STUDIES ON LYSOLECITHIN IN HUMAN PLASMA AND … · platelet aggregation initiated by five different...
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STUDIES ON LYSOLECITHIN IN HUMAN PLASMA AND ITS EFFECTS ON BLOOD PLATELET BEHAVIOUR. by MICHAEL PAUL TRAFFORD GILLETT, B.Sc., (Sp. Hons.). A Thesis submitted for the Degree of Doctor of Philosophy of the. University of London July 1974 St. Mary's Hospital Medical School London W2 1NY
Transcript of STUDIES ON LYSOLECITHIN IN HUMAN PLASMA AND … · platelet aggregation initiated by five different...
STUDIES ON LYSOLECITHIN IN HUMAN PLASMA AND
ITS EFFECTS ON BLOOD PLATELET BEHAVIOUR.
by
MICHAEL PAUL TRAFFORD GILLETT, B.Sc., (Sp. Hons.).
A Thesis submitted for the Degree of Doctor of
Philosophy of the. University of London
July 1974 St. Mary's Hospital Medical School
London W2 1NY
ABSTRACT
The presence of lysolecithin (L.P.C,) in human plasma and serum
has been confirmed. During the incubation of plasma at 370 there was
a substantial conversion of lecithin (P VC.) to L.P.C. by an enzyme
with identical characteristics to lecithin:cholesterol acyl transferase.
The concentrations of cholesterol and of individul phoSpholipid
classes have been determined in plasma samples taken from 77 apparently
healthy individuals and 76 male patients with atherosclerotic diseases,
Significant differences in the relative and absolute concentrations of
L.P.C. were found between different populations. In healthy--
individuals the plasma levels of L.P.C. were lower in women than in
men, and lower in the younger age groups studied. The relative and
absolute concentrations of L.P.C. were lower in men suffering from
chronic ischaemic heart disease and peripheral arterial disease when
compared with age-matched healthy men. The lowest levels of L.P.C.
were, however, associated with patients suffering from acute myocardial
infarction or acute ischaemic heart disease, studied within 48 hours
of the onset of chest pain. In a further study significantly
decreased relative concentrations of L.P.C. were found in platelets
and erythrocytes as well as in the plasma of chronic ischaemic
patients.
The effects of purified phospholipids on platelet and
erythrocyte behaviour in vitro have been studied. Erythrocyte
sedimentation and packing rates were reduced after exposure of blood
to L.P.C. and whole blood viscosity was increased. Irreversible
platelet aggregation initiated by five different aggregating agents
was inhibited by L.P.C.
3 WWI
Following intravenous administration of heparin in man'the rate
of formation of L.P.C. in plasma was increased, and this change was
associated with reduced irreversible platelet aggregation and
erythrocyte sedimentation. Heparin added in vitro had no similar
effects which suggested that increased formation of L.P.C. was
responsible for the alterations in platelet and erythrocyte -
behaviour.
Significantly decreased plasma levels of L.P.C. were found
associated with increased irreversible platelet aggregation or
increased erythrocyte packing rates in women taking oral contraceptives
or women during pregnancy.
The inhibitory effect of L.P.C. on platelet aggregation coupled
with the association of low plasma levels of L.P.C. in several
populations known to have an increased risk of thrombo-embolic
disease suggests that plasma L.P.C. may have a thrombo-protective
role in man.
ACKNOWLEDGEMENTS
The author would like to express his thanks to
Dr. J.D. Billimoria, M.Sc., Ph.D., D.Sc., F.R.I.C. and
Dr. B.M.M. Besterman, M.A., M.D., F.R.C.P., for the help
and encouragement that was given throughout the course of this work.
The author is grateful for financial assistance received
from the St. Mary's Hospital Research Fund and the Wellcome Trust
during the course of this study. The author is indebted to
Professor A. Neuberger, C.B.E., M.D., Ph.D., F.R.C.P., F.R.c.Path.,
F.R.S., Professor W.S. Peart, M.D., F.R.C.P., F.R.S. and
Professor V. Wynn, M.D., M.R.C.P., F.R.C.Path, who have provided
laboratory facilities during the course of this investigation.
The author is grateful to Mrs K. Porter for the preparation of
the electron micrographs illustrating the effects of lysolecithin
on platelet morphology.
The author would like to thank the physicians and surgeons of
St. Mary's Hospital, London W.2. for allowing him to study their
patients, and also to thank all those individuals who gave blood
samples for the present study.
ABBREVIATIONS.
The following abbreviations were used throughout this thesis.
P.E.
L.P.E.
P.C.
L.P.C.
P.A.
P.S.
P.I.
G.P.C.
A.D.P.
5-H. T.
P.R.P.
P.F.P.
E.S.R.
E.P.R.
T.L.C.
L.C.A.T.
Tris
1', 2' diacyl-sn-glycero-3-phosphaylethanolamine
(3-sn-phosphatidylethanolamine)
1' or 2' monoacyl-n-glycero-3-phosphorylethanolamine
(lysophosphatidylethanolamine)
3-sn- phosphatidylcholine (lecithin)
1' or 2' monoacyl-sn-glycero-3-phosphorylcholine
(lysolecithin)
Phosphatidic acid
3-sn-phosphatidylserine
3-sn-phosphatidylinositol
3-sn-glycerophosphorylcholine
3', 5' adenosine diphosphate
5-hydroxytryptamine (serotonin)
Platelet rich plasma
Platelet free plasma (platelet poor plasma)
Erythrocyte sedimentation rate
Erythrocyte packing rate
Thin layer chromatography
Lecithin:cholesterol acyl transferase
(E.C. 2. 3. 1. 43.)
2-Amino-2-hydroxymethylpropane-1,3-diol.
The following abbreviations were used in tables and figures
in this thesis.
Sph. Sphingomyelin
T.P.L. Total phospholipid
Col. Collagen
D.O.C. Sodium deoxycholate
C.T.A.B. Cetyl trimethylammonium bromide
C.P.C. Cetyl pyxidinium chloride
o.d. optical density
- 7
CONTENTS,
Page
Introduction 24
General Introduction 25
Historical Review of Plasma Lysolecithin 30
Clinical studies of plasma 33
phospholipids
Formation of lysolecithin in plasma 37
Phospholipid Effects on Platelet Function 41
Object of Study 43
Clinical Material, Biological Techniques and
Analytical Techniques 45
Clinical Material
46
Introduction and discussion of experimental
protocol. 46
Healthy control population, 48
Peripheral arterial disease. 49
Chronic ischaemic heart disease, 49
Acute myocardial infarction. 49
Intravenous administration of heparin in man. 50
Women taking oral contraceptive preparations. 51
Blood samples obtained from women during
pregnancy. 53
Biological Techniques 54
1. Techniques for studying the effects of
lysolecithin on erythrocyte behaviour in vitro 54
Introduction 54
(a) Erythrocyte sedimentation
55
(b) Erythrocyte packing rates
58
CHAPTER 1
CHAPTER 2
Section 1
Section 2
Page
2. Methods for studying platelet function in vitro 61
Introduction 61
(a) Platelet aggregation method. 62
Apparatus for studying platelet
aggregation. 62
Preparation of aggregating agents. 63
Quantitation of platelet
aggregation . 68
Preparation of phospholipid
suspensions. 73
(b) Platelet adhesiveness method, 75
Section 3 Analytical Techniques 77
1. Lipid separation techniques
Introduction 77
(a) Separation of plasma phospholipids by
one-dimensional thin layer
chromatography. 79
Extraction of plasma phospholipids. 79
Detection and identification of
lipids. 82
(b) Separation of erythrocyte and platelet
phospholipids by two dimensional thin
layer chromatogftphy. 84
(c) Separation of free cholesterol and
cholesteryl esters by thin layer
chromatography. 89
Page
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CHAPTER 3
2. Quantitation of phospholipid mass and
derivation of plasma phospholipid
concentrations 93
Introduction 93
(a) Determination of phospholipid mass
and derivation of total plasma
phospholipid concentration. 94
(b) Determination of the proportional
distribution of phosphorous in
phospholipids separated by .thin
layer chromatography. 97
3. Determination of plasma cholesterol
concentrations and radio-isotopic assay of
chlesterol esterification.
99
Introduction 99
(a) Method for the determination of the
concentration of total cholesterol
in plasma. 100
(b) Radio-isotopic assay of cholesterol
esterification. 101
Results 108
Section 1 Formation of lysolecithin in incubated human plasma/09
Introduction 109
(a) Time course of lysolecithin
formation in incubated plasma, 111
(b) Effect of temperature on lysolecithin
formation in plasma. 113
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(c) Effect of pH on lysolecithin
formation in plasma, 114
(d) Inhibition of lysolecithin
formation in plasma. 116
(e) Lysolecithin formation in
incubated serum lipoprotein
fractions. 118
(f) A comparison of the phospholipid
content of lipoprotein fractions
isolated from unincubated serum or
from serum previously incubated at
37°C.
Discussion
Section 2 Plasma cholesterol and individual phospholipid
concentrations in the healthy population and in
123
127
patients suffering from ischaemic heart disease
and peripheral arterial disease 130
(a) Analysis of plasma cholesterol and
phospholipid concentrations in the
healthy population. 131
(b) Analysis of plasma cholesterol and
phospholipid concentrations in men
suffering from acute myocardial
infarction, chronic ischaemia and
peripheral arterial disease. 135
(c) Analysis of the relative concent-
rations of individual phosphlipids
of plasma, erythrocytes and platelets
in healthy men and in men suffering
from ischaemic heart disease. 140
• Page
Discussion, • 140
Section 3 Effects of lysolecithin on erythrocyte
behaviour in vitro
147
Introduction
147
(a) Effect of lysolecithin on
erythrocyte sedimentation. 1.48
(b) Sedimentation of erythrocytes
in incubated plasma. 152
(c) Effects of lysolecithin on
erythrocyte flexibility and whole
blood viscosity. 155
Discussion. 157
Section 4 Effects of purified phospholipids on blood
platelet function in vitro
(a) Direct effects of purified
phospholipids added to stirred
platelet rich plasma.
(b) Effects of purified phospholipids
on platelet aggregation initiated
by adenosine diphosphate.
(c) Effects of purified phospholipids
on platelet aggregation initiated
by 5-hydroxytryptamine.
(d) Effects of purified phospholipids 1
on platelet aggregation initiated
by adrenaline.
(e) Effects of purified phospholipids
on platelet aggregation initiated
by throMbin or collagen.
158
159
165
170
175
180
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(f) Inhibition of the platelet
release reaction by lysolecithin. 185
(g) A comparison of the effects of
saturated and polyunsaturated
lysolecithin fractions on platelet
aggregation. 191
(h) Effects of lysolecithin and
lecithin on the retention of
platelets by exposure to glass
surfaces. 194
Section 5 Influence of small doses of heparin administered
intravenously in man on plasma lysolecithin
formation, erythrocyte behaviour and platelet
function. 197
Introduction 197
(a) Lysolecithin formation in pre- and
post-heparin plasma. 198
(b) Lecithin:acyl cholesterol transferase
activity in pre- and post-heparin
plasma. 201
(c) Effect of protamine sulphate on the
formation of lysolecithin in
incubated pre- and post-heparin plasma. 202
(d) Effect of intravenous heparin
administration on dextran-stimulated
erythrocyte sedimentation. 203
(e) Effects of intravenous administration
on platelet aggregation. 205
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Page
(f) Effects of heparins derived
from different tissues on
plasma lysolecithin formation
and platelet aggregation. 209
(g) Effects of heparin in vitro on
plasma lysolecithin formation,_
erythrocyte sedimentation and
platelet aggregation. 211
• Discussion. 213
Section 6 An analysis of plasma phospholipid levels and
platelet aggregation in women taking oral
contraceptive preparations. 217 '
Section 7 An analysis of plasma phospholipid levels and
erythrocyte behaviour in pregnant women. 224
CHAPTER 4 General Discussion 231 r
Conclusion 242
APPENDIX A comparison of the effects of other surface-
active agents with those of lysolecithin on
platelet aggregation. 243
REFERENCES 254
Publications submitted in support of Thesis.
1, Inhibition of irreversible platelet aggregation
by lysolecithin, Abstracts of the II Congress of
The International Society on Thrombosis and
Haemostasis, Oslo, Norway, 1971, p.174.
2. Inhibition of platelet aggregation by
lysolecithin, Atherosclerosis, 1971, 14 : 323-330.
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Page
3. Altered plasma-lysolecithin levels and
platelets, Lancet, 1972, i : 141.
4. Increased lysolecithin formation in human
plasma after intravenous administration of
heparin, Clin. Sci., 1972, 43 : 1P-2P
5. A comparison of the effects of saturated
and polyunsaturated lysolecithin fractions on
platelet aggregation and erythrocyte
sedimentation, Atterosclerosis, 1972, 16:89-94.
6. Heparin effects on irreversible platelet--
aggregation, Lancet, 1972, ii 282-283.
7. Influence of lysolecithin on platelet
aggregation initiated by 5-hydroxytryptamine,
Nature New Biology, 1973, 241': 223-224.
8. Heparin effects on plasma lysolecithin
formation and platelet aggregation,
Atherosclerosis, 1973, 17 : 503-513.
9. Inhibition of human platelet aggregation by
a benzothiazine derivative, Sudoxicam (CP 15,973)
given in vitro and in vivo. Abstracts of the IV
Congress of the International Society on Thrombosis
and Haemostasis, Vienna, Austria, 1973, p.405.
10. Effects of heparin derived from different tissues
on plasma phospholipids and platelet aggregation,
Lancet, 1973, ii : 1204-1205.
11. A possible thrombo-protective role for plasma
lysolecithin in man, Abstract submitted to III
Troms0 Seminar in Medicine (Lipids and Thrombosis) -
Troms0, Norway, June, 1974.
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LIST OF TABLES.
Contents Table Page
1, Composition of some oral contraceptive 52
preparations.
2. Fatty acid composition of saturated and
polyunsaturated fractions of lysolecithin. 74
3. Replicate analysis of plasma phospholipid
concentrations separated on duplicate thin
layer chromatography. 99
4. Lysolecithin formation in plasma incubated
at different temperatures for six hours.. 113
5. Inhibition of lysolecithin formation in
plasma exposed to urea or para-hydroxy-
mercuribenzoate. 117
6. Scheme to demonstrate the preparation of
serum lipoprotein fractions using the Beckman
model L2 ultracentrifuge. 119
7. Lysolecithin formation in incubated lipoprotein
fractions and mixtures. 121
8. A comparison of the phospholipid content of
serum lipoprotein fractions isolated from pre-
incubated and post-incubated serum, 124
9. Relative concentrations of individual plasma
phospholipids of healthy men and women. 132
10. Absolute concentrations of plasma cholesterol
and individual phospholipids in healthy men and
women. 133
Table
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Contents Page
Relative concentrations of individual
phospholipids in the plasma of men
suffering from ischaemic heart disease,
peripheral arterial disease and acute
myocardial infarction compared with healthy
age-matched controls. 136
Absolute concentrations of plasma cholesteld
and individual phospholipids in men suffering
from ischaemic heart disease, peripheral
arterial disease and acute myocardial
infarction compared with age-matched healthy
11.
12.
controls. 138
13. Relative concentrations of phospholipids in
plasma, erythrocytes and blood platelets in
apparently healthy men and in men suffering
from chronic ischaemic heart disease. 141
14. Relative concentrations of individual phospho-
lipids of erythrocytes and blood platelets. A
comparison of previously published results with
those of the present study. 145
15. Effects of lysolecithin and lecithin on platdbt
adhesiveness. 195
16. Effects of intravenous heparin administration on
lysolecithin formation and lecithin degradation
in incubated plasma. 200
17. Effects of intravenous heparin administratibn
on lysolecithin formation and lecithin:cholesterol
acyl transferase activity. 201
Table
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Contents
Page
18. Effect of protamine sulphate (1 mgml. -1)
on the formation of lysolecithin in
incubated pre- and post-heparin plasma. 202
19. Effects of hog-mucosal and ox-lung heparins
on plasma lysolecithin formation and platelet
aggregation induced by collagen. 210
20. Plasma cholesterol and phospholipid levels and
lysolecithin-formation during the menstrual
cycle of untreated women and women treated with
low-progestogen and high-progestogen oral
contraceptives. 219
21. Plasma concentrations of cholesterol and Tbospho-
lipids in pregnant and non-pregnant women. 226
22. Mean values for erythrocyte sedimentation and
packing rates and whole blood viscosity in
pregnant and non-pregnant women. 228
- 18 -
LIST OF FIGURES.
Figure
Title Page
1. Effect of high molecular weight dextran
fractions on erythrocyte sedimentation
rate. 57
2. • Effect of different concentrations of
dextran on erythrocyte sedimentation rate. 59
3. (i) Adenosine diphosphate-induced
platelet aggregation. 65
(ii) Adrenaline-induced platelet
aggregation. 66
(iii) Collagen-induced platelet
aggregation. 67
4. Schematic aggregation recordings illustrating
the methods for quantitation of platelet
aggregation, 70
5. Dose-response curves• relating the inhibitory
effect of an experimental drug (SudoxicamR )
on the rate of secondary or irreversible
platelet aggregation. 72
6. Separation of plasma phospholipidsby one-
dimensional thin layer chromatography. 85
7. Separation of erythrocyte phospholipids by
two-dimensional thin layer chromatography. 90
8. Separation of lipid classes by one-dimensional
thin layer chromatography. 92
Figure
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Title Page
Calibration curve for the estimation of
phosphorous mass by a modification of
Bartlett's method. 96
Calibration curve for the estimation of
cholesterol mass by the method of Leffler. 102
Influence of enzyme concentration on the
esterification of free cholesterol.. 107
Increase in the concentration of lyso-
lecithin during the incubation of plasma
9.
10,
11.
12.
at 37
112
13. Formation of lysolecithin at different pH
values during the incubation of plasma at
370
115
14. Inhibition of erythrocyte sedimentation by
exposure of blood to lysolecithin. 149
15. Effect of saturated and polynsaturated
lysolecithin fractions on erythrocyte sedimentation
behaviour. 150
16. (i) Inhibition of erythrocyte sedimentation
in plasma pre-incubated at 370. 153
(ii) Correlation of lysolecithin formation
in incubated plasma with the inhibition
of erythrocyte sedimenhtion in
incubated plasMa. 153
17. (i) Effect of lysolecithin on erythrocyte
packing during centrifugation. 156
(ii) Effect of lysolecithin on whole blood
viscosity measured at different shear
rates, 156
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19.
20.
21.
22.
Title Page
(i) Effect of adding sphingomyelin (S.M.)
and phosphatidylserine (P.S.) to
stirred platebt rich plasma. 160
(ii) Effect of adding lysolecithin to
stirred platelet rich plasma. 160
(i) Electron micrograph of centrifuged
platelets from normal platelet rich
plasma. 163
(ii) Electron micrograph of centrifuged
platelets from platelet rich plasma
exposed to lysolecithin. 164
(i) Inhibition of secondary platelet
aggregation initiated by adenosine
diphosphate. 167
(ii) Dose response curve for the
inhibition by lysolecithin of
secondary platelet aggregation
initiated by adenosine diphosphate. 167
Effect of lysolecithin added to platelet rich
plasma at different times during adenosine
diphosphate induced platelet aggregation. 169
Effect of phospholipid preparations added to
platelet rich plasma on reversible platelet 1
aggregation initiated by 5-hydroxytryptamine. 171
Figure
18,
23. Inhibition of irreversible platelet aggregation
initiated by 5-hydroxytryptamine by pre-
incubation of platelet rich plasma with
lysolecithin. 172
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Title Figure Page
Inhibition of irreversibb platelet
aggregation initiated by adrenaline
by pre-incubation of platelet rich
plasma with lysolecithin.
Dose response curves for the inhibition
by lysolecithin of the second phase of
platelet aggregation initiated by different
concentrations of adrenaline.
Effect of lysolecithin added to platelet
rich plasma at different times during
adrenaline-induced platelet aggregation.
Inhibition of thrombin-induced platelet
aggregation by pre-incubation of platelet
rich plasma with lysolecithin.
Inhibition of collagen-induced platelet
aggregation by pre-incubation of platelet
rich plasma with lysolecithin.
24.
25.
26.
27.
28.
176
177
178
181
182
29. (i) Inhibition of adrenaline-induced platelet
release reaction by pre-incubation of
platelets with lysolecithin.
(ii) Inhibition of collagen-induced platelet
release reaction by pre-incubation of
platelets with lysolecithin.
30. Aggregation of platelets previously inhibited
with lysolecithin by subsequent exposure to
186
187
adenosine diphosphate or adrenaline. 188
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Figure Title Page
31. Effects of saturated and polyunsaturated
lysolecithin fractions on collagen-
induced platelet aggregation. 192
32. Plasma phospholipids of unincubated and
incubated pre- and post-heparin plasma
samples separated by thin layer chrom-
atography. 199
33. Reduction of dextran-stimulated
erythrocyte sedimentation following intra-
venous administration of 2,500 units of
heparin. 204
34. Typical platelet response to collagen and
adenosine diphosphate before and fifteen
minutes after intravenous administration of
2,500 units of heparin. 207
35. Rates of irreversible platelet Eggregation
initiated by collagen or adrenaline before and
after intravenous administration of 2,500units
of heparin. 208
36. Inhibition of collagen-induced platelet
aggregation by hog-mucosal or ox-lung heparins
added to platelet rich plasma in vitro. 212
37. Changes in plasma lysolecithin concentrations
and in the rate of collagen-induced platelet
aggregation during the menstrual cycle of women
taking oral contraceptives. 222
- 23-
Figure Title Page
38.
40.-
41.
42.
43.
44.
Correlation between plasma lysoleciihin
concentrations and erythrocyte packing
rate in pregnant and non-pregnant women. 230
Relationship between plasma lysolecithin
level and risk of thrombo-embolic disease. 235
Summary of relationship between altered
plasma lysolecithin levels in vitro and in
vivo and alterations of platelet and
erythrocyte behaviour. 240
Aggregation of platelets initiated by (i)
digitonin and (ii) saponin. 246
Inhibition of digitonin-induced platelet
aggregation by lysolecithin. 248
Inhibition of digitonin-induced platelet
release reaction by Rogitine. 249
Inhibition of irreversible platelet
aggregation initiated by (i) adenosine
38.
diphosphate and (ii) adrenaline by pre-
incubation of platelet rich plasma with
sodium deoxycholate. 251
45. (i) Inhibition of platelet aggregation
initiated by adenosine diphosphate
by cetyl pyridinium chloride. 253
(ii) Inhibition of collagen-induced
platelet aggregation by cetyl
trimethylammonium bromide. 253
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CHAPTER 1
INTRODUCTION
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GENERAL INTRODUCTION
Acute coronary occlusion often resulting from coronary arterial
thrombosis is the greatest single cause of death in industrialized
Western society, -_Atherosclerosis of the coronary arteries is
associated with at least 95 percent of all cases of acute coronary
thrombosis and the contributory and predisposing aetiological
factors for these diseases are essentially the same (Friedberg, 1966).
The mechanism of atherogenesis is complex: it is the summation
of factors acting on the arterial wall from the blood and those
that result from changes in the arterial wall itself. An important
characteristic of human atherosclerotic lesions is that they comprise
two major elements: (i) lipid deposition (athero-) and (ii) fibrotic
organisation (-sclerosis), Any hypothesis concerned with the
pathogenesis of atherosclerosis must take into account both features.
A number of different theories have been proposed to explain the
fibrosis in aterosc/erotic lesions including platelet and fibrin
encrustation (Duguid, 1952) and the haemodynamic stress of pulsatile
blood flow (McDonald, 1960). However, the major sclerogenic factor
in atherosclerosis is probably due to the presence of large amounts of
cholesterol, particularly saturated cholesteryl esters,, within the
lesions. This view is supported by the induction of proliferative
changes and fibrosis following sub-cutaneous implantation of free
cholesterol and saturated esters of cholesterol (Abdulla et al, 1967).
There is evidence that the lipid deposition in atherosclerosii
may occur by more than one mechanism. Much of the lipid may be
deposited as the result of the infiltration and trapping of low
density lipoproteins from the blood. In the infiltration theory,
low density lipoprotein is considered to enter the arterial wall
- 26 -
from the lumen and due to the instability of its molecular structure,
it is thought to shed its lipids during passage through the inner
arterial wall (Page, 1954). This concept was supported by immuno-
electrophoretic identification of lipoprotein within the inner
arterial wall (Oro et al, 1961) and, subsequently, by histo-
immunological studies (Kao and Wissler, 1965) and immunofluorescent •
techniques (Walton and Williamson, 1968). However, the infiltration
theory of lipoprotein was compromised when it was shown that the
influx ratio of free/ester cholesterol into the arterial wall in
vivo (Newman and Zilversmit, 1962) and in vitro (Hashimoto and
Dayton, 1966) did not correspond to the concentration ratio of these
lipids in low density lipoproteins. It has been suggested that lipo-
proteins may leak into the damaged atherosclerotic arterial wall
(Adams et al, 1968) but in addition, it is now evident that there is
certainly some synthesis of lipid in the arterial wall. Under some
circumstances lipid-laden mono-nuclear leukocytes (lipophages) from
the blood may'also play an important role in the mechanism of
atherogenesis (see review by Wissler and Vesselinovitch, 1968).
It is now widely accepted that atherosclerosis is a disease of
disordered lipid metabolism, although our knowledge of the
mechanisms involved is still incomplete. Consequently studies of
the plasma lipids and.of lipid metabolism have become the most
important area of research into the causes of coronary arterial
disease.
Pickering (1964) has emphasised the importance of thrombosis
in acute myocardial infarction as the factor immediately responsible
for the occlusion of the coronary circulation. The adhesion and
aggregation of blood platelets to form a white body or thrombus,
- 27 -
which was capable of occluding the lumen of small arteries has been
known since the late nineteenth century (Robb-Smith, 1967). However,
the function of blood platelets was little studied until the
comparatively recent development of suitable methods for measuring
platelet adhesiveness (Wright, 1942 : Hellam, 1960) and platelet
aggregation (Born, 1962 :.O'Brien, 1962). Widespread acceptance of
these and other techniques has resulted in our knowledge of an
impressive array of factors influencing platelet behaviour (see
review by Mustard and Packham, 1970) without solving the fundamental
problem of the mechanism by which circulating individual platelets
are transformed in such a way that they become adherent to each other.
The comparative importance of intravascular platelet aggregation
and disordered lipid metabolism in the pathogenesis of coronary
arterial disease, has not yet been fully established. Most studies
relating to the pathogenesis of the disease, tend to emphasise one
hypothesis at the expense of the other. However, it is quite
possible that both factors play their part in the development of the
disease and indeed, may be inter-related. Epidemiological studies
(Malmros, 1950 : Strigm and Jensen, 1951 : Thomas et al, 1960) indicate
that high dietary intake of fat is associated with increased morbidity
and mortality from ischaemic heart disease and with an increased risk
of thromboembolic complications of the disease. Experimental dietary
studies (McDonald and Edgill, 1958 : Mustard and Murphy, 1962 :
Nord0y, 1965) have shown that both the quantity and type of dietary
fat can influence platelet behaviour as well as altering the plasma
lipid profile. Lowering of plasma lipid concentrations by
administration of the drug ethylchlorophenoxyisobutyrate (clofibrate)
has been reported to decrease platelet adhesiveness (Carson et al, 1963 :
Chakrabarti and Fearnley, 1968). The evidence for platelet behavioural
- 28 -
changes associated with alterations of the plasma lipid pattern is
attractive but as Pickering (1964) has stressed, the weakness of
the lipid theory of acute myocardial infarction is that it does not
account for the formation of the thrombus.
Recently, however, it has been suggested that an abnormal aspect
of platelet behaviour associated with ischaemic heart disease and
peripheral arterial disease may be directly due to a specific
alteration of the plasma phospholipids (Bolton et al, 1967), The
electrophoretic mobility of human platelets is changed by agents which
cause platelet aggregation (Hampton and Mitchell, 1966a): in low
concentrations these agents increase mobility and in higher concent-
rations these agents cause a decrease. Platelets from healthy subjects
were found to be equally sensitive to A.D.P. and noradrenaline in that
their maximum mobility was induced by incubation with 0,05 pg ml,
of either agent. Platelets from patients with ischaemic heart disease
or peripheral arterial disease differed in their sensitivity to A.D.P.
and noradrenaline, in that their maximum mobility was induced by 0.005
pg m1,-1 of A.D.P. but by 0.05 pg ml.-1 of noradrenaline (Hampton and
Mitchell, 1966b). Bolton and co-workers found that a similar abnormal
sensitivity to A.D.P. could be induced in normal platelets on exposure
to plasma from patients with ischaemic heart disease. They postulated
the presence of a transferable factor in the plasma of patients with
arterial disease and found it to consist of two components, one of
which was a lipoprotein-bound 1', 2', diacyl-sn-glycero-3-phosphoryl-
choline (lecithin, P.C.). The second component was labile and
thought to be an enzyme converting P.C. to 1' or 2' monoacyl-sn-
glycero-3-phosphorylcholine (lysolecithin, L.P.C.) which was
ultimately responsible for abnormal platelet sensitivity to A.D.P.
- 29 -
Hampton and Bolton (1969) later showed directly that L.P.C.
increased the sensitivity of normal platelets to A.D.P. suggesting
that L.P.C. may have thrombogenic properties.
Plasma concentrations of,L.R.C._in patients presenting with
acute myocardial inArction have been shown to be significantly
decreased from normal (Marinetti et al, 1959 : Berlin et al, 1969),
in contrast to what might have been expected on the basis of the
platelet electrophotetic mobility experiments (Bolton et al, 1967).
It was therefore of interest to study the formation of plasma L.P.C.
and the plasma_ concentrations of phosholipids in the healthy
population and in patients suffering from ischaemic heart disease
and peripheral arterial disease, and to confirm the results for
myocardial infarction. The effects of phospholipids and particularly
L.P.C. on platelet aggregation were largely unknown and have therefore
been investigated. Finally, the effects of pharmaceutical preparations,
including heparin and oral contraceptives, on plasma L.P.C. and
platelet function have been studied.
- 30-
Historical Review of Plasma Lysolecithin,
A general account of the chemistry and properties of the
lysolecithirs is beyond the scope of this thesis. However,, a
general review has been given by Robinson (1961) and the
pharmacological properties of lysolecithins have been briefly
discussed by Ansell (1965). The present review will be confined
to giving an account of the work which established the normal
presence of L.P.C. in human plasma and of its metabolism in the
blood,
Fahraeus (1921) first reported the increased suspension stability
of erythrocytes in incubated blood although it was not until fifteen
years later that formation of L.P.C. during incubation of blood was
put forward to account for the decreased sedimentation of erythrocytes
'o (Bergenhem and Fahraeus, 1936). Up until the late nineteen-fifties,
it had been generally assumed that the major phospholipids of plasma
were l'2' diacyl-sn-glycero-3-phosphorylethanolamine (phosphatidyl-
ethano/amine, P.M, lecithin (P.C.) and sphingomyelin, (Deuel,
1951-1955). After the introductioad reliable chromatographic
methods for the separation and determination of individual phospholipids
these fractions have attracted a renewed interest.
Lysolecithin was first shown to be a normal constituent of human
plasma and serum by Phillips (1957, 1959a) and was later found to be
a major phospholipid fraction in rat plasma (Newman et al, 1961).
Phillips reported that some seven percent of the total serum phospho-
lipid was L.P.C. and this was later confirmed by Gjone et al (1959a)
and Vogel et al (1962).
- 31 -
The distribution of L.P.Q. in serum lipoprotein fractions
was shorn to be non-uniform with the largest percentage being found
in the protein fraction of density greater than 1.21 g ml-1 (Phillips,
1959b). Only 14 percent of the total L.P.C. was found in the high
density lipoprotein fraction and very little L.P.C. was associated
with either the low or very low density lipoproteins, The discovery
of relatively high concentrations of L.P.C. in the protein. fraction
(d> 1.21) was unexpected, although this fraction had earlier been
shown to contain a significant amount of phospholipid (Havel_ et al,
1955). Subsequently the presence of L.P.C. and a smaller amount of
P.C. in the high density infranatant of serum was confirmed and data
were presented showing that L.P.C. was transported in the serum bound
to albumin (Switzer and Eder, 1965). These authors found less L.P.C.
associated with lipoproteins (d < 1.21) than had earlier been reported.
Data for the recovery of T.P.L., P.C. and L.P.C., was consistent with
some conversion of P.C. to L.P.C. during ultracentrifugation
suggesting that some of the L.P.C. that was found in the lipoprotein
fractions may have been an artefact. Glomset (1963) had previously
also shown that L.P.C. concentration increased at the expense of P.C.
during the ultracentrifugation of serum,
The fatty acid composition of individual plasma phospholipid
fractions has received only limited attention in contrast to the
repeated determinations of total plasma phospholipid fatty acid
composition. Early reports of the fatty acid composition of human
plasma L.P.X. gave the percentage of saturated fatty acids as 76,5 per-
cent (Gjone et al, 1959b), 76 percent (Tattrie and Cyr, 1963) and
64.3 percent (Williams et al, 1966). Approximately twice as much
palmitoyl-L.P.C. than stearoyl-L.P.C. was reported by these authors.
- 32-
These data were consistent with the formation of plasma L.P.C. by
removal of the unsaturated fatty acid from the 2-position of plasma
P.C. However, a more recent study has reported that 44 per cent of
the fatty acids in plasma L.P.C. were unsaturated which suggests
that plasma L.P.C. does not entirely arise from the enzymic removal
of the unsaturated fatty acid from the 2-position of plasma P.C.
(Phillips and Dodge, 1967).
- 33 -
Clinical Studies of Plasma Phospholipids
The analysis of individual phospholipids in biological fluids has
been considered difficult (Portman, 1970). Consequently much of the
clinical investigational work on plasma concentrations of individual
phospholipid fractions has been confined to examination of healthy
subjects. Recent studies of the normal population have indicated that
mean L.P.C. levels were lower in women than in men (Berlin et al, 1969a
Wittiger, 1973) and that absolute concentrations of L.P.C. (but not the
relative concentrations), increased significantly with age for both sexes,
Low plasma levels of L.P.C. and sphingomyelin have been reported in
patients with acute or chronic liver disease (Gjone and Mendeloff,
1963 : Gjone and Orning, 1966) and L.P.C. levels were especially low in
cases of biliary cirrhosis. By contrast, elevated concentrations of
L.P.C. and sphingomyelin were demonstrated in the plasma of patients
presenting with nephrotic syndrome (Nye and Waterhouse, 1961). Very
high plasma levels of L.P.C. have also been found associated with
acute pancreatitis (Gjone and Mendeloff, 1963). Raised serum L.P.C.
levels have been reported in Tay-Sachs, Spielmayer-Vogt and Niemann-
Pick diseases (Spiegel-Adolf et al, 1967).
The association of ischaemic heart disease and atherosclerosis
with raised plasma levels of cholesterol and/or triglycerides has
received considerable attention which has resulted in several systems
of classification of specific hyperlipoproteinaemias (Fredrickson et
al, 1967 : Strisower et al, 1968 : Billimoria et al, 1971). Diagnostic
use of these classifications has bedome important in the development
of rational therapeutic treatment of both familial and acquired
disoiders of lipid metabolism (Beaumont et al, 1970). However, there
have been very few and partly contradictory reports of plasma phospho-
lipid separation in relation to hyperlipoproteinaemia or associated
- 34
ischaemic heart disease and atherosclerosis. An early report by
Nothman and Proger (1962) indicated that the plasma cephalins were
increased in hyperlipidaemic patients. Hypercholesterolaemic
patients have since been shown to have raised plasma levels of all
phospholipids except L.P.C. (Christian et al, 1964) although Vikrot
(1965) demonstrated elevation of all phospholipid classes including
L.P.C. in similar material. In a recent study, Kunz et al (1970)
reported a significant elevation of plasma P.E. and P.C. in patients
with type IV (Fredrickson) hyperlipoproteinaemia. Levels of sphingo-
myelin and L.P.C. were decreased in these patients and L.P.C. levels
in patients presenting with clear clinical evidence of arterial
disease were significantly lower than for patients without vascular
complications. Kunz and Stummvoll (1971) have extended this study
and have reported that elevated plasma levels of P.E. were more
closely correlated with peripheral arterial disease than elevated
levels of any other plasma lipid fraction.
Decreased levels of plasma L.P.C. have been demonstrated in some
cases of acute myocardial infarction when compared with the fasting
values of two healthy subjects (Marinetti et al, 1959). A larger
study of plasma phospholipid levels in 22 cases of acute myocardial
infarction has recently been reported (Berlin et al, 1969b) and
significantly decreased L.P.C. levels were found in all patients
studied within 3 days of infarction. Repeated investigations of
these patients indicated that recovery from the acute stages of the
disease was accompanied by a return to near-normal levels of L.P.C.
Cigarette-smoking which is widely accepted as being a contributory
factor in ischaemic heart disease has not been shown to significantly
alter plasma levels of individual phospholipids (Pozner and Billimoria,
1970).
- 35 -
A marked increase in the absolute concentrations in plasma of
total phospholipid, P.E., P.C., cholesterol and triglycerides and
an absolute decrease in L.P.C. concentrations were observed during
pregnancy (Vikrot, 1964 : Svanborg and Vikrot, 1965a). The alterations
in phospholipid concentrations were directly correlated with the
duration of pregnancy and disappeared shortly after delivery (Svanborg
and Vikrot, 1965b). Similar changes in the plasma lipids and phospho-
lipids were found after the administration of normal clinical dosages
of oestrogen (ethinyl estradiol) to Bophorectomised women (Svanborg
and Vikrot, 1966). Long term treatment of women with oral contraceptives
has been reported to similarly influence plasma phospholipid levels
(Brody et al, 1968). Svanborg (1968) has reported on the effects of
the oestrogen, ethinyl estradiol, administered alone or in combination
with progestogens. Just as in pregnancy oestrogen administration raised
plasma T.P.L., P.E., and P.C. and lowered the plasma L.P.C. level,
norethisterone acetate, a derivative of 19-nortestosterone, influenced
plasma phospholipids in a direction opposite to that produced by
ethinyl estradiol. However, megesterol acetate, a 17-alpha-hydrexy-
progesterone, had no apparent influence on the phospholipids. More
recently evidence for cyclical (short-term) lowering of plasma L.P.C.
has been reported by Nicholls et al (1971) in women during oral
contraceptive therapy. These results emphasise the need for
investigators to make all observations on the effects of oral
contraceptives at carefully defined times.
Summary
The original discovery of L.P.C. as a plasma constituent
(Phillips, 1957) has teen confirmed in later studies although there
have been few clinical investigations of the plasma levels of L.P.C.
- 36 -
or other phospholipids. Kunz and Stummvoll (1971) have specifically
linked raised plasma P.E. with arterial disease although decreased
plasma L.P.C. levels have been demonstrated in patients with acute
myocardial infarction (Berlin et al, 1969b) and with established
peripheral arterial disease (Kunz et al, 1970). The risk of thrombo-
embolic disease has been shown to be increased during pregnancy and
in women receiving oral contraceptive therapy (B8ttiger and
Westerholm, 1971) and in both cases decreased plasma L.P.C.
accompanied by raised levels of other lipids and phospholipids has
been demonstrated (Svanborg, 1968). The clinical significant of
decreased plasma L.P.C. is unknown and deserves further study in
view of the apparent association between low plasma L.P.C., presence
or risk of thromboembolic disease and direct alterations in platelet
behaviour after their exposure to L.P.C. (Hampton and Bolton, 1969).
- 37 -
Formation of lysolecithin in plasma
The metabolism of the phospholipids in the blood has recently
been reviewed by Polonovski (1972) who recognised two main reactions
leading to the formation of L.P.C. in human plasma, These reactions
involve the conversion of lipoprotein-bound P.C. to L.P.C. by the
lecithin:cholesterol acyl transferase and post-heparin lecithinase
enzymes, The existence of a third enzyme, the plasma phospholipase,-
reported by Polonovski to be activated following the addition of
trypsin to blood, is of doubtful physiological significance.
Lecithin cholesterol acyl transforase (EC 2.3.1.43)
The esterification of plasma cholesterol was first described
by Sperry (1935) who postulated that phospholipids might provide the
necessary fatty acids for the reaction. It was initially proposed
that plasma contained a phospholipase B which hydrolysed P.C. to
provide the fatty acids for a coupled cholesterol esterase reaction
(Le Breton and Pantaleon, 1947). Subsequently a transient increase
in plasma L.P.C. and ultimately of free choline was demonstrated in
plasma incubated at 370 over 72 hours, suggesting that the reaction
was more complicated than originally:proposed (Etienne and Polonovski,
1960). Ultimately an acyl transferase mechanism was proposed for the
esterification of cholesterol on the basis of experiments which
demonstrated that labelled cholesteryl esters were formed in plasma
incubated with linoleoyl - 14C - P.C. but not in plasma incubated
with non-esterified linoleic acid - 14C. (Glomset et al, 1962). The
reaction has since become known as lecithin:cholesterol acyl
transferase (L.C.A.T.) and Glomset (1962) has reported that the fatty
acid was transferred from the 2-position of P.C. An extensive
review of L.C.A.T, has been presented by Glomset (1968) who also
discusses the possible physiological roles of the reaction.
- 38 -
Lecithin:cholesterol acyl transferase has usually been studied
by measuring cholesterol esterification, particularly by radio-
isotopic assay, and very few investigations have included measurement
of L.P.C. formation. One explanation for this is that only about one-
half of the predicted amount of L.P.C. Can be demonstrated in human
plasma after incubation (Glomset, 1963). Glomset (1968) has
suggested that this may indicate the presence of an L.P.C. hydrolyzing
enzyme in plasma. However, this lack of stoichiometry remains as an
objection to the L.C.A.T. reaction as it was originally conceived.
An enzyme, almost certainly identical to L.C.A.T. was described
and partially purified by Adlkofer et al (1968) who utilized the
inhibitory effects of L.P.C. on red cell sedimentation as an assay of
enzyme activity. During a six hour incubation of normal plasma Berlin
et al, (1969a) estimated the mean L.P.C. formation to be around 40 n mol
1-1
hour1 The mean L.P.C. formation in incubated plasma from patients
suffering from acute myocardial infarction was significantly lower than
for age-matched healthy subjects, and this suggested that decreased
formation of L.P.C. in vivo may have been responsible for decreased
L.P.C. concentrations in this group of patients (Berlin et al, 1969b).
Post-heparin Lecithinase
The intravenous administration of heparin or heparinoids in man
causes the release of clearing factor or lipoprotein lipase and this
enzyme has been the subject of several reviews (Robinson and French,
1960 : Robinson, 1963). More recently it has been shown that post-
heparin plasma contains a phospholipase which at first appeared to be
unique in its attack on P.E. (Vogel and Zieve, 1964), but was later
shown to also hydrolyse exogenous P.C. substrates (Zieve and
Doizaki, 1966 : Vogel and Bierman, 1967). The enzyme was found to
have similar characteristics to lipoprotein lipase and Doizaki and
Zieve (1968) have suggested that the two enzymes may be identical.
- 39 -
Berlin et al (1969c) have since shown that the rate of formation
of L.P.C. from endogenous plasma P.C. was increased in post-heparin
plasma. Berlin did not distinguish between post-heparin L.P.C.
formation and the L.C.A.T. reaction although Vogel and Bierman (1967)
had previously distinguished between the two on the basis of
different positional specificity. L.C.A.T. had been shown to
remove the fatty acid from the 2-position of P.C. (Glamset, 1962)
whereas Vogel and Bierman demonstrated that post-heparin lecithinase
removed the fatty acid from the 1-position of P.C. substrates.
In addition to activating post-heparin lecithinase, intravenous
heparin has recently been demonstrated to inhibit L.C.A.T. activity
when plasma triglyceride concentrations were high enough to produce
non-esterified fatty acid (N.E.F.A.) concentrations in excess of
1000 p equiv. L 1 (Rutenberg et al, 1973).
Removal of lysolecithin from the pasma.
