or

(b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points (crosses). The solid line represents the

calculated fit. Spectral parameters are as following: pulse width = 6.5 ps ( 90•‹ flip angle ), sweep width = 50,000 Hz,

delay between pulses = 2.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans = 20,000, line broadening = 5 Hz.

Figure 4.9 (a): 31 P NMR spectrum of SPMICOlapo-HDL3 reconstituted particles at 25 "C (single Lorentzian).

Figure 4.9 (b): 31P NMR spectrum of SPMICOlapo-HDL3 reconstituted

particles at 25 "C (sum of two Lorentzians).

Figure 4.10: 31 P NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3 reconstituted lipoprotein particles at 30 "C. The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points (crosses). The solid line represents the

calculated fit.

Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 50,000 Hz,

delay between pulses = 2.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans = 20,000, line broadening = 5 Hz.

Figure 4.10 (a): 31P NMR spectrum of SPMICOlapo-HDL3 reconstituted

particles at 30•‹C (single Lorentzian).

Figure 4.10 (b): 31P NMR spectrum of SPMICOlapo-HDL3 reconstituted

particles at 30 "C (sum of two Lorentzians).

Figure 4.1 1 : 3lP NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3

reconstituted lipoprotein particles at 45 "C.

The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points (crosses). The solid line represents the calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 50,000 Hz,

delay between pulses = 2.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans = 20,000, line broadening = 5 Hz.

Figure 4.11 (a): 3 1 ~ NMR spectrum of SPMICOlapo-HDL3 reconstituted particles at 45 "C (single Lorentzian).

Figure 4.11 (b): 31P NMR spectrum of SPMICOlapo-HDL3 reconstituted

particles at 45 "C (sum of two Lorentzians).

Table 4.3 : 3' P NMR linewidths of sphingomyelinlcholesteryl oleatelapo-

HDLR recombinant lipoproteins at selective temperature (about

Temperature

("C)

Broad component

(Hz)

narrow component

(Hz)

% broad

component

* Uncertainties of the linewidths are 10 %.

between the major components of biomembranes or lipoproteins.

31P NMR is one of the most powerful tools for exploring lipid-protein

interactions. This technique is only sensitive to the behavior of the phospholipid

headgroups, and it is the phospholipid headgroup with its ionic charges that is

best suited for interacting with the surface of the protein that also contains ionic

charges. Significant attractive force could be imagined between phospholipid

headgroups and protein. In contrast, for hydrocarbon chains, the only possible

interaction with protein are between two hydrophobic surfaces in a hydrophobic

medium which are governed by hydrophobic forces and weak attractive (van der

Waals) forces.

Another distinct advantage of 31P NMR is that it is non-perturbing and

does not require labeling. This is important since changes in the structure of

phospholipid headgroups by introduction of labels may significantly alter the

behavior of the labeled phospholipid (Yeagle, 1990).

4.4 A Lateral Diffusion Study of SPMICO Microemulsions

The representative 31P NMR spectra of sphingomyelin/cholesteryl oleate

microemulsions at several different viscosities at 25 "C are shown in Figure 4.1 2.

The linewidths, given in Table 4.4, are obtained from computer fits of the spectra

to a single Lorentzian functions.

A plot of (Av - C) versus q is shown in Figure 4.13. At low viscosity or

intermediate viscosity, the linewidths increase with the increases in viscosity.

Figure 4.12: 3lP NMR spectra of sphingomyelinlcholesteryl oleate microemulsions at several different viscosities at 25 "C. The spectra were simulated by an iterative least-squared fit to a single Lorentzian lineshape function (four parameters) of the spectral data points (crosses). The solid line represents the calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width =

50,000 Hz, delay between pulses = 1.5 s, dataset= 2k zero filled to 4K, dwell time = 10 ps, number of scans 2 30,000.

Figure 4.12(a): The 31P NMR spectrum of SPMICO microemulsion at a

viscosity of 0.98 centipoise.

