Lecitinas 1

Phosphatidylcholine
Biochemical and Clinical Aspects

of Essential Phosphol ipids
Editor: H. Peeters
Contributors
G. Assmann . V. Blaton . R. Blendin . D. E. Bowyer
P. F. Davies' AJ. Day B. Declercq E. Decoopman
C. Desreumaux . P. Dewailly . H. Ditschuneit
H. H. Ditschuneit . A M. Ehrly . J. M. Fox' J. C. Fruchart
G. Fuchs . D. Hegner' J. Halzl . A K. Horsch
A N. Howard' K. Hudson' H. Kaffarnik . J. Klemm
H.-U. Klar . D. Lekim . P. Mares 'J. Patelski . H. Peeters
L. Samochowiec . J. Schneider' J. Skofepa
F. Soetewey . K. Szyszka . H. Todorovicova
E. Tvrzicka . D. Vandamme
With 81 Figures
Springer- Verlag
Berlin Heidelberg New York 1976
Dr. Huben Peelers

SimonSlevin lnstituut voor Wetenschappelijk Onderzoek.
8- 8000 Brugge. Belgiu m
Proceeding s o f aSymposium held at the Simon Stevin Institute

15-18 Nov. 1975 in Brugge/ Belgium
IS BN-1 3: 978-3-642-66426-7
001: 10.1007/978-3-642-66424-3
e -IS BN-13: 978-3-642-66424-3
This work is WbjKttO copy.ight_All ''IIht5 a &served. whet he. the whole of pan of
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dete. mi ne.:! bVag'Hmen t with the pub lisher.
by Spr inger Verlag Berlin Heidelberg 1976
The use of .egistered names. t'adema rks. etc. In th is publicatioo does nOl imply.
I ven in Ihe absenel of II specific stalement. Ihal ' llc h names a' l l. empI ho m lh8 'elevan l
ptotec tiv8 1aws and regllla!ions and Iheralora hH lor genera l lise.
Preface
A symposium was centered around the unsaturated phosphatidylcholine molecule and organized in order to assemble and coordinate theoretical views with facts and results. The presence of
a high percentage of essential fatty acids in unsaturated phosphatidylcholine gave rise to the essential phospholipid concept.
An overview of the biological significance of phospholipids
and a review of a specific phosphatidylcholine-related enzyme,
namely LCAT or lecithin cholesterol acyl transferase, open
these proceedings. The simultaneous use of the synonyms - lecithin and phosphatidylcholine - was solved throughout the published material by a preferential use of the more precise chemical terminology of phosphatidylcholine.
A set of papers centered around the pharmacology of polyunsaturated phosphatidylcholine (PU-PC) or essential phospholipids
(EPL) is followed by reports on its therapeutic effects. Further
papers deal with the metabolism of the arterial wall and the
presence of phospholipid related enzyme systems. Some hemodynamic related effects are dealt with in the last section.
These proceedings could be edited within a few months thanks
to the active cooperation of the authors. The editors are
grateful to acknowledge this rather unusual performance which
tends to prove the interest of all participants in this symposium. It seems logical to presume that the topic itself is
an important one and that the meeting was timely organized.
At this point we wish to express our gratitude to the Nattermann
Company, Cologne, for their generous support and to their collaborators for the excellent organization of this meeting, including the final preparation of the manuscript.
The Town Corporation of Brugge, the Board of Directors and
the staff of the Simon Stevin Institute have also contributed
to the success of a stimulating scientific symposium on essential phospholipids.
These proceedings are intended as a summary of the information
available today and still more as a starting point for future
work in the field of phospholipids and their application as a
therapeutic agent.
Brugge, March 1976
H. PEETERS
Contents
GLOSSARY
A Glossary of Essential Phospholipids, Lipids, and

Lipoproteins
by J.M. FOX.
With 3 Figures
INTRODUCTORY REVIEWS
The Biological Significance of the Plasma Phospholipids

by H. PEETERS.
With 13 Figures
10
Lecithin-Cholesterol-Acyl-Transferase.
-Lipoprotein and Phospholipid Substrate Specificityby G. ASSMANN
34
PHARMACOKINETICS OF ESSENTIAL PHOSPHOLIPIDS
On the Pharmacokinetics of Orally Applied Essential

Phospholipids (EPL)
by D. LEKIM.
With 5 Figures
48
Pharmacokinetic Studies on Phosphatidylcholine and

Phosphatidylinositol
by J. HOLZL.
With 7 Figures 66
Plasma Removal of Intravenous Essential Phospholipids
in Han
by P. DEWAILLY, E. DECOOPMAN,
C. DESREUMAUX and J.C. FRUCHART. With 1 Figure
80
Effect of Essential Phospholipids on the ATPases and

on the Fluidity of Liver Plasma Membranes
by D. HEGNER.
With 4 Figures
87
ESSENTIAL PHOSPHOLIPID THERAPY IN DYSLIPEMIC STATES
Effects of Essential Phospholipids on the CarbohydrateInduced Hypertriglyceridemia

by H. DITSCHUNEIT, H.-U. KLOR and H.H. DITSCHUNEIT 98
Influence of EPL on Lipolysis in Vitro and in Vivo
by K. SZYSZKA.
With 7 Figures 115
VIII
The Human Plasma Lipids and Lipoproteins Under Influence

of EPL-Therapy
by V. BLATON, B. DEQLERCQ, D. VANDAMME,
F. SOETEWEY and H. PEETERS.
With 2 Figures
125
New Analytical Approach to the Study of Hyperlipidemia
by J. SKOREPA, P. MARES, H. TODOROVICOVA
and E. TVRZICKA.
With 1 Figure 133
PHOSPHOLIPID METABOLISM IN THE ARTERIAL WALL
Influence of Essential Phospholipids on Serum Lipids

and Lipid Metabolism in the Aortic Wall.
-Experiments in ;~ormal and Atherosclerotic Rabbi tsby A.K. HORSCH, K. HUDSON and A.J. DAY.
Wi th 11 Figures 140
Effect of EPL on the Metabolism of Lipids in the
Arterial Wall
by D.E. BOWYER and P.F. DAVIES. With 5 Figures
160
Effect of EPL on the Lipid Metabolism of the Arterial

Wall and Other Tissues
by A.N. HOWARD an J. PATELSKI. With 6 Figures 187
Arterial Metabolism of Cholesteryl Esters -Mechanism
of Action of Polyunsaturated Phosphatidylcholineby J. PATELSKI.
With 5 Figures . 201
On the Action of Essential Phospholipids in Experimental
Atherosclerosis
by L. SAMOCHOWIEC.
With 4 Figures
211
ESSENTIAL PHOSPHOLIPIDS IN RED CELLS AND THROMBOCYTES
Influence of Essential Phospholipids on the Flow

Properties of the Blood
by A.M. EHRLY and R. BLEND IN 228
The Treatment of Arterial and Venous Circulatory
Disturbances with EPL
by J. KLEMM.
With 5 Figures 237
Influence of Essential Phospholipids on Human Platelet
Aggregability
by J. SCHNEIDER, G. FUCHS and
H. KAFFARNIK.
With 2 Figures
244
subject Index . 249
List of Contributors
ASSMANN, G., Abteilung fur Klinische Chemie und Zentrallaboratorium der Universitatsklinik, D-5000 Kaln 41, Germany
BLATON, V., Simon-Stevin-Instituut voor Wetenschappelijk Onderzoek, B-8000 Brugge, Belgium
BLENDIN, R., Klinikum der Johann-Wolfgang-Goethe-Universitat,
Zentrum der Inneren Medizin, Abteilung fur Angiologie,
D-6000 Frankfurt a.M. 70, Germany
BOWYER, D.E., Department of Pathology, University of Cambridge,
Cambridge, England
DAVIES, P.F., Department of Pathology, University of Cambridge,
Cambridge, England
DAY, A.J., Department of Physiology, University of Melbourne,
Victoria, Australia
DECLERCQ, B., Simon-Stevin-Instituut voor Wetenschappelijk
Onderzoek, B-8000 Brugge, Belgium
DECOOPMAN, E., Laboratoire de Physiopathologie des Lipides,
Institut Pasteur, F-59012 Lille Cedex, France
DESREUMAUX, C., Laboratoire de Physiopathologie des Lipides,
DEWAILLY, P., Laboratoire de Physiopathologie des Lipides,
DITSCHUNEIT, H., Med. Universitatsklinik, D-7900 Ulm, Germany
DITSCHUNEIT, H.H., Med. Universitatsklinik, D-7900 Ulm, Germany
EHRLY, A.M., Klinikum der Johann-Wolfgang-Goethe-Universitat,
Zentrum der Inneren Medizin, Abteilung fur Angiologie,
D-6000 Frankfurt a.M. 70, Germany
FOX, J.M., Medizinische Fakultat der Universitat des Saarlandes,
D-6650 Homburg and A.Nattermann & Cie. GmbH, D-5000 Kaln 30,
Germany
FRUCHART, J.C., Laboratoire de Physiopathologie des Lipides,
FUCHS, G., Medizinische Poliklinik der Universitat, D-3550 Marburg, Germany
x
HEGNER, D., Institut flir Pharmakologie der Universitat,
D-8000 Mlinchen 22, Germany
H5LZL, J., Institut flir Pharmazeutische Arzneimittellehre der
Universitat, D-8000 Mlinchen 2, Germany
HORSCH, A.K., Medizinische Universitatsklinik, D-6900 Heidelberg, Germany
HOWARD, A.N., Department of Medicine, University of Cambridge,
Addenbrooke's Hospital, Cambridge, England
HUDSON, K., Department of Physiology, University of Melbourne,
Victoria, Australia
KAFFARNIK, H., Medizinische Poliklinik der Universitat,
D-3550 Marburg, Germany
KLEMM, J., Rontgen-Abteilung der II.Medizinischen Universitatsklinik, D-8000 Mlinchen, Germany
KL5R, H.-U., Med. Universitatsklinik, D-7900 Ulm, Germany
LEKIM, D., A.Nattermann & Cie. GmbH., Chemische Forschung und
Entwicklung, D-5000 Koln 30, Germany
~
MARES, P., 4th Chair of Internal Medicine, Faculty of Medicine,

Charles University, Prague, CSSR
PATELSKI, J., Lipid Metabolism Laboratory, Department of Biochemistry, Medical Academy, Poznan, Poland
PEETERS, H., Simon-Stevin-Instituut voor Wetenschappelijk
SAMOCHOWIEC, L., Department of Pharmacology, Pomeranian Medical
Acad~my, Szczecin, Poland
SCHNEIDER, J., Medizinische Poliklinik der Universitat,
D-3550 Marburg, Germany
SKOREPA, J., 4th Chair of Internal Medicine, Faculty of Medicine,
Charles University, Prague, CSSR
SOETEWEY, F., Simon-Stevin-Instituut voor Wetenschappelijk
SZYSZKA, K., Department of Pharmacology, Pomeranian Medical
Academy, Szczecin, Poland
TODOROVICOVA, H., 4th Chair of Internal Medicine, Faculty of
Medicine, Charles University, Pra~ue, CSSR
TVRZICKA, E., 4th Chair of Internal Medicine, Faculty of
Medicine, Charles University, Prague, CSSR
VANDAMME, D., Simon-Stevin-Instituut voor Wetenschappelijk
Glossary
Fig. 1: Stuart model of 1.2dilinoleoyl phosphatidylcholine viewed over the three methyl groups of the
choline moiety folded to the front. while the two linoleoyl residues point to the background
A Glossary of Essential Phospholipids,

Lipids, and Lipoproteins
J. M. Fox
Medizinische Fakultat der Universitat des Saarlandes,
0-6650 Homburg and A. Nattermann & Cie.GmbH, 0-5000 Koln 30, Germany
ESSENTIAL PHOSPHOLIPIDS
1,2-dilinoleoyl phosphatidyl choline (Fig.1) is the prototype

of a polyunsaturated phosphatidyl choline (PU-PC) molecule
(Fig. 2) :
1 pos.
H C-O-C
21
2 pos.
C-O-C-H
/H3
H C-O-P-O-CH-CH-N-C H
2
0e
Fig.
C H3
3 pos.
Since in these phospholipid molecules the acyl moieties consist

of essential fatty acids - particulary linoleic acid - they have
been named "essential" phospholipids, an abbreviation denoting
this particular characteristic of these special phosphatidyl
cholines.
As a pharmaceutical product, essential phospholipids (EPL) are
extracted from soybeans and purified to contain 94 to 96 per
cent of phosphatidyl choline (EPL purissimum). The gross chemical analysis of EPL purissimum shows 3.8 % phosphorus, 14.9 %
choline, 69.0 % fatty acids, and the following molar ratios are
found:
phosphorus
choline
fatty acids
= 1.00;---------choline
fatty acids
2.02: ---------phosphorus
2.00
The fatty acid composition as determined by gas chromatography

after phospholipase A2 cleavage (1) is presented in Table 1
Table 1:
Fatty acid composition (molar %) of soybean polyenyl phosphatidylcholine (EPL) as
determined after phospholipase A2 hydrolysis using gaschromatography on a 200 cm column of EGS

or EGSSX at 175
fatty acid
according to ( 1 )
in 1-position
in 2-position
total
C 16
24.0
1.7
12.9
C 18
7.9
1.0
4.4
10.9
10.0
10.5
C 18
C 18
52.4
80.6
66.5
C 18
4.7
6.7
5.7
LIPIDS
Animal fat contains three families of polyunsaturated fatty

acids which are synthesized starting from the plant fatty acids:
oleic (C18:1), or linoleic (C18:2), or ~-linolenic acid, respectively (2,3):
1)
C18:1 (69)
C18:2 (66,9) - -.... C20:2 (68,11)
C20: 3 (65,8,11)
2)
C18:2 (69,12)
C18:3 (66,9,12)
C20:3
----~~ C20:4 (65,8,11,14) = arachidonic acid
3)
C18:3 (69,12,15)
C18:4 (65,9,12,15) - -... C20:4C20:5
C22:5 ----. C22:6
(~8,11,14)
All these fatty acids are incorporated into animal phospholipids,

preferentially in the 2-position (Fig.2), but also into cholesteryl esters (4,5). Only the fatty acids of the second group
show considerable essential fatty acid activity and are therefore regarded to be of major importance.
The fatty acid pattern in phospholipids and cholesteryl esters
greatly influences membrane properties and function (6) and
lipid transport and metabolism.
The following abbreviations are frequently used:
acyl = fatty acid residue
C18:2 (69,12) = fatty acid containing 18 carbon atoms and two
double bonds in the 9- and 12- position counted
from the carboxyl group of the molecule
C, Ch
CE, ChE
EFA
EPL
FA
FFA
FC, FCh
GPC
cholesterol
cholesteryl ester
essential fatty acid
essential phospholipid
PU-PC
fatty acid
free fatty acid
free cholesterol
glycerophosphorylcholine
Backbone of PC, where the
fatty acids are split off
and replaced by OH
LEC
= lecithin = PC
LPC, LPI etc. = lyso-PC, lyso-PI etc.
= PC, PI etc. with one fatty acid replaced by OH
OH-PC, OH-PI etc. lyso-PC, lyso-PI etc.
PA
phosphatidic acid = 1,2-diacyl glyceryl phosphoric acid
PC
phosphatidyl choline = PA esterified with choline
PE
phosphatidyl ethanolamine = PA esterified with
ethanolamine
PI
phosphatidylinsositol = PA esterified with
inositol
phospholipid = PA-esters and Sph
PL
PS
phosphatidyl serine = PA esterified with serine
PU-PC
polyunsaturated (polyenyl) phosphatidyl choline
S, Sph
sphingomyelin = acylester of sphingosine
TG
triglyceride = 1,2,3 - triacyl glycerol
LIPOPROTEINS
Plasmalipoproteins are classified according to density, to ultracentrifugal flotation (7), to electrophoretic mobility and to
families of equal apoliproteins (8). The characteristics of lipoproteins with respect to their qlassifications are summarized
in Fig. 3 using data of SEIDEL (9).
The following abbreviations are frequently used:
LP
lipoprotein
apolipoprotein = protein moiety of LP
Apo LP
Apo A, Apo A-I, Apo A-II, Apo B, Apo C = apoprotein classes
and subclasses
very low density LP
VLDL
low density LP
LDL
high density LP
HDL
flotation rate (7)
Sf
CLASS
VLDL
DE NSITY 0 9
mg/ml
0,95
10 5
400
LDL
1,006
1.0 63
HDL
1.21
ULTRACENTRIFUGAL
FLOTATION
LlPIDELECTROPHORESIS
Chylomicrons Pre - ~ - LP
DIAMETER
nm
100 - 1000
M.W.
10 9
(lvIPOSlTJ 0 N
~Protein
DTG
30 - 70
1010
~-LP
cx- LP
15 - 25
7,5-10
2 .10 6
1 65-4.10 3
89
~Ch
~P~L--Em~~Uill]lliillWWlliWllilllliWllilli]illW
ApoLP
Fig.
A,B,C
3: Characteristic properties of serum lipoproteins in man
B,(A)
A,(B,C)
REFERENCES
1 - LEKIM D. und BETZING H.: Der Einbau von EPL-Substanz in
Organe von gesunden und durch Galaktosamin geschadigten
Ratten. Arzneim.Forsch. (Drug Res.) ~, 1217-1221 (1974)
2 - KLENK E.: The metabolism of polyenoic fatty acids. Advan.
Lipid Res.: i, 1 (1965)
3 - MEAD J.F.: Synthesis and metabolism of polyunsaturated
fatty acids. Fed. Proc. 20, 952 (1961)
4 - BOTTCHER C.J.T., WOODFORD F.P., TER HAAR ROMENY-WACHTER
C.CH., BOELSMA-VAN HOUTE E. and VAN GENT C.M.: Fatty acid
distribution in lipides of the aortic wall. Lancet!, 1378
( 1960)
5 - KLEIN P.D.: Polyunsaturated fatty acid composition of cholesterolesters in rat liver and plasma. Arch. Biochem. ~,
238 (1957)
6 - HEGNER D. and PLATT D.: Effect of essential phospholipids
on the properties of ATPases of isolated rat liver plasma
membranes of young and old animals. Mechanisms Ageing Developm. i, 191-200 (1975)
7 - LINDGREN F.T., ELLIOTT H.A. and GOFMAN J.W.: Ultracentrifugal characterization and isolation of human blood lipids
and lipoproteins. J. Phys. Chern. ~, 80 (1951)
8 - ALAUPOVIC P.: Recent advances in metabolism of plasma lipoproteins. Progr. biochem.Pharm. i, 91 (1968)
9 - SEIDEL D.: Plasma-lipoproteine Funktion und Charakterisierung. In: Fettstoffwechselstorungen, edited by G. SCHETTLER,
Thieme, Stuttgart, p. 24-47 (1971)
Introductory Reviews
The Biological Significance of

the Plasma Phospholipids
H. Peeters
Simon-Stevin-Instituut voor Wetenschappelijk Onderzoek, B-8000 Brugge, Belgium
CONTENTS
PART
I: The Phospholipids in Dyslipoproteinemia
Human Hyperlipoproteinemia
A.
1.
2.
3.
B.
Total plasma phospholipids

The plasma phospholipid pattern and the S/PC ratio
The phospholipid pattern of individual lipoproteins
Experimental dietary-induced hyperlipoproteinemia in

the chimpanzee
1. Total plasma phospholipids
2. Plasma phospholipid patterns
3. The phospholipid pattern of individual lipoproteins
C.
PART
Conc lusion
II: The Role of Phospholipids in the Lipoprotein Structure
A.
Relationship between primary apoprotein structure and

phospholipid content
B.
Reassembly of apoprotein with phospholipid
C.
Conclusion
PART III: Phospholipids and the Lipases
PART
IV: The Therapeutic Potential of Phospholipids
REFERENCES
11
INTRODUCTION
Plasma hyperlipoproteinemia has been associated with atherosclerosis and it is generally accepted that each type of hyperlipoproteinemia is related to a raise of one or more lipids
within the lipoproteins.
Among the biochemical parameters available for the study of
spontaneous and experimental atherosclerosis the plasma total
and free cholesterol, the percentage esterified cholesterol and
the plasma triglycerides have been extensively described while
the plasma phospholipids were mostly neglected. A different type
of evaluation of the hyperlipemic state is being derived from
the plasma lipoprotein pattern which takes into account the lipid
distribution over the lipoproteins. The quantitative evaluation
of the apoprotein moiety of the lipoproteins has not yet been
introduced into the clinical field. Anyhow the electrophoretic
classification is not ready to lose its interest because of the
analogies existing between the electrophoretic and ultracentrifugal lipoprotein patterns.
As a new source of information in the screening of the hyperlipoproteinemia we would like to introduce the phospholipid pattern. The relative sophistication of the methodology (1), requiring a chromatographic separation technique and a quantitative analysis of the individual fractions may act as a handicap
against the introduction of this pattern as a screening parameter. But another problem, and probably the real one, is the lack
of a full rational interpretation of the phospholipid pattern.
The literature available at this moment is still somewhat contradictory (2-18). Thus more time and attention are required at
the border-line between clinical chemistry and clinical medicine
in order to clarify syndromes and relationships in health and
disease starting from the information to be gained from phospholipid patterns.
At this moment there already exists a large stock of information in the field of human and experimental atherosclerosis,
from which we can evaluate the significance of the phospholipid
pattern in the diagnosis of atherosclerosis. We shall summarize
and compare information derived from the phospholipid pattern,
from the phospholipid concentration and from the relationship
of the phospholipids to the other lipid classes as well in man
as in experimental atherosclerosis.
In its second part, this paper mentions the role of phospholipids
in the lipoprotein structure. A third part is concerned with the
function of phospholipids in some lipoprotein-related enzyme
systems. Lastly the therapeutic potential of a specific type of
phospholipids, namely polyunsaturated phosphatidylcholine (EPLLIPOSTABIL) or PU-PC will be introduced.
12
This paper interrelates the work performed at this laboratory

over the years especially by Dr.sc.V. BLATON, Dr.sc.H.Y. ROSSENEU
and Hr. F. SOETEWEY with the senior technical help of W. DE
KEERSGIETER, D. VANDAMME, B. DECLERCQ and R. VERCAEt-1.ST.
PART I: THE PHOSPHOLIPIDS IN DYSLIPOPROTEINEMIA

A. Human Hyperlipoproteinemia

In order to evaluate the potential clinical significance of
plasma phospholipid values a group of control patients was followed over a period of two years. (These controls belong to a
cohort of Belgian postal workers examined for the prevalance of
atherosclerosis by a study group directed by M. VASTESAEGER.)
The results given in Fig. 1 demonstrate that some fluctuation
in the plasma phospholipid content is observed but the deviations
do not exceed the normal range and are of the same order of magnitude as for the clinical control serum.
mg/.Pl
300
0 - - 0 __
......
_ ,," . . . o~
",01973
1972
n=10
200
F M A M J
A 5 0 N 0
month
210
range
CCS17
Fig.
1:
n= 8
5
5
4
6
weeks
The plasma phospholipid concentration of 10 patients followed during 2 years
The total plasma phospholipid concentration in control and hyperlipoproteinemic patients is given in Table 1. In the human hyperlipoproteinemia the total plasma phospholipid value is not significantly related to a specific type of hyperlipoproteinemia and
13
Table 1:
Plasma lipids and lipid ratios in normal and hyperlipoproteinemic patients

n
T.CHOL.
TG
PL
32
190 + 5
86 -+ 6
229 + 5
0.83 + 0.02
o .45 -+
IIa
25
298 + 10
+ 4
279 + 9
1 .07 +
- o . 02
0.35 + 0.02 0.321 +

-
lIb
14
299 + 7
182 + 8
288 + 7
1 .04 + 0.03
0.61 + 0.03 0.304 + 0.012

-
IV
16
267 + 17
318 + 34
304 + 15
0.87 + 0.03
1.20 + 0.10 0.206 + 0.008
TYPE
101
C/PL
TG/C
S/PC
- o .004
0.02 0.252 +
o . 010
T. Chol . Total Cholesterol; TG Triglycerides ; PL .' Phospholipids

C/PL
Cholesterol/PhoSpholipid ratio; TG/C Triglycerid/Cholesterol ratio
S/Pc Sphlngomyelin/Phosphatldylchollne ratio.
therefore it is mostly neglected. However if the cholesterol to

phospholipid ratio or C/PL is considered, important information
is available as, in general, the C/PL ratio is increased in type
II. The ratio varies from slightly above one in type II to around
0.83 in normals, and is low and nearly normal in type IV. These
data are obtained from mg% estimates with an average M.W. for PL
of 775.
2. The plasma phospholipid pattern and the S/PC ratio
As there is no relationship between the total plasma phospholipid value and the type of hyperlipoproteinemia special attention
was devoted to the PL subclass pattern. The major plasma phospholipids are phosphatidylcholine (PC) and sphingomyelin (S), followed by a group of minor components such as lysophosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine. The phospholipid profile is stabile over one year
in spite of a slight elevation of lysophosphatidylcholine in the
summer months, which may possibly be ascribed to a slight enzymatic hydrolysis of PL during sample handling as demonstrated
for a control group followed over a period of 12 months (Fig. 2).
When the PL pattern is related to the hyperlipoproteinemia (Fig.
3) a relative increase of sphingomyelin against a decrease of
phosphatidylcholine is observed in type II and an inverse reaction in type IV. These results point towards the significance of
the S/PC ratio in function of the type of hyperlipoproteinemia.
In connection with these results a comparison of the S/PC ratio
was made between groups of clearly defined types of hyperlipoproteinemia (Table 1). The results indicate no significant difference of the S/PC ratio between type IIa and lIb, but a significant increase above normal in type II and a significant decrease of the ratio in type IV. Furthermore the S/PC ratio is
in fairly good correlation with total cholesterol, C/PL, triglycerides and TG/C ratios (Table 2). The best correlation was
found with C/PL and an individual plot is given in Fig. 4. From
these results it can be concluded that the S/PC ratio is a help-
14
%PL
75
PC
( n = 10 I
mean value
20
SE
~+-
+s
10
0
Fig.
::
:i~--=!::=! ~~;PC
J
Pin
PSer
0 Month
2 : The plasma phospholipid subclasses of 10 patients fOllowed during 1 year
"IoP L
70
65
o control
type lIa n=12
13 type IV n= 12
60
20
15
10
OI~"-""""""'---L..OIrI. . . . . ra:,~~
.
PC
Fig.
3:
SOH-PC
PEt
Pin
PSe,
The plasma phospholipid profiles in normal and hyperlipoproteinemic patients
15
ful parameter in the differential diagnosis between type lIb

and type IV, which may be difficult on the basis of cholesterol
and triglyceride values alone. At this point in our studies we
like to establish the following values for the S/PC ratio namely: Normals 0.25, Type IIA 0.32, Type lIB 0.30 and Type IV 0.20.
Table 2:
The correlation coefficients between S / PC and plasma lipids and lipid ratios
TC
Correlation
coeff.
( n = 87 )
For abbreviations
P
S/PC
0.4
0.411"
S/PC
:I:
TG
x Type IV (n=16)
of 8
0.011
~
j;
Normal (n=32)
o Ty pe II (n =39 )
0.370"
see table 1.
<0.01
0.3
PL
~~
0a010
.....
Co
.,.: /-" 0
.. If:.
x
x t x
./".
x
x
x
IS/PC = 0.55 C/PL - 0.25
x
0.2
r = 0.703
0.1
{,/u
0.5
Fig.
4:
1.0
1.5
The relation between S / PC in human plasma
C/PL
TG/C
C/PL
0.703:1:
O.sOg:l:
16

A further differentiation of individual plasma phospholipids can
be obtained by considering the phospholipid distribution inside
the alpha and beta lipoproteins after their electrochromatographic separation (Table 3). Each lipoprotein fraction shows its
own phospholipid profile and the highest sphingomyelin content
is to be found in the (3 -lipoproteins.
Table 3:
Percent phospholipids in human plasma lipoproteins obtained by electrochromatography
ALPHA
% PL
II
Lyso PC
BETA
N
II
7.7
8.8
2.6
4.0
11 .0
15.1
24.9
26.2
PC
74.6
67.7
64.8
64.1
PI+PS
2.6
2.1
2.8
1.8
PE
4.1
6.3
4.9
4.0
S/PC
0.15
0.22
0.38
0.41
Lyso PC
Lysophosphatidylcholine
PC
Phosphatidylcholine
PS
Phosphat idyl serine
I
I
PI
Sphingomyelin
Phosphatidyl-inositol
PE = phosphatidylethanolamine
In type II patients the sphingomyelin concentration is increased

in both lipoproteins, however most in the oc-lipoproteins. It is
out of this observation that the elevated SjPC ratio in the total plasma of type II patients has to be interpreted. Regarding
the data we should observe that the mean age of the control group
is about 20 years, while the type II patients are about 60 years
old and this means that the differences observed in the phospholipid ratio between controls and type II are partly due to atherogenicity and partly to age. Even then, as these two effect.s
cannot be isolated, we may conclude that the introduction of the
sjpc ratio is justified as a new parameter in the differential
diagnosis of dyslipemia.
17
B.
Experimental Dietary Induced Hyperlipoproteinemia in the Chimpanzee
During the induction of a hyperlipoproteinemic state in the

chimpanzee, either by feeding a cholesterol- and butter- enriched
diet for the hyperbetalipoproteinemia of type II or by a sucrose
diet for the induced hyperprebetalipoproteinemia of type IV, a
check of the parameters used in the human hyperlipoproteinemia
became available (19-24).
Fig. 5 shows how two well defined types of hyperlipoproteinemia
were obtained in these animals and Table 4 summarizes the plasma lipids of the three groups of animals, one under control diet
and two under regime. An increased C/PL ratio was obtained in
type II whereas the C/PL ratio was normal in type IV and this
parallels the observations in man.
2. Plasma phospholipid patterns
Considering the relative sphingomyelin to phosphatidylcholine
concentration in the two types of induced hyperlipoproteinemia
there is excellent agreement between the human disease and the
dietary induced hyperlipoproteinemia in the chimpanzee (Table 5
Fig. 5: Diet induced hyperlipoproteinemia in chimpanzees: Plasma lipoproteins after control diet (a ),
after high fat ( cholesterol + butter) diet ( b ). and after high carbohydrate ( sucrose) diet ( c )
18
Table 4:
Plasma lipids of chimpanzees fed control (N), fat (II ) and sucrose (IV) diets
PL
TG
C/PL
T.CHOL.
259 + 21
60 + 10
295 + 18
0.88 + 0.02
II
606 + 76
65 + 16
459 + 45
1 .31
IV
231 -+ 1 6
122 + 19
275 + 16
0.84 + 0.02
TYPE
For abbreviations
+
- 0.09
see table 1.
PLASMA PHOSPHOLIPIDS
Type
20
Hum. Chimp.
II
IV
15
10
~~I,------~,------~,~-65
70
75 Of. PC
Fig. 6.: Percent concentration of plasma sphingomyelin and phosphatidylcholine in man and chimpanzee.
Normals, Type II and Type IV are being compared. In the chimpanzee, type II and IV are dietary induced
and Fig. 6). This observation is in favour of the introduction

of the S/PC ratio as a screening parameter for atherosclerosis
in man.
Concerning the individual lipoprotein phospholipid subclasses
(Table 6) the comparison between the human pathology and the
19
Table
5:
Plasma phospholipid pattern of chimpanzees fed control, fat and sucrose diets
DIET
Control
Fat+cholesterol
4.1
-+
0.4
5.3 + 0.8
16.6 + 0.5
19.2 + 0.4
PC
70.1
PL
Lyso PC
0.5
67.
-+
-+
-+
0.3
1 .6
0.2
0.5
0.6
6.7
PS
1 .4 + 0.3
1.3
PE
6.
+ 0.3
5.1
0.24
76.1
2.1
S/PC
13.
1a
2.2 + 0.3
a -+
1 .8 + 0.2
PI
-+
Sucrose
0.29
+ 0.6
-+ 1.2
-+ .0.4
-+ 0.2
-+ 0.8
0.17
DIET
% PL
LP
Lyso-PC
alpha
6.8 + 0.3
beta
4.2
prebeta
S
5.1 + 1 .0
0.5
2.4 + 0.2
beta
18.2 + 0.9
21.2 + 1.7
17.0 + 0.3
.:
11 .4 + 0.2
alpha
68.1 + 1.3
67.0
1.0
74.9 + 1 .5
beta
67.9 + 1.5
62.1 + 1.1
70.4 + 0.9
77.7
-+
-+
-+
-+
-+
0.1
0.7
alpha
3.9 + 0.4
2.8 + 0.9
2.9
beta
1 .6 + 0.5
3.0 + 0.5
2.2
0.5
0.1 + 0.1
7.3 + 1.0
6.0 + 0.6
0.8
4.9 + 0.5
0.3
0.0
0.4
alpha
1.3 + 0.4
1.3 + 0.2
0.8
beta
0.9
0.2
1.1 + 0.2
0.5 + 0.2
-+
alpha
7.8 + 0.8
beta
7.2
prebeta
SIPC
5.3
9.8 + 0.3
prebeta
PE
5.7 + 0.4
15.4 + 0.9
prebeta
PS
0.4
-+
6.2 + 0.4
12.0 + 0.1
prebeta
PI
-+
-
sucrose
n = 4
alpha
prebeta
PC
Fat
n = 6
Control
n = 4
1.4
7.2
-+
-
7.9
-+
0.5
0.13
alpha
0.18
0.23
beta
0.27
0.34
0.24
0.15
prebeta
For abbreviations
see table 3
Table 6:
Phospholipid
distribution in the
plasma lipoproteins
of chimpanzees fed
control, fat and
sucrose diets
20
dietary induced atherosclerosis was

there is an increase of the percent
alpha as in beta lipoproteins which
tions in the human. We have no such
also positive. In type II

sphingomyelin as well in
is similar to the observadata for type IV in man.
Conclusion
From these experimental results one can conclude that phospholipids are becoming a growing source of information regarding
problems of atherosclerosis as well in man as in experimental
animals. This is true for the plasma C/PL ratio and is especially valid for the S/PC ratio in plasma and in alpha lipoproteins.
PART II: THE ROLE OF PHOSPHOLIPIDS IN THE LIPOPROTEIN STRUCTURE
A. Relationship between primary apoprotein structure and phospholipid content
The clinical importance of hyperlipoproteinemia has led to detailed studies of the structure and metabolism of the plasma
lipoproteins. As the apolipoproteins are unique in their ability
to bind phospholipids we can speculate about the interrelationship between their phospholipid binding capacity and their primary structure. In this context we should refer to the differences in phospholipid pattern of alpha and beta lipoproteins
and especially in their sphingomyelin to lecithin ratio as described in the Tables 3 and 6 in the previous section. In order
to relate the phospholipid pattern to the amino acid composition
of the apoproteins we shall compose data of the HDL apoprotein
from man, chimpanzee, baboon and rhesus (24-29).
Table 7: Phospholipid pattern of ultracentrifugal high density lipoproteins
man and primates
HDL
PC
Lyso-PC
Sph
74.4
2. 9
13.2
Chimp.
798
0.4
Baboon
81 .2
B 7.7
PI
(HDL) (1.063 1.210) of
PS
PEt
2.4
0.8
3.1
3.1
12.5
0.7
0.2
6.4
o. 7
5. 9
3.6
0.4
8.3
3.6
3.8
11
3. B
Other PL
(1.063-1.210)
Human
Rhesus
For abbreviations
a
: Skipski
(30)
see table 3.
b: Scanu
(31)
21
Let us start with a brief description of the distribution of

the main HDL-phospholipid fractions in these four animals. From
Table 7 it appears that the phosphatidylcholine and sphingomyelin content in the four human and non human primates are quite
different. Man and chimpanzee have a lower phosphatidylcholine
content whereas baboon and rhesus HDL contain relatively less
sphingomyelin. As we know that baboon and rhesus are characterized by a high amount of high density lipoproteins in comparison to man and chimpanzee, it is tempting to relate the low
sphingomyelin to phosphatidylcholine ratio in the former animals
to their apoproteins.
In the former
namely apoA-I
lated on DEAE
The main peak
four species HDL comprises two major apoproteins,

and apoA-II (32). These apoproteins have been isocellulose using a Tris gradient as shown in Fig. 7.
on the DEAE pattern corresponds to the apoA-I pro-
BABOON APO-HDL
0.10
0.05
'------10.01
1500
E
c:
o
CHIMPANZEE APO - HDL
co
N
iii 0.6
w
u
~ O.
m
0::
0.10
0.05
gO.2
m
HUMAN APO-HDL
Fig.
7:
DEAE - cellulose chromatography of apoHDL of man, chimpanzee and baboon (29)
22
tein which accounts for about 60% of the apoA-I molecule in man
and chimpanzee and for about 70% in the baboon. In all three species considered, apoA-I is very similar both qualitatively and
quantitatively.
The second polypeptide, apoA-II, represents 20% of apoHDL in man
and about 12% in chimpanzee. In the baboon the apoA-II is eluded
at a different volume from the DEAE column which means that it
presents some difference with the two other species. Actually
the main difference between man and baboon apoA-II appears to
be that the human peptide is present in the dimeric form whereas in the baboon it is a monomer. The monomeric-dimeric form of
apoA-II is accompanied by other small differences in amino acid
composition as shown in Table 8.
The difference between the dimeric apoA-II in man and chimpanzee
and the monomeric apoA-II in the baboon and rhesus is the presence of arginine and the absence of half-cystine in the baboon
and rhesus apoA-II which is substituted by serine suggesting that
the disulfide bridge is not required for the lipid binding functions of the protein (Fig. 8). Additional human to monkey sub-
Table 8:
Amino acid composition of apoA II in man, chimpanzee, baboon and r.hesus monkey
Amino Acid
Human
chimp.
115"
LYS
baboon
rhesus
117
96
104
HIS
ARG
13
13
TRP
ASP
41
65
55
52
THR
82
78
76
78
SER
82
65
81
65
221
GLU
198
195
221
PRO
52
52
51
52
GLY
43
39
27
26
78
ALA
68
65
80
CYS/2
15
13
VAL
77
65
84
91
13
MET
15
26
18
ILE
15
13
LEU
103
104
104
104
TYR
45
52
45
52
PHE
50
52
49
52
~:
mol/1000 mol of amino acids
a:
Shore and Shore (1966)
b:
Scanu et a1-
c:
OEAE-fraction II
d:
Edelstein et al.
(1974)
(33)
(34)
(1972)
(35)
23
,"-,,''='''-''''-''''--Y' "--1""-'"
AA sequence of HDL apo A-II in man

and of DEAE Fr. II in baboon and rhesus
22
23
24
50
51
S7
Fig.
8:
The amino acid sequences of HDL apoA II in man in comparison to that of baboon and rhesus (38)
stitutions are Lys-3-Glu-3, Ser-4o-Ala-40, Ile-53-Val-53,

Gly-71 -Asp-71, and the insertion of arginine between positions
71 and 72 (31, 36 ,37 ,38)
These small differences in amino acid composition and sequence
are probably to be significantly related to the differences in
phospholipid transport in these species as is reflected by a
different sphingomyelin/phosphatidylcholine ratio. Man and
chimpanzee must have a similar lipid transport and lipid metabolism as the bulk of their lipids are carried by low density
lipoproteins, while in baboon and rhesus high density lipoproteins are more actively involved in phospholipid and neutral
lipid transport.
B. Reassembly of apoprotein with phospholipid
In order to establish the relationship between primary structure

and phospholipid binding capacity of the apoproteins we performed
in vitro reassembly of these major apoproteins with phospholipids
(39). For this purpose we used two main techniques, 1. microcalorimetry which encompasses the measurement of the heat of binding phospholipids to the apoproteins followed by 2. gradient
ultracentrifugation through which the complex can be isolated
from either free protein or lipid at the appropriate density.
24
Table 9: Influence of the lecithin chain length on the apparent binding enthalpy H B, the number of mole.
bound phospholipid per mole apoprotein n, and the binding enthalpy per mole phospholipid HB / n, for the
interaction between apoHDL and phospholipids (39)
Phospholipid
Hb
(kcal/mole)
HB/n
Lyso-PC
73
28
0.38
Di-caproyl-L- -PC
97
- 43
0.44
Di-lauroyl-L- -PC
112
- 52
- 0.46
Di-myristoyl-L- -PC
132
-320
- 3.43
-6H
50 Kcallmole apo HDL
di-C 12 :O'PC
di-C8:0PC
.-....-_----0-- OH- PC
150 mole P\../mole apo HDL

Fig. 9: Enthalpy of binding short - chain synthetic PC's to apoHDL as a function
of the lipid / protein molar ratio
Fig. 9 shows the enthalpy changes observed during the reassembly

of synthetic PC's with apoHDL. The influence of the acyl chain
length on the heat of binding and on the complex composition is
shown in Table 9. Increasing acyl chain length increases both
the number of phospholipid molecules bound to one mole of apoprotein and the maximal heat of binding expressed in kcal/mole
of apoprotein and indicates that the hydrophobic contribution
is an important factor in the stabilization of the apoprotein
by phospholipid.
25
Q Kcal/mole protein
200
150
10
50
50
100
150
mole DMPC/mole protein
Fig. 10: Enthalpy of binding dimirystoyl PC to apoA - I (x), apoA -" (.), and to an equimolar
apoA - I / apoA -" mixture (0) as a function of the phospholipid / protein molar ratio
Subsequent studies were performed on isolated human apoA-I and

apoA-II HDL proteins (Fig. 10) (40-44). ApoA-II presents a regular binding behaviour for DMPC (synthetic dimyristoyl phosphatidylcholine) whereas in apoA-I the binding is characterized
by an endothermal process at low phospholipid to apoprotein ratios. This has been attributed to some rearrangement of the protein structure induced by the initial phospholipid binding. As
the two apoproteins always appear in conjunction in the HDL molecule the investigation was extended to the study of the lipid
binding properties of apoA-I/apoA-II mixtures containing the
two apoproteins in various proportions. In Fig. 11 the heat of
binding dimyristoyl phosphatidylcholine or DMPC to apoA-I/apoAII mixtures containing various ratios of the two apoproteins is
plotted against the molar ratio of apoA-I in the mixture. The
theoretical straight line connects the heat of DMPC binding to
100% apoA-I or 100% apoA-II. The observed experimental curve
deviates from the theoretical line at every ratio of the two
apoproteins and the deviation is maximal at a 1/1 ratio,indicating that this point corresponds to the most stable association
between these apoproteins. This was confirmed by the phospholipid binding curve to an equimolar apoA-I/apoA-II mixture which
lies very close to the experimental curve observed for apoHDL
(Fig. 11). These experiments strongly suggest that within the
native HDL the two apoproteins are associated, at least in the
presence of lipids, and that the binding of phospholipid to
these peptides is strongly cooperative.
26
Q Kcallmole protein
llQ Kcal/mole protein
15
llQ= Qexp-Qtheor.
Ol~--------~--------~--
0.5
AI/AI+AII
Fig. 11: Enthalpy of binding DMPC to apoA - I and apoA - II mixtures as a function of the mole fractions
of apoA - I. A: straight line represents ideal behaviour and may be substracted from the experimental curve
above, giving in B the excess enthalpy of binding of DMPC to apoA - I and apoA - II mixtures
-6H
180 Kcal/mole apo A-II
160
..A----..,,--- human monomer (meas.l
baboon mono.
(meas.l
2
100
120
mole DMPC/mole apoA-1I
Fig.
12:
Enthalpy of binding DMPC to human and baboon apoA -,II
27
These reassembly experiments should be extended to the binding

of neutral lipids, such as cholesterolesters and triglycerides
to these two apoproteins. However the evidence available provides
a first approach to the mechanism of phospholipid binding which
is the initial step involved in total lipid binding. Indeed
differences in amino acid composition of the apoproteins influence the phospholipid binding as suggested by Fig. 12, where
the enthalpy of binding DMPC to human and baboon apoA-II are
compared. The monomeric form of baboon ApoA-II is characterized
by a lower enthalpy than its human counterpart which was here
recalculated as a monomer. This strongly suggests that the small
amino acid differences between both species such as the apparition
of arginine in baboon and rhesus are meaningful for the phospholipid binding.
C. Conclusion
We conclude that phospholipid binding to either apoA-I, apoA-II

or to a mixture of the apoproteins is not at all a random process, but is determined by the primary and secondary structure
of these apoproteins.
Such studies are the stepping stones towards an interpretation
of the phospholipid data presented in Part I.
PART III:
PHOSPHOLIPIDS AND THE LlPASES
Lipoprotein lipase~ (LPL) are a group of enzymes which hydrolyze

triglycerides in the presence of lipoproteins or certain apoproteins. Bovine milk lipoprotein lipase is an easily accessible
source of enzyme. To obtain the same amount of enzyme activity
as is present in one liter skim milk, one would need at least two
liters of rat post-heparin plasma or more than 10 liters of human
post-heparin plasma or 60 kg of hen adipose tissue. Furthermore
the milk lipoprotein lipase is more stabile than the tissue enzyme and is therefore more important for experimental work. Affinity chromatography of dialyzed skim milk on heparin-sepharose
yields an enzyme preparation of casein-free milk lipoprotein
lipase with about a 2.000 fold increase in specific activity.
Gel electrophoresis of this fraction on SDS gels reveals a pattern with a major band coinciding with a high enzyme activity.
This fraction with a molecular weight of 62.000 stains for carbohydrate in the PAS reaction and has the properties of a typical lipoprotein lipase. In the absence of serum it has a very
low activity and is almost completely inhibited by 1.0 M NaCI
(45,46)
Although co-factor plasma peptides of this enzyme have been intensively studied, surprisingly little attention was given to
the role played by phospholipids during the hydrolysis of triglyceride emulsions by lipoprotein lipase. The effects of phosphatidylcholine and other individual phospholipids on milk lipo-
28
protein lipase and on the enzyme derived from rat epididymal

tissue have been studied (47-49). Using a well defined artificial triglyceride emulsion and a purified enzyme the effect of
chain length and of the double bond in the phosphatidylcholine
molecule was investigated. The enzymatic activity decreased
markedly when the chain length of the saturated acyl chains in
the PC molecule was increased. The effect of phospholipids on
the enzyme activity was strongly dependent on the fatty acid
composition of the molecule. The LPL activity was more increased
by unsaturated than by saturated phosphatidylcholine so that it
can be stated that essential phospholipids (EPL) play an important role in triglyceride metabolism. Furthermore we presume
that the phospholipids interact rather with the substrate than
with the enzyme through the presence of the apoproteins.
Next to the effect on the lipoprotein lipase activity, the type
of phospholipid plays an important role in the phospholipase and
in the LCAT-activity, not as an activator but as a true substrate.
The esterification of plasma cholesterol by the LCAT enzyme is
related to the degree of unsaturation of phosphatidylcholine in
the HDL lipoprotein. The resulting polyunsaturated cholesterolesters can result in an increased removal of cholesterol in hyperlipemic patients as demonstrated by the studies about the
effect of PU-PC on the plasma lipid and lipoproteins in type II
hyperlipoproteinemia as shown in Part IV of this paper.
Studies on experimental animals have shown that an intravenous
injection of PU-PC results in a decrease of the cholesterolestersynthetase to cholesterolester-hydrolase ratio in the aorta.
Such a result can be considered favorable as it may reduce the
formation of aortic lesions (50).
Conclusion
From the results described above it follows that given phospholipid classes and also their specific fatty acid composition are
important factors in the enzymatic catabolism of the plasma and
tissue lipids.
PART IV:
THE THERAPEUTIC POTENTIAL OF PHOSPHOLIPIDS
The evaluation of the therapeutic potential of phospholipids is

derived from studies with polyunsaturated phosphatidylcholine
(PU-PC) extracted from soybeans (LIPOSTABIL). Diets high in polyunsaturated fatty acids have long been considered as standard
preventive measures to be taken in cases of atherosclerosisprone patients with high cholesterol levels. Now soybean phosphatidylcholine with its high polyunsaturated fatty acid content
seems to hold out hope for basic treatment of the disease. While
phosphatidylcholine from animal sources contains only about 40%
of polyunsaturated fatty acids, soybean PC contains 65% linoleic
acid. This essential phospholipid is known to have a blood lipid
29
lowering effect and to reduce the accumulation of cholesterolesters in the intima.

