Analysis of intravenous lipid emulsions and their effect on cellular structure and function

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Analysis of Intravenous Lipid Emulsions

and Their Effect on Cellular Structure

and Function

Phillip David Whitfield

A thesis submitted for the degree o f Doctor o f Philosophy (Ph.D) in the

Faculty o f Life Sciences o f the University o f London

Biochemistry, Endocrinology and Metabolism Unit Institute o f Child Health

University College London Medical School

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List of Figures

Page

Chapter 1

1-1 Basic structure of glycerophospholipids and some polar head groups 20

l-2a Molecular structure of sphingomyelins 21

l-2b Molecular structure of glycosphingolipids 21

1-3 Structures of triglyceride-rich and phospholipid-rich particles 23

1-4 Possible role of phospholipids in TPN-associated cholestasis 25

1-5 Structure of the major naturally occurring phytosterols 27

1-6a Structure of cyclopentanoperhydrophenanthrene 28

1 -6b Structure of cholesterol 28

1 -7 Possible role of phytosterols in TPN-associated cholestasis 31

Chapter 2

2-1 Structure of plasma membranes 36

2-2a Structure of 1,6-diphenyl-1,3,5-hexatriene 40

2-2b Measurement of membrane fluidity using a fluorescent probe 40

2-3 Quantitation of phytosterols and cholesterol by GC-MS 45

2-4 GC trace of plasma from an infant with severe TPN-associated cholestasis 47

2-5 Typical standard curve used for quantitation of sitosterol by GC-MS 48

2-6 Correlation between membrane fluidity and membrane PC 52

2-7 Correlation between membrane fluidity and membrane C/PC ratio 53

2-8 Correlation between membrane fluidity and membrane total sterol/PC ratio 54

2-9 Correlation between membrane fluidity and plasma total sterol/PC ratio 57

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Chapter 3

3-1 Molecular structure of 2-hydroxypropyl-p-cyclodextrin 67

3-2 Effect of time on the loading of phytosterols in RBC using HPC 71

3-3 RBC cholesterol concentration during incubation with HPC solutions 72

3-4a Time profile of RBC haemolysis in the presence of HPC pre-lysis 73

3-4b Time profile of RBC haemolysis in the presence of HPC post-lysis 73

3-5a Effect of varying HPC concentration on cholesterol efflux from RBC 76

3-5b Effect of varying HPC concentration on haemolysis of RBC 76

3-6 Overview of the classical bile acid synthesis pathway 78

3-7 Proposed scheme of biliary lipid secretion in humans 81

3-8 Gas chromatogram of bile acids synthesised by Hep 0 2 cells 85

3-9 Mass spectra of bile acids from Hep 0 2 cells 86

3-10 Time course for HPC-mediated cholesterol efflux from Hep 0 2 cells 90

3-11 Cellular retention of cholesterol following incubation with HPC 91

3-12 Lipoprotein systems for transporting cholesterol in humans 93

3-13 Correlation between phytosterol solubility and solvent polarity index 97

3-14 Correlation between HPC-mediated cholesterol efflux and RBC haemolysis 102

Chapter 4

4-1 Effect of 0-50%IL2o on cholesterol uptake of Hep 0 2 cells 111

4-2 Effect of 0-2 0%IL2o on cholesterol uptake of Hep 0 2 cells 112

4-3 Time course of cholesterol uptake by Hep 0 2 cells 113

4-4 Effect of 0. 1-2%IL2o on cholesterol uptake by Hep 0 2 cells 115

4-5 Effect of preincubation with 2%IL2o on cholesterol uptake by Hep 0 2 cells 116

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4-7 Time course of Hep G2 cholesterol uptake from media preincubated +/- IL20 126

4-8 Effect of phospholipid fraction of IL20 on cholesterol uptake by Hep G2 cells 128

4-9 Effect of 10, 20 and 30% Intralipid on cholesterol uptake by Hep G2 Cells 131

4 -10a Correlation between cholesterol uptake by Hep G2 cells and PC 135

4 -10b Correlation between cholesterol uptake by Hep G2 cells and phytosterols 135

4-11 Effect of phytosterols on cholesterol uptake by Hep G2 cells 137

4-12 Time profile of Intralipid-mediated cholesterol efflux from Hep G2 cells 139

Chapter 5

5-1 Ion evaporation: the theoretical basis of electro spray ionisation 147

5-2a Schematic representation of product ion scan 149

5-2b Schematic representation of precursor ion scan 149

5-2c Schematic representation of neutral loss scan 149

5-3 a ESI-MS positive ion spectra of phospholipids from 20% Intralipid 154

5-3b ESI-MS positive ion spectra of phospholipids from 20% Lipofundin S 154

5-4a ESI-MS negative ion spectra of phospholipids from 20% Intralipid 155

5-4b ESI-MS negative ion spectra of phospholipids from 20% Lipofundin S 155

5-5a Positive ion product spectra of PC species m/z 760 157

5-5b Positive ion product spectra of PC sodium adduct m/z 782 157

5-6 Identification of [M+H]^ species of PC and SM in 20% Intralipid 159

5-7 Negative ion ESI-MS/MS of PE 18:0/20:4 in 2 0% Intralipid, m/z 766 160

5-8 Negative ion ESI-MS/MS of isobaric species at m/z 742 162

5-9 CID of [M-15]' SM 16:0 (m/z 687) 163

5-10a CID of glycoconjugate m/z 712 165

5-10b CID of glyco [M+Cl]' adduct at m/z 748 165

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List of Tables

Page

Chapter 2

2-1 Details of Patients Receiving TPN 42

2-2 Concentration of Erythrocyte Membrane Lipids in Patients Receiving TPN 51

2-3 Concentration of Plasma Lipids in Patients Receiving TPN 56

Chapter 3

3-1 Concentrations of Bile Acids Synthesised by Hep G2 Cells 87

3-2 Solubility o f Phytosterols in Organic Solvents 96

3-3 Incorporation of Phytosterols into PCS from Organic Solvents 99

Chapter 4

4-1 Solvent Extraction of [^"^CJCholic Acid and [^'^CJCholesterol 118

4-2 Separation of Radiolabelled Lipids from Media of Hep G2 Cells 120

4-3 Concentration of Lipids in Intralipid Preparations and Their Fractions 133

Chapter 5

5-1 ESI-MS/MS Analysis of Phospholipid Species in 2 0% Intralipid 152

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Acknowledgements

So many people, both at work and in play, have helped me with this project that it is

simply not possible to name everyone. However, I would like to take this opportunity

to personally thank them all for their input. In particular, I am very grateful to Kevin

Mills for all his help in the laboratory and David Stanton for his contribution. I would

also like to thank Andrew Johnson for his help with the tandem mass spectrometry and

John Rainer for his assistance with the cell culture.

A very special thanks goes to Eamonn O’Driscoll and Lucy Hackett and I am also

indebted to my parents, brother and his family for their support during my university

years.

I gratefully acknowledge the Child Health Appeal Trust (CHRAT) for the financial

support of the project.