In normal healthy man the plasma concentrations of L.P.C. are
kept within a narrow range (Berlin et al, 1969a), Several studies
have demonstrated that L.P.C. has a short half-life in plasma and
was rapidly incorporated into P.C. by the liver, heart and other
tissues (Stein and Stein, 1964 : Stein and Stein, 1965 : Portman et al,
1970). Uptake of L.P.C. was not necessarily restricted to the tissues
and metabolic pathways for the conversion of L.P.C. to P.C. or G.P.C.
have been demonstrated in erythrocytes (Mulder et al, 1965 : Mulder
and Van Deenan, 1965), polymorphonuclear leukocytes (Elsbach et al,
1968)1 1965) and platelets (Cohen,/Elsbach et al, 1971). The formation of
L.P.C. has not been demonstrated in intact platelets or erythrocytes
suggesting that the L.P.C. which participates in these reactions
probably originates from the plasma. If these pathways are normally
active then the formed elements of blood must exchange P.C. for
plasma L.P.C. in order to keep the cellular-content of P.C. constant.
- 40-
Switzer and Eder (1965) have discussed this role of L.P.C. as one
mechanism for the renewal of lipoprotein P.C. depleted by the
L.C.A.T. reaction.
Summary
The evidence for two enzymatic reactions capable of forming
L.P.C. in plasma has been reviewed. Of these enzymes at least one ,
L.C.A.T., is probably a physiologically important source of plasma __
L.P.C. since in a recently discovered inborn error of metabolism,
characterised by familial plasma L.C.A.T. deficiency, the plasma
levels of L.P.C. were decreased (Gjone and Norum, 1968 : Torsvik
et al, 1968). Finally the removal of L.P.C. from the plasma by
tissues and possibly by blood platelets, erythrocytes and
leukocytes has been briefly reviewed. Plasma levels of L.P.C. are
probably kept within the normal range by a balance between formation
and removal from the plasma by the tissues and blood cells.
Alterations in the rate of L.P.C. formation or utilization could
result in decreased or elevated plasma L.P.C. levels, both of
which have been reported in certain disease states.
- 41 -
Phospholipid effects on platelet function
Platelet function can only be studied in aqueous media
such as plasma and this limitation presents a major obstacle
when the effects of water-insoluble substances such as lipids
are to be studied. However, the sodium salts of fatty acids
are water-soluble and their effects on platelet aggregation have
been studied. Haslam (1964) demonstrated that the sodium salts
of palmitate, stearate, arachidate, behenate and lignocerate
when added to platelet rich plasma (P.R.P.), initiated irreversible
platelet aggregation and release of platelet A.D.P. These effects
of certain fatty acids have been confirmed by later investigators
(Mustard and Packham, 1970). The effects of plasma lipoproteins
-on platelet function.have also been-investigated-and beta
lipoproteins have been demonstrated to enhance pdatelet adhesion
and platelet aggregation induced by A.D.P. (Farbiszewski and
Worowski, 1968). Because beta-lipoproteins have been shown to be
increased in patients with advanced coronary artery disease
(Besterman, 1957), the effects of this lipoprotein on platelets may
be of pathological significance although other high molecular weight •
proteins containing no lipid have similar effects on platelets in
vitro.
There have been very.few studies of phospholipids and platelet
function although there have been several investigations of phospho-
lipids and blood coagulation (Poole and Robinson, 1956 : Billimoria
et al, 1965). Kerr and co-workers (1965) have prepared phospho-
lipid suspensions by adding saline with vortex-mixing to solutions
of phospholipids in ether which was subsequently evaporated. These
authors found that P.A., P.E., and P.S. added to stirred platelet
rich plasma (P.R.P.) initiated reversible platelet aggregation,
- 42-
whereas a crude mixture of sphingomyelin and L.P.C. initiated
irreversible aggregation. Exposure of P.R.P. to P.C. had no
direct effect but this phospholipid inhibited platelet aggregation
induced by stearic acid. A different effect of P.S. on platelet
function has been reported by Nishizawa et al (1969) who found
that P.S. inhibited platelet aggregation induced by collagen or
thrombin.
Evidence for the effect of L.P.C. on increasing the sensitivity
of platelets to A.D.P. has been reviewed above (General Introduction).
Subsequently some effects of L.P.C. on thrombus formation at the
site of minor injuries to cortical arteries in rabbit, have been
described (Bolton et al, 1969). Topical application of L.P.C.
solution to the injury produced a single white thrombus, although
A.D.P. similarly applied induced recurrent thromboembolism at the
site of the injury. This effect of L.P.C. was probably due to
release of A.D.P. by lysis of the tissues at the site of injury.
However, L.P.C. infused into rabbits appeared to inhibit A.D.P.-
induced thrombus formation since replacement of the L.P.C. infusion
by saline resulted in greater thrombus formation at the injury site
when A.D.P. was topically applied.
In summary, the effects of phospholipids on platelet function
has been poorly studied. In particular, the effects of L.P.C. on
platelet function require further study since although topical or
local application of L.P.C. to damaged arteries was thrombogenic,
its infusion into rabbits limited or inhibited thrombus formation
in vivo. In the present study the effects of L.P.C. and other
phospholipids on platelet aggregation and adhesiveness have been
studied in detail.
-43-
Object of Study
The presence of L.P.C. in plasma is well established (Phillips,
1957) although the physiological role of this potentially hazardous
plasma phospholipid is unknown. L.P.C. may increase the
sensitivity of blood platelets to the aggregating agent, A.D.P.,
(Hampton and Bolton, 1969) in patients with ischaemic heart disease.
There have been few quantitative studies of individual plasma
phospholipids in thromboembolic or ischaemic heart disease, although
patients suffering from acute myocardial infarction have been found
to have significantly decreased plasma L.P.C. levels (Berlin et al,
1969b). Furthermore, the plasma levels of L.P.C. are also reduced
in several populations which are subject to an increased risk of
thromboembolic disease including peripheral vascular disease
(Kunz et al, 1970), women who are pregnant (Vikrot, 1964 : Svanborg
and Vikrot, 1965a) or who are taking oral contraceptives (Brody et al,
1968). It was, therefore, of interest to determine the influence of
L.P.C. on platelet aggregation and adhesiveness in vitro and to assess
whether alterations of plasma L.P.C. in vivo were reflected by
changes in platelet and erythrocyte behaviour. In view of the
limited data available on plasma L.P.C. levels and formation in
either the healthy or at risk populations, it was first thought
necessary to, compare the levels of plasma phospholipids and L.P.C.
formation in healthy subjects and in patients suffering from acute
myocardial infarction, peripheral arterial disease and chronic
ischaemic heart disease.
To achieve the objects discussed above it was necessary to
undertake three different types of study and to examine the results
conjointly in order to assess any possible relationship between
plasma L.P.C. levels and alterations in blood platelet function.
- 44 -
The three types of study were planned to answer the following
questions which have all been previously raised in the introduction:
(i) Are plasma L.P.C. levels abnormal in patients with an
increased risk of thromboembolic disease such as those
suffering from acute myocardial infarction, chronic
ischaemic heart disease and peripheral atherosclerosis -
or in women during normal pregnancy? -
(ii) Does L.P.C. alter blood platelet function in vitro at
concentrations similar to those found normally in plasma?
(iii) Are acute changes in plasma L.P.C. levels in response to
oral contraceptive therapy or heparin administration
accompanied by changes in platelet function which are
consistent with the observed effects of L.P.C. on plates
in vitro?
- 45 -
CHAPTER 2
CLINICAL MATERIAL, BIOLOGICAL TECHNIQUES
AND ANALYTICAL TECHNIQUES
- 46-
Section 1
CLINICAL MATERIAL
Introduction and discussion of experimental protocol
A major objective of this thesis has been to study the plasma
concentrations of individual phospholipids in healthy men and in
patientwsuffering from ischaemic heart disease or peripheral
arterial disease. In addition measurements of lysolecithin
formation in incubated plasma have been made.
In planning a study such as this thesis which compares any
biochemical or biological parameters of a diseased population with
those of an apparently healthy population, there are several
important considerations to be taken into account. Firstly, rigid
criteria based on clinical history, examination and laboratory
investigation must be adopted for the diagnosis of the disease which
is to be studied. Secondly, the healthy control population must be
strictly matched with the patient group as regards age and sex.
Thirdly, a study of this type is best conducted on a double-blind
basis in order to eliminate or at least reduce, bias on the part of
the investigator. These are the important considerations although
anomalies such as drug-therapy, normal seasonal variations of
experimental parameters, effects of smoking and different exercise
habits must all be excluded in order to achieve a meaningful
comparison. Finally, the numbers of patients within each group must
be sufficient to allow statistical assessment of any differences
revealed during the study.
The diseases which have been studied in this thesis were
peripheral arterial disease, chronic ischaemic heart disease and acute
myocardial infarction. Diagnosis of each disease was made by the
physician or surgeon responsible for the patients. Thus the diagnosis
- 47 -
of each patient was kept separate from the investigator and in the
case of acute myocardial infarction, true double-blind conditions
were achieved since the final diagnosis was disclosed only after
the collection of blood samples for phospholipid analysis had been
carried out. Thus, of the total number of patients from whom blood
samples were collected, only about one half were eventually
confidently diagnosed as acute myocardial infarction. The remainder
except for a small number of uncertainties, constituted a fourth
group of patients characterised by symptoms of acute ischaemic
heart disease but without evidence of myocardial infarction.
All patients studied were men who were in middle-age or older,
(45-65 years) and blood samples were taken once only. Approximately
twenty patients were included in each disease-group to enable
statistical evaluation of the results. A similarly sized group of
middle-aged men free from symptoms or clinical history of ischaemic
heart disease or peripheral arterial disease, were included as a
control population. Neither patients nor controls were fasted before
blood collection and instead samples were collected at least three to
four hours after a moderate breakfast. Any lipaemic plasma specimens
from the control group were excluded from the study. To avoid any
possible error due to seasonal variation in plasma phospholipid levels,
'all blood samples were collected during the colder part of the year
(October - March). Data were collected from both patients and control
subjects on smoking habits, drug-therapy and occupation or exercise
habits,
- 48-
Healthy control population
Current medical opinion favours the view that widespread pre-
atherosclerotic lesions are present in even the young healthy
population and it is well known that the symptoms of ischaemic
heart disease often appear suddenly in previously healthy individuals.
It is therefore apparent that there is a strong possibility that
'previously symptomless individuals selected as healthy controls for
comparison with patients suffering from ischaemic heart disease,
may themselves subsequently present with the disease. Therefore any
control group can only properly be referred to as the apparently
healthy population.
There have been only a few studies of plasma phospholipid levels
even in the apparently healthy population (Berlin et al, 1969a :
Bottiger 1973a). It was therefore of interest to include analysis of
plasma phospholipids in both men and women of different age groups
in this thesis. The following four groups of apparently healthy
individuals have therefore been studied under the conditions already
discussed:
(i) Young women aged 16 - 30 years, who were neither pregnant.
nor taking oral contraceptive preparation since these would have
interfered with plasma phospholipid metabolism (Svanborg, 1968).
Women in this group were medical students, laboratory technicians
or secretarial staff.
(ii) Young men aged 18 - 30 years who were either office
employees, medical students or laboratory technicians.
(iii) Older women aged 45 - 60 years, whose menopausal status
was not evaluated since no changes in plasma phospholipid levels have
been reported in a comparison of menopausal and menstruating women
(Hallberg et al, 1976). Although some of the women in this group
were secretarial staff or laboratory workers, it was necessary to
include some women with clinical evidence of mild rheumatic heart
disease but who showed no symptoms of ischaemic heart disease.
- 49 -
(iv) Older men aged 45 - 65 years who were employed in a
variety of occupations by British Rail (Paddington).
All individuals in the control groups were questioned about
smoking habits, drug-therapy and family history of heart disease.
Peripheral arterial disease
• Sixteen male patients with a clinical history of chronic
peripheral arterial disease as diagnosed by the presence of
severe leg claudication, were included in the study. Patients in
this group were not receiving any drug therapy at the time of
venepuncture as they were about to enter a double-blind clinical
trial of a new drug unconnected with the present thesis.
Chronic ischaemic heart disease
Twenty-five male patients with a clinical history of chronic
ischaemic heart disease, as characterised by the presence of severe
anginal pain of more than one year's duration, were included in this
group, All patients showed evidence of ischaemic electro-cardiographic
changes and had a history of angina, and one third of the individuals
had sustained a myocardial infarction more than six months prior to the
time of blood collection. None of the patients in this group were
taking drugs such as clofibrate which lower plasma lipid levels
although a few patients had been prescribed propranolol or hypo-
tensive agents. Approximately one half of the total group were
cigarette smokers.
Acute myocardial infarction
Blood samples from more than forty patients admitted to the
hospital coronary intensive care unit with suspected acute
myocardial infarction, were coalected within 48 hours of the onset
of pain. Subsequent clinical and laboratory investigation confirmed
- 50-
the diagnosis of acute myocardial infarction in twenty-one
individuals on the basis of electro-cardiographic changes and
raised serum enzyme levels. Of the remaining patients, seventeen
had a history of previous myocardial infarction or ischaemia,
although there was insufficient evidence to warrant diagnosis of
myocardial infarction at the time they were studied. Consequently --
these patients have been included in a fourth group and designated
acute ischaemic heart disease.
In addition to a comparison of plasma phospholipids in patients
suffering from ischaemic heart disease and in healthy individuals,
this thesis has included studies of plasma phospholipid levels and
blood platelet or erythrocyte behaviour in patients receiving
intravenous heparin administration and in women who were pregnant
or were taking oral contraceptive preparations. Details of
patients included in these groups are given below.
Intravenous administration of heparin in man;
The effects of small doses of heparin adminstered intravenously
in man have been studied with reference to changes in plasma phospho-
lipid levels and platelet and erythrocyte behaviour. The patients
selected for heparin administration had no symptoms of ischaemic
heart disease although they were undergoing routine right heart
catheterisation to investigate congenital or rheumatic heart disease.
In this study all patients were pre-medicated with diazepam at least
two hours prior to catheterisation which was conducted without
anaesthesia. No patients in the study group had taken analgesics
or other drugs likely to interfere with platelet function for at
least 48 hours prior to blood collection.
- 51 -
During routine catheterisation, heparin is normally
administered in very low concentrations in order to prevent
blood coagulation within the catheter. However, in this study,
blood was collected through the catheter prior to heparinisation
of the patient. Subsequently either 2,500 or 5,000 units of
heparin were administered through the catheter and flushed into
the circulation with saline. The post-heparin blood sample was
collected through the catheter fifteen minutes later.
The heparin given in most of these studies was derived from
hog-mucosal sources and was obtained from Weddel Pharmaceuticals
or Riker. A few studies were conducted to compare the effects of
a different heparin prepared from ox-lung sources (Upjohn).
Women taking oral contraceptive preparations
Jackson (1973) has listed twenty-five different combination
oral contraceptive preparations representing twenty-three different
formulations as currently•available in the United Kingdom. Most of
the preparations contained 50 ug of oestrogen in combination with a
variable amount of progestogen (0.5 - 4 mg.) and were supplied as a
course of twenty-one tablets, Svanborg (1968) has indicated that
oestrogens and progestogen have opposite effects on plasma phospholipid
concentrations. It was therefore of importance in this study to
concentrate on just a few of the preparations available, The
preparations which have been included in the study were arbitrarily
grouped into low- and high-progestogen oral contraceptives (table 1).
- 52 -
Table 1
Composition of some oral contraceptive preparations
Group Proprietary Name Generic Name
Low progestogen
MINOVLAR 21 ) ( Norethisterone acetate 1 mg. (7) MINOVLAR ED ) ( Ethinyloestradiol 0.05 mg.
(2) ORTHONOVIN 1/50 ( Norethisterone 1 mg.
(1) OVULEN 50
( Mestranol 0.05 mg,
( Ethynodiol diacetate 1 mg. ( Ethinyloestradiol 0.05 mg.
High progestogen -
(3)
(2)
GYNOVLAR 21
ANOVLAR 21
( Norethisterone acetate 3 mg, ( Ethinyloestradiol 0.05 mg,
( Norethisterone acetate 4 mg, ( Ethinyloestradiol 0.05 mg.
A total of twenty-two women were included in this study of
whom seven were not taking oral contraceptives, five were taking •
high-progestogen and ten were taking low-progestogen oral
contraceptive preparations. The total numbers of volunbers available
for this study was limited and this has resulted in the inclusion of
women who had been taking oral contraceptive preparations for over a
year as well as women taking their first course of tablets.
Mid-morning blood samples for both platelet aggregation and•
phospho-lipid studies were taken from each individual on day-4 and
day-25 of the same menstrual cycle. These samples were taken at least -
three hOurs after the last meal and so could not be considered as
fasted samples.
- 53 -
Blood samples obtained from women during pregnancy
For ethical reasons partly relating to the volume of blood
samples required for platelet studies, it was not possible to plan
a longitudinal investigation of platelet function and plasma
phospholipids during pregnancy. However, relatively small blood
samples were required for an investigation of red cell behaviour
and therefore a study of plasma phospholipid levels in individual
pregnant women was combined with an investigation of erythrocyte
flexibi ]ity.
Twenty-five pregnant women with gestational periods ranging
from nine to thirty-nine weeks were included in this study. The
women selected (average age, 24 years) were either attending
ante-natal clinics or had been hospitalized at the onset of
labour. Sixteen women students and technicians (average age, 22
years) who were neither pregnant nor receiving oral contraceptive
preparations were included in the study as a contri group. Of
this latter group; only six individuals provided blood samples
for investigation of erythrocyte behaviour. Each subject was
seen once only on the day that blood samples were taken.
- 54
Section 2.
BIOLOGICAL TECHNIQUES
(1) Techniques for studying the effects of lysolecithin on
erythrocyte behaviour.
Introduction.
The haemolytic effect of lysolecithin (L.P.C.) has long been
known and this property of the compound is reflected in its
trivial name. Recent work has shown that L.P.C. species containing
a saturated fatty acid such as the stearoyl- and palmitoyl
compounds were very much more effective haemolysins than were
unsaturated and polyunsaturated L.P.C. analogues (Gottfried and
Rapport, 1963 : Reman et al, 1969). The effects of L.P.C. on
erythrocytes other than that of haemolysis have been less well
characterised although BergenheM and Ahraeus (1936) suggested that
L.P.C. stabilised red cell suspensions and inhibited the sedimentation
of the cells. Adlkofer et al (1968) made use of this property of
L.P.C. in their investigation of L.P.C. formation during the
incubation of plasma. These workers added high molecular weight
dextran to blood or to suspensions of erythrocytes to increase the
sedimentation rate (E.S.R.) from a few mm.hour-1
to between 50 -
100 mm.hour-1. Having increased the E.S.R. it was then possible to
quantitate the inhibitory effect of L.P.C. and to use this method
to assay the formation of L.P.C. under experimental conditions.
The effect of the dextran was to promote red cell aggregation
(rouleaux formation) by its adsorbance to the red cell membrane or
surface-coat. The inhibitory effect of L.P.C. was to prevent
rouleaux formation by altering the erythrocyte shape from biconcave
discs to spherocyte or echinocyte forms which were unable to
- 55 -
orientate themselves into aggregates. Sterescopic electron
microscopy has shown that the exposure of erythrocytes to
increasing concentrations of L.P.C. did alter their morphology
and produced echinocytes (Piper et al, 1972). A similar
transformation of erythrocyte shape has been described by
Feo (1973) when red cells were re-suspended in incubated plasma
showing that L.P.C. formed during incubation of plasma was
effective in altering red cell properties.
Since the effect of sub-haemolytic exposure of erythrocytes
to L.P.C. changes the shape of the cells, one would expect that
other erythrocyte properties such as packing of cells during
centrifugation and the morphological contribution of the cells to
blood viscosity, would also be changed by exposure to L.P.C. Simple
methods have been used to investigate these possible effects of
L.P.C. However, besides the investigation of L.P.C. effects on
erythrocyte behaviour in vitro, similar methods have been used to
study erythrocytes in blood samples thought to contain altered levels
of L.P.C. such as after heparin administration (Berlin et al, 1969c)
or samples taken during pregnancy (Svanborg and Vikrot, 1965a),
(a) Erythrocyte sedimentation
The method used to investigate the effect of L.P.C. on erythrocyte
sedimentatbn was a modification of that described by Adlkofer et al
(1968). The Westergren sedimentation apparatus was used for this
method and consisted of glass sedimentation tubes graduated for
20 cm, with mm, divisions and a rigid stand fitted with spring clips
(top) and silicone rubber pads (bottom) to hold the tubes in a vertical
position. The apparatus was set up on a horizontal bench and the
alignment of the tubes into a vertical position was checked with a
plumb line.
- 56 -
Blood was collected by clean venepuncture from apparently
healthy volunteers and anticoagulated with lithium sequestrene
(8.4 rags per 10 ml.). The plasma was removed after centrifugation
at 1500 g for 15 minutes. The buffy coat was removed from the
erythrocytes together with any remaining plasma and the erythrocytes
were washed once with 0.9 percent saline and collected by
centrifugation.
In the original Adlkofer method dextran of mean molecular
weight 96,000*
was added to the red cell suspension at a final
concentration of 2 percent immediately before filling the
sedimentation tubes. Dextran of this molecular weight was
unavailable and two commercially available dextran solutions
intended for intravenous transfusion, were tested, These were
"Dextraven 150" (mean molecular weight, 150,000) and "Dextraven
110" (mean molecular weight, 110,000) produced by Fisons and both
consisted of a 6 percent solution of dextran in 0.9 percent saline,
In an initial.study the effect. of both dextran preparations
on the B.S.R. was tested at a final concentration of 2 percent by
mixing 0.5 ml of erythrocytes with 0.5 ml of plasma and adding 0.5 ml
of dextran solution just prior to filling the sedimentation tubes,
Duplicate sedimentation tubes were filled and clamped in position.
The E.S.R. was recorded every 5 minutes, The presence o- either
dextran fraction caused a rapid increase in the sedimentation rate but
the effect was more pronounced with "Dextraven 150" (Fig. 1). However,
the addition of dextran at a final concentration of 2 percent was not
satisfactory since it produced trailing of macroscopic red cell
aggregates which made it difficult to read the E.S.R. values.
Subsequently these workers used dextran fractions of higher molecular
weight (Personal communication - G.Ruhenstroth-Bauer).
- 57 -
120 IJ
Dext raven 150"
"Dextraven 110"
60
Control 1 • 1
20 40 Minutes
Figure 1: Effect of high molecular weight dextran fractions on
erythrocyte sedimentation rate.
The dextran fractions were of mean molecular weight 110,000
and 150,000. In each test 0.5 ml. of 67. dextran solution was
mixed with 0.5 ml plasma and 0.5 ml of washed erythrocytes.
- 58 -
The effects of lower concentrations of dextrans were
investigated by mixing.0.5 ml red cells with 0.7 ml of plasma
and 0.3 ml of 6 percent, 4 percent or 2 percent dextran
solution. The effect of dextran on the speed of erythrocyte
sedimentation was found to depend upon its concentration
(Fig. 2.). There was no evidence of trailing phenomenon at -
dextran concentrations of 0.4 percent and since this concentration
of "Dextraven 150" produced a satisfactory E.S.R., it was decided
to use it for the experimental tests. At this final concentration
of "Dextraven 150" (0.4 percent) the E.S.R. was 50 - 100 mm.hour 1
for most blood samples and the assay was reproduceable provided
that the dextran was added immediately befbre setting up the test,
b) Erythrocyte packing rates
Blood samples for the measurement of erythrocyte packing
rates and whole blood viscosity were anticoagulated with heparin
(15 units ml-1). Each test was started one hour after venepuncture,
Erythrocyte packing rates were measured using the method of
Rampling and Sirs (1973). Microhaematocrit tubes were filled with
0.05 ml of blood for each estimation of the erythrocyte packing
rate. One end of each tube was sealed in a gas flame and the tube
placed in the rotor of a Hawksley centrifuge. The centrifuge was
operated for 2 minutes connected to a supply voltage of 80V (600g),
and then for 3 minutes at 230V (12,000g). Measurements were made
of the haematocrits, with a Hawksley reader, after each run. If
the packed cell haematocrits obtained at 600g and at 12,000g are
H1
and H2 respectively, then the rate of packing R40, can be
calculated for a 40 percent packed cell haematocrit using the formula
R = 0.5 (100 - H) 0.42 (H2 - 40). 40
- 59 -
20 40 Minutes
Figure 2: Effect of different concentrations of dextran on
erythrocyte sedimentation rate.
The curves represent the effect of different concentrations
(g/100 ml) of Dextran (150,000) on the sedimentation of washed
erythrocytes.
- 60-
Rampling and Sirs report that the typical values of R40 were
9.0 %min,-1
for healthy individuals and up to 30% min,-1, or
down to 1% min,-1 in exceptional cases with pathological blood
specimens,
Whole blood viscosity was measured with a Brookfield model
LVT cone on plate type viscosity meter, The apparatus was
maintained at 37° by circulating water. In a typical estimation
of viscosity the apparatus was filled with blood and the machine
set to rotate at the lowest shear rate. Four estimations of -.
viscosity were made at each shear rate working progressively up
to the highest shear rate. The mean values of readings taken at
each shear rate were recorded. After each blood sample the apparatus
was thoroughly rinsed with distilled water and carefully dried
with cellulose tissue,
- 61 -
(2) Methods for studying platelet function in vitro
Introduction
Because of the experimental evidence showing the primary role
of platelets in thrombus formation, many methods have been described
to measure platelet function in vitro. Of these methods the most
important have beep those which measure the adherence of platelets
to surfaces (platelet adhesiveness or "stickiness") (Wright, 1941
Hellem, 1960) and those which measure the adherence of platelets to
each other (platelet aggregation) (Born, 1962 : O'Brien 1962).
Other tests such as the estimation of platelet electrophoretic
mobility (Hampton and Mitchell, 1966a) have been utilized to a lesser
degree. These tests are all relatively crude and although platelet
adhesion and aggregation almost certainly play a central role in
thrombosis our understanding of the mechanisms involved in vivo is
incomplete, O'Brien (1969) has suggested that by using several of
these empirical tests together and enquiring into the mechanisms
occurring in vivo results of value might be yielded to both the
clinician and the platelet-physiologist.
The central investigation of this thesis concerns the effects
of phospholipids, particularly L.P.C., on platelet function both in
vitro and in vivo. Although platelet electrophoretic mobility
measurements have shown that L.P.C. increases the sensitivity of
platelets to A.D.P. (Hampton and Bolton, 1969), the effects of L.P.C.
or other phospholipids on platelet function have not been studied by
means of a variety of tests. In this present investigation the
effects of L.P.C. and other phospholipids on plateleg aggregation
initiated by five different aggregating agents and,on platelet
adhesiveness have been studied.
-62_
(a) Platelet aggregation method
Blood samples for platebt aggregation tests were collected
by clean venepuncture without stasis from apparently healthy
individuals of both sexes. None of the blood donors had taken
any preparation *containing acetylsalicylic acid (aspirin B.P.)
or any Other drug known to affect platelet function (Mustard and.
Peckham, 1970) for a period of two weeks prior to blood
collection. Nine volumes of blood were mixed with one volume of
0.9 percent saline containing 3.2 grams percent of trisodium
citrate in a siliconised glass centrifuge tube. The blood samples
were centrifuged at 150 g for 15 minutes in either an M.S.E. or
International bench centrifuge. After centrifugation the platelet
rich plasma (P.R.P.) was removed using a siliconised Pasteur
pipette and stored in a siliconised glass vial at room temperature
(20 - 22°) until required.
Apparatus for studying platelet aggregation
Platelet aggregation was studied by the optical density method
(Born, 1962 : O'Brien, 1962), which utilizes the fall in optical
density of stirred P.R.P. as an index of platelet aggregation.
The apparatus used was similar to that described by Mills and
Roberts (1967). It consisted of a specially modified nepholometer
(EEL Instruments) in which the sample compartment was surrounded
by a copper water-jacket through which water at 37° could be pumped
from a large reservoir. A rotating magnet was fitted beneath the
sample compartment in order that P.R.P. samples could be stirred
at 1000 r.p,mo by means of a small plastic-coated flea-magnet
contained within the sample tube. Light could be shone through
the sample and through a neutral density filter (600 nM) placed
in front of the photoelectric cell. Light transmitted through
the sample compartment could be measured by an internal meter or
alternatively the photo-cell output could be coupled with a
- 63-
10 mV pen recorder (Vitatron) by means of a simple switch mechanism.
The range of the recorder was adjusted so that a pooled sample of
P.R.P. from five subjects registered 20 percent transmission and
pooled platelet free plasma (P.F.P.) registered 100 percent trans-
mission. The adjustment of the instrument was periodically checked
byycomparing the optical density readings of a series of neutral
density filters on the internal meter with the values on the pen-
recorder scale, Minor adjustment of the nepholometer sensitivity
was sufficient to correct any discrepancy between the internal
reading and the output registered by the pen-recorder. Samples of
P.R.P. (1 ml.) were transferred with a siliconised pipette to
individual siliconised round cuvettes containing small flea-magnets.
Preparation of aggregating agents
The five aggregating agents used in the experiments described
below were A.D.P„ (disodium salt, Sigma), 5-hydroxytryptamine (5-H.T.)
(creatinine sulphate complex, Sigma), adrenaline (hydrogen tartrate,
B,D.H„), bovine collagen (Sigma) and bovine thrombin (Parke-Davis).
Solutions of A.D.P. and 5-H.T. were prepared monthly by
dissolving pre-weighed amounts of either compound in 0,9 percent
saline. The solutions were diluted with 0.9 percent saline to give
working solutions at final concentrations of 100 n mol ml.-1 (A.D.P.)
and 400 n mol ml, --1 (5-H.T.) which were stored at -20o for up to four
weeks.
Adrenaline was prepared daily as a solution in 0,9 percent saline
with a final concentration of 250 n mol ml.-1
Fresh ampoules of thrombin were used daily and diluted with
0.9 percent saline to give a final concentration of 20 units ml,-1
- 64-
A suspension of bovine collagen fibres in saline was prepared
by grinding freeze dried bovine tendons with 0.9 percent saline .
and acid-washed sand as an abrasive agent (Evans et al, 1968).
The grinding procedure took approximately three hours after which
the mixture was allowed to stand for five minutes. The supernatant
was removed and filtered through several layers of muslin to remove
any remaining coarse particles. The filtrate which contained
approximately 1 gram of collagen per 100 ml. was sealed into 5-ml.
glass ampoules and stored at 40 for up to two years without loss
of aggregating activity. Before use each ampoule was subjected to
vigorous vortex-mixing. The collagen suspension was diluted with
9 volumes of 0.9 percent saline to give a working suspension.
In a typical experiment 1 ml. samples of P.R.P. were warmed at
37o for three minutes and transferred to the sample compartment of
the aggregation apparatus. With the pen-recorder running, aliquots
(10 -. 50 Ills) of aggregating agent were injected below the surface
of the stirred P.R.P. using a Hamilton microliter syringe. Platelet
aggregation was recorded as the decrease of optical density (o.d.)
with time.
Platelet aggregation initiated by A.D.P. was dependent upon the
concentration of A.D.P. (Fig. 3 (i)). As the concentration of A.D.P.
was increased, reversible aggregation was superceded by biphasic
irreversible aggregation. The platelet response to 5-H.T.
(10 p.mol.m1-1)was similar to the reversible aggregation initiated
by low concentrations of A.D.P. except for one sample of P.R.P. which
exhibited biphasic platelet aggregation.
0-5
0
o.d.
20
- 65-
ADP
Minutes
Figure 3: (i) Adenosine diphosphate (A,D,P,)-induced platelet
aggregation,
The aggregation curves represent the effects of
increasing concentrations of A.D.P. on aggregation. The
concentrations of A,D,P, are shown in n. mol. ml.-1
- 66-
0.4 Adrenaline
Minutes
Figure 3:(ii) Adrenaline-induced platelet aggregation.
The curves represent the effects of increasing final
-1 concentrations (n. mol. ml.) of adrenaline.
- 67 -
0 6 Collagen -
136004..0104*"..**00•446.6**AvosoopioftikAA 0
5
o.d.
Minutes
Figure 3:(iii) Collagen-induced platelet aggregation..
The curves demonstrate the effect of increasing amounts of
collagen suspension (Ills) added to I ml of stirred P.R.P.
- 68 -
Platelet aggregation initiated by adrenaline was always
biphasic, but the rate of aggregation increased with increasing
concentrations of adrenaline (Fig. 3 (ii)). Platelet aggregation
initiated by collagen was irreversible and occurred one to two
minutes after the addition of collagen to stirred P.R.P. As the
concentration of collagen was increased, theie was a shortening
of the lag phase and an increase in both the rate and extent of
aggregation (Fig. 3 (iii)). Platelet aggregation initiated by
thrombin (0.2 unit ml.-1) resembled that produced by exposure of
platelets to collagen except that there was no lag phase and
aggregation was immediate.
Platelet aggregation curves produced in response to all five
aggregating agents were reproduceable providing that they were
performed between one and two hours after venepuncture. Platelet
aggregation tended to be increased with shorter lag phases in samples
tested more.than two hours after blood collection. Some samples
of P.R.P. left at ambient temperature for more than two hours
exhibited spontaneous aggregation when stirred.
Quantitation of platelet aggregation
More light passes through a suspension of aggregated platelets
than through single dispersed platelets, and it has been shown that
the rate of decrease of single platelets when forming aggregates is
proportional to the change in optical density (O'Brien, 1962).
Measurements of optical density changes from aggregation curves can
therefore be used to quantitate aggregation. O'Brien et O. (1966)
have described methods for quantitating platelet aggregation initiated
by four different aggregating agents (A.D.P., 5-H.T., adrenaline and
collagen). Simil'ar methods have been adopted in order to quantitate
the effects of phospholipids on platelet aggregation initiated by
similar aggregating agents including thrombin. However, O'Brien
did not make measurements of the secondary phase of aggregation
induced by adrenaline or A.D.P., although he did quantitate the
rate of irreversible aggregation initiated by collagen. Therefore,
an analogous method for estimating the rate of secondary (irreversible)
aggregation initiated by adrenaline or A.D.P. has been included.
Figure 4 shows typical aggregation curves produced by the different
aggregating agents and the parameters measured for each type of
response.
Reversible aggregation
The typical response of stirred platelets to 5-H.T. or low
concentrations of A.D.P. was to reversibly aggregate. In such cases
the crude optical density change (A) representing the extent of
aggregation was recorded and no estimations were made for either
the rate of aggregation or of disaggregation.
Biphasic platelet aggregation
Stirred P.R.P. exposed to adrenaline or certain concentrations
of A.D.P. produced ,a characteristic biphasic aggregation response.
The crude optical density change (Al) representing the extent of the
first phase of aggregation was recorded. The rate of aggregation
(o.d. min.-1
) was recorded for the second (irreversible) phase of
aggregation by measuring the slope of the steepest part of the
tracing (B).
Irreversible aggregation,
The optical density of stirred P.R.P. exposed to collagen did not
begin to increase until after a delay of one or two minutes. This
time interval (C) was recorded and has been termed the collagen reaction
time or lag phase. There was no lag phase before thrombin-induced
platelet aggregation. The rate of collagen- or thrombin-induced
I
- 70 -
Reversible A.D.F? 5-HT)
Bi phasic (ADP 8c
adrenaline.)
I rreversibte ( collagen &
thrombin.)
• PRP alone.
Figure 4: Schematic aggregation recordings illustrating the
methods for quantitation of platelet aggregation.
-71 -
aggregation (o.d. min.-1) was measured by estimating the slope of
the steepest part of the aggregation tracing (B1). If necessary,
the extent of aggregation initiated by collagen or adrenaline was
quantitated by measuring the crude optical density change four
minutes after the addition of aggregating agent.
Amplitude of oscillations in transmitted light.
The recording of light transmitted through a sample of stirred
P.B.P. showed characteristic oscillations in intensity. The
amplitude of these oscillations has been attributed to the degree
of platelet assymetry (disc-shape) (O'Brien, 1965), In some
experiments the amplitude (D) of the oscillations was recorded and
compared with the amplitude (D1) after the addition of a phospho-
lipid preparation to the stirred P.R.P.
Inhibition of platelet aggregation.
Inhibition of platelet aggregation was calculated by comparing
the rate of aggregation in the presence of inhibitor with the rate
of aggregation of a control sample of P.R.P. Such control samples,
in which saline was substituted for inhibitor, were included in
every experiment both at the beginning and end of each run of samples.
This enabled the inhibitory effect of a reagent to be expressed as
percentage inhibition and typical dose-response curves could be
constructed. A set of dose-response curves relating the inhibitory
effect of an experimental drug (SudoxicaP), Gillett et al, 1973) on
the rate of secondary or irreversible platelet aggregation induced
by A.D.P., adrenaline and collagen is shown in figure 5, and
demonstrates the validity of comparing aggregation rates as a means
of estimating inhibitory activity. .
- 72 -
100-
0
50-
0 05 5 50 500
[Inhibitor] nmol mr.1 Figure 5: Dose response curves relating the inhibitory effect of an
experimental drug (Sudoxicam (R))on the rate of secondary or
irreversible platelet aggregation.
Mean percentage inhibition 4 S.D. is shown for aggregation
initiated by A.JT.P. (1 n mol ml.-1) (81----m) (6 studies), adrenaline
(2.5 n mol ml.-1) (0---o) (5 studies) and collagen (10 pis ml.-1)
(0---40 (6 studies).
- 73 -
Preparation of phospholipid suspensions
The following phospholipid preparations were obtained from
Koch Light Chemicals:
3-sn-phosphatidylethanolamine (P.E., bacterial origin)
lysophosphatidylethanolamine (L.P.E., from egg) .
3-sn-phosphatidylcholine (P.C., from egg)
lysophosphatidylcholine (L.P.C., from egg)
3-sn-phosphatidylserine (P.S., from bovine brain)
sphingomyelin (from bovine brain)
3-sn-phosphatidylinositol (from bovine brain)
Synthetic L-dioleoyl-P.E. and L-dipalmitoyl-P.E. were obtained
from Dr. Billimoria, Westminster Hospital School of Medicine, London,
The purity of each compound was checked by thin-layer
chromatography using chloroform:acetone:methanol:acetic acid:water
(50:20:10:10:5 v/s) to develop the chromatograms. All compounds
exhibited a single band on the chromatogram when it was exposed to
iodine vapour except sphingomyelin which showed its characteristic
double band.
Lipid suspensions were prepared daily by adding a pre-weighed
amount of each phospholipid to either 0.9 percent saline or to 5 per-
cent human albumin (Lister Institute) dissolved in 0.9 percent
saline to give a phospholipid concentration of 20 p,mol.ml.-1
Except for L.P.C. which was readily soluble all other phospholipids
required ultrasonication in order to produce an homogenous suspenSbn.
The phosphorus content of each preparation was determined by the
method of Bartlett (1959) in order to confirm the molar concentration
of each compound.
- 74 -
A saturated fraction and a polyunsaturated fraction of L.P.C.
were obtained from Dr. H. Genthe, Natterman International, Cologne.
These fractions were dissolved in 0.9 percent saline before use,
and had a fatty acid composition as shown in Table 2.
Table 2:
of saturated and Fatty acid composition
polyunsaturated fractions of lysolecithin
Lysolecithin fraction Percentage composition of fatty acids
16:0a
18:0 18:1 18:2 --18 :3
Saturated
Polyunsaturated
23.70 76.30 0 0 0
7.76 2.68 9,27 72.72 7.57
a. Fatty acids have been abbreviated in the usual way, e.g.
16:0 carbon chain of 16 atoms with no double bands.
Glycerophosphorylcholine (G.P.C.) (cadmium chloride complex,
Sigma) was included with the phospholipids because of its close
structural relationship to L.P.C. For use it was dissolved in
0.9 percent saline at a final concentration of 20 r.mol.ml.-1
and passed through a mixed-bed ion exchange column to remove the
cadmium chloride protecting group.
The effects of some surface-active compounds on platelet
aggregation have been studied and the results have been included
in Appendix 1. Details of the surface-active compounds studied will
be given here since they were used in a similar way to the phospho-
lipid preparations already described. Digitonin (Fisons) was
dissolved daily, in 0.9 percent saline to give solutions of 2 p.mol.
-1 -1 and 20 u,mol.ml. ml.
final concentration. Saponin (B.D.H.)
- 75 -
was dissolved in 0,9 percent saline at a final concentration of
40 mg ml.-1 Sodium deoxycholate (Sigma) was dissolved in 0.9 percent
saline to give a final concentration of 40 p mol. m1.1 Cetyl-
pyridinium chloride (CPC) (Koch-Light) and cetyltetrammonium bromide
(CTAB) (Koch-Light) were dissolved in 0.9 percent saline at fina3.
concentrations of 10 F mol ml. -1
(B) Platelet adhesiveness method
--
The most widely studied aspect of platelet behaviour derives
from their property of adhesion to glass, usually expressed as
"platelet stickiness". This was originally measured by counting
residual platelets in blood after it had been swirled in a glass flask
(Wright, 1941), A later method was devised by Hellem (1960) in
which citrated blood was passed through a plastic tubing containing
packed glass beads and the platelet adhesiveness was calculated from
the fall in platelet count of the blood which had traversed the unit,
Recently the Wright and Hellem methods have been freshly
evaluated (Besterman et al, 1971) and it was shown the wide range of
results and poor reproduceability of the methods, limited their use
in clinical investigation. A modification of the Hellem method was
proposed by Besterman and termed the "initial stickiness method".
This method has been shown to provide good definition between various.
groups of patients with ischaemic heart disease.
The "initial stickiness method" was used to study the effect of
phospholipids (L.P.C. and P.C.) on platelet adhesiveness. The glass
bead units consisted of 3.75 grams of acid-washed ballotini beads
(Jencons No 8) contained in 13 cm. lengths of ESCO polythene tubing
(intemal diameter 5 mm.). The ballotini was held in position
between layers of nylon mesh which were kept in position by
two short lengths of narrow tubing inserted into the unit.
- 76 -
Blood (9 vols) was anticoagulated with one vol. of 3.2 percent sodium
citrate and tested fifteen minutes after venepuncture. About 3 ml.
of blood was taken up into narrow plastic tubing and the ends of the
tubing clamped. One end was connected to a mechanically driven
syringe containing liquid paraffin and the other end to the glass
bead unit. The clamps were removed and the blood pushed through the
unit. The syringe was driven at such a speed that the transit time,
through the unit, was 23 seconds and the first five drops of blood
emerging from the unit were collected on a piece of wax sealing
film (Parafilm). From this pooled blood the final platelet counts
were taken, and compared with the initial blood platelet count.
Initial and final blood samples were diluted with 5 percent
procaine solution and platelets were enumerated according to the
•method of Brecher and Cronkite (1950) using a phase contrast
microscope (Leitz). Single blood counts were taken from each of
two aliquots of diluted blood and the mean result was taken.
The "platelet stickiness" was calculated by expressing the
difference between the initial and final counts (the number of
platelets removed) as a percentage of the initial platelet count.
-77 -
Section 3,
ANALYTICAL TECHNIQUES
(1) Lipid Separation techniques
Introduction.
Techniques for the separation of both polar and neutral lipids
by adsorption chromatography date from the work of Trappe (1940) and
Borgstrom (1952) on the increasing adsorption of a series of lipids
(sterol esters, triglycerides, non-esterified fatty acids and phospho-
lipids) to various activated adsorbants. Alumina (Hanahan et al,
1951), magnesium silicate (Rice and Osler, 1951) and silicic acid-
impregnated paper (Lea et al, 1955) have all been used with various
mobile phases in attempts to obtain reproduceable fractionation of
biological phospholipids. Several of these methods were compared by
Lea et al and these authors found that silica gave better separation
and higher recoveries of P.C. and L.P.C. than other adsorbants.