Significant results have been reported after intravenous administration of soybean phosphatidylcholine to patients with type II
and type IV hyperlipoproteinemia (51-54). After daily injections
of 20 ml (corresponding to 1 g EPL) during 14 days, the patients
showed a marked lowering of plasma lipids especially of plasma
cholesterol. The drug also significantly reduced the concentration of beta lipoproteins (LDL). Next to a normalization of the
lipid and lipoprotein concentration, PU-PC also has a favorable
effect on the fatty acid profiles not only of cholesterolesters
due to possible changes in the substrate specificity of the LCAT
enzyme, but also of the plasma phosphatidylcholine fraction as
shown in Fig. 13. Next to the decrease of the absolute FA concentration there is a relative increase of polyunsaturated fatty
acids, which means a normalization of the fatty acid profile of
the circulating phosphatidylcholine in plasma.
From the lipid and lipoprotein data, however, it follows that
the type II hyperbetalipoproteinemia was more influenced than
type IV hyperprebetalipoproteinemia. The lipoprotein lipids and
namely the ~-cholesterol were selectively influenced so that a
faster removal of excess cholesterol could be seen as drug effect.
A longitudinal peroral therapy with EPL maintains a lower level
of plasma cholesterol, the main result being a shift from the
16:0
mg%
2 4
2
16:1
18:0
D%
18:1
18:2
20:4
~ mg%
-1 -2
-2 -4
-3 -6
p<O.OI
(lIt)p<O.OS
Fig. 13: Favorable effect of intravenous

hyperlipoproteinemia (52)
pu PC
on the fatty acid pattern of plasma PC in type II
30
saturated and mono-unsaturated fatty acids to polyunsaturated

fatty acids especially in the cholesterolesters (55). In both
types of hyperlipoproteinemia similar results were observed.
Next be the effect on enzymes and on lipid metabolism, -turnover,
and, -resorption, there are also a number of other important functions ascribed to phospholipids, which are of importance in vascular disease, such as an effect on the haemodynamic properties,
with a role in platelet aggregation, and a reduction in blood
viscosity (56,57), which also may be of interest in the study
of fat-embolism (58,59).
Conclusion
Although numerous chemical agents actively inhibit the formation

of atherosclerotic plaques in animals, only phosphatidylcholine,
one of the major plasma phospholipids, has been shown to cause
regression of experimental atherosclerosis (60). The need for an
agent to reverse atherosclerosis remains acute. By the fact that
up to now no other agent has been demonstrated to reverse clinically demonstrable atherosclerosis, more extensive investigations
must be performed to interprete and complete the results existing
in order to further justify the potential of polyunsaturated phosphatidylcholine as a useful compound in the treatment of atherosclerosis. The clinical significance of phospholipids is now being better understood than ever and the use of polyunsaturated
phospholipids in the therapy of hyperlipoproteinemia is promising indeed.
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22 - BLATON V., VANDAMHE D., VASTESAEGER M., MORTELHANS J. and
PEETERS H.: Exp. Mol. Pathol., 20, 132 (1974)
23 - BLATON V., VANDAM}lli D. DECLERCQ B. and PEETERS H.: The
A.rterial wall in Atherogenesis, Int. Symp. Hontecatini
Terme, April 1973, C. Cavallero, ed., Piccin Med. Books,
145 (1975)
24 - BLATON V., VERCAEMST R., VANDECASTEELE N., CASTER H. and
PEETERS H.: Biochemistry, ll, 1127 (1974)
25 - PEETERS H., VERCAEHST R. and BLATON V.: IIIrd Int. Syrnp.
Atherosclerosis, West-Berlin, Oct. 1973, 877 (1974)
26 - PEETERS H.: IIIrd Int. Symp. Atherosclerosis,
Oct. 1973, 633 (1974)
~vest-Berlin,
27 - BLAT ON V. and PEETERS H.: Abstr. Paper Vth Int. Symp. Drugs
Affecting Lipid Metabolism, Milan, Sept. 1914
32
28 - BLATON V., VERCAEMST Jr. R., ROSSENEU H., MORTEL.\1ANS J.,

JACKSON R.L., GOTTO A.M. and PEETERS H.: Biochemistry, in
press
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30 - SKIPSKI V., BRACKALY V., BARCLAY R., FETZER V., GOOD J.
and ARCHIBALD F.: Biochem. J., 104, 340 (1967)
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J. Biol. Chem., 248, 7648 (1973)
32 - MORRISET'l' J., JACKSON R. and GOT'rO A.: Ann. Rev. Biochem.,
!i, 183 (1975)
33 - SHORE V. and SHORE B.: Biochemistry,
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351, 341 (1974)
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7653 (1973)
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PEETERS H.: Chem. Phys. of Lipids, 11, 203 (1974)
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IRCS Medical Science: Biochemistry, i, 374 (1975)
41 - SOETEWEY F., ROSSENEU H., PEETERS H. and BROWN V.:
IRCS Medical Sciene: Biochemistry, i, 375 (1975)
42 - ROSSENEU 1-1., SOE'rEWEY F., LIEVENS J. and PEETERS H.:
IRCS Medical Science: Biochemistry, i, 376 (1975)
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Biochim. Biophys. Acta, in press
44 - ROSSENEU M., SOETEWEY F., aIDDELHOFF G., PEETERS H. and
BROWN W.V.: Biochim. Biophys. Acta, in press
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Metabolism, Obesity and Diabetes Mellitus, Georg Thieme
Verlag, 1974, p. 23
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D. and PEETERS H.: FEBS Letters,
ii,
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47 - CHUNG J., SCANU A. and REMAN F.: Biochim. Biophys. Acta,

296, 116 (1973)
48 - LA ROSA J., LEVY R., HERBERT P., LUX S. and FREDRICKSON D.:
Biochim. Biophys. Acta, Commun., il, 57 (1970)
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.il, 541 (1971)
50 - HOWARD A. and PATELSKI J.: Atherosclerosis, 20, 225 (1974)
51 - PEETERS H., BLATON V., DECLERCQ B. and VAND~~E D.: Abstr.
Paper of the 4th Int. Symp. on Drugs Affecting Lipid
Hetabolism, Philadelphia, USA, Sept. 1971, p. 93
52 - PEETERS H., BLATON V., SOETEWEY F., DECLERCQ B. and
VANDAI~E D.: Hunch. t1ed. Wschrift, .l..22, 1358 (1973)
53 - BLATON V., VANDfu~E D. and PEETERS H.: Verh. Deutsch. Ges.
Innere Hed., .:?!' 1242 (1972)
54 - BLATON V., DECLERCQ B., VANDAHHE D., SOETEWEY F. and
PEETERS H.: EPL Meeting, Brugge, Nov. 1975, in press
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Drugs Affecting Lipid Hetabolism, Hilan, Sept. 1974, in press
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in "Phospholipids", ed. SOHOCHOWIEC and J. WOJCICKI, 1973,
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60 - STAFFORD W. and DAY C.: Artery,
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106 (1975)
~,
51
(1970)
Lecithin -Cholesterol-AcylTransferase
Lipoprotein and Phospholipid Substrate Specificity
G. Assmann
Abteilung fur Klinische Chemie und Zentrallaboratorium der Universitatsklinik,
0-5000 Kbln 41, Germany
Abstract: The transfer of a fatty acid moiety from phosphatidylcholine to cholesterol is a pivotable reaction in cholesterol
metabolism and transport, and is potentially related to formation and regression of atherosclerosis. This reaction is mediated in plasma by the plasma lecithin:cholesterol acyltransferase (LCAT). The precise mechanism of the intermolecular fatty
acid transfer has not yet been revealed so far. However, the
LCAT-enzyme is much more effective with polyunsaturated phosphatidylcholine substrates than with saturated ones. This substrate-specific difference might reflect the preferential interaction of apoprotein A-I with unsaturated phospholipids. The
present review is concerned with these and further aspects to
the lipoprotein- and phospholipid substrate requirements of the
LCAT-enzyme.
INTRODUCTION
Plasma lecithin:cholesterol acyltransferase (EC 2.3.1) (LCAT)

catalyzes the transfer of an acyl group from the B-position of
phosphatidylcholine to the 3 B-hydroxyl group of cholesterol
(1,2). The esterification reaction is of major importance in
cholesteryl ester formation in plasma and in the catabolism of
plasma lipoproteins (3-6). The acyltransferase is of interest
for several reasons:
1) The esterification of cholesterol in serum mediated by the
LCAT is an integral part of metabolism and transport of cholesterol.
2) The turnover of all the components of the serum lipoproteins
is secondarily affected by the activity of LCAT.
3) There is ample evidence that the LCAT-mediated reaction plays
a major role in maintaining proper concentrations of cholesterol
in all cell membranes, thus potentially being related to the
formation of atherosclerosis.
4) An inborn error of metabolism (LCAT deficiency) has been
discovered recently in which the LCAT-mediated reaction is
miSSing. The gross perturbation of lipoprotein structure and
metabolism in this disease reflects the role of this enzyme in
the maintainance of normolipidemia (7-13).
35
5) In patients with parenchymal liver disease, the esterification of serum cholesterol is decreased; this abnormality roughly
parallels the severity of the hepatic disorder.
Despite the interest given to this enzyme, little is yet known
of the precises mechanism of the intermolecular fatty acid transfer reaction. Some aspects of the lipoprotein- and phospholipid
substrate requirements of the LCAT-enzyme are discussed in this
article.
LIPOPROTEIN SUBSTRATE SPECIFICITY
Several authors have suggested that the enzyme preferentially

reacts with the cholesterol and phosphatidylcholine transported
by HDL but does not significantly react with those of VLDL and
LDL (1,14). Host evidence in support of this hypothesis has been
obtained from experiments in which isolated human plasma lipoproteins have been incubated with partially purified LCAT preparations. In these in vitro studies, HDL, in particular the
smaller HDL subfractions, and VLDL have been identified as the
preferred substrates of the LCAT reaction (15). Further, binding
studies of semipurified LCAT to soluble or agarose-coupled lipoproteins have inferred that the affinity of HDL the LCAT is
related to the ability of these lipoproteins to form reactive
complexes with the enzyme (3,16). Also formation of complexes
of LCAT and phosphatidylcholine dispersions has been employed
as a purification technique for the enzyme (17). On the basis
of these data it has been suggested that the enzyme circulates
in the plasma as a HDL complex (4). The liver is the major
source of HDL (18,19). Nascent HDL isolated from rat hepatocytes,
from the Golgi apparatus, and from rat liver perfusate appear
as discs of about 4.5 nm diameter as demonstrated by electron
microscopy (20). These nascent particles contain protein, phospholipid, and cholesterol, but are deficient in cholesteryl
esters. This finding provides suggestive evidence that the LCATcatalyzed esterification of HDL-cholesterol occurs after release
of the nascent particles into circulation.
The exclusive role of HDL as a true substrate for the LCAT reaction has been questioned by MARCEL et al. (21,22). In their
experiments, the addition of HDL or HDL3 had no effect on LCAT
activity in normal plasma and the addition of HDL2 was inhibitory to the reaction. In contrast, the addition of triglyceriderich lipoproteins (chylomicrons and VLDL) stimulated the rate
of the LCAT reaction in whole plasma (21). It has been pointed
out, however, that the observed stimulation of the LCAT reaction by chylomicrons and VLDL does not necessarily exclude participation of HDL in the reaction and might be explained by ~e
veral mechanisms:
1) unesterified cholesterol readily equilibrates among lipoproteins and both chylomicrons and VLDL loose phosphatidylcholine
to HDL during lipolysis (23-26). Concomitantly, there may be also
an exchange of VLDL triglycerides with HDL cholesteryl esters
36
(27). Thus, as surface cholesterol and phosphatidylcholine on

HDL are converted to cholesteryl esters and lysophospha.tidylcholine, a net transfer of cholesterol and phosphatidylcholine
from the triglyceride-rich lipoproteins to HDL might occur.
2) lipid transfer is accompanied by redistribution of specific
peptides (C-apoproteins) between lipoproteins (28-30). It has
been demonstrated that after fat ingestion the concentration of
C-apoproteins in the triglyceride-rich lipoproteins doubles and
that this increase in C-apoprotein concentration is t~e result
of a transfer from HDL (31). Whether the C-apoproteins are transferred alone or in association with a complerrent of cholesterol,
cholesteryl esters, and phospholipid, is still undetermined.
As a result of these transfer reactions HDL may indirectly diminish the unesterified cholesterol and phosphatidylcholine and increase the apoC-content of triglyceride-rich lipoproteins. Therefore, a dual role as substrate and as operator in the LCAT reaction might reflect one of the major functions of HDL in plasma.
Recent advances in the chemistry of li~oproteins and apolipoproteins have given further support to the fundamental role of HDL
in the LCAT reaction. The activity of partially purified LCAT is
highly dependent on the presence of apoA-I, the major apoprotein
of dDL (32,33). The degree of activation appears to depend upon
the nature of the hydrocarbon chains of the phospholipid, in that
apoA-I is more effective with unsaturated than saturated phosphatidylcholine substrates (33). In contrast, apoC-I, also being
identified as an LCAT activator pept.ide (33), gives similar activation with both saturated and unsaturated phosphatidyl choline (33). The molecular mechanism of apoprotein-mediated LCAT
activation is not yet understood. Several types of activation
may be operative: 1) specific protein-protein interaction bebleen the LCAT enzyme and the apoprotein may facilitate the penetration of the enzyme at the interfacial surface and promote
specific interaction of the enzyme with its lipid substrates,
2) activation as a cofactor of the enzyme, and 3) activation
as a primary acceptor of the lysolecithin and / or cholesteryl
ester reaction products (33).
Elucidation of the nature of the LCAT-catalyzed fatty acyl transfer requires detailed knowledge of the mode of interaction between proteins and lipids in lipoprotein macromolecules. Recently, some aspects of lipid-lipid and lipid-protein interactions in lipoproteins became unraveled.
In the quarternary structure of HDL, the polar head groups of
both phosphatidylcholine and sphingomyelin are located in the
outer shell of a spherical micellar particle (34). Free cholesterol intercalates with the phospholipid monolayer and its hydrophilic portion must be visualized in the interfacial region.
Such steric arrangement of the 3B-hydroxyl group of cholesterol
and the charged carbonyl atom of the B-fatty acid of phosphatidylcholine at the interfacial surface region might facilitate
the interaction of the LCAT enzyme with water-insoluble lipids

and the fatty acid transfer reaction might occur within short
topographical distance.
37
The interiors of the micelles, unlike the peripheries, are not

in intimate contact with the aqueous phase, but rather occupy
a region more akin to the interior of an oil droplet. It has
been inferred from 13C-NMR studies that the cholesterol nucleus
of the esters resides in the center of the sphere with the fatty
acid residues interacting with the apoprotein and the phospholipid monolayer (35).
Therefore, cholesteryl ester formation is expected to alter both
the surface and the central core region of the micellar particles, significantly influencing the physical properties of HDL as
a result of LCAT action. The location of the apoproteins in the
quarternary structure of HDL and the specific sites of apoA-IapoA-II and of apoA-I-LCAT enzyme interactions are not completely understood (35-38). Assignment of a preferential surface location to the apoproteins in HDL is supported by several observations:
1) based on low angle X-ray scattering, both HDL2 and HDL3 have
an electron-rich (polar) outer region and a relatively electronpoor central (nonpolar) region. The outer shell of HDL2 has a
radius of 1.4 nm and an electron density near that expected for
hydrated polar head groups of phospholipids and the hydrated
hydrophilic areas of the apolipoproteins (39-43).
2) The majority of the E-amino groups of lysine in HDL are susceptible to succinylation (44); fluorescence spectroscopy studies
indicate that 70% of the tryptophane and 75% of the tyrosine residues are exposed to the solvent (45); part of the apoprotein
in HDL is accessible to proteolytic cleavage (46-48).
3) Examination of the secondary structure of apoproteins reveals
hydrophilic and hydrophobic surface areas, referred to as amphipathic helices (37,49,50). Based on modelbuilding studies, SEGREST
et al. (50) have indentified at the polar face of amphipathic
helix areas an unusual topographical distribution of charged amino
acid residues. The positively charged lysine and arginine residues
are oriented toward the lateral edges of the interface between the
polar and nonpolar faces, while glutamic and aspartic acids are
localized to the center of the polar face. This topographical arrangement led to the hypothesis that there may be electrostatic
interaction between the positively charged choline groups of the
phospholipid with the aspartic and glutamic acid side chains,
and between the negatively charged phosphates and the arginine
or lysine residues (50). The presumed electrostatic interaction
of phospholipid head groups and zwitterionic amino acids has been
suggested as a principle mechanism for lipid-protein interaction
in lipoproteins. JACKSON et al. (51) have extended this concept
into a model for HDL in which the long axis of the fatty acyl
chains of phosphatidylcholine are oriented perpendicular to the
~-helical segment of the apoprotein at the surface area. In the
proposed model, at least part of the phospholipid head groups
at the surface area is covered by apoproteins and is not freely
accessible to the LCAT enzyme in the aqueous environment.
Recent evidence, however, indicates that instead
interaction between phospholipid head groups and
hydrophobic forces between fatty acid chains and
of major importance for the structural integrity
of hydrophilic
apoproteins,
apoproteins are
of lipoproteins.
38
Using 13C-labeled phospholipids for NMR spectroscopy experiments,

direct evidence tor the hydrophobic interaction between apolipoproteins and the terminal segments of linoleic acid in B-position of phosphatidylcholine and oleic acid in sphingomyelin has
been obtained (35,52). For the choline- 13 C-methyl group of phosphatidylcholine and sphingomyelin in reassembled lipoproteins,
however, no decrease in motional freedo~ as compared to liposomes could be observed, thus excluding the importance of ionic
interaction (35,53).
Despite considerable progress in the knowledge of HDL structure,
however, the precise topographical arrangement of the apoproteins within the micellar lipid arrangement and the role of the
observed hydrophobic interaction of apoproteins and phosphatidylcholine in their relation to LCAT activity remain subjects
for further investigation.
Besides apoproteinA-I and C-I, the "thin-line" protein (apoD or
apoA-III) and the "arginine-rich" protein might be of functional
importance in LCAT activity. KOSTNER has found, that apoA-III,
isolated from human HDL, activates LCAT (54), while OLOFSSON
and GUSTAFSON (55) have suggested that it might be a specific
carrier of lysolecithin formed after the action of LCAT on HDL.
UTEru~NN et al. (56) have identified a protein (apoprotein E)
similar or identical to the "arginine-rich" protein (57) in an
abnormal HDL from patients with LCAT deficiency. The authors
suggest that apoE normally exchanges between HDL2 and VLDL and
propose that this mechanism might explain the activation of LCAT
by the addition of triglyceride-rich lipoproteins to plasma.
The molecular architecture of chylomicrons, VLDL, and LDL, which
might also participate in the LCAT reaction, is less well understood. It is generally assumed that triglycerides and cholesteryl
esters are core constituents of these lipoprotelns, while cholesterol, phospholipid, and protein have been assigned to the surface area (58-62). Recent studies that LDL arises from degradation of VLDL by a process involving removal of triglycerides and
replacement by cholesteryl esters to stabilize these particles
as they diminish in size (28,63,64). To what extent cholesterol
is removed from VLDL during lipolysis or remains as cholesteryl
esters in VLDL remnants (LDL) is not known. Regardless of the
exact mechanism, the simultaneous action of lipoprotein lipase
and LCAT ultimately determine the surface-volume changes in the
catabolism and interconversion of triglyceride-rich lipoproteins.
PHOSPHOLIPID - SUBSTRATE SPECIFICITY
The phospholipid substrate requirements of the LCAT enzyme have

been mostly studied with phospholipid liposomes and partially
purified enzyme preparations. Using natural phospholipids from
several sources, significant cholesteryl ester synthesis has been
observed only with the phosphatidylcholine substrate (65). It has
been suggested that the basis for this selectivity may lie in a
39
requirement for a positively charged nitrogen adjacent to the

reactive 2-acyl residue (66). FIELDING (66) has reported that
methylation of the amine greatly increases the rate of transesterification. The lack of reaction of LCAT with sphingomyelins
or with the ether analogue of dipalmitoyl lecithin indicates the
specificity for the carboxyl group in B-position. A variety of
PC acyl donors can serve as substrates for the enzyme and it
appears that the rate of fluidity of the substrate acyl chains
determines the rate of transesterification (33,67). The enzyme
is much more effective with unsaturated than saturated phosphatidylcholine substrates (33,67,68). Recent evidence suggests
that these substrate-dependent differences in transesterification reflect the preferential interaction of apoprotein A-I with
unsaturated phospholipids (33). It has been postulated that increasing the fluidity of the phospholipids promotes penetration
of the bilayer by apo-protein at the interfacial surface and
that subsequent apoprotein-enzyme interaction facilitates enzyme-substrate complex formation.
Further elucidation of this dual function of apoprotein A-I or
C-I and the hypothetical two-step mechanism in LCAT activation
requires enzyme preparations free from apoprotein and lipid contaminants. Such preparations are not yet available in large
amounts. The physiological relevance of phosphatidylcholinecholesterol liposomes in investigations of phospholipid substrate
specificity must be questioned, since the physicochemical properties of these particles are different from native lipoproteins.
LIPID TRANSPORT DISORDERS
Despite increasing evidence regarding the role of HDL, apoproteins and phospholipids in the LCAT reaction, the physiological
importance of this enzyme and its function in the interconversion of lipoproteins and cholesterol turnover are not immediately apparent. The discovery of familial HDL deficiency (Tangier
disease) and familial LCAT deficiency have provided unique opportunities to study some these aspects and have given insight into
the metabolic relationship of HDL and LCAT.
Tangier disease was initially described by FREDRICKSON et ala (69).
It is a rare autosomal disorder of plasma lipid transport characterized by abnormal tissue deposits of cholesteryl esters. Cholesteryl ester storage is mainly confined to cells of the reticuloendothelial system and leads to an enlargement of the liver,
the spleen, or of lymph nodes, further to alterations in the
cornea, in the intestinal mucosa, and possibly in blood vessels.
Enlarged, yellowish tonsils in childhood are regarded as pathognomonic of this disorder (70).
Low plasma cholesterol concentrations (25-80 mg %), elevated triglyceride concentrations, fasting chylomicronemia, absence of
the usual high density lipoproteins and markedly reduced levels
of LDL occur in most patients (70).
40
In the past, much attention has been focussed on the nature of

residual HD. LUX et ale (71) have demonstrated that apoA-I and
apoA-II occur in small amounts in Tangier plasma, their ratio
being 1:12 rather than the 3:1 ratio present in normal plasma.
Recent evidence suggests that the residual apoA-I and apoA-II
are not integral constituents of HDL, but occur separated from
each other in Tangier plasma (72,73). The majority of apoA-II is
confined to the 1.063 - 1.21 glml ultracentrifugal density region, while apoA-I is almost exclusively present in the density
> 1.25 glml ultracentrifugal fraction. ApoA-II has been determined to be the only apoprotein constituent of an abnormal lipoprotein with~-electrophoretic mobility, representing the residual HDL in Tangier plasma (72). Whether the primary defect in
Tangier disease resides with altered apoprotein structure causing a lack of apoA-I integration into the quarternary structure of the HDL particle is still undertermined.
One would expect that the absence of the usual HDL and the profound change in apoprotein composition is associated with an impairment of LCAT activity and disturbed interconversion of lipoproteins. However, measurement of LCAT activity in Tangier plasma (74-77) have firmly established that significant cholesterol
esterification occurs and it appears that VLDL and LDL can alternatively serve as substrates in the reaction (74). Therefore, an
absolute phospholipid substrate requirement of HDL for the LCAT
reaction must be questioned.
The amounts of apoA-I in Tangier plasma might be sufficient for
LCAT activation. Radioimmunoassay measurements of apoproteinA-I
have shown that this apoprotein is present in amounts of approx.
1 mg/100 ml Tangier plasma (73). Maximum in vitro activation of
LCAT by apoA-I with egg yolk phosphatidylcholine requires approx.
5.6 mg/100 ml (33).
The consequences of failure to maintain normal HDL in Tangier
plasma are mostly seen in signs of defective triglyceride catabolism. Abnormal composition of chylomicrons (78), low concentrations of triglyceride-rich LDL (74,78), and elevated plasma
triglyceride concentrations occur in most patients. On normal
or high fat diets, grossly abnormal lipoproteins of approx.
100 nm diameter have been detected in the HDL ultracentrifugal
density region in several patients (79). These particles, rich
in cholesteryl ester, were absent or very diminished on low fat
diets.
The C-apoproteins in Tangier plasma are severely reduced and
almost exclusively confined to the density < 1,019 glml KBr ultracentrifugal fraction (73, 80). This reduction in C-apoproteins in plasma and the impairment in the recycling of these
proteins netween HDL and chylomicrons and VLDL is expected to
significantly alter non-enzymatic lipid transfer between these
lipoproteins.
It is likely, therefore, that absence of HDL does cause incomplete chylomicron and VLDL catabolism and accumulation of abnormal lipoproteins rich in cholesteryl esters. Whether such rem-
41
nants of lipoproteins are taken up by the cells of the reticuloendothelial system and subsequently give rise to the intracellular cholesteryl ester storage is still a matter of debate.
Alternatively, a function of HDL concerned with regular removal
of cholesterol from peripheral cells and transport to the liver
might be impaired. Recent tissue culture studies may give support
to such a hypothesis, since HDL and its apoproteins, when complexed with phospholipid, can enhance the efflux of cellular
sterol to the medium (81). No doubt, revelation of the molecular
mechanism of cholesteryl ester storage in Tangier disease will
assist in defining the physiological role of HDL and LCAT in
cholesterol transport and metabolism.
Another mutant in cholesterol metabolism is LCAT deficiency. A
primary deficiency of LCAT has been originally described in 1967
(7,8,82). The disease is thought to be inherited as a single autosomal recessive trait (83). The major clinical features include
corneal opacities, anemia, and proteinuria (10). All of the patients have markedly reduced levels of plasma cholesteryl esters
and lysolecithin as well as increased concentrations of plasma
cholesterol and phosphatidylcholine. In homozygous patients, LCAT
activity is absent from plasma (10).
A variety of plasma lipid and lipoprotein changes apparently occur
secondary to LCAT deficiency. The most significant changes include the presence of LP-X, of VLDL particles with abnormal electrophoretic mobility and of two kinds of abnormal HDL particles
(4,12,13,84-89). Further, low concentrations of apoA, apoB, and
the "thin-line" polypeptide have been reported (87-89). When isolated by ultracentrifugal flotation, lipoprotein particles from
the LDL and HDL density region are more heterogenous in size and
contain excess unesterified cholesterol and phosphatidylcholine
as compared to their normal counterparts (12,85). LDL has been
subfractioned into 3 populations by chromatography on 2% agarose
(12): A large molecular weight subfraction emerges with the void
volume and contains mainly free cholesterol, phosphatidylcholine,
and triglycerides. With electronmicroscopy, this fraction appears
as large flattened structures with 90 - 120 nm in diameter. It
has been suggested that these abnormal particles represent aggregated bilayers of cholesterol and phospholipid, which in the absence of LCAT dissociate from VLDL.
Another LDL subfraction has a lipid composition identical to that
of lipoprotein-X. It contains apoA-I, apoA-II, C-apoproteins, the
"thin-line" protein and albumin. The origin of lipoprotein-X in
the plasma of patients with LCAT deficiency is unexplained, since
none of the patients had any signs of cholestasis as jugded from
liver function tests and liver biopsy.
The third LDL subfraction emerges from the agarose column in the
same position as normal LDL and appears similar in electronmicroscopy. It contains more cholesterol, phosphatidylcholine and
triglycerides as its normal counterpart, causing a lower density
and higher flotation rate of these particles.
42
Alpha lipoproteins in the plasma of patients with LCAT deficiency

are quantitatively reduced and have been separated into two subfractions by Sephadex G-200 chromatography (85, 86). A major
large molecular weight fraction is characterized by disc-shaped
structures 4 nm thick and 15-20 nm in diameter, appearing mainly
in stacks or rouleaux-formation. It has been proposed that the
"stacked disc" appearance, upon electron microscopy, is caused
by the lack of cholesteryl esters. A minor subfraction of smaller
molecular weight contains particles with 4.5-6.0 nm in diameter.
The origin of the abnormal lipoproteins in the patients' plasma
is still speculative. GLOMSET has suggested (4) that part of
the free cholesterol- and phosphatidylcholine-rich particles is
generated from the surface of chylomicrons and larger VLDL particles when the latter are attacked by lipoprotein lipase. It
has been further proposed that the LCAT reaction normally prevents these particles from accumulating by promoting nonenzymatic transfer of free cholesterol and lecithin to HDL (4). As in
the case of Tangier disease, one might assume that some of the
lipids in tissues of LCAT deficiency patients represent deposits
of abnormal circulating lipoproteins. It can be anticipated that
future understanding of the various lipid-transport disorders
will resolve some of the mysteries of structure and function of
plasma lipoproteins.
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326, 406 (1973)
Pharmacokinetics of Essential
Phospholipids
On the Pharmacokinetics of Orally

Applied Essential Phospholipids
(EPL)
D. Lekim
A. Nattermann & Cie. GmbH., Chemische Forschung und Entwicklung,
0-5000 Koln 30, Germany
Abstract: Pharmacokinetic studies were performed in male and

female WISTAR rats in order to determine the portion of intact
absorption of orally applied polyunsaturated phosphatidylcholine
(PU-PC)J i.e. essential phospholipids (EPL). For this purpose
1.2-dilinoleoyl phosphatidylcholine labeled with either 3H or
14C in the fatty acid and with 14C in the choline moieties and
the doubly labeled compound were synthesized. The radioactive
material was added to EPL. An oral dose of 70 mg/kg was given
to the animals after a starvation period of 16 h. The animals
were sacrificed at various intervals after dosing J exsanguinated
and their organs were removed for lipid extraction and radioactivity determination. Special investigations were performed
using the lymph cannulation technique and whole body autoradiography. Further J radioactivity decorporation via faeces J urine
and expired air was measured.
The results are the following: The absorption of EPL from the
gut was protrahated J because a pool of phospholipids was developing in the intestinal wall. In lymph-cannulated animals more
than 90% of the total absorbed acyl label and approximately 50%
of total absorbed choline label appeared in the lymph chylomicrons J these ratios being independent of the time after dosing.
The choline label was quantitatively found in the lymph PC fraction. Thus J around half of the orally applied PU-PC was absorbed
as 1-lyso-PC (with the 1-position remaining intact during absorption). The absorbed l-lyso-PC was immediately reacylated to give
"intact" PU-PC J since no labeled lyso-PC was detected in the
lymph and the 2-position was no longer labeled. This absorption
yield of around 50% "intact" reacylated pu-pc of the total absorbed radiolabel was independent of time. Thus J from the total
absorption yields the following percentages of "intact" PU-PC
absorbed from a single oral dose of EPL were measured: 5% after
3.5 hJ 17% after 6.5 hJ 26% after 9.5 h and around 50% after 19 h.
INTRODUCTION
Peroral application of a therapeutic agent against chronic diseases or hereditary malfunctions is an essential prerequisite
for a modern drug. Polyunsaturated phosphatidylcholine, PU-PC,
49
(or essential phospholipids, EPL) has been successfully used

intravenously and perorally in the therapy of dyslipidemias
(e.g. 1,2,3) and liver diseases (e.g. 4,5). Intravenously applied
EPL further revealed a special potency in causing regression of
experimentally induced atherosclerosis (6,7), but the possibility of an peroral anti-atherosclerotic therapy was questioned,
though (8).
A number of investigations showed that a considerable percentage,
if not the major portion, of the radioactivity of orally applied
radio-labeled phosphatidylcholine (PC) was found in the phospholipid fraction, particularly in PC, of the plasma and of several
organs after intestinal absorption (9,10,11,12). These experiments performed in mice, rats rhesus monkeys, and in humans
showed basicly the same results. However, since differences between the administered and the resorbed PC were observed concerning the ratio of the acyl-label to phosphorylcholine-Iabel,
and because of the difficulties to estimate the influences of
the phospholipid metabolism, the question has never been answered
beyond doubt, \'lhether and to what extent polyunsaturated PC is
absorbed from the gut as an intact molecule or - if hydrolysed is re-esterified. The aim of the present study, therefore, \vas
to try an answer particulary to this point, using synthetic dilinoleoyl phosphatidylcholine radioactively labeled at up to
three different localizations in the molecule.
After a study of the absorption and distribution of EPL for general orientation and for comparison with previous work, the predominant problem to solve was to get immediate access to the
phospholipids after the intestinal absorption. This was achieved
by lymph cannulation. Finally, the incorporation in different
organs and the metabolism were studied to extend the evidence
gained by the lymph cannulation experiments.
METHODS
The methods used already have been extensively described (13-16)

and, therefore, will be summarized briefly in the present study.
1.
Materials
Dilinoleoyl phosphatidylcholine labeled with 14C either in the