Finally, I would like to thank my supervisors, Peter Clayton and David Muller for their

considerable encouragement, knowledge, and of course patience as they have expertly

guided me throughout the course of my study. Their efforts have been appreciated

enormously. I must also finally thank them for their continued financial support over my

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Abbreviations

ACAT acyl coenzyme A: cholesterol acyltransferase

AST aspartate aminotransferase

BCA bicinchoninic acid

BSA bovine serum albumin

CA cholic acid

CDCA chenodeoxycholic acid

CID collision-induced dissociation

CM chylomicron

CMR chylomicron remnant

DAPI 4 ’, 6-diamidino-2-phenylindole

DHCA 3a, 7a-dihydroxy-5 (3-cholestan-26-oic acid

DMSO dimethyl sulphoxide

DPH 1,6-diphenyl-1,3,5 -hexatriene

DPM disintegrations per minute

ESI-MS electrospray ionisation-mass spectrometry

ESI-MS/MS electrospray ionisation-tandem mass spectrometry

ESR electron spin resonance

PCS foetal calf serum

GC-MS gas chromatography-mass spectrometry

GL glycolipid

Hb haemoglobin

HDL high-density lipoprotein

HEPES N-2-hydroxyethylpiperazine-N’ -2-ethanesulphonic acid

HMG-CoA 3-hydroxy-3 -methylglutaryl-coenzyme A

HPC 2-hydroxypropyl-p-cyclodextrin

3a-HSD 3a-hydroxysteroid dehydrogenase

IDE intermediate-density lipoprotein

IE Intralipid

ISTD internal standard

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Lp-X lipoprotein-X

MDR multidrug resistance

MEM Eagle’s minimum essential medium

NDCA nordeoxycholic acid

NEAA non-essential amino acids

NMR nuclear magnetic resonance

PBS phosphate buffered saline

PC phosphatidylcholine

PC-TP phosphatidylcholine-transfer protein

PE phosphatidylethanolamine

PIP phosphatidylinositol monophosphate

PIP2 phosphatidylinositol bisphosphate

PLRP phospholipid-rich particles

PS phyto sterols

RBC red blood cells

SCP-2 sterol carrier protein-2

SIM selected ion monitoring

SM sphingomyelin

TGRP triglyceride-rich particles

THCA 3a ,7 a , 12a-trihydroxy-5 P-cholestan-26-oic acid

TPN total parenteral nutrition

VLDL very-low-density lipoprotein

v/v volume to volume

w/v weight to volume

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Abstract

Total parenteral nutrition (TPN) offers the chance of survival to children who have had

extensive gut resections or suffer from severe gut failure. However, in infants it is often

associated with serious complications including cholestatic liver disease. The aetiology

of these complications remains unclear although it has been suggested that the lipid

emulsions used in TPN may be responsible. Commercial fat emulsions are typically

made up of a complex mixture of soybean oil-derived long chain triglycerides emulsified

with natural phospholipids and contain significant quantities of sterol contaminants in

the form of plant sterols and cholesterol. The hypothesis underlying this study was that

components of the lipid emulsions, in particular plant sterols and/or phospholipids, may

play a role in the development of TPN-associated cholestasis. The following methods

were established and validated during the course of this project i) the measurement of

membrane fluidity of erythrocyte ghosts by fluorescence polarography, ii) the

quantification of lipids from both cells in tissue culture and patient samples using gas

chromatography-mass spectrometry, iii) an in vitro system to study the effect of lipid emulsions and their component fractions on cholesterol uptake and efflux using cultured

Hep G2 cells and iv) the characterisation of the structure of phospholipids in lipid

emulsions by electro spray ionisation-tandem mass spectrometry. Results showed a) the

accumulation of phytosterols in plasma and erythrocyte membranes of patients receiving

TPN and a positive correlation between erythrocyte membrane lipid composition and

membrane fluidity, b) the TPN lipid emulsions had a dose-dependent inhibitory effect on

cholesterol uptake by Hep G2 cells, c) the phospholipid-rich particles of the lipid

emulsion which also contain high concentrations of phytosterols were primarily

responsible for producing the effects noted above and d) electrospray ionisation-tandem

mass spectrometry could be used to make comparisons of the polar head group, acyl

substituents and the positional placement of the fatty acid side chains of the

phospholipids found in the lipid emulsions frequently used in TPN regimes.

In conclusion, these results indicated that a component or components of the lipid

emulsions may interfere with cellular lipid homeostasis in vivo and may thus play a role in the development of TPN-associated cholestasis. Further work is required to

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1.4 Composition of Total Parenteral Nutrition 15

1.4.1 Carbohydrates 16

1.4.2 Amino Acids 16

1.4.3 Lipids 16

1.4.4 Vitamins, Minerals and Trace Elements 16

1.4.5 Electrolytes and Water 16

Chapter 1 General Introduction

Page

. 1 Total Parenteral Nutrition 14

2 History of Total Parenteral Nutrition 14

3 Clinical Indications for Total Parenteral Nutrition 14

5 Complications of Total Parenteral Nutrition 17

6 Specific Components of Intravenous Lipid Emulsions 19

6.1 Phospholipids 19

6.2 Structure o f Phospholipids 19

6.3 Structure of Phospholipid-Containing Particles 22

6.4 Metabolism of Phospholipid-Containing Particles 22

.6.5 Possible Adverse Effects of Phospholipid-Containing Particles 24

6 .6 Phytosterols 26

6.7 Structure of Phytosterols 26

6 .8 Metabolism of Phytosterols 29

6 .9 Possible Adverse Effects of Phytosterols 29

1.7 Aims of the Present Study 32

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1.1 Total Parenteral Nutrition

Total parenteral nutrition (TPN) can be used as a method to provide nutrients to

seriously ill infants unable to tolerate enteral feeding. This form of nutritional support,

however, is not without risks and is associated with serious complications in particular

cholestatic liver disease (Kerner, 1991). Cholestasis is defined as the diminution or

arrest of the flow of bile. The aetiology of this complication still remains poorly

understood. The hypothesis underlying this study was that a component or components

of the lipid emulsions used in TPN may play a role in the development of TPN-

associated cholestasis.

1.2 History of Total Parenteral Nutrition (Reviewed by Wretlind, 1992)

The successful development of TPN, in a modem sense, was only initiated in the late

1930’s. The first major step towards this therapy was taken in 1939 by the U.S.

physicians Elman and Weiner who successfully infused protein hydrolysates into adult

patients. This was repeated a few months later in infants by Shohl and colleagues. It

soon became apparent, however, that these mixtures failed to provide sufficient calories

to efficiently metabolise the amino acids. At the time, the only non-protein energy

source was glucose but there was a limit to the amount that could be supplied

intravenously. It either had to be administered in very large volumes or as a

concentrated solution which often resulted in damage to peripheral veins. High-calorie,

isotonic fat emulsions appeared to be a more realistic option. However, many of the

early emulsions caused significant side effects in man and it was not until the early

1960’s that safe intravenous fat emulsion became commercially available. From there it

was a small step to include vitamins, electrolytes and trace elements in the fat emulsions

and solutions of amino acids and glucose. In 1968 the use of long-term TPN became a

clinical reality when Wilmore and Dudrick demonstrated that a central venous catheter

could be used to administer the TPN solutions intravenously.

1.3 Clinical Indications for Total Parenteral Nutrition

Following its inception the benefits of TPN in a few disorders readily became apparent.