With the advent of thin layer chromatography (T.L.C.) using so
called "open columns", several authors published methods for
separating phosphoglycerides (Wagner et al, 1961 : Vogel at al,•1962
and others). These early attempts utilized Silica Gel G (Merck and Co.)
containing a CaSO4 binder and although good separations of P.E., P.C.,
sphingomyelin and L.P.C. were obtained, minor phospholipids such as
phosphatidylserine phosphatidylinositol (P.I.) and phosphatidic
acid (P.A.) co-chromatographed with other phospholipids in the neutral
or acidic solvents used. The introduction of a commercially available
binder-free silicic acid (Silica Gel H, Merck and Co.) gave a good
separation of P.E., P.S., P.I., P.C., sphingomyelin and L.P.C.
(Skipski et al, 1963). However, no one-dimensional T.L.C. method
could separate all classes of phospholipids adequately and two-
- 78 -
dimensional methods have been introduced (Skidmore and Enteman, 1962
and others). Slice these early attempts to adapt T.L.C. methods to
lipid separation and analysis, there have been many additional one-
dimensional and two-dimensional methods described both for
phospholipids and neutral lipids (Freeman and West, 1965 : Skipski
et al, 1965) using Silica Gel G or H.
The main object of this thesis has been the investigation of
L.P.C. in human plasma, its formation in incubated plasma and its
quantitation in healthy subjects and in patients with ischaemic
heart disease. A method was required which would separate effectively
and reproduceably, the main classes of plasma phospholipids and
which would enable relatively large numbers of plasma specimens to be
analysed. This could best be achieved by adoption of a one-dimensional
T.L.C. method which would separate P.E., P.C., sphingomyelin and
L.P.C. Although several one-dimensional T.L.C. methods have been
described which would also separate Mhor phospholipid components of
plasma such as P.I., P.A., and P.S., (Skipski et al, 1963 : Kunz
et al, 1970) it was decided to ignore the minor phospholipids of
plasma which are present in trace amounts which would make
quantitation difficult. However, P.S. and P.I. are major components
of all membrane phospholipids and since it was of interest to
briefly investigate erythrocyte and platelet phospholipids in
healthy individuals and in patients with ischaemic heart disease,
a method for their separation had also to be included in this study.
Since the number of studies on erythrocyte and platelet
phospholipids was much smaller than the number of studies on plasma
phospholipids, two different separation methods have been used. A
two-dimensional T.L.C. separation of phospholipids from erythrocytes
and platelets was used which was based on the work of Rouser et al (1966).
- 79 -
For convenience, the one-dimensional T.L.C. method for separation
of plasma phospholipids was adapted to utilize one of the solvent
mixtures also required for the two-dimensional method.
(a) Separation of plasma phospholipids by one-dimensional thin-
layer chromatography.
Extraction of plasma phospholipids.
The majority of lipids in tissues are in intimate association
with non-lipids that help stabilise water-lipid interactions, Before
the lipid fraction can be solubilised in organic solvents, the non-
lipid components (proteins, inorganic salts, polysaccharides etc.)
must be removed. There are many regimes developed for extraction
of lipid from lipoprotein complexes. They generally rely on a
denaturant, e.g. methanol, ethanol or acetone to destroy the
tertiary structure of the proteide, with a lipid solvent such as
chloroform to solubilise the released lipids.
In this study, the system used was a modification of that of
Foich et al (1957), using a mixture of chloroform (Fisons Ltd.,
analytical grade) and methanol (Fisons Ltd., analytical grade)
(2:1 v/v). 0.5 ml. samples of plasma or serum were pipetted into
20 mls. of chloroform-methanol mixture contained in a glass
centrifuge tube fitted with a ground glass stopper. The mixture
was agitated violently for several minutes and allowed to stand
for a further fifteen minutes at 4o A 10 ml, aliquot of 0.05 M
KCL solution was added to the extraction mixture which was shaken
and allowed to stand for a further thirty minutes at 4°. The
tubes were centrifuged, after counter balancing, for ten minutes at
1800 r.p.m. (45,000 g-min) in a laboratory centrifuge at 10°C. A
two phase system formed with a film 'of denatured protein at the inter-
face. The tubes were allowed to come to room temperature and the
phases were examined. If the bottom (chloroform) phases appeared
cloudy the tubes were recentrifuged.
- 80-
The upper (aqueous-nethanolic) phase and denatured protein
were removed from the chloroform extract of lipids (lower phase)
by aspiration. Using this system the lipids from 0.5 ml. of plasma
or serum were extracted into a final volume of 14 mis of the
chloroform phase. Aliquots of this extract were used for
determination of total phospholipid and total cholesterol and
for separation of individual phospholipids by one-dimensional T.L.C.
Preparation of thin layers of Silica Gel H.
20 x 20 glass plates (Shandon) were soaked in 10 percent (wt/v)
sodium hydroxide solution for 12 hours. The plates were removed,
rinsed thoroughly in tap water, INHCL, tap water (twice), deionised
water (twice) and driri in an oven at 100°C. Plates were prepared
in batches of 5 on the day before use. When cool 5 plates were
laid with end plates, onto a bed (Desaga) and wiped once with -
diethylether using clean cellulose tissues.
50g of silica Gel H (Merck) was weighed into an acid-washed,
250 ml ground glass stoppered conical flask. 100 ml. of deionised
water was added and the flask shaken for several minutes.
The slurry was quickly transferred to the trough of a 20 cm
applicator (Desaga) and the plates were spread as usual. 0.25 mm.
layers were prepared routinely. The plates were transferred to a
horizontal rack and_left,to dry in air. When required, plates were
transferred to an oven and activated at 110oC for 30 - 60 minutes.
Plates were removed from the oven, cooled on a glass tile and used
immediately,
Using a bridging template parallel grooves were cut every 2 cms
across the plate in the direction of development to divide it into
ten lanes.
- 81 -
Sample application
5 mis of lipid extract were transferred to an acid-cleaned
25 ml pear-shaped flask (Quickfit). The extract was evaporated
to dryness in vacuo at 25-300C on a rotary evaporator and care
was taken not to allow the extract to boil during the procedure.
The vacuum was released by flushing the flask with oxygen-free
nitrogen, after which the extract was visible as a yellowish
droplet in the apex of the flask. 25 xls of chloroform were added
to the flask using a Hamilton microliter syringe, and the re-
dissolved extract was applied to the silica gel thin layer as a
2 cm streak, 2 ems from the lower edge of the plate using a second
Hamilton syringe. The rotary evaporation flask was washed three
times with 25 pls of chloroform and the washings were added to the
material already applied to the plate.
In this way up to eight samples could be applied to a single
plate leaving a blank lane at each edge.
Solvents
Chloroform (Fisons, analytical grade)
50 vols.
Acetone (Fisons, analytical grade)
20 vols.
Methanol (Fisons, analytical grade)
10 vols.
Glacial acetic acid (Fisons, analytical grade)1) vols.
Deionised or glass redistilled water 5 vols.
The solvent mixture was made up immediately before use in 100 ml
volumes, In practise the organic solvents were measured out
separately and mixed in a clean measuring cylinder. 4 mls of water
were added to the mixture and the remaining water was added dropwise
until the solvent mixture just failed to resolve into single phase,
At this point one drop of glacial acetic acid was added and a single
phase mixture obtained, This procedure ensured that the solvent
sy-stem'remained in phase equilibrium under different atmospheric
conditions.
- 82 -
Development
Rectangular glass tanks were lined with Whatman No. 1 paper
some 4 hours before chromatography and were wetted with the solvent
mixture. One hour before chromatography the solvents were rejected
and 100 mls of fresh solvent was added to give a liquid depth of
about 1 cm. Tanks were equilabrated at room temperature.
Plates were chromatographed by ascending chromatography
immediately after sample application. The solvent was allowed to
ascend to within 2 or 3 ems of the top of the plate. The plate was
removed from the tank and immediately dried in a current of warm air.
For routine visualization of the lipids the plates were briefly
exposed to iodine vapour in a closed chamber as described below,
Detection and Identification of Lipids
A general lipid detection test and specific tests for amino-
nitrogen and phosphorous were used to detect separated plasma
phospholipids. The identification of resolved phospholipids was
made by comparison with standard phospholipids co-chromatographed
on the same thin layer plate.
1. General test for lipids
Iodine crystals (B.D.H. resublimed) were placed in a
chromatography tank. When rapid or intense iodination was required
for e.g. photographic recording of chromatogram, the tank was placed
on a warm surface. After exposure the iodine was allowed to
evaporate overnight at 400.
2. Test for phosphorous.
A modification of the Dittmer and Lester (1964) molybdic acid
reaget was used,
Solution I: 40.1 g Mo02, was boiled in 1 litre of 25N H2SO4
(reagent grade),
- 83 -
Solution II: 500 ml of solution I was boiled gently while 1.78 g
of powdered molybenum was added. The solution was cooled after
15 minutes and decanted.
Working solution: Equal volumes of solution I and solution II were
mixed and then diluted with 2 volumes of water.
This working reagent was applizi to the dry T.L.C. plate as a
fine spray. Phospholipids produced a blue colouration after
several minutes. The colour could be intensified and background
quenched by exposing the T.L.C. plate briefly to a jet of steam.
3. Test for amino-nitrogen
0.3 g Ninhydrin (B.D.H.) was dissolved in 95 ml. acetone, 5 ml.
of redistilled collidine added. The spray was stable for about one
month when kept at 4°C.
This spray produced a red-purple colouration to the amino-
nitrogen of P.E., L.P.E., and P.S. in the cold within ten minutes of
spraying. Greater sensitivity could be obtained by either heating
at 90°C for 10 minutes or by steaming the chromatogram.
Detection procedure
In the initial studies of separation of phospholipids from plasma'
extracts, standard phospholipid preparations (P.E., P.C., and L.P.C. -
Koch Light and sphingomyelin - Sigma) were chromatographed in
adjacent 2 cm lanes to the extracted phospholipids. These plates
were run in triplicate. One plate was exposed to iodine vapour
and the positions of the phospholipid bands and standards were marked.
The other two plates were sprayed with the phosphorous reagent and
ninhydrin respectively. A comparison of the Rf values and staining
properties of the dandards enabled the separated phospholipids to be
identified as L.P.C., sphingomyelin, P.C., and P.E. (in order of
increasing Rf value). No other phosphorous - or ninhydrin-positive
- 83-
Solution II: 500 ml of solution I was boiled gently while 1.78 g
of powdered Molybenum was added, The solution was cooled alter
15 minutes and decanted.
Working solution: Equal volumes of solution I and solution II were
mixed and then diluted with 2 volumes of water.
This working reagent was applid to the dry T.L.C. plate as a
fine spray. Phospholipids produced a blue colouration after
several minutes. The colour could be intensified and background
quenched by exposing the T.L.C. plate briefly to a jet of steam.
3. Test for amino-nitrogen •
0.3 g Ninhydrin (B.D.H.) was dissolved in 95 ml. acetone, 5 ml.
of redistilled collidine added. The spray was stable for about one
month when kept at 4°C.
This spray produced a red-purple colouration to the amino-
nitrogen of P.E., L.P.E., and P.S. in the cold within ten minutes of
spraying. Greater sensitivity could be obtained by either heating
at 90oC for 10 minutes or by steaming the chromatogram.
Detection procedure
In the initial studies of separation of phospholipids from plasma:
extracts, standard phospholipid preparations (P.E., P.C., and L.P.C. -
Koch Light and sphingomyelin - Sigma) were chromatographed in
adjacent 2 cm lanes to the extracted phospholipids. These plates
were run in triplicate. One plate was exposed to iodine vapour
and the positions of the phospholipid bands and standards were marked,
The other two plates were sprayed with the phosphorous reagent and
ninhydrin respectively. A comparison of the Rf values and staining
properties of the standards enabled the separated phospholipids to be
idedtified as L.P.C., sphingomyelin, P.C., and P.E. (in order of
increasing Rf value). No other phosphorous - or ninhydrin-positive-
- 84 -
spots were visible on the plates although there was a strong iodine-
staining band on the solvent front (neutral lipids) and occasionally
the origin was lightly stained with iodine vapour.
Routinely developed T.L.C. plates were exposed to iodine vapour
for a few minutes until the P.E., P.C., sphingomyelin and L.P.C.
bands were visualized. The plate was then removed and lines were
scored across the plate to separate all bands of the same phospho-
lipid into equal sized areas of silica gel, The blank lanes were
divided into similar areas,
Cutting
The waste areas below and above the resolved phospholipid
bands were removed from the plate by scraping with a piece of
scrubbed cellulose film-base, and the exposed glass was careiily
cleaned with a solvent soaked pad.
A number of glazed paper squares cut from weighing paper
(Gallenkamp) were taken. Using a clean piece of film-base, each
band was carefully scraped onto a clean paper using passes of the
scraper over the plate. The silica gel removed was then set aside
for chemical quantitation.
Results
Figure 6 shows a typical T.L.C. plate on which plasma phospho-
lipids have been separated for analysis.
Analyses of phosphorous distribution on replicate thin layer
chromatograms of plasma phospholipids have been given with details
of the method for estimating phosphorous below.
(b) Separation of erythrocyte and platelet phospholipids by two-
dimensional thin-layer chromatography.
The method used has been described by Rouser et al (1966).
Solvent front neutral lipids
Phosphatidyl-ethanolamine (PE)
Lecithin (PC)
___Sphingomyelin
Lysiecithin (LPC)
Orig in
•■•■•••■•■
- 83 -
Figure 6: Separation of plasma phospholipids by one-dimensional thin
layer chromatography.
The chromatogram shows phospholipids separated from four plasma
samples before and after six hours incubation at 370.
The plate was developed by ascending chromatography with
chloroform, acetone, acetic acid, methanol, water (50:20:10:10:5, by
vol.) and the bands were visualised by exposure to iodine vapour.
- 86 -
Extraction of erythrocyte lipids
Fresh blood was anticoagulated with Lithium EDTA (9.4 mgs
per 10 ml.) and centrifuged at 150 g for 10 minutes. The super-
natant (P.R.P.) was removed to a siliconised centrifuge tube with
siliconised Pasteur pipette. The partially packed erythrocytes
were centrifuged at 1500 g for 10 minutes and the plasma removed
to a clean sample tube to await extraction. The red cells were
washed with 0.9 percent saline twice and collected by centrifugation
at 1500 g for 15 minutes,
0.5 ml of packed erythrocytes were added to 5 ml„ of methanol
in a stoppered centrifuge tube. After 10 minutes, during which the
erythrocytes lysed and their proteins denatured, 10 mis Of chloroform
were added to extract the lipids. The tube was violently agitated and
left for 15 minutes. It was then treated as for the extraction of
plasma lipids.
Extraction of platelet lipids
10 mis of P.R.P. or less if necessary, were centrifuged at 1500g
for 10 minutes. The plasma was removed to be pooled with the plasma
already obtained from the erythrocyte preparation, The platelet
residue was resuspended in 0.9 percent saline containing 1 mg
sodium EDTA per ml, and re-centrifuged to obtain a mass of washed
platelets.
2.5 mis of methanol were added to the platelet button. A few
anti-bumping granules were added, and the tube stoppered and mixed
with a vortex mixer. After 10 minutes 5 mis of chloroform were
added to extract the lipids. The tube was left for 15 minutes and
then treated as for tile extraction of plasma lipids.
- 87 -
Preparation of T.L.C. plates
20 x 20 layers of Silica Gel H (0.25 mm) were prepared and
activated as described above with the exception that no grooves
were marked on the surface of the plate.
Sample application
2.5 ml aliquots of lipid extract were taken to dryness on a
rotary evaporator. The vacuum was released by flushing with
oxygen-free nitrogen, and the extracts were redissolved in 10 -
15 pis of chloroform. The redissolved extract was applied to the
chromatogram as a single spot in the bottom left hand corner of
the T.L.C. plate at a position which was 2 cm from either edge.
The evaporation flask was washed twice with 10 - 15 pls of
chloroform and the washings were also gplied to the chromatogram.
Solvents
(1) Chloroform (Analytical grade) 65 vols
Methanol (Analytical grade) 35 vols
28 percent aqueous ammonia 5 vols
(2) Chloroform 50 vols
Acetone (Analytical grade) 20 vols
Methanol 10 vols
Glacial acetic acid (Analytical grade) 10 vols
Deionised water 5 vols
The solvent mixtures were made up immediately before use
in 100 ml. volumes. .
Development
Separate rectangular glass chromatography tanks for each
solvent mixture were lined with Whatman No. 1 paper some 2-4 hours
before chromatography and were wetted with the solvent mixtures.
- 88 -
One hour before chromatography the solvents were discarded and
100 ifs of fresh solvent was added to give a depth of about 1 am.
Tanks were equilabrated, at room temperature in a fume cupboard.
Each plate was first chromatographed by ascending chromatog-
raphy in solvent 1 immediately after sample application. The solvent
was allowed to ascend to within 1 cm of the top edge of the plate.
The plate was removed from the tank and immediately dried in a
stream of nitrogen in the fume cupboard. The plate was then turned
:through 900 in an anti-clockwise direction and re-chromatographed
in solvent 2 to within 1 cm of the top edge of the plate. The plate
was dried in a current of warm air. For routine visualisation of
the separated lipids the plates were briefly exposed to iodine
vapour in a closed chamber.
Detection and identification of phospholipds
The phospholipids were detected by spraying with phosphorous
reagent or by exposure to iodine. Standard phospholipid preparations
(P.I., P.S., P.C., L.P.C., P.E., Koch Light and sphingomyelin -
Sigma) were run on separate chromatograms. The phospholipid spots
separated from platelet or erythrocyte extracts were identified by
comparison with the chromatograms of the standard preparations. The
identity of the P.E. and P.S. spots was confirmed by spraying with
ninhydrin.
Routinely developed T.L.C. plates were exposed to iodine vapour
for a few minutes until the P.E., P.S., P.C., P.I., Sphingomyelin and
L.P.C. spots were visible. The positions of these spots were marked
by scoring the surface of silica around them. The iodine was then
allowed to evaporate overnight.
- 39 -
Cutting
Areas of silica gel corresponding to phospholipid spots and
a similar number of blank areas were removed by scraping and
- carefully transferring to squares of glazed paper to await
chemical analysis.
Results
Figure 7 shows a typical eeparation of erythrocyte phospho-
lipids. Neutral lipids (triglycerides, free and esterified
cholesterol and non esterified fatty acids) and P.E., P.C., P.S.,
P.I., Sphingomyelin (S) and L.P.C. were readily identifiable.- In
addition two further spots were sometimes visible. These spots gave
weak responses to both iodine and phosphorous reagent. One of
these spots probably represented phosphatidic acid (P.A.) and the
other may have been diphosphatidyl glycerol. These spots were not
included in the analysis,
(c) Separation of free cholesterol and cholesteryl esters by
thin layer chromatography.
The separation of free cholesterol and cholesteryl ester used
in this study was based on the method of Skipski.et al (1965) for
the fractionation of neutral lipids by T.L.C.
Solvents
Petroleum ether (40-600) (Fisons, analytical grade) 90 volumes
Diethyl ether (Fisons, analytical grade) 10 volumes
Glacial acetic acid (Fisons, analytical grade) 1 volume
The solvent mixture (101 mls) was prepared freshly immediately
before each chromatography session.
17eutrat PE
fib ."
• • • a*
PC
P1- (z2z)
S ezz'
LPC22)
0
■•■ /
?PA. • ,.
• ,
PS
- 90 -
1
Figure 7: Separation of erythrocyte phospholipids by two-dimensional
thin layer chromatography.
The lipid extract was applied on a single spot in the bottom
left hand corner of the plate (0) and the chromatogram was
developed with solvent 1 (chloroform:methanol:28% ammonia - 65:35:5)
and subsequently with solvent 2 (chloroform:acetone:methanol:acetic
acid:water - 50:20:10;10:5) in the directions indicated.
- 91 -
Plate preparation
20 x 20 cm plates were layered with an 0.25 nun layer of
Silica Gel H and activated as described above. After activation
the plate was scored with a series of parallel lines in the
direction of development in order to mark out a series of 2 cm
lanes for sample application.
Method
Rectangular chroMatography tanks were lined with Whatman No. 1
paper and wetted with the solvent mixture about one hour before
chromatography. Immediately before chromatography the solvent in
the tank was replacedstth a fresh aliquot to give a solvent depth
of 1 cm. 4 ml aliquots of plasma lipid extracts were evaporated to
dryness in vacuo and redissolved in 10-15 Ills of chloroform. The
samples and washings from the evaporator flask were applied using a
Hamilton microliter sypIringe to give a 2-cm. band 2 ems above the
lower edge of the plate.
The plate was developed by ascending chromatography until the
solvent front was some 2 cms from the upper edge of the silica layer.
The plate was dried in a current of warm air and the lipid bands
located after a brief exposure to iodine vapour.
Results
Lipid bands were identified by comparison of their Rf value with
those of standard lipids co-chromatographed on adjacent lanes of the
plate. The following standard lipids were chromatographed: free
cholesterol and cholesteryl palmitate (Sigma), triolein and palmitic
acid (B.D.H.) and egg lecithin (Koch Light).
'figure 8 shows, a typical separation of plasma lipids by T.L.C.
and their identification by comparison with the Rf values of
standard lipids.
- 92 -
3- 6
Solvent front
110 • Cholesteryl esters
• - Triglycerides
▪ Non-esterified fatty acids
▪ Free cholesterol Phospholipid/origin MOP 11111/111
Figure 8: Separation of lipid classes by one-dimensional thin layer
chromatography.
The lipid standards were Cholesteryl palmitate (1), triolein (2),
palmitic acid (3), cholesterol (4) and lecithin (5). Lane 6 shows the
separation of lipid classes extracted from plasma.
The plate was developed by ascending chromatography with petroleum
ether, diethyl ether, acetic acid (90:10:1, by vol.) and the bands
visualised by exposure to iodine vapour.
- 93 -
Cutting
Routinely developed chromatograms were very lightly exposed
to iodine vapour and the positions of cholesteryl ester and free
cholesterol were marked. These areas of silica gel were removed
by scraping in the usual way. Duplicate chromatograms for each
sample were run and the silica gel set aside for chemical or
radio-isotopic assay.
2. Quantitation of Phospholipid Mass and Derivation of Plasma
Phospholipid Concentrations
Introduction
Phospholipid mass may be estimated by dye-binding techniques
(Harris and Gambol, 1963) or by estimation of the residual inorganic
phosphate (Pi) remaining after wet oxidation of the phospholipid
sample. The latter technique is the method in general use and
enables phospholipid mass to be expressed as pg Lipid-P (pgP), This
unit is independant of the molecular weight of a given phospholipid
species, a function which may not be.known if the sample under assay
is a mixture of several molecular species.
Phosphate quantitation involves the production of a soluble
phosphomolybdate complex from the residual Pi and the subsequent
reduction of this complex to colloidal molybdenum blue, The blue
colour is assayed against standard Pi samples using a spectro-
photometer. Reduction under different conditions will produce
different intensities of colouration from a common phosphate
standard, A number of methods of varying sensitivity have been
developed using for example, 2,4-diamino phenol (Allen 1940), 1-
amino-2-naphol-2-sulphonic acid (Fiske and Subbarow, 1925), ascorbic
acid (Lowry et al, 1954) and stannous chloride (Berenblum and Chain,
(1939) as reducing agents.
- 94 -
In this study, a method was required to estimate total plasma
phospholipid concentrations and also the relative concentrations of
individual phospholipids after their separation by T.L.C. These
samples contained 0.5 - 10 pgP. A modification of the method of
Bartlett (1959) using the Fiske and Subbarow reducing agent has
been used, in which sulphuric acid digestion has replaced the
perchloric acid oxidation of the phospholipid sample. The advantage
of this method was that the modified digestion procedure appreciably
shortened the length of time required for the assay.
(a) Determination of phospholipid mass and derivation of
total plasma phospholipid concentration.
Reagents
1. Pi standard: 0.129 m mol potassium dihydrogen ortho-
phosphate containing 4 pg Pi ml.-1
2. Sulphuric acid (Fisons analytical reagent).
3. Hydrogen peroxide (100 volumes) (Fisons analytical reagent).
4. Sodium molybdate
4.3g Na2
Mo04. 2H20 in 1000 mis deionised water,
5. Fiske and Subbarow Reducing Reagent.
7.5g sodium metabisulphite, 0.5 g sodium sulphite, and
0.125g 1-amino-2-napthol-4-sulphonic acid (Eastman Kodak)
were finely ground together in a mortar and made to 100 ml
with deionised water. This reagent was stored for up to
5 days at 4o in an amber glass bottle.
Method
Duplicate 1 ml. aliquots of plasma lipid. extract were pipetted
into can pyrex tubes fitted with ground glass necks (Quickfit).
The extracts were evaporated to dryness on a 1000 water bath and
after cooling, 0.3 ml of concentrated sulphuric acid was added.
- 95 -
The tubes were heated for 10 minutes on an electric digestion rack,
The tubes were cooled and two drops bf hydrogen peroxide were added
and the tubes reheated on the rack for a further 15 minutes. The
tubes were then allowed to cool before 7 mis of sodium molybdate
solution were added. The tubes were mixed and examined to ensure
that all contents were colourless* Any tubes showing a yellow
colouration indicated that not all of the peroxide had been destroyed,
If this did happen, then the assay for affected samples had to be
repeated with fresh lipid extract. Finally 0.4 ml of reducing
reagent was added and the tubes mixed and loosely stoppered, The
colour was developed by immersing the tubes in boiling water for 10
minutes. The blue colouration which resulted was stable for 24 hours,
but in practise it was read immediately after the tubes had cooled
to ambient temperature. The tubes were read against a water blank
at 830nm in 1-cm, glass curettes in a Unicam SPG00 spectrophotometer,
Reagent blanks and a standard Pi sample were included in each assay.
Results
The calibration curve given in figure 9 shows that Beer's. Law
is obeyed up to a concentration of 10 pgP.
The total phospholipid concentration of the plasma extract could
be calculated in igP from the calibration curve or from the optical
density developed from a standard amount of P. In this study the
total plasma phospholipid has been expressed in pmolml.-1 since the
plasma phospholipids are almost exclusively molecular species
containing one phosphorous atom per molecule. Thus the micro-molar
concentration of phospholipid can be calculated simply by dividing
the ugP by the atomic weight of phosphorous(31).
- 9.G -
5 10 pg P
Figure 9: Calibration curve for the estimation of phosphorous
mass by a modification of Bartlett's (1958) method,
- 97 -
The slope of the calibration curve (9.2 pg P/O,D, unit) was
used to calculate the concentration of P per ml. of extract,
Since 14 mis of extract were prepared from 0,5 ml of plasma,
the plasma concentration of total phospholipid was riven by the
equation:-
0.D. x 9.2 x 14 -1 Concentration _ p mol ml, 31 x 0.5
(b) Determination of the proportional distribution of
phosphorous in phospholipids separated by thin-layer
chromatography.
Phosphorous was determined directly in sulphuric acid digests
of the silica gel. The direct digestion of phospholipid in the
presence of silica has been criticised by Williams et al (1969)
who stated that the presence of silica during digestion and
colour formation depresses the colour intensity. Other authors,
e.g. Rosenthal and Ching-Hsien Han (1967), have shown that under
their conditions, silica does not quench,
Method
Phospholipid fraction; identified as described elsewhere, were
scraped from /I.L.C. plates and transferred to pyrex digestion tubes.
Blank areas of silica from the T.L.C. plate were taken of equal
area to the phospholipid bands. Heavily loaded phospholipid
fractions (i.e. P.C.) were sometimes sub-divided into two digestion
tubes. Inorganic phosphate standards equivalent to 4 ugP and reagent
blanks all without silica gel were prepared.
0,3 ml of concentrated sulphuric acid was added to all sample
tubes which were then, placed into a sand-bath heated to 200°C and
digested for 20 minutes, The tubes were then cooled and two drops
of hydrogen peroxide added. The tubes were then re-digested for
20 minutes. At the end of this period, the tubes were coaled and
-98-
7 mls of 0.43 percent sodium molybdate were added and the tubes mixed.
Finally 0.4 ml of Fiske and Subbarow reagent were added to each tube
which was mixed and loosely stoppered.
Colour was developed by immersing the tubes in boiling water for
ten minutes. The tubes were cooled by immersion in a cold water bath
and afterwards centrifuged at 2,500 r.p.m. in a bench centrifuge for
ten minutes, Aliquots of the clear supernatants were carefully removed
by decanting and the colour was assayed against a water blank at 830 nm
in a Unicam SP 600 instrument.
Calculation.
From the summated 'P' values for all of the phospholipid bands,
a proportional distribution of the phosphorous on the plate among the
phospholipid classes was derived, The total plasma lipid concentration,
proportioned according to this distribution; gave the plasma concent-
ration of each phospholipid class as p mol ml.-1
Results
1. Preliminary experiments showed that all phosphorous recovered
from the T.L.C. plate was confined to the four phospholipid bands
identified as P.E„ P.C., sphingomyelin and L.P.C.
2. The recovery of P from the T.L,C. plate was calculated from
the summated 'P' values for all phospholipid bands relative to the
concentration of P in the unchromatographed extract, Recovery was
usually between 90-95 percent.
3. Replicate samples of plasma lipid extracts chromatographed
on the same or different T.L.C. plates showed=very close agreement (Table 3).
Conclusion
The plasma concentrations of individual phospholipid classes could
be determined reproduceably by direct digestion and phosphorous analysis
of phospholipid fractions scraped from T.L.C. plates.
- 99 -
Table 3:
Replicate analysis of plasma phospholipid concentrations
on duplicate thin layer chromatograms
Fraction
Percentage of total phospholipid
Plate 1 Plate 2
Solvent front 0 0 0 0 -
P.E. 4.0 4.7 3.7 3,9
P.C. 68.2 68.4 68,9 69.3
Sphingomyelin 19.2 18.9 19.4 18.9
L.P.C. 7,6 8.0 7.8 7.8
Origin 0 0 0 0
3. Determination of Plasma Cholesterol Concentrations and Radio-
Isotopic Assay of Cholesterol Esterification
Intrliduction
The Liebermann-Burchard colour reaction has long been used in
the chemical estimation of cholesterol. This reaction consists of
the development of a blue-green colouration when cholesterol is
mixed with acetic anhydride and sulphuric acid. Of the many
cholesterol methods employing the Liebermann-Burchard reaction,
those of Bloor (1916) and of Schoenheimer and Sperry (1943) have
been the most widely used. However, all of these methods have the
disadvantage that the final colour is unstable,
The more recently described colour reaction of an acetic acid-
ferric chloride reagent with cholesterol (Zlatkis et al, (1953)),
does produce a stable colouration, This method has been used by
Zak et al (1954) in the estimation of both free and total cholesterol.
- 100 -
This method has been modified by Leffler (1960) for use with
isopropanol- or chloroform-extracts of cholesterol. This method
has been used in the present study to determine the plasma
concentration of total cholesterol.
(a) Method for the determination of the concentration of
total cholesterol in plasma,
The concentration of total cholesterol in chloroform extracts
of plasma has been determined by the method of Leffler (1960).
Reagents
1. Isopropanol (Fisons reagent grade - 99 percent),
2. Sulphuric acid (Fisons Analytical grade) (specific gravity 1.84),
3. Zlatkis-Zak Reagent modified according to Leffler,
50 mg of FeC13 6H20 dissolved in /00.m1. of glacial acetic acid.
The reagent was kept in an amber glass bottle at room temperature
and is stable for at least one year.
4. Cholesterol standard,
100 mg. cholesterol (Sigma) dissolved in 100 ml of isopropanol
to give stock solution. 2 mis of the stock solution were
diluted with isopropanol to 25 mis to give the working solution.
One ml. of the working standard contains 0.08 mg, of cholesterol,
Method
Duplicate 1 ml, aliquots of plasma lipid extract were evaporated
to dryness in pyrex tubes. One ml. of isopropanol was added to
redissolve the extract, Two mis. of ferric chloride reagent were
added to each tube, and the contents were mixed. Two mis of sulphuric
acid were added to each tube from a burette and the tubes stoppered and
immediately mixed by Inverting five times. The tubes were allowed to
stand and the colouration developed within minutes. Reagent blanks and
cholesterol standards were similarly treated.
- 101-
Ten minutes after mixing the contents of the last tube, the
colour was read against a water blank at 540 nm in a Unlearn SP 600
spectrophotometer.
Calculation
The cholesterol working standard contained 0.08 mg, of
cholesterol per ml. Using this value and incorporating the
appropriate dilution factors the total cholesterol concentration
was given by
0.D. (unknown) x 0,08 x 14 x 100 mgs per 100 ml, 00. standard 0.5
For comparison with phospholipid concentration the plasma concentration
of total cholesterol was converted to F mol ml.-1 by a division
involving the molecular weight of cholesterol,
Results
The calibration curve for cholesterol masses of up to 0.18 mg
was linear (Fig. 10), The equivalent plasma concentration of total
cholesterol• was 448 mgs per 100 ml for a cholesterol mass of 0.16 mg.
Discussion
The method was rapid and enabled total cholesterol concentrations
of up to 448 mg per 100 mi to be determined without dilution of the
plasma. However, plasma samples with an expected total cholesterol
concentration above 400 mgs per 100 ml, were diluted accordingly with
0.9 per centsaline before extraction.
(b) Radio-isotopic Assay of Chiesterol Esterification
In studying the effects of intravenous administration of heparin
in man, it was necessary to differentiate between increased L.P.C.
formation arising from increased L.C.A.T. activity or from a separate
enzymatic reaction, ":For this reason in a small number of studies,
the activity of L.C.A.T. measured by radio-assay was compared with
L.P.C. formation in incubated samples of pre- and post-heparin
plasma.
- 102 -
010 020 Cholesterol mgs.
Figure 10: Calibration curve for the estimation of cholesterol
mass by the method of Leffler (1960).
- 103 -
The method of Glomset and Wright (1964) for the radio-isotopic
assay of L.C.A.T. was adopted.
Reagents
1. 7-3H-cholesterol - albumin suspension
This was prepared by the method of Porte and Havel (1961). 5 gm.
of fat-free bovine albumin (Cohn fraction V, Miles-Davis) were
dissolved in 100 mls of 0.9 percent saline. This solution was heated
at 60°C for 30 minutes to destroy any transferase activity present,
The solution was allowed to cool and 1 ml. of ethanol containing
0.1 mCi of 7-3H-cholesterol (The Radiochemical Centre, Amersham) was
rapidly injected below the surface of the solution by means of a
tuberculin syringe fitted with a fine hypodermic needle. The solution
was subsequently warmed to 30°C and warm air was blown over the surface
until the odour of ethanol was absent. 2-ml. aliquots of the suspension
were stored at -25°C for up to 3 months.
2. Heat-inactivated plasma substrate
600 mis of three week old human plasma (anticoagulated with standard
acid-citrate-dextrose for transfusion) was centrifuged at 1500 g for 15
minutes to remove any residual cells or fibrin clots. The supernatant .
plasma was heated at 60°C for 30 minutes to destroy L.C.A.T. activity
and recentrifuged. 5-ml. aliquots were stored at -25°C for up to 2 months.
3. Scintillation fluid
All chemicals and solvents used were scintillation grade
reagents obtained from Nuclear Enterprises, Edinburgh, 100 gm.
Napthalene, 7 gm. 2,5-diphenyloxazole and 0.3 gm. of 1,4,-bis-2-
(4-methy1-5-phenoxazoloy1)-benzene were dissolved in dioxane and made
up to a volume of 1 litre. The fluors were dissolved under mild
ultrasonic agitation in an ultrasonic bath. 48 gm. Cab-O-Sil
- 104 -
(Koch Light Ltd.) was dispersed in the scintillation fluid. 200 mis
of distilled water was added and the final scintillation fluid
subjected to further ultrasonic mixing. The fluid was stored at
room temperature in a sealed flask, and was reshaken before use.
Method
The assay procedure was as follows: the incubation was
performed in stoppered erlenmeyer flasks at 37°C in a Grant
shaker. The incubation mixture consisted of 0.1 ml, 3H-cholesterol-
albumin suspension, 0.8 ml of heatal plasma substrate and 0.1 ml, of
fresh test plasma. After 6 hours incubation, 0.5 ml of the
incubation mixture was pipetted into 20 mis of chloroform-methanol
(2/1 v/v), The extraction procedure was identical to that
described elsewhere for the preparation of plasma lipid extracts.
0.5 ml of the contents of a control flask containing 0.1 ml of
0.9 percent saline in place of test plasma was similarly extracted
after 6 hours incubation at 37oC. Under these assay conditions less
than 5 percent of the plasma free cholesterol became esterified during
the 6-hour assay period.
Counting method
Cholesterol and cholesteryl ester fractions adsorbed onto silica
gel after separation by T.L.C. were scraped onto polished paper and
transferred to polythene counting vials. 15 mis of scintillatern were
added and the sealed vial was subjected to two minutes ultrasonication
at the surface of an ultrasonic bath filled with a 2 percent Decon
solution. The vials were then mixed on a vortex-mixer, wiped and
placed in the counting chamber of a Packhard Tri-Carb 3380 instrument,
mattained at 6°C. The vials were equilabrated for 30 minutes before
counting commenced.
- 105 -
The instrument was set to count in the 'red' (tritium) channel.
Duplicate counts were taken for a minimum counting period of 20
minutes and the automatic external standardisation (A.E.S.) ratio
reoorded for each sample. -The second counting cycle followed the
first after one full revolution of the vial conveyor belt, Preset
background counts were automatically substracted during counting,
Differences in coUting geometry due to irregularities of vial
position in the counting chamber were minimised by meaning the
duplicate counts and duplicate A.E.S. ratios. After the completion
of the two cycles of counts, 1 ml. of an internal 3H-standard •
(13,900 distintegrations min,-1) was added to selected vials with
different A.E.S. ratios. These vials were recounted under standard
'conditions and their A.E,S. ratios were automatically recorded. The
counting efficiency of the internal standards was plotted against the
A.E.S. ratio to give a calibration curve. From this curve the
counting efficiency of each sample vial could be determined given the
A.E.S. ratio.
Calculation
Results were expressed as the percentage of free cholesterol
esterified during the standard incubation period. Alternatively,
the number of u mols of cholesterol esterified per ml. assay medium
per hour incubation could be determined from the specific activity
of the free cholesterol from the T.L.C.plate.
DiscusSion
Very little radio-activity was recovered from the triglyceride
or non esterified fatty acid fractions scraped from T.L.C. plates.
Mean recovery of radio-activity in the cholesterol and cholesteryl
ester fractions was 96 percent.
- 106 -
The percentage of radio-activity recovered in the cholesteryl
ester fraction increased linearly with the amount of fresh plasma
added to the incubation mixture for up to 100 ils ml. -1 (fig. 11).
This showed that the method was able to assay differences in the
concentration of L.C.A.T. and the results were in good agreement
with those published by Glomset and Wright in the original method.
- 107 -
50 100 pts fresh plasma
Figure 11: Influence of enzyme concentration on the esterification
of free cholesterol.
The enzyme was added as uls of fresh plasma per 0.9 ml of heat
inactivated plasma - 3H-cholesterol substrate mixture.
-10S-
CHAPTER 3
RESULTS
- 109 -
Section 1
Fonnation of lysolecithjn in incubated human plasma
Introduction
Two reactions have been recognised for the formation of
L.P.C. in plasma. In normal plasma the lecithin : cholesterol
acyl transferase (L.C.A.T.) enzyme is thought to control the
formation of cholesteryl esters and L.P.C. (see review by Glomset,
1968). Following the in vivo heparinisation of blood a second
enzymatic reaction leading to the formation of lyso-phosphoglycerides
in plasma has been described (Vogel and Zieve, 1964 : Zieve and
DOizaki, 1966 : Vogel and Bierman, 1967).
The properties of L.C.A.T. have generally been studied either
on the basis of the change in unesterified cholesterol concentrations
following the incubation of plasma or serum for 24 - 72 hours at 37°,
or else by radio-isotopic assay of cholesterol esterification.
Only in a very few studies of L.C.A.T. have the changes in P.C. and
L.P.C. concentrations been measured (Glomset, 1963 : Vogel and
Bierman, 1967). However, the properties of a plasma "L.P.C.-releasing
enzyme" which was almost certainly identical to L.C.A.T. have been
described by Adlkofer et al(1968). These authors did not chemically
measure the concentrations of L.P.C. during the enzyme reaction
but made use of the inhibitory properties of L.P.C. on erythrocyte
sedimentation to demonstrate semi-quantitatively - the formation of
L.P.C. during the incubation of plasma or serum.
One of the main objects of the present thesis has been the
measurement of individual phospholipid concentrations and the
estimation of L.P.C. formation-in plasma obtained from healthy
individuals and from patients suffering with atherosclerotic diseases.
- 110 -
For this reason a preliminary study was necessary in order to
characterise L.P.C. formation in incubated plasma and to define
the conditions for the routine estimation of the reaction. The
formation of L.P.C. iA plasma has been measured by means of thin
layer chromatography and the results have been compared with the •
known properties of L.C.A.T. and with the results of Adlkofer.
Section 1(a)
Time course of lysolecithin formation in incubated plasma
Experimental details and Results
Samples of serum or plasma (anticoagulated either with lithium
E.D.T.A. or sodium citrate) were prepared from blood taken from
apparently healthy volunteer subjects. The samples were placed in
stoppered conical flasks for incubation at 37° in a water bath fitted
with a mechanical shaking device. Samples of plasma or serum were
extracted into chloroform:methanol before and at various times during
incubation. The total and individual phospholipid concentrations
of pre- and post-incubated samples were determined.
The concentration of L.P.C. increased linearly for the first
six hours of incubation (Fig. 12) and then the reaction slowed
until no further increase in L.P.C. concentration could be detected
after 24 hours incubation. The rate of lysolecithin formation was
not significantly different in serum or in citrated plasma compared
with E.D.T.A. plasma. There was a very close correlation between the
increase in L.P.C. concentrations and the decrease in P.C. levels
during incubation (r = 0.980, for the analysis of 24 incubated
plasma samples). No significant changes in the concentrations of
total phospholipid, P.E., or sphingomyelin were apparent after
incubations of up to 24 hours.
- 112-
0.6
-5 E a
02 I I
0 1 3 4 Hours
Figure 12: Increase in the concentration of lysolecithin during the
incubation of plasma at 370.
The figures in parenthesis indicate the number of determinations
made at each time interval. The mean plasma concentrations of L.P.C.
+ one standard deviation are shown. _
- 113-
Section 1 (b)
Effect of temperature on lysolecithin formation in plasma
Experimental details and Results
The formation of L.P.C. was determined in samples of plasma
incubated for six hours at 4o, 25o, 37
o and 47o The results of
three such experiments are shown in table 4.
Table 4
Lysolecithin formation in plasma incubated at different
temperatures for six hours.
Incubation temperature
(°C)
L.P.C.
1.
-1 -1, formation (p mol.L. .hr. 2
2. 3.
4 0 5 0
25 18 40 25
37 48 62 52
47 8 27 12
Maximal formation of L.P.C. occurred in plasma incubated at
37o
There was no detectable L.P.C. formation in plasma kept at
o . 4 in two out of the three experiments, and the activity was
reduced to 50 percent at 25° and 30 percent at 47°.
- 114 -
Section 1 (c)
Effect of pH on lysolecithin formation in plasma
In two experiments freshly prepared citrated plasma was sib-
divided into six separate samples. Using a pH meter the pH values
of three samples of plasma were adjusted to 5.2, 6.5 and 7.1 by
adding 0.2 N HCL dropwise, The pH of two other aliquots of plasma
were adjusted to 8.4 and 9.0 respectively by the addition of
0.2 N NaOH. Physiological saline was added to the plasma samples
in order to compensate for dilution incurred during pH adjustment.
Finally the pH values of all six plasma samples were checked and
found to be 5.2, 6.5, 7.1, 7.6, 8.4 and 9.0.
Each plasma sample was incubated at 37o for 24 hours and plasma
lipids were extracted before and after incubation. The concentrations
of total and individual phospholipids were determined in the usual
way. The results were expressed as the rate of L.P.C. formation
per litre per hour.