1- or in the 2-position acyl-, or in the choline moiety, and
dilinoleoyl PC labeled with 3H in the acyl- and with 14C in the
choline moieties were synthesized according to (15,16). All
products were purified by silicic acid chromatography and identified by DC, IR, NMR and as. The purity of the labeled compounds was at least 98%. In particular, the specificity of the
label in the 1- or in the 2-position of the PC was analyzed by
phospholipase A hydrolysis. The specificity for the 1-position
was better than 96% and for the 2-position better than 99%. The
labeled PC was added to essential phospholipids from soybean
50
(EPL) dissolved in soybean triglycerides. Sunflower mono- and

diglycerides were added to obtain a clear free flowing solution
of different specific activity.
2.
Absorption Studies in Intact Animals
Male and female Wistar rats (200 - 250 g) were starved for 16
hours before oral application of 70 mg/kg labeled EPL. The rats
were kept in metabolic cages, the excreted urine and faeces and
the exspired 1 4C02 were collected and their radioactivity was
determined.
The animals were killed by exsanguination,the blood was collected and the organs were immediately removed. The lipids were exhaustively extracted with chloroform / methanol (2:1 v/v). The
lipid mixtures were separated by silicic acid chromatography.
The pure PC fraction was dissolved in ether and subjected to
phospholipase A (Crotalus terr. terr., BOEHRINGER Mannheim)
hydrolysis over night. The resulting fatty acids and lyso-PC
were separated by silicic acid chromatography and esterified or
transesterified with 5% HCl in methanol. The fatty acid methylesters were analyzed by gas-liquid chromatography, stationary
phase EGSS-X on KIESELGUR. The radioactivity was measured as
described previously (14).
3.
Lymph Cannulation Experiments
Rats were starved 24 h before the operation. Under urethane anesthesia (20% solution, 5 ml/kg) a polyethylene cannula was
inserted into the abdominal part of the thoracic duct according
to the technique of BOLLMAN et al. (17). The animals received
water via gastric tube during the experiments. Lymph fluid was
collected for periods of 1 h up to 24 hours. It was immediately
extracted with chloroform-methanol (2:1 v/v) and the lipid extracts were analyzed.
4.
Autoradiography
Wistar Rats (about 150 g) were starved over night. 70 mg/kg EPL
was applied through a stomach tube, with 2.02 mCi 1,2 - (9,10,
12,13-) 3 H4 -dilinoleoyl PC per animal as tracer. The animals
were killed in deep urethane anesthesia by an acetone-dry ice
cooling at -72C. The preparation was cut at -1SoC (slice thickness 90 urn) the slices were lyophilized at -30C and exposed to
G-5 Film (Ilford).
The dry slices were pressed together with two standards of 3Hpoly-n-butyl-methacrylate of 50 ~Ci/g and 500 ~Ci/g and kept at
-20C for 47 days. Autoradiograms were made simultaneously from
inactive slices to control chemographic effects. These controls
were not blackened.
51
RESULTS
1.
Absorption and Distribution of Radiolabel of Orally Applied Labeled Essential Phospholipids (EPL)
For general orientation and for comparison with previous investigations the absorption of radiolabeled EPL from the gut was
measured using a synthetic dilinoleoyl phosphatidylcholine as a
tracer labeled with 3H in the fatty acids- and with 14C in the
choline moieties.
100
50
f
'p
-- --- --- -
10
12
18
--- --9+
24
Fig. 1: Incorporation of a single orally applied dose of 70 mg / kg of labeled EPL as measured by the rate
of disappearance of radioactivity from the gastro - intestinal tract. 4 male and 4 female rats were sacrificed at
each interval after dosing and the 3H_ and 14C - radioactivity (open and closed symbols, respectively) was
determined. Label: 1,2- (9,10,12,13 - 3 H4 ) dilinoleoyl . sn - glycero - 3 - phosphoryl (N - 14CH3 )choline, 186 x 10 6 dpm 3H and 5.5 x 10 6 dpm 14C. Data in per cent of the administered (means.:t S. D. )
52
The rate of disappearence of the radioactivity from the digestive

tract was considered as a good measure of the absorption rate,
since after 24 hours the radioactivity recovered from the faeces
on a whole amounted to only 3.6 + 0,5% of the administered dose.
Fig. 1 summarizes the results. The disappearance of both the 3Hand the 14C-radioactivity from the gastro-intestinal tract was
quite similar over the whole time period studied. It was not
significantly different for male and female animals (4 determinations each). The time constant of decrease of intestinal radioactivity, i.e. the time constant of incorporation into the body,
as estimated from Fig. 1, is roughly 240 min. More than 90% of
the orally applied 3H- and 14C-radio-label was absorbed from
the intestinal lumen after 24 hours, but a certain amount of the
radioactivity was still present in the intestinal wall (see also
Fig. 3).
The accumulation of radioactivity in the lipid fraction of the
blood and of the liver is depicted in Fig. 2. The radioactivity
in the lipid fraction accounted for more than 98% of the total
absorbed radio-label and was, therefore, representative for the
30
20
10
/
0 __
0 _____ .....
if
'1/
--/,./J.--t:.~
--\il
ii
'/1l'/
/'
--_
12
-- -- - _
18
-I!.
24
t[h] Fig.
2:
Accumulation of 3H_ and 14C - radioactivity (open and closed symbols, respectively) in the lipid
fractions of rat blood (triangles) and liver (circles). Experimentals as in Fig. 1
53
Stomach
Brain
1", '.,
"
Intestinum
-" ..
'
( 0)
( b)
Intestinum
Stomach
Brain
Liver
Fig. 3: Whole body autoradiography of rats 6 h (a) and 24 h (b) after an oral dose of 70 mg / kg
EPL labeled with 1,2 (9, 10, 12, 13 3 H4 ) dilinoleoyl . PC
54
incorporated radioactivity. Fig. 2 shows an increase of radioactivity during the first 6-8 hours. The general time course of
3H- and 14C-uptake is rather similar, however the percentages
are different: After 8 hours the liver contained 30% of the
applied 14C-radioactivity, but only around 10% of the applied
3H-radioactivitYi the corresponding figures for blood were 8
and 4%, respectively, revealing that part of the phosphatidylcholine must have been disintegrated. After 8 hours a decrease
in radioactivity was observed both in the blood and in the liver
with approximate half lives of
To a lesser extent the radio-label was ubiquitarily distributed

over those organs studied in addition: lungs, spleen, kidneys,
heart, and brain. All contained a small (but significantly above
background) percentage of both 3H- and 14C-radioactivity over
the whole period of 24 hours. No significant differences were
observed between male and female animals.
A semiquantitative estimation of the body distribution of the
absorbed radio-label was obtained using autoradiography 6 and
24 hours after oral administration of EPL labeled with 3H in
the fatty acid moieties (Fig.3). 6 hours after application, the
major portion of the radio-label was still found in the digestive
tract (lumen and wall). The radioactivity in the liver and, interestingly, in the heart muscle was significantly higher than
in other organs (Fig. 3 a). 24 hours after EPL-application, the
intestinal wall (but not the lumen) and the liver contained the
major portion of the radio-label. The other inner organs are
also blackened to a small but significant extent, whereas the
brain and the spinal cord apparently did not contain 3H-radioactivity from the acyl groups (Fig. 3 b).
2. The Mechanism of Intestinal
pc Absorption as
Revealed by Lymph Cannulation Experiments
The major problem investigating intestinal phospholipid absorption is to get immediate access to the absorbed molecules and,
thus, to gain evidence on their integrity. This was achieved by
lymph cannulation, collecting the total lymph transported via
the thoracic duct after oral application of dilinoleoyl phosphatidylcholine labeled with 3H in the fatty acid and with 14C in
the choline moieties.
Table 1 represents a balance of 3H- and 14C-radioactivity measured 6.5 hours after oral EPL-administration. As also shown
above, at this point the major portion of the radioactivity was
still present in the intestinal tract (lumen and wall). Almost
90% of the absorbed 3H -radioactivity was found in the accumulated lymph, less than 3% of 3H could by-pass the lymph system
and reach the liver, and around 9% of 3H were lost, i.e. probably exchanged for non-radioactive IH in the body H2 0 (11) distributed over the organism or excreted (Table 1, left). This means
that at least 90% of the fatty acid moieties of the applied EPL
take their way into the body via the thoracic duct and that,
55
therefore, the search for absorbed intact or re-esterified PC

and the estimation of the disintegrated portion of PC may be
confined to the investigation of the collected lymph. The advantage of this technique is that the lipids in the lymph as drained
of the body are not further subjected to any metabolism and thus
reflect the true input pattern after intestinal absorption.
A higher percentage of the 14C-radioactivity (i.e. the choline
moiety) by-passed the lymph path\vay and was uptaken by the liver
during lymph cannulation (Table 1, right). This was due to nonacyl degradation products (such as glycerophosphorylcholine,
phosphoryl-choline, or choline) of PC cleaved during or after
intestinal resorption and not transported with the lymph.
Table 1: Balance of total radioactivity administered to lymph-cannulated rats as a single oral dose of
70 mg I kg EPL, labeled with 1,2- (9,10,12,13- 3 H4 ) - dilinoleoyl - sn - glycero - phosphoryl - (N- 14CH3 )choline. Average results from 6 male rats measured 6.5 h after dosing
3H -
Total
administered
radioactivity
10 3 dpm
% of
dosis
228 000
100
14C - radioactivity
% of the
absorption
10 3 dpm % of
dosis
3 940
% of the
absorpti on
100
163 700
71 ,9
2 590
65,8
Total
absorption
64 300
28,1
100
1 350
34,2
100
Lymph
chylomicrons
56 400
24,8
88,2
684
17 , 4
51 ,0
Liver
1 790
0,8
2,9
492
12,5
36,5
Hissing
6 110
2,5
8,9
174
4,3
12,5
Intestinal
lumen + wall
Table 2:
Percentage of absorbed 3H_ and 14C - radioactivity in lymph chylomicrons as obtained from
experiments similar to that of Table 1 (data for 6.5 h are taken from table 1).
Time after
dosing (h)
Percentage of absorbed radio-activity

3H
l4C
6,5
88,2
51,0
9,5
91 ,2
49,4
95,1
68,3
19
56
The percentage of the total absorbed 3H- radioactivity present

in the lymph (mean: 92%) was independent of time within 20 hours
after dosing. Similarly, the percentage of the absorbed 14C-radioactivity in the lymph (mean: 56%) did not differ significantly with time (Table 2).
The distribution of the 3H- and 14C-ra.dioactivity over the lymph
lipids vs. time was analyzed. The results are collected in Table
3. Only two radioactively labeled lipid fractions were detected
in the lymph: phosphatidylcholine (PC) and triglycerides (TG).
No significant radio-label was found either in lyso-PC or in
free fatty acids. Further, the 14C-radioactivity was located
~uantitatively in the PC fraction only. The distribution of the
H-radioactivity over the two lipid fractions, PC and TG, remained practically constant during the whole period of observation: about 24% in PC and 76% in TG. The 3H/ 14C-ratio observed
in the PC fraction varied only in narrow limits between 17.2
and 24.3. The 3H/ 14C-ratio in the applied PC was 58:1.
Chylomicron PC samples from single rats collected at different
periods were pooled, purified and subjected to phospholipase
A hydrolysis. Unexpectedly, an unequal distribution of the 3Hradioactivity between the two fatty acid moieties was found:
about 90% of the radioactivity was located in the 1-position
and only about 10% in the 2-position (Table 4), whereas the 3Hlabel in the orally applied PC was equally distributed. This
means that during absorption almost exclusively the fatty acid
in the 2-position was split off, while the 1-position remained
intact, and, that the intermediate 1-lyso-PC immediately was
reacylated to PC, since no labeled lyso-PC could be detected in
the lymph chylomicrons.
The percentage of orally applied PC-radioactivity accumulated in
the lymph chylomicrons after intestinal absorption was measured
at different times after dosing (Table 5, left). The values at
0.5 and 3.5 h were obtained from experiments described in Table
3, the later values are from experiments described in Tables 1
and 2. Absorbed radioactivity occurred first in the lymph 30 minutes after dosing and increased continuously to 75.5% of the
applied 3H and to 57.2% of 14C after 20 hours. From these values
the total amount of reacylated "intact" PC accumulated in the
lymph was calculated (Table 5, right), assuming that on the average 24% of 3H are related to 3H-mono-Iabeled absorbed PC and
that 100 % of 14C represent solely absorbed PC (Table 3). As
will be discussed below the 3H-Iabel leads to an underestimation
of the absorption because of label exchange with body water;
after a correction for 3H exchange (by arithmetically increasing
the portion of 3H to reach 3H/1 4 C = 29:1, see DISCUSSION) the
absorption yields calculated from the 3H-radioactivity are very
close to those obtained from 14C, which obviously more closely
represent the true values.
57
Table 3:
Distribution of the 3H - and 14C - radioactivity in the lipid fractions of the lymph chylomicrons in
lymph - cannulated male rats ( n = 7) after a single oral dose of 70 mg / kg EPL labeled as in Table 1
(3H 14C = 58 : 1 ). After 0,5 h the lymph was collected for periods of 1 h. The only labeled lipid fractions
were PC and TG. No labeled Iyso - PC, no labeled free fatty acids were detected. 100 % of the 14C_
radioactivity was located in the PC - fraction, practically 0 % were found in the TG - fraction
Time after
Phosphatidylcholine
dosing (h)
% of 3H
Neutral lipids
3H / llfC
% of 3H
1 ,5
23,5
24,3
77,5
2,5
24,0
17 , 2
76,0
3,5
24,5
20,4
75,5
4,5
25,0
19,7
75,0
5,5
24,0
17 ,9
76,0
6,5
24,5
20,9
75,5
Table 4:
Molecular distribution of the 3H - radio - label in the fatty acid moieties of absorbed phosphatidyl-
choline in the lymph after a single oral dose of 70 mg / kg 3H - labeled EPL (50 % 3H in 1 -, 50 % 3H in
2 - position). Lymph PC samples from 4 male lymph - cannulated rats were purified and subjected to phospholipase A hydrolysis
Percentage of 3H-radiolabel in the

1 - position
2 - position
89.3 + 6.2
10.7 + 6.3
Table 5: Percentage of the orally administered radioactivity accumulated in the lymph chylomicrons at various
intervals after dosing of labeled EPL and the percentage of absorbed .. intact" polyunsaturated phosphat idyl choline. The values for the absorbed PU - PC were calculated from the accumulated 3H - radioactivity on the
base of the results of Table 3 (x) and with the additional correction for 3H - exchange with body water (+)
to end up with 3 H/ 14C = 29 (see DISCUSSION, part 2). 100 per cent of the absorbed 14C - radioactivity
are found in the PC fraction (Table 3). Experimental conditions as in Table 3 (0.5 and 3.5 h) and as in
Table 1 (6.5 - 19.0 h)
Time
after
dosing
(h)
0.5
3.5
6.5
9.5
19
Absorbed "intact"
PU-PC
(% of appl. dose)
Accumulated
radioactivity
(% of appl. dose)
3H.
0.1
7.5
24.8
35.4
74.5
llfC
0.1
5.0
17.3
27.5
57.2
3H
(x)
3.6
11. 9
17 .0
35.7
3H (+)
0.1
5.1
17 .3
24.7
51.8
1lfC
0.1
5.0
17 .3
27.5
57.2
58
3.
Investigations on the Metabolization of Orally Applied EPL
Supporting evidence for the conclusions derived from the results

of the lymph cannulation experiments was obtained by investigating the metabolic fate of orally applied essential phospholipids.
As mentioned above more than 98% of the radio-label incorporated
into the liver after oral EPL-application was located in the liver
lipids. Its distribution over the different lipid fractions was
analyzed 6 hours after dosing by silicic acid- and thin-layer
chromatography. The results are presented in Table 6.
The 3H-label vTas mainly distributed in three fractions: PC and
lyso-PC, PE, and TG. There is, in the male animals, a slight
but statistically significant higher incorporation of the fatty
acid label in the PC fraction with a concomitant lower de novo
synthesis of triglycerides than in the female ones. This sex
specific difference was also observed in rats after intravenous
instead of oral application of EPL (14). The lYC-radioactivity
was located solely in the PC- and lyso-PC fractions, sphingomyelin was not labeled to any significant extent in both cases.
A similar pattern of 3H- and lYe-distribution (as shown in
Table 6 for the liver) was found in the lipids of the lungs, the
spleen, the kidneys, the heart, and the brain, i.e. 100% of lyC
and 40-60% of 3H being located in the PC- and lyso-PC fractions.
Moreover, this pattern was independent of the time after oral
dosing.
Table 6: Distribution of the 3H and 14C radioactivity in the liver lipid fraction of male and female rats
determined 6 h after a single oral dose of 70 mg / kg EPL, labeled with 1,2 (9, la, 12, 13 3 H14 )
. dilinoleoyl . sn glycero 3 . phosphoryl (N 14 CH3 ) choline, 186 000 000 dpm 3H and
5 500 000 dpm 14C. Means.. S.D. (n = 4, each)
male
lipid
fraction
% of 3H
neutral lipids
26,7 + 2,1
phosphatidic acid
cardiolipine
phosphatidyl
ethanolamine
11 ,8 + 3,8
phosphatidyl
choline
60,9 + 2,1
lyso-PC
0,6
female
% of lYC
100
% of 3H
29,7 + 0,8
% of lYC
5,3 + 4,3
9,3 + 0,6
53,9 + 2,0
0,8
100
59
Table 7: Molecular distribution of the 3H - radio - label in the fatty acid moieties of the liver phosphatidyl choline fraction in rats after a single oral dose of 70 mg / kg EPL, labeled as in Table 6. Data in per cent of
total label (means of n animals)
Sex
Time after
dosing
(h)
male
female
mean + S.D.
Percentage of
3 H-Iabel
in the
1-position
2-position
77
23
96
88
12
95
96
95
93.3 + 5.2
6.7 + 5.2
15
In addition, the position of the radio-label in the PC- and

lyso-PC fractions of the liver was studied. The PC fractions
were purified by silicic acid chromatography and subjected to
phospholipase A hydrolysis. The result is presented in Table 7.
In the isolated liver PC fractions of male and female animals
on the average 93% of the 3H-radioactivity was located in the
1-position of the glycerophosphorylcholine backbone and only 7%
in the 2-position. This pattern is fairly independent of time
after oral dosing and is very close to the results obtained in
the lymph chylomicrons.
Further support for the results: that the 1-position of the EPLmolecule remains intact during and after intestinal absorption,
was obtained from the investigation of the respiratory air. For
this purpose dilinoleoyl phosphatidylcholine labeled with 14C
either in the 1-, or in the 2-position, or in the choline moiety
was orally applied and the 14C0 2 from the exspired air was measured for 6 hours. The appearance of 14C in the respiratory air
is a measure for the break-down of the PC-molecule during or
after intestinal resorption and subsequent oxidation of the degradation products (fatty acid- or GPC mOieties). As shown in
Fig. 4, the break-down of the choline moiety is very small (1.8%
14C after 6 h); also the fatty acid in the 1-position is fairly
little attacked (7.7% 14C after 6 h), however in the 2-position
the degradation rate is high (25% 14C after 6 h).
60
30
20
t)
II)
~
0
... 10
,....--.
...,u
....
)'
'---'
/
.t
......-::;l
,,- /
____ 4
-----_.
...... ---
} 2
--I
---
_______ t _______ t
t[h] Fig. 4: Cumulative amounts of respiratory 14 C02 collected after a single oral dose of 70 mg / kg labeled
with 14C in the acyl - 1 - position ( 1 ). the acyl - 2 position (2), and in the 3 - position (choline) (3).
Mean values in per cent of the administered dose measured in 2 male plus 2 female animals for each labeled
PC species. The standard deviations for the cumulated values after 6 h were.. 4 % ( 1 ). .. 7 % (2), and
..:': 11 % of the mean. The exact labeling was
1) 1 - ( 1 - 14C ) - linoleoyl - 2 - (9, 10, 12, 13 - 3 H4 ) - linoleoyl - sn - glycero - 3 - phosphorylcholine;
37300000 dpm 3H and 620000 dpm 14C
2)
1 - ( 9, 10, 12, 13 - 3 H4 ) - linoleoyl - 2 - ( 1 - 14C) - linoleoyl - sn - glycero - 3 - phosphorylcholine;

66900 000 dpm 3H and 1 730000 dpm 14c
3)
1,2- (9, 10, 12, 13 - 3 H4 ) - dilinoleoyl - sn - glycero - 3 - phosphoryl - (N - 14CH3 ) - choline;

186 000 000 dpm 3H and 5 500 000 dpm 14C
DISCUSSION
1.
Hydrolysis and Reacylation of Essential Phospholipids in Intestinal Absorption
Evidence has been presented (Table 4, Table 7, Fig. 4) that a

high portion of orally applied essential phospholipids is hydrolyzed only in the 2-position of the GPC-backbone to form 1-acyllyso-PC which is subsequently reacylated in the intestinal mucosa.
61
FA
] -----)"-------1..
Phospholipase A
E.C.3.1.1.4
Lysophospholipase
E.C. 3.1.1.5
FACoA
1 - Lyso-PC
------~----"----.-~
Lyso-PC acyl
transferase
E.C. 2.3.1.2.3
FA
rpcl
LJ
------------- I I
Kennedy pathway
Glycerophosphorylcholine diesterase
E.C. 3.1.4.2
TG
GP+@j
Fig. 5: Pathways of PC - metabolism in intestinal absorption. The framed compounds were measured in the
lymph cannulation experiments
This result is most important, because the 2-position is preferentially re-esterified with unsaturated fatty acids (18). Therefore, the reacylated PC, which enters the body via the thoracic
duct, is "intact" PU-PC.
The possible main metabolic pathways for PC in intestinal absorption as postulated by several authors (19,20) are depicted
in Fig. 5. PC is hydrolyzed to a 1-acyl lyso-PC by pancreatic
phospholipase A2 Part of this 1-acyl lyso-PC is absorbed in the
mucosal cells and is reacylated to form PC by the lyso-PC: acyl
transferase. The other part of the 1-acyl lyso-PC is further
hydrolyzed in the intestinal tract by lysophospholipase form
GPC. The fatty acids split off are also absorbed and enter the
KENNEDY pathway to form triglycerides before appearing in the
lymph chylomicrons. GPC is either directly absorbed or hydrolyzed by a phosphodiesterase to glycerophosphate plus choline.
GPC and its break-down products are the only PC moieties which
are not transported via the thoracic duct. The data presented
compile sufficient evidence to give an assessement of the importance of each of these pathways in the intestinal absorption
of essential phospholipids. All the enzymes necessary for the
described reactions have been found in the intestinal lumen or
mucosa.
2.
The Percentage of Intact Essential Phospholipids in the Absorbed Lipids
On the base of the above discussed experimental evidence on the

mechanism of intestinal PC resorption of re-esterified "intact"
PU-PC in the resorbed lipids can be estimated. This estimate has
to be derived from the following data:
62
1) The orally applied dilinoleoyl phosphatidylcholine was equally

labeled with 3H in the 1- and 2-position fatty acids (1H+, 2H+)
and with 1 4 C in the choline (C+) moiety. The ratio 3H/ 14C was
58: 1
2) After absorption, 24% of the H-label was found in the PCand 76% in the TG fraction of the lymph chylomicrons (Table 3).
Most of the PC was no longer doubly labeled, but had a single
label in the 1-position only. The average ratio 3H/ 14C was
21:1 (Table 3).
3) On the average around 50% of the total absorbed 14C-label
is found in the lymph PC fraction (Tables 2 and 3).
As was shown in Tables 2 and 3 these percentages are independent
of time and therefore reflect the average absorption process.
Thus, starting with hypothetically 100 mM of orally applied
labeled phosphatidylcholine (1H+, 2H+, C+) the following equation
would roughly fit the data (LPC = lyso-PC, GPC = glycerophysphorylcholine, FFA = free fatty acid):
100 PC ( 1H+, 2H+, c+) - - .
+
+
50 LPC
50 GPC
150 FFA
50 LPC are reacylated with unsaturated fatty acids from the pool
to form 50 PC (1H+, c+). 150 FFA (H+) are incorporated into de
novo synthesized triglycerides (TG). Both 50 PC and 150 FFA (in
TG) appear in the lymph, while 50 GPC (partly disintegrated)
enter the body via the blood. In the lymph chylomicrons the distribution of 3H would then be 50:150 (PC/TG) or 25:75 as it was
observed.
Further in the reacylated PC (1H+, C+) practically half of the
3H-label is present as against to the orally applied PC (1H+,
2H+, C+). Therefore the ratio 3H/ 14C is expected to be reduced
from originally 58:1 to 29:1. The actual value found is 3H/ 14C =
21:1. This ratio is somewhat too low. The most probable explanation is that part of the 3H-label in the fatty acid moieties
was exchanged for non-radioactive IH from body water, a process
that was demonstrated to occur by extensive studies on rats under
comparable conditions and in rhesus monkeys (11,12). This "exchanged" radio-label would account for the missing portion of
the 3H radio-activity not being transported with the lymph (see
Table 1). It wbuld not affect the ratio 3H (PC) / 3H (TG)
24:76 in the lymph, since the exchange for IH of the body water
should equally occur in the fatty acid moieties of PC and TG as
well.
3.
The Total Percentage of Essential Phospholipids Absorbed after Oral Application
Since for the first time the integrity of orally applied polyunsaturated phosphatidylcholine after absorption was demonstrated,
it was possible to calculate the total amount of PU-PC absorption
in rats (Table 5). It is approximately 5% after 3.5 h, 17% after
6.5 h, 26% after 9.5 h, and 50% after 19 h. These values apply
for narcotized rats starved for 24 h before oral dosing. A similar
63
percentage of absorption (20% after 6 h) in rats starved for

14 h before dosing was estimated from bile cannulation experiments comparing intraportal and intraduodenal injection of labeled
PU-PC (10). Comparable qualitative results !'lere obtained in rats
fed ad libitum (11).
The relevance of these results for the human may be derived from
comparing extensive pharmacokinetic studies in rats (11), rhesus
monkeys (12), and man (21) after single oral dose of essential
phospholipids labeled with 3H in the acyl- and with 14C in the
choline moieties. In all 3 cases the plasma phospholipids showed
the same general time course after a single oral dose: an increase of the PC concentration to a maximum after 6-8 hours and a
protrahated decrease. Values up to 5% of oral PC-radioactivity / I
plasma were reached in man (21).
A protrahated absorption of EPL from the gut was observed, obviously due to a storage of lipids, within the intestinal wall
(Fig. 3). Such a lipid pool was already described by other investigators (10,11). The protrahated absorption of orally applied
EPL, may be of major importance, since it could explain the finding that equal oral and intravenous doses of EPL exert comparable lipid lowering effects in experimental Triton-hyperlipidemia (13). The plasma levels are high after injection, but EPL
is rapidly removed from the plasma in rats (14) as well as in
humans (21,22), while comparably low, but long-lasting plasma
levels are achieved after oral administration.
REFERENCES
- YASUGI T. et al. mult.: Effects of EPL on serum lipid.
J.New Remedies & Clinics ~, 691 (1973)
2
- BLATON V., DECLERCQ B., VANDAi'4.'VlE D., SOETEWEY F. and
- HEVELKE G., GROTT E., X1ACHALKE K. und ZAPPE R.: Zur \<iir-
- NUTING D., DOHN P. und REIKOWSKI J.: Die Wirkung hoch-
- WALLNOFER H. and HANUSCH M.: "Essentielle" Phospholipide

zur Behandlung von Lebererkrankungen. Med.Monatschr. ~,
331-336 (1973)
PEETERS H.: The human plasma lipids and lipoproteins

under influence of EPL therapy. In: Phosphatidylcholine Biochemical and clinical aspects of essential phospholipids;
edited by H. PEETERS, Springer, Berlin-Heidelberg-New York,
(1976)
kung essentieller Phospholipide auf den Blutfettgehalt des
Menschen. Folia Angiologica Suppl., Vol. VI, 80-87 (1975)
dosierter i.v. und oraler Zufuhr essentieller Phospholipide auf den EiweiB- und Fettstoff,lechsel sowie Enzymaktivitaten chronisch Leberkranker. Verh.Dtsch.Ges.Innere
Ned.~, 1389-1392 (1972)
64
- SAMOCHOWIEC L., KADLUBOWSKA D. and ROZEWICKA L.: Investigations in experimental atherosclerosis. Part 1: The
effects of phosphatidylcholine (EPL) on experimental atherosclerosis in white rats. Atherosclerosis 23, 305-317 (1976)
SAMOCHOWIEC L., KADLUBOWSKA D., ROZEWICKA~., KUZNA W. and
SZYSZKA K.: Investigations in experimental atherosclerosis.
Part 2: The effect of phosphatidylcholine (EPL) on experimental atherosclerotic changes in miniature pigs. Atherosclerosis Q, 319-331 (1976)
- STAFFORD W.W. and DAY C.E.: Regression of atherosclerosis

effected by intravenous phospholipid.Artery 1, 106-114 (1975)
- DAY C.E.: Obstacles to the clinical investigation of phospholipids, Artery 1, 100-102 (1975)
- HOLZL J.: Pharmacokinetic studies on phosphatidylcholine

and phosphatidylinositol. In: Phosphatidylcholine - Biochemical and clinical aspects of essential phospholipids,
edited by H. PEETERS, Springer, Berlin-Heidelberg-New York,
(1976)
10 - HOLZL J. and WAGENER H.: tiber den Einbau von intraduodenal

appliziertem 14C/32p Polyen-Phosphatidylcholine in die Leber von Ratten und seine Ausscheidung durch die Galle.
Z. Naturf. 26b, 1151-1158 (1971)
11 - CHASSEAUD L.F., DOWN W.H., WILLIAHS J.H., SACHARIN R.H.
and FRANKLIN E.R.: The metabolic fate of 3H: 14C-essential
phospholipid (EPL) in the rat. Res.Rep. NTN4/75379
Huntington Research Centre, 1975, unpublished
12 - CHASSEAUD L.F., DOWN W.H. and SACHARIN R.M.: The metabolic fate of 3H: 14C-essential phospholipid (EPL) in the
rhesus monkey. Res.Rep. NTN 5/75497, Huntington Research
Centre, 1975, unpublished
13 - LEKIM D. and GRAF E.: Zur Pharmakokinetik und antihyperlipamischen Wirkung oral applizierter EPL-Substanz bei
Ratten. Arzneimittelforschung~, (1976), in press
14 - LEKIM D., BETZING H. and STOFFEL W.: Incorporation of
complete phospholipid molecules in cellular membranes of
rat liver after uptake from blood serum. Hoppe-Seyler's
Z. Physiol. Chern. 353, 949-964 (1972)
15 - STOFFEL W., LEKIH D. and TSCHUNG T.S.: A simple chemical
method for labelling phosphatidyl choline and sphingomyelin in the choline moiety. Hoppe-Seyler's Z. Physiol.
Chern. 352, 1058-1064 (1971)
16 - LEKIH D. and BETZING H.: Synthesis of labelled phosphatidylN,N-dimethylethanolamine and phosphatidylcholine. HoppeSeyler's Z. Physiol.Chem. l2!, 1490-1492 (1973)
65
17 - BOLLMAN J.L., CAIN J.C. and GRINDLAY J.H.: Techniques for

the collection of lymph from the liver, small intestine or
thoracic duct of the rat. J.Lab.Clin.Med. 33, 1349-1352
(1948)
-18 - SCOW R.O., STEIN Y. and STEIN 0.: Incorporation of dietary
lecithin and lysolecithin into lymph chylomicrons in the
rat. J.Biol.Chem. 242, 4919-4924 (1967)
19 - PARTHASARATHY S., SUBBAIAH P.V. and GANGULY J.: The mechanism of intestinal absorption of phosphatidylcholine
in rats. Biochem.J. 140, 503-508 (1974)
20 - VAN GOLDE L.M.G., VAN DEN BOSCH H., SLOTBOOM A.J. and
SCHERPHOF G.L.: Synthesis and metabolism of labelled monoacyl-sn-glycero-3-phosphorylcholines. Progr.biochem.
Pharmacol. 2, 129-148 (1969)
21 - WAGENER H.: Preparation, Distribution and Turnover of Tritium-Labelled "Essential" Phospholipids (EPL). In: Phospholipids - Biochemistry, Experimentation, Clinical Application; edited by G. SCHETTLER, Thieme, Stuttgart, (1972)
22 - DEWAILLY P., DE COOPMAN E., DESREUMAUX C. and FRUCHART J.C.:
Plasma removal of intravenous essential phospholipds in man.
In: Phosphatidylcholine - Biochemical and clinical aspects
of essential phospholipids; edited by H. PEETERS, Springer,
Berlin-Heidelberg-New York, (1976)
Pharmacokinetic Studies on
Phosphatidylcholine
and Phosphatidylinositol
J. Holzi
Institut fur Pharmazeutische Arzneimittellehre der U niversitat,
D-8000 Munchen 2, Germany
Abstract: Pharmacokinetic studies of intravenously injected and

orally applied radioactively labeled phosphatidylcholine (PC
and phosphatidylinositol (PI) were performed in rats and mice.
The double labeling was achieved by germinating soybeans in a
solution of the precursors 14C-acetate, -glycerol, -choline,
-inositol and 32p-phosphate and subsequent extraction and separation of the phospholipids. The injected PC Was rapidly eliminated from the plasma, the rate of elimination depended on
the physicochemical state of the PC and - in the case of suspension - on the injected dose. After 15 min. 80%, after 75 min.
92%, and after 10 hours 99% of the injected sonicated PC were
removed from the plasma, and incorporated into the liver, the
spleen, and the lungs. The injected PC was preferably bound to
the a -lipoprotein fraction. Injected lyso-PC was reacylated in
the plasma. A transacylation of fatty acids from exogenous PC
to cholesterol was observed. - Injected PI was as rapidly eliminated from the plasma as PC. Lyso-PI was not reacylated in
the plasma or in the liver. - Polyenyl-PC was incorporated also
into the brain. The amount was 0.32% after 15 min. and 0.20%
after 75 min. From ~easure~ents of the labeling of individual
fatty acids it was concluded that the permeation of PC thl'ough
the blood-brain barrier takes place as lyso-PC.
Orally applied PC is resorbed by the intestinal wall to a considerable extent as intact ?C or as lyso-PC which is subsequently reacylated. The 3H/3 2p ratio excludes a complete degradation
and l'esynthes,is of PC after resorption. Lyso-PC is found in the
intestinal wall after oral PC administration, but all radioactive label was then detected only in the P-fraction of the plasma and the liver, and no labeled lyso-PC was observed. PI, however, apparently is uptaken to a very low extent, most of the
substance being degraded during resorption.
INTRODUCTION
The use of labeled phosphatidylcholine (PC) for the investigation of the absorption and the metabolism of phospholipids (PL)
is of great advantage. Chemically labeled PC can be produced by
cleaving the fatty acid from position 2 of the molecule with the
67
aid of phospholipase A and subsequent reacylation with labeled

fatty acid (1,2,3) or by the introduction of a labeled methyl
group into a cemethylated PC molecule (4). Our technique of labeling of phospholipids, however was based on a biological method, na~ly ger~ination of soybeans in the presence of 14C- or
32P-precursors (5). The phospholipids thus obtained were separated from each other, and phosphatidylcholine (PC) and phosphatidylinositol (PI) were selected for the use in the experiments.
PI was chosen in addition to PC because of its high metabolic
rates in different organs (6,7,8), particularly in the liver
and in the brain, and because of the high content of arachidonic
acid in these organs showing a great metabolic exchange for PI
(9). Phosphatidylethanolamine (PE) and phosphatidic acid (PA)
were also labeled during soybean germination, but they are less
favorable for pharmacokinetic studies: PE induces to some extent
blood clotting, and therefore distribution studies after injection of the labeled material are subjected to considerable systematic errors; PA causes exitus of the animals after intravenous injection probably due to binding of calcium ions, which
results in complete insolubility of PA, which then appears to
be deposited in the lungs, since only there radioactivity can
be detected.
Our predominant interest was the distribution of the injected
phospholipid, particularly its incorporation into the brain.
Previously we had observed that PC penetrated the blood-brain
barrier, and we suggested that the passage of the PC would occur
in the chemical form of a lyso-PC (10). This postulate has been
experimentally confirmed by other investigators since (11,12),
and therefore was of main interest for further detailed investigation.
METHODS
1. Labeling of Phosphatidylcholine (PC) and Phosphatidylinositol (PI)
The labeling of the phospholipids was perfor~ed by germination

of soybeans in the presence of radioactive precursors. 1 or 10
mCi of 33p_ or 32p-phosphate, of 14C-acetate, -glycerol, -choline, or -inositol (dissolved in 10 ml H20) were added to 10 g
of soybeans. After soaking, the soybeans were germinated on cellulose for 48 hours and subsequently freeze-dried. The neutral
lipids were removed by homogenization in acetone, the phospholipids were extracted with chloroform-methanol. PC was isolated
by chromatography on alumina, PI was separated on a silicic acid
column.
The amounts of different precursors incorporated into the soybean phospholipids are listed in Table 1. The highest incorporation was achieved using acetate. Glycerol was incorporated into all phospholipids; choline and inositol were only incorporat-
68
Table 1:
Labeling yields of phospholipids ( means of 3 determination) in soybean extracts after
germination in the presence of various precursors
[%
Labeling yield
Precursor
14C _ acetate
33p_ or 32p
PC
PE
PI
7,8
3,7
2,7
1, 3
0,8
glycerol
2,0
choline
2, 1
of PL]
inositol
phosphate
3,2
1, 4
4,9
0,9
ed into PC and PI respectively. The phosphate moiety was labeled with 32 p or 33p and, starting with the highest specific
activity of 3.7 mCi/g soybean tolerated without radio-toxic effects, specific labeling of 22 mCi/roM PC, 27 mCi/mM PE, and 13
mCi/mM PI could be achieved.
The fatty acid composition and the grade of labeling of the phospholipids after soybean gerJY1ination in 14C-acetate solution
under 02-atmosphere are shmvn in Table 2. The fatty acid composition was identical to that of the so-c~lled "essential" phospholipids (EPL-NATTERMANN), while PI contained an almost equal
percentage of palmitic and linoleic acid. In PC the highest
grade of labeling was achieved in oleic acid; 75% of the label
was localized in the unsaturated fatty acid. This is typical of
acetate incorporation under 02-atmosphere, wheras exposure to
air during gemination results in a higher labeling of saturated
fatty acids.
Table 2:
Fatty acid composition (a) and 14C - labeling yield (b) of phosphatidyl choline (PC) and
phosphatidylinositol (PI). Data given in per cent. 10 mCi 14C - 1 - acetate (specific activity 50 mCi / mM )
were applied to 10 g of soybeans. Germination for 36 h under O2 - atmosphere at 20 0 C
PC
PI
PC
PI
16:0
13
39
18
65
18:0
18: 1
11
44
16
18:2
66
41
31
18:3
--
--
69
In PI 74% of the label was localized in the saturated fatty

acids. Labeling of linoleic acid could not be achieved during
soybean germination.
2. Preparation of Labeled Lyso PC and Lyso PI Using Phospholipase A2
10 mg of labeled PC was dissolved in 10 ml ether and 0,1 ml phospholipase A2 (E.C. 3.1.1.4) (BOEHRINGER, Mannheim, Germany) plus
0,05 ml 1M CaCI were added. After shaking for 24 h the lyso-PC
was centrifuqed.
10 mg labeled PI was dissolved in 10 ml ether and 2 mg bee venom
(MACK, Illertissen, Germany) in 10 mg H20 was added. After shaking for 12 h the water phase was separated and evaporated. From
the residue the lyso-PI was extracted with methanol and purified
on a silicic acid column with chloroform, chloroform-methanol
(95:5) and methanol.
The radioactivity distribution after the incorporation of 14Cacetate with respect to the 1- and 2-position of the phospholipid molecules was revealed by phospholipase A2 cleavage and the
fatty acid pattern was determined by radio-gas-chromatography.
3. Procedures of Separation and of Radioactivity Detection
Plasma or organs were removed from the animals and weighed. Samples of 1 g freeze-dried tissue were extracted three times with
20 ml chloroform-methanol (2:1) in a Sorvall Omnimixer (5 min.
10.000 U/min.). The centrifuged solutions were combined and
shaked with water; the lipid phase was evaporated and the residue solved with 10 ml chloroform-methanol.
Thin-layer chromatography (TLC) of the phospholipids was performed on silica gel 60 F 2 54 (E. MERCK, Darmstadt, Germany)
usina the solvents (1) chloroform-methanol-water (65:25:4), (2)
chloroform-methanol-7N ammonia (120:40:7), (3) butanol-acetic
acid-water (4:1:5), upper phase, for separation of lyso-PI from
PI, and (4) hexane-ethyl ether-acetone (90 10:1) for separation
of cholesterol esters.
The radioactive labeling of the separated compounds was detected by autoradiography by exposing the thin-layer plates (or
paper strips from electrophoresis) to X-ray film (Structuric
D 10, Gevaert, Belgium, or Osray T 4). The time of exposure depended on the radioactivity present in the chromatography spots
It was in the range of a few hours (20 000 dpm/spot) to several
days (10 dpm/spot). An example is shown in Fig. 1.
The fatty acid patterns of the different phospholipids were determined by radio-gas-chromatography as described elsewhere (13).
Fig. 2 shows a gas chromatogram of the soybean-PC after a 48
hours' germination under 02-atmosphere at 20 0 C.
The incorporation of labeled lipids into different lipoprotein
fractions was studied using paper electrophoresis, because this
technique allows to analyze relatively high volumes of plasma.
70
fro n t
TG
CL
PE
PC
PI
Ac
start
Fig . 1: Autoradiogram of the acetone extract (a) and the chloroform

methanol extract (b) of soybeans after incorporation of 14C . acetate
4. Animals and Intravenous Application of the Phospholipids
Male Dawley rats and mice were used for the experiments. Doses
of 0.5 mg PCjg body weight were injected into the tail veins, if
not indicated otherwise. Doses of 0.5 mg PIjg were not tolerated
in any case, and therefore had to be reduced. Lyso-phospholipids
exert high hemolytic activity and lead to death of the animals.
Therefore compounds of high specific activities had to be used
and the doses were limited to a maximum of 0.05 mgjg.
Bile cannulation was performed as previously described (5). The
rats were narcotized with 0.3 mgj100 g body weight Nembutal.
Fig .
2:
Fatty acid pattern of soybean PC as determined
by radio gas chromatography after germination

' - - - - - - - - - - - - - --
for 48 h under light and

2 , atmosphere at 20 0 C.
- - - - Smooth line = normal gas chromatogram for comparison
71
RESULTS
1. Pharmacokinetics of Intravenously Injected Phospholipids
a. Plasma levels
Intravenously injected phospholipids were rapidly removed from
the plasma. Thus, already 5 minutes after the injection only a
rather low percentage of the applied phospholipids was left.
The rate of removal depended on the physico-chemical state of
the injected material, so that 30% of the PC sonicated in H20,
only 6% of the PC in suspension, but 60% of the PC solubilized
with desoxycholate (DOC) were found in the plasma 5 minutes
after the injections (Table 3). After 10 hours 99% of the sonicated PC were eliminated. Lyso-PC was reacylated in the plasma:
75 minutes after the injection of comparable doses of lyso-PC
as well as of sonicated PC the same amount of PC was found in
the plasma. Lyso-P, however was slowly reacylated in the plasma, so that after 75 minutes half of the radioactivity was detected as lyso-PI and half as PI.
Table 3: Plasma concentration of various phospholipids in mice at different times after intravenous injection.
Data in per cent of the injected doses (see METHODS). Means of 3 determinations each; PC: DOC = 25 : 14;
PI as sonicated suspension
Tillie
I' C
Ultrason.
'5 m
15
35
75
2,5 h
6,2
10
22
38
Suspension
P C L P C
+DOC
P I
60
20
21
17
8
5
1,5
1,2
--
The percentage of the injected PC found in the plasma after 5

minutes depended on dose in the case of the PC-suspension, but
was independent of dose in the case of the PC-DOC solution
(Table 4). Obviously a great portion of the suspended PC is very
quickly eliminated from the plasma, as lipid particles of great
diameters are retained in the liver and the lungs.
72
Table 4:
Plasma concentration of PC in mice depending on the injected dose as measured 5 min after the
intrevenous injection. Data in per cent of dosage (n = 3 )
mg PC
per
35
g mouse
So [HUon
Suspension
I'C:DOC
1.8
26
6")
4.2
13
66
12
73
18
65
36
25: 14
The binding or incorporation of the injected PC to the plasma

lipoproteins was studied using paper electrophoresis and measuring the radioactivity of each fraction (Table 5). As expected, the highest percentage of radioactive PC was found in the
a-lipoprotein fraction. It increased during the first 75 minutes, and was decreased after 315 minutes, while the albuminbound radioactivity was enhanced simultaneously. This was due
to degradation of the PC and incorporation of the products,
lyso-PC and free fatty acids, into the albumin fraction. The
distribution of the PC over the lipoprotein fractions was not
significantly influenced by the presence of DOC.
Table 5:
Percental distribution of 14C - radioactivity on the plasma lipoproteins in mice at different times
after intravenous injection of 0.5 mg 14C - polyenyl - PC / g body weight (0.5 mCi / mM) a = sonicated
suspension, b = solution in DOC (PC: DOC = 25 : 14)
( n = 3, each)
Chylomicrons
12
~-Lipoproteins
a-Lipoproteins
Albumin
min
16
74
9
12
,)
79
80
11
64
75 min
a
b
,)
3 1 2 min
a
15
73
700
500
3
300
1.00
500
600
100
200
300
1.00
500
600
Fig. 3: Transacylation of fatty acids from exogenous PC to cholesterol in mice. 14C - radioactivity vs. time
after the intravenous injection of 223 nCi 14C - polyenyl - PC / g body weight (specific activity 0.37 mCi / mM).
Lett: total plasma radio activity; right: radioactivity in the plasma cholesteryl ester fraction (units:
10 dpm. minutes)
The trans acylation of fatty acids from exogenous PC to cholesterol was estimated by measuring the radioactivity of the cholesteryl ester fraction (Fig. 3). Its maximum radioactivity was
observed inbetween 75 and 155 minutes after the injection of
labeled PC. At this period 10 per cent of the 14C-fatty acids
were bound to cholesterol.
b. Phospholipid distribution in organs
The temporal distribution of labeled polyunsaturated PC in different organs of mice after intravenous injection is depicted
in Fig. 4. The rapid removal from plasma coincides with an increase of the radioactivity in the liver (max. after 3 h) and
in the spleen (max. after 1 h). There is a decrease observed in
the lungs either due to rapid metabolism or to decorporation.
Comparative studies were made for PC or PI and lyso-PC or lysoPI, respectively. Table 6 summarizes the percentages of the in1.0
0/0
Fig: 4: Distribution (% of dosis) of labeled polyenyl - PC in various organs in mice after an intravenous
injection of 0.5 mg PC / 9 body weight (specific activity 0.5 mCi / mM )
74
Table 6: Comparative organ distribution in mice (% of dosis) of various 32p - lapeled phospholipids at
several intervals after the intravenous injection. PC, LPC, PI and LPI as described in the METHODS
section; hydro PC = completely hydrated (saturated) PC. PC, PI, hydro PC as sonicated suspensions.
The LPI found in the lungs and the spleen was totally found as PI
I'C
I.PC
hydr.PC
PI
15 min
75 min
315 min
14
29
24
10 (PC)
2,5
16
20
Lung
15 min
75 min
24 (PC)
1 (LPC)
7,5
Spleen
15 min
75 min
7
15
In.jcetcu. 1'1.
Li ve r
Ll'l
7,7
7,0
2,6
3,3
0,35
0,44
4,3
5,/1
0,11
O,L5
( 1.1' r )
( 1,1'
I)
jected phospholipids at certain intervals after the application.