This was followed by widespread enthusiasm for the use of TPN in a broad spectrum of

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ever performed. Thus, TPN is a powerful therapeutic tool, for which the indications

have never been accurately defined. Over recent years, however, significant advances

have taken place in the assessment of nutritional requirements as well as in the design of

regimes and in the preparation of energy sources and nutrients (Elia, 1995).

Whenever possible, nutrition should be administered via the enteral route and TPN

should only be considered when the gastrointestinal tract is compromised and it is

judged that the patient will be unable to absorb adequate amounts of nutrients. There

are, however, no absolute indications as to when TPN should be started, decisions have

to be based upon the clinical condition of the patient. As far as possible the parenteral

diet should be based on a normal oral diet, although normal energy requirements will

not be sufficient in certain diseases. Every surgical procedure or stress situation

interferes in one way or another with normal physiological processes of the body.

Consequently it is impossible to establish a “standard formula” for all patients. The

actual metabolic state, the prevailing clinical situation, the general condition and

possible other underlying conditions of the patient must be taken into account (Heird

and Winters, 1975). Therefore, specific TPN regimes have to be tailored to the differing

needs of each individual patient.

This thesis is concerned solely with the effects of TPN administration in children.

Infants have special metabolic needs which must be addressed when providing TPN for

them. Throughout childhood, but especially the low birthweight neonate, energy stores

are low and so it is essential to provide sufficient nutrients to prevent malnutrition.

Children also require nutrients for normal growth and development, processes which

involve the synthesis of macromolecules, such as proteins, carbohydrates and lipids.

These requirements are typically increased during periods of nutritional stress resulting

from illness. Therefore, unless there is provision of a well balanced and adequate supply

of nutrients permanent cellular and functional deficits could well arise (Evans and

Cockburn, 1991).

1.4 Composition of Total Parenteral Nutrition

All the major classes of nutrients are included in a paediatric TPN regime. Fats and

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carbohydrates serve as the energy sources whilst amino acids furnish the vital

components for the synthesis of body protein. Carbohydrates and amino acids are

usually given in the form of a combined, commercially prepared solution, whilst the

lipid element is administered as a fat emulsion.

1.4.1 Carbohydrates

Glucose, in the form of dextrose is the carbohydrate of choice for parenteral use since it

is readily utilised by many cells and tissues such as erythrocytes, brain and cardiac

muscle. It also decreases the osmotic pressure of the carbohydrate component.

1.4.2 Amino Acids

A balanced mixture of crystalline L-amino acids (including the essential amino acids) is

given to infants at a rate that will promote growth. Several amino acid solutions suitable

for the very young child are commercially available.

1.4.3 Lipids

Lipids are normally administered in the form of energy-rich fat emulsions which provide

a concentrated source of calories and essential fatty acids. Commercial preparations

given include Intralipid (Upjohn-Pharmacia Ltd., Milton Keynes, UK), Lipofundin (B.

Braun Medical Ltd., Aylesbury, UK) and Ivelip (Clintec, Slough, UK).

1.4.4 Vitamins, Minerals and Trace Elements

The requirements of vitamins, minerals and trace elements in TPN have been poorly

characterised. They are usually provided as two separate supplements. The water

soluble vitamins and minerals are added to the solution of amino acids and dextrose;

and the fat soluble vitamins are administered with the lipid emulsion.

1.4.5 Electrolytes and Water

Finally, consideration must be given to the adequate replacement of water and

electrolytes. Providing no secondary disturbances exist in water-electrolyte balance,

substitution of electrolytes in TPN should be performed in concentrations

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1.5 Complications of Total Parenteral Nutrition

Although the technique of TPN has been a major clinical advance, promoting the

normal growth of very small and ill infants, complications can occur especially with

prolonged TPN in very young patients. The major complication of long-term TPN is

cholestasis (Peden et al., 1971). Cholestatic liver disease is a frequent problem which /fleets up to 50% of infants who have been fed intravenously for over 2 months (Bell et

ah, 1986). In a small number of cases it may progress to severe hepatic dysfunction and death from liver failure. This sequence of events is the most common reason for

children being considered for combined gut-liver transplantation.

TPN-associated cholestasis is most likely to occur in preterm infants. This may be due

to the fact that the premature infant has a diminished synthesis rate and pool size of bile

salts (Watkins et a l, 1975). Further predisposing factors include a disturbed enterohepatic circulation which also reduces the bile salt pool size (Balistreri et al.,

1983) and the loss of enteral feeding which reduces the hormonal and neural stimuli of

hepatic bile secretion and gall bladder contraction (Lucas et al., 1983). Although it is recognised which patients are most likely to develop TPN-associated cholestasis, there

is much less agreement about which components of the TPN are responsible. The

situation is extremely complex and is almost certainly multifactorial.

A great deal of attention has been focused on the possible effects of excess amino acids.

Vileisis et al. (1980) noted that infants receiving a higher intake of amino acids as part o f their TPN regime (3.6 versus 2.3g/Kg/day) experienced an earlier onset and more

severe course of cholestasis. Individual amino acids have also been implicated. For

example, intraperitoneal injections of tryptophan into suckling rats have been shown to

be hepatotoxic (Merritt et al., 1984). Conversely, Cooper et al. (1984) reported extremely low plasma concentrations of taurine in three neonates who developed

progressive TPN-associated cholestasis. They argued that the absence of taurine

impaired the conjugation of bile acids producing the observed liver disease.

Amongst other suggestions it has been proposed that cholestasis may be caused by an

increased production of lithocholic acid. Fouin-Fortunet et al. (1982) found that in

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adult patients who developed hepatic abnormalities during TPN, lithocholic acid

comprised between 7 and 15% of total biliary bile acids compared to less than 1% in

patients who remained unaffected. In other studies it has been suggested that

deficiencies of antioxidants such as vitamin E might expose the liver to damage by

increased concentrations of free radicals (Berger et al., 1985) whilst the possibility has been raised that TPN solutions containing high levels of trace metals may contribute to

hepatic dysfunction. For instance, Klein et al. (1987) showed that intravenous administration o f aluminium to piglets induced cholestatic liver disease.

Another view is that the lipid emulsions used in TPN could be involved. La Scala et al.

(1993) demonstrated that the bile flow of TPN fed rats receiving a lipid emulsion was

significantly lower (10.14 + 1.27|Lil/min) than in control animals (15.61 + l.Blpl/min).

Commercial fat emulsions consist in general of soybean triglycerides stabilised with egg

phospholipids and rendered isotonic with glycerol. Lipid emulsions also contain

significant quantities of sterol contaminants in the form of cholesterol and phytosterols.

Phospholipid deposits have been described in hepatocytes of patients with TPN-

associated cholestasis which are thought to reflect the intrahepatic accumulation of

phospholipids provided by TPN fat emulsions (Degott et al., 1988). Gerard-

Boncompain et al. (1992) reported the development of cholestasis in long-term TPN patients following a change in lipid emulsion. Differences in the sodium oleate content,

size of the lipid particles and phospholipid purification process of lipid emulsions were

suggested to explain this observation. Finally, excess phyto sterols delivered from lipid

emulsions have been implicated in the development of cholestatic liver disease. Clayton

et al. (1993) found a correlation between high plasma phytosterol concentrations and infants with severe TPN-associated cholestasis. Furthermore, the accumulation of

phytosterols in neonatal piglets has been associated with a significant inhibition of both

bile acid excretion and bile acid stimulated bile flow (Iyer et al., 1998).