The formation of L.P.C. was maximal at pH values of 7.1 and 7.6
whilst very little L.P.C. formation occurred at pH values of 5.2 and
9.0 (fig. 13).
- 115 -
0 I
5 6 7 8 pH
Figure 13: Formation of lysolecithin at different pH values
during the incubation of plasma at 370.
- 116 -
Section 1 (d)
Inhibition of lysolecithin formation in incubated plasma
Inhibition of lysolecithin formation by urea
Urea was added to plasma to give a final concentration of
3 mol L. -1 One sample of urea-treated plasma was dialysed at 40
for three hours against three changes of 0.1 M HCL - tris buffer
(pH 7.4). A second sample of urea-treated plasma and a control
sample of plasma were stored at 40 for three hours during the
dialysis of the other sample, Subsequently all three samples of
plasma were incubated at 370 for 24 hours and plasma lipids were
extracted before and after incubation. The concentrations of
total and individual phospholipids were determined in the usual way.
The results were expressed as the rate of L.P.C. formation per litre
per hour,
The formation of L.P.C. was completely inhibited in the presence
of 3 M urea. However, dialysis of urea-treated plasma to remove the
urea prior to incubation restored full activity (table 5).
Inhibition of lysolecithin formation by para-hydroxymercuribenzoate
Para-hydroxymercuribenzoate (p-H.M.B.) was added to plasma at
a final concentration of 1 - 2 ?imol ml.-1 The p-H,M.B.-treated
plasma samples together with a control sample of the same plasma were
incubated at 370 for 24 hours. The formation of L.P.C. was
measured for each plasma sample,
The formation of L.P.C. was ompletely inhibited in plasma
exposed to p-H.M.B. at concentrations of 1 - 2 pmol ml. (table 5).
- 117 -
Table 5
Inhibition of lysolecithin formation in plasma exposed
to urea or para-hydroxymercuribenzoate.
(Results of two representative experiments are shown).
Experiment . -1
L.F.C. formation .(p melia-re. )
Control plasma 19
plasma + urea (3 mol L 1) 0
plasma + urea : dialyzed 19
Control plasma 24
plasma + p-H.M.B. (1 p mol ml.-1) 0
-1, plasma + p-H.M.B. (2 p mol ml. ) 0
- 118 -
Section 1 (0)
Lysolecithin formation in incubated serum lipoprotein fractions
Blood samples obtained from healthy volunteers who had fasted
for 12 - 16 hours was allowed to clot in unsiliconised glass tubes. .
After clot retraction had occurred the tubes were centrifuged at
1500 g for 15 minutes to collect the serum. Lipoprotein fractions
were prepared for the serum samples by a procedure similar to that
described by Adlkofer et al (1963). The preparative procedure is
outlined in table 6.
Preparation of very low density lipoproteins
One volume of serum was mixed with 0.1 volume of an Na Br /
Na Cl solution of relative density (d) 1.140 gm. ml.-1 Pre-soaked
nitro-cellulose centrifugation tubes (Beckman) were filled with .
6.5 mis of the serum mixture and closed by means of metal screw
caps fitted with rubber gaskets. The tubes were loaded into the
40.3 rotor of a Beckman model L2 preparative ultracentrifuge. The
rotor was centrifuged at 40,000 r.p.m. for 18 hours at a temperature
of 12 - 140 After the centrifugation the tubes were individually
removed and placed in the Beckman tube slicing apparatus. The tubes
were sliced in such a way as to leave the floating lipoproteins and
1.5 ml of supernatant separate from the rest of the tube. This
fraction was removed by pasteur pipette and stored at 40 until
required. It contained the very low density lipoproteins (d <1.019).
The next 2 mls of solution were removed by Pasteur pipette and
discarded. The remaining infranatant solution from the tubes was
collected in a measuring cylinder.
- 119 -
Table 6
Scheme to demonstrate the preparation of serum lipoprotein fractions
using the Beckman model L2 preparative ultracentrifuge.
1 vol, serum + 0.1 vol. NaBr/NaC1 solution
-1 (relative density, d, = 1.140 gm. ml. ).
6.5 mis per centrifuge tube
Centrifugation at d =
1.019 at 40,000 r.p.m.
at 12 - 14o for 18 hours.
top 1.5 mis = Intermediate 2 mis Infrnatant mixed with
Very low density discarded equal vol, of NaBr/NaC1
lipoproteins solution (d =1.105 -1
-1
(d <1.019 gm.ml. ) Centrifugation at d =
1.063 at 40,000 r.p.m.
at 12 - 14o
for 18 hrs.
top 1.5 mis =
Low density lipo-
proteins
(1.019<d<1.063gm.m1-1
)
Intermediate 2 mis Infranatant mixed with
discarded. equal vol. of NaBr/NaC1
solution (d =1.333gm.m1-1 )
Centrifugation at d = I
1.200 at 40,000 r.p.m.
at 12 - 14o for 24 hrs.
top 1.5 mis = Intermediate zone
High density lipo-
proteins
(1.063<d<1.200 gm.m1-1)
Infranatant (1.5 mis)
= Very high density lipo-
proteins -1 (d ) 1.200 gm.ml. )•
- 120 -
Preparation of low density lipoproteins
An equal volume of an NaBr/NaC1 solution (d = 1.:05 gm.mlo-1)
was added to the pooled infranatant from the previous centrifugation.
The material was placed in nitrocellulose centrifugation tubes and
re-centrifuged at 40,000 r.p.m. for 18 hours at 12 - 140C in the 40.3
rotor of the L2 machine.
After centrifugation the upper 1.5 mis of each tube was sliced
and the concents removed by pipette and stored at 40. This fraction
contained the low density lipoproteins (d 1.019 - 1.063 gm.ml. 1 )
The intermediate zone (2 mis) of each tube was discarded and the
remaining infranatant was collected in a clean measuring cylinder.
Preparation of high density and very high density lipoproteins
An equal volume of an NaBr/NaC1 solution (d = 1.333 gm. ml.-1)
was mixed with the infranatant fraction prepared from the previous
centrifugation. This material was centrifuged at 40,000 r.p.m. for
24 hours at 12 - 140C. After centrifugation the upper 1.5 mis of
solution was removed from each tube by slicing. This fraction
contained the high density lipoproteins (d 1.063 - 1.200 gm. ml. )
and was stored at 40C until required. The intermediate zone of 305 mis
per tube was carefully removed by pipette and discarded. The remaining
infranatant (1.5 mis per tube) contained the very high density lipo-
proteins which had to be dispersed with the aid of a glass rod before
they could be removed.
Each lipoprotein fraction was placed in a dialysis sac made from
Viskirg tubing and dialyzed overnight at 4° against four changes of
0.1 r HCL Tris buffer (pH 7.4).
After dialysis equal aliquots of each lipoprotein fraction were
mixed together to give a pooled fraction and each of six possible
paired combination3of lipoproteins. The lipoprotein mixtures and
individual aliquots of each lipoprotein fraction were diluted with
- 121 -
saline to their original concentrations in serum. Each fraction
was incubated at 37o
for 6 hours and phospholipid concentrations
were determined in each fraction before and after incubation. The
results of a typical experiment are shoWn in table 7 and have been
expressed as the formation of L.P.C. per ml per hour.
Table 7
Lvsolecithin formation in incubated lipoprotein fractions and mixtures
Lipoprotein fractions or mixture , L.P.C.formation (pmol.L.-1 hr.-1
)
Very low density lipoproteins (V.L.D.L.) 0 .
Low density lipoproteins (L.D.L.) 0
High density lipoproteins (H.D.L.) 2
Very high density lipoproteins (V.H.D.L.) 2
Pooled fractions 18
V.L.D.L. + L.D.L. 0
V.L.D.L. 4- H.D.L. 0
V.L.D.L. + V.H.D.L. 0
L.D.L. + H.D.L. _ 3
L.D.L. + V.H.D.L. 10
H.D.L. + V.H.D.L. 20 .
As will be seen from examination of the data there were no
appreciable amounts of L.P.C. formed in any single lipoprotein fraction
when incubated at 37o
alone. However, moderate activity was recovered
in the pooled sample of lipoprotein fractions and also in combinations
of low or high density lipoproteins with the very high density fraction.
The activity recovered from high density lipoproteins incubated with
very high density lipoproteins was quantitatively similar to that of the
- 122 -
pooled fraction, whereas the low density fraction plus the very high
density fraction, had only about 50 percent of the activity of the
pooled fraction. The data suggested that the substrate for the
reaction (P.C.) was located in both the low and high density fractions
and that the enzyme was part of the very high density infranatant.
The low and high density lipoproteins contained similar concentrations
of P.C. and the very high density fraction contained very little P.C.
(see similar data in table 8). This raises the question of why the
recovery of L.P.C. formation in the low density lipoproteins plus
infranatant should be much lower than for the high density lipo-
proteins plus infranatant, when the concentrations of P.C. were similar.
One explanation might be that high density lipoprotein-bound P.C. is
the preferred substrate for the enzyme reaction. Such a preference
could be related to the fatty acid composition of the high density
lipoprotein-bound P.C. or more likely, it would be due to the
organisation and size of the high density lipoproteins. An alternative
explanation of the difference in L.P.C. formation between low or high
density lipoprotein fractions incubated, may be that the enzyme becomes
distributed in both the high and very hign density fractions during
the ultracentrifugation procedure. This would account for the higher
activity of the high density fraction incubated with the infranatant
than for the low density lipoproteins incubated with the infranatant.
However, this explanation seems unlikely since the amount of L.P.C.
formed during the incubation of high density lipoproteins alone was
very low and would doubtfully explain the large difference in L.P.C.
formation between high or low density lipoproteins incubated with
high density infranatant.
- 123 -
Section 1 (f)
A comparison of the phospholipid content of lipoprotein fractions
isolated from unincubated serum or from serum previously incubated
0 at 37 .
Blood was taken from each of three healthy volunteer subjects
and allowed to clot in order to produce serum. Each serum sample
was divided into two portions which were incubated at 370 or stored
at 40 respectively for six hours. A 0.5 ml aliquot of each serum
sample was extracted into 20 mis of chloroform methanol (2:1 v/v)
after the six hours and the total and individual phospholipid'con-
centrations were determined.
An aliquot (12 mis) of serum sample was used as the starting
material for the preparation of lipoprotein fractions using the method
described above. Each lipoprotein fraction was dialyzed overnight
against four changes of 0.1 M Tris buffer (141 7.4) at 4° and sub-
sequently diluted ivith 0.9 percent saline to the volume of the
initial serum sample (12 ml). A 0.5 ml. aliquot of each diluted
lipoprotein fraction was extracted into chloroform-methanol for
eventual phospholipid analysis.
The tidal and individual phospholipid concentrations of all serum
and serum lipoprotein fractions are shown in table 8.
- 124 -
Table 8
A Comparison of the phospholipid content of scrum lipoprotein fractions
isolated from pre-incubated and post-incubated serum.
(Results are expressed as r mol phospholipid per ml. scrum.
EX PRE-INCUBATED SERUM EX POST-INCUBATED SERUM
1. WHOLE SERUM
Study TPL PE PC Sph. LPC TPL PE PC Sph. LPC
1 3.00 0.19 1.97 0.60 0.24 2.96 0.17 1.73 0.57 0.44
2 2.30 0.16 1.42 0.47 0.25 2.36 0.14 1.16 0.52 0.54
3 3.00 0.08 1.86 0.78 0.28 2.90 0.10 1.56 0.75 0.49
2. VERY LOW DENSITY LIPOPROTEINS
Study TPL PE PC Sph. LPC TPL PE PC Sph. LPC
'1 0.72 0.01 0.51 0.17 0.03 0.71 0.02 0.50 0.16 0.03
2 0.29 0 • 0.20 0.09 0 0.31 0 0.21 0.08 0.02
3 0.33 0 0.18 0.13 0.02 0.21 0 0.11 0.10 0
3. LOW DENSITY LIPOPROTEINS
Study TPL PE PC Sph. LPC TPL PE PC Sph. LPC
1 0.83 0.09 0.46 0.23 0.05 0.81 0.09 0.44 0.22 0.06
2 0.84 0.05 0.59 0.18 0.02 0.80 0.06 0.52 0.20 0.02
3 0.88 0.06 0.45 0.34 0.03 0.90 0.07 0.44 0.35 0.04
- 125 -
Table 8 continued
A Comparison of the phospholipid content of serum lipoprotein fractions
isolated from pre-incubated and post-incubated serum.
(Results are expressed as r mol phospholipid per ml. serum).
EX PRE-INCUBATED SERUM EX POST-INCUBATED SERUM
1. WHOLE SERUM
Study TPL PE PCB), Sph. LPC TPL PE PC Sph. LPC
1 3.00 0.19 1.97 0.60 0,24 2.96 0.17 1,78 0.57 0.44
2 2.30 0.16 0.42 0.47 0.25 2.36 0.14 1.16 0.52 0.54
3 3.00 0.08 1.86• 0.78 0.28 2.90 0.10 1.56 0.75 0.49
4. HIGH DENSITY LIPOPROTEIN
Study TPL PE PC Sph. LPC TPL PE PC Sph. LPC
1 1.19 0.04 0-89 0.18 0.08 0.94 0.04 0.63 0,18 0.09
2 0.80 0.10 0.53 0.15 0.02 0,•.74 0.08 0.44 0.20 0.02
3 1.17 0.02 0.84 0.28 0.03 0.98 0.02 0.68 0.22 0.06
5. VERY HIGH DENITY LIPOPROTEINS
Study TPL PE PC Sph. LPC TPL PE PC Sph. LPC
1 0.29 0.04 0.08 0.02 0.15 0.49 0.03 0.10 0.03 0,33
2 0.27 0 0 0 0.27 0.42 0 0.02 0 0.41
3 0.27 0 0.02 0 0.25 0.50 0 0.04 0.02 0,44
- 126 -
As has been described above, the concentration of L.P.C. in
incubated serum was greater than in unincubated serum and there was
a reduction in the P.C. content of incubated serum. There were no
significant differences in total phospholipid, P.E. or sphingomyelin
concentrations between incubated and unincubated serum samples.
The phospholipid composition of both the very low density
lipoproteins and the low densith lipoprotein fractions were not
significantly different for,the preparations obtained from incubated
serum when compared with those from unincubated serum. The total
phospholipid and P.C. concentrations of high density lipoprotein
fractions were decreased in the preparation isolated from incubated
serum. The concentrations of P.E., sphingomyelin and L.P.C. were,
however, not significantly different between high density lipoprotein
fractions isolated from unincubated or incubated serum. The total
phospholipid and L.P.C. concentrations were increased in the very
high density lipoprotein fractions isolated from pre-incubated serum
when compared with the fractions from nnn-incubated serum. The increase
in total phospholipid or L.P.C. for the very high density fraction was
quantitatively similar to the decrease in total phospholipid or P.C.
of the high density lipoprotein fraction. The data suggests that
during the incubation of serum some high density lipoprotein-bound
P.C. is converted to L.P.C. which leaves the high density lipoproteins
and becomes associated with the very high density fraction,
The major phospholipid of the very high density lipoprotein
fraction of both unincubated and post-incubated serum was L.P.C.
This very high density fraction contained nearly all of the L.P.C.
found in plasma, Furthermore, there appeared to be more L.P.C.
recovered from the lipoprotein fractions (particularly those of
- 127 -
unincubated serum) than was originally present in the whole serum
sample. This suggests that conversion of P.C. to L.P.C. occurred
during the ultracentrifugation procedure, a point which has already
been suggested by Slyitzer and Eder (1965).
Discussion
The formation of L.P.C. from P.C. in incubated human plasma has
been confirmed and some aspects of the reaction have been characterised.
Previously this reaction has been studied by Adikofer et al (1968) who
utilized the effect of L.P.C. on erythrocyte suspension stability as
a semi-quantitative measure of the reaction.' In the present study the
levels of L.P.C. and P.C. in plasma or serum have been measured
chemically. Very similar results to those of Adikofer have been found
despite the difference in assay methods. In particular, the inhibitory
effects of urea and para-hydroxymercuribenzoate on L.P.C. formation
have been confirmed. The pH optima of the reaction was found to be
in the weakly alkaline range (pH 7-8) which was similar to the results
reported by Adikofer. Furthermore, the separation of serum lipo-
protein fractions by ultracentrifugation at increased density indicated
that the enzyme activity was found in the very high density lipoprotein
-1 fraction (d) 1.200 gm. ml. ) and that lipoproteins of the high and
possibly low density fractions were the substrate for the reaction.
These results were again similar to those previously described,
The properties of the enzyme responsible for L.P.C. formation in
plasma or serum incubated at 37° were consistent with the characteristics
of lecithin:cholesterol acyl transferase (L.C.A.T.) (Glomset and Wright,
1964 : Glomset, 1968).
- 128 -
Comparison of the phospholipid concentrations of lipoprotein
fractions prepared from unincubated serum confirmed the presence of
most of the..serum L.P.C. in the very high density fraction (Phillips,
1959a : Switzer and Eder, 1965). The content of L.P.C. in this
fraction accounted for most of the total phospholipid. When the
phospholipid concentrations of lipoprotein fractions prepared from
post-incubated serum were examined, a decrease in high density
lipoprotein - P.C. and an increase in very high density lipoprotein -
L.P.C. were found. This finding was quantitatively consistent with
the formation of L,P.C. during the pre-incubation of serum in vitro
and suggested that during the incubation of serum there was a specific
conversion of high density lipoprotein-bound P.C. to L.P.C. and its
subsequent relocalisation with the very high density lipoproteins.
This observation supports the hypothesis of Switzer and Eder who have
suggested that L.P.C. is transported in the blood as an albumin-bound
complex.
When freshly prepared lipoprotein fractions were mixed together
and incubated at 37o
for some hours,.lysolecithin formation occurred
only in mixtures containing very high density lipoprotein and either
high- or low density fractions. This suggested that the reaction
substrate was found in both high- and low density lipoproteins:
However, comparison of lipoprotein fractions prepared from either pre-
or post-incubated serum indicated that either high density lipoproteins
were the preferred substrate, or that if low density lipoprotein-
bound P.C. had been utilized, it had subsequently been replaced with
P.C. from the high density lipoproteins. This latter interprdation
seems unlikely and therefore direct conversion of high density
lipoprotein-bound P.C. would appear to be the most likely mechanism
for the L.C.A.T. reaction.
- 129 -
The formation of L.P.C. in plasma was linear with regard to
incubation times of up to six hours. Therefore, in the subsequent
studies of L.P.C. formation in healthy and pathological plasma
specimens,- an incubation time of six hours-has been allowed. Such
a procedure results in the formation of relatively large amounts
of L.P.C. but still allows the results to be expressed as the
rate of L.P.C. formation per hour.
- 130 -
Section 2
Plasma cholesterol and individual phospholipid concentrations
in the healthy population and in patients suffering from
ischaemic heart disease and peripheral arterial 'disease.
Introduction
Only a very few studies of individual plasma phospholipids in
the healthy population or in clinbal patients have been reported.
A brief review of previous work in this field has been given in the
introductory chapter of the present thesis. Almost without exception,
all previous studies have been limited either in sample size or else
only selected individual phospholipid fractions have been analysed.
Furthermore, some reports of differences in individual phospholipid
concentrations between different populations have been contradictory.
The present investigation was formulated largely in an endeavour to
resolve some of these differences, and to investigate any abnormalities
in the plasma phospholipids of men suffering from ischaemic heart
disease, peripheral arterial disease and acute myocardial infarction.
For these reasons relatively large numbers of healthy and diseased
individuals have been studied. The total sample recorded during this
study consisted. of 77 apparently healthy individuals and 76 male
patients suffering from either ischaemic heart disease, peripheral
arterial disease or acute myocardial infarction. Furthermore, a sample
of 12 male patients suffering from ischaemic heart disease and 12
healthy male volunteers have been included in an additional study of
the phospholipid composition of plasma, erythrocytes and blood platelets.
For convenience the analysis of the study has been set out in three
sections dealing with the healthy population, clinical patients and
lastly, the phospholipid composition of plasma and blood cells.
- 131 -
Section 2 (a)
Analysis of plasma cholesterol and phospholipid concentrations
in the healthy population.
The plasma concentrations of total cholesterol, total phospho-
lipids, P.E., P.C., sphingomyelin and L.P.C. have been determined in
samples obtained from 77 apparently healthy individuals. The population
studied consisted of 21 young women aged 16 - 30 years, 8 older women
aged 45 - 60 years, 23 young men aged 18 - 30 years and 25 older men
aged 45 - 65 years.
The phospholipid concentrations of both fresh plasma and plasma
incubated at 37o for six hours were determined in order to estimate
the rate of L.P.C. formation during the incubation of plasma.
Phospholipids have been recorded as both relative concentrations
(percentage of total phospholipid) and as absolute concentrations
(p mol. m1.-1).
The analysis of the relative concentrations of P.E., P.C.,
sphingomyelin and L.P.C. of the four groups is showlin table 9.
Differences between mean relative concentrations have been
statistically compared for men and women of similar age-groups and
between men or women of different age-groups. There were no
significant differences between women of different age-groups or
between men of different age-groups. There were, however, significant
sex differences between the relative concentrations of P.C. and L.P.C.
in both age-groups. The relative concentration of P.C. was higher
and the relative concentration of L.P.C. was lower in women when
compared with men of similar age.
The mean absolute concentrations of cholesterol and of phospholipids
in plasma from four groups of healthy individuals are shown in table10.
The mean values for L.P.C. formation during the incubation of plasma
at 37o are also shown in table 10.
- 132-
Table 9
Relative concentrations of individual plasma phospholipids
of healthy men and women
Relative concentrations of individual phospholipids (70)
+ standard deviation _
Group and age-range .P.E. P.C. Sph. L.P.C.
WOMEN (16-30 years) 3.9 + 1.7 - 70.0 + 3.7 _ 19.5 + 3.1 _ 6.8 + 1.4 _
(N=21)
WOMEN (45-60 years) 3.7 + 0.4 69.0 + 1.3 20.9 + 1.0 6.1 + 1.1 (N =8)
MEN (18 - 30 years) 3.8 + 1.5 66.7 + 3.5 21.0 + 2.4 8.4 + 1.3 (N=23)
MEN (45 - 65 years) (N=25)
4.1 + 1.0 '"
65.9 + 3.1 - 22.1 + 2.7 -
7.8 + 1.3 -
Comparison
Statistical significance of differences between groups ('p' value)
P.E. P.C. Sph. L.P.C.
Young women
Young women
Older men -
Older men -
- older women
- young men
young men
older women
N.S.
N.S.
N.S.
N.S.
N.S.
p <0.01
N.S.
p <0.01
r.s.
N.S.
N.S.
N.S.
N,S.
p <0.001
N.S.
p<0.005.
Table 10
Absolute concentrations of plasma cholesterol and
individual phospholipids in healthy men and women
Group and age range Cholesterol
Plasma concentrations p mol ml.-1 + standard deviation
L.P.C. LPC
formation, pmol L h
Total P.L. P.E. P.C. Sph.
WOMEN (16-30 years) (N=21)
WOMEN (45-60 years) (N=8)
MEN (18-30 years) (N=231
MEN (45-G5 years) (N=25)
5.40+ 0.91-
5.9G+ 0,61
5.65 + _ 1,10
6.47+ 0,74
2.92+ 0.37
3.45+ 0.27
2.96 + _. 0,36
3.43F _ 0:49
0.11+ 0.05
0.1-2+ 0.02
0.11+ 0.03
0.14+- .... 0.03
2.04+ 0.24
2.33+ 0.25
1.98+ - 0.32
2.26+ 0.30
0.57+ 0.10
' 0.713+ 0.09
0.62+ _ 0009
0.75+ 0.12
0.19+ 0.04
0.22+ _ 0.03
C.25+ 0.03
0.27+ 0,03
48+ 9
51+ a
51+ 14
47+ .... 8
Statistical significance of differences between groups ('p' value)
LPC Comparison Cholesterol Total P.L. P.E. P.C. Sph. L.P.C. formation
.-. Young women - older women N.S. p \ 0.001 N.S. p<b.02 p <0.001 p (0.02 N.S.
Young women - young men N.S. N.S. N.S. N.S. N.S. p 0.001 N.S.
Older men - young men p <0.01 p <0.001 p(6.02 p<6.01 1j.050 <0.1 0.05(p <0.1 N.S.
Older men - older women p (0.05 N.S. N.S. N.S. N.S;.. p(0.001 N.S.
- 134 -
.The total cholesterol concentration was significantly higher
in older men compared with the concentration in age-matched women
or younger men. No significant elevation of the plasma total
cholesterol was apparent in older women compared with younger women.
Plasma total phospholipid concentrations were significantly
increased in older men and women compared with younger men and women,
and correspondingly the plasma concentrations of all individual
phospholipid fractions were higher in older men and women compared
with younger men and women. Comparison of differences in the
absolute concentrations of individual phospholipids between men and
women of similar age-groups, showed that the L.P.C. levels were
significantly lower in women. There were no significant differences
in the rates of L.P.C. formation of any group.
In some cases statistically significant differences in the
relative concentrations of P.C. between different populations were
not reflected by the comparison of absolute concentrations. For
example, the mean relative concentrations of P.C. in young women
was significantly greater (p <0.01) than for age-matched young men,
Whereas the difference in absatte concentrations of P.C. was not
significant. This was explained by the fact that the total phospho-
lipid concentration was slightly higher in young men than in young
women. Similarly as explained above, there were no significant
differences in the relative concentrations of individual phospholipid
fractions between young and older men or between young and older
women. However, the total phospholipid concentrations were higher in
both older/ aR older women when compared with younger men and younger
women and consequently there were significant increases in the
absolute concentrations of all phoSpholipid fractions in the older
age groups.
- 135 -
Section 2 (h)
Analysis oil plasma cholesterol and phospholipid concentrations in
men suffering from acute myocardial infarction, chronic ischaemic
heart disease and peripheral arterial disease
The plasma concentrations of total cholesterol, total phospho-
lipids, P..E., P.C., sphingomyelin and L.P.C. have been measured in
groups of patients suffering from atherosclerotic diseases. The
populations studied included 24 men suffering from chronic ischaemic
heart disease, 16 men presenting with chronic peripheral arterial
disease, 20 men who had suffered acute myocardial infarction within
the preceding 48 hours and 15 men admitted to the coronary intensive
care unit with suspected acute myocardial infarction who were later
diagnosed as acute ischaemic heart disease patients not suffering
from acute myocardial infarction. The results of these investigations
were compared with those of a control group consisting of 25
apparently healthy age-matched male volunteer subjects.
Relative concentrations (percentage of total phospholipid) and
-1 absolute concentrations (p mol ml. ) of individual plasma phospholipids
have been recorded.
The analysis of the relative concentrations of individual phospho-
lipids is shown in table 11. Compared with the healthy control
population the relative concentrations of L.P.C. were significantly
decreased in all patient groups. The plasms relative concentrations
of P.C. were increased in all patient groups but only in the case of
chronic ischaemic patients, was the increase in relatiVe concentration
of P.C. statistically significant when compared with the control group.
There were no significant differences in the relative concentrations
of P.E. or sphingomyelin.
- 136-
Table 11
Relative concentrations of individual phos2holipids in the plasma
of men suffering from ischacmic heart disease, peripheral arterial
disease and acute myocardial infarction compared with healthy age-
matched controls.
Relative concentrations (;10) + standard deviation
GROUP MEAN AGE (years)
P.E. P.C. Sph. L.P.C.
Healthy controls 54 4,1+1.0 65.9+3.1 22.1+2.7 _ 7.8+1.3 (N=25)
Chronic I.H.D. 55 4.6+1.1 68.0+2,5 21.4+1.9 _6.0+1.4 (N=24) .
N.S. p<0.02 N.S. p<0.001
Peripheral arterial disease
60 4.1+1.5 66.6+4.5 22.6+1.2 6.7+1.3
(N=16) N.S. N.S, N.S. 130.02
Acute myocardial infarction
(N=20)
53 4.5+1.6
N.S.
67:5+3.8
N.S.
23.6+3.7
N.S.
4.3+1.5
p<0.001
Acute I,H.D. 54 4.54.-1.8 67.5 +3.6 22,8+2.6 5.2+1.8 (N=15)
N,S.- N.S. N.S. p<0.001
- 137 -
The mean absolute concentrations of cholesterol and of
individual phospholipids in the plasma of patients and healthy
subjects, are shown in table 12.
Only in the case of men suffering from chronic ischaemic heart
disease was the plasma total cholesterol concentration significantly
higher than for the control population. The total plasma phospho-
lipid concentration was just significantly lower (p <0.05) in
patients suffering from acute myocardial infarction or peripheral
arterial disease. There were no significant differences in total
phospholipid concentrations of patients suffering from acute or
chronic ischaemic heart disease when compared with the healthy control
group.
The plasma concentration of P.E. was significantly higher in
patients suffering from ischaemic heart disease compared with the
controls but was unaltered in the cases of patients suffering from
peripheral atheroma, myocardial infarction and acute ischaemic,
The plasma concentrations of P.C. were not significantly different
in the patient groups compared with the controls except in the case
of patients suffering from acute myocardial infarction who had lower
levels of P.C. There were no significant differences in sphingomyelin
concentrations in any patient group compared with the control
population.
The most consistent differences in individual plasma phospho-
lipid concentrations were the significantly lower levels of L.P.C.
in all patient groups. The L.P.C. levels were moderately decreased
in patients suffering from chronic ischaemic and peripheral arterial
diseases and dramatically lower in patients presenting with acute
myocardial infarction and acute ischaemia. The rates of L.P.C.
Table 12: Absolute concentrations of plasma cholesterol and individual phospholipids in men suffering
from ischaomic heart disease, peripheral arterial disease and acute myocardial infarction compared with
age-matched healthy controls.
Group Cholesterol
Plasma concentrations (p mol.m1-1 ) + standard deviation
L.P.C. LPC foriatiin p mol.L h .
Total P.L. P.E. P.C. Sph.
Healthy controls (N=25)
Chronic I.H.D.
(N=24)
Peripheral Arterial disease (N=16)
Acute myocardial infarction (N=20)
Acute I.H.D.
(N=15)
6.47+0.74
7.73+ 1.54 p <0.001
6.58+ 1.11
N.S.
6.07+ 2.06-
N.S.
6.64+ 1.39
N.S.
3.43+0.49
3.64+ 0.62-
N.S.
3.12+ 0.39_ p <0.05
2.99+ - 0.77
p 0.05
3.12+ 0.63
N.S.
0.14+0.03
0.17+ 0.05 p <0.05
0.13+ 0,04
V.S.
0.14+ - 0.08 N.S.
0.15+ 0.04-
N.S.
2.26+0.30
2.45+ 0.45-
N.S.
2.08+ 0,33
N.S.
1.99+ 0.50 p 0,05
2.12+ 0.47
N.S.
0.75+0.12
0.79+ 0.14
N.S.
0.70+ 0.14
N.S.
0.70+ 0.17 .
N.S.
0,68+ 0.16
N.S.
0.27+0.03
0.20+ 0.04 p <0,001
0.21+ 0.04 p <0.01
0.13+ 0.05 p <0.001
0.16+ 0.06 p <0.001
47 + 8 .....
• 51 + _ 12 N.S.
46 + .... 8 N.S.
39 + .... 8
p 0.01
43 + _ 15 N.S.
- 139 -
formation on incubated plasma samples taken from patients suffenbg
from acute myocardial infarction were significantly lower than for
the control population or for patients suffering from chronic
ischaemia or chronic peripheral atherosclerosis.
- 140 -
Section 2 (c)
Analysis of the relative concentrations of individual phospholipids
of plasma, erythrocytes and platelets in healthy men and in men
suffering from chronic ischaemic heart disease.
The relative concentrations of individual phospholipids in plasma,
erythro9ytes and blood plateits have been determined in samples taken
from 12 male patients suffering from chronic ischaemic heart disease
and 12 age-matched male volunteer subjects who were apparently
healthy.
The analysis of results is shown in table 13. The only consistent
difference in the relative concentrations of individual phospholipids
in plasma, erythrocytes and blood platelets between healthy controls
and patients suffering from chronic ischaemic heart disease, was in
the concentration of L.P.C. The relative concentrations of L.P.C. in
plhsma, erythrocytes and platelets was significantly lower in patients
suffering from chronic ischaemic heart disease. There were no
significant differences between controls and ischaemic patients in
plasma, erythrocyte and platelet concentrations of P.E., P.C., P.I.,
P.S., and sphingomyelin.
Discussion
Plasma cholesterol and phospholipid concentrations have been
analysed in 77 apparently healthy subjects and in 76 patients
suffering from atherosclerotic diseases. The relative concentrations
of individual phospholipid fractions in plasma, erythrocytes and
blood platelets have been determined in 12 healthy subjects and in
12 patients suffering from ischaemic heart disease.
- 141 -
Table 13: Relative concentrations of phospholipids in plasma,
erythrocytes amd blood platelets in apparently healthy men and
in men suffering from chronic ischaemic heart disease.
PLASMA
GROUP P.E. P.C. P.I. P.S. Sph. L.P.C.
Healthy males 3.9+ 70.0+ - - 20.4+ 5.7 + (51 years) 0.7 1.1 1.2 0.9 N=12
Ischaemic males 3.94- 70.0+ - - 21.7+ 4.4+ (50 years) 1.1 +4.8 _ 3.4 1.8 N=12
Significance 'p' value N.S. N.S. N.S. p<0.05
ERYTHROCYTES
GROUP P.E. P.C. P.I. P.S. Sph. L.P.C.
Healthy males 30.7+ 33.1+ _ 0.4+ - 7.8+ . - 28.4+ - 1.6+
(51 years) 1.5 2.4 0.7 3.7 1.7 1.0 N=12
Ischaemic males 29.2+ 31.5+ 1.1+ 9.2+ - 28.2+ _ 0.7+ _
(50 years) 2.4 2.2 1.0 2.7 1.6 0.7 N=12
Significance 'p' value N.S. N.S. N.S. N.S. N.S. p <0.05
PLATELETS GROUP P.E. P.C. P.I. P.S. Sph. L.P.C.
Healthy males 27.1+ 39.7+ 4.1+ 9.0+ 19.0+ 1.1+ (51 years) 2.0 1.8 1.2 1.3 1.4 0.6 N=12
Ischaemic males 26.4+ 40.1+ 4.3+ 9.5+ 19.3+ 0.5+ (50 years) 2.8 4,0 3.0 2.5 2.6 0.6 N = 12
Significance t p' value N.S. M.S. N.S. N.S. N.S. p0.05
- 142 -
Normal values
There was no significant difference in the concentrations of
total cholesterol and total phospholipid between men and women of
the same age. The values were increased, however, in older men
and women compared with younger men and women. This was in
agreement with previously published results (Adlersberg et al, 1956).
Recent records of individual plasma phospholipid concentrations
in the apparently healthy population have usually been confined to
small numbers of individuals (Marinetti et al, 1959 : Berlin et al,
1969a) or else the individuals have not been differentiated
according to their age or sex (Phillips and Dodge, 1967 : Kunz et al,
1970). An investigation of 100 normal subjects differentiated into
10 groups according to age and sex (BLIttiger, 1973a) has only reported
values for total phospholipid, P.C. and L.P.C, These reasons have
made it difficult to compare the present results with the earlier
work.
The relative and absolute concentrations of P.E. in plasma
samples taken from apparently healthy individuals included in the
present study, were higher than those recently reported by Berlin et
al (1969a) and Kunz et al (1970). However, other recent reports of
normal P.E. concentrations (Jones and Ways, 1967 : Phillips and
Dodge, 1967, and Cooper and Gulbrandsen, 1971) were similar to those
given above, Both the relative and absolute concentrations of P.C.
and sphingoilyelin reported above agreed well with those given in
previous studies except those given by Wittiger (1973a) which were
up to ten percent lower than the widely accepted values. The relative
and absolute concentrations of L.P.C. were found to be higher in men
than in women which confirmed the results of Berlin et al (1969a)
and Bdttiger. The absolute connntrations of L.P.C. were higher in
- 143 -
older men and women compared with younger men and women respectively.
This supported BOttiger's findings but failed to confirm the results
of Berlin et al who reported that plasma L.P.C. concentrations were
higher in young men in comparison with older men.
There were no significant differences in the rates of L.P.C.
formation in incubated plasma obtained from healthy men or women.
This suggested that differences in L.P.C. concentrations between men
and women and between different age-groups, were not due to
differences in the rate of formation of L.P.C. in plasma.
Pathological values
The most consistent difference in the phospholipid composition of
plasma obtained from healthy men and from men suffering from athero-
sclerotic disease, concerned the L.P.C. fraction. Both the relative
and absolute concentrations of L.P.C. were lower in men suffering
from chronic ischaemic heart disease, peripheral arterial disease,
acute myocardial infarction and acute ischaemic heart disease. The
lowest values for the absolute concentration of L.P.C. were found in
men suffering from acute myocardial infarction and acute ischaemie.
These results were in agreement with previously published data which
reported low values of L.P.C. in acute myocardial infarction
(Marinetti et al, 1959 : Berlin et al, 1969b) and in vascular disease
(Kunz et al, 1970). No previous study has been made of individual
plasma phospholipids in men suffering from ischaemic heart disease.
It was apparent that the lowest L.P.C. levels were found in acute
rather than chronic clinical conditions. It is possible that low
L.P.C. levels were a biological response to the stress of cardiac
pain in the acute diseases. However, Berlin reported that the lowest
L.P.C. levels were not consistently associated with those cases of
- 144-
acute myocardial infarction exhibiting the most pronounced shock
symptoms or pain.
The rates of L.P.C. formation in incubated plasma were lowest in
patients with acute myocardial infarction, but were not significantly
different from those of the healthy population in the case of patients
suffering from acute or chronic ischaemic heart disease and peripheral
arterial disease. This suggests that the low L.P.C. levels in
patients with an acute infarction may have been partly due to a
decrease in the rate of L.P.C. formation in vivo.
The absolute concentrations of plasma P.B. have been reported to
be decreased in acute myocardial infarction (Berlin et al, 1969b) and
increased in peripheral arterial disease (Kunz et al, 1970). The
results of the present study did not confirm these previous results,
although a significant increase in the P.B. fraction was found in
patients suffering from chronic ischaemic heart disease.
Relative concentrations of phospholipids in blood cells.
Several recent reports have shown that L.P.C. was present as a
minor phospholipid component of erythrocytes (Dodge and Phillips, 1967
Cooper and Gulbrandsen, 1971, and Piper of al, 1972) and of blood
platelets (Cooper and Gulbrandsen, 1971). These previous results are
compared with those of the present study in table 14, which also
includes a recent analysis of normal platelet phospholipid composition
reported by Nordtly and Gjone, 1971.
The results of the present study confirm the presence of L.P.C.
as a normal phospholipid constituent of both plates and erythrocytes.
The relative concentrations of P.E., P.C., P.S., P.1., sphingomyelin
and L.P.C. in normal erythrocytes and platelets reported in the present
study were in good agreement with previously published figures.
- 145 -
Table 14: Relative concentrations of individual phospholipids of
erythrocytes and blood platelets, A comparison of previously •
published results with the present study.
INVESTIGATION
Relative concentrations
P.E. P.C. P.S.
leerythrocyte phospholipids(%)
LPC Other P.I. Sph.
Dodge and Phillips, 1967 27.5 29.2 14.8 0.6 25.4 1.0 1.5
Cooper and Gulbrandsen, 29.7 34.1 10.8 - 23.8 1.7 - 1971
Piper et al, 1972 26.6 29.8 12.7 4.8 23.5 1.3 1.3
Present study 30.7 32.1 7.8 0.4 27.4 1.6 -
Relative concentrations of platelet phospholipids %
INVESTIGATION P.E. P.C. P.S. P.I. Sph. LPC Other
Cooper and Gulbrandsen, 1971
25.0 51.8 5.8 - 15.7 1.6 -
Nordoy and Gjone, 1971 31.6 44.8 6.8 3.0 13.8 - -
Present study 27.1 39.7 9.0 4.1 19.0 1.1 -
- 146-
The comparison of the relative concentrations of erythrocyte
and platelet phospholipids between apparently healthy men and
patients suffering from ischaemic heart disease revealed a
--difference in L.P.C. -concentrations-which resembled that described
above for plasma phospholipids. In the comparison of 12 healthy
men and 12 patients with ischaemic heart disease, the relative
concentrations of L.P.C. in plasma, erythrocytes and platelets,
were significantly lower at the 5 percent level_ in patients
suffering from chronic ischaemic heart disease. No othr significant
differences were found between patients and controls. This Wes an
apparently new finding that relatively low levels of L.P.C. in
erythrocytes and platelets were associated with decreased plasma
levels of L.P.C. and suggested that perhaps the L.P.C. content of
blood cells derives from the plasma L.P.C. pool.
- 147 -
Section 3
Effects of Lysolecithin on Erythrocyte Behaviour in vitro.
Introduction
It has long been known that L.P.C. was a powerful haemolytic
agent, and more recently it has been shown that L.P.C.fractions
containing saturated acyl groups were more active'haemolysins than
un the correspondingAsaturated or polyunsaturated.compounds (Gottfried
and Rapport, 1963 : Roman et al, 1969). The effects of sub-haemolytic
concentrations of L.P.C. on erythrocyte behaviour have been less well
studied, although L.P.C. has been shown to inhibit erythrocyte
sedimentation (Adikofer et al, 1968) and to alter red cell morphology
(Piper et al, 1972). Sub-haemolytic concentrations of L.P.C. have
also been shun to induce cell fusion, including the fusion of
erythrocytes (Poole et al, 1970). On the basis of such fusion
experiments Lucy (1970) has proposed a model for the interaction of
L.P.C. with biological membrans. According to this hypothesis L.P.C.
is thought to induce a phase change in the membrane from a bimolecular
leaflet to a micellar structure. Lucy has suggested that such a
mechanism would account for erythrocyte shape. changes and haemolysis
caused by exposure of the cells to L.P.C. and to have other important
effects on the function of other types of cells.
In the present study typical experiments are described in
which the effects of L.P.C. exposure on erythrocyte behaviour have
been studied by relatively simple techniques to determine
erythrocyte sedimentation rates (E.S.R.) and erythrocyte packing
rates (E.P.R.). In addition the viscosity of whole blood has been
measured after its exposure to L.P.C. and the results are discussed
in terms of altered erythrocyte morphology.
- 148 -
Section 3(a)
Effect of lysolecithin on erythrocyte sedimentation
The effect of L.P.C. on erythrocyte sedimentation has been
studied using high molecular weight dextran as an agent promote
high rates of sedimentation.
Experimental details and results.
In a typical experiment washed red cells were re-suspended in
a mixture of autologous plasma and 0.9 percent saline containing
dextran (molecular weight 150,000) to give an haematocrit of 35 per-
cent. The final concentration of dextran was 0.4 percent. An
aliquot of L.P.C. solution (10 - 50 p1) or of 0.9 percent saline
was then added to each 1.5 ml, of re-suspended erythrocytes and
after gentle mixing the sedimentation tubes were filled and clamped
in position. Sedimentation readings were noted at regular intervals
of time until the control tubes had sedimented 50 m.m. Duplicate
tubes were set up for each concentration of L.P.C. tested and
inhibition of E.S.R. was calculated with reference to the control
results. Exposure of erythrocytes to L.P.C. inhibited the E.S.R.
in a dose-dependant fashion (Fig. 14) and at concentrations up to
0.5 p mol ml.-1
there was no macroscopically obvious haemolysis in
the supernatant plasma, although inhibition was maximal.
In a further series of experiments the effects of saturated
L.P.C. and polyunsaturated L.P.C. were compared. Figure 15 shows
the mean E.S.R. curves for samples of erythrocytes exposed to
-1 either saturated or polyunsaturated L.P.C. (0.24 r mol mi. ) and
for a control containing no added L.P.C, Saturated L.P.C. inhibited
the E.S.R. although there was no difference in the E.S.R. of the
control and the erythrocytes exposed to polyunsaturated L.P.C.