PC and PI showed a similar distribution in the organs. The highest percentage of the radioactivity of lyso-PC was observed in
the lungs (25%), but only 1% were found as lyso-PC and 24% as
reacylated PC. All lyso-PC incorporated into the liver was reacylated to PC after 75 minutes. In contrast, no lyso-PI was
reacylated in the liver. There was a slow reacylation of LPI in
the plasma as mentioned above.
c. Phospholipid incorporation into the brain
Previous investigations demonstrated the incorporation of intravenously injected polyenyl-PC into the brain (10). For comparison the uptake of other phospholipids was studied. The results
are collected in Table 7. Polyenyl-PC amounted to 0.32 and 0.20
per cent after 15 and 75 minutes, respectively. Lyso-PC was 3
times as much, fully saturated PC, however, was not detected or
was 0,01 per cent. PI was found to a rather high amount at 15
minutes, but decreased quickly with time. Only very little lysoPI was uptaken. To investigate the permeation of the PC molecules from the blood to the brain, doubly labeled compounds were
used and the radioactivity incorporated into the PC fraction of
the plasma, of the brain and of the liver was measured (Table
8). 3 hours after the injection of diacyl-glycero-2-3H-~hospho
ryl_32p-choline, approximately 7% of the applied radioactivity
were found in the plasma-, 0,2% in the brain-, and 10% in the
liver-PC. The ratio 3H/ 32 p was little different from that of
the injected PC. However, the PC synthesized from glycero-phosphate or from glycerol plus phosphate contained in the plasma
and in the brain) up to 5 times more 3H- than 3 2 P-radioactivity
as compared with the injected material, in the liver this ratio
was inbetween 1,3 and 1,8.
75
Table 7: Uptake of intravenously injected phospholipids (PL and Lyso PL) into the brain of mice.
Dosages: 0.5 mg PL / 9 body weight as sonicated suspension and 0.033 mg Lyso . PL / 9 in solution (n = 3 )
Injected
Phospholipid
Uptake
Polyenyl-PC
LPC
0.32
0.20
1.00
0.75
(% of dose)
75 min
15 min
0.01
Distearyl-PC
PI
0.80
0.15
LPI
0.002
0.002
Table 8:
Incorporation of labeled compounds into the plasma, the brain, and the liver of mice. The radio
activity ( 10 6 dpm, means of 3 determinations) was measured 3 h after the intravenous injection
Measured Radioactivity
Injected
Compound
Injected
Activity
(+ )
(]lCi)
3H
158
ratio
10
1.0
ratio
100
1.0
G + P
ratio
100
1.0
PC
GP
(+)
PC
GP
G+P
ratio
Plasma-PC
32 P
149
1.06
Brain-PC
3H
32p
Liver-PC
3H
32 P
0.060 0.052
1.16
315 227
1. 38
0.206 0.041
5.0
0.326 0.060
4.9
44
57
1.30
0.131 0.027
4.9
0.052 0.015
3.4
327 180
1. 82
Diacyl-glycero-2- 3H-phosphoryl-32p-choline
Glycero-2- 3H-phosphate- 32 p
Glycerol-2- 3H +
3H/32p
Phosphate- 32 p
Comparing the amounts of labeled PC, it is seen from Table 8

that PC-labeling is due to injection of labeled PC and that in
vivo synthesis of labeled PC from labeled degradation products
of injected PC would give an unimportant contribution to the
total PC measured. Furthermore, from in vivo synthesis a nearly unaltered ratio of 3H/32p, as was observed after PC-injection, cannot be expected according to the results of Table 8.
76
A possible exchange of cleavage of fatty acids from the injected
PC-molecules during incorporation into plasma-, liver-, and brain
PC was studied with the aid of triple 14C-fatty acid: 32 p-Dhosphate-Iabeled PC. The 14C-label was equally distributed in the
1- and 2-position. Three hours after the injectiori only a little
loss of 14C-radioactivity was observed for the 2-position in the
plasma- or in the liver-PC, meaning that the PC was incorporated
practically intact (Table 9). However, the brain-PC revealed a
considerable decrease of the radioactivity in the B-Dosition SUqgesting that there the fatty acid was split off from the PC-molecules during penetration of the blood brain barrier.
Table 9:
Distribution of radioactivity in labeled PC isolated from various organs in mice" The 14C - radio-
activity in the 1 - and in the 2 - position of the PC molecule was measured 3 h after an intravenous injection
of diacyl- l4C - glycero - phosphoryl- 32p - choline
C14-a La.
P}:.!
C14( a f.a.+ 0 f.a.)
C14- 0 f.a.
2.0
1.00
Plasma-PC
2.0
1.20
I i v c r-PC
2.55
4.20
1.37
PC injected
BI"ain-PC
6.50
~'.C
14 - a r .a
50.0
,)4.,)
:;8.7
8 6'()
P-q
)-
C 1ll- :X r .a .
'I . ()
3 ~ 8:2
3.79
'1.7
The uptake of PI from the blood into the brain was measured by
autoradiography after thin-layer chromatography in comparison
with the uptake into the liver. 75 min. after 32 P-PI-injection
the phospholipid distribution was nearly the same in the liver
and in the brain (Fig. 5). A complete degradation of the Pi
during the passage through the blood-brain barrier is, therefore,
excluded.
fron t
PE
PC
PI
5 tar t
Fig_
5:
Autoradiogram of the phospholipid fraction of
mouse liver and brain after intravenous injection of 8 mg

32p _ phosphat idyl inositoL I ncubation time: 75 min
77
2. Pharmacokinetics of Orally Applied Phospholipids
Resorption studies of orally applied phosphatidylcholine were

performed by separation and autoradiography of the lipids in the
intestinal wall and in the liver in comparison with the administered labeled material (Fig. 6). In the intestinal wall as in
the liver the predominant amount of radioactivity was found in
intact PC. However also a considerable amount of lyso-PC was
observed in the intestinal wall, but not as much in the liver.
A minor portion of radioactivity was found in inorganic phosphate (start) and in the triglycerides (front). Consequently,
a minor part of the applied labeled PC was degraded during resorption but the major portion was uptaken either as intact PC
and or as lyso-PC, which then was reacylated to PC after resorption.
f ron t
TG
PE
PC
sta rt
Fig.
6 : Autoradiogram of 14C. 32PC and of the phospholipid
fractions of mouse intestine and liver after oral administration

of 0,5 mg / kg (0,5 mCi / mM) doubly labeled polyene PC
The bile cannulation technique was applied to investigate the

time course of resorption of PC. In a first series 14C_32p_PC
was injected into the vena portae and the radioactivity in the
excreted bile was determined \Fig. 7, top). The ratio 3H/32p
was reversed from > 1 to a value <1 after 2 hours. The maximum
radioactivity excretion was found inbetween 3 and 4 hours after
the injection. A total of 1% of the injected material was excreted in the bile within 6 hours. In a second series, the
doubly labeled PC was applied into the duodenum. After intestinal resorption a total of 0,2% of the applied labeled material
was excreted in the bile within the first 2 bours (Fig. 7, bottom).
The excretion is more protrahated in comparison to the intraportal injection, obviously due to a slow intestinal resorption.
The 3H/32P-ratio is practically not changed during the first 6
hours, suggesting that the PC was not greatly degraded during
resorption.
78
x 103 dpm
11.
12
10
8
6
I.
Fig.
7:
Comparison of the 14C and 32p - radioactivity
( dpm) measured in the bile of bile - cannulated male rats after

intraportal (top) and peroral (bottom) administration of
equal doses of doubly labeled polyenyl - PC. 1,7 flCi 14C_
and 1,7 fJCi 32p - label were administered in both cases
32p-PI was orally administered for comparison, and the radioactivity incorporated in PI and PC was measured in the stomach
and the intestine and in the liver. The results are collected
in Table 10. In contrast to PC, no lyso-PI at all was found in
the intestinal wall. The percentage of PI found in the liver
was very low in comparison with the values observed for PC
after peroral PC application.
Table
10:
32p - radioactivity found in the PI - and PC - fractions of the intestinal tract (lumen + wall)
and of the liver in mice after oral administration of 115 nCi / g body weight of 32p - phosphat idylinositol
( specific activity: 0.2 mCi / mM )
Intestine and Stomach

PI
75 min
375 min
20:!:.7
3,4:!:.1,3
Liver
PC
PI
PC
2,8:!:.0,3
0,03:!:.0,005
0, 13:!:.0,02
1,6:!:.0,1
0, 12:!:.0,01
0, 9:!:.0, 2
REFERENCES
- ROBERTSON A.F. and LANDS W.E.M.: Biochemistry
1,
- VAN DEENEN L.L.M.: J.Amer.Oil Chemistrs' Soc.
ii,
- DE HAAS G.H. and VAN DEENEN L.L.M.: Rec.Frav.Chim. 80, 951

(1961)
804
257
(1962)
(1968)
79
- STOFFEL W., LEKIM D. and TSCHUNG T.S.: Hoppe Seyler's Z.

Physiol.Chem. 352, 1052 (1971)
- HOLZL J. und WAGNER H.: Z.Naturf. 26b, 425 (1971)
- HOKIN L.E. and HOKIN M.R.: J.gem.Physiol. 44, 61 (1960)

Nature (London) 190, 1016 (1961)
--
- HOLZL J. und WAGNER H.: Biochem.Z. 339, 327 (1964)
- WAGNER H., LISSAU X., HOLZL J. und HORHAMMER L.: Lipid Res.
1, 177 (1962)
- BAKER R.R. and THOMPSON W.: Biochim.Biophys.Acta 270, 489

(1972)
10 - HOLZL J.: II.Intern.Meet. of Intern.Soc. of Neurochem.

Milano, 1969
11 - ILLINGWORTH D.R. and PORTMAN O.W.: Biochem.J. 130, 557 (1972)
12 - DHOPESHWARKAR G.A., SUBRAMARIAN C. and MEAD J.F.: Lipids
753 (1973)
13 - HOLZL J.: Z.Anal.Chem. 252, 137 (1970)
14 - SCHERER R.: Untersuchungen tiber den Abbau von markiertem
Lecithin in Humanserum. Dissertation Mtinchen 1972
~,
Plasma Removal of Intravenous

Essential Phospholipids in Man
P. Dewailly, E. Decoopman, C. Desreumaux and J. C. Fruchart
Laboratoire de Physiopathologie des Lipides, Institut Pasteur,
F-59012 Lille Cedex, France
Abstract:
Essential phospholipids intravenously injected in
man (n = 20) caused a rapid increase of the plasma lipid phosphorus to 108% (p < 0~001) within the first 2 minutes~ followed by a
decrease to the control level within the subsequent 10 minutes.
Simultaneously~ the percentage of linoleic acid in plasma lecithins was en larged during the first two minutes J ' but remained increased (p < 0.05) for at least 40 minutes. The increase of linoleic acid was similar in the lecithins of different plasma lipoproteins~ but did not occur in plasma phosphatidyl-ethanolamines.
ObviouslYJ the excess of phospholipids was quickly removed from
the plasma J but either part of the plasma lecithins as a whole or
their fatty acids were exchanged for the injected moieties.
INTRODUCTION
Essential phospholipids - LIPOSTABIL 1 - mainly consist of soybean

lecithins rich in linoleic acid (18:2) and were recently used
intravenously in the treatment of fat embolism (1) and hypercholesterolemia (2). Moreover, some workers have shown an antiatherosclerotic effect of intravenous polyunsaturated lecithins
( 3)
In order to investigate the plasma removal of such phospholipids,

we studied in man the quantitative and qualitative modifications
of plasma phospholipids after intravenous administration of LIPOSTABIL.
METHODS
Plasma Lipid Phosphorus and Lecithins
Twenty control subjects (20-50 years old) received in the fasting

state one intravenous injection of LIPOSTABIL containing 0.2 ml/kg
1NATTERMANN Company, Cologne, Germany
Relative fatty acid composition of LIPOSTABIL (% of weight) :
16:0 13%, 18:0 3.6%, 18:1 7.5%, 18:2 66.9%, 18:3 9%.
81
or 10 mg/kg of phospholipids, respectively. Venous blood samples

were taken before and after injection at the following times:
2, 5, 10, 20, 30, 40 and 60 minutes.
Plasma lipids were extracted by the method of Folch. Total lipid
phosphorus was measured in the extract as described by MISSON et
al. (4). Phospholipids were separated with thin layer chromatography on silica gel G (Merck) with chloroform: methanol: NH 40H
7N (115/45/7.5 , v/v/v). After revelation of the spots by Rhodamine 6 G, the plasma lecithins were recovered. Preparation of
fatty acid methyl esters was performed directly on these fractions (5). The methyl esters were analyzed by gas lbquid chromatography on standard column of D.E.G.S. (10 %) at 185 C. These
fatty acids were used to calculate percentages: 14:0, 16:0, 16:1,
18:0, 18:1, 18:2 and 20:4.
Lecithins of VLDL + LDL and HDL
A same injection of LIPOSTABIL was used in five control subjects

to study the incorporation of linoleic acid (18:2) into lipoprotein lecithins. VLDL and LDL were precipitated using calcium
chloride and heparin as described by BURSTEIN et al. (6). Lecithins of both fractions, VLDL + LDL and HDL, were separated as
mentioned above. After methylation, gas liquid chromatography
was performed in order to establish the percentages of fatty acid
methyl esters.
Plasma Phosphatidyl - Ethanolamines
In the same study the plasma phosphatidyl-ethanolamines and lecithins were analyzed. Phosphatidyl-ethanolamine (P.E.) and phosphatidyl-choline (P.C.) were separated by silicic acid column
chromatography (7) and isolated with thin layer chromatography
as described. Fatty acid methyl esters from P.E. and p.C. were
analyzed by gas liquid chromatogra~hy.
RESULTS
Plasma Lipid Phosphorus and Content of Linoleic Acid (18: 2) in Plasma Lecithins (Fig. 1).
A significant increase (p < 0,001) of plasma lipid phosphorus

is found at 2 and 5 minutes after the injection of LIPOSTABIL.
In the period after 10 minutes the level of lipid phosphorus does
not Significantly differ from the control.
Similarly, there is a significant increase (p <0,05) of the content of linoleic acid (18:2) in total plasma lecithins two minutes
after injection. This increase remains statistically significant
after 40 minutes.
82
% Ph.
110
105
100
% 18: 2
30 ]
~p. ..
,..
...
I"
20
.... ... 5 - 10
20
30
40
60'---
Fig. 1: Values of plasma lipid phosphorus (Ph.) after intravenous administration of LlPOSTABIL
( % established from control values ( 100 % ) obtained before injection) and linoleic acid ( 18 : 2 )
percentages in plasma lecithins. (Mean.. standard error from 20 experiments)
Linoleic Acid in Lecithins of Lipoproteins (Table 1 )
The percentage of linoleic acid increases significantly in the

two plasma lipoprotein fractions: VLDL + LDL and BDL. The increase is of the same order in the different linonroteins and
remains constant for at least 60 minutes.
iJDL
LDL
VLDL
FRACTIONS
0,2 :!: 0,2

0,2 :!: 0,2
0,2 + 0,2
20
40
60
0,022
0,012
0,2 :!: 0,2

0,2 :!: 0,2
40
60
20
0,2 :!: 0,2

0,2 :!: 0,2
10
:!: 0,1
0,1 :!: 0,2
0,1
30,8 :!: 2,3
0,2 + 0,2
10
:!: 2,3
30,5 :!: 1,3
29,1
29,8 :!: 2,8

2,6 :!: 0,5
1,9 :!: 0,8

2,0 :!: 0,6
2,0 :!: 0,6

2,1 :!: 0,5
29,3 :!: 2,3

29,4 :!: 2,8
2,6 :!: 0,7
2,2 :!: 0,9
1,9 :!: 0,8

2,2 :!: 0,8
1,8 + 0,8
-
2,1 :!: 0,7

2,0 :!: 1,2
16 :
33,6:!: 2,7
30,4 :!: 1 ,1
30,6 :!: 2,4
30,1 :!: 2
30,0 :!: 1,6
33,3 :!: 1,9
0,3 :!: 0,2
16 :
0,2 + 0,2
14 :
(nin)
TIMFS
0
:!: 1,5
9,2 :!: 2,6
:!: 5,5
31,2
29,7 :!: 4,8
15,0 :!: 1,9

14,6 :!: 1,6
:!: 2,5
12,2 + 2,6
10,2 :!: 2,8
10,2 :!: 1,4

10,1 :!: 1.7
9,9 :!: 1,7
22,4 :!: 3,8

1 1 ,4 :!: 3,5
5,2::
10,5 :!: 1,4
31,7 :!:
8,9 :!: 2,6

9,4 :!: '2,1
:!: 1,9
31,9 :!: 5,3

31,3 :!: 6,2
12,4
20 :
29,2 :!: 5
30,0 :!: 5,3

30,3 :!: 5,5
22,9 :!: 3,5

11,4 :!: 2,1
30,5 :!: 4,9~
9,3 + 1,6
30,5 + 4,5
9,6 :!: 1,9
18 :
:!: 1,6
:!: 1,3
14,6 + 1,5
15,6
15,4
15,6
15,1 +
- 1,4
15,1 :!: 1,1
15,1 + 1,5
15,8
18 :
14,7 :!: 1,4

14,7 :!: 2,2
11,8 :!: 2,4

11,8 + 2,3
14,3 :!: 2,2

11,8 :!: 2,4
12,4 :!: 1,6

13,0 :!: 2
12,9 + 1,9
12,9 :!: 1,9
14,2 :!: 2,2

12,8 :!: 2,1
18 :
Table 1: Time Variations of fatty acid percentages in lecithins from plasma VLDL + LDL and HDL after injection of LlPOSTABIL
(means.:':. S.E.M., n = 5 )
en
2:
.... N.
'-
(P.
S.
0,018
r:. )
CI::PI.ALHIS
(P.C. )
LECIThINS
1,6 :!: 1,4
60
0,6
1,2
40
:+.:
1,7
20
10
0,7
1,4 :!: 0,7
2,0 :!: 1
2,4 :+.: 0,4
:+.:
0,1 :!: 0,2
60
40
0,2 :!: 0,3

0,2 :!: 0,2
0,2 :!: 0,4

0,1 :!: 0,2
0,1 :!: 0,2
14
20
10
TH'ES
(win)
0,2
20,9 :!: 2,9
24,8 :!: 1,5

22,9 :!: 4,3
2,6 :!:
2,8 :!: 0,4

2,6 :!: 0,4
:+.:
22,4 :!: 2,2

3,6
2,7 :!: 0,4

2,8 :!: 0,5
2,4
:+.:
23,7 :!: 2
22,0
1,6 :!: 0,8
1,9 :!: 1,1

1,7 :!: 0,8
1,9 :!: 0,7
2,2 :!: 0,5

1,5 :+.: 0,6
16 :
30,7 :!: 2,9
31,2 :+.: 3
30,1 :!: 2,1
30,4 :!: 1,5
34,5 :!: 2,2

30,1 :!: 1,4
16 : 0
17,5
:+.:
4,2
17,0 :!: 4,6

18,0 :!: 2,4
17,6 :!: 3,3
18,0 :!: 2,3
17,0 :!: 3,4
12,7 :!: 2
12,5 :!: 0,3
12,7 + 1,4
:+.:
1,6
:+.:
12,9
:+.:
3,1
12,5 +
- 0,5
13,3 :+.: 1 , 1
12,2
12,4 :!: 0,7

12,0 :!: 1,2
15,2 :!: 1,3

15,0 :!: 0,5
15,0
15,9 :!: 1,5
15,1 :!: 1,6

15,2 :!: 1,4
18 :
14,3 :!: 1,5
12,6 :!: 2
12,3 :!: 1,2
18 :
:+.:
:+.:
4, 1
16,5 :!: 5,8
18,1
10,3
:+.:
28,1
23,9
:+.:
:+.:
4,5
6,8
5,5
5,9
3,2
5,9
8,9 :!: 1,2

9,1 -+ 1,9
8,7 :!: 1,7

7,7 :!: 1,8
9,9 :!: 2,8
20
18,2 :!: 4,2

24,5 :!:
15,5 :!: 2,5:::: 27,7 :!:
15,9 :!: 3
25,9 :!:
18,1 + 2,7 23,1 :+.:
29,8 :!: 5,8
30,7
31,7 :!: 6,3

31,0 :!: 5,5
23,0 :!: 3,3

31,8 :+.: 5,8::
18 : 2
Time variations of fatty acid percentages in plasma lecithins and cephalins after injection of LlPOSTABIL (means-- S.E.M., n ~ 5)
FRACTIONS
Table
85
linoleic Acid in Plasma Cephalins (P.E.) (Table 2)
After intravenous injection of LIPOSTABIL the percentage of linoleic acid in the phosphatidyl-ethanolamine fraction is not enhanced. Thus, the increase of 18:2 percentage is specific of
lecithins.
DISCUSSION
Phospholipids are important constituents of the plasma lipoproteins. Most of them are synthetized in the liver. Blood phospholipids are transformed by circulating enzymes to lysolecithins
inducing a modification of the lipoprotein structure. The transformation essentially is mediated by the lecithin-cholesterolacyl-transferase or by phospholipases. The lysolecithins are
rapidly attached to cell membranes in various tissues, e.g. red
cells and endotheliums (8).
Our results show that the plasma lipid phosphorus increases rapidly after an intravenous injection of LIPOSTABIL and returns to
normal values after ten minutes. Surprisingly, the linoleic acid
in lecithins remains significantly increased for at least 40
minutes (Fig. 1). Moreover, the increase of linoleic appears to
be similar in all lecithins of different plasma lipoproteins and
does not occur in plasma phosphatidyl-ethanolamines (Tables I
and II)
These results clearly indicate that the excess of lecithin is
quickly removed from the plasma after injection of LIPOSTABIL.
However, the permanent increase of linoleic acid in lecithins
leads to the conclusion that either part of the plasma lecithins
are exchanged as a whole for the injected phosphatidylcholine or
an exchange of their fatty acids has taken place. To solve this
question studies of a possible intermediate lysolecithin in the
plasma and of the lecithin structures are intended.
REFERENCES
1 - WATTEL F., SCHERPEREEL P., SEZILLE G. et FRUCHART J.C.

Utilisation des phospholipides polyinsatures dans Ie traitement de l'embolie graisseuse. Lille Medical 29, 870-874,
(1974)
2 - BLATON V., VAND~~E D. and PEETERS H.
The effect of essential phospholipids on plasma lipid and
fatty acids in hyperlipidemia. Verh. Deut. Ges. Inn. Med.
78, 1 - 4, (1972)
3 - STAFFORD W.W. and DAY CH.E.
Regression of atherosclerosis effected by intravenous phospholipid. Artery 1, 106-114, (1975)
86
4 - HENRY J. et CHATELAIN S.
Application de la reaction de HISSON au dosage du phosphore
lipidique serique. Ann. BioI. Clin. 22, 143-148, (1964)
5 - BOWYER D.E., LEAT W.M.F., HOWARD A.N. and GRESHAM G.A.
The determination of the fatty acid composition of serum
lipids separated by thin-layer chromatography and a com~a
rison with column chromatography. Biochim. Biophys. Acta
70, 423-431, (1963)
6 - BURSTEIN M. et SAMAILLE J.
Nouvelle methode de separation et de dosage des lipoproteines
de faible densite. Ann. BioI. Clin. 12, 23-34 (1959)
7 - ROUSER G., KRITCHEVSKY G. and YAMAMOTO A. In: G.V. MARINETTI
(Ed.), Lipid chromatographic analysis. Arnold, London, 1967,
p.99
8 - POLONOVSKI J.
Metabolism of phospholipid in the blood. In: G. SCHETTLER
(Ed.), Phospholipid biochemistry, Experimentation and Clinical
application. Thieme, Stuttgart 1972, pp. 14-18
Effect of Essential Phospholipids

on the ATPases and on the Fluidity
of Liver Plasma Membranes
D. Hegner
Institut fur Pharmakologie der Universitat, D-8000 Munchen 22, Germany
Abstract: Essential phospholipids (EPL), rich in dilinoleoylphosphatidylcholine, were applied orally to young (40 - 80 d)
and old (?80 - 800 d) rats. The double 14C/ 32 P-labelled dilinoleoyl-phosphatidylcholine was incorporated to different degrees
into the organs of the animals and particularly into plasma membranes of liver cells. Old animals incorporated a higher amount
of radioactivity than young ones. The 14C/ 32 p ratio of the incorporated radioactivity was lower than in the administered EPLsubstance. The activity of the (Na+-X+)-ATPase of rat liver plasma
membranes of old animals increased after pretreatment with EPL.
The thermostability of the Mg++- and (Na+-X+)-ATPases was increased in old animals treated with EPL. EPL further caused a
significant decrease of the cholesterol content and an increase
of membrane fluidity parallel with the amount of incorporated
EPL. This increase of fluidity was also found in spin-labeled
liposomes consisting either of dipalmitoyl phosphatidylcholine
or of total lipids from isolated rat liver plasma membranes and
of EPL. The range of the lipid:EPL ratio tested was between
100 : 1 and 1 : 100 (w / w). The minimal concentration of EPL
causing an increase in membrane fluidity detectable by the spin
label technique was 2 per cent of weight of the total lipid in
the liposomes.
INTRODUCTION
It is well known, that the physical state of lipids in biological membranes has an important influence on protein-lipid interactions and on the activity of membrane-bound enzymes. For the
functioning of (Na++K+)-ATPase it could be shown that the fluidity of fatty acid chains as well as the polarity of the head
groups of lipids appear as a response to the ~aximum activity
of membrane-bound enzymes (1). The fluidity of the membranes,
however, depends on the degree of unsaturation of the fatty acid
chains of the me~brane lipids and on the content of cholesterol.
Cholesterol was shown to have a condensing or rigidifying effect
on the hydrocarbon chains of unsaturated fa.tty acids (2). Thus
the content of cholesterol within the lipid core of a biological
membrane should affect the transport activity.
88
Age-dependent changes of lipid composition modifying the propeties of plasma membranes have previously been described. In the
rat liver, the total phospholipids and the content of stearic
acid was increased whereas linoleic acid decreased in old animals
as compared with young ones (3). There is also some evidence that
the content of cholesterol is increased in several tissues during
aging (4). As demonstrated by LEKIM, STOFFEL and BETZING (5)
essential phospholipids (EPL) applied intravenously are incorporated into membranes of liver cells. The substance has a great
potency in changing cholesterol and lipid composition in serum
and tissue. It \vas further shown that EPL is the most effective
substance in the reactivation of latent mitochondrial succinic
oxidase activity and confirms that unsaturated fatty acids with
a chain lenqth of C16-C18 and a double bond in the middle of the
chain are most effective reactivators of the Ca++-dependent
ATPase (6). Recently it has been shown (7) that EPL diminished
the peroxydative degradation of polyunsaturated fatty acids which
could also take place in aged biological membranes.
In the light of these observations we studied the influence of
orally applied EPL on properties of rat liver plasma membrane
ATPases and the physicochemical state of lipids in young and old
rats.
METHODS
Male and female Wistar rats, 40-80 and 780-800 days old, were
used in the experiments. 14C/32P labeled EPL was applied orally
in sin ale dosps of 100 mg/100 g body weight on two successive
days before the preparation of subcellular particles. 14C-racioactivity was equally distributed among the fatty acids in positions 1 and 2 of the lecithin molecules. The solvent used for
14C/32p-EPL was oleum olivarum. Plasma membranes were isolated
from the livers using the procedure described by COLEI>1AN et ale
(8). 14C/32p-EPL used in these experiments was a generous gift
from Dr. H~LZL. The methods used for enzyme determinations and
spin label measurements have been previously described (9).
RESULTS
Before the in vitro studies on rat liver plas~a membranes were

undertaken, it was established, that orally applied EPL was incorporated into the membranes. Table 1 summarizes the results of
a 12 h-experiment. The distribution of radioactivity shows that
the 14-C-labeled fatty acids and 32P-labeled phosphorylcholine
are predominantly located in the liver, in the intestine and the
lung. The hi~h radioactivity located in the plasma membrane fraction, in microsomes, and mitochondria indicates that both 1 4C
and 32p radioactivity is incorporated into liver cell membranes.
(The enrichment of radioactivity in plasma membranes as compared
with liver homogenate is about 2.0). Related to the protein con-
9509
7815
4579
33922
28279
78480
68851
11826
27561
479
8477
18620
10095
11149
6942
2402
Brain (grey matter)

Spleen
Intestine
Blood
Faeces
Aorta
Muscle
Liver homogenate (+) 62404

Mitochondria (+)
53962
Hicrosomes (+ )
78046
Plasmamembranes (+)
80596
0.2 mg EPL
0.4 mg EPL
611
5514
18362
12065
17039
8273
5267
3871
26852
14728
7119
7157
Liver
Lung
Kidney
Heart
40 d
14C
Label:
1800 d
14C
Organs
Age:
P
13135
26055
91037
106929
166627
222259
21384
8192
3358
1028
10759
35804
25380
37676
21359
20283
15008
77313
78338
157120
153103
21765
9556
6447
1058
17205
34763
22625
30270
15969
13464
8112
32.
32
40 d
or
800 d
Radioactivity [dpm/100 mg wet weight
14
0.44
0.36
0.50
0.45
0.68
0.50
0.47
0.36
0.9
1.05
0.44
0.82
0.71
0.58
0.32
0.53
0.53
0.56
0.52
0.39
0.48
14C/ 32 p
40 d
0.52
0.64
0.72
0.47
0.42
0.52
0.40
0.71
0.45
0.35
0.48
cj3 2. P
800 d
dpm/100 mg protein (+) ]
Distribution of radioactivity in organs of rats of different age after oral administration of 14C . 32p. polyenyl phosphatidylcholine. The animals were
sacrificed 12 h after oral administration of PC. The values represent the averages from 4 animals in each case.
Table 1:
co
co
90
tent, old ani~als incorporated co~parably higher amounts of radioactivity into plasrea membranes than young animals except for the
microso~al fraction. The 14C/32P ratio in the pure labeled EPL
substance administered is 1.0. In all the investigated orqans and
subcellular fractions this ratio is lower than expected from the
applied EPL. This suqqests that a partial hydrolysis of the lecithin molecules possibly occurs before resorption or after incorporation. The amount of fatty acid (14C) incorporated is lower
than that of phosphorylcholine or lysophosphorylcholine (32 p ).
Wether reacylation takes place within the me~brane was not studied
so far. Considerable differences in the ratio between young and
old animals were not observed. More detailed investigations were
carried out on isolated rat liver plasma membranes.
Table 2:
Effect of orally administered EPL on the specific activities of Mg ++ . ATPase and (Na + K +)
ATPase iSOlated from rat liver. "
Age
[d]
800
70
28
= control group,
EPL
= treated group.
Means.. S. E. M.
+ +
++
Na -K -Mg ATPase
r19 ++ -
ATPase
+ +
Na -K ATPase
9J
EPL
EPL
9J
EPL
55.2
55.3
70.1
75.9
14.9
20.6
7.4
4.6
3.0
+4.2
4.2
0.4
28.0
2.6
34.2
2.4
6.2
0.2
Table 2 shows the specific activities of the Mg++-ATPase and the

(Na+-K+)ATPase in isolated rat liver plasma membranes of old and
young animals. In 800-day-old animals the enzyme activities are
twice as high as those in young animals. This was also shown in
other tissues and was explained by the presence of isoenzymes of
ATPases with a lower specific activity during the first postnatal
weeks. The more interesting observation is that the (Na++-K+)-,
but not the Mg++-ATPase, could be further activated significantly after oral pretreatment with EPL. A possible explanation would
be that this is a response to the modification of the membrane
properties, particularly in the molecular environment of the
ATPase by the incorporated essential fatty acids.
Total phospholipid and cholesterol contents of isolated rat liver
plasma membranes of young and aged male rats are shown in Table
3. Phospholipids, calculated from total phospholipid phosphorus,
were significantly different in young and aged animals. The
total cholesterol is significantly higher in aged animals than
in young ones. After oral treatment of old animals with EPL, the
phospholipid content is significantly increased, whereas the
91
Table 3.:
Content of phospholipid and of cholesterol in liver plasma membranes in young and old male rats.
The phospholipid content was calculated from the total lipid phosphorus; the molar ratio of cholesterol/inorganic
phosphorus (PL Pi) of phospholipid was calculated setting the MW at 386. Mean values.. S.E.M. of 5
different membrane preparations. n = number of determinations. Il = controls, EPL = animals treated with
100 mg / 100 g body weight per os on two successive days
Age [ d]
Treatment
80
Phospholipid
% of total lipid
780
EPL
780
QJ
QJ
50.4 + 5.6
41.0 + 4.1
44.5 + 4.2
20
20
12
6.6 + 2.2
9.7 + 0.3
7.2 + 0.2
15
16
n
Total Cholesterol
% total lipid
n
0.264
Cholesterol
,uH/ jUM PL-P i
11
0.479
0.373
content of cholesterol is lowered. This results could confirm

experiments of GOLDENBERG et ale (10) that deficiency or sufficiency in the dietary content of essential fatty acids of rats
changes the allosteric and conformational properties of the
ATPases. We therefore carried out some experiments to estimate
the age-dependent thermostability and also the influence of
orally given EPL of plasma membrane ATPases.
100
Control membranes
~~
lJ
tI
] 70
70
.e
....0
40
0
Fig.
1:
Na~ WMg..'ATPase
Mg':!ATPase
50
100
40
Effect of incubation time at 37
150
50
TOO
min
150
on the activity of Mg ++- and (Na + . K +). ATPases in rat liver
plasma membranes of 80 day old rats. Total activity was taken as 100 %. Averages of three measurements
from each membrane preparation are given. Five animals were used for each membrane preparation
92
Fig. 1 shows the inactivation of the Mg++- and the sodium-potassium dependent ATPase vs. incubation time at 37c in rat liver
plasma membranes from 80-day-old rats. In young animals both the
Hg++- and the (Na+-K+)-ATPase show inactivation after 50 minutes
of incubation. The total enzyme activity was set to 100%. There
is no difference in EPL-treated and untreated animals.
Po lyeny I PC-treated
Control membranes
b
:~ 70
70
litl
o-------<l
__
.....
o
.0
.0
~L~~~~~~~~~~
Fig.
50
100
-r
150
Na~K~Mg'!ATPase
Mg+'!ATPase
50
100
150
min
2:
Effect of incubation at 37 DC on the activity of Mg ++ . and (Na + K +). ATPases in rat liver plasma
membranes of 780 - day old male rats. Averages of three measurements from each membrane preparation
are given. Five animals were used for each membrane preparation. Experimental conditions are described
under Material and Methods and in the legend of Fig. 1.
Different results are obtained with male 780 days old animals
(Fig. 2). In controls, the enzyme activities show a rapid inactivation at the beginning of the incu~ation. The enzyme activity
after oral pretreatment with EPL did not decline during the incubation time which was chosen. In female animals the results are
Polyenyl PC-treated
Control membranes
100
100
-<>--~-~
.0
:~
H
'070
11
.....
0
'0
l'
Fig. 3: Effect of incubation time at 37 0 C on the activity of Mg ++- and (Na + - K + ) - ATPases of 780day - old female rats. Averages of three measurements from each membrane preparation are given. Five animals
were used for each membrane preparation. Experimental conditions are described under Material and Methods
and in the legend of Fig. 1.
93
similar (Fig. 3) but the stabilizing effect of EPL treatment is

smaller. As reported in several investigations, the interrelationship between the physical state of plasma membrane lipids
and ATPase activity depends on cholesterol and on the unsaturated
fatty acid composition.
The spin labeling technique has been shown to provide information
on changes of the physical state of membrane lipids (11). The
stearic acid spin label J (12.3), where the nitroxide group is
located near the polar head group of the molecule, shows anisotropic motion in the lipid core of a membrane. The motion parameter a denotes the mean angular deviation between the nitroxide
2pw orbital axis and the center axis of oscillation of the long
axis ~ of the label molecule. Greater freedom of motion or higher
fluidity of the membrane, respectively, is related with greater
values of a
The label J (1.14) can reflect the membrane fluidity in the more
apolar region of the membrane. For this spin label the rotational
correlation time (lc) as a measure of the motional freedom, can
be calculated. The h~gher the fluidity of a membrane, the smaller
is the value for (1 c ).
Table 4: Changes of the membrane fluidity by EPL in rat liver plasma membranes as a function of age and
temperature as reflected by different stearic acid spin labels incorporated into the plasma membranes. The angular
deviation was calculated for the label J (12,3), and the rotational correlation time 'i:', for the label J (1.14).
Means.:!:. S.E.M., n = 4 - 6
a [ 0]
[ns]
Age
Treat-
Temp.
[ d]
ment
roc]
25
27,0
+ 0,2
2,19 + 0,04
37
29,2
+ 0,35
1 ,58 + 0,05
25
25,93 + 0,22
1 ,78 + 0,03
37
29,02 + 0,02
1 ,46 + 0,02
25
27,00 + 0,09
2,20 + 0,07
37
28,90 + 0,13
1,82 + 0,03
25
27,37 + 0,02
-
2,0
37
29,92 + 0,13
1 ,64 + 0,02
80
80
EPL
800
800
EPL
(12,3)
(1,14)
P = 0,05
+ 0,03
0,01
0,05
94
23
------.
't
EPL 0
PC 100
10
20
90
30
80
70
40
60
,.O...--.J
100
31
,,/'"
~_--------------e
........
,.
~
2
27
1:
EPL 0
TL 100
20
80
40
60
60
40
80
20
100
0
Lipid composition (w/w)--..