As a result of these observations the hypothesis was formulated that phyto sterols and/or

phospholipids may be important factors in the development of TPN-associated

cholestasis. These components of intravenous lipid emulsions will now be examined

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1.6 Specific Components of Intravenous Lipid Emulsions

1.6.1 Phospholipids

1.6.2 Structure of Phospholipids (Gurr and Harwood, 1991)

The large variety of phospholipids whicl

glycerophospholipids and sphingolipids.

The large variety of phospholipids which exisy can be divided into two broad groups,

Glycerophospholipids

These lipids consist of a hydrophobic moiety derived from diglyceride which is linked

via a phosphodiester bridge to a hydrophilic polar head group (Figure 1-1). The two

fatty acyl side chains of the glycerophospholipids are esterified at the 5« -l and sn-2

positions of the glycerol backbone whilst the phosphate group is attached to sn-2>. This terminal phosphate can itself be esterified to one of several polar head groups which

define the specific class to which a glycerophospholipid belongs. The most common

head groups include the bases choline and ethanolamine and alcohols such as inositol.

Apart from differences in their functional head groups glycerophospholipids also display

considerable heterogeneity in their fatty acid composition.

Sphingolipids

Sphingolipids constitute a class of lipids with a common sphingosine or a related

sphingoid long chain base to which various groups are attached (Figure 1-2).

Sphingosine is an amino alcohol containing an 18 carbon hydrocarbon chain and one

double bond between C4 and C5. Both of the amino and hydroxyl functional groups in

this backbone can be substituted. Acylation of the amino group o f the sphingosine with

a variety of fatty acids yields a ceramide, and estérification of the primary hydroxyl

group of a ceramide to phosphocholine produces sphingomeylin (Figure l-2a).

Alternatively the hydroxyl group may be conjugated to a carbohydrate chain giving rise

to a glycosphingolipid (Figure l-2b). Glycosphingolipids possess one or more sugars.

These can vary in size and complexity from a monosaccharide such as glucose or

galactose up to oligosaccharides containing several sugar residues.

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o

sn-\

Ri — C — O — CH,

O

sn-2

R? — C — O — CH

O

sn-3

C H . — O

O — X

OH

X

_{CH2CH2N(CH3)3}

_Choline

CH2CH2NH3

Ethanolamine

OH

HO

OH

Inositol

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a)

H3C (CH2)

H

H H

1 2 C = C C

c

CH2

— o

H

OH NH

0 = C

R

O

G CH2CH2N (CH3)3

OH

b)

H

H3C

(CH2)

i

2 c = c

H

H H

■CH, O

OH NH

0 = C

Ri

HEXOSE U N n(S )

(23)

1.6.3 Structure of Phospholipid-Containing Particles

Phospholipids are added to lipid emulsions to facilitate the dispersion of the soybean

triglyceride droplets and to ensure the stability of the emulsion (Bach et al.^ 1996). A single layer of phospholipid molecules surrounds a droplet of triglycerides, thereby

shielding it from the aqueous exterior and forming what is referred to as a triglyceride-

rich particle (TGRP) (Figure 1-3). Commercial lipid emulsions contain more

phospholipid than is strictly necessary to emulsify the triglycerides. These excess free

phospholipids form vesicular-like bilayers known as phospholipid-rich particles (PLRP)

(Figure 1-3). Although composed primarily of phospholipid, PLRP are also reported to

contain small amounts of other lipid species (Ferezou et a l, 1994). Thus, lipid emulsions contain two distinct pools of phospholipid.

1.6.4 Metabolism of Phospholipid-Containing Particles

Metabolism o f Triglyceride-Rich Particles

In vivo the large TGRP (250-500nm) can acquire cholesterol esters and apoprotein (apo)-E from circulating plasma lipoproteins and are then metabolised in a similar

manner to dietary chylomicrons (Bryan et a l, 1976). Most of the triglycerides are first hydrolysed by lipoprotein lipase and then the remaining triglycerides, phospholipids and

cholesterol ester of the resultant TGRP remnants are taken up by the liver where they

are degraded.

Metabolism o f Phospholipid-Rich Particles

The metabolism of the PLRP (80-100nm) is poorly understood. It is believed that on

infusion the PLRP acquire apo-CII and apo-E before being partially hydrolysed by

lipoprotein lipase. Some of the liberated phospholipids are taken up by high-density

lipoproteins (HDL) and are eventually removed from the circulation by the liver. HDL

have a limited capacity for handling this phospholipid and long-term TPN has been

associated with reduced levels of HDL (Hajri et a l, 1990). Therefore, in TPN patients PLRP may not be cleared very rapidly from the circulation and this could result in their

accumulation in the plasma. The progressive accumulation of exogenous phospholipids

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Triglyceride-Rich Particle (TGRP)

.KV

E

'%

M s

:u ; E

\

i

I

4C-.: U i

K-.^.fc

„

^ ^

E % . E

< ^ c E

u s y

OCm

s

m

n

I

Phospholipid

Lysophospholipid

Free Cholesterol

Free Phytosterol

Esterified Phytosterol

Triglyceride

Diglyceride

Monoglyceride

Free Fatty Acid

Phospholipid-Rich Particle (PLRP)

« c ^

Figure 1-3. Structures of triglyceride-rich and phospholipid-rich particles.

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behave as liposomes and attract free cholesterol from blood vessels and tissues (Wang

et al., 1996). These cholesterol-enriched particles may also act as precursors for the pathological lipoprotein, lipoprotein-X (Lp-X) (Untracht, 1982). Lp-X which has a

density very similar to that of low-density lipoproteins (LDL) is comprised of equimolar

quantities of phospholipid and free cholesterol. Varying concentrations of apo-CII and

-GUI are also associated with these abnormal particles. Lp-X-like bodies have been

identified in the plasma of rats following Intralipid administration (Breckenridge et al.,

1979), whilst Hajri et al. (1990) have demonstrated that infusion of the pure PLRP fraction in rats very rapidly induces Lp-X formation. Lp-X has also been reported to

accumulate in the circulation of preterm neonates maintained on TPN with lipid (Griffin

et a l, 1979). Interestingly, the plasma concentrations of this abnormal lipoprotein falls once the lipid infusion has ceased.

1.6.5 Possible Adverse Effects of Phospholipid-Containing Particles

Some of the pathological effects of phospholipids may be explained by their interaction

with cell membranes. For example, an alteration in the relative amounts of

phospholipids and changes in their fatty acid composition have been shown to occur in

the erythrocyte membranes of rats infused with Intralipid (Innis, 1989). Furthermore,

the PLRP element of lipid emulsions may substantially modify the composition of red

blood cells by depleting them of free cholesterol (Carpentier, 1989) which could

adversely affect the properties of the membrane. Similarly, it has been suggested that

TPN lipid emulsions can alter the lipid profile of intestinal brush border and microsomal

membranes in piglets (Van Aerde et a l, 1997).