O
- 149 -
01 02 03 04 05 LPC Concentration Nmolml.
Figure 14: Inhibition of erythrocyte sedimentation by exposure of
blood to lysolecithin.
50-
AC) . 0
80 4 AO
AO DO
A° Ao
40
40
40 4° 0
AO 40
400 4 40
.4040 480
0 con•••••••""""a 00 o (43° cti,
eg swap coli•
- 150 -
0 40 Minutes
Figure 15: Effect of saturated and polyunsaturated lysolecithin
fractions on erythrocyte sedimentation behaviour.
coo control (no added L.P.C.).
••fs saturated L.P.C.. (0.24 p mol. ml.-1)
AAA polyunsaturated L.P.C. (0.24 }1 mol. ml.-1)
-151-
Discussion
The effect of exposure of erythrocytes to sub-haemolytic
concentrations of L.P.C. was to inhibit the E.S.R. This confirms
the findings of Adlkofer et al (1968)-and other workers. Piper
et al (1972) have shown by scanning electron microscopy that
exposure of erythrocytes to L.P.C., at concentrations similar
to those inhibiting the E.S.R., changed the morphology of the cells
from characteristic biconcave discs to echinocytic shapes. Such
cells would be very unlikely to aggregate into rouleaux and
consequently the sedimentation rate would be reduced.
Although it has not been reported previously, it was clear
from the results that saturated L.P.C. inhibited E.S.R. and that
the polyunsaturated fraction was inactive. This corresponds with
the lack of, or reduced haemolytic activity of polyunsaturated L.P.C.
(Gottfried and Rapport, 1963 : Reman et al, 1969). Differences in
the effects of L.P.C. species may be explained with recourse to
Lucy's argument (1970) for membrane changes induced by L.P.C. This
proposed that phase changes in the membrane lipoprotein organisation
occur following penetration of the membrane by L.P.C. The conformation
of a given L.P.C. molecule would be important in this process and this
suggests that whilst saturated L.P.C. species have the necessary
shape for membrane penetration and induction of micellar rearrangement,
polyunsaturated L.P.C. molecules do not. Differences in the degree
of binding of L.P.C. species to plasma-albumin might also explain
the different effects of saturated and polyunsaturated L.P.C., since
only "free" L.P.C. causes haemolysis. However, this explanation
appears unlikely because the observations on the different relative
haemolytic effects of saturated and unsaturated L.P.C. were made in
the absence of plasma proteins (Gottfried and Rapport, 1963 :
Roman et al, 1969).
- 152 -
Section 3(b)
Sedimentation of erythrocytes in incubated plasma
The sedimentation rate of erythrocytes has been measured after
their resuspension-in plasma pre-incubated at 370 for-up to six hours.
Experimental details and Results
In a typical experiment erythrocytes were separated by
centrifugation and washed once with 0.9 percent saline. They were
subsequently stored at 40 for up to six hours. The plasma was
0 incubated at 37 for up to six hours and aliquots were removed at
hourly intervals for determination of plasma L.P.C. levels and for
storage at 40 until the incubation was completed. Samples of plasma
and erythrocytes were then allowed to warm up to ambient temperature
(210). Duplicate 0.5 ml. samples of washed erythrocytes were then
• mixed with 0.7 ml. of incubated plasma and 0.3 ml. of 2 percent
dextran solution. After gentle mixing the sedimentation tubes were
filled and clamped in position. When the control tubes containing
unincubated plasma had sedimented 50 mm. the readings from the other
tubes were recorded and the percentage inhibition of the E.S.R. was
calculated.
Inhibition of the E.S.R. increased linearly with the incubation
time of plasma (Fig. 16 (i)) for incubations of up to six hours.
There was a significant correlation (p < 0.01) between the percentage
inhibition of the E.S.R. and the increase in the concentration of
plasma L.P.C. during the incubation (Fig. 16 (ii)).
Discussion
The formation of L.P.C. in incubated plasma has been described in
Section 1 of this chapter, and has been shown to increase linearly with
incubation time for up to six hours. The results presented here showed
that the sedimentation rate of erythrocytes resuspended in incubated
plasma, was inhibited. Furthermore, there was a significant correlation
- 152-
Section 3(b)
Sedimentation of erythrocytes in incubated plasma
The sedimentation rate of erythrocytes has been measured after
their resuspension in plasma pre-incubated at 370 for up to six hours.
Experimental details and Results
In a typical experiment erythrocytes were separated by
centrifugation and washed once with 0.9 percent saline. They were
subsequently stored at 40 for up to six hours. The plasma was
0 incubated at 37 for up to six hours and aliquots were removed at
hourly intervals for determination of plasma L.P.C. levels and for
storage at 40 until the incubation was completed. Samples of plasma
and erythrocytes were then allowed to warm up to ambient temperature
(210). Duplicate 0.5 ml. samples of washed erythrocytes were then
mixed with 0.7 ml. of incubated plasma and 0.3 ml. of 2 percent
dextran solution. After gentle mixing the sedimentation tubes were
filled and clamped in position. When the control tubes containing
unincubated plasma had sedirnented 50 mm. the readings from the other
tubes were recorded and the percentage inhibition of the E.S.R. was
calculated.
Inhibition of the E.S.R. increased linearly with the incubation
time of plasma (Fig. 16 (i)) for incubations of up to six hours.
There was a significant correlation (p <0.01) between the percentage
inhibition of the E.S.R. and the increase in the concentration of
plasma L.P.C. during the incubation (Fig. 16 (ii)).
Discussion
The formation of L.P.C. in incubated plasma has been described in
Section 1 of this chapter, and has been shown to increase linearly with
incubation time for up to six hours. The results presented here showed
that the sedimentation rate of erythrocytes resuspended in incubated
plasma, was inhibt.ted. Furthermore, there was a significant correlation
- 153 -
0 2 4 Hours.
20 40 60 %Inhibn- of ESIR:
Figure 16: (i) Inhibition of erythrocyte sedimentation in plasma pre-
incubated at 370.
(ii) Correlation of lysolecithin formation in incubated plasma
with the inhibition of erythrocyte sedimentation in
incubated plasma.
- 154 -
between L.P.C. levels of incubated plasma and the inhibition of
the E.S.R. This indicated that L.P.C. formed during incubation
of plasma from_the plasma P.C. was effective in altering erythrocyte
behaviour. Significant correlations between L.P.C. levels of
unincubated plasma and clinical E.S.R. have recently been claimed
(BOttiger, 1973b : Berlin et al, 1973), and there seems no doubt
that plasma L.P.C. levels do influence the behaviour of erythrocytes.
These findings support those of Adlkofer et al (1963) who
measured plasma L.P.C. formation and enzyme activity by means of
dextran-promoted E.S.R.
- 155 -
Section 3 (c)
Effects of lysolecithin on erythrocyte flexibility and whole blood
viscosity.
Experimental details and Results.
In a typical study blood was collected from a healthy volunteer
and anticoagulated with heparin (15 units ml.-1). Each blood
specimen was divided into three samples and L.P.C. solution was
added to two of the samples at final concentrations of 0.15
p mol ml.-1 and 0,30 p mol ml.-1: 0.9 percent saline was added to
the third sample as a control.
Erythrocyte flexibility.
The effect of L.P.C. on erythrocyte flexibility was assessed by
the method of Sirs (1968). Duplicate capillary tubes were filled
from each blood sample and then sealed by heating the tube tip in
a flame. All six capillary tubes were then loaded into the micro-
haematocrit centrifuge which was connected to an 80 v supply (600g).
The centrifuge was started and then stopped every two minutes over
a ten minute period for the haematocrits to be measured. The mean
haematocrit values plotted against centii;ugation time are shown in
figure 17 (1). The erythrocyte packing rate (E.P.R.) value for each
sample can be calculated from these curves. The erythrocyte packing
curves for L.P.C.-treated samples were shallower than for the control
indicating that the E.P.R. had decreased. The E.P.R. provides an
index of erythrocyte flexibility since completely rigid cells cannot
be packed by centrifugation (Sirs 1968). Thus exposure of erythrocytes
to L.P.C. decreases the flexibility of the cells.
Blood viscosity
Blood and plasma viscosity measurements were performed at 370
using a Brookfield model LVT blood viscosity meter. The mean
results of four measurements of viscosity at shear rates of 6, 12,
- 156 -
100-
INM
Haem
a toc
rit (
%)
30
14 Centrifuge time (min)
612 24 48 Shear rate (se61)
Figure 17: (i) Effect of lysolecithin on erythrocyte packing during
centrifugation.
(ii)Effect of lysolecithin on whole blood viscosity measured
at different shear rates.
e--s Control; 0-0
o 0
L.P.C. (0.15 p mol. ml.-1)
-1 (0.30 F mol m1.)
- 157 -
24 and 43 sec.-1
were recorded. Results for whole blood viscosity
were converted to the values applying to a standard hacmatocrit of
40 percent.
Plasma viscosity was unchanged in samples prepared from blood
containing added L.P.C. However, whole blood viscosity was
significantly increased following exposure of the blood sample to
L.P.C. (Fig. 17 (ii)). The increase in blood viscosity was most
apparent at the lowest shear rate (6 sec.-1).
Discussion
The effects of L.P.C. on haemorrheology have not been described
elsewhere. Rampling and Sirs (1973) have given the value of the
E.P.R. as about 9 % min.-2 for healthy subjects, although variations
of from 1 to 30 % min.-1 have been recorded in exceptional cases.
The decrease of E.P.R. caused by L.P.C. is probably due to its action
on red cell shape, since echinocytic or spheroid cells would be
expected to be much more rigid than normal discoid erythrocytes.
In this respect L.P.C. probably had a similar effect tq'chat of
formaldehyde which also decreased the E.P.R. and increased erythrocyte
rigidity (Sirs, 1969). Similarly the increase in blood viscosity
induced by L.P.C. was most likely due to changed red cell morphology
since it had no effect on plasma viscosity. The effect of L.P.C. on
blood viscosity was more pronounced at low shear rates at which red
cell morphology would be expected to play an important role in
determining blood viscosity than at higher shear rates.
The influence of altered levels of L.P.C. in vivo on red cell
properties has been investigated in women during pregnancy (Section
7).
- 158 -
Section 4
effects of purified phospholipid preparations on blood platelet
function in vitro.
Typical experiments are described in which samples of P.R.P.
or whole blood exposed to- purified phospholipid preparations were
tested for platelet aggregation and platelet adhesiveness. In the
case of the platelet aggregation studies phospholipids have been
tested for their direct effeds on stirred platelet rich plasma
(P.R,P.) as well as on platelet aggregation initiated by a range of
chemically dissimilar aggregating agents including adenosine diphosphate
(A.D.P.), 5-hydroxytryptamine (5-11.T.), adrenaline, thrombin and
collagen.
- 159 -
Section 4 (a)
Effects of purified phospholipids added to stirred platelet rich plasma
Experimental details and Results
In a typical experiment freshly prepared P.R.P. was divided into
1 ml. aliquots which were separately warmed at 37° for three minutes
and then transferred to the sample compartment of the aggregation
apparatus. The P.R.P. sample was maintained at 37° and stirred
whilst its optical density was continuously recorded. After one
minute an aliquot (10 - 50 pls.) of a phospholipid preparation was
added and its effects on the platelet suspension were recorded.
Phospholipids were tested at final concentrations of up to 1.0 - 1.5
p mol ml.-1
; suitable control.s were recorded in which aliquots of
0.9 percent saline or 5 percent human albumin solution were sub-
stituted for the phospholipid preparation.
Sphingomyelin and Phosphatidylserine. The addition of sphingomyelin
or P.S. to stirred P.R.P. at concentrations of 1.5 p mol ml.-1
initiated platelet aggregation as recorded by increases in the
intensity of the light transmitted by the P.R.P. Aggregation
initiated by P.S. was slight and spontaneously reversed after approx-
imately one minute; aggregation induced by sphingomyelin was
irreversible (fig. 18 (i)).
Lysolecithin. The addition of L.P.C. (0.5 p mol -1
) to stirred
P.R.P. reduced the amplitude of the oscillations in the intensity of
the light transmitted through the P.R.P. (fig. 18 (ii)).
Other phospholipids. The exposure of stirred P.R.P. to other phospho-
lipids including P.E., P.If, P.C., G.P.C. or L.P.E., at final
concentrations of up to 1 p mol.ml.-I
had no consistent effects on
the light transmitted through the sample.
C o nt ro 1 1prnot SM
1.5 plot PS
- 160 -
Minutes
02 4
o.d.
05 prribt LPC
0
Minutes Figure 18: (i) Effect of adding sphingomyelin (S.M.) and
phosphatidylserine v.P.S.) to stirred platelet rich plasma.
(ii) Effect of adding lysolecithin (L.P.C.) to stirred platelet
rich plasma.
- 161 -
Discussion
Exposure of platelets to high concentrations-of sphingomyelin
or P.S. resulted in platelet aggregation which was irreversible in
the case of sphingomyelinntreated_platelets. The concentration of
phospholipid required to demonstrate aggregating activity was higher
than the normal plasma level of the individual phospholipid,
particularly so in the case of P.S. Consequently the effect of P.S.
was probably of no physiological importance, although high concent-
rations of sphingomyelin were found in atheromatous plaques (Smith,
1965), and sphingomyelin-platelet interaction may play a role in the
pathology of atherosclerosis. Although these results partially
confirmed those of Kerr et al (1965) they did not confirm their
observations of P.E.-induced platelet aggregation even though several
different synthetic and natural P.E. fractions were tested.
The reduction in amplitude of the oscillations in the intensity
of light transmitted through P.R.P. caused by L.P.C. was characteristic
of a change in platelet shape from discocyte to spherocyte (O'Brien
and Heywood, 1966). A similar change in erythrocyte shape after
exposure of blood to L.P.C. is well known and has recently been
studied with the aid of steroscopic electron-microscopy (Piper et al,
1972). Hampton and Bolton (1969) were unable to show a shape change
in L.P.C.-treated platelets when they were examined by phase-contrast
microscopy. In an endeavour to demonstrate such a shape change in
platelets exposed to L.P.C. two aliquots of P.R.P., one of which had
been exposed to L.P.C. (0.4 11. mol mL -1) were centrifuged at 1500 g
for ten minutes and stained with osmic acid prior to investigation
by routine electron microscopy by The Experimental Pathology Department,
St. Mary's Hospital, London W.2. Unfortunately, using this technique
- 162 -
of fixing platelets after centrifugation resulted in extensive
damage to the untreated platelets, many of which showed extensive
loss of cytoplasmic contents. This was in contrast to the L.P.C.-
treated platelets which appeared relatively undamaged and more
loosely packed than did the control platelets. This difference was
most striking (fig, 19) particularly since both preparations were
treated in identical fashion except for the exposure of one
preparation to L.P.C. A possible explanation for this difference
would be to suggest that L.P.C.-treated platelets were spherical
since by analogy with the erythrocyte experiments described in Section
3, they would not pack down easily during centrifugation and would
consequently suffer less mechanical damage than the untreated
control platelets.
- 133 -
Figure 19: (i) Electronmicrograpn of centrifuged platelets from
normal platelet rich plasma.
(Magnification x 13,700).
- 16.4 -
Figure 19: (ii)Electronmicrograph of centrifuged platelets from
platelet rich plasma exposed to lysolecithin.
(Magnification x 13,700).
- 165 -
• Section 4 (b)
Effects of purified phospholipid preparations on platelet aggregation
initiated by adenosine diphosphate.
Experimental details and Results.
In a typical experiment 10 - 20 ml. of freshly prepared P.R.P. was
divided into a number of 1-ml. aliquots. Depending upon the
concentration of A.D.P. to which they are exposed, platelets can show
a range of aggregation responses including reversible and biphasic
irreversible aggregation (fig, 3 (1)). For each experiment it was
therefore necessary to determine the correct concentration of A.D.P.
required to produce the desired result.. This was done by trial and
error using the first few aliquots of P.R.P. Subsequently pre-warmed
aliquots of P.R.P. were transferred to the aggregation apparatus where
they were stirred, maintained at 37° and the transmitted light was
continuously recorded. After one minute 10 - 50 pl. of phospholipid
preparation (0.2-1.0 p mol.) was added to the P.R.P. using a microliter
syringe. This was followed one minute later by the addition of the
pre-determined concentration of A.D.P. and platelet aggregation was
recorded. To minimise any possible variation in platelet response
due to ageing of the P.R.P. all experiments (including those of
subsequent sections) were performed only between the first and second
hours after venepuncture. At least two control samples of P.R.P.
treated with 0.9 percent saline in place of phospholipid were
recorded, one at the start and one at the finish of each experiment.
-1 Lysolecithin. At concentrations of up to 1 pmol ml. L.P.C. had no
effects on reversible A.D.P.-induced platelet aggregation, nor did it
affect the minimum concentration of A.D.P. required to initiate
irreversible platelet aggregation.
-166-
When higher concentrations of A.D.P. were used to initiate
biphasic, irreversible aggregation L.P.C. (0.2-0,7 1mol ml.-1)
inhibited the second phase of aggregation without altering the
magnitude of the first phase (fig. 20 (i)). The degree of
inhibition was proportional to the log. of the concentration of
L.P.C. (Fig. 20 (ii)) and at the higher concentrations of L.P.C.
platelets deaggregated slowly. No difference in the inhibitory
activity of L.P.C. was apparent when it was dissolved in 5 percent
human albumin solution in place of 0.9 percent saline.
Other phospholipids. None of the other phospholiptd preparations
tested, including P.C., P.E„ P.S., P.I., Sphingomyelin, G.P.C.
and L.P.E., at final concentrations of up to 1 1.t mol ml,-1 affected
the reversible or irreversible aggregation of platelets exposed to
A.D.P.
Discussion
Only L.P.C. of the phospholipids tested, had any effect on
A.D.P.-induced platelet aggregation. Whilst it inhibited secondary or
release-phase aggregation it did not inhibit reversible aggregation
which suggests that L.P.C. does not directly prevent platelet-platelet
adhesion. The L.P.C. concentrations inhibiting seandary aggregation
were slightly higher than the observed plasma concentrations
(Section 2) but were similar to those of plasma incubated at 37° for
several hours.
In a subsequent series of experiments L.P.C. was added to
platelets at different times before and after the addition of A.D.P.
Although the inhibitory effect of L.P.C. was unaltered by pre-
incubation of the P.R.P. with L.P.C. for at least ten minutes, tho time
at which L.P.C. could be added after A.D.P. to inhibit the second
(ii)
Minutes
100
CIO
inhibition
(I )
- 167 -
06
o.d.
0.2
•1 •2 .3 -4 -5
LPC Concentration prnolint. Figure 20 (1): Inhibition of secondary platelet aggregation initiated by
adenosine diphosphate by pre-incubation of platelet rich plasma
with lysolecithin. The final concentrations of L.P.C. are shown -1
as F mol. mi. (ii): Dose-response curve for the inhibition by
lysolecithin of secondary platelet aggregation initiated by
adenosine phosphate.
- 168 -
phase of aggregation was critical, Provided that the L.P.C. was
added during the initial phase of A.D.P. induced aggregation it
was still effective as an inhibitor of the second phase of
aggregation (fig. 21). However, if it was added to P.R.P. after
the completion of the initial phase it was ineffective in inhibiting
the subsequent secondary phase of aggregation.
Secondary aggregation of platelets induced by exposure of
platelets to A.D.P. has been attributed to the release of platelet
constituents including A.D.P. which actually initiate the second
phase of aggregation (Haslam, 1967 : Mills et al, 1968) and it seems
likely that L.p.c. blocks the platelet release reaction rather than
inhibits directly the aggregation of platelets.
-169-
07
0-4 ad.
0.7
04
i.
ADP i
.
LPC -ADP 1 1 ....L....,\....„
1
iii.
ADP LPC
ADP
iv.
L.PC
M inutes Figure 21: Effect of lysolecithin added to platelet rich plasma at different
times during adenosine diphosphate-induced platelet aggregation.
(i) Control (no added L.P.C.).
(ii) L.P.C. added 30 sec. before A.D.P.
(iii) L.P.C. added during first phase of platelet aggregation.
(iv) L.P.C. added at the start of the second phase of aggregation.
- 17 -
Section 4 (c)
Effects of purified phospholipid preparations on platelet aggregation
initiated by 5-hydroxytryptanine.
Experimental details and Results.
The experimental details of these experiments were similar to those
described in Section 4(b) except that 5-FLT. was used as an aggregating
agent at a final concentration of 10 n mol ml.-1
At this concentration
5-H.T. initiated reversible platelet aggregation except in one
experiment in which biphasic platebt aggregation similar to that
induced by A.D.P. was recorded.
Phosphatidylserine, Phosphatidylethanolamine and Sphingomyelin. When
P.R.P. was pre-incubated with P.S. or P.E. (1 p mol.m1.-1) the effect
of 5-H.T, was potentiated and aggregation was not reversed after five
minutes. Both synthetic (di-oleoyl-P.E.) and a fraction of P.E. from
a bacterial source were effective in potentiating 5-H.T.-induced
aggregation. Potentiation of 5-H.T. -induced aggregation was seen in
samples previously aggregation by F.S., although there was no
potentiation of 5-H.T.-induced aggregation in P.R.P. samples previously
aggregated by sphingomyelin (fig, 22).
Lysolecithin. The addition of 5-H.T. to one sampb of P.R.P. resulted
in biphasic platelet aggregation which was similar to that produced
by A.D.P. Pre-incubation of aliquots of this sample of P.R.P. with
L.P,C. at concentrations greater than 0.15 r mol. ml,-1 abolished the
second phase of 5-H.T.-induced aggregation and inhibited the first
phase also at higher concentrations (fig. 23). At concentrations of
L.P.C.. below 0.15 p molml,-1 the first phase of 5-H.T.-induced
aggregation was unaltered but the rate of secondary aggregation was
reduced. Further studies showed that L.P.C. could also affect normal
06,PS 5-HT
- PE 5-HT 061
- 171 -
0-61
06] SM 5-HT
od.
Minutes Figure 22: Effect of phospholipid preparations added to platelet rich plasma
on reversible platelet aggregation initiated by 5-hydroxytryptamine.
Sphingomyelin (S.M.), phosphatidylserine (P.S.) and phosphatidyl-
ethanolamine (P.E.) were added to P.R.P. at a final concentration of
1 u mol.m1-1 prior to adding 5-H.T.
- 172 -
07
ad.
01 Minutes
Figure 23: Inhibition of irreversible plaid et aggregation initiated
by 5-hydroxytryptamine by pre-incubation of platelet rich
plasma with lysolecithin.
P.R.P. was pre-incubated with L.P.C. (final concentration in F mol mi.-1)
and 5-H.T. was added at the mark at a final concentration of 10 n mol.ml.-1
- 173 -
reversible 5-H.T.-induced aggregation. Low concentrations of L.P.C.
(0.1 - 0.2 p mol. ml.-1) potentiated reversible 5-H.T.-induced
aggregation, and at higher concentrations (0.3 - 0.5 p mol.ml. -1)
the potentiation was less marked. Concentrations of L.P.C. above
0.5 F mol ml.-1 inhibited reversible aggregation initiated by 5-H.T.
in a dose-dependent fashion.
Other phospholipids. None of the other phospholipids tested (P.C.,
P.I., L.P.E., and G.P.C.) had any consistent effects on reversible
5-H.T.-induced platelet aggregation.
Discussion
Platelet aggregation initiated by 5-H.T. was potentiated by
pre-treatment of P.R.P. with P.S. and P.E. resulting in irreversible
aggregation. However, the concentrations of P.S. and P.E. which had
this effect were very much higher than the normal concentrations of
these phospholipids in plasma.
One sample of P.R.P. exhibited irreversible platelet aggregation
when treated with 5-H.T. although Born (1968) has reported that
exposure of human platelets to 5-H.T. does not result in a release
reaction and that aggregation initiated by 5-H.T. was small, rapidly
reversed and never entered the second phase which can be induced in
human platelets by A.D.P. This argument has been shown to be incorrect
since 5-H.T. can in some cases induce biphasic platelet aggregation
suggesting that 5-H.T. also initiates the platelet release reaction.
Besterman and Gillett (1973) have published figures which showed that
5-H.T. did initiate biphasic aggregation in a small percentage of
platelet samples from normal subjects and patients with arterial
disease. The effect of L.P.C. on biphasic aggregation initiated by
5-H.T. was to inhibit the second phase of aggregation, probably by
blocking the platelet release reaction. However L.P.C. also inhibited
- 174-
the first phase of platelet aggregation at concentrations which
were higher than those inhibiting the second phase. This was
confirmed in normal samples of P.R.P. which did not give biphasic
aggregation and L.P.C. was found to potentiate reversible 5-H 4T.-
induced platelet aggregation at low concentrations and to inhibit
it at higher concentrations.
Platelet aggregation initiated by 5-H.T. differs from that
induced by A.D.P. since it is closely linked with the active
accumulation of 5-H.T. by the platelets by a mechanism which involves
specific 5-H.T. receptor sites (Born and Gillson, 1959 : Born, 1968)
It has been suggested thatexposure of cell membranes to L.P.C. leads
to "micelle" formation within the membrane lipoprotein structure
(Lucy, 1970). Low concentrations of L.P.C. may cause only slight
changes in the platdbt membrane organisation which might expose
more 5-H.T. receptor sites and potentiate 5-H.T. uptake and
associated pltelt aggregation. As the concentration of L.P.C. is
increased, so one would expect the extent of the membrane re-
organisation to increase, and this may progressively shut off or
block the 5-H.T. receptor sites and consequently inhibit 5-H.T.-
induced aggregation.
- 175 -
Section 4 (d)
Effects of purified phospholipid-preparations on platelet
aggregation initiated by adrenaline
Experimental details and Results
The experimental details for these experiments were identical to
those described for Section 4 (b) with the exception of the change of
aggregating agent. The final concentration of adrenaline added to
P.R.P. in these experiments was usually 2,5 - 5.0 n mol ml.-1
which
always resulted in the initiation of biphasic platelet aggregation
(Fig. 3 (ii)).
Lysolecithin. Exposure of platelets to L.P.C. at concentrations of
0.1-0.5 p mol ml. inhibited the second phase of adrenaline-induced
aggregation without affecting the magnitude of the first phase
(Fig. 24). The degree of inhibition was dose-dependant on the
concentration of L.P.C. If the concentration of adrenaline was
increased then the inhibitory effect of L.P.C. on the second phase
of aggregation was reduced (fig. 25).
L.P.C. was only effective as an inhibitor of the second phase
of adrenaline-induced aggregation if it was added before the
adrenaline or during the first phase of aggregation. It was
ineffective if added immediately before the second phase of
aggregation (Fig. 26).
In one experiment L.P.C. was added to citrated blood at a final
concentration of 0.4 p mol ml.-1
before the preparation of P.R.P.,
which was subsequently tested for adrenaline-induced platelet
aggregation, The second phase of aggregation initiated by adrenaline
(1 n mol ml,-1) was inhibited when compared with a.-similar sample of
P.R.P. prepared from an untreated sample of the same blood specimens.
- 176 -
0,6 ITC adrenaline
I
Minutes Figure 24: Inhibition of irreversible platelet aggregation initiated
by adrenaline by pre-incubation of platelet rich plasma with
lysolecithin.
Aliquots of P.R.P. were pre-incubated for one minute with L.P.C.
(final concentrations shown in p mol. ml.-1) and then challenged with
adrenaline (2.5 n mol. ml.-1)
- 177 -
1001
Inhibition
.05 •1 .2 .3 LPC Concentration p mot mt.
Figure 25:Dose response curves for the inhibition by lysolecithin of the
second phase of platelet aggregation initiated by different concentrations
of adrenaline. 111-411 adrenaline, 1.25 n mol. ml.-1
adrenaline, 2.5 n mol. m1.-1 410----0 adrenaline, 3.75 n mol.ml.-]
Minutes
Adren.
L PC. Adren.
1 Adren.
LPC iii. Adren.
LPC
iv.
0-5
02
0-5
o.d.
0-5
05
0-2
MOM
- 178 -
Figure 26: Effect of lysolecithin added to platelet rich plasma at
different times during adrenaline-induced platelet aggregation..
- 179 ^
Other phospholipids. Adrenaline-induced platelet aggregation was
unaffected by preparations of other phospholipids (including P.C.,
P.E., P.S., P.I., Sphingomyelin, G.P.C., and L.P.E.)
Discussion
The inhibitory effect of L.P.C. on the secondary phase of
adrenaline-induced aggregation resembled the effect described above
for A.D.P.-induced secondary platelet aggregation. As in the case
of A.D.P.-induced secondary aggregation adrenaline has also been
shown to release platelet A.D.P. which is probably responsible for
the second phase of aggregation (O'Brien, 1964: Haslam, 1967).
Thus the inhibitory effect of L.P.C. appears to result from the
inhibition or blockade of the platebt release reaction.
- 180 -
Section 4 (e)
Effects of purified phospholipid preparations on platelet aggregation
initiated by thrombin or collagen.
Experimental details and Results
The experimental details for these experiments were identical to
those described in Section 4 (b) except that thrombin (final
concentration 0,2 units ml.-1
) or a suspension of bovine collagen
were used as aggregating agents. Both aggregating agents always
initiated irreversible platelet aggregation although in the case of
collagen-induced aggregation there was a lag-time of one or two
minutes before response.
Lysolecithin: Pre-incubation of P.R.P. with L.P.C. (0.2-0.7 mol ml.-1)
for one minute inhibited both thrombin- and collagen-induced platelet
aggregation in a dose-dependent fashion. Collagen-induced aggregation
was abolished at the higher concentrations of'L,P.C. (fig. 28) but
increasing concentrations of L.P.C. revealed an underlying biphasic
nature to thrombin-induced aggregation without inhibiting the first
phase of aggregation (Fig. 27).
Other phospholipids:Pre-incubation of P.R.P. with P,C., P.E., Sphingomyelin,
P.S., P.I., G.P.C. and L.P.E. for one to five minutes had no effect on
platelet aggregation initiated by either thrombin or collagen.
Discussion.
Of the phospholipids tested, only L.P.C. had a consistent effect
on platelet aggregation. As in the case of irreversible aggregation
initiated by A.D.P. and adrenaline the effect of L.P.C. was to inhibit
secondary aggregatinn only. In all of these experiments inhibition of
irreversible platelet aggregation could be demonstrated by exposure of
platelets to concentrations of L.P.C. which were similar or slightly
07 Thrombin
Qd.
0.3- 0.5
01
- 181 -
Minutes Figure 27: Inhibition of thrombin-induced platiet aggregation by
pre-incubation of platelet rich plasma with lysolecithin.
Aliquots of P.R.P. were pre-incubated with L.P.C. (final
concentrations shown in p mol. ml.-1
) for one minute after which
aggregation was initiated by addition of 0.2 I.U. of thrombin.
0 LPC Collagen 5
0 1
- 182 -
Minutes Figure 28: Inhibition of collagen-induced platelet aggregation by
pre-incubation of platelet rich plasma with lysolecithin.
Aliquots of P.R.P. were pre-incubated with L.P.C. (final
concentrations shown as p molml.-1
) for one minute after which
aggregation was initiated by addition of 10 ils of collagen
suspension.
- 183 -
higher than the normal plasma concentrations (Section 2). Like
secondary aggregation initiated by A.D.P. and adrenaline, both
thrombin and collagen are thought to initiate aggregation as a
result of causing the platelets to release some of their constituents
including A.D.P. (Zucker and Borrelli, 1962 : Gainter et al, 1962 :
Hovig, 1963 : and Mills et al, 1968). Irreversible platelet
aggregation initiated by four chemically distinct aggregating agents
known to initiate a platelet release reaction (Mustard and Packham,
1970) was inhibited by L.P.C. in each case. Since reversible platelet
aggregation was unaffected by L.P.C. it seems very probable that the
inhibitory action of L.P.C. is on the platelet release reaction rather
than on the adhesion of platelets one to another.
Nishizawa et al (1969) have claimed that P.S. inhibited platelet
aggregation induced by collagen, thrombin and gamma-globulin-coated
particles. This effect of P.S. was believed to have been due to
inhibition of the platelet release reaction and thus resembles that
described above. However, in the experiments described above no
inhibitory effect of P.S. could be demonstrated on either thrombin-
or collagen-induced platelet aggregation. No explanation for this
difference can be suggested other than the unlikely one of chemical
differences between P.S. fractions investigated. However, since
the P.S. fraction used in the above experiments initiated platelet
aggregation itself it would be very unlikely to act as an inhibitor
of aggregation also.
It has generally been assumed that thrombin initiates irreversible
aggregation of platelets and that this occurs in a single stage reaction.
However, the inhibitory effect of L.P.C. on thrombin-induced aggregation
revealed that thrombin-induced aggregation was biphasic but without
- 184 -
the presence of inhibitor the two phases could not be distinguished.
Furthermore, in the presence of L.P.C. at concentrations of 0.3 -
0.5 p mol ml.-1 thrombin initiated reversible platelet aggregation,
which was not inhibited by further increases in L.P.C. concentration.
This observation supports the findings of Haslam (1967) who described
reversible thrombin-induced aggregation of human platelets as a process
occurring only in the absence of fibrin formation.
- 185 -
Section 4 (f)
Inhibition of the platelet release reaction by lysolecithin
Experimental details and Results
In a typical experiment P.R.P. was pre-incubated for one minute
with sufficient L.P.C. to completely inhibit irreversible platelet
aggregation initiated by A.D.P., adrenaline or collagen. Three
minutes after the addition of aggregating agent the stirring magnet
was removed from the P.R.P. which was then centrifuged at 1500 g for
five minutes. The supernatant platelet free plasma (P.F.PA) was
removed and aliquots (50 - 200 pi) were added back to fresh samples
of stirred P.R.P. and a recording of the transmitted light was
obtained in the usual way. Control samples of P.R.P. exposed to
A.D.P., adrenaline or collagen were allowed to aggregate for three
minutes after which the pre-determined amount of L.P.C. was added.
These samples were centrifuged under identical conditions to those
pre-treated with L.P.C. Aliquots of the supernatant plasma
(P,F.P ) were added back to fresh stirred samples of P.R.P.
Exposure of fresh samples of P.R.P. to aliquots of P.F.Dt
prepared from aggregated P.R.P. always resulted in platelet
aggregation which was either reversible or breversible. Exposure of
fresh P.R.P. to aliquots of P.F.P.A prepared from P.R.P. inhibited
with L.P.C. failed to produce platelet aggregation (fig. 29 (i) and
(ii).
In a second type of study samples of P.R.P. were exposed to
sufficient L.P.C. to completely inhibit irreversible aggregation
initiated by A.D.P. or adrenaline and were then exposed to a second
addition of aggregating agent. In all cases L.P.C.-treated platelets
were found to be still responsive to the second aggregating stimulus
provided by A.D.P. or adrenaline (fig. 30).
- 18G-
Adrenaline 0.7
ad.
0.1-1
A Pre-treated with LPC.
B. LPC added after
PFP aggregation
071 PFP 13
0.1- 10011 I I
Minutes Figure 29 (1): Inhibition of adrenaline-induced platelet release reaction
by pre-incubation of platelets with lysolecithin.
Platelet free plasma (P.F.P.) prepared from aggregated (B) or
inhibited (A) P.R,P. was added back to fresh samples of stirred P.R.P.
- 187 -
Collagen A. Pre-treated
with LPC 0-5
ad.
• IM
B. LPC added after aggregation.
200p
PFPD
-- 05 .4._ 0_5011 "\i"-"11001
200p1 I
Minutes Figure 29 (ii): Inhibition of collagen-inchiced platelet release reaction
by pre-incubation of platelets with lysolecithin.
Platelet free plasma (P.F.P.) prepared from aggregated (B) or
inhibited (A) P.R.P. was added back to fresh samples of stirred P.R.P.
(2.5 n mol.m1.-1) -1 (2.5 n mol.ml. )
ADP+LPC ADP 0.5-
(1 n mol. ml.-1)
+LPC (0.5 p mol. m1.-1)
.11•8=1111111•11111.1111171•1111
Mit
o.d.
01-
ADP Adrenaline pan
Minutes Figure 30: Aggregation of platelets previously inhibited with lysolecithin by subsequnt exposure to
adenosine diphosphate or adrenaline.
- 189 -
Discussion
Fresh samples of P.R.P. were not aggregated by supernatant plasma
prepared from P.R.P. pre-treated with L.P.C. and subsequently also
with an aggregating agent. This was in marked contrast to the
aggregating effects of supernatant plasma prepared from pre-aggregated
P.R.P. post-treated with L.P.C. Clearly this difference was not due.to
transfer of L.P.C. to the fresh P.R.P. since both samples of super-
natant plasma contained the same amount of added L.P.C. For similar
reasons the aggregating effect of P.F.P.B when added to fresh P.R.P.
was not due to the transfer of the original aggregating agent which
would in any case be too dilute to initiate aggregation. Furthermore,.
aggregation initiated by P.F.P.B prepared from collagen-aggregated
P.R.P. was both immediate and reversible and therefore not due to the
transfer of collagen which is known to initiate only irreversillth
aggregation and that only after a lapse of several minutes. Thus the
aggregating effects of the control supernatant plasma samples must be
due to the release of intrinsic platelet aggregating agents during
irreversible aggregation and that in the case of P.R.P. pre-treated
with L.P.C. to inhibit irreversible aggregation, no such release
reaction occurred. Thus the effect of L.P.C. was to inhibit the
platelet release reaction which provides the aggregating stimuli for
irreversible aggregation (O'Brien, 1964 : Haslam, 1967). This
conclusion was supported by the observation that platelets pre-treated
with L.P.C. to inhibit irreversible aggregation were sensitive to
further exposures to A.D.P. or adrenaline.
Perhaps the inhibition of the platelet release reaction by
L.P.C. could be best measured directly by an estimation of the
amounts of platelet A.D.P. or 5-H.T. or other platelet constituents
liberated daring platelet aggregation. However, the apparatus or
- 190-
methods to make this possible were unavailable. Nevertheless, the
effect of the platelet release reaction in vitro is to initiate
irreversible aggregation (O'Brien, 1964 : Haslam, 1967) and therefore
exposure of fresh platelets to plasma in which platelets have been
irreversibly aggregated or inhibited, can be used to demonstrate
the presence or absence of the platelet release reaction. Recent
work reported by Joist et al (1973) has confirmed the inhibition of
irreversible platelet aggregation by L.P.C. (Besterman and Gillett,
1971) and these workers also confirmed the inhibition of collagen-
or thrombin-induced platelet release reactions by L.P.C.
- 191 -
Section 4 (g)
A comparison of the effects of saturated and polyunsaturated
lysolecithin fractions on platelet aggregation.
Experimental details and Results.
The experimental details for this study were similar to those
described for Section 4 (b), and A.D.P., adrenaline and collagen
were used as aggregating agents, The composition of the saturated
and polyunsaturated fractions of L.P.C. has already been given
(table 2).
-1 At final concentrations of 0.4 - 1.0 F mol ml. saturated L.P.C.
inhibited the second phase of A.D.P.- and adrenaline-induced platelet
aggregation and inhibited collagen-induced aggregation. Polyunsaturated
L.P.C. had no inhibitory effect on platelet aggregation except at
concentrations of 0.8 u mol ml.-1
or above when slight inhibition of
i3reversible aggregation was observed (fig. 31).
Discussion
The results indicate that there was a considerable difference in
inhibitory activity of saturated and polyunsaturated L.P.C. fractions
on platelet aggregation. Polyunsaturated L.P.C. produced only slight
inhibition of irreversible platelet aggregation at high concentrations
(0.8 - 1.0 i mol ml.-1). Saturated L.P.C. was more effective in
inhibiting irreversible platelet aggregation initiated by A.D.P.,
adrenaline or collagen and because the polyunsaturated L.P.C. fraction
contained eleven percent of saturated L.P.C. its weak inhibitory
activity could probably be attributed to this contamination with
saturated L.P.C. Ideally this study should have been repeated with
synthetic L.P.C. analogues of homogenous composition. However, such
compounds were not commercially available.
100
50
0
- 192 -
1 I
02 06 10 [LPC]( p mot mil. )
Figure 31: Effects of saturated and polyunsaturated lysolecithin
fractions on collagen-induced platelet aggregation.
0.----110 saturated L.P.C. fraction,
polyunsaturated L,P,C. fraction;
- 193 -
Nevertheless it was apparent from the results that inhibitory
activity was associated with the surface activity of L.P.C. Several
investigations of the haemolytic properties of L.P.C. fractions with
different acyl-side chains showed that stearoyl-L.P.C. and palmitoyl-
L.P.C. were the most potent haemolysins and that unsaturated L.P.C.
fractions had much weaker activity (Gottfried and Rapport, 1963 : Reman
et al, 1969). The experimental effects of saturated and polyunsaturated
L.P.C. were also in agreement with these results (Section 3) indicating
that only saturated L.P.C. has profound effects on cell behaviour.
Differences in the effects of saturated afilpolyunsaturated L.P.C. on
red cell properties have been discussed above (Section 3) and this
application of Lucy's (1970) model for membrane penetration by L.P.C.
would apply equally to platelet membranes.
- 194 -
Section 4 (h)
Effect of lysolecithin on platelet adhesiveness.
The effect of exposing whole.blood to L.P.C. or P.C. on the
retention of platelts during the passage of blood through columns
of packed glass beads has been investigated.
Experimental details and Results.
In a typical study three 5-ml. aliquots of fresh citrated blood
were taken and 0.5 ml. of 0.9 percent saline or 0.5 ml. of L.P.C. or
P.C. suspension were added. The final concentration of L.P.C. or
P.C. added was 0.3 F moi ml.-1 Initial platelet counts were made
from duplicate diluting pipettes for each. sample. The blood samples
were then passed at a constant flow rate through a cOlumn of packed
glass beadso and the first five drops of blood to emerge from each
sample were pooled. Duplicate platelet counts were made from
different pipettes for each sample of emergent blood. The results
were expressed as platelet 'adhesiveness' calculated as the
percentage of blood platelets retained by passage through the glass
beads.
Exposure of blood to L.P.C. significantly reduced the platelet
adhesiveness although P.C. had no effect (table 15).
- 195-
Table 15.
Effects of lysolecithin and lecithin on platelet adhesiveness
Study No. Initial count (x 10 3)
Platelet adhesiveness (70 Control +L.P.C. +P.C.
1 179 31 19 33
2 102 47 44 49
3 228 49 34 47
4 204 40 21 42
5 183 35 13 32
Mean + WO. - 40+8 ..._ 26+ 12 12 41 + 8
Significance(P-value) - - 0.05<p<0.1 NS
Discussion
The concentration of L.P.C. added to whole blood in this study
was sub-haemolytic to ensure that no A.D.P. was released by damage
to erythrocytes. This concentration of L.P.C. significantly reduced
the retention of platelets in the glass bead columns. This result
was in agreement with the effects of L.P.C. on platelet aggregation
in vitro. Since L.P.C. only inhibits secondary platelet aggregation
and, the platelet release reaction it seems likely that L.P.C.
reduces platelet 'adhesiveness' by inhibiting the platelet release
reaction of platelets ddhercEtng to glass surfaces. Such a mechanism
would prevent a chain reaction of platelet aggregation initiated
by platelet release of A.D.P. and other agents.
Sumary
It has been shown that saturated L.P.0 blockelthe platelet
release reaction and inhibited irreversible platelet aggregation
initiated by A.D.P., 5-H.T. adrenaline, thrombin and collagen and
reduced platelet adhesiveness. A polyunsaturated fraction of L.P.C.
- 196 -
was ineffective in inhibiting platelet aggregation and adhesiveness.
No other phospholipid which was tested including P.C., P.E., L.P.E.,
P.S., P.I., G.P.C., and sphingomyelin demonstrated any inhibitory
activity on platelet aggregation, although at high concentrations
P.S. and sphingomyelin initiated platelet aggregation.
- 197 -
Section 5
Influence of small doses of heparin administered intravenously in man
on plasma lysolecithin formation, erythrocyte sedimentation and platelet
aggregation.