Fig,
4:
J ( 12.3)
J ( 12.3)
Effect of essential phospholipids on molecular mobility of spin label

in liposomes. The outer hyperfine splitting Til
or ex. respectively of the spin label probe
is plotted vs. the composition of the liposomes. (PC = dipalmitoylphosphatidylcholin; EPL =
essential phospholipids; TL
=total
lipids of isolated rat liver plasma membranes)
The spin labels were incorporated in isolated plasma membranes

of young and aged EPL treated and untreated animals after exchange from bovine serum albumin.
Table 4 shows that the motion parameter of the label J (1.14)
in which the paramagnetic centre is located whithin the apolar
region of the membrane lipids reflects higher fluidity of the
membrane in young compared with old animals. The treatment of
old animals with EPL provokes a significant increase of membrane
fluidity if compared with untreated controls. The label J (12.3)
which could reflect changes in membrane fluidity near the polar
head groups of the lipid core shows only a small but not significant increase of a between EPL treated and untreated aged
animals.
95
Recently we have performed experiments on liposomes in order to

discover the minimal concentration of EPL in an artifical membrane consisting of dipalmitoyl-phosphatidylcholine or total
lipids of plasma membranes of aged animals which can increase
membrane fluidity to an extent measurable by the label J (12.3).
The outer hyperfine splitting parameter Tn of the spin label
probe (12.3) is plotted versus the composition of the lipid
vesicles (Fig. 4). The hyperfine splitting T, is related to
the freedom of motion of the nitroxide group, greater freedom
of motion being associated with smaller values of T,
The
results show that the critical concentration of EPL which increased the membrane fluidity, reflected by this spin label, is
about 2% EPL of the total lipid weight in liposomes.
DISCUSSION
Summarizing these results we may assume that incorporation of

additional unsaturated essential fatty acids into rat liver
plasma membranes of old animals results in changes of the physical properties of the membranes. In addition, this modified
physical state could appear in response to changes in the nature
of the lipid-protein interactions and in general to changes in
the properties of membrane-bound enzymes.
The results of the experiments on temperature stability show a
time-dependent inactivation of both Mg++- and sodium+potassium
activated ATPase at 37C. The activity vs. time relationship
shows that ATPases from plasma membranes of old animals are more
rapidly inactivated than those of young animals. In all preparations from old animals pretreated with oral EPL, the ATPase
are stabilized. These results may be interpreted in several different ways:
1. The incorporated EPL substances have free radical scavenging
properties and prevent lipid peroxydation and indirect inactivation during the incubation period.
2. The incorporated EPL substance prevents changes in conformation of the ATPases effected by proteolytic or lipolytic
membrane destruction during incubation of the membrane suspension.
3. The EPL substance changes the physical state of the lipids
in the molecular environment of the ATPase to a more fluid
state and therefore influences the stability, possibly the
conformation of the enzyme complex. The exact reason for the
phenomenon of ATPase-stabilization needs further examination.
However these preliminary experiments support the view that
in old animals membrane-bound ATPases can be modified by oral
EPL.
96
REFERENCES
1 -
KIHELBERG H.K. and PAPAHADJOPOULOS D.: Phospholipid requirement for (Na+-K+)ATPase activity. Biochem. Biophys.
Acta 282, 277 (1972)
2 -
van DEENEN L.L.H.: Phospholipide, Beziehungen zwischen

ihrer chemischen Struktur und Biomembranen, Naturwissenschaften, ~, 484 (1972)
3 -
BRINGS R.G., PETERSON B.J., THOHPSON J.H. and SLATER R.B.:

Lack of effect of chronic nicotine administration on fatty
acid distribution in the liver, testis and adipose tissue
of male Fischer-344 rats. J. Lipid. Res. li, 688 (1973)
4 -
ADAMS C.H. and ABDULLA Y.H.: Polyunsaturated phospholipids

and experimental atherosclerosis in G. SCHETTLER (ed.)
Phospholipide, G. Thieme Verlag Stuttgart, 1972, p. 44
5 -
LEKIM D., BETZING H. and STOFFEL W.: Incorporation of

complete phospholipid molecules in cellular membranes of
rat liver after uptake from blood serum. Z. Physiol. Chern.,
353,949 (1972)
6 -
HASSELBACH W.: Unsaturated fatty acids as reactivators of

the calcium-dependent ATPase of delipidated sarcoplasmic
membranes, Eur. J. Biochem. ~, 63 (1973)
7 -
LEKIM D. und BETZING H.: Der Einbau von EPL-Substanz in

Organe von gesunden und durch Galactosamin geschadigten
Ratten. Arzneimittel-Forsch. ~, 1217 (1974)
8 -
COLEMAN R., MITCHEL R.H., FINEAN J.B. and HAWTHRONE J.N.:

A purified plasma membrane fraction isolated from rat liver
under isotonic conditions. Biochim. Biophys. Acta, 135, 573
(1967)
-
9 -
HEGNER D. and PLATT D.: Effect of essential phospholipids on

the' properties of ATPases of isolated rat liver plasma membranes of young and old animals. ~1echanisms Ageing Development i, 191 (1975)
10 - GOLDENBERG A.L., FARIAS R.N. and TRUCO R.F.: Allosteric

transitions and membrane-bound ATPase from rat tissues. The
effect of rat deprivation on the allosteric inhibition by
fluoride. Biochem. Biophys. Acta 291, 489 (1973)
11 - CHAPMAN D.: Biological t.1embranes. Academic Press, New York
1968, p. 125
12 - HEGNER D., SCHUMMER U. and SCHNEPEL G.H.: The interaction
of a lytic peptide melittin, with spin labeled membranes.
Biochem. Biophys. Acta, ~l, 15 (1973)
Essential Phospholipid Therapy in

Dyslipemic States
Effects of Essential Phospholipids

on the Carbohydrate -Induced
Hypertriglyceridemia
H. Ditschuneit. H:-U. Klor and H. H. Ditschuneit
Med. Universitatsklinik. 0-7900 Ulm. Germany
Abstract: Hypertriglyceridemias playa very important role as a

risk factor of atherosclerosis. at least in Westeuropean countries. To assess the therapeutic potency of polyunsaturated phosphatidylcholine or essential phospholipids (EPL) the drug was
tested in the experimental model of carbohydrate-induced hypertriglyceridemias. 10 healthy volunteers were subjected to the
following dietary and therapeutic regimen: normal diet (control).
carbohydrate induction. carbohydrate induction plus EPL (3 g
daily per os). and normal diet + EPL after an intermission of
3 weeks. During the carbohydrate induction the test persons
received 240 g sucrose plus 180 g skimmed milk powder in addition to a normal carbohydrate-~ontaining diet (corresponding
to a daily total intake of 5-8 g carbohydrate/kg body weight).
Plasma triglyceride (TC) and cholesterol (Ch) levels from fasting blood samples werg determined. The lipoprotein fractions
Were isolated by zonal-ultracentrifugation and their lipids
were separated by thin-layer chromatography. After acid hydrolysis and methylation. the fatty acid aompositions of th6 lipoprotein lipids were determined by gas chromatography.
The main results are the following: EPL diminished significantly the carbohydrate-induced rise of plasma triglycerides (TC).
occurring particularly in the VLDL-fraction. The plasma cholesterol levels were lowered by carbohydrate induction and this
tendency was supported by EPL. The fatty acid patterns in the
phospholipids and triglycerides were significantly shifted from
saturated to polyunsaturated fatty acids in VLDL. LDL and HDL
as well due to sole EPL treatment. The ratios of saturated to
polyunsaturated fatty acids during carbohydrate induction plus
EPL were below the control values. though not significantly different in several cases. but they were significantly below the
values measured during carbohydrate induction without EPL. Thus,
EPL is capable of compensating or eVen overcompensating for the
effects of carbohydrate induction on the fatty acid patterns of
phospholipids and triglycerides.
99
INTRODUCTION
Hyperlipoproteinemias count among the most important risk factors

of atherosclerosis. According to recent results of investigations
by BIERMAN and ALBERS (1) and by ROSS and GLOMSET (2) the atherosclerotic process is initiated by proliferation of smooth muscle
cells in the intima and by enhanced formation of mucopolysaccharides, collagen and elastic fibres. This proliferation is promoted
by LDL and by VLDL as could be demonstrated in smooth muscle cell
cultures.
By these investigations the great importance of plasma cholesterol and of triglyceride for the pathogenesis of athersclerosis
was proven in vitro. In our country hypertriglyceridemias play
a very important role as a risk factor (3): In a series of 130
patients with myocardial infarction elevated triglyceride levels
were found in 50%, but pure hypercholesterolemia in only 4% of
the patients. This distribution can not be interpreted as a definite proof for a greater atherogenic potency of elevated triglycerides, because it is caused by the higher incidence of hypertriglyceridemia in the general population.
In the prevention of atherosclerosis equal attention should be
payed to hypercholesterolemia and to hypertriglyceridemia as
well.
An elevation of triglyceride levels can be easily induced in
normals by the administration of a carbohydrate-rich diet (4).
METHODS
In order to assess the triglyceride lowering effect of EPL, we

investigated the influence of 3,0 g of this drug p.o. on the
carbohydrate-induced hypertriglyceridemia in 10 normal volunteers.
The first experimental period without treatment served to find
out the day of maximum triglyceride values.
240 g sucrose and 180 g skimmed milk powder were given daily
in addition to a normal, carbohydrate-containing diet. The
daily intake of carbohydrate varied between 5 and 8 g per kg
body weight. Blood was taken every day before breakfast for
determination of cholesterol according to LEVINE and ZAK (5)
and of triglycerides according to KESSLER and LEDERER (6).
Before and 2 days after beginning of the carbohydrate induction,
the lipoprotein fractions were isolated by zonal-ultracentrifugation and the lipoprotein lipids were separated by thin-layer
chromatography as described by DITSCHUNEIT and KLOR (3). After
acid hydrolysis and methylation, the fatty acid compositions of
100
the phospholipids, the triglycerides and of cholesteryl esters

were determined in a Varian gas chromatograph on a DEGS column
at a constant temperature of 1800 C.
In order to assess the effect of essential phospholipid per se
on the fatty acid composition of lipoprotein lipids, the 10 volunteers received 3 g EPL daily per os under a normal diet 3 weeks
after termination of the last carbohydrate induction. After 5
days of treatment fasting blood samples were withdrawn for fatty
acid analysis.
The total dietary and therapeutic regimen was as follows: control
- 5 d carbohydrate diet (period I) - 16 d intermission - control
(period 0) - 5 d carbohydrate diet (period II) - 12 d intermission
- 4 d EPL-treatment solely - 5 d carbohydrate diet plus EPL-treatment (period III) - 20 d intermission - EPL-treatment solely
(period IV).
RESULTS
The maximum rise of plasma triglyceride was reached on the second day (Table 1). In the second test period the same peak value
was found and more blood was withdrawn on the second day for lipoprotein and fatty acid analysis.
The plasma cholesterol decreased somewhat under the carbohydraterich diet in the first as well as in the second experimental
period (Table 2). Analysis of the triglyceride content of VLDL,
LDL and HDL showed that the increase in plasma triglycerides
was due to an increase of VLDL triglycerides from 26.5 to 50.0
mg% (Table 3). The total cholesterol content is slightly increased in the VLDL and slightly decreased in the LDL (Table 4).
During the third period under the same dietary regimen as before, EPL was given 3 times 1 g daily per os to the same test
persons. The administration of the drug was started already 4
days before the beginning of the third test period in order to
guarantee a maximum therapeutical effect.
This time, the increase in plasma triglycerides on the carbohydrate-rich diet was much smaller than without EPL (Table 1).
While the maximum rise was 49% and 65% excess in the two preceeding control periods and while on day 5 there was still an
increase of 60% as compared to the starting level, the plasma
triglycerides increased to only 33.5% (peak value) and to 8% on
the fifth day under administration of EPL. EPL had no significant effect on plasma cholesterol (Table 2).
Similar changes could be observed by separate determination of
the triglyceride content in VLDL, LDL and HDL. In the control
period the VLDL triglycerides increased by 90% (Table 3), while
under EPL-treatment there was only a 55% rise (Table 5). The
cholesterol content of the different lipoprotein fractions showed
no change under the EPL-treatment (Table 6).
50
80
38
40
55
50
85
70
73
40
53
38
10
35,7
II
III
IV
VI
VII
VIII
IX
10
38
43
81,6
10
55
70
70
27
105
90
103
78
73
55
93,0
10
73
110
90
70
115
105
108
63
98
98
86,6
10
53
78
73
68
110
130
98
73
105
78
83,3
68
70
70
105
85
102
83
79,9
10
58
50
63
80
100
98
85
65
100
60
50
105
75
118
130
88
75
150
65
9
90
10
91,9
103
128
84,0 -
72
83
113
78
60
85
88
100
68
105
95
95
90
55
113
75
85
88
115
103
109
75
77,5 79,6 103,5
10
48
90
88
75
95
100
50
63
78
68
4,9 4,9 6,1 5,5 3,9 6,3 5,7 4,9 5,8 7,4 5,7 5,4 5,18,4 7,8 6,4 5,4-
71,2 56,3
43
108
53
45
80
55
68
73
35
55
72
98
73
50
65
78
88
SEM
82,0
10
73
103
83
65
90
80
93
63
85
25
Period III
15,7 15,3 19,2 17,5 12,4 18,5 17,815,6 18,3 23,3 15,3 17,1 16 ,0 19 , 9 2 3 ,8 30,2 16 , 2 -
83,8
10
10
89,1
80
91
83
63
113
65
83
60
98
100
75
110
75
123
68
108
93
73
75
93
Period I
62,8
10
53
58
73
83
63
50
80
53
Patient
No.
Period II
Plasma triglyceride levels Img %) of 10 normals under 2 periods ( I + II) of carbohydrate induction (5- 8 9 / kg BW / day) and under a third period
with additional 3 9 EPL per os daily
Table 1:
170
141
10
175
178
150
10
176
22,1 23,3 21,9 21,3 20,4 18,7 21,3 20,7 26,3 28,521,320,6 22,o 19,5 28,9 23,1 21,2 -
VIII
IX
SD
SEM
162
163
178
VII
160
10
143
160
154
193
10
174
154
152
158
245
153
10
134
152
144
153
192
156
10
136
156
170
162
193
169
167
10
152
143
176
196
205
159
162
10
141
147
150
151
207
173
167
10
146
142
154
157
221
178
155
151
10
136
121
143
125
108
154
169
148
10
121
135
144
152
193
176
162
138
10
132
123
128
170
180
135
157
153
138
130
132
147
10
122
154
140
132
141
10
125
131
122
137
185
159
148
151
_~'~~'~ _ 6,9
159
129
150
143
216
131
152
151
196
158
172
168
192
172
6,~ _~,5
164
10
154
160
159
156
216
175
159
6,9 8,6 6,9 6,7 6,4 5,9 6,7 6,5 8,4 9,o 6,7 6,5
170
175
209
235
172
171
179
-I
224
197
190
152
163
VI
160
165
160
133
176
158
177
156
193
198
198
145
107
150
130
137
132
117
119
152
126
151
137
160
164
150
102
IV
162
201
157
120
177
150
116
Period III
184
146
172
183
145
146
III
141
190
180
157
131
Period II
157
119
158
165
II
123
Period I
172
156
Patient
No.
Table 2: Plasma total cholesterol (mg %) of 10 normals under 2 periods ( I + II ) of carbohydrate induction (5 - 8 9 / kg BW / day) and under a third period
with additional 3 9 EPL per os daily
103
Table 3: Effect of a carbohydrate rich diet (58 9 / kg BW / day) on triglycerides (mg %) in VLDL,
LDL and HDL of 10 normal volunteers
Period II
Patient
No.
Initial value
Peak value
VLDL
LDL
HDL
VLDL
LDL
HDL
45,0
12,5
3,25
62,5
16,25
6,25
II
18,75
18,75
4,5
66,25
21,25
6,25
III
12,5
23,75
2,5
16,25
23,75
4,5
IV
40,0
17,5
4,5
63,75
23,75
8,25
60,0
17,5
3,25
53,75
17,5
5,75
VI
13,75
32,5
2,5
60,0
30,0
5,0
VII
23,75
11 ,25
2,0
35,0
VIII
30,0
16,25
2,0
45,0
17,5
3,75
18,75
3,25
66,25
18,75
6,25
5,75
31,25
17,5
3,25
10
10
50,0
19,5
5,3
17,4
5,7
1,54
IX
8,75
10,0
15,0
10
10
26,25
18,4
SD
17,2
6,1
10
3,35
1,2
8,75
3,75
10
104
Table 4:
Effect of a carbohydrate rich diet (58 g / kg BW / day) on total cholesterol (mg %) in
VLDL, LDL and HDL of 10 normal volunteers
Patient
No.
Period II
Peak value
Initial value
VLDL
LDL
HDL
VLDL
LDL
HDL
11,00
82,50
12,00
13,00
79,00
11,00
II
8,00
75,00
24,00
24,50
71,50
17,75
III
3,00
113 ,50
15,25
6,50
103,00
21,25
IV
15,50
105,50
19,50
18,50
104,00
24,25
17,00
108,00
10,75
21,00
130,00
15,75
VI
9,50
124,00
10,25
14,50
123,00
14,75
VII
2,00
100,00
5,50
11,50
38,00
14,50
VIII
6,50
26,00
12,00
13,50
82,50
16,50
IX
3,00
80,50
24,50
17,00
63,50
20,00
8,50
62,50
44,50
13,00
68,50
14,75
10
10
10
10
10
9,10
94,80
17,80
15,30
86,30
17,10
4,60
19,20
11,20
5,10
28,40
3,86
x
SD
10
105
Table 5: Effect of carbohydrate induction (5 - 8 9 / kg BW / day) in the presence of 3 9 EPL per day on
the triglycerides (mg %) in VLDL, LDL and HDL of 10 normal volunteers
Patient
No.
Period III
Initial value
VLDL
LDL
Peak value
HDL
VLDL
LDL
HDL
33,15
13,75
3,25
77 ,5
11 ,25
6,25
II
45,0
12,5
8,75
52,5
17,5
8,75
III
27,5
31,25
3,25
53,75
26,25
5,75
IV
33,75
13,75
5,0
22,5
20,0
7,5
60,0
16,25
6,25
58,75
18,75
3,75
VI
40,0
28,75
7,5
58,75
32,5
7,5
VII
33,75
17,5
5,75
38,75
17,5
7,5
VIII
46,25
16,25
7,0
72 ,5
15,0
5,5
IX
36,25
16,25
7,5
78,75
18,75
8,75
21,25
13,75
6,25
62,5
11,25
8,75
10
10
10
10
37,75
17,0
58,6
18,9
10
6,05
10
6,95
SD
10,8
4,8
1,8
18,9
6,5
1,7
SEM
3,4
1,5
0,57
5,9
2,1
0,54
106
Table 6: Effect of carbohydrate induction (5 - 8 9 / kg BW / day) in the presence of 3 9 EPL per day
on the total cholesterol (mg %) in VLDL, LDL and HDL of 10 normal volunteers
Period III
Patient
No.
Peak value
Initial value
VLDL
LDL
HDL
VLDL
LDL
HDL
8,0
94,0
12,5
19,0
71,0
22,5
27,5
65,5
30,0
22,5
53,0
36,0
III
9,5
125,5
18,25
16,5
98,5
24,5
IV
10,5
29,5
23,75
7,0
88,5
53,0
18,5
111,0
18,0
19,5
93,5
13,7
VI
17,5
180,0
24,0
17,5
110,5
26,0
VII
13,0
102,0
20,25
21,5
80~5
26,5
VIII
15,0
99,0
25,0
21,0
84,5
23,0
IX
12,5
90,0
25,5
24,0
24,0
25,0
10,0
72,0
37,75
18,0
61,0
37,75
10
10
10
10
10
10
x-
14,2
103,0
23,5
18,65
82,5
29,1
SD
5,8
32,0
7,0
4,7
17,2
10,8
SEM
1,8
10,1
2,2
1,45
5,4
3,4
I
II
107
The results of this investigation demonstrate that a daily oral

dose of 3 g EPL significantly diminished the carbohydrate-induced
rise of plasma triglyceride in normals. Gas chromatographic
analysis of the fatty acids of lipoprotein lipids showed significant changes as well.
Under carbohydrate induction the palmitic acid of the VLDL phospholipids increased from 31.6 to 34.7%, while the other fatty
acids remained at the same level. Under EPL treatment linoleic
and arachidonic acid increased and palmitic and stearic acid
decreased. Carbohydrate induction markedly diminished this effect
of EPL, althouqh linoleic and arachidonic acid still remained
significantly elevated (Table 7).
The changes in the fatty acid pattern became more obvious when
the ratio of saturated to polyunsaturated fatty acids was calculated. This ratio increased during carbohydrate induction from
1.96 to 2.10, decrease under EPL treatment to 1.12 and rose again
under EPL plus carbohydrate induction to 1.6.
The fatty acid pattern of the phospholipids in the LDL also
changed under carbohydrate induction plus EPL. In this fraction,
also a marked increase in linoleate content could be demonstrated
which was almost abolished by carbohydrate-rich diet. The ratio
of saturated to unsaturated fatty acids was 1.98 under a normal
diet, rose up to 2.03 under carbohydrate induction, decreased
under EPL to 1.68 and increased again under carbohydrate induction plus EPL to 1.89, which is still below the starting value.
Thus, the effect of EPL on the fatty acid pattern of the LDL
phospholipids resembles that on the VLDL phospholipids (Table 8).
Principally the same changes could be observed in the fatty
acid pattern of HDL phospholipids (Table 9). The ratio of saturates to polyunsaturates showed a decrement under EPL from 2.2
to 1.46, while the carbohydrate induction brought this ratio
almost back to the starting level.
The fatty acid composition of the cholesterol esters in VLDL,
LDL and HDL was only minimally changed during the test diet and
the EPL-treatment (Tables 10-12). Nevertheless, EPL increased
the linoleate content in the cholesteryl esters of VLDL, LDL
and HDL, while the ratio of saturates to polyunsaturates remained almost the same.
The fatty acid pattern of the triglycerides in VLDL, LDL and HDL
was most markedly altered (Tables 13-15). Carbohydrate induction
led to a decrease of polyunsaturated fatty acids and thereby induced an increase of the ratio of saturates to unsaturates in
the VLDL from 2.03 to 2.34, in the LDL from 1.02 to 2.37 and in
the HDL from 2.16 to 3.06. EPL administration caused an increment
in the linoleate and a decrement in the oleate and palmitate content such that the ratio decreases to 1.76 in the VLDL, to 1.45
in the LDL and to 1.76 in the HDL. This shift in favor of the
polyunsaturates was diminshed by simultaneous carbohydrate induction, although the initial ratios were not reached in all
lipoprotein classes. Therefore, a more favorable ratio than
108
Table 7:
Fatty acid pattern of VLDL - PL
Weight , fatty acids
Treatment
P
Diet EPL
14:0
1411
16:0
1611
18:0
1811
1812
20:3
20:4
::!:SEH
1.5
0.3
0.8
0.1
31.6
2.4
2.3
0.4
19.9
1.0
16.9
1 1
19.4
1 1
1.7
0.2
4.7
0.6
0.5
0.1
0.3
0.1
34.7
0.4
19.9
1.5
5.4
1.8
0.2
1.3
17.7
1.1
19.0
::!:SEM
1.1
0.3
0.6
0.4
0.1
0.3
0.1
26.6
1.2
0.3
0.0
16.9
1.2
16.3
0.8
31.3
1.5
0.4
0.4
5.9
0.8
0.7
0.1
0.5
0.1
32.2
2.7
0.8
0.2
18.1
0.5
15.8
1.0
21.9
1.3
0.9
0.4
7.0
0.8
NS
NS
NS
NS
- - ,
II
- ,
IV
,
::!:SE:l
III
,
:!:SEM
Comparison
(p. value)
0
II
IV
II
III
Table 8:
IV
III
0.005 0.005 0.005 NS
0.005 NS
0.005
0.025 0.005 0.005 0.005 0.005 0.005 0.005

1410
14:1
1610
16: 1
18:0
1811
1812
20:3
20:4
1.0
!SEH 0.2
0.6
0.1
34.8
0.9
1.6
0.2
18.9
1.0
15.6
0.8
19.9
0.8
1.8
0.2
3.8
0.4
- - ,
II
0.005 0.005 NS
Fatty acid pattern of LDL - PL
Treatment
P
Diet EPL
0
0.5
0.1
0.3
0.3
0.2
18.3
0.9
19.2
0.0
37.7
2.0
14.9
:!:SI::H
0.7
1.2
2.0
0.4
5.0
0.6
0.2
0.4
0.0
31.3
1.5
0.8
0.1
19.6
13.5
0.7
24.2
0.7
0.9
0.4
5.1
0.9
0.6
37 .1
0.9
16.5
15.0
20.3
1.8
4.7
0.7
0.5
1 1
!SEa
+
,
:!:SEH
0.1
0.9
0.3
0.2
1.1
0.1
0.2
0.6
0.6
Comparison
(p. value)
0
II
IV
II
III
0.005 0.005 NS
NS
NS
NS
0.005 NS
0.005 0.005 0.005 0.00
NS
0.005 0.005 NS
NS
0.025 NS
0.005
NS
109
Table 9:
Fatty acid pattern of HDL - PL
Weight , fatty aCids

1410 14:1 1610 16: 1
18:0
18:1
18:2
20:)
20:4
0.9
0.1
0.8
0.1
34.2
2.)
1.0
0.1
20.5
1.5
17.5
0.6
20.5
1.5
0.9
0.2
).)
:!:SEM
!SE~l
0.5
0.0
0.4
0.0
36.4
0.7
0.1
0.1
15.8
0.7
15.8
0.)
20.1
0.7
2.7
0.2
6.4
0.4
!SEH
0.3
0.0
0.5
0.1
31.0
1.7
0.9
0.1
18.2
0.9
15.)
0.6
26.0
1.3
0.6
0.2
5.8
0.7
+SEI\
0.4
0.1
0.3
0.1
35.8
1.2
0.8
0.1
20.0
1.2
14.7
0.5
19.0
0.9
0.8
0.5
5.2
0.5
Treatment
P
Diet EPL
- - ,
- ,
II
IV
III
0.5
Comparison
(p. value)
0
II
IV
II
III
TablelO:
- - ,
- ,
NS
0.005 0.005 0.005 0.005 0.005
0.005 0.005 0.005 0.005 0.005 0.005
IV
III

1411 1610 16:1 18:0
14:0
1811
1812
2014
:!:SEM
4.4
0.8
2.9
0.5
12.)
2.2
6.)
0.5
2.0
0.4
23.6
1.3
42.1
2.4
6.4
1.2
:!:SEII
2.0
0.2
0.7
0.1
14.6
0.7
5.4
0.7
1.5
1.1
27.9
1.2
42.7
1.5
4.2
0.9
3.0
:!:SEII 0.2
1.6
0.1
11.4
0.7
7.0
0.4
1.1
0.0
23.9
1.8
44.5
2.4
6.0
0.7
1.4
0.3
12.0
0.9
6.1
0.7
0.9
0.2
24.7
1.1
45.5
2.4
6.9
1.6
II
0.005 NS
0.005 0.005
Fatty acid pattern of VLDL - CE
Treatment
P
Diet EPL
0
0.005 0.005 0.005 0.005 NS
2.5
:!:SEM 0.3
Comparison
(p. value)
0
II
IV
II
III
0.005 0.005 NS
!liS
0.005 NS
0.005 0.005 NS
0.005 NS
NS
0.005
0.025 NS
0.005 0.005 0.005
110
Table 11: Fatty acid pattern of LDL - CE
Treatment
P
Diet EPL
1410
1411
1610
16:1
1810
1811
1812
2014
1.5
0.2
0.4
0.0
15.6
1.3
4.1
0.6
0.3
0.0
24.6
0.8
50.9
1.8
2.6
0.6
!:SE/I
1.5
0.2
0.6
0.1
15.0
1.0
4.6
0.4
1.7
0.1
27.8
1.5
46.8
2.3
2.0
0.9
!:SEl4
2.5
0.5
1.2
0.3
12.9
1.3
8.9
0.7
0.2
0.1
27.0
1.2
42.8
3.3
1.1
1.0
:!:SEI4
1.5
0.2
1.6
0.3
13.7
0.6
5.7
0.7
0.2
0.1
22.9
1.3
50.5
1.7
3.9
0.5
NS
NS
0.005 0.005 0.005 NS
- - ,
!:SEI-t
II
IV
III
Comparison
(p. value)
0
II
IV
II
III
0.005 0.005 0.005 0.005 0.005 0.005

0.005 0.005 0.005 0.005 0.005 0.005
Table 12: Fatty acid pattern of HDL - CE
Treatment
P
Diet EPL
IV
III
1411
1610
16.1
1810
1811
1812
2014
!:SEM
3.4
0.6
1.2
0.2
19.0
1.7
4.7
0.7
0.9
0.2
28.5
2.2
39.1
3.6
3.2
0.8
!:SDI
2.0
0.6
1.0
0.4
20.7
2.4
6.5
0.9
0.5
0.1
27.9
2.2
37.5
4.5
3.9
0.5
!:SE11
2.3
0.3
1.2
0.2
14.5
1.8
9.1
0.6
0.3
0.0
27.5
2.1
41.9
3.8
3.2
0.2
1.9
0.4
1.0
0.3
21.0
2.5
5.1
1.2
3.1
0.7
22.0
1.2
44.6
4.0
1.3
0.2
NS
0.005 0.005 NS
NS
NS
0.005 0.005 0.005 NS
NS
NS
- ,
- ,
II

14.0
,
:!:SEH
Comparison
(p. value)
0
II
IV
II
III
NS
0.005 0.005 0.005 0.005 0.005
111
Table 13:
Fatty acid pattern of VLDL - TG
Treatment
p
Diet EPL
,
+SJ:;~I
II
IV
III
,
+SJ::M
,
+SJ:;N
%
:!:SEI!
14:0
14: 1
1610
16: 1
1810
1811
1812
2014
1.7
0.4
0.8
0.3
27.1
1.4
4.1
3.7
0.5
46.6
1.5
16.0
0.7
0.0
0.5
1.4
0.3
0.6
0.1
26.6
0.9
6.7
0.9
2.3
0.4
49.5
2.0
12.6
0.8
0.3
0.2
1.5
0.4
0.9
0.1
26.4
1.8
7.6
1.2
3.3
0.6
42.6
2.2
16.1
1.8
1.6
0.2
2.7
0.8
0.4
0.1
27.8
3.8
4.3
0.5
6.9
0.8
37.6
2.7
19.4
2.5
0.9
0.3
NS
0.005 0.005 0.005 0.005 NS
NS
0.005 NS
NS
0.005 0.005 0.005 0.005 0.005
0.0
COr:lparison
(p. value)
0
II
IV
II
III
Table 14:
- -
II
IV
III
0.005 NS
0.005
Fatty acid pattern of LDL - TG
Treatment
P
Diet EPL
0

14: 1 1610 1611 18:0
14:0
18:1
18:2
20:4
1.5
0.1
0.3
0.0
26.5
1.3
2.9
0.1
2.7
0.3
50.9
1.3
15.2
1.6
0.0
0.0
:!:SEM
1.7
0.4
0.6
0.1
24.5
1.6
5.8
0.9
4.4
0.7
50.1
1.7
11.8
1.7
1.1
0.9
:!;SEM
1.7
0.2
0.4
0.1
22.4
1.3
5.5
0.7
4.8
0.5
45.5
1.3
17.2
0.9
2.6
0.4
1.0
0.1
0.2
0.0
23.2
1.4
5.8
0.7
4.4
0.7
45.9
2.7
18.8
1.9
0.7
0.2
NS
0.005 0.005 NS
+SEl-I
%
,
,
+SE."i
COMparison
(p. value)
0
II
IV
II
III
0.005 0.005
0.005 0,005 0.005 0.005 0.005 0.005

NS
NS
NS
0.005 0.005 NS
112
Table 15:
Fatty acid pattern of HDL - TG

14: 1 16:0 1611 18:0
Treatment
P
Diet EPL
- ,
- ,
II
IV
III
14:0
1811
1812
20:4
0.0
0.0
:!:SEM
2.1
0.6
0.7
0.1
27.7
1.3
3.7
0.4
3.8
0.7
46.5
1.5
15.5
1.6
:!:SEM
2.0
0.3
0.7
0.1
28.3
1.5
4.8
0.3
4.6
0.4
48.2
3.1
10.9 0.5
1.0
0.1
2.3
0.2
0.9
0.1
21.9
1.1
8.5
0.9
6.1
0.6
43.1
1.9
15.8
1.2
1.4
0.5
2.7
0.8
0.4
0.1
27.3
3.8
4.3
0.5
6.9
0.8
39.4
2.7
18.1
2.5
0.9
0.3
NS
0.005 0.005 NS
,
:!:St!1
,
:!:SEM
Comparison
(p. value)
0
II
IV
II
III
0.005 0.005
0.005 0.005 0.005 0.005 NS

NS
NS
0.005
0.005 0.005 0.005 0.005
under a normal diet was reached, even though a carbohydraterich diet was given. The simultaneous administration of EPL under
carbohydrate induction led to a ratio of 1.48 in the VLDL, of
1.46 in the LDL and of 1.94 in the HDL.
DISCUSSION
Ingestion of more than 7 g of carbohydrate per kg body weight

leads to an increase in plasma triglycerides and VLDL levels
even in normals (4,7). According to LEES and FREDRICKSON (4) the
triglyceride levels are almost doubled under carbohydrate-rich
diets consisting mainly of sugar. Although we gave comparable
amounts of carbohydrate (5-8 g daily) the triglyceride levels
did not rise that rouch in our experiment. Two reasons may account
for the lesser extent of carbohydrate induction. We did not administer formula diets but rather a carbohydrate supplement to
a relatively normal diet. This may have resulted in less fat
restriction than in the experiments of LEES and FREDRICKSON (4).
In addition to that, we did not continue the experiment for several weeks as was the case in the carbohydrate inductions of
113
other authors. The maximum rise observed on the 2nd day may only
be a first, relatively low peak which might be followed by a
still higher rise in plasma triglyceride after a week or two.
Nevertheless, in our experiment as well, the most characteristic
change induced was a marked increase in VLDL concentration. Thus,
even a short term carbohydrate induction period in normals seems
to be a suitable model for testing the effectiveness of triglyceride lmlering drugs.
In our experiment 3 g EPL p.o. daily were able to reduce the
maximum rise in plasma triglyceride from 60% to 33% after only 6
days of drug administration (4 days before the onset of the induction period and another 2 days until the maximum rise was
reached). The maximum rise in VLDL triglyceride was 55 instead of
90% under the EPL-treatment.
There are several possibilities to explain the effect of EPL on
the triglyceride levels. Essential fatty acids decrease plasma
triglyceride and cholesterol in both normals and hyperlipemic
patients. Triglyceride production and, thereby, VLDL synthesis
in the liver is reduced when the dietary linoleate rises. The
reason for this is that linoleate is preferentially incorporated
into phospholipids thus reducing the fatty acid pool available
for triglyceride formation in the liver (8). In our experiments
carbohydrate induction produced marked changes in the fatty
acid pattern of the lipids in plasma lipoproteins. In all lipid
classes, but most markedly in the triglycerides, an increase in
palmitic, stearic and oleic acid and a concomitant decrease in
linoleic and arachidonic acid was observed. This shift from the
unsaturated towards the saturated acids which paralleled the rise
in plasma triglyceride was abolished by the adimistration of EPL.
The ratio of saturated to polyunsaturated fatty acids was about
the same before the carbohydrate induction and after carbohydrate
induction in the presence of EPL. Thus, the favorable ratio of
saturated to unsaturated fatty acids in the plasma lipids under
EPL treatment seems to be related to the decrease in VLDL triglyceride production in the liver, i.e. it seems to reflect the
shift of the incorporation of liver fatty acids from triglyceride
into phospholipids which was demonstrated by NESTEL and BARTER
(8)
On the other hand, BAGDADE et al. (9) have shown that a diet
rich in linoleate is capable of increasing the removal of VLDLtriglycerides by activating the lipoprotein lipase system. As
was shown by BLATON and PEETERS (10), EPL exhibits a similar
stimulating effect on the activity of lipoprotein lipase. The
combined effect of EPL on both triglyceride production and removal may therefore account for its triglyceride lowering potency.
The cholesterol lowering effect of EPL which has been shown by
PEETERS et al. (11) could not be evaluated in our experimental
model, because the carbohydrate induction per se resulted in a
significantly lowered cholesterol level.
114
REFERENCES
- BIERMAN E. and ALBERS J.: Biochim. Biophys. Acta, 388,

198-202 (1975)
2
- ROSS R. and GLOMSET J.: Science 180, 1332-1339 (1973)
- DITSCHUNEIT H.H. and KLOR U.: Dietrich Steinkopf-Verlag,

Darmstadt, 1973, S. 79
- LEES R.S. and FREDRICKSON D.S.: Clin. Res.
- LEVINE J. and ZAK P.: Clin. chim. Acta,
- KESSLER G. and LEDERER H.: Automation in Analytical

Chemistry, New York, p. 341, 1965
- GLUECK C.J., LEVY R.I. and FREDRICKSON D.S.: Diabetes

739 (1969)
- NESTEL D. and BARTER W.:
- BAGDADE J.D., HAZZARD W.R. and CARTIN J.:

1020 (1970)
Metabolism~,
1l,
~,
327 (1965)
381 (1964)
~,
1 (1970)
Metabolism~,
10 - BLATON V. and PEETERS H.: 3rd International Atherosclerosis

Congress; G. SCHETTLER, A. WEIZEL edts., Springer, Berlin,
1974, p. 565
11 - PEETERS H., BLATON V., SOETEWEY F., DECLERCQ B. and
VANDAMME D.: MUnch. Med. Wschr. 115, 1358 (1973)
Influence of EPL on Lipolysis

in Vitro and in Vivo
K. Szyszka
Department of Pharmacology. Pomeranian Medical Academy. Szczecin. Poland
Abstract: Mechanisms of the lipolytic activity of essential phospholipids (EPL) were studied by incubating samples of epididymal
adipose tissue from WISTAR rats with EPL or/and norepinephrine
and/or theophylline in a Krebs-solution (pH 7.2, T = 37 o C). The
levels of free fatty acids and of glycerol were measured in the
tissue and in the incubation medium, the ATP content of the
tissue was determined and the cAMP was isolated.
EPL (10- 6 -10- 3M) did not influence the basic spontaneous lipolytic activity in adipose tissue, but inhibited - dependent on
dose - the increase of lipolytic activity induced by norepinephrine (10- 6 -10- 4 M), or theophylline (3xlO- 3M) or dibutyryl
cAMP (10-3M). EPL reduced the decrease of ATP content in adipose
tissue caused by norepinephrine or theophylline. Further, EPL
inhibited the increase of cAMP synthesis elicited by norepinephrine or norepinephrine + theophylline.
The lipolytic activity of EPL was studied also in vivo by administering EPL (750 mg/kg, i.p.) to rats 120 or 240 min before
sacrificing them. Further, the effect on the stimulation of lipolysis was investigated by additionally injecting 0.5 mg/kg
norepinephrine i.p. 30 min before sacrificing.
EPL markedly increased the lipolytic activity in vivo as measured
by the levels of free fatty acids and of glycerol in the blood
serum. Also an inhibition of the increased lipolysis due to the
action of norepinephrine was observed.
From the discrepancy in the effects of EPL in vitro and in vivo
it is hypothesized that EPL activates the lipoprotein lipase
and the hepatic lipase, but inhibits the hormone-sensitive lipase in adipose tissue.
INTRODUCTION
Adipose tissue is a simple and effective system for studying the

action of hormones and drugs in lipolysis (1). Incubation of adipose tissue in vitro with lipolytic hormones (noradrenaline, NEi
or dibutyryl cyclic M4P, cAMP) results in an increase of the
rate of release of free fatty acids (FFA) and glycerol (2).
116
It has been shown that the action of lipolytic hormones in adipose tissue is associated with an increase in the synthesis of
cyclic AMP, and that inhibitors of phosphodiesterase (e.g. theophylline) can mimic the action of the lipolytic hormones (3,4).
Increase of cAMP concentration occurs parallel to rapid ATP consumption.
ATP is nesessary in the lipolytic process not only for the synthesis of cAMP, but also for conversion of inactive lipase into
its active form (2,5). HmvARD et al. (6) disclosed, after intravenous injection of polyunsaturated phosphatidylcholine (EPL;
NATTERMANN) an increase in the activity of lipase in the blood
plasma and in the liver of rabbits.
For these reasons, the effect of EPL on lipolysis and on cAMPas well as ATP-Ievels in adipose tissue in vitro and the influence on lipolysis in vivo were studied. Further the effect
of EPL on lipolysis induced by NE, theophylline, and dibutyryl
cAMP was investigated.
METHODS
1.
Effect of Lipolysis in vitro
Epididymal fat was obtained from fed WISTAR rats (200-250 g)

under ether anaesthesia. The fat pads were immediately divided
into pieces of 100 10 mg and placed into a Krebs-solution
(pH 7.2, bicarbonate buffer) containing 2.5% 80vine albumin
(fraction V, Sigma), and were incubated at 37 C for 150 min in
a metabolic shaker. EPL dissolved in 95 0 ethanol was added to
the incubation. The same volume of ethanol (0.05 ml) was added
to the control samples. The presence of ethanol did not alter
the spontaneous lipolysis. At the end of the incubation period
0.1 ml of 2.5 N H2S04 was introduced into each assay to stop
the reaction. Free fatty acids (FFA) were determined in the incubation medium, accoroLng to DOLE (7). Glycerol was determined
by the enzymatic method of EGGSTEIN (8).
In a further set of experiments the effect of EPL on the kinetics of lipolysis was measured. Samples of 200 10 mg of epididymal fat from normally fed rats were incubated in a metabolic
shaker at 37 0 C in 2 ml of a Krebs-solution (pH 7.2, bicarbonate
buffer) containing 3.5% bovine albumin. After 30 min incubation
the norepinephrine (NE) or theophylline (TP) was added.
At intervals of 0, 30, 41, 60 and 90 min after the beginning
of the experiments, the levels of FFA and glycerol were determined in the medium according to (7,8) and the ATP content in
the adipose tissue was measured with the aid of lucipheril-Iucipherase (Sigma) liquid scintillation counting.
117
The influence of EPL on the content of cyclic adenosine monophosphate (cAr-iP) in adipose tissue was measured as follows: Fat
pads (200 + 10 mg) removed from rats were pooled for random
distribution, weighed and placed into 1.90 ml of a Krebs-solution
(pH 7.2, bicarbonate buffer) containing 2.5% bovine albumin
plus EPL. After incubation in a metabolic shaker at 37 0 C for
30 min, norepinephrine and/or theophylline were added (each
dissolved in a volume of 0.05 ml of 0.9% NaCl) and the assays
were further incubated for 11 min at 37 o C. At the end of the
incubation period, tissue and medium were immediately separated
by vacuum filtration. cAMP from the tissue and the medium was
isolated according to the method of FASSIHA et al. (9).
2.
Effect of EPL on Lipolysis in vivo
The rats were pretreated with a single dose of 750 mg/kg EPL i.p.
120 or 240 min before sacrificing, and 30 min before sacrifice
the lipolysis was stimulated by an i.p. injection of 0.5 mg/kg
norepinephrine. EPL was injected as a 10% suspension in 0.9%
NaCl, and NE in saline solution.
In a further experiment, rats fasting for 24 hours were injected
750 mg/kg EPL i.p. (10% saline suspension) 120 min before sacrifice. The following serum parameters were determined:
FFA according to DOLE (7), glycerol according to EGGSTEIN (8),
cholesterol according to WATSON (10), triesterified fatty acids
(TEFA, practically triglycerides), by the method of COMERTY et
al. (11), and glucose by the GOD enzymatic method according to
SCmlIDT et al. (12) using the BOEHRINGER test set.
RESULTS
1.
Effect of EPL on Lipolysis in vitro
The release of free fatty acids and of glycerol as two indicators of the lipolytic activity in adipose tissue was measured
with various concentrations of EPL present in the incubation. A
similar set of experiments was performed after hormonal stimulation of lipolysis. The results are summarized in Figs. 1 and
2: EPL (after 150 min incubation) did not influence the spontaneous release of free fatty acids, but exerted an inhibitory
effect on the spontaneous glycerol release. EPL depressed - increasingly with dose - the stimulated release of both FFA and
glycerol induced by the action of NE, TP and dibutyryl cAMP as
well.
The influence of EPL in the kinetics of spontaneous and stimulated release of FFA and of glycerol is shown in Fig. 3. There
were no significant deviations from the controls caused by EPL
as such. However, EPL depressed the stimulatory effect of NE
and of TP.
118
~lill 0ED 00 ::ll!l 0DDD