The potential role of phospholipids in the development of TPN-associated cholestasis is

shown in Figure 1-4. Excess phospholipids from PLRP and TGRP may accumulate in

hepatocytes and induce hepatic damage and dysfunction. The PLRP may also attract

free cholesterol from cell membranes and be transformed into Lp-X. The loss of

cholesterol would be expected to alter the membrane fluidity of the cell. Alternatively

Lp-X might directly interfere with the function of hepatocytes. Such effect(s) could be

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p T G R /

t Phospholipids in Hepatocytes

Lipid Emulsions

PLRP

▼

Lp-X

Hepatocyte Damage

Hepatic Dysfunction

Removal o f Cell Cholesterol

K>

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1.6.6 Phytosterols

Phytosterols are lipids found in plants. They are not synthesised by man and so their

concentration in plasma varies with diet. Important dietary sources of phytosterols

include corn, beans, nuts, and plant oils. During the manufacture of intravenous lipid

emulsions, phytosterols are added with the soybean triglycerides. The phytosterols are

present in both the free and esterified form. Little is known about the distribution of the

sterols between the phospholipid-containing particles but it is thought that esterified

phytosterols are located in the hydrophobic core of the TGRP whilst free phytosterols

are mostly associated with the phospholipid monolayers of both TGRP and PLRP.

1.6.7 Structure of Phytosterols (Nes and McKean, 1977)

A wide variety of phytosterols have been described and identified all showing a similar

structure and biology. The phytosterols are structural derivatives of cholesterol and like

cholesterol comprise two units, a nucleus and a side chain. The most frequently found

phytosterols found in nature are sitosterol, campesterol and stigmasterol. The structures

of the major phytosterols are shown in Figure 1-5.

All the phytosterols examined in this thesis have a nucleus which has the same basic

structure as that found in steroids i.e. a fixed fused ring system of three cyclohexane

rings the so-called A,B,C rings and a terminal cyclopentane ring (D ring). This

configuration is referred to as the cyclopentanoperhydrophenanthrene nucleus (Figure

l-6a). In phytosterols and cholesterol the nucleus possesses a double bond at C5 and a

hydroxyl group on C3 (Figure l-6b).

Phytosterols generally differ from one another and cholesterol only in the size and

configuration of their side chain. The side chain of the phytosterols typically ranges

from eight carbon atoms in the C27 phytosterols to ten carbon atoms in the C29

phytosterols although occasionally it is longer. Compared to cholesterol, sitosterol and

stigmasterol both have an extra ethyl group at position C24. In addition, stigmasterol

possesses a double bond at C22. Fucosterol and iso-fiicosterol are also C29 sterols but

with an ethylidene substitutent at the C24 position, whilst campesterol is a C28 sterol

(28)

Campesterol

H O HO

Sitosterol Stigmasterol

HO HO

Fucosterol Iso-fucosterol

Figure 1-5. Structure of the major naturally occurring phytosterols.

(29)

a)

c

D

A

B

Cyclopentanoperhydrophenanthrene Nucleus

b)

HO

Cholesterol

(30)

1.6.8 Metabolism of Phytosterols

Phytosterols are normally poorly absorbed by man. In healthy adults, normally less than

5% of dietary phytosterols are absorbed, whereas 50% of dietary cholesterol is

absorbed (Salen et al., 1970). Human infants appear to have a higher phytosterol absorption than adults. The amounts of phytosterols in a child’s diet depends upon the

relative amounts of animal fats and vegetable oils ingested. Low concentrations of plant

sterols are found in breast milk and cows milk, whereas soybean based infant formulae

contain relatively high concentrations (Mellies et a l, 1976; 1978). Phytosterols given orally impair cholesterol absorption and they have been used successfully for the

treatment o f hypercholesterolaemia (Lees et a l, 1977). The low intestinal uptake of phytosterols is, however, selective, for example campesterol is absorbed more easily

than sitosterol. It has been suggested that this uptake of sterols in humans is related to

differences in the structure o f their side chain (Heinemann et al., 1993). The small amounts of phytosterols that are absorbed in healthy individuals are concentrated in the

liver and excreted in bile (Lin et al., 1984). The liver, however, has a limited capacity for handling phytosterols which may prove problematic when large concentrations of

phytosterols have to be eliminated (Bhattacharyya etal., 1991). There is also some loss through the skin.

Studies have been performed examining the conversion of sitosterol into C24 bile acids.

Salen et al. (1970) reported that cholic and chenodeoxycholic acids were formed from intravenously administered radiolabelled sitosterol in humans at approximately the same

rate as from cholesterol. However, subsequent investigations in man did not confirm

this metabolism (Boberg et al., 1990). In rats it has been reported that sitosterol is metabolised into novel di- and trihydroxylated C21 bile acids rather than being converted

into C24 bile acids (Lund et al., 1991). Phytosterols have also been shown to inhibit the conversion o f cholesterol to bile acids (Boberg et al., 1989) and to interfere with the solubilisation of cholesterol in bile (Bjorkhem and Skrede, 1989).

1.6.9 Possible Adverse Effects of Phytosterols

Like cholesterol, plant sterols affect the packing o f membrane layers and are also

capable of displacing cholesterol from cell membranes. Since differences exist between

(31)

the cholesterol and phytosterol side chains incorporation of phytosterols into cell

membranes can affect membrane structure and, therefore, alter the physical properties

of the membrane. For example, in one study it was shown that when rats were fed on a

diet containing 3% sitosterol and 2% campesterol for 21 days, the phytosterols

displaced the cholesterol from liver microsomal membranes and led to a decrease in

membrane fluidity (Leikin and Brenner, 1989).

A condition which provides some insight into some of the possible adverse effects of

phytosterols is hereditary phytosterolaemia, a rare inborn error o f metabolism which

was first described by Bhattacharyya and Connor (1974). The exact metabolic basis of

this disorder has yet to be elucidated but the available evidence suggests that there is an

excessive intestinal absorption coupled with reduced hepatic excretion of phytosterol.

The condition is characterised by high plasma levels of the plant sterols, sitosterol,

campesterol and stigmasterol, and the accumulation of these phytosterols in blood cells

and tissues. Tendon and tuberous xanthomas develop and episodes of haemolytic

anaemia and thrombocytopenia have also been described (Bjorkhem and Skrede, 1989).

Some patients also show reduced hepatic activity of cholesterol 7a-hydroxylase (Shefer

et a i, 1988) the rate limiting step of bile acid synthesis.

A hypothesis for the contribution of phytosterols in the development of TPN-associated

cholestasis is proposed in Figure 1-7. It is suggested that phytosterol accumulation may

interfere with bile acid synthesis in infants receiving TPN. Because many of these

patients already have a reduced bile acid synthesis rate, reduced pool size and disrupted

enterohepatic circulation, bile acid secretion may be suppressed to the point where

cholestasis occurs. These children may also have bile which has a high concentration of

phytosterol. In this situation precipitation of the phytosterol may occur leading to the

formation of sludge and stones in the biliary tree which in turn further reduces bile flow.