Introduction
Berlin and co-workers (1969c) have demonstrated a significant
increase in L.P.C. formation in incubated plasma collected within
minutes of intravenous administration of 5,000 or 2,500 units of
heparin. Patients suffering from ischaemic heart disease and acute
myocardial infarction have low plasma levels of L.P.C. (Section 2,
P.P..135-9). The pathological significance of low levels of plasma L.P.C.
in ischaemic heart disease is unknown although L.P.C. acts as an
inhibitor of platelet aggregation in vitro (Section 4, p.p.165-9),
A study of platelet aggregation before and during heparin treatment
may provide data on the effects of altered L.P.C. metabolism in vivo
on platelet function. Accordingly the effects of small doses of
heparin administered to patients free of ischaemic heart disease have
been studied, including L.P.C. formation, L.C.A.T. activity,
erythrocyte sedimentation and platelet aggregation. A comparison of
the effects of heparin prepared from hog-mucosa and from ox-lung has
been attempted and the in vitro effects of both heparin preparations
on L.P.C. formation, erythrocyte sedimentation and platelet aggregation
have been studied. The results of these investigations are discussed
in relation to the therapeutic and prophylactic properties of heparin
in thromboembolic disease.
- 198 -
Section 5 (a)
Lysolecithin formation in pre-and post-heparin plasma.
Experimental details and Results
In a typical study blood samples were taken immediately before
and fifteen minutes after intravenous administration of 2,500 or
5,000 units of mucous heparin. Plasma phospholipids were estimated
in both samples before and after six hours incubation at 37° without
added substrates. Figure 32 shows a typical T.L.C. separation of
pre- and post-heparin plasma phospholipib from one .such representative
study in which 5,000 units of heparin were administered.
There were no consistent differences in total phospholipid or
individual phospholipid concentrations between pre- and post-heparin
plasma samples prior to incubation, although in about half of the post-
heparin samples there appeared to be a slight increase in L.P.C.
concentrations of 0.02 - 0.05 F mol ml.-1
All post-heparin samples,
including two from studies in which only 1,000 units of heparin were
administered, demonstrated increased formation of L.P.C. and increased
degradation of P.C. after incubation-. There were no consistent changes
in total phospholipid, P.E and sphingomyelin concentrations during
incubation of pre- and post heparin plasma. The results of 9 studies
of 2,500 units of heparin and 11 studies of 5,000 units of heparin are
shown in table 16. Both concentrations of heparin caused quantitatively
similar increases in lecithinase activity suggesting that there was no
direct relationship between heparin dosage and activity of post-heparin
L.P.C. formation.
- 19f) -
4+ 3
---- Solvent front
P.E.
P.C.
Sphingomyelin 0111•■••••■
L.P.C.
Origin
L J Figure 32: Plasma phospholipids of unincubated and incubated pre- and
post-heparin plasma samples separated by thin layer chroNatography.
The chromatogram was developed in chloroform:acetone:methanol:acetic acid:
water (50:20:10;10:5, by vol.) and the phospholipid bands visualised by exposure
to iodine vapour.
I. Unincubated pre-heparin plasma. 3. Unincubated post-heparin plasma.
2. 6 hour-incubated pre-heparin plasma. 4. 6-hour-incubated post-heparin
plasma.
- 200 -
Table 160
Effects of intravenous heparin administration on lysolecithin
formation and lecithin degradation in incubated plasma.
Post-heparin plasma samples were taken 15 minutes after
administration of heparin.
Heparin dosage L.P.C. formation P.C. degradation
(units) (F mol/L/h.) (F mol/L/h)
2, 500
Pre-heparin 41 + 10E1 45 + 13 _ _
Post-heparin 62 + 10 59 4. 11 _ ..._
N =. 9 p <0.001b 0.01 < P < 0.02
5,000
Pre-heparin 33 + 9 38 + 11 _ _
Post-heparin 59 +13 60 + 13 _ _
N = 11 p <0.001 p <0.001
a. means + standard deviation
b, statistical significance calculated from Student's t-test.
- 201 -
Section 5 (b)
Lecithin : cholesterol acyl transferase activity in pre- and post-heparin
plasma.
Experimental details and Results.
In seven studies in which 2,500 units of mucous heparin were
administered intravenously to patients the rates of L.P.C. formation
during incubation of pre- and post-heparin plasma were compared with --
the L.C.A.T. activity measured by radio-isotopic assay of the same
samples.
The mean formation of L.P.C. during incubation of post-heparin
plasma was increased by 50 percent. There was no significant
difference in the rate of cholesterol esterification in post-heparin
plasma (Table 17), These results indicate that increased L.P.C.
formation in incubated post-heparin plasma was not due to increased.
L.C.A.T. activity.
Table 17: Effect of intravenous heparin administration on lysolecithin
formation and lecithin:cholesterol acyl transferase activity.
L.P.C. formation
(p mol/l/h.)
Cholesterol esterification
(p mol/l/h.)
Pre-heparin
Post-heparin
N = 7
43 4- 11a
64 4 13 _
0.001<p <0.01°
71 4. 10b
_
73 4- 11 _
NS
a Means 4- standard deviation
b Means 4. standard error of the mean
c Statistical significance calculated from Student's t-test
- 909 -
Section 5 (c)
Effect of protamine sulphate on the formation of lysolecithin
in incubated pre- and post-heparin plasma.
Experimental details and Results.
In five studies in which 5,000 units of heparin were administered
intravenously to patients, the rates of L.P.C. formation in pre7 and
post-heparin plasma incubated with the heparin antagonist, protamine
sulphate, (1 mg ml.-1) were compared. Control samples of plasma were
incubated with an aliquot of sterile water in the place of protamine
sulphate solution,
Mean L.P.C. formation was increased by 58 percent in incubated
post-heparin plasma without added protamine sulphate. By contraat,
no significant increase in L.P.C. formation was demonstrated in
post-heparin plasma incubated with protamine sulphate when compared
with similarly treated samples of pre-heparin plasma (Table 1B),
-1 Table 18: Effect of protamine sulphate (lmg ml. ) on the formation of
lysolecithin in incubated pre- and post-heparin plasma.
L.P.C. Formation (p mo1/1/h.)
Control + Protamine sulphate
Pre-heparin 38 + 9a 41 + 13 — —
Post-heparin 60 + 15 42 + 10 _
N = 5 0.02 <P <0.0513 N.S.
a Means + standard deviation
b Statistical significance calculated from Student's t-test
- 203-
Section 5 (d)
Effects of intravenous heparin administration on dextran-stimulated
erythrocyte sedimentation.
Experimental details and Results.
In six studies in which either 2,500 or 5,000 units of heparin
were administered intravenously the dextran-stimulated E.S.R. was
measured using pre- and post-heparin blood samples. 0.5 ml, of
packed washed erythrocytes was resuspended in 0.7.ml. of autologous
plasma and 0.3 ml. of a 2 percent solution of dextran (average molecular
weight 150,000) in 0.9 percent saline. Duplicate sedimentation tubes
for pre- and post-heparin blood were set up and read at 10 minute
intervals for up to two hours.
In all six studies there was a significant reduction in the E.S.R.
in the post-heparin samples when compared with pre-heparin erythrocytes
resuspended in pre-heparin plasma. Fig. 33 shows the E.S.R. curves
for pre- and post-heparin blood in one typical study.
5°1 o Pre -
0 Post-
30 Minutes
- 204 -
Figure 33: Reduction of dextran-stimulated erythrocyte sedimentation
following intravenous administration of 2,500 units of heparin.
The post heparin blood sample was taken 15 minutes after
intravenous administration of 2,500 units of heparin.
- 205 -
Section 5 (e)
Effects of intravenous heparin administration on platelet aggregation
Experimental details and Results
In a typical experiment blood samples were taken immediately
before and 15 minutes after intravenous administration of 2,500 units
of mucous heparin. Samples of pre- and post-heparin P.R.P. were
prepared under identical conditions as regards speed and time of
centrifugation. Estimates of P.R.P. platelet concentration were
performed on every sample. Several concentrations of collagen,
adrenaline or A.D.P. were added to separate samples of pre-heparin
P.R.P. and a series of concentration-dependant aggregation curves
were recorded. The same concentrations of aggregating agent were
then tested on post-heparin samples of P.R.P. The same times of
testing, relative to time of venepuncture, were rigorously observed
in these comparative aggregation studies.
No significant variation between pre- and post-heparin platelet
counts was observed, and in all studies there was less than 5 percent
variation between individual pre- and post-heparin counts.
Both the initial rate and extent.of collagen-induced platelet
aggregation after four minutes were reduced in all post-heparin P.R.P.
samples when compared with pre-heparin samples tested under identical
conditions. By contrast, reversible A.D.P.-induced platelet aggregation
in post-heparin P.R.P. was unaltered or even slightly potentiated
(Fig. 34). In these studies it was noted that the reduction in post-
heparin aggregation was more apparent at concentrations of collagen
that initiated a maximal decrease in O.D. in pre-heparin P.R.P. which
was consistent with maximal irreversible aggregation. If the
- 206 -
concentration of collagen was further increased, but without increasing
the rate or extent of aggregation in pre-heparin P.R.P., the difference
in post-heparin aggregation was less apparent than at concentrations of
collagen that were just sufficient to initiate maximal aggregation of
pre-heparin P.R.P. Similarly if the concentration of collagen tested
on pre-heparin P.R.P. was reduced then there was very little difference
in the aggregation response in post-heparin P.R.P. (Fig, 34). At the
optimal concentration of collagen the reduction in the initial rate of
post-heparin aggregation was found to be consistent and statistically
significant (0.001< P<.0.01). The results of eleven studies with
collagen and three with adrenaline are shown in figure 35.
-1
PRE-HEPARIN
ner; pollagen %-iw ir&eitiosomber2sramirmodes.:1 0
20
ad.
0
30 -- 40 [it m
POST-HEPARIN Collagen
ADP ADP 0.6 10
20 2.0
mot ml 0-31
- 207 -
Minutes Figure 34: Typical platelet response to collagen and adenosine
diphosphate before and 15 minutes after intravenous administration
of 2,500 units of heparin.
0.4
0.2
- 208 -
collagen adrenaline
Minutes post-heparin
Figure 35: Rates of irreversible platelet aggregation initiated
by collagen or adrenaline before and after intravenous administration
of 2,500 units of heparin,
- 209 -
Section 5 (f)
Effects of heparins derived from different tissues on plasma lysolecithin
formation and platelet aggregation.
Experimental details and Results
Five studies were performed in which 2,500 units of heparin derived
from ox-lung (Upjohn Co.) were administered intravenously in place of
the hog-mucosal heparin (\Veddel Pharmaceutical or Riker Laboratories)
used in the previous studies. Plasma lysolecithin formation and
collagen-induced platelet aggregation were assessed as in previous
studies.
Plasma L.P.C. formation in post-lung-heparin samples was increased
by 30 - 50 percent compared to preheparin samples. The initial rate
of collagen-induced aggregation was reduced by a mean value of 50 per-
cent in post-lung-heparin P.R.P. These results are summarised in
table 19 and compared with the effects of hog-niucosal heparin recorded
in previous studies. From this data it was clear that there were no
significant differences between the effects of hog-mucosal and ox-lung
heparins on the parameters studied.
- 210-
Table 19$
Effects of hog-mucosal and ox-lung heparins on plasma lysolecithin
formation and platelet aggregation induced by collagen.
Hog-mucosal heparin
0 hour 15 min.
Ox-lung heparin
0 hour 15 min,
L.P.C. formation 41 + 10a
..... 62 + 10 47 + 7 64 + 8 _
p mol/l/h, (9) (9) (5) (5)
Mean differences +21 N.S.b
+17
Initial rate of collagen-induced aggregation
0.285 + 0.180 + 0,245 + 0.120 +
0.D./Min. (12) (12) (5) (5)
Mean differences -0.105 N.S. -0.125
a Mean values + standard deviation are shown with the number of
studies indicated in parentheses.
b No significant differences between effects of the two different
heparin preparations.
- 211 -
Section 5 (g)
Effects of heparin in vitro on plasma lysolecithin formation,
erythrocyte sedimentation and platelet aggregation.
Experimental details and Results.
Mucous heparin (1-10 units ml.-1) was added directly to aliquots
of normal (pre-heparin) plasma which were then incubated at 37° for
six hours. L.P.C. formation was compared in control plasma containing
no heparin and the heparin-treated samples. There was no obvious
difference either in L.P.C. formation or P.C. degradation in the
heparin treated samples compared with the controls.
The effect of mucous heparin on dextran-stimulated erythrocyte
sedimentation was investigated by adding heparin (1-10 units ml.-1)
to washed erythrocytes resuspended in autologous plasma containing
dextran. Sedimentation readings of duplicate E.S.R. tubes4rere
recorded at 10 minute intervals. No consistent effect of heparin on
the E.S.R. was noted, although in several tests 10 units ml.-1 of
heparin increased the E.S.R. relative to that of untreated resuspended
erythrocytes.
The effects of both hog-mucosal and ox-lung heparins on platelet
aggregation initiated by collagen or adrenaline were investigated.
Aliquots of P.R.P. were pre-incubated with heparin (0.5 - 100 units
ml.-1
) for one minute at 370 and then exposed to aggregating agent.
At concentrations of 5 units ml.-1
and above heparin inhibited
collagen-induced irreversible aggregation and the secondary phase of
adrenaline-induced aggregation. The effect was dose-dependant and
similar for both preparations of heparin (Fig. 36).
- 212 -
1001
0
50
c
0
0
1 10 100
[Heparin] I.Urre
Figure 36: Inhibition of collagen-induced platelet aggregation by
hog-mucosal or ox-lung heparins added to platelet rich plasma in vitro.
Mean % inhibition (.4- standard deviation) at different heparin
concentrations is shown for four studies with hog-mucosal heparin (0
and four studies of ox-lung heparin (0----0).
- 213-
DISCUSSION
These studies have demonstrated increases in both the formation
of L.P.C. and degradation of P.C. after intravenous administration of
2,500 or 5,000 units of heparin in man. The results confirm the
findings of Berlin et al (1969c) and are quantitatively similar. No
direct dependance of post-heparin lecithinase activity on heparin
dosage was apparent in the dosage range studied (1,000 - 5,000 units)
which may indicate that the enzyme is activated at a threshold
dosage of heparin, which Berlin suggests may be as low as 500 units.
Radio-isotopic assay of L.C.A.T. demonstrated that cholesteryl
ester formation and L.C.A.T. activity were not stimulated by heparin
administration. Since L.P.C. formation in native (pre-heparin) plasma
results from L.C.A.T. activity and was unaffected by heparin added in
vitro the increased formation of L.P.C. after heparinisation was not
due to activation of L.C.A.T. but to the release or activation of a
distinct enzyme in vivo. Incubation of post-heparin plasma with the
heparin antagonist, protamine sulphate, inhibited only the increased
L.P.C. formation and did not affect pre-heparin rates of L.P.C.
formation. This suggests that the enzyme responsible for post-heparin
L.P.C. formation is distinct from L.C.A.T. and is similar to lipo-
protein lipase, and Doizaki and Zieve (1968) have claimed that the
two post-heparin enzymes are one and the same.
The increased formation of L.P.C. in post-heparin plasma, which
presumably also occurs in vivo, might be expected to alter the plasma
concentrations of P.C. and L.P.C. in non-incubated post-heparin plasma.
Although sligit decreases and increases in the plasma levels of P.C. and
L.P.C. were observed in some post-heparin samples, the effect was not
consistent. It is probable that raised L.P.C. levels in response to
- 214 -
heparinisation if they occur at all, are extremely transient because
of the rapid metabolism of plasma L.P.C., for example, by pathways
described in erythrocytes (Mulder et al, 1965 : Mulder and Van Deenan,
1965), leukocytes (Elsbach et al, 1965) and platelets (Elsbach et
al, 1971).
Despite the absence of significant increases in L.P.C. levels
in nonincubated post-heparin plasma, the rate of irreversible
platelet aggregation and the E.S.R. were significantly reduced
after heparinisation. These effects were not due to the direct
action of heparin on platelets or erythrocytes, since heparin added
in vitro at equivalent concentrations to those in vivo ( 1 unit
ml.-1) had no effect on platelet aggregation or the E.S.R. Only at
concentrations of 5-100 units ml.-1 did heparin inhibit irreversible
platelet aggregation initiated by collagen or adrenaline. This was
in agreement with earlier observations of heparin effects on
platelets (O'Brien et al, 1969). Exposure of plates to L.P.C. in
vitro inhibited irreversible platelet aggregation initiated by
collagen and adrenaline, among other aggregating agents, but did not
affect reversible A.D.P.-induced aggregation (Section 4). L.P.C.
also inhibited dextran-stimulated erythrocyte sedimentation in
vitro (Section 3). There is thus a close similarity between the
direct effects of L.P.C. in vitro and the indirect effects of heparin
in vivo on platelet aggregation and on the E.S.R. Sire heparin in
vivo increases plasma L.P.C. formation there is a strong possibility
that changes in platelet and erythrocyte behaviour might result from
altered L.P.C. metabolism. Furthermore, it has been suggested that
post-heparin lecithinase is identical with lipoprotein lipase
(Doizaki and Zieve, 1968) which is known to be adsorbed onto platelet
- 215 -
membranes (Smith and Barboriak, 1967). Thus it appears likely that
post-heparin L.P,C, formation may occur at the platelet surface or
in the immediate plasmatic environment of the platelets. Such a
mechanism might then explain decreased irreversible platelet
aggregation after heparinisation, even in the absence of raised
plasma levels of L.P.C.
A comparison of the effects of hog-mucosal and ox-lung heparins
did not reveal any differences in the effects of these preparations
on post-heparin lecithinase activity or irreversible platelet
aggregation. Recent reports have been at variance over the -
relative potency of different heparins. Two studies which compared
the number of heparin units neutralized by protamine sulphate as a
function of heparin source and potency, showed significant
differences (Novak et al, 1972 : Lowary et al, 1971). A third more
comprehensive study showed differences tht were not significantly
consistent (Bangham and Woodward, 1970). Novak et al also claimed
that treatment of patients with ox-lung heparin resulted in decreased.
A.D.P.-induced aggregation when compared with the effects of hog-
mucosal heparin, although no differences in collagen-induced
aggregation were noted. The results of the present study do not
confirm Novak's findings and suggest that there is no difference in
the effects of hog-mucosal and ox-lung heparins on platelet
aggregation either in vivo or in vitro. Furthermore, double-blind
cross-over trials also failed to show any difference in the anti-
coagulant activity of these heparin preparations (Gomez-Perez, 1972 :
Baltes et al, 1973) and their biological activities are probably
identical.
- 216-
Summary
Intravenous administration of 2,500 or 5,000 units of hog-mucosal
or ox-lung heparin increased plasma L.P.C. formation by 30 - 70 percent
bt activating an enzyme distinct from L.C.A.T. Intravenous heparin
administration reduced the dextran-stimulated E.S.R., and inhibited
irreversible platelet aggregation initiated by collagen or adrenaline,
although it did not affect reversible A.D.P.-induced aggregation.
Heparin added to blood in vitro at concentrations similar to those
achieved in vivo did not affect L.P.C. formation, E.S.R. or platelet
aggregation. The indirect effects of heparin in vivo on E.S.R. and
platelet function are exactly similar to the effects of L.P.C. on
E.S.R. and platelet function in vitro. It has been suggested that
altered L.P.C. metabolism after heparin administration may be
responsible for inhibited E.S.R. and irreversible platelet aggregation.
- 217 -
Section 6
An analysis of plasma phospholipid levels and platelet aggregation
in women taking oral contraceptive preparations.
Introduction
It is now widely accepted that the use of oral contraceptive
preparations containing an oestrogen-progestogen.combination is
associated with an increased risk of thromboembolic disease
(British Medical Research Council, 1967 :BUttiger and Westerholm,
1971), Deep venous thrombosis, often with pulmonary embolism, has
been most frequently reported although there have been significant
reports of arterial thrombosis including cerebral, coronary and
retinal arterial thrombosis.
Increased platelet aggregation has been reported in women taking
oestrogen-nrogestogen oral contraceptives (Poller at al, 1969) but
not in women taking preparations with only progestogenic activity
(Poller et al, 1972). Long term oral contraceptive therapy has been
shown to decrease plasma L.P.C. levels (Brody et al, 1966) and
Svanborg (1968) has demonstrated that plasma L.P.C. levels are
decreased in women taking oestrogens but that progestogenic
compounds had essentially an opposite effect to oestrogens on plasma
lipids. More recently oral contraceptive therapy has been shown to
cause cyclical variations in the plasma L.P.C. level (Nicholls et al,
1971) although these workers did not distinguish between oral
contraceptives with either high or low progestogen content. L.P.C.
inhibits irreversible platelet aggregation in vitro (Section 4) and
decreased plasma levels of L.P.C. in oral-contraceptive treated-women
might be expected to alter platelet function. In this connection
- 218 -
the role of decreased plasma L.P.C. levels as a contributory factor
leading to an increased risk of thromboembolic disease can not be
excluded. The work described below is an analysis of plasma
phospholipid levels and collagen-induced platelet aggregation
during the menstrual cycle of non-treated women and women taking
widely prescribed oral contraceptive preparations.
Experimental details and Results.
A total of twenty-two women were included in this study of
,who whom seven/were not taking oral contraceptive preparations acted as
controls. The remainder consisted of ten women taking low-progestogen
preparations (e.g. Minovlar) and five women taking high-progestogen
preparations (Anovlar or Gynovlar) (see table 1: Chapter 2).
Mid-morning blood samples were taken from each woman at least
three hours after her last meal on day-4 and day-25 of the same
menstrual cycle. Total,cholesterol, total and individual phospho-
lipids were estimated in plasma samples before and after incubation
at 37o for six hours. P.R.P. was prepared in the usual way from
citrated blood samples and platelet aggregation induced by 10 p1 of
standardised collagen suspension was recorded exactly one hour after
the time of venepuncture. In a few cases sufficient P.R.P. was
available for aggregation initiated by A.D.P. at a final concentration
of 1 n mol ml.-1 to be recorded. Platelet counts for P.R.P. were not
performed but instead the optical density of the P.R.P. as recorded
on the correctly adjusted aggregation apparatus was used as an index
of platelet concentration.
The analysis of plasma cholesterol levels, phospholipid
concentrations and plasma L.P.C. formation is shown in table 20.
No significant changes in plasma cholesterol, total or individual
phospholipids or plasma L.P.C. formation between samples from day-4
Table 20; Plasma cholesterol and phospholipid levels and lysolecithin formation during the menstrual cycle
of untreated women and women treated with low-progestogen and high-progestogon oral contraceptives.
Group Age
Day (years) Cholesterol
-1, ) Plasma concentrations (II mol ml.
T.P.L. P.E. P.C. S.M. L.P.C.
LPC formation
p mol 1-1hr.-1
CONTROL 20.6 +
5.60 + 1.19
5.67 + 1.28
N.S.
5.91 + 1.05
6.34 + 1.10
N.S.
5.75 + 1.76 _
5.53 + 1.53
N.S.
2.92 + 0.59
2.86 + 0.42
N.S.
3.14 + 0.45
3.36 + 0.41
0.2 <P <0.3
3.14 + 0.53 .._
3.02 + 0.35
N.S.
0.12 + 0.05
0.12 + 0.05
N.S.
0.15 + 0.04
0.18 + 0.05
0.1 <P < 0.2
0.13 + 0.03 ....
0.12 + 0.04
N.S.
1.97 + 0.41
1.89 +
N.S.
2.19 + 0.37
2.41 + 0.37
0.1 <P< 0.2
2.13 + 0.33 _
2.03 + 0.26
N.S.
0.63 + 0.15
0.67 + 0.13 ..... N.S.
0.59 + 0.08
0.63 + 0.08
M.S.
0.69 + 0.12 ....
0.68 + 0.10
N.S.
0.20 + 0.05
0.20 + 0.05 - N.S.
0.21 + 0.03
0.14 + 0.03
p <0.001
0.19 + 0.02 ....
0.20 + 0.02
N.S.
40 + 10 ....
43 + 10
N.S.
45 + 10 ..._
55 + 10 ..... 0.026)0.05
53 + 15 ....
50 + 15 .._ N.S.
1.0
Day-4
N = 7
Day-25
Significance (P-value)
LOW PROGESTOGEN 22.3+ 3.4-
Day-4
N = 10
Day-25 -
Significance (P-value)
HIGH PROGESTOGEN 22.2+ 2.EC
Day-4
N = 5
Day-25
Significance (P-value)
- 220 -
and day-25 of the menstrual cycle were found for the untreated women
(controls) or for the women taking oral contraceptives with a high-
progestogen content. By contrast, changes in all parameters except
plasma sphingomyelin concentraions were found_in women taking oral
contraceptives with a lo'-progestogen content. Plasma cholesterol,
total phospholipd, P.E., P.C. and L.P.C.-formation were all
increased in this group of women at the time they were actively
taking their preparations when compared with their pre-treatment or
between-treatment values. Furthermore, there was a very significant
reduction of plasma L.P.C. during low-progestogen oral-contraceptive-
therapy.
No significant changes in platelet concentrations were apparent
between day-4 and day-25 for any of the three study groups. Collagen-
induced aggregation was not significantly changed at day-25 when
compared with day-4 for both the antrol and highprogestogen groups.
No changes in A.D.P.-induced aggregation were found in P.R.P. samples
from three control subjects and two women from the high-progestogen
group tested at day-4 and day-25. In contrast to these results, there
was a significant increase in the rate of collagen-induced platelet.
aggregation on day-25 when compared with day-4 in the group of women
taking low-progestogen oral contraceptives (Fig. 37). Furthermore,
in the low-progestogen group three samples of P.R.P. from a total of
six tested for A.D.P.-induced aggregation on day-4 exhibited
reversible platelet aggregation compared to only one sample from six
tested on day-25. This suggested a parallel increase in A.D.P.-
induced irreversible aggregation accompanying increased collagen-
induced aggregation.
- 221 -
Discussion
The results showed that oral contraceptive therapy with
preparations containing low-progestogen (e.g. Minovlar) increased
plasma cholesterol, total phospholipid, P.E. and P.C. and reduced
the plasma L.P.C. level. No similar changes occurred in untreated
women or in women taking oral contraceptive preparations which
contained three or four times more progestogen than did the low-
progestogen oral contraceptives. These results agree with those
previously published (Svanborg, 1968). The results also indicate
the antagonistic effect of oestrogens and progestogens on plasma
lipids. In the case of the high-progestogen oral contraceptive,
the oestrogenic and progestogenic effects on plasma lipids were
balanced since no net changes were found. However, in the low-
progestogen oral contraceptives the oestrogenic effect on plasma
lipids was apparent. There was no significant difference in plasma
phospholipids on day-4 for all three groups of women despite the
fact that some individuals had been taking oral contraceptive
preparations for more than a year previously. This supports
Nicholls (1971) observation that plasma L.P.C. levels varied
cyclically during the treatment cycle.
There were no changes in platelet aggregation in both the
control and high-progestogen groups although there was an obvious
association between decreased L.P.C. levels and increased platelet
aggregation in the group treated with low-progestogen oral
contraceptives (Fig. 37), Although these changes may have been
coincidental, there was a strong possibility that decreased L.P.C.
levels due to oestrogen-therapy may have influenced platelet function.
P< 0.001
0.3
0
- 292 -
P(0.005
4 25 4 25 4 25
Control. Minoviar. Gynovtar.
Figure 37.
25 4 25 4 25
Changes in plasma lysolecithin concentration and in the rate
of collagen-induced platelet aggregation during the menstrual
cycle of women taking oral mntraceptives.
- 223 -
This would be expected if L.P.C. acts as an inhibitor of platelet
aggregation in vivo as it does in vitro since any decrease in
plasma L.P.C. might be expected to result in increased irreversible
platelet aggregation. The results from this study suggest that
such a mechanism does apply during low progestogen oral contraceptive
therapy and consequently may contribute towards the thrombogenic
effects of oral contraceptive therapy.
- 224 -
Section 7
An analysis of plasma phospholipid concentrations and of erythrocyte
flexibility during pregnancy.
Introduction.
Significant alterations of both relative and absolute concentrations
of plasma phospholipids have been reported during normal pregnancy
(Svanborg and Vikrot, 1965a), The plasma concentrations of total
phospholipid, P.C. and P.E. were raised in a fashion which was
dependant upon the duration of amenorrhoea, whilst L.P.C. levels
declined during pregnancy and were 50 percent below normal at full
term. There was a return to normal phospholipid levels within days of
delivery (Svanborg and Vikrot, 1965b). Svanborg (1968) has attributed
the changes in plasma phospholipid levels during pregnancy to the
metabolic effects of high plasma oestrogen levels,
It has previaasly been shown that L.P.C. in vitro inhibits platelet
aggregation (Section 4) and decreases erythrocyte sedimentation and
flexibility (Section 3). Thus decreased plasma levels of L.P.C.
occurring during pregnancy might be expected to alter platelet and
erythrocyte properties, and indeed, to possibly contribute towards
the increased risk of thromboembolic disease associated with pregnancy,
An ideal investigation into the possible effects of altered
L.P.C. levels during pregnancy would include measurements of platelet
aggregation and erythrocyte flexibilith concurrently with
determination3of the plasma phospholipid concentrations in a group of
women studied at intervals during normal pregnancy. However, for
ethical reasons a longitudinal study of this nature was not possible
since it would necessarily involve the collection of large samples
of blood for platelet aggregation studies. Consequently the present
study has been restricted to an investigation of erythrocyte properties
- 225 -
and plasma phospholipid levels in blood samples taken from different
women at various stages during pregnancy. However, alterations of
the plasma phospholipid levels have been confirmed and these findings
have been correlated with erythrocyte behavioural changes during
pregnancy.
Experimental details and Results
Twenty-five pregnant women (average age, 24 years) who were
attending hospital ante-natal clinics or who had been admitted to
hospital at the onset of labour, were included in this study.
Sixteen women students and technicians (average age, 22 years) who
were neither pregnant nor receiving oral contraceptive treatment
were included in the study as a control group. Of this latter
group, only six subjects provided blood samples for tests of
erythrocyte behaviour. Each subject was seen once only when blood
samples were taken. Platelet free plasma was prepared from an
aliquot of fresh blood and used for estimations of cholesterol,
total and individual phospholipid concentrations. Further aliquots
of blood were used for the dtermination of clinical E.S.R.
(Westergren), whole blood viscosity measured at a shear rate of
6 sec.-1 and erythrocyte flexibility (E.P.R.). The E.P.R. results
were adjusted to values corresponding to an haematocrit of 40
percent.
The mean values of plasma cholesterol, total and individual
phospholipid concentrations for bbth the control group and the
pregnancy group are shown in table 21. The absolute concentvtions
of cholesterol, total phospholipid, P.E., P.C., and sphingomyelin
were all significantly higher in the group of pregnant women as
compared with the control group. Conversely, the plasma level of
L.P.C. was significantly reduced during pregnancy, Furthermore,
Table 21: Plasma concentrations of cholesterol and phospholipids in pregnant and non-pregnant women
Group Total cholesterol
p mol ml,-1 Total phospholipid
p mol ml.-1 Individual phospholipids p mol ml,-1
P.E. P.C. Sph. L.P.C.
Non-pregnant
N = 16 4.92 +1,18 -- 2.70 + 0,33 _ 0.11 + 0,04 1.87 + 0.28 0.56 + 0.14 0.19 + 0.03
Pregnant
N = 25 6.73 + 1.25 3.34 + 0.31 - 0.17 + 0.04 2.33 + 0.23 0.70 + 0.09 0.12 + 0.03
Significance
(p - value) p <0.001 p <0.001 p <0.001 p <0.001 p <0,005 p < 0.001
1st trimester 5,26 3.07 0.14 2,16 0.62 0.14 N = 5
2nd trimester 6.97 3,36 0,16 2.38 0,68 0.12 N = 8
3rd trimester 7.20 3.43 0.19 2,37 0.76 0.10 N = 12
- 227 -
the mean values of cholesterol, total phospholipid, P.E., P.C.
and sphingomyelin during pregnancy were higher in each successive
trimester and a similar but opposite trend for L.P,C, levels was
alSo apparent (Table 2l),
The mean E.S.R. and E.P.R. values were signifiantly increased
for pregnant women compared with the control group, and the mean
values of these measurements increased in successive trimesters
(Table 22). The mean whole blood viscosity measured at low shear
rate was significantly lower in the group of pregnant women although
there was no apparent trend to suggest that the decrease was_closely
linked with the duration of pregnancy.
Discussion
This investigation has shown that plasma cholesterol, total and
individual phospholipid concentrations were significantly altered
during normal pregnancy and the present results were quantitatively
in good agreement with previously published values (Svanborg and
Vikrot, 1965 a: Svanborg, 1968). Furthermore, the relationship
between duration of pregnancy and decreased L.P.C. levels and
increased plasma cholesterol, total phospholipid, P.E., P.C. and
sphingomyelin has been confirmed. ■
It is well known that the E.S.R. is increased from the end of
the first trimester of pregnancy until three or four weeks post-
partum (Wintrobe, 1936). The E.S.R. results presented in this study
did reflect this change although, overall, the E.S.R. values were
high both for control subjects and for pregnant women. This may
have been due to acceleration of the E.S.R. by the heparin used as an
anticoagulant in place of the usual sodium citrate. The effects of
pregnancy on the E.P.R. and on blood viscosity have not been reported
elsewhere although the preeent results were in good agreement with
- 228.-
Table 2T2
Mean values for erythrocyte sedimentation and packing rates and
whole blood viscosity in pregnant and non-pregnant women.
Group Clinical E.S.R.
(mm. hour-1)
E.P.R. 1,
(% min.- )
Blood viscosity (centipoises)
Non-pregnant 15 4. 7 9.0 4- 1.9 9.7 + 1.0 — N = 6
Pregnant 58 + 26 14.2 I- 3.0 7.5 + 0.7 — N = 25
Significance
(p-value)
p <0,001 p <0.001 p <0.001
1st trimester 39 11.9 7.3
N = 5
2nd trimester 61 13.7 7.7
N = 8
3rd trimester 65 15.5 7.4
N = 12
- 999 -
those of a larger study yet to be published (Myers and Dupont,
personal communication), The results showed that the E.P.R.
increased relative to the duration of pregnancy which suggests
that the erythrocytes become more flexible as pregnancy progresses
(Sirs, 1970). Since the effect of L.P.C. added to blood in vitro
was to render the erythrocytes less flexible and to decrease the
E.P.R. (Section 3), it was of interest to attempt a correlation
between altered E.P.R. and L.P.C. levels in both the control and
pregnancy subjects. There was a striking and significant negative
correlation (p (0.001) between L.P.C. level and E.P.R. (Fig. 38).
This finding suggests that there is a definite relationship between
reduced plasma L.P.C. and erythrocyte flexibility, although other
factors such as altered plasma protein levels may also influence
erythrocyte behaviour. The exposure of blood to L.P.C. also
influenced blood viscosity and inhibited the E.S.R. (Section 4).
However, there was no apparent close correlation between L.P.C. levels
and either viscosity or E.S.R. which suggests that factors other than
decreased plasma L.P.C. levels contribute towards the alterations of
erythrocyte properties during pregnancy.
Since a significant negative correlation between plasma L.P.C.
levels and E.P.R. has been demonstrated in the present study, it is
very probable that a similar negative relationship may exist between
L.P.C. level and altered platelet behaviour sincelexposure of plates
to L.P.C. in vitro decreased platelet aggregation. Furthermore, the
association of altered irreversible platelet aggregation and altered
plasma L.P.C. levels after the administration of small doses of
heparin (Section 5) or of oestrogen-progestogen oral contraceptives
(Section 6) suggests that this argument may indeed be valid.
[1..PC]
010
- 230 -
r = 0.790
a a a
a
o a
3
0
10 15 20 EPR
Figure 38:
Correlation between plasma lysolecithin concentrations
and erythrocyte packing rate in pregnant and non-pregnant
women.
- 231 -
CHAPTER 4
GENERAL DISCUSSION
and
CONCLUS ION
- 232-
General Discussion
The results for the plasma phospholipid levels reported in the
present study include investigations of the apparently healthy
population, of patients with various pathological conditions, of
women during pregnancy or oral contraceptive therapy and of healthy
individuals studied before and after intravenous heparin admin-
istration, As such, the results represent the largest study made to
date of the concentrations of individual phospholipid fractions in
human plasma. Several of the pathological groups investigated,
including both acute and chronic ischaemic heart disease, have not
previously been studied and there has been a paucity of similar data
with respect to acute myocardial infarction and treatment of women
with oral contraceptives.
The study has confirmed that the plasma concentration of L, P.C.
in peripheral arterial disease (Kunz et al, 1970), acute myocardial
infarction (Berlin et al, 1969b), pregnancy (Svanborg and Vikrot,
1965a) and women taking certain types of oral contraceptives (Brody
et al, 1968), were significantly decreased. The effect of intravenous
heparin administration on the rate of formation of L,P,C, in incubated
plasma (Berlin et al, 1968c) has been confirmed, An increased plasma
level of P.E. has been reported in patients suffering from vascular
disease (Kunz et al, 1970) although this was not verified in the
present study,
Decreased plasma L.P.C. levels were the most consistent differences
in the phospholipid profiles of male patients suffering from peripheral
arterial disease, ischaemic heart disease and acute myocardial
infarction, The lowest absolute levels of L,P,C. were associated
with patients studied within 48 hours of the onset of proven myocardial
- 233 -
infarction or acute chest pain (acute ischaemic heart disease) for
which a thrombotic episode was the most probable cause. In all of
the cases of acute myocardial infarction and in many of the patients
assigned to the acute ischaemic heart disease group, there was
evidence of decreased formation of L.P.C. in plasma incubated in
vitro which suggested that decreased levels of L.P.C. in these
patients may have been partially due to decreased formation in plasma
in vivo. Patients suffering from chronic ischaemic heart disease
and chronic peripheral arterial disease did not exhibit any
differences in the rate of L.P.C. formation in incubated plasma
although the plasma concentrations of L.P.C. were significantly
decreased.
Low plasma levels of L.P.C. were found both in women taking low7
progestogen oral contraceptives and in women during normal pregnancy.
It is well known that both pregnancy and oral contraception are
associated with an increased risk of thromboembolic disease in
women (British Medical Research Council, 1967 ; Blittiger and Westerholm,
1971). It is therefore evident that low plasma levels of L.P.C. are
present in several different populations known to have an increased •
risk of thromboembolic disease (peripheral arterial disease, chronic
ischaemic heart disease, pregnancy and oral contraception) as well as
in patients suffering from a recent acute thrombotic episode (acute
myocardial infarction, acute ischaemic heart disease), Furthermore,
it has been shown that intravenous heparin administration in man
increases the formation of L.P.C. in plasma in vitro and presumably
also in vivo. As well as its routine usage as an anticoagulant in
established thromboembolic disease, heparin has, in clinically small
doses, been used successfully as a prophylactic agent in the prevention
- 233 -
infarction or acute chest pain (acute ischaemic heart disease) for
which a thrombotic episode was the most probable cause. In all of
the cases of acute myocardial infarction and in many of the patients
assigned to the acute ischaemic heart disease group, there was
evidence of decreased formation of L.P.C., in plasma incubated in
vitro which suggested that decreased levels of L.P.C. in these
patients may have been partially due to decreased formation in plasma
in vivo, Patients suffering from chronic ischaemic heart disease
and chronic peripheral arterial disease did not exhibit any
differences in the rate of L.P.C. formation in incubated plasma
although the plasma concentrations of L.P.C. were significantly.
decreased.
Low plasma levels of L.P.C, were found both in women taking low-
progestogen oral contraceptives and in women during normal pregnancy.
It is well known that both pregnancy and oral contraception are
associated with an increased risk of thromboembolic disease in
women (British Medical Research Council, 1967 ; BUttiger and Westerholm,
1971). It is therefore evident that low plasma levels of L.P.C. are
present in several different populations known to have an increased
risk of thromboembolic disease (peripheral arterial disease, chronic
ischaemic heart disease, pregnancy and oral contraception) as well as
in patients suffering from a recent acute thrombotic episode (acute
myocardial infarction, acute ischaemic heart disease), Furthermore,
it has been shown that intravenous heparin administration in man
increases the formation of L.P.C. in plasma in vitro and presumably
also in vivo. As well as its routine usage as an anticoagulant in
established thromboembolic disease, heparin has, in clinically small
doses, been used successfully as a prophylactic agent in the prevention
- 234 -
of thromboembolic disease (Williams, 1971 : Kakkar et al, 1971).
There is thus an apparent association between the level of L.P.C.
in blood (or of its formation) and risk of thromboembolic disease.
This has been summarised in figure 39 in which a low plasma level of
L.P.C. indicates an increase in risk whereas an increase in L.P.C.
formation or in L.P.C. concentration points to a decreased risk of
thromboembolic disease.
Further results included in this thesis suggest that the
association between altered L.P.C. levels and high or low risk of
thrombotic disease is not merely coincidental, It was found that
exposure of blood platelets to L.P.C. at concentrations only slightly
higher than those found normally in plasma (0.2 - 0.5 p mol ml.-1)
inhibited irreversible platelet aggregation initiated by several
different aggregating agents including A.D.P„ adrenaline, thrombin
and collagen. Irreversible platelet aggregation initiated by these
aggregating agents is thought to occur as a result of the release of
A.D.P. and other substances from granular bodies within the platelets
themselves (Haslam, 1964). Subsequent experiments indicated that the
effect of L.P.C. was to block or inhibit this 'release reaction', and
this effect of lysolecithin has recently been confirmed by Joist et
al (1973), Other phospholipids were investigated although none showed
any inhibitory effects on platelet function.
The inhibitory effect of L.P.C. on platelet aggregation was first
reported by Besterman and Gillett, (1971) although other interactions
between L.P.C. and blood platelets had been previously described.
Kirschmann and co-workers (1963) showed that L.P.C. was strongly
adsorbed to the platelet mambrane. It was later shown that treatment
of platelets with L.P.C. altered their electrophoretic properties
(Hampton and Bolton, 1969) and mimicked the abnormal platelet electro-
L.C.A.T.
PROPHYLACTIC THERAPY FOR DEEP VENOUS THROMBIS -
Low risk of
thromboembolic
disease
ACUTE ISCHAEMIC HEART DISEASE
OR ACUTE MYOCARDIAL
INFARCTION
decreased
formation
Thromboembolic
disease
present
'ow
intravenous increased heparin :)0 formation
ANTICOAGULANT THERAPY IN THROMBOEMBOLIC DISEASE
v low plasma levels
post-heparin lecithinase
L.P.C. < A
P.C.
CHRONIC ISCHAEMIC.HEART DISEASE, PERIPHERAL ARTERIAL DISEASE, PREGNANCY OR ORAL CONTRACEPTIVE THERAPY
High or increased • risk of thromboembolic disease
Figure 39: Relationship between plasma lysolecithin level and risk of thromboembolic disease,
- 236 -
phoretic behaviour found in patients suffering from ischaemic heart
disease and peripheral vascular disease (Hampton and Mitchell, 1966b),
and in women taking oral contraceptive preparations (Bolton et al,
1968). These results suggested that there were perhaps increased
amounts of L.P.C. present in the blood of these groups of patients
and that the action of L.P.C. might be thrombogenic. - However, it
has been shown both in the present study and in earlier investigations
that decreased plasma levels of L.P.C. occur in ischaemic heart
disease, vascular disease and in pregnancy. The association of low
levels of plasma L.P.C. in various pathological or physiological
conditions associated with an increased risk of thromboembolic
disease may indicate that the inhibitory effect of L.P.C. on platelet
aggregation is important in vivo.
As well as its effect on blood platelets L.P.C. has been shown
to inhibit both erythrocyte sedimentation and erythrocyte packing
during centrifugation. Furthermore, L.P.C. has been shown to be
adsorbed to erythrocyte membranes (Klibansky et al, 1962 :
Klibansky and de Vries 1963). The action of L.P.C. on platelets and
on erythrocytes is due to its surface active properties since a
polyunsaturated (and non-haemolytic) fraction of L.P.C. (Roman et al,
1969), had no apparent inhibitory effects on either platelet
aggregation or on erythrocyte sedimentation.