60~
10
100
1000
EPl.Theophyliine
EPL+ NE (10)
10
100
1000
EPL+NE + Theophylline
Dono !DDoo
40
40rn
20
NE
20
10
100
0'"-...."..0....
1000
EPL+ NE (100)
:~Pl' Dibutyryl
=lU DOn
o
____
__
~~~~_L_
10
100
10
1000
5D
100
1000
100
1000
EPL - - . .
Fig. 1: Influence of EPL on lipolysis in epididymal adipose tissue in vitro: Release of FFA (JUEq / g fresh
tissue) into the incubation medium vs. concentration of EPL (f-JM ).
NE:
10 jUM and 100 ,AIM, TP:
3 mM, DBcAMP:
:~ ~Pl' Th~phy~ne ~
EPl
l
0.4lll
0.2
o.SUl
o
...
III
c:>
000
10
100
EPL + NE (10)
1000
0 . 2 l l loPh.
10
100
O.1W~p
NE
~::lR
1000
:~ n~' DibutyrYolcAMP
0.3
0.1
5 mM
100
1000
EPL-'
EPL+ NE
(100)
03
0.1
10
100
1000
EPL -+Fig.
2:
Influence of EPL on lipolysis in epididymal adipose tissue in vitro: Release of glycerol (tuM / I / g
fresh tissue) into the incubation medium vs. concentration of EPL (jUM ). Details see Fig. 1
119
Control
28
QJ
::J
III
III
TP 3mM
NE lO11M
EPL lmM
EPL+TP
_ ........ EPL+NE
24
20
16
.c
....
III
01
cr
/;'/
//7./
b _..../
~/
12
:l.
......~--"c(.
<f
u.
u.
(a)
0
60
90 min
60
90 min
0.5
0.3
o
....
QJ
U
>.
C>
0.1
( b)
Fig. 3: Kinetic effect of EPL on lipolysis in vitro: Release of FFA (jUEq / g fresh tissue) and of glycerol
( JlM / I / g fresh tissue ) vs. time
The influence of EPL (1, 0.1, 0.01 roM) on the NE-induced stimulation of lipolysis was studied in more detail by measuring the
changes of relative FFA release depending on the concentration
of NE present in the incubation (Fig. 4). At concentrations of
NE below 1 jUM a slight stimulation of lipolysis by EPL was observed independent of dose. At higher concentrations a marked
dose-depending depression of the stimulation of lipolysis caused
by EPL was measured.
120
0/0
90
80
70
OJ
g> 60
+-'
C
OJ
OJ
0...
LL...
.,
50
40
30
20
10
o.m
0.1
10
100
Concentration of noradrenaline [/.tMJ

Fig.
4 : Dependence of noradrenaline induced lipolysis in vitro on the concentration of EPL : Percental
release of FFA into the incubation medium
The ATP content in adipose tissue was reduced during stimulated

lipolysis as compared with the respective control values at the
different measuring intervals . Taking these control values at
spontaneous lipolysis as 100%, the ATP content was reduced to
about 40% after 60 min and to about 25% after 90 of incubation
under the experimental conditions chosen (Fig. 5).
The effect of EPL on the content of cyclic AMP in adipose tissue
is depicted in Fig. 6. A depression by 18% and by 46% of the increases in cAMP induced by NE or by NE plus TP, respectively, was
observed, while there was no significant change caused by EPL
alone.
2.
Effect of EPL on Lipolysis in vivo
A marked increase of free fatty acids or glycerol in the blood

serum of rats was observed after i.p. injection of EPL.
A decrease of the NE-induced rise of lipolytic activity due to
EPL occurred after 2 h, no effect could be detected after 4 h,
and an additional stimulation of lipolysis was seen after 24 h
for rats in the fasting state (Fig. 7).
121
140
120
Control
100 I------~------.;..;......;..--
................
80
;-a..
TP 3mM " .
NE lOpM .......... " . ,
60
EPL+ TP
............... ... _.~PL lmM
... _ ._-
.......... ..........
'
..... ..... "
............. ....
~ '" .......... .
.~~... ~.~=
. . ...--.....
.::....---
EPL + NE
40
20
o~--~---~----~----~~~--
60
Fig .
5:
25
15
10
Kinetic effect of EPL on the content of ATP
Totol content of cAMP
Content of cAMP in adipose tissue
~ Content of cAMPin incubation medium
5
0,5
90 min
% of control values ) in adipose tissue in vitro
II
IL
~ 0,4
"0
0,3
0,2
0,1
Fig.
Con tro l
EPLlmM
NE IOpM
NE lOpM
+TP 3 mM
EPL.NE
6 : Influence of EPL on the content of cyclic AMP in adipose tissue in vitro
EPL+NE+ TP
122
EPL-2 h
1800
EPL-l,h
EPL-2h
Fasted-24h
~ 1400
~1000
u.
u.
600
100
50
40
30
=-
r:t~rn. . .m~DJ~DJL------....L..rn--L-...L.L.[1J~
..
~
C
o
u
Fig.
7:
-'
0..
w
+ ---1
wo..
zw
.....;
C
0
u
-.J
0..
......J
wo..
zw
0
W
u.
+
0....1
wo..
u.w
Effect of EPL on lipolysis in vivo
DISCUSSION
In general, EPL does not affect the spontaneous lipolysis in

adipose tissue in vitro,but inhibits this process induced by
norepinephrine, theophylline, and dibutyryl cM1P. EPL also
diminishes the decrease of ATP in adipose tissue elicited by
NE or by theophylline. EPL depresses the increase of cAMP synthesis triggered by NE, or by NE plus theophylline as well.
EPL causes a significant increase in lipolysis in vivo. However,
it is also possible to observe in vivo an inhibitory action of
EPL exerted after 2 h on lipolysis induced by NE. EPL, further,
seems to intensify the process of lipolysis caused by starvation.
The observed discrepancies in the effects of EPL on lipolysis
in vitro and in vivo might be associated with the activation of
various lipases (13,14). Exposure of adipose tissue in vitro to
lipolytic hormones (or to dibutyryl cAMP) increases the rate
of FFA release and the tissue concentration of hormone-sensitive
lipase activity, and decreases simultaneously the tissue concentration of lipoprotein lipase (15). BLATON and PEETERS (16)
demonstrated that EPL acts as an activator of lipoprotein lipase
of cow-milk in vitro. Thus, it may be suggested that EPL might
inhibit the hormone-sensitive lipase activity in adipose tissue
123
and/or activate the lipoprotein lipase (15). HOWARD et ale (6)

reported similar results concerning the effects of the heparinreleased lipoprotein lipase and an increase of lipase activity
(in the plasma, the liver and in the aortic wall) after intravenous injection of LIPOSTABIL in normal and atherosclerotic
rabbits.
During starvation a marked decrease is seen in lipoprotein lipase
concentration in adipose tissue and an increase in the rate of
FFA release (15). Thus, EPL, while activating lipoprotein and
hepatic lipases, may intensify the lipolytic effect of starvation
in vivo which was observed in our experiment.
In conclusion, it seems that EPL acts as an activator of both
the lipoprotein lipase and hepatic lipase, but inhibits the
hormone-sensitive lipase in adipose tissue.
ACKNOWLEDGEMENT
These investigations were performed at the Institute of Pharmacology University of Padua, Padua, Italy. The author appreciates
the technical assistance of Mr. G.F. Daniel.
REFERENCES
- FAIN J.N.: Pharmacol.Rev. 25, 67 (1973)
2
- FASSINA G.: Life Sci.
- BUTCHER R.W., BAIRD C.E. and SUTHERLAND E.W.: J.Biol.Chem.

243, 1705 (1968)
- VAUGHAN 1-1. and STEINBERG D.: J.Lipid Res.
- FASSINA G., DORIGO P. and GAl ON R.M.: Pharmacol.Res.Corom.

~, 1 (1974)
- HOWARD A. N., PATELSKI J. and WALIGORA Z.: in "Phospholipids"

- Proceeding of the International Symposium on Phospholipids,
Szczecin, September 11-12, Editions L. SAMOCHOWIEC and
J. WOJCICKI 1972, p. 55
- DOLE V.P.: J.Clin.lnvest. 35, 150 (1956)
- EGGSTEIN M.: Klin.Wschr.
- FASSINA G., GAION R. ~-1. and DORIGO P.: Biochem. Pharmacol.
D,
225
~,
8 (1973)
ii,
Clin.Chem.Acta~,
193 (1963)
267 (1966)
(1972)
10 - WATSON D.:
i,
637 (1960)
124
11 - COMERTY H.V. et al.: Clin.Chem.
2,
37 (1961)
12 - SCHMIDT et al.: Handbuch des Diabetes mellitus E.F. PFEIFFER

u.a., J.F. Lehmanns Verlag, Mlinchen, Bd. II, 1971
13 - FIELDING C.J.: in "Atherosclerosis III" Eds. G. SCHETTLER
and A. WEIZEL, Springer Verlag, Berlin-Heidelberg-New York
1974, p. 545
14 - LA ROSA J. C., LEVY R.1., WINDMUELLER H. G. and FREDRICKSON
D.S.: J.Lipid Res. 11, 356 (1972)
15 - STEINBERG D. and KHOO J.C.: in "Atherosclerosis III"
Editions G. SCHETTLER and A. WEIZEL, Springer Verlag,
Berlin-Heidelberg-New York 1974, p. 550
16 - BLATON B. and PEETEHS H.: in "Atherosclerosis III" Editions
G. SCHETTLER and A. WEIZEL, Springer Verlag, Berlin-Heidelberg-New York 1974, p. 565
The Human Plasma Lipids

and Lipoproteins Under Influence
of EPL-Therapy
V. Blaton. B. Declercq. D. Vandamme. F. Soetewey and H. Peeters
Simon-Stevin-Instituut voor Wetenschappelijk Onderzoek. B-8000 Brugge. Belgium
Abstract: The effect of polyunsaturated phosphatidylcholine

(PU-PC) on the biochemical types of hyperlipoproteinemia and
the induced changes in the individual lipoprotein classes were
investigated. 350 male and female hyperlipoproteinemic patients
were screened by a panel of clinical and biochemical parameters.
The selected patients belonged to type IIa and b (n=55) and type
IV (n=3?). 16% of the cohort members had hypertension, 16% had
cardiovascular complications, 23% had cerebral symptoms and 14%
had peripheral atherosclerosis. 31% of the group Were obese and
9% of them were diabetic. Only patients with the same hyperlipoproteinemic type taken twice at an interval of 14 days were retained for statistical evaluation. 92 patients were intravenously
treated during 14 days with 10c mg PU-PC/day and 69 patients were
further treated perorally with 3x2 caps/day (1800 mg/d) for 106
days.
On agarose electrophoresis low density lipoproteins were decreased after intravenous PU-PC therapy and 25% of the lipoprotein
patterns in type II Were normalized. The plasma lipids, except
triglycerides, Were significantly decreased. From regression analysis of the data it has been statistically proved that an aVerage of 38% of the excess plasma cholesterol was eliminated. Plasma
cholesterol esters and phosphatidylcholine showed a significant
increase of linoleic acid accompanied by a decrease of cholesterol oleate. Type II hyperlipoproteinemia seems to be more receptive than type IV-patients. Low ahd high density lipoproteins
are both decreased but their lipid load is differently modified
during the intravenous therapy. The prolonged peroral effect of
PU-PC keeps the plasma lipids significantly lower than the starting values, but to a lesser extent than during the intravenous
therapy. Polyunsaturated fatty acids increase further and more
than ur.der the intravenous therapy.
INTRODUCTION
The main aim of our study was to investigate the preferential

influence of polyunsaturated phosphatidylcholine (PU-PC) on the
biochemical types of the hyperlipoproteinemia and to establish
the changes in the individual lipoprotein classes. Advantages
126
and disadvantages of the peroral and intravenous therapy will

be described. In the EPL ampoules used, PC is the major component (96%) and is slightly contaminated with OH-PC due to the
hydrolysis of PU-PC during aging. PU-PC was present in the ampoules in a complex form with desoxycholic acid in a 1/1 ratio.
LIPOSTABIL capsules contain 84% PU-PC, 2% OH-PC, 13% PE and 1%
OH-PE in the presence of 10% triglycerides (1). In the PU-PC
ampoules 65% of the fatty acid content is linoleic acid which
is significantly lower in the capsules due to the presence of
triglycerides.
PATIENTS AND METHODS
Male and female hyperlipoproteinemic patients were screened by

clinical and biochemical observations as described in earlier
papers (2,3). The selected patients showed a hyperbetalipoproteinemia (type II) or a type IV hyperprebetalipoproteinemia.
About 60% of the patient group was younger than 65 years. A
siJldlar percental distribution of the hyperlipoproteinemic types
was found in relation to age and sex.
16% of the cohort members had clinical symptoms of hypertension,
16% had cardiovascular complications, 23% had cerebral symptoms
and 14% of the group had peripheral atherosclerosis. 31% of the
group was obese and 9% of them were diabetic.
From a group of 350 suspected patients only 93 followed the
established experimental therapy procedure and 85 patients
showed the same lipoprotein pattern at both control pOints taken
at an interval of 14 days. For statistical evaluation only these
patients with the same hyperlipoproteinemic type at their two
control points were retained. A similar percental distribution
of the two types of hyperlipoproteinemia were found with regard
to sex and age.
The intravenous therapy with 1000 mg PU-PC/day (20 ml LIPOSTABIL)
was given during 14 days. 69 patients were further treated perorally with 3x2 caps/day (LIPOSTABIL FORTE, 300mg/capsule) during
106 days. There was no dietary prescription.
Methods and techniques used for the separation and quantitation
of plasma lipids, lipoproteins and fatty acids were previously
described (4,5). The individual differences of the start values
and those resulting from samples taken during treatment were
calculated. The effect of the therapy was evaluated by statistical analysis of these differences on a IBM call 360 time sharing
system.
127
RESULTS
1. The I nfluence of I ntravenously Injected EPL on Plasma Lipids and Lipoproteins
More than 25% of the lipoprotein patterns of patients with type

II hyperlipoproteinemia were normalized after 14 days of intravenous EPL therapy. In type IV hyperprebetalipoproteinemia, the
beta-lipoproteins were decreased and 10% of the patterns were
normalized. The plasma lipids, except triglycerides, were significantly decreased in concentration, however, a percental increase
of triglycerides was observed. The C/PL ratio and the CE/FC ratio
remained unchanged, however, a percental increase of PC was observed. The main results are the following:
1. Decrease of cholesterol (C) and phospholipids (PL)
(p
0.01)
2. Decrease of 18:1/18:2 especially in cholesterol esters (CE)

3. Similar results in type II and type IV
4. Selecti vi ty on lipoprotein (LP) classes: C/PL in DC. LP increased
C/PL in (3 LP decreased
As it was our aim to follow the preferential influence of PU-PC
on the biochemical types of hyperlipoproteinemia, the changes of
plasma lipids and plasma fatty acids in patients with type II
and type IV hyperlipoproteinemia were statistically evaluated.
A similar effect of PU-PC on both types was observed, however,
type II was more influenced. A negative correlation coefficient
(r=-0.57) as evaluated between the decrease of plasma cholesterol
and the mean mmol/l cholesterol value at the starting points demonstrates that the degree of effect on plasma cholesterol is a
function of the excess of plasma cholesterol (3). Furthermore
from the regression line it was statistically proved that a mean
of 38% of the excess of plasma cholesterol was eliminated. The
effect of PU-PC on the plasma fatty acid profile can be summarized as a significant change in 16:0, 18:1 and 18:2 fatty acids,
particularly in the cholesterol esters.
The preferential influence of intravenously injected PU-PC on
the subclasses of plasma lipoproteins was investigated in 7 preselected patients with type II hyperbetalipoproteinemia. From
EDTA plasma samples high and low density lipoproteins were
separated by ECG and individual lipids were analysed. As well
for alpha- as for betalipoproteins there was a lipid lowering
effect under the intravenous therapy. However, there was only
a significant decrease of total cholesterol and cholesterol esters
in the betalipoproteins and a significant decrease of phospholipids in alphalipoproteins. TG are unaffected in both lipoproteins (Fig. 1).
128
E
E
...
TC
(J\
befo re
ex: af ter
1.00
D f3
CE
200
ex
(SI
before
P after
p <0.05 on ind od iff.
I.
I.
FC
200
100
100
E
2
200
TG
PC
PL
200
2
100
Fig. 1: The effect of intravenously injected EPL on the plasma lipoproteins in type II hyperlipoproteinemia (n = 7 I
Regarding the changes of fatty acids in both lipo~roteins, there

was only in the alphalipoproteins a significant decrease of palmitic acid and an increase of 18:2, which was related to an increase of cholesterol linoleate.
2. The Effect of a Prolonged Peroral EPL Therapy on the Plasma Lipids and Plasma Fatty Acids
The effect of the prolonged peroral therapy on the plasma lipids

of type II and type IV hyperlipoproteinemia is given in Table 1.
The long-time peroral effect of PU-PC, summarized in Table 2,
was preferentially observed for free cholesterol (FC) , keeping
total plasma cholesterol (TC) values still significantly lower
than the starting values but to a lesser extent than after the
intravenous therapy. The C/PL ratio was further decreased. Important results were the significant decrease of free cholesterol
as well in type II as in type IV hyperlipoproteinemia and the
percental increase of esterified cholesterol in type IV hyperprebetalipoproteinemia.
+
++
TC
FC
CE
TG
PL
p~0.05
p< 0.01
Lipid
63.6 0.5
18.5 0.4
45.1 0.7
9.3 0.5
27.1 0.4
'l'ype II
Start
- t start
-2.4 0.7 ++
-1.1 0.5 +
-1.30.7
+1.0 0.6
+1.4 0.4 +++
(n=26)
t 120 d
Mean difference
Mean difference
t 120 d - t start
57.3 1.0
16.0 0.5
41.30.9
15.9 1.0
26.8 0.6
+0.3 0.7
-0.4 0.8
-1.6+0.7+
+1.2 0.9
+0.1 0.9
Type IV (n=16)
Start
Table 1: The effect of a prolonged peroral EPL therapy on the plasma lipids in hyperlipoproteinemic patients. Stat. values and mean
differences after a treatment of 120 days (14 d intravenous + 106 d oral). Data given in molar per cent
I\J
co
130
Table 2:
The long-time peroral effect of EPL on the plasma lipids.
Hyperlipoproteinemia of
Type IV
Type II
Decrease of TC and FC
p <0.05
Decrease of FC
p< 0 . 05
Increase of PL
p<0.01
Increase of % CE
p< 0.05
Further decrease of C/PL
p < 0.01
Increase of
in CE
~:p-FA
Decrease of
p<0.01
and A: 1-FA in CE p< 0.01
~:o
The prolonged peroral therapy increased the amount of cholesterollinoleate which is however more prounounced than after the
intravenous therapy. Fig. 2 establishes the negative correlation coefficient between the plasma oleate and the plasma
linoleate (18:1/18:2 ratio) from patients before and after EPL
therapy.
618:1118:2
0.4
co
0.2
o
o
oco
0
-0.2
R= -0.64
o
o
o
00
00
0
0
0
00
-0.4
~e-'4-:---------:0'-:'8:--------....Ll.-2-18:1I18:2 start
Fig. 2: The effect of EPL on the ratio of plasma oleate to Iinoleate (18: 1/18: 2) in hyperlipoproteinemic patients
DISCUSSION
Our r.esul ts show several lipid lowering effects of PU-PC. Important to note is the significant decrease of total cholesterol after the long-time peroral EPL therapy both in type II
and in type IV hyperlipoproteinemia, and especially of free
cholesterol, accompanied by an increase of esterified cholesterol in type IV hyperlipoproteinemia.
131
The mechanism of the cholesterol lowering effect of EPL as proposed by ADAMS (7), which is related to an increased LCAT activity, is supported by the percental increase of unsaturated
cholesterol esters in low and high density lipoproteins of the
plasma, especially by the significant increase of cholesterol
linoleate in high density lipoproteins which is a better substrate for the LCAT activity. Such a process is further activated by the increase of cholesterolesterase in plasma as shown
in the baboon by HOWARD (8).
A similar phenomenon was described by WOLIGORA et ale (9) in
the aorta where after EPL therapy the ratio cholesterolesterase/
cholesterolsynthetase was increased. The increased cholesterol
esterification found after peroral therapy is in agreement with
the results obtained by SKOREPA et ale (10). Despite milk lipoprotein lipase (11) and liver lipases (8) are activated by PU-PC,
there is no effect on the plasma triglycerides.
Regarding the individual plasma lipoproteins, the low and the
high density lipoproteins are affected, resulting in a decrease
of the ratio of low to high density lipoproteins and in a normalization of the lipoprotein pattern, especially in type II
hyperlipoproteinemia (3). The effect on the plasma lipoproteins
may be also related to the presence of bile salts needed for the
solublization of PU-PC for intravenous injection.
The prolonged EPL-therapy is especially characterized by the
decrease of plasma saturated and mono-unsaturated fatty acids,
especially cholesterol palmitate and oleate. Furthermore, the
favourable increase of cholesterol linoleate obtained after
peroral administration of EPL may be related to the continuous
absorption of unsaturated fatty acids in the intestine, similar
to that observed by diet, but without disadvantages of high caloric intake.
Conclusively, these results underline the therapeutical value
of polyunsaturated phosphatidylcholine in hyperlipoproteinemic
patients.
Acknowledgements
We wish to thank Dr. W. Van Belle, Dr. P. De Jaegere,
Dr. E. Lust, Dr. P. Van Eeckhoutte, Dr. E. Van der Stichelen
and Dr. A. Hongenaert for their collaboration.
132
REFERENCES
- BLATON V., VANDAMME D. and PEETERS, H.

Verhandl. Deutsch. Gesellsch. Innere Med.,
(1972)
~,
1242-1245
- PEETERS H., BLATON V., SOETEWEY F., DECLERCQ B. and

VANDAMME, D.
MUnch. Med. Wochenschr., 115, 1358-1362 (1973)
- BLATON V., SOETEWEY F., VANDAMME D., DECLERCQ B. and

PEETERS, H.
Atherosclerosis (1976) in press
- BLATON V. and PEETERS, H.

In.: Blood lipids and lipoproteins, G. NELSON, ed.
J. Wiley and Sons, New York, Chapt. ., 369-431 (1971)
- PEETERS H., BLATON V., DECLERCQ B., HOWARD A.N. and

GRESHAM, G.A.
Atherosclerosis, 1, 283-290 (1970)
- BLATON V., VAND~rnE D., DECLERCQ B., SOETEWEY F. and

PEETERS, H.
Lancet (1976) in press
- ADAMS, C. and ABDULLA, Y.

In: Phospholipids, ed. G. SCHETTLER, Georg Thieme Verlag,
Stuttgart, 44-49 (1972)
- HOWARD A. and PATELSKI, J.

Atherosclerosis, 20, 225 (1975)
- WOLIGORA Z., PATELSKI J., BROWN D. and HOWARD, A.

Biochem. Pharmacol., li, 1 (1975)
10 - SKOREPA J., FUCIK M., MARES P. and TODORIVICOVA, I.

Atherosclerosis, III, ed. G. SCHETTLER and A. WEIZEL,
895 (1974)
11 - BLATON V., VANDAMME D. and PEETERS, H.
FEBS Letters, 44, 185 (1974)
New Analytical Approach to the

Study of Hyperlipidemia
J. Skorepa, P. Mares, H. Todorovicova and E. Tvrzicka
4th Chair of Internal Medicine, Faculty of Medicine, Charles University,
Prague, CSSR
Abstract: Polyunsaturated phosphatidyl choline (PU-PC) reduces

significantly (p < 0.01) the total plasma cholesterol in hypercholesterinemic patients after a therapy of 8 weeks. The polyunsaturated fatty acids in the plasma cholesteryl esters are
increased. It is shown that gas liquid chromatography (GLC) analysis of the lipid profile is particularly suituable for further
investigation of cholesteryl ester metabolism in plasma.
INTRODUCTION
In the last years numerous data have been accumulated, which demonstrate the metabolic differences between cholesterol and cholesteryl esters (1-4). The amphiphilic molecules of free cholesterol are essential constituents of cellular biomembranes. Together with phospholipids and with protetns they form the outer
layer in lipoprotein particles. The lipophilic cholesteryl ester
molecules are a storage form of cholesterol in the cell. Together
with triglycerides they are a component of the hydrophobic core
in lipoproteins, where cholesteryl esters represent a trans~ort
form of cholesterol.
The metabolic relations are much better understood after the
discovery of LCAT (lecithin:cholesterol acyltransferase, EC 2.3.1
group) (5, 6). Considerable biochemical information on the metabolism of cholesteryl esters has been obtained from the studies
of patients with the rare disease, genetically determined, known
as familial LCAT deficiency (7).
According to the present knowledge the unesterified cholesterol
which is a surface component of very low density lipoproteins
(VLDL) is non-enzymatically transferred to high density lipoproteins (HDL). After the transferation it is esterified and the
cholesteryl esters are transferred to the core of VLDL and LDL
(4). This transfer is non-enzymatic as well. Further metabolic
processes take place intracellularly. The rate of intracellular
metabolism is controlled by highly specific molecular receptor
sites on the cell surface (8, 9).
134
EFFECTS OF PU - PC IN HYPERCHOLESTERINEMIA
In a previous study (10) it was shown that polyunsaturated phosphatidyl choline was able to reduce significantly the plasma
cholesterol values in hypercholesterinemic patientA. Simultaneously the amount of polyunsaturated fatty acids in the plasma
cholesteryl esters increased. The effect of a PU-PC therapy
(1.8 g/d per os for 8 weeks) in 12 hypercholesterinemic patients
is summarized in Table 1.
Table 1:
Effects of PU - PC on Plasma cholesterol and cholesteryl esters (CE) after a therapy of 8 weeks
in 12 hypercholesterinemic patients. Means.. S.D. Significance according to the paired t - test
Total
Cholesterol
CE
Cholesteryl
linoleate
(mg/dl)
(mM/l)
(mM/I)
Polyunsaturated CE
% of total CE
Before
PU-PC
319 + 79
3.4 + 0.7
1.6 + 0.5
47.6
After
PU-PC
282 + 69
3.5 + 0.8
1.9 + 0.5
60.7
Significance
p < 0.01
n.s.
p <
0.01
It was suggested that free cholesterol decreases as a result of

an increased rate of esterification which dependS on .the supply
of a phosphatidyl choline containing linoleic acid at its sn-position 2 (l-acyl-2-linoleoyl-sn-glycerol-3-phosphatidyl choline).
The increase of the cholesteryl linoleate production could be demonstrated after the intravenous administration of a phosphatidyl
choline solution containing PU-PC as the main component (Table 2).
Table 2: The effect of intravenously injected PU - PC on the level of cholesteryllinolate in plasma. 500 mg of polyunsaturated
phosphatidyl choline for intravenous use dissolved in 10 ml volume (LiPOSTABIL. Nattermann) diluted by 10 ml of 5% were
injected slowly during 10 minutes. The lipids were extracted from the plasma according to the method of FOLCH (12). After
thin-layer chromatography the fatty acids of the isolated cholestervl ester fraction were analyzed with GLC
Time after injection

(min)
o
60
120
180
Cholesteryl linoleate
(mM/l)
0.34
0.33
0.45
0.65
135
--::-=
I
~J_
"
r-
I '"
.. -:::
"
- ::.-=-
--
-T- - ,---,.-- - ,-I
--
. --
1-
l=- -
- -
I"
.~
1.f=
- r--::
~-
I"
:- 1-=
f-
-.
.-
----
t--
f-
...
--."
.-
'--
I--
-l-
-=-. - .....
.....
=-=
-f-
~-
~::-
t".- =:=:=':': f-;;.
.:.
:~
.'"'- -'=
l+- r--
;:: I;:..-:
r-- ...-.
- - --
-:-
--f-I--
1-4-
I=~
----
9_1_
- I--- - :
r-
I=:::
6
.,8
t-- ==
- 1-- - 3rfL7'ff
10
= =:..
~~
-" ....
\. 0;;;1
-
.. :,--
--..
~ -c--
+-
f-
I-
r-
--= t::::::=:
~
5
r--
'-- r=
- I---
r- "=- 1=
--=.:= r= ....
I==::
l~~
IW
f-
--
-- - --
t--::
1-' -;... .
"
-~.
+- .-: -=-t=-:
r- -=-
- :==-=
~
~-
I-
Fig. 1: Lipid profile of human plasma as measured by gas - liquid - chromatography_ Identification of peaks:
1 =
free cholesterol , 2
47, respectively,
cholesteryl butyrate,
3 - 6 = cholesteryl esters of the carbon no. 41, 43, 45,
7 - 10 = triglycerides of the carbon no.
48, 50, 52 , 54,
respect ively
For the study of these metabolic processes it is necessary to

assess the level of unesterified cholesterol and the one of
cholesteryl esters simultanously. The method must be sensitive
and accurate, because the change of lipid levels produced by
LCAT is very small (about 1oojUffiol/l/h). Therefore the gas liquid
chromatography (GLC) method proposed by KUKSIS (11) was modified
to enable the examination of lipid profile. From one GLC pattern
it is possible to assess the level of unesterified cholesterol,
cholesteryl esters and triglycerides. It is possible to determine
the levels of individual molecular weight fractions of cholesteryl esters and triglycerides as well (Fig. 1).
The accuracy of the method was calculated from 25 duplicate analyses according to the formulaljLd 2 /2n ',where d is the difference
between the duplicates and n is the number of samples. The method-
136
ological error was 0.5 mg/dl that is 13)UM/l for the unesterified
cholesterol, 1.6 mg/dl that is 25)UM/l for the cholesteryl esters,
and 2.2 mg/dl for the triglycerides.
To study the ~ipid transfer mediated by LCAT, the plasma was incubated at 37 C and the lipid profile was determined at zero time
and after 60 and 180 minutes, respectively. The results of one
representative determination are summarized in Table 3. It appears
that the values for the unesterified cholesterol were reduced and
that the level of cholesteryl esters was increased. Stoichiomet.ric
evaluation after 60 min of incubation demonstrates the agreement,
as the deficit of unesterified cholesterol is 0.11 roM and the increase of esters is 0.11 roM. The deficit of unesterified cholesterol after 180 min of incubation was 0.29 mM, while the increase
of cholesteryl esters was 0.30 mmol.
Table 3. Esterification of plasma cholesterol in vitro
Time
(min)
Free cholesterol
(mM/l)
Cholesteryl esters
(mM/l)
Triglycerides
(mg/dl)
2.38
2.27
2.09
5.28
5.39
5.58
203
202
203
0
60
180
The lipid transfer which is simultaneously developing in HDL lipoproteins results in a small increase of cholesteryl esters. Other
esters of cholesterol formed in HDL are probably transferred into
lipoproteins of lower density (LDL, VLDL), where they replace
triglycerides, which on the other hand are transferred to HDL.
Non-esterified cholesterol is esterified in HDL,but appeares to be
continuously supplemented by the transfer of surface cholesterol
of VLDL particles. This might be the cause why the resulting
amount of unesterified cholesterol in HDL did not change (Table 4)
Table 4. Esterification of HDL cholesterol in vitro
Time
(min)
0
60
180
HDL-free
cholesterol
CJuM/l)
0.34
0.34
0.31
HDL CE
(fJl'1/l )
1.03
1.08
1 11
HDL
triglycerides
(mg/dl)
15
19
20
CONCLUSION
Phospholipids appear to greatly influence cholesterol esterification, which in turn plays an essential role in lipid metabolism. The development of new sensitive methods for the investigation of these processes might be useful for the study of the
pathogenesis of hyperlipidemias as well as for their therapy.
137
REFERENCES
- GLOMSET J.A., J. Lipid Res.
~,
155 (1968)
- Mc CONATHY W.J., ALAUPOVIC P., CURRY M.D., !1AGNANI H.N.,

TORSVIK H., BERG K. and GJONE E.: Biochim. Biophys. Acta 326,
406 (1973)
- GJONE E.: Scand. J. Clin. Lab. Invest.

(1974)
- GLOMSET J.A., NORUM K.R., NICHOLS A.V., FORTE T., KING W.C.,
ALBERS J., MITCHELL C.D., APPLEGATE K.R. and GJONE E.: Scand.
J. Clin. Lab. Invest. 33, Suppl. 137, 165 (1974)
- GLOMSET J.A.: Biochim. Biophys.
- GLOMSET J.A.: Biochim. Biophys. Acta 70, 389 (1963)
- GJONE E. and NORUM K.R.: Acta Med. Scand. 283,107 (1968)
- BROWN M.S. and GOLDSTEIN J.L.: Proc. Nat. Acad. Sci.

(1974)
- GOLDSTEIN J.L. and BROWN M.S.: J. BioI. Chem. 249, 5153 (1974)
11,
Acta~,
Suppl. 137, 73
128 (1962)
21,
788
10 - SKOREPA J., MARES P. and TODOROVICOVA H.: Cas. Lek. Ces. 113,
784 (1974)
11 - KUKSIS A.: Fette, Seifen, Anstrichmittel 75, 517 (1973)
12 - FOLCH J., ASCOLI I., LEES M., MEATH J.A. and Le BARRON F.H.:
J. BioI. Chem. 226, 833 (1951)
Phospholipid Metabolism in the

Arterial VV all
Influence of Essential Phospholipids

on Serum Lipids and Lipid Metabolism
in the Aortic W"all
Experiments in Normal and Atherosclerotic Rabbits
A. K. Horsch, K. Hudson and A. J. Day
Medizinische Universitatsklinik, 0-6900 Heidelberg, Germany and
Department of Physiology, University of Melbourne, Victoria, Australia
Abstract: A deficiency of essential fatty acids in the atherosclerotic artery leads to the formation of saturated cholesterol
esters which are sclerogenic. The presence of essential phospholipids (EPL) within the arterial wall should favor the formation
of non-sclerogenic polyunsaturated cholesterol esters which are
readily removed from the vessel wall. Therefore~ the effects of
EPL (40 mg/kg/day) in the cholesterol-fed rabbit on serum and
aortic lipids in vivo~ and on the arterial lipid synthesis in
tissue cultures of arteries of the same animals were investigated.
The EPL was intravenously injected 3 times weekly in parallel to
the cholesterol diet; the control animals received a corresponding injection of saline. Blood samples were taken after 4 and 8
weeks of treatment~ then the aortas were removed and placed into
a tissue culture. The explants were pulse-labelled during a 24 h
incubation either with 3H-EPL~ llfC-acetate and 32p-phoshate or
with 3H-oleic and l4C-linoleic acid~ respectively. After this
pulse label incubation the explants were removed~ one third of
them were used for uptake studies and two thirds of them were
reincubated in a non-radioactive medium to determine the labelled
lipids released from the explants. - After 4 and 8 weeks the ratio
of linoleic:oleic acids in serum phospholipids and serum triglycerides in the EPL treated animals differred significantly from
the controls. The LCAT activity was lower in the EPL treated
group. - No significant differences in the arterial lipid composition were observed. The incorporation of 3H-EPL into cholesterol
esters was significantly higher in the atherosclerotic artery.
3H-EPL cholesterol esters were removed from arterial tissues after
8 weeks of EPL treatment. l4C-acetate. 3H- o l eic and llfC-linoleic
labelled cholesterol esters were also removed. The phospholipid
synthesis from l4C-acetate was significantly depressed by the EPL
treatment.
INTRODUCTION
The experimental atherosclerosis induced by cholesterol feeding

in the rabbit is a well established model for the study of art.erial wall metabolism in atherosclerosis. Despite morphological
differences in atherosclerotic lesions in man and rabbit the lipid metabolism of the arterial wall is very similar at comparable
141
stages of the disease, and the study of this rabbit model is

therefore relevant for the situation in man (1,2,3). This is
especially true for tissue culture experiments, where comparable
conditions can be choosen.
The lipid accumulation in the arterial wall by the influx of
serum cholesterol and its subsequent esterification on one hand
and by the endogenous phospholipid- and fatty acid synthesis on
the other is associated with the progression of atherosclerosis.
Fatty acid synthesis in the atherosclerotic artery seems to operate by chain elongation of acyl units with acetyl CoA (4), whereas only a small proportion of long chain and unusual polyunsaturated fatty acids is synthesized (5,6). Consequently, saturated
fatty acids are incorporated in all locally synthesized ,lipids,
including cholesterol esters and phospholipids. This deficiency
of essential fatty acids in the atherosclerotic artery thus promotes the atherosclerotic process by the formation of saturated
cholesterol esters, which are sclerogenic. At this stage, lecithin
which acts as a donator of fatty acids for the esterification of
cholesterol, contains predominantly saturated fatty acids and
therefore also contributes to atherogenesis.
There is some evidence that the presence of essential phospholipids (EPL) within the arterial wall might favor the formation
of the non-sclerogenic polyunsaturated cholesterol esters which
are more readily removed from the vessel wall (7). These considerations led us to investigate the effect of EPL in the cholesterol-fed rabbit on 1. serum and aortic lipids in vivo and
2. on arterial lipid synthesis in tissue culture with the same
arteries.
METHODS
The experimental procedure was briefly the following: 18 weeks

old male New Zealand albino rabbits were fed cholesterol for 8
weeks (1 g of cholesterol and 3 ml of peanut oil in 100 g of
rabbit pellets, normal fed rabbits received the unprepared pellets).
EPI was sonicated in water and 200 mg were injected intravenously
three times weekly. The total amount administered per animal was
5.4 g which corresponds to a daily dose of 40 mg/kg body weight,
and the treatment was started together with the cholesterol feeding. The control group received the same diet and, at the same
time-intervals, intravenous injections of an equal volume of
saline. Individual blood samples were taken 4 and 8 weeks after
the beginning of the experiment for lipid analyses and determination of LCAT-activity (8).
After 8 weeks, the thoracic aorta was removed, the grade of atheroma assessed (9) and the artery dissected into 24 pieces of
similar size. Duplicates of these explants were randomly choosen
and placed in tissue culture dishes containing Eagle's basal
142
medium with Earle's balanced salt solution and homologous serum

(2:1/v:v). Before the incubation of the explants, the desired
isotopes had been added to the tissue culture medium that was
then distributed into the tissue culture dishes.
The experimental plan for this tissue culture is given in Fig. 1:
The explants were incubated for 1, 3 and 5 days with daily changes of the medium. At day 1, 3 and 5 one third of the explants
(4 duplicates at each time interval) was taken down.
Preparation of the artery

Dissection into 24 explants
Randomizlltion of the ex plants
Incubation in duplicates
with:
Precursors
Removal of the medium
Repeated WASH I N::ncubation in "cold" medium
8 explants
(medium is changed daily and pooled>

2 days
4 days
8 explants
8explants
ta ken down, washed repeated ly, int i rna and med ia

homogenizpd and extracted.
Fig. 1: Tissue culture of thoracic (normal and atherosclerotic) aortas of rabbits
At the first incubation period of 24 hoqrs the explants were

pulse-labelled either with 3H-EPL, 14C-acetute and 32p-phosphate
or with 3H-oleic- a.nd 14C-linoleic acid respectively. After this
24 hour incubation the radioactive medium was removed, all explants were washed thoroughly several times and two thirds of
them reincubated for further two or four days in non-radioactive
medium. The medium was changed every day and the removed medium
143
was pooled for determination of the labelled lipids released from

the explants.
The viability of the tissues was checked by their glucose uptake. Fig. 2 shows that the glucose uptake of both normal and
atherosclerotic explants is linear and that the glucose utilization of the atherosclerotic explants is higher than that of
c
~
~
a.
01
.....E
<II
0
U
:J
01
1,20
1,00
01
z
0
;::
<{
0,80
0,60
If)
::;
;::
0.40
0.20
::::>
If)
-- --
::::>
--'
(!)
J..
..3
5
days
atherosclerotic
- -- normal
Fig. 2: Glucose utilisation by aortic explants from cholesterolfed (n = 161 and from normal (n=41 rabbits (means ~ S.E.M.I
the normal explants. At the end of the different incubation

periods, the explants were washed very thoroughly several times
again, the intima and the media were stripped off and homogenized
and the lipids extracted (10). The protein content of the extracted tissue was assayed (11) and the content of phosphorus,
triglyceride, cholesterol and cholesterol ester in the lipid
extract was determined (12, 13, 14). The lipids were seoarated
on neutral lipid- and phospholipid TLC and the fatty acid composition of different lipids was determined after methylation
on 15% diethylene-glycol-succinate columns at 185 0 C using an
F&M Model 5750 Gas Chromatograph. A more detailed description of
the experimental procedures wiil be reported elsewhere (14).
144
RESULTS
Serum Lipids
Table 1 shows the effect of EPL treatment after 4 and 8 weeks on

serum lipids and on LCAT-activity compared as to the control
group. There is obviously no significant difference between the
two groups. After 8 weeks, the LCAT-activity was slightly higher
in the control group.
Table 1: The effect of essential phospholipid (EPL) in cholesterol-fed rabbits (n=6) on serum lipids (mg/l00 ml
serum) and on LCAT activity (mg esterified cholesterol /100 ml serum / 6h). Means . S.E.M.
8weeks fed
4weeks fed
1.23
25.5
2040
100
2640
194
31
520
24
550
75
767
129
1742
60
1883
1.39
78.7
0.37
77.1
0.35
77.8
1.39
50.4
0.7
67.3
19.0
110
10
175
89
3.22
0.05
3.63
Q29
6.43
0.55
8.17
0.78
1.47
24.5
924
132
27
206
629
117
75.2
Triglyceride
LCAT Activity
0.81
12.1
759
131
-Free
195
-Ester
-'Yo Ester
lipid Phosphorus
11.5
Cholesterol-Total
3.19
134
The fatty acid composition of serum phospholipids, serum triglycerides and serum cholesterol ester is given in the Tables
2, 3 and 4. The ratio 18:2/18:1 is significantly higher at 4
and 8 weeks in the EPL treated group for phospholipids and triglycerides and after 4 weeks of treatment also for cholesterol
esters. In this latter lipid fraction also the content of palmitic
acid is significantly lower than in the control group after 8
weeks of treatment. All other fatty acids are not significantly
influenced by the treatment.
145
Table 2: The effect of essential phospholipid (EPL) on the distribution of fatty acids in serum phospholipids in
cholesterol-fed rabbits (n=6, means:t S.E.M.)
Phospholipid Fatly Acids
8week
4 week
Fally Acid
EPL
16,0
24.5 t 0.79
25.6 t 0.54
25.5 ! 0.49
27.2 t 0.88
18,0
16.5 t 0.35
16.0! 0.57
15.9 t 0.30
15.5 t 0.63
18, 1
14.7
0.46
16.3! 0.50
15.0 ! 0.45
15.9 t 0.42
18 ' 2
36.9 t 0.32
35.1 ! 0.82
36.6 ! 0.63
34.8 t 0.63
20,4
6.4 t 0.53
6.4 ! 0.51
6.0! 0.46
5.7 t 0.28
EPL
Saline
Saline
Ratio:
a2.51~
a-a
0.085
a2.17! 0.101
0.072
a2.48~
a2.20! 0.088
pcO.05
All others n.S.
Table 3: The effect of essential phospholipid (EPLI on the distribution of fatty acids in serum triglvcerides in cholesterol-fed
rabbits (n=6, means:!: S.E.M.1
Serum Triglxceride
Fattl Acids
4week
Fatty Acid
U'l
Saline
~J'.1
week
Salin..!
14, 0.
1.1
0..16
1.4 ! 0..18
1.2 ! 0..12
1.5
0..22
16 ' 0.
33.8
2.35
31.7 ! 1.58
32.0. ! 2.0.5
29.2
1.26
18' 0.
6.0.
0..84
6.0. ! 0..73
5.3 : 0..75
5.0.
0..39
18'
31.4
1.52
35.5 : 1.54
32.2 !
1.44
35.0.
0..63
18 ' 2
27.7
2.33
25.2
1.32
28.9 : 0..87
28.0.
o..72! 0..0.5
o..9o.! 0..0.5
1.0.2
Ratio:
0..90.: 0..11
a-a
pc 0..0.5
b- b
P c 0.0. 1
o..SO. 0..0.2
146
Table 4: The effect of essential phospholipid (EPLI on the distribution of fatty acids in serum cholesterol esters in cholesterolfed rabbits. (n=6. means ~ S.E.M.1
Cholesterol Ester Fattr: Acids
4 week
Fatty Acid
week
Saline
1.05
813.2 : 0.32
"14.3 : 0.29
0.19
4.8 : 0.28
5.8 : 0.35
0.50
4.5 : 0.48
3.2 : 1.05
1.14
49.8 : 0.50
50.0
0.94
25.8 : 0.58
24.4 : 0.84
o. 18
1.9 : 0.12
Saline
18,0
18.8! 0.37
15.8
18 ' 1
4.4:t 0.92
5.8
18' 0
5.9:t 0.52
4.7
18 ' 1
b48.7:t 0.32
bOO. 7
18' 2
24.7: 1. 14
23.0
18' 3
1.4: 0.34
1.1 ! 0.29
0.53: 0.02
"0.48: 0.02
1.9 :
0.58
Ratio:
18,2/18' 1
8 -.
0.05
b-b
pc
0.01
0.52: 0.14
0.49:
om
All others n.s.
Arterial Lipids
The arterial lipids are nearly identical in the two groups after
8 weeks of treatment (Table 5) and also cholesterol and cholesterol ester content is very similar. There is only a slight difference in atheroma grade but the difference is statistically not
significant.
147
Table 5: The effect of essential phospholipids IEPLI on the content and concentration of protein and lipid of thoracic aortic