Finally, the accumulation of phytosterols in hepatocyte membranes may lead to

(32)

Lipid Emulsions

Phytosterol Accumulation

Phytosterols in B ile

Biliary Sludge/Stones

Reduced B ile Flow

Inhibits Bile Acid Synthesis Effect on Cell Membranes

Reduced Bile Acids in Bile

Hepatocyte Damage

(33)

1.7 Aims of the Present Study

The principal aim of the present study was to examine the hypothesis that TPN-

associated cholestasis is caused by either the phospholipid and/or phytosterol

component(s) of lipid emulsions. This hypothesis was explored by a series of ex vivo

and in vitro experiments which involved:

1. The measurement of membrane fluidity by fluorescence polarography of erythrocyte

ghosts from patients receiving TPN and with cholestatic liver disease.

2. The quantification of lipids from both cells in tissue culture and patient samples using

gas chromatography-mass spectrometry.

3. The development of a tissue culture system using cultured Hep G2 cells to study the

effect of intravenous lipid emulsions and their component fractions on cholesterol

uptake and efflux.

4. The characterisation of the structure of phospholipids in intravenous lipid emulsions

(34)

Chapter 2 Effect o f Intravenous Lipid Emulsions on the Fluidity

o f Erythrocyte M embrane Ghosts

Page

2.1 Introduction 34

2.2 Ceil Membranes 34

2.3 Regulation o f Membrane Fluidity 3 7

2.4 Theory of Membrane Fluidity 38

2.4.1 1,6-Diphenyl-1,3,5-Hexatriene 38

2.4.2 Fluorescence Polarisation 38

2 .6 Methods 41

2.6.1 Blood Samples from Patients Receiving Total Parenteral Nutrition 41

2.6.2 Preparation of Erythrocyte Membrane Ghosts 43

2.6.3 Protein Determination of Erythrocyte Membrane Ghosts 43

2.6.4 Determination of Membrane Fluidity 44

2.6.5 Gas Chromatography-Mass Spectrometry of Phytosterols and Cholesterol 44

2.6.6 Measurement of Choline Containing Phospholipids 49

2.6.7 Statistical Analysis 49

2.7 Results 50

2.7.1 Erythrocyte Membrane Lipids 50

2.7.2 Plasma Lipids 55

2.8 Discussion 59

(35)

2.1 Introduction

The fluidity of biological membranes is mainly determined by their lipid composition.

Modification of the membrane lipid components may alter the structure and the fluidity

o f a membrane and as a result interfere with the function of membrane bound enzymes,

expression of receptors, cell growth and a host of other cell processes (reviewed by

Spector and Yorek, 1985). The aim of this series of experiments was to determine

whether a correlation existed between the membrane fluidity of erythrocytes and

phospholipid/ phytosterol concentrations in patients receiving long-term TPN.

2.2 Cell Membranes

The mammalian plasma membrane is composed of a lipid bilayer consisting of

phospholipids, glycolipids and sterols with protein molecules either partially embedded

or completely traversing the lipid matrix (Singer and Nicolson, 1972) (Figure 2-1).

Lipids

Phospholipids are the most abundant lipids of cell membranes. A common feature

characteristic of all types of phospholipids is their amphipathic nature. The

phospholipids of cell membranes are arranged in a bilayer such that the hydrophilic

polar head group in each layer faces an aqueous environment (extra- or intracellular

space), and the hydrophobic acyl chains arrange themselves in the middle of the bilayer.

This bilayer forms the basic structure of the membrane.

The phospholipids in a membrane bilayer can exhibit the random motions characteristic

of a liquid phase. The fatty acid tails of the molecules “wiggle” about such that the

bilayer is flexible. Furthermore, the phospholipid molecules can diffuse sideways within

their own monolayer and as a result two neighbouring phospholipids in the same

monolayer can change places with one another. However, flip-flop movements of

phospholipids between the two halves of the bilayer are infrequent as testified by the

asymmetric distribution that exists across the cell membrane. Phosphatidylcholines and

sphingomyelins are generally located in the outer monolayer of the cell membrane whilst

phosphatidylethanolamines tend to be found in the inner leaflet. Glycolipids which make

(36)

exclusively in the outer leaflet with their sugar groups exposed to the extracellular

space.

Cholesterol is the most abundant of the sterol lipids. The cholesterol molecule

intercalates itself in the phospholipid bilayer such that the hydroxyl group is facing

towards the aqueous interface whist the bulk of the molecule extends into the

hydrophobic core of the bilayer and interacts with fatty acid chains of the phospholipids.

Thus the steroid nucleus sterically blocks large motions of the phospholipid molecules

and affects their mobility.

Proteins

The proteins of biological membranes behave as specific functional molecules. They can

serve, for example, as ion pumps, gates, receptors, energy transducers and enzymes.

Membrane proteins can be classified into two categories according to their association

with the lipid bilayer. Peripheral or extrinsic membrane proteins are loosely bound to

the membrane and can be removed by relatively mild procedures that leave the

membrane intact. Examples of extrinsic membrane proteins include antigens. Integral or

intrinsic membrane proteins are the second class of membrane protein. These proteins

are embedded in the lipid bilayer of the membrane. Some only just penetrate the bilayer,

others penetrate nearly half way whilst others span the entire lipid bilayer and may even

traverse the bilayer several times. These proteins can only be removed by disruption of

the membrane with treatments such as detergent. Some integral proteins act as

transmembrane transporters or ion channels for example the sodium-glucose co

transporter.

Proteins as well as lipids can change position in the membrane. A protein can diffuse

laterally or rotate about its axis but it does not move from one side of the membrane to

the other i.e. it does not flip-flop. Indeed once a membrane protein has been inserted

into a membrane it usually retains its orientation indefinitely and thus membranes are

both structurally and functionally asymmetric.

(37)

Extracellular Space

Extrinsic Protein

Giycolipid

Phospholipid Cholesterol

Intrinsic Protein Cytosolic Space

U)

(38)

2.3 Regulation of Membrane Fluidity

The phospholipid composition of membranes is known to affect their fluidity. For

example, increasing the concentrations of phosphatidylcholines relative to

sphingomyelins results in increased membrane fluidity (Borochov et al., 1977). In mammals, phospholipids commonly possess one saturated and one unsaturated fatty

acyl chain of between 16-24 carbons in size. The length and degree of unsaturation of

these fatty acyl side chains is another major determinant of membrane fluidity (Cooper,

1977). Long chain fatty acids are able to interact more strongly (with one another) than

shorter ones and, therefore, produce less fluid membranes. This is also true of saturated

fatty acids. However, with unsaturated acyl chains the presence of cis double bonds introduces kinks which prevents the close packing of the fatty acids and thus enhances

fluidity.

Cholesterol also plays a key role in controlling membrane fluidity. It has been shown

that an increase in cholesterol concentrations relative to phospholipids decreases

membrane fluidity in both liposomes and biological membranes (Cooper et at., 1978). Thus, the cholesterol/phospholipid (C/PL) ratio has a major influence upon the physical

properties of a membrane. Mature red blood cells (RBC) are not capable of de novo

cholesterol and phospholipid synthesis and thus lack the specific adaptive processes

necessary to control membrane fluidity. Instead they depend on the lipid composition of

plasma lipoproteins (Cooper, 1977). The cholesterol and phospholipids from

lipoproteins readily exchange with their counterparts in membranes. As a result of this

exchange any changes in circulating plasma lipid composition are associated with

corresponding changes in the erythrocyte membrane composition which may affect the

membrane fluidity of the cell. The lipid emulsions used in TPN differ from the plasma

lipoproteins that normally bathe the cells in the body in that they contain significant

quantities of phytosterols whilst their phospholipids possess a greater proportion of

unsaturated fatty acids. Thus, in TPN patients receiving infusions of fat emulsions any

disturbance in the plasma lipid profile is likely to be reflected in both the composition

and membrane fluidity of their RBC. This makes the erythrocyte an ideal cell to study.