An investigation of the importance of altered L.P.C. concentrations
in vivo on changes in platelet and erythrocyte properties is not without
difficulties. However, the problem has been investigated by studying
platelet aggregation and red cell behaviour during treatment with
heparin or oral contraceptives and during pregnancy when the
concentrations of L.P.C. in plasma are altered.
- 237 -
The increased formation of L.P.C. found in post-heparin plasma
was accompanied by decreased irreversible platelet aggregation
initiated by collagen or adrenaline and also by decreaselerythrocyte
sedimentation when-compared-with pre-heparin samples for the same
individual. Comparable concentrations of heparin (0.5 - 2 units ml.-1)
added to P.R.P. or erythrocytes in vitro did not decrease either
platelet aggregation or erythrocyte sedimentation. This showed that
the decrease in plait aggregation and erythrocyte sedimentation
was not due directly to the presence of heparin but to some indirect
effect. The inhibitory effect of L.P.C. on red cell and platelet
properties suggests that the indirect effect of heparin which
decreased both aggregation of platRets and erythrocyte sedimentation,
was mediated by the increase in L.P.C. formation in the post-heparin
plasma. However, in many post-heparin plasma samples the absolute
concentrations of L.P.C. were no higher than those of pre-heparin
plasma. Smith and Barboriak (1967) have produced evidence thich
suggests that post-heparin lipase may be adsorbed to platelet membranes
and if as Doizaki and Zieve (1968) have asserted, the post-heparin
L.P.C. forming enzyme is similar to the lipase, it too may be
associated with the platelet membrane. If this is confirmed, then
the enzyme might have a direct effect on platelet membrane phospho-
lipids or their immediate plasmatic environment. Such a mechanism
may explain decreased platelet aggregation in the absence of raised
plasma levels of L.P.C.
The effects which have been described in this study relating to
certain types of oral contraceptive are essentially the reverse of
the effects of heparin. The plasma concentration of L.P.C. decreased
in women taking low-progestogen dosage combination oral contraceptives
- 238 -
such as 'Minovlar' or 'Ovulen 50', Significantly the decrease in
plasma L.P.C. concentrations during active treatment with these
preparations vas matched by a significant increase in the rate of
irreversible platlet aggregation. Both effects were related to
usage of the low-oestrogen oral contraceptives since no similar
changes in either L.P.C. concentrations or in platelet aggregation --
were found in women taking high progestogen oral contraceptives
('Anovlar/IGynovlar') or in a control group of women studied under
similar conditions.
The plasma levels of L.P.C. were measured in a group of women
at different stages during normal pregnancy and it was found that
whilst the concentrations of other lipids and phospholipids were
increased, the levels of L.P.C. were lower than for a gropp of
healthy non-pregnant women* Although this was not a longitudinally
planned study the decrease in plasma L.P.C. was found to be related
to the gestational period and the lowest L.P.C. concentrations were
found in women at full-term, It was not ethically possible to
obtain sufficient blood for a parallel study of platelet aggregation
and instead, measurements of erythrocyte properties were included.
The rate of packing of erythrocytes during centrifugation and the
clinical erythrocyte sedimentation rate were both increased during
pregnancy and the highest values were associated with women during
the last trimester of pregnancy. Both sedimentation and packing
rates are inhibited by L.P.C. in vitro and an attempt was made to
correlate these properties with the plasma L.P.C, levels in pregnant
and non-pregnant women. The result was a highly significant
correlation between the L.P.C. concentrations and the erythrocyte
packing rate (r = 0.790 : p <0.001) in the pregnant women and non-
- 239 -
pregnant women taken as one group. This is strong evidence for
diminished levels of L.P.C. in plasma leading to an increase in
the erythrocyte packing rate which in effect represents an increase
in erythrocyte flexibility (Rampling and Sirs, 1972). Clearly if a
close relationship between L.P.C. levels and erythrocyte behaviour
exists, as has also been indicatal by the work of B8ttiger (1973b)
and Berlin et al (1973), then it is just as probable that platelet
behaviour is at least partially influenced by plasma L.P.C. levels.
A study of the phospholipid composition of erythrocytes and
platelets in healthy men and men with chronic ischaemic heart
disease has been described in the present thesis. The relative
concentrations of L.P.C. in erythrocytes and in platelets as well
as in the plasma of patients suffering from ischaemic heart disease,
were significantly lower than in normal controls, This would be
expected if the L.P.C. corient of these formed elements in blood
has its origin in the plasma L.P.C. pool. Such a mechanism
which would explain altered erythrocyte and platiet behaviour caused
by alterations in the plasma L.P.C. pool either in vitro or in
myocardial infarction and pregnancy or in response to heparin or
oral contraceptive treatment, is summarised in figure 40.
If in the plasma there is a continuous conversion of P.C. to
L.P.C. by the action of L.C.A.T., then part of the L.P.C. formed
may be steadily adsorbed by both the erythrocyte and platelet
membranes. This would explain the presence in these membranes of
enzymes converting L.P.C. to P.C. or G.P.C. (Mulder et al, 1965
Mulder and Van Deenan, 1965 : Elsbach et al, 1971), which may be
part of an overall transport mechanism for the renewal of phospho-
lipids both within the membranes and the plasma lipoproteins,
INCREASED E.S.R. AND E.P.R.
HIGH PLASMA
EVELS OF LPC
•04.
Pregnano
Acute M.I. INCREASED IRREVERSIBLE
> PLATELET. AGGREGATION DECREASED IRREVERSIBLE PLATELET AGGREGATION
L PC added in vitro
HIGH ERYTHROCYTE
LEVELS C*L.P.C. NV INCREASED FORMATION OF PLASMA L.P.C.
LOW ERYTHROCYTE
LEVELS OF L.P.C.
LOW PLASMA
LEVELS OF LPC
LEVELS OF LPC
DECREASED E.S.R. AND E.P.R.
IV. Heparin
HIGH PLATELET
/LEVELS OF LPC '1
DECREASED FORMATION 1 OF PLASMA L.P.C. 4
LOW PLATELET
Figure 40.
Summary of relationship between altered plasma lysolecithin levels in
vitro and in vivo and alterations of platelet and erythrocyte behaviour.
- 241-
No attempt was made in the present study to investigate
platelet aggregation in patients suffering from myocardial
infarction or ischaemic heart disease. However, abnormalities
in plataet function_ have been described in -patients with ischaemic _ _
heart disease (O'Brien et al, 1966). More recently it has been
shown that platelets from patients suffering from acute myocardial
infarction are aggregated by lower concentrations of collagen than
are normal platelets (Salky and Dugdale, 1973). Such an abnormality
in platelet function in acute myocardial infarction is identical to
that described in the present study for increased collagen-induced
aggregation in women taking low-progestogen oral contraceptives.
The role of plasma L.P.C. as a factor which influences blood
platelet behaviour has been discussed in the present thesis. Indeed,
the action of L.P.C. may be described as thrombo-protective since it
would explain not just the association of low levels of plasma L.P.C.
with thromboembolic disease, but also the prophylactic effects of
heparin therapy on the incidence of thrombosis. This also provides a
mechanism for relating a disorder of lipid metabolism to the patho-
genesis of thrombosis. Previously, much attention has been focussed
on the incidence of raised plasma concentrations of cholesterol and
triglyceride (hyperlipoproteinaemia) in ischaemic heart disease.
Whilst raised plasma lipid levels undoubtedly contribute to the
development of atherosclerosis and the narrowing of the coronary
arteries, there is no known mechanism to suppose that increased
cholesterol and triglyceride concentrations could alter blood platelet
behaviour and so contribute directly to thrombosis. It seems possible
that decreased plasma levels of L.P.C. may be part of a more general
disorder of lipid metabolism involving hyperlipoproteinaemia (Kunz
et al, 1970) and if so then altered plasma lipid concentrations could
be implicated in both atherosclerosis and subsequent thrombosis.
- 242 -
CONCLUSION
The plasma concentrations of lysolecithin (L.P.C.) have been
shown to be significantly decreased in patients suffering from
acute myocardial infarction, ischaemic heart disease or-peripheral
arterial disease and also in women who were either pregnant or
taking oestrogenic oral contraceptives. In each case low plasma
levels of L.P.C. were associated with patient populations known to
have an increased risk of thromboembolic disease.
The significance of altered levels of plasma L.P.C. with relevance
to thrombosis, has been studiedAnd it has been shown that L.P.C. added
to preparations of blood platelets inhibited irreversible platelet
aggregation. Furthermore, alterations of the plasma L.P.C. concent-
ration or of its rate of formation following the administration of
oestrogenic oral contraceptives or of hepath were reflected by changes
in blood platelet function. Thus decreased plasma levels of L.P.C.
in women taking oral contraceptives were associated with increased
irreversible platelet aggregation. Intravenous administration of
heparin in man increased the formation of L.P.C. in plasma and
decreased platelet aggregation,
These results suggest that the association of low plasma levels
of L.P.C. in populations at risk from thromboembolic disease may be
explained by L.P.C. having some thrombo-protective properties or
function in man.
- 243 -
APPENDIX
A comparison of the effects of other surface-active agents with
those of lysolecithin on platelet aggregation.
- 244 -
Introduction
The experiments described in the main part of the present thesis
have demonstrated the inhibition of the platelet release reaction and
of irreversible platelet aggregation by lysolecithin (L.P.C.). Because
L.P.C. is a strongly surface-active agent it was of interest to
investigate the effects of other surface-active compounds. The
choice of which surface-active agents to investigate has been confined
to substances which have already been studied for effects on platelet
function.
Davey and LUscher (1968) have investigated the effects of a number
of lysosome-activating (surface-active) substances on the release'of
nucleotides, amino acids and proteins from washed platelet preparations
at.37o
Digitonin, deoxycholate and the detergent Triton X-100 all
liberated platelet contents indiscriminately and differed in this
respect from the thrombin-induced platelet release reaction which
occurs without lysis (Grette, 1962). Davey and LUscher incubated
washed and resuspended platelets with bee venom (from Trimersurus
okinavensis) and showed that this resulted in the considerable release
of platelet nucleotides and amino acids. They concluded that the
phospholipase contained in the venom extract had caused the formation
of lysophosphorylglycerides (L.P.C.) which were responsible for
lysing the platelet membrane.
More recently Hampton and Nicholls (1972) have investigated the
effects of a variety of anionic, cationic and non-ionic detergents
on platelet function. Many of these substances including sodium
dodecyl sulphate (S.ILS.), fatty acids, polyoxyethylene sorbitan
mono-laurate and mono-oleate (Tween 20 and Tween 80), and cetyl
trimethylammonium bromide (C.T.A.B.) induced abnormal platelet
sensitivity to A.D.P. as judged on the basis of platelet electro-
Ohoretic mobility changes. The detergent cetyl pyridinium chloride
- 245 -
(C.P.C.) had no effect on platelet sensitivity to A.D.P. The same
compounds exhibited different effects when tested on platelet
aggregation initiated by A.D.P. or noradrenaline. Platelet
aggregation was inhibited by C.P.C. and to a less marked extent by
C.T.A.B., whereas S.D.S. prolonged platelet aggregation and
inhibited platelet disaggregation. Palmitic acid, Tween 20 and
Tween 80 had no apparent effects on platelet aggregation.
These findings suggest that different surface-active agents have
variable effects on platelet function. In the present study the
effects of digitonin, sodium deoxycholate, Triton X-100, C.P.C. and
C.T.A.B. have been studied and compared with the effects of L.P.C. on
platelet aggregation.
Experimental details.
The effects of surface-active substances on platelet aggregation
were studied using the apparatus and methods described elsewhere.
The surface-active agents were dissolved in 0.9 percent saline and.
added to pre-warmed samples of P.R.P. which was continuously stirred
at 37oC. This was followed twenty seconds later by the addition of
aggregating agent (A.D.P., adrenaline, or collagen). One tube in
each series was utilized as a control with the addition of 0.9 per-
cent saline in place of the surface-active agent. Usually three
experiments of each type were performed.
Results and Discussion.
(1) Digitonin.
The effects of direct addition of digitonin to stirred P.R.P.
were dependant upon the final concentration of digitonin added to
P.R.P. (fig, 41 (1)). Low concentrations of digitonin ( 0.04 mol ml.-1)
completely reduced the amplitude of the oscillations in the light
transmitted through stirred P.R.P. which suggested that the mean platelet
- 245 -
(C.P.C.) had no effect on platelet sensitivity to A.D.P. The same
compounds exhibited different effects when tested on platelet
aggregation initiated by A.D.P. or noradrenaline. Platelet
aggregation was inhibited by C.R.C. and to a less marked extent by
• C,T.A.B., whereas S.D.S. prolonged platelet aggregation and
inhibited platelet disaggregation. Palmitic acid, Tween 20 and
Tween 80 had no apparent effects on platelet aggregation.
These findings suggest that different surface-active agents have
variable effects on platelet function. In the present study the
effects of digitonin, sodium deoxycholate, Triton X-100, C.P.C. and
C.T.A.B, have been studied and compared with the effects of L.P.C. on
platelet aggregation.
Experimental details.
The effects of surface-active substances on platelet aggregation
were studied using the apparatus and methods described elsewhere.
The surface-active agents were dissolved in 0.9 percent saline and
added to pre-warmed samples of P.R.P. which was continuously stirred
at 37oC. This was followed twenty seconds later by the addition of
aggregating agent (A.D.P., adrenaline, or collagen). One tube in
each series was utilized as a control with the addition of 0.9 per-
cent saline in place of the surface-active agent. Usually three
experiments of each type were performed.
Results and Discussion.
(1) Digitonin.
The effects of direct addition of digitonin to stirred P.R.P.
were dependant upon the final concentration of digitonin added to
P.R.P. (fig. 41 (i)). Low concentrations of digitonin ( 0.04 p mol ml.-1)
completely reduced the amplitude of the oscillations in the light
transmitted through stirred P.R.P. which suggested that the mean platelet
- 246 -
(1)
071
o.d.
02
Digitonin (prnot m(.1)
iii) Saponin Minutes
Control
01 Minutes
Figure 41: Aggregation of platelets initiated by (i). digitonin and
(ii) saponin.
05
o.d.
- 247 -
shape had been changed from discoid to spherical (O'Brien and Heywood,
-1, 1966). Higher concentrations of digitonin (0.05.- 0.2 p mol ml. I
induced maximal platelet aggregation after an initial delay period
which was concentration dependant. The aggregation curves recorded
were similar to those normally obtained when platelets were aggregated
by collagen or connective tissue preparations, and on removal from
the aggregation apparatus macroscopic platelet clumps were clearly
visible in the plasma. At the highest concentrations tested (0,5 -
1.0 p mol ml.-1) digitonin caused a spontaneous decrease in optical
density which was not characteristic of platelet aggregation because
the curves recorded no oscillations in amplitude due to the movement
of platelet aggregates within the light path. The optical density
change was only about 60 percent of that expected for maximal platelet
aggregation and the samples were turbid when viewed against a dark
background. Phase-contrast microscopy failed to revealcny intact
platelets in the samples, suggesting that complete lysis had occurred.
Treatment of P.R.P. with saponin (a mixture of glycosides related to
digitonin) initiated platdbt aggregation but differed from the action
of digitonin in that saponin-induced aggregation was reversible or
bi-phasic and resembled that induced by A.D.P. (fig. 41 (ii)).
Platelet aggregation initiated by digitonin was inhibited by
L.P,C, at final concentrations of 0.5 p mol ml.-1 (fig. 42) and by
O the alpha-adrenergic blocking drug Rogitine (phentotamine) (fig. 43).
In one experiment samples of P.R.P. were pre-treated with Rogitine
to inhibit platelet aggregation induced by digitonin (0.05 p mol m1.-1).
The samples were centrifuged to prepare P.P.P. which was then added
back to fresh, stirred P.R.P. This was compared with P.F.P, prepared
O from P.R.P. exposed to Rogitine only after maximal aggregation had been
r Digitonin
02-A
- 248 -
Minutes
Figure 42: Inhibition of digitonin-induced platelet aggregation
by lysolecithin.
Aliquots of P.R.P. were pre-incubated with L.P.C. (final
concentrations shown as p mol. ml.-1
) for 30 seconds before
initiating aggregation by addition of digitonin at a final
concentration of 0.05 F mol. ml.-1
- 249 -
A. 0.8 Pre-treated
with Rogitine
o.d. B. Rogitine added after
03 aggregation
Minutes rut 10011 PFPA "]
't PFP 0. 1001 B
0.5
Figure 43: Inhibition of digitonin-induced platelet release
reaction by Rogitine.
Digitonin
- 250 -
initiated by digitonin. The plasma from aggregated platelets
(P.P.P.B) initiated platelet aggregation, whereas plasma from
inhibited platelets (P.R.P.A) did not. Since phentolamine inhibits
the platelet release reaction (0'Brien,_1953 Mills, and Roberts,
1967) it follows that the exposure of platelets to digitonin (0.05 -
0.2 p mol ml.-1
) causes a release reaction similar to that induced
by adrenaline or thrombin.
These results differ from the effect of digitonin on platelets
reported by Davey and LUscher who described the non-specific release
of platelet constituents after exposure to digitonin. Clearly at
the high concentrations tested in this experiment and in Davey and
Llischer's study digitonin lyses platelets releasing many different
substances from platelets. However, at lower concentrations digitonin
aggregates platelets and induces a release reaction which is similar
to that initiated by adrenaline, A.D.P. and collagen and which can be
inhibited by normal inhibitory substances such as L.P.C. or alpha-
blockers.
Pre-incubation of P.R.P;. with digitonin at concentrations below
the threshold for aggregation did not affect aggregation initiated
by A.D.P., adrenaline or collagen. This shows that there was no
similarity between effects of L.P.C. and digitonin on platelet function.
Sodium dooxycholate
Pre-treatment of P.R.P. with sodium deoxycholate at final
concentrations of 0.5 - 2.0 p mol ml.-1
inhibited the second phase
of A.D.P.-induced aggregation (fig. 44 (i)) and adrenaline-induced
aggregation (fig. 44 (ii)).
Sodium deoxycholate had a similar inhibitory effect to that of
L.P.C. on irreversible platelet aggregation except that higher
concentrations of deoxycholate were required to maximally inhibit the
second phase of aggregation.
- 251 -
ADP (DOC prnot
10
o.d.
Adrenaline 051
Minutes Figure 44: Inhibition of irreversible platelet aggregation initiated
by (i) adenosine diphosphate (1 n molml.-1) and (ii)
adrenaline (2.5 n mol.ml.-1) by pre-incubation of platelet
rich plasma with sodium deoxycholate.
- 252-
Triton X-100
Exposure of stirred platelets to the detergent at concentrations
above 0.5 mg ml.-1 lysed platelets as judged by the fall in optical
density of stirred P.R.P. after the addition of Triton X-100.
Cetyl pyridinium chloride and Cetyl trimethylammonium bromide.
Pre-incubation of platelets with either C.P.C. or C.T.A.B. for
twenty seconds inhibited platelet aggregation initiated by A.D.P. or
collagen (fig. 45). Both detergents were effective inhibitors of
both the first and second phases of A.D.P.-induced aggregation at
concentrations in the range of 0.1 - 0.5 p mol ml.-1.
Although the inhibitory effect of both detergents on collagen-
induced aggregation, resembled that described for L.P.C. the effect
on A.D.P.-induced aggregation differed in that C.P.C. and C.T.A.B.
unlike L.P.C., inhibited the first phase of aggregation.
Conclusion
Surface-active substances including L.P.C., deoxycholate, digitonin,
C.P.C. and C.T.A.B. have effects on platelet function in vitro other
than causing platelet lysis. However, the effects of different
surfactants were variable, including initiation of plat et aggregation
or the inhibition of platelet aggregation induced by other agents.
Of the compounds investigated only deoxycholate had an effect which was
similar to that of L.P.C. although inhibition of secondary platelet
aggregation required higher concentrations of deoxychiate than of L.P.C.
- 253 -
CO 0.6 ADP
CPC pmol ml?
0-10 0-05
o.d.
0.3
(ii) Cot.
06 CTAB mot mi..1
0.50
o.d. 0.25
02
Minutes Figure 45: (i) Inhibition of platelet aggregation ihitiated by adenosine
diphosphate by cetyl pyridinium chloride (C.P.C.).
(ii) Inhibition of collagen-induced platelet aggregation by
cetyl trimethylammonium bromide (C.T.A,B.).
- 254 -
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Atherosclerosis Elsevier Publishing Company, Amsterdam — Printed in The Netherlands
323
INHIBITION OF PLATELET AGGREGATION BY LYSOLECITHIN
E. M. M. BESTERMAN AND M. P. T. GILLETT Department of Cardiology, St. Mary's Hospital, London, 1V2 (Great Britain)
(Received January 14th, 1971)
SUMMARY
The effects of purified phospholipids on platelet aggregation initiated by ADP, adrenalin and collagen have been studied. The only phospholipid to have a consistent effect was lysolecithin. Lysolecithin inhibited the second phase of both ADP and adrenalin induced aggregation, and abolished the aggregation response caused by collagen. The inhibition was dose-dependent on the lysolecithin concentration and did not alter the initial aggregation response of platelets to ADP or adrenalin.
The possible mechanism and significance of this inhibition, and its relevance to the problem of arterial disease, is discussed.
Key words: ADP — Adrenalin — Collagen — Lysolecithin — Phospholipids — Platelet aggregation — Platelet release reaction
INTRODUCTION
Abnormalities of the plasma lipids and of platelet behaviour have dominated aetiological studies of ischaemic heart disease in recent years. FREDERICKSON et al.1 and STRISOWER et a1.2 have classified these lipoprotein abnormalities. The most consistent lipid abnormality is an increase in plasma cholesterol. However, no direct relationship has been found between platelet function and raised cholesterol levels in atherosclerosis. HAMPTON AND MITCHELL3 have shown an abnormal facet of platelet behaviour that is related to coronary and peripheral arterial disease. Their measure-ments of platelet electrophoretic mobility showed that the platelets from patients with arterial disease are more sensitive to ADP than are those from control subjects. Since ADP is thought to play a central role in the aggregation of platelets, both in vitro and in vivo, these authors' findings of altered platelet behaviour might suggest a thrombotic tendency in such patients.
This abnormal platelet reactivity has been linked to a complex system, in-
a Atherosclerosis, 1971, 14: 323-330
324 E. M. M. BESTERMAN, M. P. T. GILLETT •
volving the plasma phospholipids4. This system is thought to release lysolecithin, enzymically, from its lipoprotein-bound precursor lecithin. The released lysolecithin is then responsible for the increased sensitivity of the platelets to ADP. HAMPTON AND BOLTON5 have since demonstrated this directly by using purified lysolecithin and normal platelets. However, contrary to expectations, the plasma levels of lysolecithin have been shown to be definitely decreased in acute myocardial inf arc-tion6,7 and in patients with clinically proven atherosclerosis and type IV hyper-lipoproteinaemia8.
This present study describes the effects of lysolecithin and other purified phospholipids on platelet aggregation.
MATERIALS AND METHODS
Blood was collected by clean venepuncture into disposable plastic syringes; 13.5-m1 samples were transferred to siliconized glass centrifuge tubes containing 1.5 ml of 3.2% (w/v) trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 150 x g. The platelet count was usually in the range 2-4 x 105/inm3.
Platelet aggregation was studied by the turbidimetric method of MILLS AND ROBERTS9 using a modified EEL nepholometer coupled with a 10 mV pen-recorder (Vitatron). The range of the recorder was pre-set so that a pooled sample of PRP gave 20% light transmission and platelet-free plasma 100% transmission. Aggregation tests were started one hour after venepuncture, and each series was usually completed with-in the subsequent hour. All tests were performed at 37°C. In a typical test, 1 ml of PRP was pipetted into a siliconized cuvette containing a plastic-coated stirring-bar. The sample was warmed to 37C° for 3 min and then transferred to the sample compart-ment. The phospholipid was added, followed 1 min later by the aggregating agent.
Subjects These were healthy volunteers, who were not taking drugs known to affect
platelet behaviour. Aggregation tests were performed on either individual samples of PRP, or on pooled samples taken from two or more subjects.
Aggregating agents Bovine collagen (Sigma) was prepared in 0.9% saline as described by EVANS
et a/.10. ADP (disodium salt, Sigma) was stored frozen at a concentration of 0.1 in 0.9% saline, and aliquots were thawed immediately before use. Adrenalin (hy-drogen tartrate salt, B.D.H.) was prepared daily as a 1 mil/ solution in 0.9% saline.
The volume of aggregating agent used was adjusted for each sample of PRP in order to obtain maximal irreversible aggregation. Under these conditions ADP and adrenalin produced biphasic aggregation; the optical density change of the first phase was measured as well as the rate of secondary aggregation. Collagen produced aggregation after an initial delay of up to one minute; this delay time was also measured.
• Atherosclerosis, 1971, 14: 323-330
PLATELET AGGREGATION BY LYSOLECITHIN 325
Phospholipids The following Koch—Light preparations were used: lecithin (egg), lysolecithin
(egg), lysophosphatidyl ethanolamine (egg), phosphatidyl ethanolamine (bacterial), phosphatidyl serine and sphingomyelin (both bovine brain). Synthetic L-dioleoyl and L-dipalmitoyl phosphatidyl ethanolamines were obtained from Dr. Billimoria, Westminster School of Medicine, Great Britain. Glycerophosphoryl choline (cadmium chloride complex) was procured from Sigma, and was passed through a mixed bed ion-exchange column before use. The purity of each compound was checked by thin layer chromatography with chloroform:acetone:methanol:acetic acid:water (50:20: 10:10:5, v/v). All compounds gave a single spot when exposed to iodine vapour. Lipid suspensions were made in 0.9% saline or in 5% human albumin by ultrasoni-cation, and the phosphorus content of each was determined by the method of BARTLETT", so as to confirm the molar concentration of each compound. Phospho- lipids were tested at final concentrations of up to 1 mill by addition of 50 or less of the suspension. Control systems, using saline or albumin solution alone, were tested in parallel for each experiment.
RESULTS
(1) Direct action of phospholipids on stirred PRP Lysolecithin. In some, but not all, experiments the addition of lysolecithin
(0.5-0.7 mill) caused a change in the oscillations of the intensity of the light trans-mitted through PRP from those that are characteristic for disc-shaped platelets to a pattern characteristic of spherical platelets. This effect lasted at least five minutes.
Phosphatidyl serine and sphingomyelin. The addition of phosphatidyl serine and sphingomyelin (at concentrations above 1 mill) produced slight platelet aggregation, which was irreversible in the case of sphingomyelin (Fig. 1).
Other phospholipids. The other phospholipids tested for their direct action on PRP had no effect.
(2) Effect of phospholipids on ADP-induced platelet aggregation Lysolecithin. Lysolecithin had no effect on reversible platelet aggregation
0. D. O'8- 07- CONTROL 0.6 10mM 5F! 0.5 I.5mN
1 MIN
Fig. 1. Aggregation of platelets initiated by sphingomyelin (SP) and phosphatidyl serine (PS). The control shows the trace of PRP to which only albumin solution has been added. Sphingo-myelin (final concentration 1 milf) caused irreversible aggregation and phosphatidyl serine (1.5 mM) slight reversible aggregation.
Atherosclerosis, 1971, 14: 323-330
ADP -
0.4 - 0.3 mM LPC
0.2 MM LPC 0.3-
0.2
0.1 MM LPC 0.I
326 E. M. M. BESTERMAN, M. P. T. GILLETT
O.D
CONTROL
1 MIN
Fig. 2. Inhibition of secondary ADP-induced aggregation by lysolecithin (LPC). The final con-centration of ADP was 10-6 M and the curves have been superimposed to show the degree of inhibition due to three different concentrations of LPC.
initiated by ADP at concentrations less than 10-6 M, nor did it affect the minimum concentration of ADP required to cause aggregation.
When higher concentrations of ADP were used to initiate biphasic platelet aggregation, lysolecithin inhibited the second phase without altering the magnitude of the first phase (Fig. 2). The degree of inhibition was dose-dependent upon the concentration of lysolecithin (Table 1). Maximal inhibition required lysolecithin concentrations in the range 0.2-0.7 mill, depending upon the concentration of ADP and the platelet count.
The inhibitory effect of lysolecithin only occurred if it was added before ADP or during the initial aggregation response. It had no effect if added after the onset of secondary aggregation (Fig. 3).
Inhibited platelets showed a tendency to disaggregate slowly, and they were still responsive to further additions of ADP. The inhibitory effect of lysolecithin was unaltered whether it was dissolved in saline or in 5% human albumin.
Other phospholipids. None of the other phospholipids affected the response of the platelets to ADP.
TABLE 1
EFFECTS OF LYSOLECITHIN ON SECONDARY PLATELET AGGREGATION INDUCED BY ADP
The concentration of ADP was 10-6 M.
Lysolecithin Rate of secondary aggregation Inhibition (nonoles/l) (O.D. tinitslmin) %)
— 0.250 - 0.06 0.160 36 0.12 0.105 58 0.18 0.085 66 0.24 0.020 92 0.36 0.000 100
Atherosclerosis, 1971, 14: 323-330
PLATELET AGGREGATION BY LYSOLECITHIN 327
. ADP
I \\III
LPC ADP * * 6.1—\\.......„.„„_
2 ADP
LPC
3
ADP
4
LPC
Fig. 3. Effect of adding lysolecithin at different times during biphasic aggregation initiated by ADP. 1. Control. The final ADP concentration was 10-6 .11; 2. Inhibition of second phase by LPC (final concentration 0.5 mili) added before ADP; 3. Inhibition of second phase by LPC (0.5 mill) added during the initial aggregation response; 4. Lysolecithin added after the onset of secondary aggregation had no inhibitory effect.
O.D. Ad rena lin
0.5-
0.4- 0.7mM LPC.
0.3- 0.4 mM LPC.
0.2-
CO NTROL
1 MIN
Fig. 4. Inhibition of adrenalin-induced secondary aggregation by lysolecithin (LPC). The final concentration of adrenalin was 2.5 x 10-5 M and maximal inhibition of secondary aggregation required a final concentration of 0.7 mmole LPC/1.
(3) Effect of phospholipids on adrenalin-induced platelet aggregation Lysolecithin. Lysolecithin inhibited the second phase of adrenalin-induced
aggregation, without affecting the magnitude of the first phase (Fig. 4). The inhibition was dose-dependent (Table 2) and its characteristics were very similar to those already noted for ADP aggregation. The delay time between the first phase of aggregation and the onset of the second phase was increased in samples in which the concentration of added lysolecithin only produced partial inhibition.
In one experiment lysolecithin was added to whole blood before preparing the PRP, which was then tested for adrenalin-initiated aggregation. The secondary aggregation response of this sample was inhibited when compared with a similar sample of PRP prepared from the same blood specimen but without added lyso-lecithin.
Other phospholipids. Adrenalin-induced aggregation was not affected by any of the other phospholipids tested.
O.D
0.7 Ob
05
04
Atherosclerosis, 1971, 14: 323-330
328 E. M. M. BESTERMAN, M. P. T. GILLETT
TABLE 2
INHIBITION OF THE SECOND PHASE OF ADRENALIN INDUCED AGGREGATION BY LYSOLECITHIN
The concentration of adrenalin was 2.5 x 10-5 M.
Lysolecithin (no
Rate of secondary aggregation (0.D. linitshnin)
Inhibition (%)
— 0.160 - 0.05 0.160 0 0.10 0.110 31 0.20 0.080 50 0.30 0.040 75 0.40 0.020 87 0.50 0.015 91 0.60 0.005 97
(4) Effect of phospholipids on collagen-induced platelet aggregation Lysolecithin. The aggregation response of platelets to suspensions of collagen
fibres was inhibited by lysolecithin (Fig. 5). The delay time between addition of collagen and the onset of aggregation was increased by lysolecithin. Lysolecithin added during this delay period was'still active as an inhibitor, but had no effect once rapid aggregation had started.
In one experiment samples of PRP were pre-incubated with lysolecithin for up to 30-min"withoucaltering its inhibitory effect on collagen-mediated aggregation.
Other phospholipids. None of the other phospholipids had any effect on platelet aggregation initiated by collagen.
0.0 0.7 0.6—
Collagen
0.5—
0.4-
0.4--
0.2—
0.1—
1 MIN Fig. 5. Inhibition of platelet aggregation induced by collagen. 50 id of collagen suspension were used to initiate the aggregation response.
Atherosclerosis, 1971, 14: 323-330
PLATELET AGGREGATION BY LYSOLECITHIN 329
DISCUSSION
Only a very few studies have been concerned with the effects of phospholipids on platelet aggregation either directly or in response to aggregating agents such as collagen. KERR et a1.12 have shown that phosphatidic acid, phosphatidyl serine and phosphatidyl ethanolamine cause reversible aggregation and that sphingomyelin causes irreversible platelet aggregation. Our results confirm their earlier findings, at least in respect of phosphatidyl serine and sphingomyelin. Sphingomyelin is one of the major lipids found in atheromatous plaques13, and it is therefore possible that its aggregating ability may play a role in the pathogenesis of atherosclerosis. NISHIZAWA14 has described the inhibition of collagen-induced aggregation of canine platelets by phosphatidyl serine. This inhibition by phosphatidyl serine closely resembled that which we have described for lysolecthin, but we were unable to confirm it in our studies on human platelets.
The inhibitory effect of lysolecithin on all types of irreversible aggregation that were studied fails to support the earlier view of BOLTON et a1.4 that lysolecithin-platelet interaction represents a thrombotic tendency in certain patients with ischaem-ic heart disease.
The fact that lysolecithin specifically inhibits irreversible aggregation caused by ADP, adrenalin and collagen suggests that it acts on a mechanism common to all three agents. It is usually accepted that irreversible aggregation induced by a variety of dissimilar agents is due to release of ADP from the platelets into the plasma. It seems possible that lysolecithin blocks this platelet release reaction. Since platelet clotting factors 3 and 4 are also made available during the release reaction15 the inhibitory effect of lysolecithin on blood clotting16 may also be mediated by its effect on the platelets.
The apparent change in platelet shape caused by lysolecithin has not been noticed in phase-contrast microscope studies5, but this would seem to be similar to its"sphering action on red cells.
The concentration of lysolecithin that inhibited aggregation was higher than the normal plasma level, but was similar to the plasma level after incubation at 37°C for several hours7,17. It is possible that the enzymic release of lysolecithin in plasma may play a role in regulating platelet behaviour, since platelets are known to in-corporate lysolecithin and fatty acids into their phospholipid18. BERLIN et al.7 have shown that both the plasma lysolecithin level and its rate of release are greatly re-
.
duced in patients suffering from acute myocardial infarction. These levels were seen to increase towards the normal range when these patients were discharged, except in one case when a further decrease was noted in a patient who suffered from a further myocardial infarct. KUNZ et al.8 have shown that the lysolecithin level is diminished in type IV hyperlipoproteinaemic patients with clinically proven vascular disease compared with type IV patients without vascular complications. Our in vitro findings suggest that the decreased lysolecithin levels in myocardial infarction and in vascular disease may indicate that platelets from such patients are more susceptible to aggre-gation and thus to thrombosis.
Atherosclerosis, 1971, 14: 323-330
330 L. M. M. BESTERMAN, M. P. T. GILLETT
REFERENCES
1 FREDERICKSON, D. S., R. I. LEVY AND R. S. LEES, Fat transport in lipoproteins. An integrated approach to mechanisms and disorders, N. Engl. J. Med., 1967, 276: 34, 94, 148, 215, 273.
2 STRISOWER, E. H., G. ADAMSON AND B. STRISOWER, Treatment of hyperlipidemias, Amer. J. Med., 1968, 45: 488.
3 HAMPTON, J. R. AND J. R. A. MITCHELL, A transferable factor causing abnormal platelet behaviour in vascular disease, Lancet, 1966, ii: 764.
4 BOLTON, C. H., J. R. HAMPTON AND J. R. A. MITCHELL, Nature of the transferable factor which causes abnormal platelet behaviour in vascular disease, Lancet, 1967, ii: 1101. HAMPTON, J. R. AND C. H. BOLTON, Effect of phospholipids on platelet electrophoretic mobility, J. Atheroscler. Res., 1969, 9: 131.
6 MARINETTI, G. V., M. ALBRECHT, T. FORD AND E. STOTZ, Analysis of human plasma phospha-tides by paper chromatography, Biochim. Biophys. Acta, 1959, 36: 4.
7 BERLIN, R., C. 0. OLDFELT AND 0. VIKROT, Acute myocardial infarction and plasma phospho-lipid levels, Acta Med. Salmi., 1969, 185: 439.
8 Ku1.1z, F., G. MATT AND H. HACKL, Plasma phospholipids in type IV hyperlipoproteinemia, Atherosclerosis, 1970, 11: 265.
9 MILLS, D. C. B. AND G. C. K. ROBERTS, Effects of adrenalin on human blood platelets, J. Physiol. ( London), 1967, 193: 443.
10 EVANS, G., N. A. PACKHAM, E. E. NISHIZAWA, J. F. MUSTARD AND E. A. MURPHY, The effects of acetyl salicylic acid on platelet function, J. Exptl. Med., 1968, 128: 877.
11 BARTLETT, G. R., Phosphorus assay in column chromatography, J. Biol. Chem., 1959, 234: 466. 12 KERR, J. W., R. PIRRIE, I. MACAULAY AND B. BRONTE-STEWART, Platelet-aggregation by
phospholipids and free fatty acids, Lancet, 1965, i: 1296. 13 SMITH, E. B., The influence of age and atherosclerosis on the chemistry of the aortic intima,
J. Atheroscler. Res., 1965, 5: 224. 14 NISHIZAWA, E., Phospholipid, blood coagulation, platelet aggregation and thrombosis,
Federation Proc., 1965, 24: 154. 15 NIEWIARONBKI, S., A. POPLAWSKI, B. LIPINSKI AND R. FARBiszEwsKi, The release of platelet
clotting factors during aggregation and viscous metamorphosis. In: Platelets in Hemostasis, Exptl. Biol. Med., 1968, 3: 121.
16 BILLIMORIA, J. D., V. J. IRANI AND N. F. MACLAGAN, Phospholipid fractionation and blood clotting, J. Atheroscler. Res., 1965, 5: 90.
17 ADLKOFER, F. W. VON, SCHIEBEL, E. ANCKER AND G. RunENSTROm-BAuER, Nachweiss, Anreicherung and Characterisierung tines Lysolecithin freisetzenden Enzyms aus mensch-lichem Serum, Hoppe-Seyler's Z. Physiol. Chem., 1968, 349: 417.
18 COHEN, P., Preliminary observations on the incorporation of 14C-labelled fatty acids into human platelet phospholipids in vitro. In: Platelets in Hemostasis, Exptl. Biol. Med., 1968, 3: 135.
Atherosclerosis, 1971, 14: 323-330
Atherosclerosis 89 Elsevier Publishing Company, Amsterdam — Printed in The Netherlands
A COMPARISON OF THE EFFECTS OF SATURATED AND POLYUNSATURATED LYSOLECITHIN FRACTIONS ON PLATELET AGGREGATION AND ERYTHROCYTE SEDIMENTATION
E. M. M. BESTERMAN AND M. P. T. GILLETT Department of Cardiology, St. Mary's Hospital, London TV.2 (Great Britain) (Received February 17th, 1972)
SUMMARY
Irreversible platelet aggregation initiated by adrenalin, adenosine diphosphate or collagen was inhibited by a fully saturated lysolecithin fraction. By contrast, similar concentrations of a lysolecithin fraction containing a very high proportion of polyunsaturated fatty acids did not affect platelet aggregation.
The sedimentation of erythrocytes in autologous plasma, containing a final concentration of 0.4 % dextran (molecular weight: 150,000), was inhibited by addition of the fully saturated lysolecithin, but not by the polyunsaturated mixture.
The differences and possible significance of these results are discussed in relation to the fatty acid composition of human plasma lysolecithin.
Key words: Erythrocyte sedimentation — Irreversible platelet aggregation — Phospho-lipids — Polyunsaturated fatty acids
INTRODUCTION
It has been previously shown that lysolecithin inhibits the secondary or irre-versible phase of platelet aggregation initiated by adenosine diphosphate (ADP), adrenalin or collagens. Treatment of platelets with lysolecithin was often accompanied by evidence of a change in platelet shape; the mechanism of inhibition was thought to be due to the blockade of the platelet release reaction by lysolecithin. Lysolecithin also affects the red cell membrane causing haemolysis and at lower concentrations, it has been shown to "sphere" red cells, prevent "rouleaux" formation and retard the erythrocyte sedimentation rate (ESR)2.
This work was supported by an allocation from the Research Fund of St. Mary's Hospital, London W.2.
Atherosclerosis, 1972, 16: 89-94
90 E. M. M. BESTERMAN, M. P. T. GILLETT
Several investigations have been made on the haemolytic activity of lysoleci-thins with different acyl side-chains. GOTTFRIED AND RAPPORT3 showed that unsatura-tion in the paraffinic chain reduces haemolytic activity, and this has been confirmed by RENAN et al.4, who also reported that alterations in the acyl chain length influence haemolysis rates. Palmitoyl- and stearoyl-lysolecithins were the most potent haemo-lysins: any increase or decrease in the chain length or the introduction of double bonds appreciably reduced the activity. Saturated lysolecithins comprise the major fraction of the total human plasma lysolecithin5-7, and consist mainly of palmitoyl- and stea-royl-lysolecithins. The predominant unsaturated lysolecithins found in human plasma were the oleoyl- and linoleoyl-compounds7. In the present study, we have studied the effects of saturated and polyunsaturated lysolecithins on platelet aggregation and on red cell sedimentation.
MATERIALS AND METHODS
Lysolecithin (1-acyl-sn-glycero-3-phosphorylcholine) samples, prepared from soya bean lecithin, were obtained from Dr. H. Gentile (Nattermann International,
1n). The composition of the two samples is shown in Table 1, and the purity of these compounds was confirmed by thin layer chromatography. Known amounts of these two fractions were dissolved in 0.9 % saline, immediately before starting each experi-ment. ADP and adrenalin solutions and suspensions of bovine collagen were prepared as previously described'. Platelet-rich plasma (PRP) samples (1 ml) were warmed at 37° for 3 min and placed in the EEL aggregation apparatus already described'. Subsequently 20 ,u1 or less of lysolecithin solution or 0.9 % saline was added using a microliter syringe. After 20 seconds 20-40 ,u1 of aggregating agent were added to initiate irreversible platelet aggregation, which was continuously recorded as an increase in light transmission at 602 nm through the PRP sample. Measurements were made of the initial aggregation response and of the initial rate of secondary aggregation.
The measurement of red cell sedimentation using the Westergren technique was modified as described by ADLKOFER et al.2. Human red cells were washed three times with two volumes of 0.9% saline; 0.5 ml aliquots were added to 0.7 ml of autologous
TABLE 1
FATTY ACID COMPOSITION OF THE LYSOLECITHIN FRACTIONS
Lysolecithin fraction % Composition a
16:0 18:0 18:1 18:2 18:3
Saturated 23.7 76.3 Unsaturated 7.76 2.68 9.27 72.72 7.57
a Component fatty acids have been abbreviated as follows: e.g. 16:0 carbon chain length, 16 atoms without double bonds; 18:1 carbon chain length, 18 carbon atoms with one double bond.
Atherosclerosis, 1972, 16: 89-94
LYSOLECITHIN AND PLATELETS 91
EDTA (1 mg/ m1) plasma; 0.3 ml of a 2 % solution of dextran (average molecular weight: 150,000) in 0.9 % saline (Fisons Ltd., Loughborough) was added to increase the ESR. Finally, 10 Jul of lysolecithin were added to give a final concentration of 0.24 prnolefml. Sedimentation readings were recorded at intervals of 1 min until the control tubes (no added lysolecithin) had sedimented 50 mm. Four experiments of this type were performed in duplicate.