Figure 4.12(b): The 3'P NMR spectrum of SPMICO microemulsion at a viscosity of 1.29 centipoise.

Figure 4.12(c): The 3 l P NMR spectrum of SPMICO microemulsion at a viscosity of 2.16 centipoise.

Figure 4.12(d): The 3 1 ~ NMR spectrum of SPMICO microemulsion at a

viscosity of 4.45 centipoise.

Figure 4.1 2(e): The 31 P NMR spectrum of SPMICO microemulsion at a viscosity of 6.01 centipoise.

Figure 4.1 2(f): The 3lP NMR spectrum of SPMICO microemulsion at a viscosity of 8.46 centipoise.

- - - -

Table 4.4: The viscosity dependence of linewidths of sphingomyelinl

cholesteryl oleate microemulsions

However, at high viscosity, the increase in the linewidth gradually levels off. This

is important since it indicates that as q increases beyond r 35 centipoise,

particle tumbling (rotation) does not significantly contribute to the linewidth.

A plot of (Av 1 ~ 2 - C) versus q-1 is shown in Figure 4.14. The solid line

represents the least square fit of the reciprocal of linewidth to the experimental

data points. In order to obtain D (lateral diffusion coefficient), the radius of the

SPMICO microemulsion particles must be known. The radius of the particle was

determined by quasi-elastic light scattering to be about 12.5 nm.

We were specifically interested in diffusion of sphingomyelin in the

microemulsions. From equation 2.21, the lateral diffusion coefficient (D) of

sphingomyelin in SPMICO microemulsions was determined to be 1.0k0.3 x 10-9

(cm 2 s-1). A comparison of diffusion coefficients for serum lipoproteins,

microemulsions, reconstituted lipoproteins and phospholipid bilayers is shown in

Table 4.5. The lateral diffusion coefficient of SPMICO microemulsions is

approximately 1.4 times smaller than that of phospholipids in low density

lipoproteins (the value for LDL at 25 "C is 1.4 + 0.5 x 10-9 cm s-1). The reason

to compare this to that of LDL is that low density lipoproteins are cholesteryl

ester rich lipoproteins. The microemulsions were prepared using cholesteryl

oleate as a neutral core, and the size of microemulsions were about the same as

that of low density lipoproteins.

The core of SPMICO microemulsions is composed of only cholesteryl

esters, which undergo a thermal liquid-crystalline to liquid phase transition over

b the range of 20 - 40 "C (Deckelbaum et al., 1977). At 25 "C, the core lipid of

I I 1 I I

1500- -

1000- -

-

0 10 X) 30 40 60

q ( centipoise )

Figure 4.13: The linewidths of 3' P NMR spectra of SPMICO microemulsions versus viscosity.

Figure 4.14: Plot of (Av - C) -1 versus q - 1 of sphingomyelin

/cholesteryl oleate microemulsions.

microemulsions is just into the phase transition. If the core-monolayer

interactions are co-operative in the microemulsions, then the solid like

cholesteryl oleate may be responsible for the slower diffusion of the

phospholipids. Another possible explanation is that the phenomenon could

happen through interdigitation of the acyl chain of sphingomyelin with cholesteryl

oleate.

SPMICO microemulsions appear to behave just like LDL. The diffusion

coefficient constant for low density lipoproteins (core lipids are mainly cholesteryl

ester) at 45•‹C is 10 times greater than that at 25 "C (Cushley et al., 1987;

Fenske et al., 1 990). Whereas in VLDL (core lipids are triglyceride-rich

lipoproteins; triglyceride over the range of 25 to 45 "C is a liquid-like phase), D

remains essentially constant with temperature going from 25 "C to 45 "C as

shown in Table 4.5 (Cushley et al., 1987; Chana et al., 1990).