(39)

2.4 Theory of Membrane Fluidity

Membrane fluidity is a complex, ill defined concept, which in general terms can be

considered as describing the relative motional freedom of lipid molecules within a

biological membrane (Cogan and Schachter, 1981). Despite the complexities, the

measurement of membrane fluidity is often used to investigate the physical dynamics of

cell membranes. There are a number of techniques available for the measurement of

specific movements of molecules in membranes that contribute towards its overall

fluidity. For example, fluorescence recovery after photobleaching is a technique used to

measure the lateral diffusion coefficients of fluorometrically labelled membrane

components (Housley and Stanley, 1990) and electron spin resonance (ESR) has been

used to determine the rate of flip-flop of nitroxide labelled phospholipids in liposomes

and erythrocytes (Houslay and Stanley, 1990). There are also a range of methods that

provide information on the overall degree of mobility or fluidity o f membrane bilayers.

For instance the melting or transition of bilayers from the crystalline to the liquid

crystalline phase with change in temperature can be followed using differential scanning

calorimetry. The mobility of molecules at various depths in membrane bilayers may be

measured using nuclear magnetic resonance (NMR) or by fluorescence polarisation

techniques that utilise labelled phospholipids or probes incorporated into a bilayer. In

this study, fluorescence polarisation using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the

probe was used as a measure of membrane fluidity.

2.4.1 1,6-Diphenyl-1,3,5-Hexatriene

Fluorescence probes (ftuorophores) are often employed to obtain information regarding

molecular interactions within membranes. One of the most common fluorescence

probes used in fluidity studies is DPH (Figure 2-2a). Like other hydrophobic probes,

DPH is insoluble in water and so it partitions into the hydrophobic interior of biological

membranes and lipid bilayers. Most of the DPH molecules orientate themselves parallel

to the phospholipid acyl chains although, a minority are orientated perpendicular to the

membrane surface (van Ginkel, 1989).

2.4.2 Fluorescence Polarisation

(40)

different directions) is a measure of the degree of movement (rotation) of an extrinsic

fluorophore. Fluorophores fluoresce when excited by light of a specific wavelength,

which corresponds to a peak in their absorption spectrum (Schachter, 1984). Excitation

of a “static” fluorophore with polarised light results in the emission of light which is

also polarised. However, once incorporated into a biological membrane the fluorophore

is free to rotate. If the probe rotates during the lifetime of this excited state then the

emitted light will be depolarised. This rotation is in turn directly influenced by the

structural order of the membrane i.e. the resistance of the membrane to the probe’s

movement (van Ginkel et al., 1986). Thus, the degree of depolarisation of emitted light can be used as a measure of the rotation of the probe which in turn is related to its ease

of movement in the membrane. Figure 2-2b is a representation of the excitation and

emission of a fluorophore such as DPH. Excitation of the fluorophore parallel to the

plane of polarisation will result in emission of light polarised in the same direction

unless the fluorophore rotates. The degree of depolarisation of the emitted light can be

measured by positioning an emission polariser both at right angles parallel (Iw ) and

perpendicular ( I v h ) to the plane of polarisation of the excitation light. Steady-state

anisotropy (rs) is the ratio of intensities of the polarised component of emitted light to

the total emitted light and is determined from the equation below;

rs = Iw - Ivh

Iw + 2 I v h

where:

rs = Steady-state anisotropy

Iv h = Intensity of light when both the excitation and emission polarisers are vertically

positioned.

Iw = Intensity of light when the excitation polariser is in the vertical position and the emission polariser is horizontal.

The steady-state anisotropy is inversely related to the fluidity of a membrane.

(41)

a)

b)

Rotation ofProbe

V

Plane ofPolarisation

Ivh

Excitation Light

Figure 2-2. a) Structure of 1,6-diphenyl-1,3,5-hexatriene.

(42)

2.5 Materials and Reagents

Isotonie lOmM phosphate buffered saline (PBS) pH7.4, Img/ml bovine serum albumin

(BSA) protein standard, bicinchoninic acid (BCA), 1,6-diphenyl-1,3,5-hexatriene

(DPH), 5a-cholestane, cholesterol, campesterol, stigmasterol, sitosterol, fucosterol and

tétraméthylammonium hydroxide in methanol were all purchased from the Sigma

Chemical Company (Poole, UK). Di-sodium hydrogen orthophosphate 2-hydrate,

sodium di-hydrogen orthophosphate 1-hydrate and copper sulphate 5-hydrate were

purchased from BDH Laboratory Supplies (Poole, UK). HPLC grade propan-2-ol and

tetrahydrofuran were bought from Merck Chemical Ltd., (Lutterworth, UK).

Tetrachloroethylene and methylbutyrate were purchased from the Aldrich Chemical

Company, (Gillingham, UK). Tri-sil was purchased from Pierce (Luton, UK) and the

phospholipid kit from Boehringer Mannheim (Lewes, UK).

2.6 Methods

2.6.1 Blood Samples from Patients Receiving Total Parenteral Nutrition

Eight patients, two male and six female, aged between 4 and 27 months were studied

(see Table 2-1 for details). All the patients were hospitalised and were receiving 20%

Intralipid. Three of the eight patients showed biochemical evidence of severe cholestatic

liver disease (aspartate aminotransferase (AST), >200Units/L [normal: 20-60]; total

bilirubin >100pM [normal: <17]). These patients were being considered for gut-liver

transplantation. Another four patients had only mild elevations of liver function tests.

One control patient was studied who was receiving TPN but who appeared to show no

signs o f liver disease. Three other controls used for the study were healthy adult

volunteers from the Biochemistry Unit, Institute of Child Health. Samples of l-2ml of

fresh venous blood were drawn from patients and control subjects into lithium heparin

tubes.

(43)

Table 2-1. Details of Patients Receiving TPN

Patient Number

Sex Age AST

(Months) (Units/L)

Bilirubin (^M)

Presenting Condition

Severe Liver Dysfunction

1 F 20 656 534 Gut failure

2 F 9 627 148 Short gut

3 F 16 403 482 Gut failure

Mild Liver Dysfunction

4 F 17 167 2 2 Enteropathy

5 M 27 62 8 Bone marrow

transplant

6 F 4 60 65 Necrotising

enterocolitis

7 F 10 81 7 Gastrectomy

Controls

8 M 23 42 12 Bone marrow

transplant

9 M

10 M Normal Adult Controls

(44)

2.6.2 Preparation of Erythrocyte Membrane Ghosts

Erythrocyte ghosts were prepared by a modification of the method of Burton et al.