intima from rabbits fed cholesterol for 8 weeks. Data is expressed as f'9 /9 aortic intima or as 1'9/ m9 of protein. respectively.
In=6. means
S.E.M.I
EPL
Protein
43.7
lipid Phosphorus
94.5
2.23
lipid (1'9/m9 protein)
SALINE
2.93
10.0
41.0
2.78
89.1
6.96
0.28
0.19
2.24
5215
1735
4508
872
122
40.2
110
19.8
Triglyceride
376
52.2
305
24.6
Triglyceride jJ9/mg protein
8.99
Cholesterol (Total)
Cholesterol (Tolal)
I'g/mg
protein
Cholesterol - Free
. Ester
7.51
1.49
0.54
1883
456
15 B 1
273
3518
1306
3223
749
. 010 Ester
60.10
3.47
64.10
ATHEROMA GRADE
225
0.63
2.75
3.05
0.66
The fatty acid composition of the different arterial lipids is

shown in the Tables 6, 7 and 8 and neither for the phospholipids
or triglycerides nor for the cholesterol ester significant differences have been obtained between the two groUDS.
Table 6: The effect of essential phospholipid IEPLI on the distribution of fatty acids in the phospholipids of the thoracic aortic
intima from rabbits fed cholesterol for 8 weeks. In=6. means + S.E.M.I
Phospholipid Fatty Acids
SALINE
Ratio
16 ' 0
31.8
99
30.0
0.91
18 ' 0
22.6
0.88
22 7
0.89
18 '
18.8
0.48
19.8
0.54
18, 2
20.5
1. 00
20.5
1.04
18 ' 2 118' 1
1.10
0.073
1.04
0.045
148
Table 7: The effect of essential phospholipid (EPL) on
t~e
distribution of fatty acids in the cholesterol esters of the thoracic
aortic intima from rabbits fed cholesterol for 8 weeks. (n=6, means:!:. S.E.M.)
Cholesterol Ester Fatty Ac ids

SALINE
.ll
14' 0
0.5
0.03
16' 0
13.5
0.66
16' 1
4.9
0.46
5.3
0.39
18,0
3.1
0.31
3.2
0.22
18' 1
55.5
1.30
55.5
0.63
18,2
22.0
0.79
19.7
0.70
0.40!
0.022
0.5
0.12
13.7 ! 0.88
0.36 ! 0.012
Table 8: The effect of essential phospholipid (EPL) on the distribution of fatty acids in the triglycerides of the thoracic aortic
intima from rabbits fed cholesterol for 8 weeks. (n=6, means:!:. S.E.M.)
Trigtyceride Fatty Acids
14,0
1.8
0.15
1.4
16,0
26.0
0.64
27.1
18' 0
11.9
0.78
13.2
0.46
lS' 1
33.6
0.63
34.3
0.94
lS,2
20.2
1.09
lS.6
0.22
20' 4
6.2
0.66
4.S
0.72
0.6
0.028
0.54!
0.016
0.17
!
1.10
Lipid Synthesis in Tissue Culture: Uptake and Removal of 3H - EPL
The uptake and subsequent removal of the 3H-fatty acid labelled

lecithin is shown in Fig. 3 and Table 9 for different lipids.
After the pulse label of 24 h, the activity in the explants decreases and labelled lipids are released into the incubation medium. This is very striking for phospholipids, but it is also
149
found for triglycerides and cholesterol esters. Cholesterol esters

are mobilized and released into the medium (Table 9), their proportion is smaller, however, than that of the other lipids.
"'-
0.9
0.6
Neutral lipid
0.3
D.6
\...
0-
0.3
,,. ./
Phospholipid
0.9
./
.'",
'" '"
----- -----"
.;/
0.06
Triglyceride
...............
.............
-.......-.-.-
0.04
0.04
0.02
Cholesterol
0.06
Ester
-'- ._.-.- ' - ' - ' -
0.02
.~
..........
. .:..:..,;.; ...:.. ..:- - - - -
-.:..' ..
~:-=
:..:-=-
5 days
_ . - EPl-treated
.... 1M of EPl
- - Saline treated
- - - 1M
of Saline
Fig. 3: Uptake and subsequent removal of 3H-EPL by arterial explants
Table 9: Uptake of 3H-EPL by atherosclerotic aortic explants (n=192) and subsequent removal of labelled lipids:
(dpm I explant and dpm I incubation medium)
1 Dayl
Neutral lipids
870711
95121
Medium
Phospholipids
698023
66071
Medium
TriglYl'erides
54758
9367
Medium
Cholesterolester
26413
7038
Medium
3 Days2
5 Days 2
381732
49788
300843
43241
552164
87060
148399
22226
270143
25453
208658
21220
406036
65619
83375
11892
44192
11061
45470
8539
3025
329
1642
231
31593
7883
33552
10677
2461
368
1532
187
1 Art i\"ity after 24h incubation with 3H- EPL

2 Activity after 2 and 4days of incubation in "cold" medium.
150
Table 10: Uptake and metabolism of 3H - acyl -labelled phosphatidylcholine in tissue culture: explants 124 units/aorta) of
atherosclerotic thoracic aortas from 8 rabbits. Data Imean + S.E.M.) are given in dpm/mg protein 1106 dpm in incubation
medium. 3H-pulse labelling 1+) for 1d followed by non radk,active incubation for 3 and 5d Ixx).
day 1+
day 3"
2317
199
1228
PHOSPHOLIPIDS
1867
164
871
TRIGLYCERIDES
143
80
NEUTRALLIPIDS
total
CHOLESTEROLESTER
day 5"
150
960
156
90
645
99
17
141
32
143
20
103
24
108
27
31
specific activity
10000
log
CE
10
PL
100
TG
10
days
----..j
pulse
label
Fig. 4: Specific activity of 3H_EPL in different lipids of atherosclerotic aortic explants In=192). Data given in dpm/mg CEo
TG. or PLl106 dpm in incubation medium
151
The same data in relation the the protein content of the tissues
are presented in Table 10: While the activity per mg of protein
is decreasing in the phospholipid fraction very considerably it
is increasing slightly in the cholesterol ester fraction. In triglycerides the activity remains on the same level, reflecting a
constant turnover in this fraction. This can also be demonstrated
by the specific activity curves plotted vs. time after the pulse
label period of 24 h (Fig. 4).
While the uptake and incorporation of 3H-fatty acid labelled lecithin into phospholipids and triglycerides is very similar in normal and atherosclerotic arterial explants, there is a significant
difference in the esterification of cholesterol with this label.
Fig. 5 shows the esterification of cholesterol in normal and
atherosclerotic explants after the 24 h pulse label followed by
non-radioactive incubation medium. In these experiments, up to
10% of the labelled EPL incorporated into li~ids was found in
cholesterol esters in atherosclerotic explants whereas less than
1% were present in cholesterol esters of the normal explants.
atherosclerotic
ex plants (n=192)
100
75
normal
explants (n=20)
C)
....
[
"'C
pulse
label
"I- Incubation in cold" medium
..
6 days
Fig. 5: Incorporation of 3HEPL into cholesterolesters in normal and atherosclerotic aortic explants
152
Uptake and incorporation of 14C . acetate
From Fig. 6 it is apparent that the control group takes up more

of the 14C-acetate than the group of explants treated with EPL,
secondly that in both groups most of the label is incorporated
into phospholipid, and finally that only a small amount of the
label is subsequently released into the medium. Obviously the
14C-acetate released from phospholipids is then incorporated
into triglycerides and cholesterol esters.
dpm
x 10. 6
Phospholipid
09
0.9
-------- . -
0.6
.-
0.3
---._.-
0.6
0.3
....
"
...........
- --
006
0.06
0.04
0.04
Cholesterol
Ester
Triglyceride
0.02
0.02
. '- -'
-. -
EPL - treated
- - Saline
treated
1M
of EPL
1M
of Saline
-'
.:.
''':
._
:.. . ..,:
5 days
Fig. 6: Uptake and subsequent removal of 14e.acetate by arterial explants in tissue culture (I M=incubation medium)
Table 11 shows that most of the labelled acetate is incorporated

into phosphatidylinositol and into lecithin and a smaller proportion into phosphatidylethanolamine and sphingomyelin. This
distribution is the same for both groups, however there is a siqnificant difference in phospholipid synthesis between the two
groups: During the 24 hour incubation, less acetate is incorporated into each of the individual phospholioid fractions, the incorporation is reduced up to 70 percent of that measured for the
controls. The most impressive change occurs in the sphingomyelin
fraction: The incorporation is reduced to 25 percent of that observed in the control group.
153
The specific activity on day 3 and day 5 is also lower in the

EPL- than in the control group. On the other hand no difference
in the rate of removal of the labelled acetate can be seen between the two groups. The effect of EPL, therefore, is a decrease
in the synthesis of all phospholipids especially of sphingomyelin.
Table 11: Effect of essential phospholipid (EPL) on the incorporation and removal of acetate from phospholipids in the thoracic
aortic intima cholesterolfed rabbits. (SM = sphingomyelin, Lee = lecithin, PI =phosphatidylinositol, PE = phosphatidylethanol-
amine). Statistical significance
* at the
Means and S.E.M.'s n=8
5%.
** at the 1% level
(Dpm/~g lipid phosphorous/10 6dpm per 2 mls incubation

medium)
DAY 1
DAY 3
DAY 5
Phospholipid
Treatment
Total PL
EPL
Control
1718+ 503
2719+ 467* *
1393+
2250+
259
364
1307+
1986+
283
207
SM
EPL
Control
661+ 159
2665+1218* *
802+ 166
2709+ 1316"
898+
2154+
213
721
Lec
EPL
Control
2877+ 566
*
3772+ 571
2250+
3345+
457
495
1909+
2877+
400
257
PI
EPL
Control
3184+ 584
4172+ 400 *
2440+
3632+
386
509
2463+
3623+
439
604
PE
EPL
Control
548+ 122
776+ 103 *
628+
973+
102
182
707+
1021+
135
122
dpm x
Phospholipid
--
0.3
0.2
0.1
.... -=.
-'
--
...
- 3
5 days
Fig. 7: Uptake and subsequent removal of 32PPO 4 by arterial explants. Symbols as in Fig. 6
154
I ncorporation of 32p - phosphate
The incorporation of 32p-phosphate is similar in both groups

(Fig. 7).
Table 12 shows this similarity also for the incorporation into
total phospholipids, into lecithin, phosphatidyl inositol and
phosphatidylethanolamine. The highest incorporation occurs into
phosphatidyl inositol in both groups. The incorporation into
sphingomyelin however is significantly lower in the EPL treated
group and the specific activity remains lower over the 5 days
period. This difference in incorporation into sphingomyelin between the two groups is of the same order as the difference in
the incorporation of 14C-acetate into this phospholipid fraction.
Table 12: Effect of essential phospholipid IEPL) on the incorporation and removal of phosphate from phospholipids in the
thoracic intima cholesterolfed rabbits. Statistical significance: at the 5%, at the 1% level
Means and S.E.M.'s n=8
(Dpm/~g lipid phosphorous/10 6dpm per 2 mls incubation

medium) .
DAY 1
DAY 3
DAY 5
Phospholipid
Treatment
Total PL
EPL
Control
412+ 76
368+ 32
545+104
708+ 70
465+100
*
675+140
SM
EPL
Control
57+ 12
198+
164+ 43
**
460+203
210+ 67
415+113* *
Lec
EPL
Control
601+ 93
501+ 72
761+124
938+ 74
580+ 95
850+185
PI
EPL
Control
2317+471
2057+269
1866+330
2279:t335
1359+211
1926+626
PE
EPL
Control
153+ 32
122+ 20
479+100
600+ 86
571+151
746+169
9t
Uptake and Removal of 3H - oleic and 14C - linoleic acid
The uptake and subsequent removal of 3H-oleic and 14C-linoleic

acid by the atherosclerotic explants is demonstrated by Fig. 8
and 9. The incorporation of these labels is lower in the EPL
treated group and significantly lower in the cholesterol ester
of this group.
155
Phospholipid
Neutral lipid
03
0.2
.......
~...
~.,
0.1
.-
~
~.-.-
...--:-.~.
Triglyceride
O.D!
~
'-
006
UOO
"
-.7"-".-,
Cholesterol Ester
0.06
0.04
0.02
5 days
-
.-
EPl - treated
- - - Saline treated
..... 1M of
EPl
- - -- 1M of
Saline
Fig. 8: Uptake and subsequent removal of 14C - linoleic acid by arterial explants
Phosphol ipi d
Neutral lipid
0.9
~
,/
0.6
-.............
0.3
/'
,/
............
.... .'
,/
..... .
....
~.
/
~.
Triglyceride
0.3
~
'-.
0.2
0.1
---Cholesterol
Ester
--.-.---
5 days
-
. -EPl-treatod
---Saline trealed
.. , 1M of EPl
- - -1M
of Saline
Fig. 9: Uptake and subsequent removal of 3H - oleic acid by arterial explants
156
These data are summarized for both 3H-oleic and 14C-linoleic acid
in Fig. 10: The ratio of oleic acid:linoleic acid does not differ
significantly between the two groups, as might be expected. But
a significant difference is found in the incorporation of both
of these precursors into cholesterol esters between the EPLtreated and the control group.
...
3(}(x)
-@10
2(}(X)
.....
c:
.~
...Q,
...
.....E
.... -._.- '-'-'~'-'-'-'
,../,.1._._. _. _. _.-1._._. _.::: ._. -+-.:-;..".
'-. -+-
'-
Control
3H
"c
days
animals
Rabbits treated with p olyunsaturat.d

lecithin (600 mg / w k )
3H I 14C Ratio:
I day
3 days
5 doys
EPL
,.' ~ 7 t 0.080
1.375 t 0.255
1.377 t 0.132
SALINE
1.501 t 0.023
1.478 :t 0.035
1.~79 t
0.024
Fig. 10: Cholesterol ester of atherosclerotic arterial explants:Uptake and removal of 3H - oleic - and 14C -linoleic acid
Removal of Cholesterolesters from Arterial Tissue
Fig. 11 gives the specific activities of the arterial cholesterol

esters endogenously labelled with 3H-EPL and 14C-acetate. It is
obvious, that the specific activity of cholesterol ester from the
EPL treated group decreases, whereas it is increasing in the control group. This is interpreted as a sign of removal of cholesterol ester from the arterial tissue after EPL treatment.
157
5000
14C
;'
,/
4000
3000
t---
,/
,/
,/
,/
--
;'
14C
2000
_-1
en
3H
a.
"'C
3H
1000
'"
.....
a.
"'C
5
days
- - EPL treated
animals (n =41 group)
- - - - control
Fig. 11: Specific activity of cholesterolester endogenously labelled with 3 H_EPL and 14Cacetate
The relevance of these findings in view of the anti atherogenic

effect of EPL is illustrated by Table 13: after the intravenous
injection of 100 mCi of 3H-fatty acid labelled lecithin in the
normal rabbit the radioactivity of the serum was determined at
different time intervals and also in different tissues after 6 h
after the injection. The activity in the serum is rapidly decreasing and most of the label is recovered in the liver. Only
a small amount of the activity is incorporated into the thoracic
aorta, but the aorta contains the relatively highest proportion
of cholesterol esters. This seems to be important as the cholesterolester of the arterial wall accumulates in atherogenesis and
the reversibility of atherosclerotic lesions appears to depend
upon the fatty acid composition of these cholesterol esters.
158
Table 13: Incorporation of 3H-fatty acid-labelled lecithin (l00,uCil into lipids of different tissues in vivo (6 h after intravenous injection)
PL
TG
CE
255616
44602
2768
23177
22785
147
2076
116
51
.
SERUM 1h p.L
55495
1081
2222
.
SERUM 6h p.L
3812
283
377
LIVER
MYOCARDIUM
AORTA
Data are given in

respectively
cpm/~g
dry defatted weight br
cp~/ml
of serum
CONCLUSIONS
After 4 and 8 weeks of cholesterol feeding the ratio of linoleic

acid/oleic acid in serum phospolipids and serum triglycerides is
significantly different in the EPL treated animals as compared
to the control group. In the serum cholesterol esters this ratio
is only significantly different at 4 weeks whereas at 8 weeks
significantly less palmitic acid is found in this lipid fraction.
The LCAT activity after 8 weeks of treatment is lower in the EPL
treated group.
No significant differences in the arterial lipid composition have
been found.
The incorporation of 3H-EPL into cholesterol esters is significantly hiqher in the atherosclerotic artery. 3H-EPL cholesterol
esters are removed from arterial tissues after 8 weeks of EPL
treatment. 14C-acetate, 3H-oleic and 14C-linoleic labelled cholesterol esters are also removed.
Phospholipid synthesis from 14C-acetate is significantly depressed by EPL treatment, especially the sphingomyelin synthesis. The
phospholipid synthesis from 32p-phosphate is not depressed by the
EPL treatment except for sphingomyelin. The depression is in this
lipid fraction of the same order as the sphingomyelin synthesis
from acetate.
REFERENCES
1 - DAY A.J., HORSCH A.K., and PROUDLOCK J.W.: Lipid metabolic
pool in subcellular fractions of rabbit and human atherosclerotic lesions. Proc. III. Int. Symp. Atherosclerosis,
Springer Berlin, Heidelberg, New York (1974) p 103-106
159
2 - HORSCH A.K., DAY A.J. and SANWALD R.: Lipidstoffwechsel

normaler und atherosklerotisch veranderter Intima in menschlichen Femoralarterien. Virchows Arch. A 361, 71-75 (1973)
3 - DAY A.J. and WAHLQVIST M.L.: The uptake and metabolism of
14C-Iabeled oleic acid by atherosclerotic lesions in rabbit

aorta. A biochemical and radioautographic study. Circ. Res.
~, 779 (1968)
4 - WHEREAT A.F.: Fatty acid synthesis in cell-free system from

rabbit aorta. J. Lipid Res. 2, 671-677 (1966)
5 - DAY A.J. and WILKINSON G.K.: Incorporation of 14C-Iabelled
acetate into lipid by isolated foam cells and by atherosclerotic arterial intima. Circ. Res. ~, 593-600 (1967)
6 - HOWARD C.F.: De novo synthesis and elongation of fatty acids
by subcellular fractions of monkey aorta. J. Lipid Res. 9,
254-261 (1968)
7 - ABDULLA Y.H., ADAMS C.W.M. and MORGAN R.S.: Differential resorption rates of subcutaneous implants of 3H-cholesterol,
various 3H-cholesterol esters, and 3H-cholesterol-I 14C-linolenate. J. Atheroscl. Res. 1, 81 (1969)
8 - GLOHSET J.A. and WRIGHT J.L.: Some properties of cholesterol
esterifying enzyme in human plasma. Biochim. Biophys. Acta
g, 266-276 (1964)
9 - DAY A.J. and WILKINSON G.K.: Severity of atherosclerosis in
rabbits in relation to serum lipids and to aorta cholesterol
content. Aust. J. Exp. BioI. Med. Sci. li, 423-432 (1956)
10 -
FOLCH J., LEES M. and SLOANE-STANLEY G.H.: A simple method

for the isolation and purification of total lipids from animal tissue. J. BioI. Chern. 226, 497-509 (1951)
11 - LOWRY O.H., ROSEBROUGH N.J., FARR A.L. and RANDALL R.J.:

Protein measurement with the Folin phenol reagent. J. BioI.
Chern. 193, 265-275 (1951)
12 - BARTLETT G.R.: Phosporus assay in column chromatography.
J. BioI. Chern. 234, 466-468 (1959)
13 - ZLATKIS A., ZAK B. and BOYLE A.J.: A new method for the
direct determination of serum cholesterol. J. Clin. Lab.
Med. .!l, 486 ( 1 953 )
14 - HORSCH A.K., HUDSON K. and DAY A.J.: in preparation
Effect of EPL on the Metabolism

of Lipids in the Arterial "Wall
D. E. Bowyer and P. F. Davies
Department of Pathology, University of Cambridge, Cambridge, England
Abstract: Intracellular accumulation of lipids, mainly of cholesteryl oleate. is the characteristic feature of the early fatty
streak lesion in atherosclerosis. By the use of experimental models of fatty streak lesions. such as the cholesterol-fed rabbit,
it has been shown that in situ synthesis can contribute as much
as 50% of the cholesteryl oleate. Since in vitro studies showed
that essential phospholipids (EPL) are able to influence the arterial lipid metabolism, EPL might be expected to reduce lipid
accumulation or eVen to promote regression of fatty streaks.
Therefore the effect of EPL on the metabolic turnover of lipids
was studied using a new technique of perfusion of the intact
rabbit aorta. Segments of the aortas were perfused in vitro
under physiological conditions of temperature, blood flow and
blood pressure. Two aspects of arterial metabolism were studied
in the normal rabbit aorta: (1) the effect of EPL on synthesis
of lipids during a one hour perfusion with 3H-acetate in normal
rabbit plasma and (2) the effect of EPL on the efflux of previously endogenously synthesized lipids during a one hour perfusion with a buffer solution containing serum albumin as a fatty
acid acceptor. The following results were obtained:
1) Following perfusion in the presence of EPL. the concentration
of lysolecithin (LPC). phosphatidyl serine (PS) and phosphatidyl
ethanolamine (PE) were significantly reduced. There were no
changes in cholesterol or other lipid fractions. This suggests
that the presence of a micellar suspension of the polar EPL in
plasma may withdraw phospholipids from the arterial wall, especially those associated with the plasma membrane.
2) Perfusion in the presence of EPL caused a significant reduction in the incorporation of acetate into the free fatty acid
(FFA) pool of the artery. In general the mean values for incorporation into other lipids were also lower, but did not reach
statistical significance. EPL may reduce synthesis of FFA or reduce the amount of newly synthesized fatty acid in the arterial
wall by favoring efflux.
3) The absolute rates of lipid synthesis as estimated from the
specific activity of the free fatty acid pool did not differ significantly between the control and the EPL groups.
4) EPL was found to increase the mole fraction of lyso-lecithin
(LPC) and cholesteryl esters (CE) metabolically labeled by the
newly synthesized fatty acid. This result reveals that EPL caused
more of the available FFA to be diverted into LPC and CE, al-
161
though the incoporation of acetate into the FFA pool of the artery ~as reduced.
5) The newly synthesized fatty acids are removed from the artery
by a buffer perfusion. This removal appears to be increased by
the presence of EPL.
INTRODUCTION
Lipid accumulation is a characteristic feature of most types of

atherosclerotic lesion, except early focal gelatinous sub-endothelialoedema (1). The composition of accumulating lipid and its
location in the arterial wall varies, however, from one morphological type of lesion to another. It has been shown by SMITH et
al. (2), and confirmed by others (3) that in human fibrous plaques
the lipid is mainly extra-cellular and in association with collagen and elastic fibers. This so called perifibrous lipid is composed mainly of sphingomyelin, free cholesterol and cholesteryl
linoleate. The similarity of its composition to that of the plasma low density lipoprotein suggests that it accumulates by deposition from the plasma. On the other hand, in fatty streaks,
lipid accumulates mainly as intracellular droplets in fat-filled
cells. This lipid is mainly cholesteryl oleate (2,4), some free
cholesterol and various phospholipids.
There are a number of possible explanations for the or~g~n of
the intracellular lipids. On the one hand, they may be derived
by uptake of extracellular lipids with preferential retention of
molecules such as cholesteryl oleate. On the other hand they may
be derived by synthesis in situ from small molecular weight precursors such as acetate and glucose, or by assembly from fatty
acids and cholesterol derived from the plasma.
With respect to phospholipids, early studies by CHERNICK et al.
(5) showed that they could be synthesized by the arterial wall.
ZILVERSrlIT et al. (6) later showed that the majority of phospholipids of fatty streak lesions are probably derived from in situ
synthesis. This conclusion has been upheld by more recent studies
(7) except for sphingomyelin, which has a very low rate of synthesis as judged by incorporation of orthophosphate and fatty
acid (8). Sphingomyelin is probably derived by deposition from
plasma followed by association with intracellular free cholesterol (9).
With regard to cholesterol, it appears that the arterial wall
synthesizes the sterol nucleus slowly if at all, depending upon
the species (10). It is however, able to catalyze the assembly
of cholesteryl esters from free cholesterol and fatty acid by a
number of mechanisms:
1) by acylation of cholesterol with fatty acyl CoA (8,11,12,13)
a reaction requiring energy derived from hydrolysis of ATP and
having a pH optimum of about 7.4.
2) by the reversal of the normally catabolic reaction of sterol
ester hydrolase (14) a reaction not requiring energy which has a
162
pH optimum of 6.2, but probably only occurs in the presence of

high concentrations of reactants.
3) putatively, by an arterial wall lecithin: cholesterol acyl
transferase (15). This mechanism has not received widespread acceptance. It probably only occurs in the presence of high concentrations of reactants or at water-oil interfaces in vitro as described by ABDULLA et ale (15), or as a result of contamination
of arterial samples with plasma enzyme. Its physiological significance remains in doubt.
Various studies have attempted to measure the importance of in
situ synthesis of lipid in contributing to its accumulation in
fat-filled cells. For example:
1) In vivo studies of cholesterol esterification in w~ole tissue.
DAYTON and HASHIMOTO (16) labeled the plasma and arterial wall
of cholesterol-fed rabbits by including the unnatural fatty acid,
elaidic acid, in the diet. The ratio of atheroma to serum elaidic
acid in cholesteryl esters was used to estimate the contribution
of in situ synthesis. A mean value of 52% was found. An objection
to this experimental approach is that elaidic acid being an unnatural trans fatty acid, might have a different rate of esterification and hydrolysis and therefore turnover, compared with
oleic acid, its natural isomer. In order to overcome this disadvantage, DAYTON and HASHHI0TO (17) also used dietary 3H-oleic
acid. In this case they found that 19% of the atheroma cholesteryl esters could be accounted for by in situ synthesis. This vTas,
however, considered an underestimate, for two reasons. Firstly,
because other arterial lipids, such as triglyceride, were labeled
and could have contributed oleic acid directly to cholesteryl
ester and secondly, because some of the labeled cholesteryl
esters synthesized in the arterial wall might have been returned
to the plasma.
2) In vitro studies of cholesterol esterification in whole tissue. These have shown that in experimental animals and birds such
as rabbits and pigeons, the rate of incorporation of exogenous
fatty acids and endogenous fatty acids synthesized from acetate
into the cholesteryl esters of fatty streaks is greater than in
normal tissue (13,18,19,20,21).
There is therefore, unequivocal evidence that phospholipid and
cholesteryl ester may be synthesized in the arterial wall and.
account for a significant proportion of the intracellular lipid
of fatty streaks. Thus modification of arterial lipid metabolism
by drugs, especially inhibition of synthesis might be expected
to reduce lipid accumulation in the face of atherogenic stimuli
such as hypercholesterolemia and hypertension, and even to promote the regression of establisheJ fatty streaks.
Previously, studies of the effect of EPL on arterial metabolism
have concentrated largely on the alteration of the activity of
arterial enzymes as measured in vitro. They have suggested that
the depression of sterol ester hydrolase and elevation of triglyceride hydrolase observed in developing fatty streaks is relieved
by injection of EPL during the induction of lesions (22,23).
163
These res~lts are, however, sonewhat equivocal. Firstly, because

of nethodological difficulties involved in measuring low activities of these enzymes, and secondly, because the nethod of the
presentation of the lipid substrates in vitro profoundly affects
the observed enzyne activity (24). Thus extrapolation of the
results to the physiological effects of EPL in the whole artery,
is difficult. In order to try to overcome this problem we have,
therefore, begun a systematic study of the effect of EPL on the
metabolic turnover of lipids of rabbit aortas by a perfusion technique of the intact artery. Segments of aorta are perfused in
vitro under physiological conditions of temperature, blood flow
and blooo pressure.
The perfusion technique permits an analysis of the synthetic capabilities of the arterial wall in the situation where the normal anatomical architecture, at least of the lumen intimal surface, is undisturbed. Thus the i:nportant role of the endothelium
in regulating transfer of metabolites between the luminal per~usate and wall is preserved. In addition to these advantages,
this technique provides a method for the investigation of a hitherto unstudied question, !'lamely, the possibility that EPL promotes efflux of arterial lipidS, synthesize6 by the artery, into
the blood.
In the experiments reported here, we have studied the effect of
EPL on two aspects of arterial metabolism in nornal rabbit aorta.
Firstly, the effect of EPL on synthesis of lipids during a one
hour perfusion with 3H-acetate in normal rabbit plasma, together
with the efflux of labeled lipids into the perfusate. Secondly,
the effect of EPL on the efflux of previously endogenously synthesized lipids, into a buffer solution containing serum albumin
as a fatty acid acceptor.
MATERIALS AND METHODS

1.
Arterial Perfusion
Aortas of New Zealand white rabbits, 12 - 16 weeks of age, were

perfused essentially as described previously (25,26). Animals
were killed by a blow on the head and the whole aorta rapidly exposed. Polyethylene cannulas (Portex, U.K.) were inserted into the
aorta, one at the level of the scar of the ductus arteriosus and
the other 2 cm above the bifurcation. The blood was washed out
with Krebs bicarbonate buffer containing 4g/dl defatted Bovine
Serum Albumin (BSA, Cohn F5action V, Armour, U.K.), gassed with
air + 5% C02 pH 7.45 at 37 C. The composition was 100 ml HaCl,
4.0 ml KCL, 1.0 ml KH2-P04, 1.0 ml MgS04 7H20, 21.0 ml NaHC03'
all 0,154 molar, further 1.5 ml of 0.11 M CaC1 2 , and 1.1 ml 0.3 M
glucose. To re-establish Ca++ ion concentration, the medium was
dialyzed against albumin-free buffer for 16 hours at 4C.
All of the side branches were ligated with 3/0 silk (Hersilk,
Ethicon, U.K.). Occasionally more buffer solution was flushed
164
through the aorta and the outside was kept Moist with 0.9% saline.
The aorta was then removed and tested for leaks by clamping the
abdominal cannula and increasing the pressure of the buffer solution to 100 rom mercury. Any leaks were closPd by ligation. Thp
aorta was then nounted in the perfusion apparatus, which is illustrated in Figure 1, and extended to its normal length as measured in vivo. Perfusion was then begun and the pressure adjusted
to 110 rom of mercury systolic and 80 rom of mercury diastolic pressure.
To pressure
ransducer
--
perfusine medium
(res rvoir)
I direction
pulsatile
Fig.
1:
of
flow
' - - - - gas in
Diagram of apparatus for perfusion of arteries at phYSiological pressure
165
2. Extraction Separation and Estimation of Arterial and Perfusate Lipids
All solvents and reagents were AnalaR reagents. Solvents were

further purified by redistillation.
Following perfusion, segments of aorta were opened and rinsed
twice with 50 ml of a wash solution, containing 10% w/w sodium
acetate (CH3COONa5 H20), and the excess wash removed with a
filter paper. Intima plus approximately one half of the media
was then stripped from the artery, weighed and frozen in 1.5 ml
0.9% w/w saline. This sample was then disrupted by freeze pressing (27) and the lipids extracted essentially by the procedure
of FOLCH et al. (28) as follows:
The pellet was placed in a weighed folded glass fiber filter disc
Olhatman GF/A) and placed in a stoppered tube containing 10 ml
methanol. 20 ml of chloroform was then added and the saMple extracted for 1 hour at room temperature with occasional swirling.
The sample in the filter disc was then extracted with a further
30 ml of chloroform : methanol (2 : 1 v/v) for 1 hour at 40 0 C.
The extracts were then combined, 12 ml of 0.9% saline added, and
the two phases allowed to separate overnight at 40 C in a tap
funnel. The lower chloroform phase was then washed with a pure
upper phase (chloroform: methanol: 0.9% saline, 3 : 48 : 47)
to remove contaminating acetate and the upper phase washed with
pure lower phase (chloroform: methanol: 0.1N HCl, 86 : 14 : 1
v/v) to prevent loss of free fatty acids. The lipid-containing
lower phases were then combined and evaported to dryness on a
rotary evaporator and stored under nitrogen in benzene at -20 0 C
until required for thin-layer chromatography. The filter discs
were then weighed to give the mass of dry defatted tissue and
then the DNA content measured by the method of BURTON (29). Thinlayer chromatographic separation was performed by a new and rapid
micro-technique (30) using 76 x 76 cm plates with 250 ~ layers
of silica gel (Camag, type DO without binder from Gallenkamp,
U.K.) impregnated with 0,25% w/w ammonium sulphate for phospholipid separation and 0.1% w/w sodium carbonate for neutral lipid
separation. The conditions for chromatography and typical separations obtained are shown in Figures 2 and 3. Following chromatography, lipids were revealed by exposure to iodine vapour and
assayed by specific micro-chemical methods for cholesterol (31)
for free fatty acid (32) for glycerides (33), and for phosphorus
(34). For radioactivity determinations, silicic acid was scraped
into 10 ml scintillator containing toluene : triton X100 : water,
100 : 50 : 15 v/v plus PPO (4 g/~) and POPOP (0.2 g/~). This
elutes even the polar phospholipids into solution, thereby preventing self-absorption losses on the silicic acid. Samples were
counted in a Nuclear Chicago Mk II scintillation counter for 10
min, or until 10,000 counts had been accumulated. All of the
data from assays and scintillation counter were processed by a
special computer programm (35), to give the mass of acetate incorporated per unit of tissue for each lipid class and also the
specific activity of each lipid (i.e. the mole fraction of each
lipid labeled by radioactive precursor).
166
Nl
PE
PE
X
PI
PI
PS
PS
lPE
PC
11
SPH
p. ethanolamine
p. serine
Iyso p. ethanolamine
lecithin
p. inositol
SPH -
sphingomyelin
LPC
lysolecithin
neutral lipids
unknown
LPE
PC
lPC
NL
origin
(p. - phosphatidyll
Solvent 1 - chloroform: methanol: acetic acid: water :: 55: 35: 3: 2 v/v

(running time - 12 min)
Solvent 2 - chloroform: methanol: acetone: acetic acid: water :: 45: 15: 16: 11 : 6 v/v
(running time - ca 20 min)
Layers
- 76 x 76 x O. 25 mm silicic acid impregnated with ammonium sulphate
Fig.
2
CE
TG
DG
C
ME
H - hydrocarbons
CE - cholesteryl esters
ME - methyl esters
NX - unknown
TG - triglycerides
DG - diglycerides
C - cholesterol
F - free fatty acids
MG - monoglycerides
MG
o -
phosphol ipids
Solvent 1 - petrol: ether: acetic acid :: 40 : 60 : 0.1 v/v

Solvent 2 - petrol : acetic acid : ethyl acetate :: 95 : 2: 2 v/v
Layers
- 76 x 76 x O. 25 mm silicic acid impregnated with sodium carbonate
Fig. 3
167
EXPERIMENTAL DESIGN AND RESULTS
1. Effects of EPL on the I ncorporation of 3H acetate into Arterial and Perfusate Lipids (Experiment I)
The incorporation of 3H-acetate into lipids was determined during

a 1 hour perfusion with heparinised rabbit plasma. Three experimental groups were set up as follows: Group 0 : No artery. 10 ml of pooled heparinised normal rabbit
plasma containing 0.68 mM 3H-acetate (3 H- U- sodium acetate, TRA.
Radiochemical Centre, Amersham, U.K. specific activity 250.AICij.
~mole) was pumped around the perfusion apparatus for 1 hour. This
group was used to control for incorporation of 3H-acetate into
perfusate lipids other than by activity of arterial enzymes, by,
for example plasma or blood cell enzyme activity, bacterial contamination, non-specific 3H-exchange.
Group 1
Artery, control. 5 aortas were perfused for 1 hour,
each with 10 ml perfusate as above.
Group 2
Artery, EPL. 5 aortas were perfused for 1 hour each
with 10 ml perfusate as above, plus EPL (NATTElli1ANN), 10% solution of EPL, added in vitro to give a final concentration of
1 mg/ml.
Table 1 shows the amount of acetate, which was found to contaminate each lipid when plasma containing 3H-acetate was extracted without having been perfused (pre-perfusate) and following
one hour perfusion without an aorta in the system (group 0). The
results are expressed as pmoles of acetate in each lipid class
per ml of perfusate. They were calculated assuming that there
was no dilution of 3H-acetate by cold endogenous acetate of the
plasma and, therefore, that the specific activity was 250 ~Ci/
~mole.
It can be seen from the pre-perfusate sample that, despite Folchwashing of the lipid extracts of the perfusate samples, some contamination of the phospholipids and neutral lipids occurred. The
amount is similar throughout all o~ the lipid classes, about 3
pmoles acetate in each lipid from 1 ml of perfusate.
When perfusate was circulated in the perfusion apparatus for 1
hour, the, activity associated with the free fatty acid (FFA)
fraction was about 60 pmoles/ml, i.e. a 20 fold increase, but
the amount contaminating the other lipids was similar to that
found in the pre-perfusate sample. This suggests that activity
may be transferred to the fatty acid, either by tritium exchange
or more likely by de novo synthesis of fatty acid by residual
blood cells, or contaminating micro-organisms. As will be shown
below, however, the amount of acetate in the free fatty acid
fraction in 1 ml perfusate perfused through an aorta is about
1,800 pmoles, i.e. 30 fold greater than the blank value.
...
Fig.
2:
Phospholipid separation by 2 dimensional thin layer chromatography on small plates
Fig.
3:
Neutral lipid separation by 2 dimensional thin layer chromatography on small plates
168
Table 1: Amount of 3H - acetate found to contaminate each lipid after separation of lipids of pre - perfusate
and post - perfusate as estimated assum ing specific activity of 250 }lei /}l mole
pM acetate/ml perfusate
Phospholipid plate
Lipid
Neutral lipid plate

Lipid
pre
post
pre
post
26.37
58.91
Origin
2.18
0.78
Origin
LPC
0.59
3.11
MG
SPH
2.56
2.14
FFA
3.10
62.34
PC
2.09
5.77
CHOL
3.04
1. 42
LPE
0.85
5.15
DG
PS
1. 71
2.72
TG
2.40
2.20
PI
ME
PE
7.39
CE
0.51
0.71
PA
0.94
0.78
19.22
13.14
NL
REST
19.42
0
20.68
156.54
6.45
30.10
REST
The concentrations of the major arterial lipids in the control

and EPL groups after perfusion are shown in Figures 4 and 5. Following perfusion in the presence of EPL, the concentration of
lysolecithin (PLC) , phosphatidyl serine (PS) and phosphatidyl
ethanolamine (PE) were significantly reduced. There were no
changes in cholesterol or other lipid fractions.
Endogenous synthesis of lipids as measured by acetate incorporated
into each lipid in the perfused arteries, is shown in Table 2 for
the control group 1 and the EPL group 2. In the EPL group there
was a statistically significant reduction in the incorporation of
169
20
0
0
18
"
plasma only
5 animals
plasma plus EPl (l mg/ml)
5 animals
16
r-
14
12
nmolesl
mg dry
4
2
o
Fig. 4:
without
significant difference P<O. 05
**
sign ificant difference P< O. 01
t-
8
6
....
....
....
..
r-
I- **-1
f:":"
r-
..,,
....
1-*-1
t-
..
......
....
..
..
....
..
......
......
..
SPH
PC
LPE
..
tl
LPC
..
.."
..
r--
..
"
"
..
"
"
t-*,
nm
..
"
PS
PI
r--
f:-:"
..
..
..
"
PE
Phospholipid concentration in aortas perfused with heparinised normal rabbit plasma with and
EPL
10
plasma only
plasma plus EPl (l mg/ml)
8
7
6
nmolesl
mg dry
4
3
r-f-
2
1
..
rb
o
FFA
Fig. 5:
without
OG
Ib
..
TG
CE
Neutral lipid concentrations in aortas perfused with heparinised normal rabbit plasma with and
EPL
170
Table 2: Lipid synthesis from 3H acetate in normal rabbit aorta during 1 hour perfusion with heparinised
normal rabbit plasma. Incorporation of acetate in pM / mg dry defatted tissue / h .. S.E.M. Specific
activity of acetate 250 pCi / pM. P = probability from unpaired T test using untransformed data or, where
appropriate, logarithmically transformed data to stabilize variances; P > 0.05 unless otherwise shown
Group 1
Group 2
Control
EPL
LPC
0.056 0.020
0.241 0.100
SPH
0.083 0.016
0.082 0.031
PC
1.046 0.404
0.435 0.204
LPE
0.459 0.341
0.079 0.026
PS
0.160 0.049
0.097 0.029
PI
0.302 0.068
0.131 0.040
PE
0.141 0.040
0.108 0.040
Total of
Phospholipids
2.27
1.17
FFA
0.551 0.082
0.253 0.039
DG
0.870 0.200
0.673 0.230
TG
0.408 0.103
0.337 0.128
CE
0.029 0.014
0.023 0.005
Total of
Neutral
lipids
1. 86
1.49
0.50
0.41
0.43
<0.05
0.36
acetate into the FFA fraction. Calculation of the total amount

of activity in the phospholipid and neutral lipid fractions of
the artery shows that the mean values were lower in the EPL group,
but the difference failed to achieve statistical significance.
This suggests that EPL may act to reduce the synthesis of FFA in
the arterial wall and/or favor efflux of newly synthesized fatty
acid. In either case the amount of complex lipid synthesized was
probably also reduced.
There are a number of possible mechanisms by which EPL may have
decreased the rate of arterial fatty acid synthesis: -
171
1) EPI might inhibit the penetration of acetate through the endothelium into the arterial wall. No measurement of the specific
activities of acetate in the perfusate and wall were made and,
therefore, this hypothesis could not be tested. It is thought
that an alteration of the fatty acid composition of cellular
~hospholipids can alter the permeability of the plasma membrane
to small molecules, and furthermore, may affect endocytosis of
large molecules. It is unlikely in this experiment, however, that
exposure of the artery to EPL for only 1 hour would cause a significant change in the fatty acid composition of the phospholipids. A reduced influx of acetate into the artery is therefore,
unlikely.
2) EPL may be taken up into the cells and inhibit either cytoplasmic fatty acid synthesis or mitochondrial fatty acid chain elongation. We have no evidence, from this experiment, for this effect.
3) The ac.di tion of EPL to the heparinised plasma used as perfusate, nay increase the concentration of FFA in the tissue and,
thereby, decrease fatty acid synthesis by inhibition of acetyl
eoA carboxylase. As was shovTn in Fig. 3, no difference was detected between the control group 1 and the EPL group 2 in the
concentration of FFA in the arteries.
1I.nalysis of the concentration of FFA in the perfusates, hO\'lever,
shows that there was a significantly higher concentration of FF]I.
in the perfusate of the EPL <Jroup compared 'Ili th the control group
after 1 hour perfusion. The analyses are shown in Table 3. Thus,
inhibition of FFA synthesis in the arteries of t.he EPL group could
have been brought about by the increased FFA in the perfusate. As
expected, the concentration of lyso-Iecithin (LPC) and lecithin
were also higher in the EPL group and either, or both, of these
could have an inhibitory effect on FFA synthesis, as discussed
above. It should be noted that the higher concentration of FFA
and LPC in the perfusate after perfusion cannot be accounted for
solely on account of that added in with the EPL. (The measured
mole percentage composition of EPL was PC 90.4%, LPC 7.3%, FFA
2.1%). Some hydrolysis of the EPL lecithin MUSt have occurred
during the perfusion with heparinised plasma. Indeed, the molar
amounts of FFA and LPC formed were approximately equivalent to
the amount of PC hydrolyzed.
It would obviously be interesting to discover which constituent
of the EPL perfusate is the most important in reducing arterial
fatty acid synthesis. This could be established with respect to