4.5 A Field Dependence Study of SPMICO Microemulsion

The field dependence of a SPMICO microemulsion was conducted at four

different frequencies (four magnetic fields), specifically:

40 MHz (Bruker SY-100)

102 MHz (our "home built" Machine )

160.2 MHz ( Bruker AMX 400)

202.46 MHz ( Bruker AMX 500 )

The 31P NMR linewidths measured at different frequencies are given in Table

4.6. The linewidth was broader at higher field strength. From equation 2.22, we

can obtain the chemical shift anisotropy.

Table 4.5: Comparison of lateral diffusion coefficients for serum

lipoproteins, microemulsions, reconstituted lipoprotein

particles and phospholipid bilayers.

VLDL

VLDL

LDL

LDL

HDL2

HDL3

egg PCITO

egg PC vesicles

SPMICO microemulsion

Broad components of

SPMICOlapo-HDL3

Narrow components of

SPMICOlapo-HDL3

a Cushley et. al. (1987);

c Cullis (1 976);

This study.

Tempe rat ure ("C)

b Chana, et al., (1 990)

d Fenske et al. (1 990)

The chemical shift anisotropy (Ao) calculated from the slope in Figure

4.1 5 (where Te is defined in equation 2.1 6) is 33 ppm. Table 4.7 shows the

comparison of the chemical shift anisotropy of different native lipoproteins as

well as reconstituted biomembrane. From Table 4.7, we can see that the

chemical shift anisotropy (Ao) value for SPMICO microemulsions is very close to

the value for egg PCfrO microemulsions at 25•‹C (or the value for egg PC

vesicles at 50•‹C). This suggests that the headgroup orientations for these three

systems are similar.

4.6 A Lateral Diffusion Study of SPMICOlapo-HDL3 Reconstituted

Lipoproteins

Representative 3lP NMR spectra of sphingomyelin/cholesteryI oleatelapo-

HDL3 reconstituted lipoproteins at different viscosities at 25 "C are shown in

Figure 4.1 6 to Figure 4.21. During the course of fitting the 31P linewidths for the

reconstituted lipoproteins, we found that at higher viscosity we were unable to

represent our data by a single Lorentzian function. A superposition of two

Lorentzians was necessary to get a reasonable fit. At lower viscosities,

especially with the sample without glycerol or with small amounts of glycerol (low

viscosity), the experimental data fit a single Lorentzian function. The two

spectral domains at lower viscosities still exist but we cannot resolve two

components at 25 "C in these spectra. This is most likely due to the fact that

the linewidths of these two spectral components are not sufficiently different at

25•‹C. This argument is supported by observations in our previous temperature

study in this thesis. At temperature below 25 "C (without glycerol present) the

data could not be represented as a single Lorentzian function. However, as

Table 4.6: Variation of linewidths of 3IP NMR spectra of SPMICO microemulsions at different NMR field

strengths.

vo2 ( Hz2 )

1.643 x 10

10.44 x 10 '5

26.24 x 10 '5

40.99 x 10 '5

Linewidth (Av 1,2 ) (HZ)

2 5 ( 6 )

4 9 ( 8 )

8 9 ( 1 2 )

1 2 8 ( 1 6 )

Table 4.7: Comparison of chemical shift anisotropy for serum

lipoproteins, microemulsions, and phospholipid bilayers.

VLDL

VLDL

LDL

LDL

HDL2

H DL3

egg P c r r o

SPMJCO microemulsions

Egg PC vesicles

Egg PC vesicles

DPPC liposomes

DPPC liposomes

a Chana, et al., (1 990);

c Parmar et al., ( 1 985 );

* This study

Viscosity

b Fenske et al. (1 990)

d Mclaughlin et. al. (1 975).

Figure 4.15: (Av - C) as a function of vO2 for SPMICO microemulsion at

25•‹C. The straight line is a least square fit to the data points.