C ^ - G O a )

(1981). Whole blood samples were centrifuged at 2000fpm for 5 minutes and the

plasma removed. The erythrocytes were then washed three times with 2ml isotonic

lOmM phosphate buffered saline (PBS) pH7.4. (Note: PBS was isotonic to body fluids

and consisted of lOmM Na2HP0 4/NaH2P0 4 buffer; 138mM NaCl and 2.7mM KCl at

pH7.4). Membrane ghosts were prepared by the lysis of 250-1 OOOjiil RBC. The

erythrocytes were diluted 1:5 volume/volume (v/v) with chilled 2.5mM phosphate

buffer pH8.0 and stirred for 15 minutes. The erythrocyte ghost pellet obtained after

centrifuging at 18000rpm for 20 minutes at 4°C was then washed and centrifuged twice

more with 5ml 1.25mM phosphate buffer pH8.0 as above. This removed the

haemoglobin i.e. pellet was creamy white to the naked eye. The red cell ghosts were

then resuspended in 500pl isotonic lOmM PBS pH7.4 gassed under nitrogen gas and

stored at -20“C. To minimise any potential effects of storage and inter-assay variation

all samples were studied on the same occasion and within 2 months of preparation.

2.6.3 Protein Determination of Erythrocyte Membrane Ghosts

This method was based on the bicinchoninic acid (BCA) method of Smith et al. (1985). The method combines the biuret reaction with unique characteristics of BCA. In the

reaction proteins react with alkaline Cu^^ to produce Cu^. Two molecules of BCA then

react with one cuprous ion to produce an intense purple colour from which the

spectrophotometric quantitation of protein in the aqueous solution can be determined.

All assays were carried out in duplicate. A standard curve was prepared containing 5-

50|ig of 1 mg/ml BSA standard in 50p,l distilled water. For samples 5pi RBC membrane

preparation was diluted to 50pl with distilled water. After the addition of 1ml BCA the

samples and standards were incubated at 37”C for 10 minutes. 20pl o f copper sulphate

solution was added and the samples incubated at 37°C for at least 20 minutes. The

absorbance was then read at 562nm and the protein concentration of the sample in

pg/pl was calculated from the standard curve.

(45)

2.6.4 Determination of Membrane Fluidity

A 2mM stock solution of DPH in tetrahydrofuran was stored in the dark at -20°C. A fresh 0.5fj,M working solution was made by diluting the stock DPH solution 1:4000

(v/v) with isotonic lOmM PBS pH7.4. This was kept in the dark at 4°C and stirred for

at least 2 hours prior to use to ensure adequate mixing. A 50pl aliquot of the red cell

ghost suspension was incubated with 2ml O.SpM DPH at 37°C for Ihour. Fluorescence

was then measured in a 1cm cuvette heated to 37“C in a Perkin-Elmer LS3 fluorimeter

using an excitation wavelength of 360nm and an emission wavelength of 430nm. Four

measurements were made with each sample i.e.vertical (0°) and horizontal (90°)

polarised excitation with either vertical (0°) or horizontal (90°) polarisation of the

emission filter. Steady-state anisotropy was then determined using the equation:

0 90) (9 0 0

Ô" W I V %

rs

0 90/ I 0 90

Blank readings of the fluorescence 5pM DPH in isotonic lOmM PBS pH7.4 without

membranes were made in an identical manner and subtracted from readings given by

DPH and membranes for each polariser setting. Due to a limited amount of material it

was not possible to take blank readings of membranes incubated in PBS without 5pM

DPH. However, validation studies previously undertaken in the Biochemistry Unit,

showed that the fluorescence produced by these samples was negligible.

2.6.5 Gas Chromatography-Mass Spectrometry of Phytosterols and Cholesterol

Plasma total (free and esterified) phytosterol and cholesterol concentrations were

measured by gas chromatography-mass spectrometry (GC-MS) by the method of

Clayton et al. (1993). The procedure which used 5a-cholestane as the internal standard (ISTD) is shown schematically in Figure 2-3.

Sterol Extraction

1ml of a saponification mixture containing 0.0372mg 5a-cholestane, mixed with 0.25ml

(46)

Plasma

Saponify (5a-choles

2nd

Extract ane, ISTD)

Total Sterols

Concentrate îind Derivatise

Analytical Sample

GO -MS

Response Ratio

Compare with (Calibration Curve

Sterol Quantitation

Figure 2-3. Quantitation of phytosterols and cholesterol by GC-MS.

(47)

was added to 2 0 0|al of plasma and then heated at 80°C for 15 minutes. The saponified

sterols were then extracted by the addition of 500|il tetrachloroethylene/methylbutyrate

1:3 (v/v) and 2ml distilled water. After centrifuging at 2000'ipm for 10 minutes, lOOpl

of the tetrachloroethylene/methylbutyrate lower phase was taken and dried down under

vacuum at 60°C.

GC-MS o f Phytosterols and Cholesterol

Trimethylsilyl ethers of sterols were made by derivatising with 200^1 of Tri-sil and

heating at 60°C for 1 hour. These derivatives are formed by replacing the active

hydrogen of the C3 hydroxyl group of the sterols with a trimethylsilyl group (Si(CH3)3).

Using a splitless technique, Ipl was injected into a Hewlett-Packard 5890 Series II gas

chromatograph containing a 0.25 mm ID 30m fused silica column lined with a 0.25p.m

film thickness of D Bl. After a delay of 2 minutes with the oven at 120°C, the oven

temperature was ramped to 200°C at 20°C/min and then to 300°C at 2°C/min. The gas

chromatograph was coupled to a Hewlett-Packard 5970 mass-selective detector and

Chem Station data system. The ionisation energy was 70eV and the source temperature

was 170°C. A typical GC trace of plasma phytosterols is shown in Figure 2-4.

Sterol Standards and Calibration Curves

ImM solutions of each sterol and internal standard were made up in propan-2-ol. A

standard curve of 5-lOOOpM was constructed by addition of the appropriate amounts of

each sterol and ISTD in a total volume of 200pl. Standards were dried down under

vacuum at 60°C, derivatised and analysed by GC-MS as described above. Quality

control was performed by running a calibration curve for each sterol with each batch of

analysis. Calibration curves based on the ion currents ratios of the sterol of interest to

ISTD were linear for concentrations upto ImM. A typical calibration curve for

sitosterol is shown in Figure 2-5. For quantitation of the sterols in samples the mass

spectrometer was operated in selected ion monitoring (SIM) mode. The following ions

were used, 5a-cholestane, mass/charge ratio (m/z) 372; cholesterol, 458; campesterol,

382; stigmasterol, 484; sitosterol, 396; and iso-fucosterol, 386. The response ratio of

(48)

4^

1.8e+5

1.6e+5

1.4e+5

1.2e+5 -(U

o

l.Oe+5 -§

1

< _8.0e+4

6.0e+4

4.0e+4

2.0e+4

-0 -r

20

ISTD

l

L

—I 1---1---1---1---- 1---1---- 1---r

25 30

- I 1---1---1---1---1---1--- 1--- 1--- 1--- 1---1---1---r

35 40 45

Time (Minutes)

^---- 1---- 1---- 1---1---1---- 1---- 1--- 1— 1---- r₁

50

r

55

—r

60

(49)

«J

c o

Q-C / 5

D

2. On

0.5-0.0

0 250 500 750 1000

Concentration of Sitosterol (|oM)

Analysis of intravenous lipid emulsions and their effect on cellular structure and function