RESULTS AND DISCUSSION
Erythrocyte sedimentation Erythrocyte sedimentation was almost totally inhibited by saturated lysoleci-
thin (0.24 dumole/m1) but not by the polyunsaturated lysolecithin at the same concen-tration (Fig. 1). At this concentration of lysolecithin there was no macroscopically visible haemolysis in the plasma supernatant.
Platelet aggregation In these experiments, collagen produced irreversible aggregation after an
initial delay of up to 2 min. This lag period was unaffected by saturated lysolecithin, but both the rate and extent of aggregation were inhibited at lysolecithin concentra-
S 0 -
40-
30-
0
20- 2
I 0 -
0 -
A*
A•
ii2is
•• 00 0000000 0008:_ _
000
Ale
gi
A A..
A.. A.
•
A. Q'
0000000000000000
a a
0 10 20 30 40 MINUTES
Fig. 1. The effects of a saturated lysolecithin and a predominantly unsaturated fraction on eryth-rocyte sedimentation. Saturated lysolecithin (0 0 0) or polyunsaturated lysolecithin (A A A) were added to a mixture containing 0.5 ml of washed red cells, 0.7 ml of autologous plasma, and 0.3 ml of 2 % dextran to give a final lysolecithin concentration of 0.24 /mole/mi. The control mixture (S • •) contained 0.9 % saline in place of lysolecithin. Mean values of duplicate determinations are plotted.
Atherosclerosis, 1972, 16: 89-94
0,2 0,4 0'6 0,8 0 1.0
100
4-5?-
0
60
0 40 I-
2 z
0
92 E. M. M. BESTERMAN, M. P. T. GILLETT
LYSOLEC I THIN p moLEOAL
Fig. 2. The effects of a saturated (• •) and a polyunsaturated ( 0 0) lysolecithin fraction on collagen-induced platelet aggregation. Percentage inhibition of aggregation plotted on the ordinate refers to the decrease in initial rate of aggregation following addition of lysolecithin, when compared with aggregation of a control sample.
tions of up to 1 ,umole/m1 (Fig. 2). By contrast, polyunsaturated lysolecithin did not inhibit aggregation except slightly at the higher concentrations tested (0.9-1 ,umole/ ml). As our polyunsaturated lysolecithin contained about 10% of saturated lyso-lecithin it-was thought probable that the inhibition by high concentrations could have been due to this contamination. Adrenalin- (Fig. 3) and ADP-initiated secondary aggregation were both totally inhibited by saturated lysolecithin (0.5-0.8 ,umole/m1) but similar concentrations of polyunsaturated lysolecithin were inactive in this respect. Increased concentrations of polyunsaturated lysolecithin (< 1 ,umole/m1) caused slight inhibition which again could have been attributed to contamination with saturated lysolecithin.
These experiments showed that there was a considerable difference between the effects of saturated and polyunsaturated lysolecithins on cell membranes. This supports the 'earlier observations3,4 that lysolecithins with unsaturated fatty acids had weaker haemolytic activities than the corresponding saturated compounds. Lucv8 has described a model for the penetration of erythrocyte membranes by lyso-lecithins involving a phase change in the membrane lipid-protein structure: such a model might apply to the interaction of lysolecithin with platelet membranes with the result that the platelet release reaction is prevented. In this respect palmitoyl- and stearoyl-lysolecithins may possess the right configurations for membrane penetration. Differences in the degree of binding of saturated and polyunsaturated lysolecithins to plasma-albumin could also explain their different effects on erythrocyte and platelet properties, for only "free" lysolecithin inhibits the ESR. However, this explanation appears unlikely because the earlier observations3,4 on the different relative haemolyt-
Atherosclerosis, 1972, 16: 89-94
O.D. - ADRENALIN
o•s-
LYSOLECITHIN AND PLATELETS 93
MINUTES
Fig. 3. The effect of a saturated lysolecithin fraction and a predominantly polyunsaturated lyso-lecithin mixture on platelet aggregation initiated by adrenalin (final concentration 2.5 x 10-6M). In the control 20 attl of 0.9% saline was added 20 sec before the adrenalin. Saturated lysolec-ithin (SAT. LPC) at a final concentration of 0.5 itmoleiml or polyunsaturated lysolecithin (UNSAT.LPC) at a final concentration of 1.1 pmoles/ml was added 20 sec before the adrenalin and the resultant aggregation curves are shown superimposed over that of the control.
is effects of saturated and unsaturated lysolecithins were made in the absence of plasma proteins.
Our earlier report of inhibition of platelet aggregation by lysolecithinl was based on studies of lysolecithin derived from egg yolk lecithin. This material differed from the saturated mixture shown in Table 1 in that it contained 60 % palmitoyl- and 30 % stearoyl-lysolecithin and it was a more potent inhibitor of irreversible platelet aggre-gation. Whether or not this apparent difference in activity is due to an increased con-centration of palmitoyl-lysolecithin will require studies using well-defined synthetic lysolecithins. Lysolecithin isolated from human plasma contains predominantly palmitoyl- and stearoyl-analogues5-7 which we have shown to be the more potent lysolecithins in inhibiting irreversible platelet aggregation. As these compounds are normally present in plasma, the decreased levels of lysolecithin reported in acute myocardial infarctions and peripheral arterial diseasen may play a contributory role in the multiple aetiology of thrombus formation.
ACKNOWLEDGEMENTS
The authors wish to thank Professor C. Adams for his helpful suggestions, Dr. H. Genthe for the generous gifts of lysolecithin preparations, and Dr. J. D. Billimoria for many helpful discussions.
REFERENCES
1 BESTERMAN, E. M. M. AND M. P. T. GILLETT, Inhibition of platelet aggregation by lysolecithin, Atherosclerosis, 1971, 14: 323.
Atherosclerosis, 1972, 16: 89-94
94 E. M. M. BESTERMAN, M. P. T. GILLETT
2 ADLKOFER, F. VON, W. SCHIEBEL, E. ANCKER AND G. RUHENSTROTH-BAUER, NaChWeiSS, Anreicherung und Characterisierung eines Lysolecithin freisetzenden Enzyms aus mensch-lichem Serum, Hoppe-Seyler's Z. Physiol. Chenz., 1968, 349: 417.
3 GOTTFRIED, E. L. AND M. M. RAPPORT, The biochemistry of plasmalogens, Part 2 (Haemolytic activity of some plasmalogen derivatives), J. Lipid Res., 1963, 4: 57.
4 RENAN, F. C., R. A. DESIEL, J. DE GIER, L. L. M. VAN DEENEN, H. EIBL AND 0. WESPHAL, Studies on the lysis of red cells and bimolecular lipid leaflets by synthetic lysolecithins, lecithins and structural analogues, Chem. Phys. Lipids, 1969, 3: 221.
5 GJONE, E., J. F. BERRY AND D. A. TURNER, Isolation and identification of lysolecithin from lipid extracts of normal human serum, Biochim. Biophys. Acta, 1959, 34: 288.
6 WILLIAms, J. H., M. KUCHNIAK AND R. F. WITTER, Phospholipids of human serum, Lipids, 1966, 1: 89.
7 PHILLIPS, G. B. AND J. T. DODGE, Composition of phospholipids and of phospholipid fatty acids of human plasma, J. Lipid Res., 1967, 8: 676.
8 LUCY, J. A., The fusion of biological membranes, Nature (London), 1970, 227: 813. 9 BERLIN, R., C. 0. OLDFELT AND 0. VIKROT, Acute myocardial infarction and plasma phospho-
lipid levels, Acta Med. Stand., 1969, 185: 439. 10 KuNz, F., G. MATT AND H. HACKL, Plasma phospholipids in type IV hyperlipoproteinaemia,
Atherosclerosis, 1970, 11: 265.
Atherosclerosis, 1972, 16: 89-94
(Reprinted from Nature New Biology, Vol. 241, No. 111, pp. 223-224, February 14, 4973)
Influence of Lysolecithin on Platelet Aggregation initiated by 5-Hydroxytryptamine PREVIOUS studies from this laboratory1'2 have shown that irreversible platelet aggregation initiated in vitro by adenosine diphosphate (ADP), adrenaline or collagen was inhibited by lysolecithin, whilst reversible ADP-induced aggregation was unaffected. This indicated that lysolecithin blocked the platelet release reaction which precedes secondary or irrever-sible platelet aggregation. Born3 has reported that exposure of
5 HT
4 0 E
Time (min)
Fig. I Inhibitory effects of lysolecithin on biphasic aggregation of human platelets induced by 5-hydroxytryptamine (5-HT). Platelet rich plasma samples were incubated for 1 min at 37° C with lysolecithin at the final concentrations shown before the addition of 5-HT (final concentration 10 gli.{) at the arrow.
human platelets to 5-hydroxytryptamine (5-HT) does not result in a release reaction and that platelet aggregation initiated by 5-HT is small, rapidly reversed and never enters the second phase which can be induced in human platelets by ADP or adrena-line. However, our results failed to confirm this and 5-HT was found on occasions to initiate biphasic aggregation resembling that induced by ADP. Table 1 shows the frequency of biphasic platelet aggregation, initiated by 5-HT, ADP and adrenaline, recorded during platelet studies of a group of normal male subjects and a group of male patients suffering from chronic peripheral arterial disease, none of whom was known to be taking drugs affecting platelet behaviour. The frequency of biphasic responses within both groups did not differ significantly and the response for individuals was usually the same when repeated at a later date. From the individual data summarized in Table 1 it was found that biphasic 5-HT induced aggregation was always accompanied by biphasic responses in parallel samples treated with ADP and adrenaline. Biphasic platelet aggregation curves initiated by 5-HT were all similar to that shown in Fig. 1 (control), in which secondary aggregation was preceded by partial disaggregation.
During this study we investigated the effects of lysolecithin on both reversible and irreversible platelet aggregation initiated by 5-HT using the methods previously described'''. The fully-saturated lysolecithin fraction used in these studies contained 23.7 % palmitoyl- and 76.3 % stearoyl-lysolecithins (Natterman, Köln) and was dissolved in 0.9 % saline before use. 5-HT (creatinine sulphate complex, Sigma) was dissolved in 0.9 saline and used to initiate platelet aggregation at a final con-centration of 10 Limoli.-1. Platelet-rich plasma (PRP) samples were preincubated with lysolecithin (0.1-1.0 timol ml.-1) for 1 min before initiating platelet aggregation. Platelet aggregation was continuously recorded as an increase in light transmission through the PRP.
Preincubation of PRP with lysolecithin at concentrations greater than 0.2 umol ml.-1 abolished the second phase of 5-HT initiated biphasic platelet aggregation and inhibited the first phase at the higher concentrations tested (Fig. 1). At concentrations below 0.2 'Imo' ml.-1 the first phase of 5-HT initiated aggregation was unaltered, but the rate of secondary aggregation was reduced. Although lysolecithin was found to inhibit irreversible 5-HT initiated platelet aggregation, the effect differed from that earlier reported for ADP or adrenaline induced aggregation in that lysolecithin also inhibited the primary response of platelets to 5-HT. Further studies showed that lysolecithin could also affect reversible 5-HT induced aggregation in the absence of a secondary aggregation phase. Low concentrations of lysolecithin (0.1-0.2 ttmol ml.-1) stimulated reversible 5-HT induced aggregation, and at higher
Table 1 Frequency of Biphasic (Irreversible) Platelet Aggregation
Frequency of biphasic platelet aggregation (% total tests)
Aggregating agent 5-HT ADP Adrenaline Final concentration AM 10 1 2.5 Normal subjects N=50 8 38 94 Arterial disease N=88 9 34 91
Platelet rich plasma from normal male subjects and in a group of male subjects suffering from chronic.peripheral arterial disease used. The frequency is showy as the percentage of the total number of subjects whose PRP gave a Rositive biphasic response for the respec-tive aggregating agent.
concentrations (0.3-0.5 umol ml.-1) the stimulation was less marked. Concentrations of lysolecithin above 0.5 Limol ml.--1 inhibited the reversible platelet response to 5-HT in a dose- dependent fashion.
This study has shown that 5-HT can initiate irreversible platelet aggregation involving the platelet release reaction and that this can be blocked by preincubation of the platelets with lysolecithin. However, lysolecithin also inhibited the first phase of 5-HT induced aggregation, and in the absence of secondary aggregation the primary response was potentiated at low concentrations of lysolecithin and inhibited at higher concentrations. This effect has not been observed in studies in which platelets were preincubated with lysolecithin before being reversibly aggregated with ADP.
Platelet aggregation initiated by 5-HT differs greatly from that induced by ADP since it is closely linked with the active accumulation of 5-HT by the platelets which involves specific 5-HT receptor sites3•4. Lucy5 has described a model for membrane "phase" changes caused by lysolecithin which lead to "micelle" formation within the membrane lipid—protein structure. Low concentrations of lysolecithin may cause slight changes in membrane organization which might expose more 5-HT receptor sites and potentiate 5-HT uptake and aggrega-tion. As the concentration of lysolecithin is increased, so the extent of the merrtrane changes would increase, and this might progressively shut off or block the receptor sites and reduce the extent of aggregation.
E. M. M. BESIERMAN M. P. T. GILLETT
Department of Cardiology, St Mary's Hospital, London W2
Received June 16. 1972.
•
i Besterman, E. M. M., and Gillett, M. P. T., Atherosclerosis, 14, 323 (1971).
2 Besterman, E. M. M., and Gillett, M. P. T., Atherosclerosis (in the press).
3 Born, G. V. R., Plenary session papers of the Twelfth Congress of the International Society of Haematology, 95 (1968).
4 Born, G. V. R., and Gillson, R. E., J. Physiol.,146, 472 (1959). 5 Lucy, J. A., Nature, 227, 813 (1970).
Printed in Great Britain by Flarepath Printers Ltd., St. Albans, Hens.
Atherosclerosis, 17 (1973) 503-513 503 © Elsevier Scientific Publishing Company, Amsterdam — Printed in The Netherlands
HEPARIN EFFECTS ON PLASMA LYSOLECITHIN FORMATION AND PLATELET AGGREGATION
E. M. M. BESTERMAN AND M. P. T. GILLETT Department of Cardiology, St. Mary's Hospital, London, W.2 (Great Britain) (Revised, received August 14th, 1972)
SUMMARY
(1) Intravenous administration of heparin (2,500 or 5,000 units) resulted in a 60-70% increase in the rate of lysolecithin formation in incubated human plasma.
(2) This effect has been shown to be due to the release or activation of a plasma enzyme which is distinct from lecithin : cholesterol acyl transferase (LCAT).
(3) Both the rate and extent of irreversible platelet aggregation initiated by collagen or adrenalin were reduced in post-heparin platelet samples. By contrast the rate and extent of reversible platelet aggregation induced by adenosine diphos-phate was unaltered or even stimulated in post-heparin platelet rich plasma.
(4) The rate of dextran-accelerated erythrocyte sedimentation was decreased in post-heparin blood.
(5) Heparin added in vitro to plasma, platelet rich plasma or whole blood, at concentrations equivalent to the in vivo levels, did not increase lysolecithin formation neither did it decrease platelet aggregation nor retard red cell sedimentation. This suggests that increased lysolecithin formation activated by heparin in vivo may influence both platelet and erythrocyte behaviour. This is discussed as a mechanism which may be relevant to the prophylactic effects of low dose heparin treatment on the incidence of deep vein thrombosis.
Key words: Erythrocyte sedimentation — Heparin — Irreversible platelet aggregation — Lysolecithin formation — Phospholipids
This work was supported by an allocation from the Research Fund of St. Mary's Hospital, London W.2, and by the Wellcome Trust.
504 E. M. M. BESTERMAN, M. P. T. GILLETT
INTRODUCTION
The administration of heparin or heparinoids causes the release of clearing factor or lipoprotein lipase into the blood stream and this enzyme has been the subject of several reviews1,2. More recently it has been shown that post-heparin plasma contains a phospholipase which at first appeared to be unique in its attack on phosphatidyl ethanolamine3, but was later shown to hydrolyze exogenous lecithin substrates4'5. This enzyme has been found to have similar characteristics to lipo-protein lipase and it has been suggested that the two are identical6. Berlin et al.7 have since shown that the rate of formation of lysolecithin from endogenous plasma lecithin is increased in post-heparin plasma. Lysolecithin inhibits irreversible platelet aggregation in vitro8 , and retards the sedimentation rate of red cells resuspended in autologous plasma containing high molecular weight dextran9. Decreased plasma levels of lysolecithin have been reported in subjects suffering from peripheral arterial disease associated with hyperlipoproteinaemialm and from acute myocardial in-farction". In this latter case there is also evidence of decreased plasma lysolecithin formation which may at least be partially responsible for the lower than normal plasma levels. Because of the known inhibitory effects of lysolecithin on platelet aggregation and red cell sedimentation, the evidence which suggests that decreased lysolecithin levels are associated with atherosclerotic arterial disease may be of clinical importance. A study of platelet and red cell behaviour in situations in which altered plasma lysolecithin metabolism can be induced may offer an opportunity for in-vestigating the importance of alterations in plasma lysolecithin concentrations in vivo. The present report describes decreased irreversible platelet aggregation and erythrocyte sedimentation changes accompanying increased lysolecithin formation in post-heparin plasma.
METHODS AND MATERIALS
Subjects Fasted male and female patients undergoing routine right heart catheterisation
were given 1,000-5,000 units of heparin intravenously. These subjects, whose ages ranged from 17-45 years, had evidence of congenital or rheumatic heart disease but no clinical signs of atherosclerotic arterial disease. They were not taking drugs known to affect platelet behaviour. Blood samples were collected in disposable plastic syringes, immediately before and 15 min after heparin administration.
For phospholipid and erythrocyte sedimentation studies 10 ml of blood were transferred to a plastic centrifuge tube containing 8.4 mg of lithium EDTA (Staynes Laboratories Ltd) and centrifuged at 3,500 g for 15 min. Plasma was removed and, after discarding the buffy coat, the packed red cells were washed once with 2 vol. of 0.9 % saline. For platelet studies, 9 vol. of whole blood were transferred to a sili-conised glass centrifuge tube containing 1 vol. of 3.2 % (w/v) tri-sodium citrate • 2H20.
HEPARIN, LYSOLECITHIN AND PLATELETS 505
Platelet rich plasma (PRP) was prepared by centrifuging citrated blood at 150 g for 15 min.
Estimation of lysolecithin formation Aliquots (0.5 ml) of pre- or post-heparin plasma were extracted with 20 ml of
chloroform—methanol (2:1, v/v) before and after a 6 h incubation at 37 °C without added substrate. The extracts were washed with 5 ml of 0.05 M KCI, and the chloro-form layer was retained. Five ml aliquots of this chloroform extract were evaporated to dryness in vacuo. These extracts were re-dissolved in 3 x 20 id of chloroform, and applied as 2 cm streaks to a 20 x 20 cm thin-layer plate (0.25 mm thickness of Silica Gel H—Merck) which had been previously activated at 120 °C for 20 min. The thin-layer chromatogram (TLC) was developed with chloroform—acetone—methanol-acetic acid—water (50:20:10:10:5 by vol.). In preliminary experiments the identity of the resolved phospholipid bands was confirmed from the Rf of authentic phospho-lipid standards (Sigma), after exposure to iodine vapour and the position of the bands had been marked. The iodine was allowed to evaporate, and the areas of silica gel corresponding to phosphatidyl ethanolamine, lecithin, sphingomyelin and lysolecithin were removed to Pyrex digestion tubes. Their phosphorus content was measured by a modification of Bartlett's method12 using suitable unstained areas of silica gel as blanks. The colour intensity was recorded at 830 nm after the removal of the silica gel by centrifugation. The total phospholipid concentration of the whole extract was similarly measured, and from the proportion of phosphorus recovered from the four phospholipid bands their concentrations in plasma was calculated. Phosphorus recovery after TLC was in all cases greater than 90 % and replicate samples agreed well for each phospholipid class. Lysolecithin formation was recorded as the differ-ence between the initial and post-incubation plasma concentrations, and was ex-pressed as /moles formed per litre of plasma per hour. LCAT activity was measured by the method described by Glomset and Wright13, in which the rate of esterification of [7-3H1cholesterol (Radiochemical Centre, Amersham) by plasma incubated at 37°C was measured. Results were expressed as iumoles free cholesterol esterified per litre per hour.
Platelet aggregation Platelet aggregation in pre- and post-heparin PRP was measured using the
turbidimetric technique and apparatus previously describeds. One ml aliquots of PRP were warmed to 37°C and transferred to the sample compartment where they were maintained at 37°C and magnetically stirred. Platelet aggregation was continu-ously recorded as a decrease in optical density (O.D.) after the addition of collagen, adrenalin or ADP. Several concentrations of collagen or adrenalin were added to separate pre-heparin PRP samples and a series of concentration-dependent aggrega-tion curves were recorded. The same concentrations of collagen or adrenalin were then tested on post-heparin samples of PRP. The same times for testing, relative to time of venepuncture, were rigorously observed in these comparative aggregation
506 E. M. M. BESTERMAN, M. P. T. GILLETT
studies. Platelet counts for PRP samples were made by phase-contrast microscopy, and were usually in the range of 3-5 x 105/mm3. No significant variation between pre- and post-heparin platelet counts was observed, and in all studies there was less than 5 % variation between individual pre- and post-heparin counts. Irreversible platelet aggregation curves for corresponding pre- and post-heparin PRP samples were compared by measuring both the initial rate of secondary aggregation over a period of 1 min or by measuring the total decrease in O.D. 4 min after the addition of aggregating agent. Similar comparisons resulted when either of these two methods was used and in this report the initial rates of secondary aggregation have been compared. In some studies ADP was used to initiate reversible aggregation in pre-heparin PRP, and this was directly compared with the aggregating effect of the same concentrations of ADP on post-heparin PRP. No comparisons of irreversible platelet aggregation induced by ADP in pre- and post-heparin PRP were made.
A series of control experiments were performed in order to investigate the direct effect of heparin on platelet aggregation. In these studies aliquots of normal (pre-heparin) PRP were incubated with heparin (0.5-10 units/m1), for 1 min before initia-ting aggregation with collagen or adrenalin. Aggregation curves were quantitatively compared with control curves (no heparin) as described above.
Erythrocyte sedimentation The measurement of red cell sedimentation using the Westergren technique was
modified as described by von Adikofer et al.U. 0.5 ml of packed washed red cells was re-suspended in 0.7 ml of autologous plasma and 0.3 ml of a 2 %, solution of dextran (average molecular weight 150,000) in 0.9 % saline (Fisons Ltd.) was added to accelerate the ESR. Duplicate tubes for pre- and post-heparin blood were read at 5 or 10 min intervals for up to 1 h.
Several studies were performed to investigate the direct effects of heparin on red cell sedimentation. Washed red cells were re-suspended in autologous plasma containing dextran and aliquots of heparin (0.5-10 units/ml) were added. The sedimentation rate of heparin-treated cells was compared with a control ESR (no added heparin).
Chemicals All solvents and chemicals used in this study were analytical grade (Fisons Ltd).
Heparin (mucous) was obtained from Weddell Pharmaceuticals and protamine sul-phate (1 % in sterile water) from Boots Pure Drug Co.
RESULTS
( I) Increased lysolecithin formation in post-heparin plasma Preliminary studies showed that lysolecithin formation was maximally in-
creased within 5-10 min of heparin administration, and that this effect lasted for at least 1 h. Table 1 shows the results of a series of studies with 2,500 and 5,000 units of
HEPARIN, LYSOLECITHIN AND PLATELETS 507
TABLE 1
THE EFFECTS OF INTRAVENOUS HEPARIN ADMINISTRATION ON LYSOLECITHIN FORMATION AND LECITHIN DEGRADATION IN INCUBATED HUMAN PLASMA WITHOUT ADDED SUBSTRATES
Post-heparin samples were taken 15 min after administration of heparin.
Heparin dosage (units)
Lysolecithin formation (pmoles/1/h)
Lecithin degradation (pnioleslilh)
2,500 Pre-heparin 41 ± 10,4 45 ± 13 Post-heparin 62 ± 10 59 ± 11
N = 9 P < 0.001b P < 0.02 5,000
Pre-heparin 33 ± 9 38 ± 11 Post-heparin 59 ± 13 60 + 13
N = 11 P < 0.001 P < 0.001
a Means ± standard deviation. B Calculated from Student's t-test.
heparin. Increased lysolecithin formation was accompanied by increased lecithin degradation, and the results for these two heparin dosages were quantitatively similar. In several studies 1000 units of heparin were administered and this also gave similar results. There was no evidence of any changes in the concentration of total phospho-lipid, sphingomyelin or phosphatidyl ethanolamine during incubation of pre- or post-heparin plasma. In some post-heparin plasma samples the initial level of lyso-lecithin was noted to be greater than the pre-heparin concentration, but this effect was not consistent.
(2) Differentiation of post-heparin lysolecithin formation front LCAT Incubation of pre- and post-heparin plasma samples with protamine sulphate
( 1 mg/ml) abolished the increase in post-heparin lysolecithin formation but did not
TABLE 2
THE EFFECT OF PROTAMINE SULPHATE (1 mg/m1) ON THE FORMATION OF LYSOLECITHIN IN INCUBATED PRE- AND POST-HEPARIN PLASMA
Post-heparin samples were taken 15 min after the intravenous administration of 5,000 units of heparin.
Lysolecithin formation ( pmolesIllhour)
no addition + protamine sulphate
Pre-heparin 38 ± 9a 41 ± 13 Post-heparin 60 ± 15 42 ± 10
N = 5 P < 0.05° • N.S.
a Means ± standard deviation. b Calculated from Student's t-test.
POST-HEPARIN PRP OLLAGEN
PRE-HEPARIN PRP
10,1. 20,1.
30,4.
40,t.
508 E. M. M. BESTERMAN, M. P. T. GILLE1T
TABLE 3
THE EFFECT OF INTRAVENOUS HEPARIN ADMINISTRATION ON LYSOLECITHIN FORMATION AND CHOLESTEROL ESTERIFICATION IN PRE- AND POST-HEPARIN PLASMA
Post-heparin blood samples were taken 15 min after the administration of 2,500 units of heparin.
Lysolecithin formation Cholesterol esterification (!moles/1/1z) (!moles/11h)
Pre-heparin 43 ± lla 71 ± 10b Post-heparin 64 + 13a 73 ± 11b
N -= 7 P < 0.01 N.S.
Mean ± standard deviation. b Mean ± standard error of the mean.
affect pre-heparin activities (Table 2). During incubation of plasma samples with protamine sulphate slight turbidity developed which may have resulted from pre-cipitation of plasma proteins. In several experiments pre- and post-heparin samples were incubated with p-hydroxymercuribenzoate (2 ,umoles/m1). This resulted in the partial or total inhibition of pre-heparin lysolecithin formation, whilst post-heparin lysolecithin formation was reduced but never totally inhibited.
There was no increase in the rate of cholesterol esterification in post-heparin plasma despite the significant increase in lysolecithin formation (Table 3).
MINUTES
Fig. 1. Comparison of platelet aggregation initiated by collagen or adenosine diphosphate (ADP) in pre- and post-heparin PRP. Identical conditions of testing, including preparation, platelet count (± 5 %), concentrations of reagents and time of testing after venepuncture, were rigorously observed in the comparison of aggregation before and after heparin administration.
04 pre- post-
04— pre- post-
0 03 0
cn
g10.2 — O
0 — COLL AGEN A DRENALIN
0
HEPARIN, LYSOLECITHIN AND PLATELETS 509
(3) Platelet studies Both the initial rate and the extent of collagen-induced irreversible platelet
aggregation after 4 min were reduced in post-heparin PRP samples compared with pre-heparin samples tested under identical conditions. By contrast, reversible ADP-induced platelet aggregation in post-heparin PRP was unaltered or even slightly stimulated (Fig. 1). This effect on collagen-induced aggregation was found to be consistent and statistically significant (P < 0.01), and in three studies with adrenalin the initial rate of secondary platelet aggregation was also reduced in post-heparin PRP (Fig. 2). In these studies a range of collagen or adrenalin concentrations was tested and it was noted that the reduction in post-heparin aggregation was more apparent at concentrations of aggregating agent that initiated a maximal decrease in O.D. in pre-heparin PRP which was consistent with maximal irreversible aggre-gation. If the concentration of collagen was further increased, but without increasing the rate or extent of aggregation in pre-heparin PRP, the difference in post-heparin aggregation was less apparent than at concentrations of collagen that were just suffi-cient to initiate the maximal aggregation response in pre-heparin PRP. Similarly, if the concentration of collagen tested on pre-heparin PRP produced only slight aggre-gation, then there was very little difference in the aggregation response in post-heparin PRP (see Fig. 1). The rates of aggregation shown in Fig. 2 are for concen-trations of collagen or adrenalin that produced maximal platelet aggregation in pre-heparin PRP.
At concentrations that were equivalent to those administered in vivo, heparin (0.5-2 units/ml) had no consistent effect on either collagen- or adrenalin-initiated aggregation. However, higher concentrations of heparin (< 10 units/nil) partly inhibited irreversible platelet aggregation.
Fig. 2. The effect of heparin administration on the initial rate of irreversible aggregation initiated by collagen and on the initial rate of secondary aggregation initiated by adrenal in . The rate of aggregation before and 15 min after heparin administration has been plotted for the concentration of aggregating agent which was sufficient to initiate maximal irreversible aggregation in the pre-heparin sample. The difference between the mean initial rates of collagen induced aggregation, shown by the horizontal bars, was statistically significant (P < 0.01: Student's t-test).
40 te—e Pre-heparin
a—a Post-heparin 3
0 77, E 20
20 40 60 80 Minutes
10
510 E. M. M. BESTERMAN, M. P. T. GILLETT
Fig. 3. The effect of heparin administration on the dextran-stimulated erythrocyte sedimentation test. 0.5 ml of washed pre- or post-heparin red cells was suspended in 0.7 ml of autologous plasma and 0.3 ml of 2% dextran (average mol. wt. 150,000) was added to increase the ESR. The mean values obtained from duplicate tubes have been plotted.
(4) Erythrocyte sedimentation In six studies, in which collagen-induced aggregation in post-heparin PRP was
decreased, the erythrocyte sedimentation rate was measured using pre- and post-heparin plasma and washed red cells. Decreased sedimentation rates were found in each case; Fig. 3 shows the results of one such study which was typical of the results obtained.
Heparin (10 units/m1) added in vitro increased the ESR, but had no effect at concentrations similar to those used in vivo (0.5-2 units/nil).
DISCUSSION
Our results showing the increased formation of lysolecithin in incubated human plasma after intravenous administration of heparin confirm the earlier work of Berlin et a1.7 and are quantitatively similar. An attempt has been made to study the enzyme responsible for this increased lecithinase activity and to compare it with lecithin: cholesterol acyl transferase (LCAT) (see review by Glomset14), which has been shown to be responsible for lysolecithin formation in normal (pre-heparin) plasma. The differing inhibitory effects of protamine sulphate and of p-hydroxymercuribenzoate on lysolecithin formation in pre- and post-heparin plasma indicated that post-heparin stimulated lysolecithin formation was not due to increased LCAT activity. This has been confirmed by direct measurements of cholesterol esterification as a means of assaying LCAT in pre- and post-heparin plasma. In a series of studies no increased cholesterol esterification in post-heparin plasma was observed despite a significant increase in lysolecithin formation. Although this post-heparin enzyme has only been partially characterized, it would seem to be similar to lipoprotein lipase as both are inhibited by protamine sulphate.
HEPARIN, LYSOLECITHIN AND PLATELETS 511
The increased formation of plasma lysolecithin after heparin, which presumably also occurs in vivo, might be expected to alter the relative concentrations of lecithin and lysolecithin in the non-incubated post-heparin sample. Although in some studies lysolecithin levels were slightly raised and lecithin levels decreased, this was not always so. It is possible that lysolecithin formed in the plasma in vivo has a short half-life due to its metabolism by enzymes described in red cells15,16, leucocytes17,18 and platelets1°.
Despite the absence of significant increases in lysolecithin levels in unincubated post-heparin plasma, we have found that both irreversible platelet aggregation and erythrocyte sedimentation were significantly reduced after heparin. This was not due to the direct action of heparin on the platelets and red cells, for heparin added in vitro at equivalent concentrations to those in vivo had no effect on aggregation or sedimentation. It thus seems probable that these changes in platelet and erythrocyte behaviour are due to an indirect effect of heparin in vivo. It is tempting to speculate that this indirect mechanism involves the release of an enzyme responsible for in-creased lysolecithin formation, as both irreversible platelet aggregation and red cell sedimentation are decreased by lysolecithin in vitro. Smith and Barboriak2° have produced evidence that suggests that lipoprotein lipase may be adsorbed onto the platelet membrane and, if post-heparin lysolecithin releasing enzyme is similar to lipoprotein lipase, it too may be adsorbed onto platelets. If this is confirmed, then the enzyme might have a direct effect on platelet membrane phospholipids or their immediate plasmatic environment. Such a mechanism may explain decreased irre-versible platelet aggregation in the absence of raised plasma levels of lysolecithin. This problem is currently being studied.
Post-operative patients have been shown to develop abnormal platelet ad-hesiveness one to two days after surgery21; this is probably related to the high risk of post-operative deep calf-vein thrombosis (DVT). Small doses of heparin admini-stered pre- and post-operatively have been shown to decrease both the increased platelet adhesiveness22 and the incidence of DVT23. Kakkar et (//.24 have also shown that small doses of heparin, administered subcutaneously before operation, are prophylactic for DVT. They have suggested that an inhibitor of activated factor X may be potentiated by small doses of heparin. However the prophylactic doses of heparin used in these studies23,24 neither prolonged the clotting time nor produced significant clinical bleeding; thus the anti-thrombotic effect does not seem to be directly due to the inhibition of the coagulation mechanism. Slack et a/.25 have correlated platelet adhesiveness with post-heparin lipoprotein lipase activity. A similar correlation might occur between platelet adhesiveness and post-heparin lysolecithin formation. This could involve a mechanism known to cause changes in platelet behaviour, namely the inhibition of irreversible platelet aggregation by lysolecithin. If this relationship exists, and the results reported in our present study suggest that it does, then it could explain the prophylactic effects of small dose heparin treatment on the incidence of DVT during and after surgical operations.
512 E. M. M. BESTERMAN, M. P. T. GILLETT
ACKNOWLEDGEMENTS
The authors wish to thank Dr. C. McCarthy for his help with this project, and Dr. J. D. Billimoria for his many helpful discussions and suggestions.
REFERENCES
1 RounsrsoN, D. S., AND J. E. FRENCH, Heparin, the clearing factor lipase and fat transport, Phar-macol. Rev., 12 (1960) 241.
2 ROBINSON, D. S., The clearing factor lipase and its action in the transport of fatty acids between the blood and the tissues, Advan. Lipid. Res., 1 (1963) 133.
3 VOGEL, W. C., AND L. ZIEVE, Post-heparin phospholipase, J. Lipid Res., 5 (1964) 177. 4 VOGEL, W. C., AND E. L. BIERMAN, Post-heparin serum lecithinase in man and its positional
specificity, J. Lipid Res., 8 (1967) 46. 5 ZIEVE, L., AND W. M. DOIZAKI, Similarities between post-heparin lipase and post-heparin phospho-
lipase, Fed. Proc., 25 (1966) 365. 6 DOIZAKI, W. M., AND L. ZIEVE, Similarities between post-heparin lipase and post-heparin phospho-
lipase, Proc. Soc. Exp. Biol. Med., 129 (1968) 182. 7 BERLIN, R., C. 0. OLDFELT AND 0. VIKROT, Increased lecithinase activity after heparin admini-
stration, Acta Med. Scand., 185 (1969) 433. 8 BESTERMAN, E. M. M., AND M. P. T. GILLErr, Inhibition of platelet aggregation by lysolecithin,
Atherosclerosis, 14 (1971) 323. 9 ADLKOFER, F. VON, W. SCHIEBEL, E. ANCKER AND G. RUHENSTROTH-BAUER, Nachweiss, An-
reicherung and Charakterisicrung eines Lysolecithin freisetzenden Enzyms aus menschlichem Serum, Hoppe-Seyler's Z. Physiol. Chent., 349 (1968) 417.
10 KUNZ, F., G. MATr AND H. HACKL, Plasma phospholipids in type IV hyperlipoproteinaemia, Atherosclerosis, 11 (1970) 265.
11 BERLIN, R., C. OLDFELT AND 0. VIKROT, Acute myocardial infarction and plasma phospholipid levels, Acta Med. Scand., 185 (1969) 439.
12 BARTLETT, G. R., Phosphorus assay in column chromatography, J. Biol. Chem., 234 (1959) 466. 13 GLOMSET, J. A., AND J. L. WRIGHT, Some properties of a cholesterol esterifying enzyme in human
plasma, Biochim. Biophys. Acta, 89 (1964) 266. 14 GLOMSET, J. A., The plasma lecithin—cholesterol acyl transferase reaction, .1. Lipid Res., 9 (1968)
155. 15 MULDER, E, J. W. 0. VAN DEN BERG AND L. L. M. VAN DEENEN, Metabolism of red-cell lipids,
Part 2 (Conversion of lysophosphoglycerides), Biochint. Biophys. Acta, 106 (1965) 118. 16 MULDER, E., AND L. L. M. VAN DEENEN, Metabolism of red cell lipids, Part 3 (Pathways for
phospholipid renewal), Biochim. Biophys. Acta, 106 (1965) 348. 17 ELSBACH, P., J. W . 0. VAN DEN BERG, H. VAN DEN BOSCH AND L. L. M. VAN DEENEN, Metabolism
of phospholipids by polymorphonuclear leukocytes, Biochim. Biophys. Acta, 106 (1965) 338. 18 ELSBACH, P., Metabolism of lysophosphatidyl ethanolamine and lysophosphatidyl choline by
homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages, J. Lipid Res., 8 (1967) 359.
19 ELSBACH, P., P. PErrts AND A. MARCUS, Lysolecithin metabolism by human platelets, Blood, 37 (1971) 675.
20 SMITH, J. C., AND J. J. BARBORIAK, Blood platelets and heparin-induced lipolytic activity, Amer. J. Physiol. 212 (1967) 1113.
21 WRIGHT, H. P., Changes in the adhesiveness of blood platelets following parturition and surgical operations, J. Pathol. Bacterial., 54 (1942) 461.
22 NEGUS, D., D. J. PINTO AND W. W. SLACK, Effects of small doses of heparin on platelet adhesive-ness and lipoprotein lipase activity before and after surgery, Lancet, i (1971) 1202.
23 WILLIAMS, H. T., Prevention of post-operative deep vein thrombosis with peri-operative sub-cutaneous heparin, Lancet, ii (1971) 950.
HEPARIN, LYSOLECITHIN AND PLATELETS 513
24 KAKKAR, V. V., E. S. FIELD, A. M. NICHOLAIDES, P. T. FLUTE, S. WESSLER AND E. T. YIN, Low dose heparin in prevention of deep vein thrombosis, Lancet, ii (1971) 669.
25 SLACK, J., J. SEYMOUR, L. McDoNALD AND F. LOVE, Lipoprotein lipase levels and platelet stickiness in patients with ischaemic heart disease and in controls, distinguishing those with an affected first degree relative, Lancet, ii (1964) 1033.
If
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THROMBOSIS RESEARCH Supplement Number 1 Vol. 4, 1974 Ptd. in USA LIPIDS AND THROMBOSIS, Tromscp Pergamon Press
A POSSIBLE THROMBO-PROTECTIVE ROLE FOR PLASMA LYSOLECITHIN IN MAN. M.P.T. GILLETT AND E.M.M. BESTERMAN. DEPARTMENT OF CARDIOLOGY, ST. MARY'S HOSPITAL, LONDON W2INY, ENGLAND.
Lysolecithin is quantitatively the third most important phospho-lipid found in human plasma where it is formed from lecithin by the action of lecithin:cholesterol acyl transferase (LCAT). Lyso-lecithin is strongly surface-active and potentially dangerous because of its haemolytic properties. Very little is known about the function of plasma lysolecithin or whether or not it plays a role in physiological or pathological changes to the blood. This report will concern the possible role of lysolecithin as one factor affecting blood platelet behaviour in vivo.
The exposure, of platelets to lysolecithin at concentrations simi-lar to those found in normal plasma, inhibits irreversible plate-let aggregation induced by adenosine diphosphate (ADP), adrenaline and collagen (Besterman and Gillett, 1971) and also by thrombin and serotonin (Besterman and Gillett, 1973a). This inhibitory activity has been shown to be due to the blocking of the normal platelet release reaction and is linked with the surface-activity of lysolecithin. Saturated lysolecithin fractions inhibited irreversible aggregation but a lysolecithin fraction containing a high percentage of polyunsaturated fatty acids, did not (Bester-man and Gillett, 1972). Similar differences were found when the haemolytic activity of different lysolecithins was studied (Re-nan et al, 1969).
Among the many known inhibitors of platelet function, lysoleci-thin is of particular interest because it is a normal constituent lipid of the blood, and its plasma level has been found to be de-creased in patients presenting with acute myocardial infarction (Berlin et al, 1969b). Results from this laboratory show de-creased levels of plasma lysolecithin in patients suffering from chronic ischaemic and peripheral arterial diseases, and still lower levels were recorded in patients during the acute stage of myocardial infarction. Decreased plasma lysolecithin levels have also been shown in pregnant women and in women taking some types of oral contraceptive preparations. It was not possible to in-clude measurements of platelet function in the pregnancy study, although a significant negative correlation was found between de-creased lysolecithin levels during pregnancy and red cell behavi-our, which was consistent with-lysolecithin effects on red cell - behaviour in vitro. In the case of women treated with oral con-traceptives, increased collagen-initiated platelet aggregation was found to correlate with decreased plasma levels of lysoleci-thin during the treatment cycle.
85
Supplement Number 1, Vol. 4, 1974
Intravenous administration of heparin (500 - 5,000 units). to man increases plasma lysolecithin formation (Berlin et al, 1969a), although this is not due to increased LCAT activity (Besterman and Gillet, 19-73b).--Irreversible platelet- aggregation induced by collagen or adrenaline was significantly decreased following heparin administration, although reversible ADP-induced aggre-gation was unaffected. Platelets exposed to heparin in vitro at concentrations equivalent to those administered in vivo, showed no functional changes and it is possible that increased lyso-lecithin formation stimulated by heparin in vivo, is responsible for the observed decreases in irreversible platelet aggregation.
Decreased plasma lysolecithin levels have been found in several populations having an increased risk of arterial thrombosis, in-cluding acute and chronic ischaemic heart disease, peripheral arterial disease and women who are pregnant or taking oral con-traceptives. Decreased irreversible platelet aggregation is associated with increased lysolecithin formation after heparin administration and by contrast, increased platelet aggregation can be correlated with decreased plasma lysolecithin in women taking oral contraceptives. Both of these latter results are consistent with the inhibitory action of lysolecithin on platelet aggregation in vitro. These results suggest that lysolecithin may have a possible thrombo-protective role in the blood and that a reduction in its concentration may be a contributory fac-tor towards intravascular platelet aggregation and subsequent thrombosis.
(This work was supported by a generous allocation from the Wellcome Fund).
REFERENCES: Berlin R., C.O. Oldfelt and O. Vikrot, Acta.med.Scand., 1969, 185:433. Berlin R., C.O. Oldfelt and O. Vikrot, Acta.med.Scand., 1969, 185:439. Besterman, E.M.M. and M.P.T. Gillett, Atherosclerosis, 1971, 14: 322. Besterman, E.M.M. and M.P.T. Gillett, Atherosclerosis, 1972, 16: 89. Besterman, E.M.M. and M.P.T. Gillett, Nature New Biology, 1973, 241;223. Besterman, E.M.M. and M.P.T. Gillett, Atherosclerosis, 1973, 17: 503. Renan, F.C., R.A. Demel, J. de Gier, L.L.M. Van Deenan, H.Eibl
Jand 0. Wesphal, Chem. Phys. Lipids, 1969, 3:221.