Figure 4.16: 31P NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3

reconstituted lipoprotein particles at 25 "C (viscosity = 0.99

centipoise). The spectra were simulated by an iterative least-squared fit to

(a) a single Lorentzian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points. The solid line represents the

calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90' flip angle), sweep width =

50,000 Hz, delay between pulses = 1.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans 2 50,000.

Figure 4.16 (a): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 0.99 centipoise (single Lorentzian), no LB.

Figure 4.1 6 (b): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 0.99 centipoise (sum of two Lorentzians), no LB.

Figure 4.1 7 : 3' P NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3

reconstituted lipoprotein particles at 25 "C (viscosity = 1.53

centipoise). The spectra were simulated by an iterative least-squared fit to (a) a single Lorenttian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points. The solid line represents the

calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90' flip angle), sweep width =

50,000 Hz, delay between pulses = 1.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans 2 50,000.

Figure 4.17 (a): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 1.53 centipoise (single Lorentzian), no LB.

Figure 4.17 (b): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 1.53 centipoise (sum of two Lorentzians), no LB.

Figure 4.18: 31P NMR spectra of sphingomyelin/cholesteryl oleate/apo-HDL3 reconstituted lipoprotein particles at 25 "C (viscosity = 2.71

centipoise). The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or

(b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points. The solid line represents the calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width =

50,000 Hz, delay between pulses = 1.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans 2 50,000.

Figure 4.18 (a): SPM/CO/apo-HDL3 reconstituted lipoproteins at viscosity 2.71 centipoise (single Lorentzian) (LB = 5Hz).

Figure 4.18 (b): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity

2.71 centipoise (sum of two Lorentzians) (LB= 5 Hz).

Figure 4.1 9: 3' P NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3 reconstituted lipoprotein particles at 25 "C (viscosity = 5.30 centipoise). The spectra were simulated by an iterative least-squared fit to (a) a single Lorenttian lineshape function (four parameters) or (b) a superposition of two Lorentzian functions (seven parameters)

of the spectral data points. The solid line represents the calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90" flip angle), sweep width = 50,000 Hz,

delay between pulses = 1.5 s, dataset = 2k zero filled to 4K, dwell time = 10 p , number of scans 2 50,000.

Figure 4.19 (a): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 5.3 centipoise (single Lorentzian) (LB = 10 Hz).

Figure 4.1 9 (b): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 5.3 centipoise (sum of two Lorentzians) (LEI= 10 Hz).

Figure 4.20: 3lP NMR spectra of sphingomyelin/cholesteryl oleatelapo-HDL3 reconstituted lipoprotein particles at 25 "C (viscosity = 9.89 centipoise). The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or

(b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points. The solid line represents the calculated fit. Spectral parameters are as following: pulse width = 6.5 pi (90" flip angle), sweep width = 50,000 Hz,

delay between pulses = 1.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans 2 50,000.

Figure 4.20 (a): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity

9.89 centipoise (single Lorentzian) (LB = 25 Hz).

Figure 4.20 (b): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity

9.89 centipoise (sum of two Lorentzians) (LB= 25 Hz).

Figure 4.21 : 31 P NMR spectra of sphingomyelin/cholesteryl oleate/apo-HDL3 reconstituted lipoprotein particles at 25 "C (viscosity = 17.24

centipoise). The spectra were simulated by an iterative least-squared fit to (a) a single Lorentzian lineshape function (four parameters) or

(b) a superposition of two Lorentzian functions (seven parameters) of the spectral data points. The solid line represents the

calculated fit. Spectral parameters are as following: pulse width = 6.5 ps (90' flip angle), sweep width = 50,000 Hz,

delay between pulses = 1.5 s, dataset = 2k zero filled to 4K, dwell time = 10 ps, number of scans 2 50,000.

Figure 4.21 (a): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity

17.24 centipoise (single Lorentzian) (LB = 50 Hz).

Figure 4.21 (b): SPMICOlapo-HDL3 reconstituted lipoproteins at viscosity 17.24 centipoise (sum of two Lorentzians) (LB= 50 Hz).

Table 4.8:

Viscosity

(CP)

0.99

1.53

2.71

5.30

9.89

17.24

32.64

The viscosity dependence of linewidths of SPMICO

microemulsions in the presence of apo-HDL at 25 "C

- -

N / 2 of

broad

component

(Hz)

w / 2 of

narrow

component

(Hz)

w , / 2 -C) -' x104 (Hz - I )

broad

temperature increased, it became progressively more difficult to resolve

separate spectral components (see table 4.3).

Table 4.8 shows the variation of linewidths versus the viscosity and

reciprocal NMR linewidths versus reciprocal of solvent viscosity at 25 "C. The

constant C was taken as 15 Hz for phosphorus NMR.

A plot of linewidth (Av 1 , ~ - C) (broad component) as a function of

viscosity is shown in Figure 4.22. At low to intermediate viscosity, the linewidths

increase as viscosity increases. However, at higher viscosity, for example at 33

centipoise, the increase in linewidth seems to level off. This phenomenon is in

excellent agreement with observation in the case of protein-free SPMICO

microemulsions. Beyond this point, the lateral diffusion will become the

dominant mechanism of the line narrowing mechanism.

A plot of (Av 1 , ~ - C)-1 ( broad component ) of SPMICOlapo-HDL3

reconstituted particles versus q-' is show in Figure 4.23. The solid line

represents the least square fit of the data points. We have ignored the data of

reconstituted particles (SPMICOlapo-HDL3) in the absence of glycerol (0.99 cp).

The reason for doing this is as following:

In the absence of glycerol, the linewidths of the two components ( broad

and narrow ) of SPMICOlapo-HDL3 reconstituted particles are too close to

be clearly and easily resolved. However, it is clear that two components do

exist at lower temperatures as described in the temperature study of

SPMICOlapo-HDL3. The difficulty in fitting and resolving the two spectral

components of the NMR spectrum therefore makes the linewidth

uncertainties larger.

The smaller linewidths in the absence of glycerol compared with those at

other viscosities cause the less accurate ( bigger uncertainty ) data points.

The least square fit routine we used cannot exclude data with the biggest

uncertainty automatically; instead, it compromises the other more accurate

data with the least accurate data, resulting in the strange situation that the

fitted straight line (double reciprocal plot) only passes through the least

accurate data points. This makes the slope and intercept of the straight line

more uncertain. The lateral diffusion coefficient (D) is very much dependent

on the ratio of the intercept and slope, especially on the intercept of the

straight line as described in equation 2.21. In order to overcome the big

uncertainty encountered in the calculation, the data obtained from glycerol

free samples were neglected.

A plot of the variation of linewidth (Av 1,2 - C) of the narrow component of

SPM/CO/apo-HDL3 versus viscosity is shown in Figure 4.24. The behavior for

the narrow component in Figure 4.24 is similar to that of the broad component as

shown in Figure 4.22.

A plot of (Av 1,2 - C)-I of the narrow component of SPMICOlapo-HDL3

reconstituted particles versus q is shown in Figure 4.25. The solid line

represents the least square fit of the data points. As explained above, the data

point in the absence of glycerol was also excluded.

Figure 4.22: The plot of linewidth of broad component of SPMICOlapo-HDL3

reconstituted lipoprotein versus viscosity.

Figure 4.23: The double reciprocal plot of ( Av - C)-1x10 4 versus q-1 of

broad component of SPMICOlapo-HDL3 reconstituted lipoprotein.

q ( centipise )

Figure 4.24: The plot of linewidth for narrow component of

SPMICOlapo-HDLg reconstituted lipoprotein versus viscosity.

Figure 4.25: The double reciprocal plot ( Av 112 - C) X 1 o4 versus q for

narrow component of SPMICOlapo-HDL3 reconstituted

lipoprotein.

The lateral diffusion constants were calculated to be 1.0 + 0.4 x 10-9 cm2

s-land 2.1 + 0.8 x 10-8 cm2 s-I for the broad and narrow components of

SPMICOlapo-HDL3 reconstituted particles, respectively. The difference in the

diffusion constant between the two components are 20 to 30 times, hence, we

can predict that the environments of the two components are quite different from

each other. The lateral diffusion constant of the broad component of

SPMICOlapo-HDL3 is very close to that of the protein-free SPMICO

microemulsion as shown in Table 4.5. Therefore, we argue that the broad

component probably corresponds to the contribution of the phospholipids in the

"protein - deficient" domain. It has been shown that populations of phospholipids

that contribute to the broad and narrow components are in the same sphere as

shown in the structure of lipoproteins (Figure 1.2). Therefore, the rotational

correlation time (T,,~) is expected to be the same for the two components

because they are in the same particle and rotate at the same speed. The

diffusion time ( T ~ ~ ~ ) for the broad component is bigger than that of the narrow

component as demonstrated in equation 2.13. From equation 2.1 1, z,-'(b) will

be smaller for the broad component compared to ~,- l(n) of the narrow

component and T,@) for the broad component will be bigger than the T,(") of

narrow component. That is consistent with the experimental results. Therefore,

we conclude that the narrow component of the spectrum of the SPMICOlapo-

HDL3 reconstituted particle corresponds to the contribution of the phospholipids

adjacent to the proteins, and the broad component of the spectrum is the

contribution of phospholipids relatively far away from the proteins.

CHAPTER 5

CONCLUSION

In this study, we report a procedure for the preparation of stable

microemulsions of low density lipoprotein size containing cholesteryl oleate (CO)

and sphingomyelin (SPM). This method represents an improvement over the

past work in terms of size homogeneity, reproducibility, stability and productivity

of sufficient amounts of material. More importantly, the procedure rids the

solution of vesicles which, being approximately the same size as

microemulsions, would be indistinguishable using alP NMR.

Analysis of the model system by 3lP NMR was used to probe lipid-lipid

and lipid-protein interactions which may be extended to native lipoproteins. The

results from the temperature dependent study have shown that the core of the

microemulsion may slightly moderate the diffusion behavior of the phospholipid.

The lateral diffusion study on the SPMICO microemulsion has demonstrated that

the diffusion constant for the SPMICO microemulsion is 2 or 3 times smaller

when compared with that of low density lipoproteins due to the cooperative

interaction between the cholesteryl oleate core and sphingomyelin. Another

possible explanation is that the heterogeneous phospholipid compositions may

slightly interfere with diffusion. The field dependence study on this system

revealed that the chemical shift anisotropy for SPMICO microemulsions is similar

to those of egg PC vesicles and egg PC/TO microemulsions of LDL size. This

suggests that the headgroup orientations for these three systems are similar.

The 3lP NMR spectra of SPMICOlapo-HDL3 reconstituted particles at

low temperature cannot be represented very well by a single Lorentzian function.

Instead a superposition of two Lorentzians is needed to represent the spectra,

indicating the presence of two phospholipid domains in the surface of the

reconstituted particles at low temperatures.

In order to understand more about the occurrence of the two domains, the

lateral diffusion study of SPMICOlapo-HDL3 reconstituted particles was

conducted at 25 "C. At higher viscosities, it is very difficult to fit NMR spectra to

a single Lorentzian function, and we need a superposition of two Lorentzians to

fit the 31P NMR spectra as in the case of temperature study of the SPMICOlapo

HDL3. The lateral diffusion constant for the broad components of the

reconstituted particles is very close to that of SPMICO protein-free

microemulsions. We concluded that the broad component is the "protein-

deficient" domain of the phospholipids on the surface of the reconstituted

particles, and that the narrow component represents sphingomyelin in contact

with apolipoprotein.

CHAPTER 6

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