LPC and FFA by a determination of their relative inhibition constants (K i ) using the perfusion technique. It would also be i~
portant to determine the Ki for fatty acids of different chain
lengths, and unsaturation. One might speculate that linoleic acid
(18: 2) \vhich is produced during hydrolysis of the lecithin of EPL,
but is normally a very small percentage of the plasma FFA, miqht
have a very different Ki from oleic (18:1) and palmitic (16:0)
acids, which are the major FFA of the plasma.
th respect to inhibition of arterial FFA synthesis, one should
further consider that inhibition might be produced as a result
of the local release of FFA from EPL in the artery without any
increase in plasma FFA. The effectiveness of such a process would
~.;ri
172
Table
3: The concentration of lipids in the perfusate following 1 hour perfusion. Concentrations in
"oM / ml, (i.e. mM) .!. S.E.M. P as in Table 2
Group 1
Group 2
Control
EPL
LPC
0.062 0.02
0.375 0.09
PC
0.310 0.02
FFA
Difference
Group 2 - 1
Composition
of added EPL
and (percentage purity)
<0.01
0.313
0.073 (7.3%)
0.966 0.18
<0.01
0.656
0.906 (90.6%)
0.801 0.09
1.099 0.12
<0.05
0.545
0.021
CHOL
0.226 0.01
0.221 0.02
CE
1.128 0.08
1.050 0.06
(2.1%)
Table 4: Lipid synthesis from endogenously synthesized fatty acid in normal rabbit aorta, and a comparison
with the rate of utilization of exogenous fatty acid. Utilization of FFA as measured from specific activity
of arterial FFA pool after 1 hour; pM / mg dry defatted tissue / h .!. S.E.M. P as in Table 2
Group 1
Group 2
Control
EPL
0.5)JE / ml
Specific
activity
FFA
pCi / pM
0.046 0.008
0.023 0.002
LPC
446 96
2799 1258
305 111
SPH
453 42
1003 490
46 22
PC
7437 1198
4657 1878
3033 904
LPE
817 155
973 223
974 204
PS
922 281
1095 315
845 167
PI
1649 235
1466 454
3583 695
PE
789 163
1172 389
1694 251
DG
4590 572
5908 2404
TG
2186 432
2430 969
3670 525
CE
175 89
194 23
256 69
<0.05
Exogenous FFA
173
depend upon the specificity of the arterial phosholipases. There

is indeed already some evidence for the more ready hydrolysis of
linoleoyl phosphatidyl choline compared with oleoyl or palmitoyl
lecithin. Such a mechanism of inhibition of arterial FFA synthesis would obviously have the important biological advantage of
not requiring an elevation of plasma FFA with its consequent undesirable effects, such as elevation of myocardial oxygen consumption and possible direct damage to the endothelium.
The endogenous synthesis of complex lipids was measured from the
specific activity of the arterial FFA pool. Acetate is mainly
converted in the arterial wall into FFA which is then incorporated into complex lipids. Thus the specific ac.tivity of the FFA
pool allows a calculation of the absolute rates of synthesis of
each lipid class. Table 4 shows the rates, in pM/mg of dry defatted tissue per hour, incorporated into each lipid as derived
from the measured specific activity of the FFA pool at the end
of the 1 hour perfusion. The specific activity of the FFA in the
EPL treated group was significantly lower than in the control
group. This is consistent with the decreased incorporation of
acetate into FFA, without change of pool size produced by EPL,
as shown above (Figures 4 and 5 and Tables 2 and 3). The rate
of the subsequent utilization of FFA was not, however, significantly affected by EPL. The rates of utilization of endogenously
synthesized FFA compare very closely with rates of utilization of
exogenous fatty acid measured in a different experiment by perfusion with 3H-oleic acid bound to serum albumin (34).
Although the synthetic capacity of the tissue for the various
lipids can be expressed per unit weight of dry defatted tissue
(Tables 2-4), it is also very informative to consider the fraction of any lipid pool which is metabolically labeled by the newly synthesized fatty acid. This gives an estimate of the 'rate
of turn over or metabolic activity' of that lipid. The results,
calculated from the specific activity of the tissue FFA pool,
are shown in Table 5.
In the phospholipids the relative rates of 'turn over' were in
accord with those measured before, using other precursors such
as glycerol (36) orthophosphate (8, 26, 37, 38, 39) and FFA (8,
39, 40, 41). Particularly notable is the low turn over of sphingomyelin, which accumulates both with age in lesion-free intima and
also in fibrous plaques. This low turn over is problably a reflection of the low activity of sphingomyelin hydrolase. Of all of
the phospholipids in the control group, phosphatidyl inositol
(PI) had the highest turn over rate. EPL significantly increased
the turn over rate of lyso-phosphatidyl choline (LPC). Among th7
neutral lipids, the turn over of cholesterol ester (CE) was significantly increased by EPL.
The results reveal that although EPL reduced the incorporation
of acetate into the FFA of the artery, it caused more of the LPC
and CE pools to be labeled during the 1 hour perfusion. At first
sight, such a result could be interpreted to mean that EPL might
174
Table 5:
Lipid synthesis from newly synthesized fatty acid in normal rabbit aorta. The mol fraction of each
lipid labeled by newly synthesized fatty acid.
Rates in nM
FFA / nM of each lipid in the artery / h
S.E.M. P as in Table 2
Group 1
Group 2
Control
EPL
LPC
0.134 0.02
3.499 1.71
SPH
0.071 0.01
0.230 0.11
PC
0.561 0.15
0.537 0.19
LPE
0.115 0.03
0.220 0.06
PS
0.197 0.07
PI
1.089 0.56
0.966 0.39
PE
0.319 0.12
0.464 0.12
TG
0.184 0.06
0.340 0.18
CE
0.112 0.06
0.669 0.17
<0.01
0.302 0.15
<0.05
Table 6: The incorporation of activity from 3H - acetate into the lipids of perfusate during a 1 hour
perfusion of normal rabbit aortas. pM acetate / ml perfusate / h. (There were no significant differences
between groups)
Group 1
Group 2
Control
EPL
Blank
(see Table 2)
LPC
5.98
1 13
7.15
1. 37
3.11
PC
17.63
4.25
25.53
9.61
5.77
229.69 101.02
62.34
FFA
344.65 40.82
TG
2.29
0.54
0.74
0.26
2.20
CE
2.10
0.54
0.68
0.26
0.71
175
lead to the accumulation of these two lipids in vivo. This would

not, however, be the case if the enhanced utilization of FFA in
syntesis of the lipids were balanced by increased catabolism. Indeed the enhanced turnover might be an expression of a stimulated
catabolism of lipid, which would be in accord with previous suggestions that EPL stimulates arterial sterol ester hydrolase (23).
The reduced activity observed in the FFA of the artery during
perfusion in the presence of EPL might be due to an enhanced
efflux of newly synthesized FFA. In order to test this hypothesis
the amounts of acetate incorporated into the lipids in the perfusate were estimated. The results are shown in Table 6. They
have been calculated as the mass of acetate in each lipid fraction. The blank values for a perfusion without artery (from
Table 1) are also shown. Most of the activity was present in the
FFA fraction although some fatty acid was found in complex lipids. This may have occurred as a result of egress of labeled
complex lipids from the artery or, more likely, as a result of
incorporation of fatty acid into complex lipids, catalyzed by
the endothelial cells. There was no significant effect of EPL
on the total amount of activity from acetate in the perfusate
or on the activity in any lipid.
It is clear that the arterial wall releases some of the newly
synthesized fatty acid into the perfusate. This finding suggests
a further mode of action of EPL in addition to inhibition of
fatty acid synthesis, namely, enhanced efflux from the arterial
wall. If this phenomenon were to be significant in vivo then arterial synthesis of complex lipids would be reduced by EPL and
their accumulation in atherosclerotic lesion might be prevented.
In order to confirm the authentic labeling of FFA with 3H from
3H-acetate, (i.e. to verify that activity is chemically associated with the product, not merely an entrained contaminant), the
FFA fraction from a number of TLC separations of both arterial
and perfusate lipids were analysed as follows: The FFA were converted to their methyloesters (ME) by heating in 5% w/w sulphuric
acid in methanol at 80 C for 1 hour. The methyl esters w~re extracted from the methanol with petroleum ether (BP 60-80 C) and
then chromatographed on TLC. The resultant ME spot was then counted by scintillation counting. The overall efficiency of this
process, checked an internal standard of 14C-oleic acid was found
to be 33%. Allowing for this recovery, 100% of the activity of
the original FFA was recovered as ME. Preliminary investigations
by gas-liquid chromatography (GLC) have shown that the synthesized fatty acids are mainly myristic (60%) and palmitic (40%)
acids.
2.
Effect of EPL on the Efflux of Lipids, Including Free Fatty Acids, from the Arterial Wall (Experiment 2 )
In this experiment normal rabbit aortas were perfused with pooled

heparinised normal rabbit plasma containing 1rnM 3H-acetate (details see above) for 1 hour in order to 'pulse' label the arterial
176
lipids. The arteries were then washed by perfusion for 1 minute

as described below and a piece of aorta sampled for analysis. The
remaining artery was then 'chase' perfused for a further 1 hour.
At the end of the 'chase' perfusion, the artery and perfusate
were analyzed as described below. Two groups containing 5 animals
per group were studied as follows:
Group 1
Wash and 'chase' perfusates were Krebs bicarbonate
buffer containing bovine serum albumin (4g/100ml) gassed with
air + 5% CO 2 as described in the Methods section.
Group 2
Wash and 'chase' perfusates were as in Group 1 plus
EPL (1 mg/ml).
The concentrations of lipids in lower arterial segments sampled
after 'pulse' and in the upper segments sampled at the end of the
'chase' are shown in Table 7. Statistical analysis by unpaired Ttests revealed, as expected, that there was no significant difference between the concentrations of lipids in the pre-chase
samples of the control group 1 and the EPL group 2. When the lipid concentrations in the 'post-chase' samples were compared with
the 'pre-chase' samples within the control group 1, and within
the EPL group 2, by paired T-test, a significant increase in LPC
was found following the chase in the control group 1. In the EPL
group 2 there was a significant fall in the concentrations of
Table 7: Concentration of lipids in lower segments of arteries sampled after .. pulse" and upper segments
after .. chase" perfusions. Mean values nM / mg dry defatted tissue 2:. S.E.M. P = Probability (as in
Table 2) that the difference between .. pre - chase" and .. post - chase" samples was due to chance
Group 1
Group 2
Control
EPL
Pre-chase
(Lower)
Post-chase
(Upper)
Pre-chase
(Lower)
Post-chase
(Upper)
LPC 0.524 0.28 1.000 0.30 <0.05 0.765 0.28 0.374 0.17
SPH 3.635 0.95 3.988 0.47
3.986 0.30 3.103 0.32 <0.01
PC
8.192 1. 94 8.583 0.74
9.855 0.55 7.834 0.75
LPE 1.843 0.42 2.551 0.67
1.902 0.41 1. 455 0.40
PS
1.730 0.43 1.945 0.29
1. 686 0.28 1.457 0.32
PI
1.558 0.59 1.244 0.20
0.155 0.22 0.950 0.25
PE
2.871 1.15 3.162 0.77
4.067 0.32 3.530 0.11
FFA 3.999 1. 35 4.340 0.71
3.837 0.66 2.233 0.76
TG
9.862 3.00 5.016 0.84
5.421 0.45 2.302 1.38 <0.01
CE
0.243 0.07 0.239 0.08
0.256 0.05 0.186 0.03
177
sphingomyelin (SPH) and triglyceride (TG). It is difficult to

assess the biological relevance of the slight fall in sphingomyelin concentration. Presumably the decrease in triglyceride
concentration reflects the hydrolysis of triglyceride by the
arterial triglyceride hydrolase. This may be activated by the
relatively non-physiological nature of the bicarbonate buffer
used in the chase perfusion and/or by the EPL.
The incorporation of 3H-acetate into arterial lipids after the
1 hour 'pulse' perfusion and at the end of the 1 hour 'chase'
perfusion is shown in Table 8. The mass of acetate per mg dry
defatted tissue incorporated into each lipid after the 1 hour
'pulse' perfusion and remaining after the further 1 hour 'chase'
are presented. As was expected, there were no significant differences in the amounts of acetate incorporated into the lipids in
the two groups during the 'pulse' perfusion. Neither could any
significant difference be demonstrated, using a paired T-test,
in the amount of activity left in each lipid class after the
chase in either the control group 1 or the EPL group 2. It was
noted however, in both groups that the mean values of the activity from acetate in FFA after the chase were lower than in the
'pre-chase' samples, and in general the mean values of the other
lipids were higher. This suggests that the activity of FFA
Table 8: Incorporation of 3H - acetate into lipids of .. pre - chase" and .. post.' chase" arterial segments.
Mean pM acetate / mg dry defatted tissue / h of pulse. Following statistical analyses by paired T tests.
there were no significant differences between the .. pre - chase" and .. post - chase" values in either group
Group 1
Group 2
Control
EPL
Pre-chase
Post-chase
Pre-chase
Post-chase
LPC 0.719 0.32
0.492 0.22
0.292 0.12
0.284 0.10
SPH 0.349 0.13
0.661 0.39
0.337 0.12
0.532 0.29
PC
5.729 1.08
7.224 3.49
5.395 2.06
6.124 2.03
LPE 0.348 0.17
0.205 0.08
0.278 0.09
0.302 0.16
PS
0.452 0.11
0.423 0.15
0.364 0.13
0.418 0.18
PI
1 .193 0.49
1.048 0.61
0.949 0.29
0.731 0.26
PE
0.857 0.32
0.939 0.40
1.176 0.58
1.104 0.36
FFA 1.723 0.43
1. 257 0.55
1.177 0.44
0.584 0.17
TG
5.319 2.23
1.924 0.70
2.034 0.70
1.798 0.61
CE
0.092 0.02
0.113 0.05
0.065 0.02
0.091 0.04
178
was falling because of removal of the hot 3H-acetate percursor
and also, because the labeled FFA pool was being depleted by
virtue of synthesis of complex lipids.
As was described for experiment 1, an estimate of the rate of
synthesis of each complex lipid from the FFA pool of the tissue
may be made from the measured value of the specific activity of
the newly synthesized FFA. This is, of course, only an estimate,
because one must assume that each lipid is labeled directly by
the FFA and also that the measured value of the specific acti-
vity of FFA is the same as that of the immediate precursor pool
of the complex lipids. Table 9 shows the values for the measured
specific activity of the arterial FFA and also the rates of incorporation of fatty acid into complex lipids, derived from these
specific activities. As expected, no significant differences were
detected between the 'pre-chase' segments of arteries of the control group 1 and the EPL group 2. Statistical analysis by paired
T-tests failed to reveal any significant change in the specific
Table 9:
the arterial
Utilization of fatty acid in complex lipid synthesis calculated from the measured specific activity of
FFA
pool.
Mean values pM
mg dry defatted tissue
No significant differences between pre and post
h of perfusion
Group 1
Group 2
Control
.:I:. S.E.M.
(see Table 8)
EPL
Pre-chase
Post-chase
Pre-chase
Specific
activity 0.149 0.093 0.076 0.025
pCi/pM
Post-chase
5
0.090 0.038 0.071 0.018
LPC
3465 1317
1278 318
1271 524
1306 557
SPH
1025 358
1828 615
1029 165
2197 1371
21244 6762
21343 5335
17816 5200
23889 9251
PC
LPE
805 173
738 139
864 102
1336 749
PS
1876 539
1695 380
1140 272
1756 868
PI
5716 2674
1820 860
3775 1080
2737 1120
PE
3609 1392
1985 368
3948 1729
4085 1831
'IG
11494 4967
7798 2809
8800 3245
7209 2818
CE
292 93
372 39
273 88
357 132
179
activities or rates of incorporation of newly synthesized fatty

acid during the 'chase ' in either the control group 1 or the EPL
group 2.
It was interesting to note that in both groups the mean values
for most phospholipids and cholesteryl ester were higher following the 'chase ' and for phosphatidyl inositol and triglyceride
lower following the 'chase ' One might speculate that if the
'chase ' perfusion could be maintained for longer and the variability of the system reduced, then one would find a relatively
slow egress of fatty acid from lipids such as sphingomyelin,
which turn over slowly, and a rapid egress from lipids such as
phosphatidyl inositol which turn over rapidly and are localized
pri~arily in membranes. We propose (see the discussion below)
that a relevant experimental model for maintenance of a long
'chase ' could be achieved with an organ culture of everted aortic
segments.
Table 10 shows the mole fraction of each lipid labeled by the
newly synthesized fatty acid, calculated from the neasured specific activity of the arterial FFA pool. There was no significant difference between the 'pre-chase ' and 'post-chase ' samples
in the control group 1. In the EPL group 2, there was a significant increase in the mole fraction of cholesteryl ester (CE)
labeled after the 'chase ' As in experiment 1, the presence of
EPL significantly increased the turn over of the cholesteryl
ester pool measured from the estimated specific activity of the
newly synthesized fatty acids.
Table 10: Mole fraction of each lipid labeled by newly synthesized fatty acid.
lipid in the artery / h of perfusion .. S.E.M. P as in Table 7
Group 1
Control
Rates in nM / nM of each
Group 2
EPL
Pre-chase
Post-chase
Pre-chase
Post-chase
LPC 4.141 1.51
1 .127 0.55
0.382 0.05
1.721 1.16
SPH 0.485 0.23
0.584 0.22
0.266 0.05
0.781 0.51
4.651 3.33
2.681 0.61
1.764 0.45
3.082 1. 11
LPE 1.388 0.97
0.551 0.19
0.554 0.12
0.935 0.50
PS
1.144 0.48
1.192 0.33
0.770 0.23
1.507 0.82
PI
3.602 2.61
2.881 0.94
3.853 1. 28
4.430 2.29
PE
1.012 0.42
0.995 0.13
1.004 0.44
1.170 0.56
TG
1.867 1. 22
2.265 1. 22
1.743 1. 67
3.886 0.67
CE
1.729 0.73
1.854 0.50
1 131 0.48
1.902 0.43 <0.05
PC
180
The estimates for mole fractions labeled are higher in this experiment than in experiment 1. It must be borne in mind, however,
that the values of the mole fraction of lipids labeled by newly
synthesized fatty acid is only an estimate, based on the speCific activity of FFA of an intima-media strip, sampled by dissection. This pool must in reality be at least two pools. Firstly,
that which is intracellular and is the direct precursor of the
complex lipids. Secondly, a pool, not available metabolically for
synthetic reactions, which is probably extracellular. Thus, the
measured specific activity of the arterial FFA pool is probably
a gross underestimate of the true specific activity of the pool
which is utilized, in the microsomes, as a precursor of complex
lipids.
The discrepancy between experiments 1 and 2 in the values for
mole fractional labeling may also be explained by the different
concentration of acetate used in the perfusates (experiment 1:
0.68 m!1i experiment 2: 1.00 m!1). At the higher concentration of
acetate the intracellular acetate pool may have been expanded,
thereby enhancing the real rate of fatty acid synthesis.
In order to see whether the presence of EPL in the 'chase' perfusate had any effect on the efflux of radioactivity or mass of
any lipid in the 'chase', the lipid concentrations of the 'pulse'
perfusate which was heparinised plasma and the bicarbonate buffer
'chase' were determined. These are shown in Table 11. As expected
there was no significant difference between the lipid concentrations in the perfusates used to 'pulse' label the arteries of the
two groups. This was important because, as had been shown in experiment 1, different concentrations of FFA might lead to different rates of arterial fatty acid synthesis and labeling of the
arterial lipids. The concentration of LPC, PC and FFA in the
'chase' perfusates of group 2, were consistent with the addition
of the EPL solution. No significant efflux of lipids as a result
of perfusion with EPL in the 'chase' could be detected.
Table 12 shows the amount of acetate which was incorporated into
the major lipids of the buffer during the 1 hour 'chase' together
with 'blank' values from Table 1. In the control group 1 activity above the background was detectable only in the FFA and CE
fractions. In the EPL group 2, there was a statistically significant increase in activity in the lecithin fraction. This result
suggests that in the presence of EPL, newly synthesized fatty acid
does leave the arterial wall and is incorporated into lecithin.
Presumably the lyso-lecithin present in the EPL is acting as the
fatty acid acceptor under the catalysis of enzymes in the endothelium. This reaction could be brought about either by a fatty acyl
CoA transferase or, possibly, as a result of the reverse reaction
of phospholipase A2 in the presence of excess lyso-lecithin. It
should be noted (see Table 11) that the concentration of lysolecithin in the 'chase' perfusate with EPL is 0.133 mM, whilst in
normal rabbit plasma is only about 0.03 m!1, i.e. about 4 times
the physiological concentration. Thus it is doubtful whether EPL
would have a significant affect on withdrawing fatty acid from
the arterial wall, by this mechanism, in vivo.
181
Table 11: Concentration of the major lipids of pulse and chase perfusate. Mean nM / ml perfusate
There were no significant differences between groups 1 and 2
Pulse Perfusate
S.E.M.
Chase Perfusate
Group 1
Group 2
Group 1
Group 2
Control
EPL
Control
EPL
LPC
0.021 0.002
0.038 0.008
Trace
0.133 0.02
PC
0.141 0.02
0.132 0.01
Trace
2.271 0.32
FFA
0.645 0.04
0.560 0.05
0.173 0.02
0.214 0.03
CHOL
0.110 0.01
0.115 0.02
Trace
Trace
CE
0.651 0.01
0.658 0.03
Trace
Trace
Table 12: Incorporation of activity from 3H-acetate into lipids of "chase" perfusate during 1 hour
perfusion with buffer (control group 1) or buffer plus EPL (EPL group 2). pM acetate / ml
" chase" P as in Table 2
Control
n
EPL
Blank
(from Table 2)
LPC
0.54
3.61
3.11
SPH
2.14
PC
5.04
LPE
PS
PI
PE
FFA
16.30
5.77
5.15
2.72
0
19.42
77 .37
97.14
62.34
TG
1. 37
1. 53
2.20
CE
3.04
2.22
0.71
182
GENERAL DISCUSSION AND CONCLUSIONS
It is widely accepted that during the development of the fatty

streak lesion of atherosclerosis, although lipid accumulates from
plasma, a significant proportion of the intracellular lipid is
also derived by assembly of the complex lipids in situ. Thus, a
beneficial reduction in arterial lipid accumulation might be
achieved by modification of arterial lipid synthesis or by enhanced catabolism and turnover. In the experiments described above;
it has been possible by the use of arterial perfusion to test
for a direct pharmacological effect of EPL on these reactions
in the arterial wall.
In this experimental approach we have been able to use a number
of novel approaches of investigating alterations in metabolic
turnover in the arterial wall in situ. Firstly, the use of the
in vitro perfusion technique per se has enabled us to look for
effects of EPL under quasi-physiological conditions of temperature, perfusate flow, and pressure. Furthermore, with this method,
in contrast to studies with tissue slices, homogenates, or enzyme
extracts, the normal anatomical arrangement and, to a certain extent, the normal physiological order are preserved. In the second
place, by the use of micro-methods of thin-layer chromatography
and chemical assay, it has been possible for the first time, as
far as we are aware, to make an estimate of the specific activity
of the newly synthesized arterial free fatty acid pool and, thereby, to estimate the relative molar utilization of fatty acid used
in synthesis of the various complex lipids. As was discussed
above, certain limitations of this approach must be accepted. One
must consider that the free fatty acids of the artery are in a
number of pools and that only a portion of the total is a direct
precursor of the complex lipids. In addition, other mechanisms,
such as acyl transfer, may contribute to complex lipid synthesis.
There is a further limitation in these experiments, which is the
variability of the values for FFA utilization. We believe that
this arises from variable penetration of precursor into the inner
layers of the intima-media, thus producing a non-uniform distribution of labeled FFA and complex lipids in these layers. It is
impossible to strip the intima-media to the level at which thp
substrate has the same activity in every experiment.
Preliminary experiments have shown that the problem of inadequate
penetration may be overcome by two approaches. Firstly, perfusion
for longer periods, up to 48 hours under sterile conditions and
secondly, by the use of segments of artery in organ culture.
Using the perfusion technique, we have been able to show the following significant effects during a 1 hour perfusion:
1) EPL added, in vitro to a perfusate of heparinised plasma, produced a significant reduction of arterial fatty acid biosynthesis
from acetate. This inhibition is probably mediated by elevation
of the perfusate free fatty acid concentration.
2) When the apparent rates of synthesis of complex lipids were
183
calculated from an estimate of the specific activity of newly synthesized fatty acid, the rates obtained were in close agreement
with measurements, obtained in a different experiment, of the utilization of exogenous fatty acids.
3) Estimation of the mole fractions of each lipid labeled by newly
synthesized fatty acid revealed that the presence of EPL in the
perfusate caused a significant increase in the 'turnover' of lysolecithin and cholesteryl esters. Exactly the same observations
were made in the second experiment in which arteries, previously
labeled by newly synthesized fatty acid, were perfused for 1 hour
with 'chase' perfusates containing EPL. EPL is clearly affecting
the flow of fatty acid through the cholesteryl ester pool. This
is in accordance with the studies of HORSCH (see this symposium)
showing that EPL produces a significant reduction of cholesteryl
esters in arterial cells grown in tissue culture in hyperlipemic
serum.
4) It was shown that some of the fatty acid synthesized in the
artery was released into the perfusion medium. Contrary to early
expectation from preliminary studies, however, EPL did not significantly increase the efflux of fatty acid to the free fatty acid
pool of the perfusates.
5) The presence of ZPL in a 'chase' perfusion lead to an enhanced
appearance of newly synthesized fatty acid in the lecithin of the
perfusate. This was assumed to be due to the presence of a higher
than physiological concentration of lyso-Iecithin in the perfusate, which would act as a fatty acid acceptor.
These studies have revealed that the presence of EPL in the perfusate of an artery in vitro may affect fatty acid synthesis and
utilization. They have shown that the turnover of cholesteryl
esters is modified. This may have a significant role in prevention of cholesteryl ester accumulation during atherogenesis. The
results of similar studies in atherosclerotic rabbit aortas are
now awaited with interest.
Acknowledgements
We wish to thank Haus Nattermann for their generous support, which
made these studies possible.
We are also grateful to Mrs. J. King for dedicated technical
assistance, to Mr. M. Pollard and Hr. R. Burgess for animal husbandry, to Hr. P. Curtis for presentation of the figures and to
ars. L.M. Wright for typing the manuscripts.
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2
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-
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N., WILSON L.: Characteristics of the cholesterol-esterifying activity in normal and atherosclerotic rabbit aortas.
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14 - KOTHARI H.V., HILLER B.F., KRITCHEVSKY D.: Aortic cholesterol esterase: characteristics of normal rat and rabbit. Biochim. Biophys. Acta. 296, 446 (1973)
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15 - ABDULLA Y.H., ADMiS C.W.M. and BAYLISS O.B.: The location
of lecithin : cholesterol transacylase activity in the
atherosclerotic arterial wall.J.Atherscler.Res.lQ,229 (1969)
16 - DAYTON S., HASHIMOTO S.: Origin of fatty acids in lipids of
experimental rabbit atheroma.J.Atheroscler.Res.~,555 (1968)
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other esterified lipids of rabbit atheroma. Atheroscler. 12,
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lipid accumulation in experimental atherosclerosis. Ph.D.
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and synthesis of fatty acids in atherosclerotic aortas of
pigeons. J. Lipid Res. ~, 739 (1968)
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of duration of cholesterol feeding on esterification of fatty
acids by cell-free preparation of pigeon aorta. Circulation
Res. l:.2, 213 (1970)
22 - PATELSKI J., BOWYER D.E., HOWARD A.N., GRESHAM G.A.: Changes
in phospholipase A, lipase and cholesterol esterase activity
in the aorta in experimental atherosclerosis in the rabbit
and rat. J. Atheroscler. Res. ~, 221 (1968)
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C.J.R. and GRESHAM G.A.: Modification of enzyme activities in
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24 - BOWYER D.E. and PEARSON J.D.: Sterol ester hydrolase. Proc.
3rd. Symposium on Atherosclerosis, edit. SCHETTLER and WEIZEL,
publ. Springer-Verlag, Berlin (1973) p 132
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B.V.: Aortic perfusion in experimental animals: a system
for study of lipid synthesis and accumulation. Progr. Biochern. Pharmacol. i, 235 (1968)
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and other cells, J. Biochem.-microbiol. Technol. Engng. 2,
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diphenylamine reaction for the colorimetric estimation of
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cholesterol and cholesteryl ester in tissue on the nanogram
level. Annal Biochem. ~, 363 (1971)
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for scintillation counter data processins ~ith optional features for users specific requirenents. Liquid Scintillation
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ethanolamine-1,2- 14 C into the major phospholipids of human
peripheral arteries. Proc. Soc. Exp. Biol. Med. 131, 880
(1969a)
37 - NAKATANI rl., SASAKI T., MIYl>.ZAKI T. and NAKAUHRA M.: Synthesis of phospholipids in arterial walls, Part 1. (Incorporation of 32p into phospholipids of aortas and coronary
arteries of various animals). J. Atheroscler. TIes. 7, 747
(1967)
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incorporation of 32p phosphate into the major phospholipids
of swine coronary and pulmonary arteries. J. Atheroscler.
Res. 10, 283 (1969b)
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arteries. Atherosclerosis (1976) To be published.
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lipids of aortic slice of rabbits, dogs, rats and baboons.
J. Atheroscler. Res. ~, 400 (1962)
41 -
BO~~ER D.E.: In 'The smooth muscle of the artery'. Proceedings of a workshop conference held in Heidelberg, 1973.
p 154. Edit. WOLF S. and WERTHESSEN N.T., New York, I'lesUI!l
Press. (1975).
Effect of EPL on the Lipid

Metabolism of the Arterial "Wall
and Other Tissues
A. N. Howard and J. Patelski
Department od Medicine, University of Cambridge,
Addenbrooke's Hospital, Cambridge, England
and Department of Biochemistry, Medical Academy, Poznan, Poland
Abstract: A study of arterial lipid metabolism has indicated

that the enzymes controlling the hydrolysis and synthesis of
cholesteryl esters plays an important role in the deposition of
lipids in the arterial wall. The hypothesis is proposed that an
increase in cholesteryl ester hydrolase and a decrease in acylCoA cholesterol acyl transfel'ase (ACAT) activities would have a
beneficial effect on preventing the deposition of cholesteryl
esters and facilitating their removal.
Polyunsaturated phosphatidylcholine or essential phospholipid
(EPLj when injected intravenously has a number of different effects on serum and tissue enzymes. Of particular interest is a
stimulation of liver phospholipase and lipase, which are considered to be substrate-induced. In the aorta the most important property of EPL is the stimulation of cholesteryl ester
hydrolase and inhibition of ACAT when given oVer several weeks.
These changes in enzyme activities may be responsible for the
beneficial prophylactic action of EPL solution on experimental
atherosclerosis in the rabbit and baboon.
INTRODUCTION
1. Mechanisms of Lipid Deposition in the Arterial Wall
The chief lipids which accumulate in the atherosclerotic lesions

in man and animals are cholesterol and its esters, and phospholipids (1-3). There is a relative increase in cholesteryl esters
compared with free cholesterol, much of the former being synthesized by esterification in situ. Fig. 1 shows the current status
of our knowledge of the metabolism of lipoproteins in the arterial wall.
Lipoproteins in the vascular lumen enter the vessel wall, chiefly
as low density and high density lipoproteins, and are broken down
by enzymes into their constituents. The arterial wall contains
phospholipases and lipases (4) which further break down the released phospholipids and lipids into components which are readily
transported out of the wall. For instance, triglycerides are lipo-
188
VAHCTLAR
LIVER
I NT I MA
LUM)<~:'Il
Apoprotpin
PL-t FA + Hasp + Phosphat!'
TO~FA + Olyc!'rol
PI, + Protein
+
C + CE
Bill'
Acids
HDL
Lipoproteins
VLDL
LDL
HDL - C
,
PREFERENTIAL
SYNTHESIS
OF
OLEATE
il
RESISTANCE
+ FA
CE
TO
HYDROLYSIS
OF OLEATE
tCAT
HDL -CE
PL=Phospholipld
TG=Trigly<.eride
F A-Fatt y add
CE=Cholesterol ester
C =(holesterol
VLDl- Very low density lipoprotein
HOL LOL -
High density lipoprotein

Low density lipoprotein
LeAT- lecithin cholestE!rol acyl
transferase
Fig.
1:
Lipoprotein metabolism in the arterial wall
lyzed to glycerol and fatty acids, the former is water soluble,

the latter combine with albumin which can diffuse readily through
the arterial wall. Likewise, the phospholipids form transportable
and water soluble products.
The situation with cholesterol and its esters is different.
These are relatively irrunobile substances which may have no specific transport system. Cholesterol itself can diffuse slowly
through cell membranes, but the esters require to be hydrolyzed
beforehand (5,6). Cholesteryl ester hydrolase and synthetase have
been demonstrated in the arterial wall (4, 7-9). There are two
synthetases, Acyl CoA cholesterol acyl transferase (ACAT) and
lecithin cholesterol acyl transferase (LCAT), present. The former uses acyl-CoA as the substrate and is very abundant; the
latter, which uses lecithin as a substrate has also been demonstrated in some species.
The chief ester which accumulates in the arterial wall is chol.esteryl oleate (1). The reason for this is two-fold: the cholesteryl ester synthetase esterifies cholesterol preferentially
with oleic acid (7-10). Secondly, the hydrolysis of cholesteryl
oleate is less than of other esters (7,8). The net effect of
these two properties act together in the sa~e direction to favor the formation of the oleate.
Whether free or esterified cholesterol is formed, depends on
the relative activities of the cholesteryl ester synthetase (S)
or hydrolase (R). This is often expressed as the SiR ratio. On
feeding a hypercholesterolemic diet SiR is increased, a factor
which would lead to the deposition of cholesteryl esters rather
than free cholesterol in the arterial wall.
189
2. The Prevention and Removal of Arterial Lipid Deposits
Given the validity of the above hypothesis, there are a number

of points of attack on the prevention and removal of cholesterol
deposition. These are:
I)
Reduction in the concentration of plasma low density (LDL)

and very low density lipoproteins (VLDL),
II) Reduction in the permeability of the vessel wall to lipoproteins,
III) Inhibition of cholesteryl ester synthetase activity,
IV) Stimulation of cholesteryl ester hydrolase and other enzyme
systems which would lead to increased hydrolysis,
V)
Increase in the concentration of high density lipoprotein
(11-13).
The first three factors affect the formation and accumulation
of cholesteryl esters; the last two the removal of cholesterol.
Little is known of the removal mechanism of cholesterol. One
hypothesis which has been proposed is that the apoprotein of
high density lipoprotein which has a high affinity for free cholesterol, enters the vessel wall and removes cholesterol which
is then esterified in plasma by the LCAT enzyme to form high
density lipoprotein. This then goes to the liver where it can
be catabolized to cholesterol, some of which may be excreted in
the bile, as free cholesterol or as bile acids.
During the last two decades, considerable efforts have been made
by many investigators to decrease the level of LDL and VLDL by
means of drugs and diet. However, it is now becoming increasingly recognised that it may be possible to prevent or remove arterial lipid deposits by other meanings than affecting just plasma lipoproteins.
INVESTIGATION OF THE EFFECTS OF EPL ON LIPID METABOLISM
1. General Action of EPL Solution on Enzymes Involved in Lipid Metabolism
Most of the experimental work has concerned the use of polyunsaturated phosphatidyl choline (EPL) in sodium desoxycholate
solution, the latter being used so as to give a water soluble
form of EPL. This preparation, when injected intravenously,
was shown to have a clearing effect on lipemic serum in subjects
having a fatty meal (14). The basis of this effect is the stimulation of serum and tissue lipase which causes a greater lipolysis of triglycerides in chylomicrons and VLDL (15). A single
injection of EPL solution in the normal rabbit gave a 50% increase in serum lipase and a 100% increase in lipase content of
190
LI PASE
HEPARIN LI POSTAB IL
SERUM
LIVER
HEART
AORTA
f 90
f 100
i 5070
t
50
50
15
70
PHOSPHOLI PASE
HEPARIN LI POSTAB IL
t 100
t 40
f 65
t 40
t
t
75
130
25
20
Fig. 2: Comparison of heparin and EPL solution (Lipostabil) in normal rabbits. Animals injected with
heparin ( 167 i. u. / kg) or EPL solution (70 mg / kg EPL / kg) and killed 15 min later. Figures given
are % change from control animals injected with saline
the liver. This effect is very similar to that of heparin (16)

except that the relative magnitudes are different (Fig.2). Heparin gives a larger increase in serum and a smaller increase
in liver. Heparin stimulated lipase consists of two components,
lipoprotein lipase (LPP) and liver lipase (LL), both of which
have been isolated in a purified form by column chromatography
(17). These two enzymes can be distinguished by incubation with
M NaCl (Table 1), in which media LPP is inhibited and LL is
stimulated. The action of EPL solution would appear to stimulate
chiefly liver lipase, since increased activity is seen with
r'l NaCl. Moreover, this would explain why EPL solution is more
active than heparin on the lipase in liver.
Studies on purified liver lipase have shown that it has phospholipase A activity (18). Both EPL solution and heparin affect
phospholipase, but the effects are different. Heparin stimulates
Table 1: Effect of M NaCI on lipase activity in rabbit liver. A single dose of 100 mg EPL / kg body weight
was intravenously injected and the animals were killed 15 min after the injection. EPL solution = EPL +
DOCA, DOCA = sodium desoxycholate (4 % )
Injection
Lipase activity (neq./mg/h)

without H NaCl with 11 NaCl
Saline
22.1
23.0
52.0
25.4
EPL solution
43.0
36.0
35.0
56.7
EPL hydrosol
46.3
35.9
79.5
47.6
191
phospholipase in serum and tissues, but EPL solution decreases

phospholipase in serum and stimulates tissue phospholipase,
especially in the liver. It is proposed that the effect on liver
phospholipase arises because of substrate stinulation. The nost
immediate effect of the injection of excess phospholipid into
an animal would be an increase in those enzymes \vhich attempt
to remove it, namely phospholipase. On the basis of this supposition, the effect of EPL solution on lipase activity can now
be more clearly understood. Since liver phospholipase and lipase can be the same enzyne, a stimulation of nhospholinase
activity would immediately involve an increase in lipase acti~
vity.
2.
Specificity of EPL
EPL solution contains sodium desoxycholate, a substance known

to influence lipolytic enzyne acti vi ties. It seer.1ed ir:tportant
to separate the possible effects of the t\VO conponents of the
mixture, and also to compare EPL with other lecithins. Groups
of rabbits were injected with EPL or egg yolk lecithin hydrosols or as a preparation in 4% sodium desoxycholate, and the
effects on liver lipase \vere determined. As shmvn in 7able 2,
sodium desoxycholate had no effect on its own, but considerably
enhanced the activity of both EPL and egg yolk lecithin (19).
Table 2:
Liver lipase activity of different lecithin preparations in normal rabbits.
Single doses of
100 mg
lecithin / kg were intravenously injected and the rabbits were killed 15 min after wards. EPL + DOCA =
EPL solution,
DOCA = sodium desoxycholate (2 ml, 4 %). (+) = significant
Injection
Lipase acti vi ty (neq. /mg/h)
% of control
Saline
EPL hydrosol
26.5
33.9 (+ )
100
127
(+)
EPL + DOCA
Egg lecithin hydrosol
50.4 (+)
190
(+ )
29.6
112
Egg lecithin + DOCA
35.2 (+ )
132
DOCA
27.6
104
(+ )
EPL either as a hydrosol or as solution had a greater activity

than respective egg yolk lecithin preparations. Thus, it is concluded that the action of EPL on liver lipase is a function of
pure EPL, but that the effect can be further improved by its solution in sodium desoxycholate.
3.
Effect of a Hypercholesterolemic Diet
Lipolytic enzyme activities are markedly changed in the aortas

of animals given experimental diets. In rabbits given a semi-
192
synthetic hypercholesterolemic diet without added cholesterol,

the phospholipase and the lipase were increased and the cholesteryl ester hydrolase decreased (20). Giving a cholesterol
containing diet (15), the najor change was a decrease in cholesteryl ester hydrolase (Fig.3). Rather surprisingly, ACAT was
also decreased to the same extent so there was no change in the
S/H ratio (Fig.4).
In the baboon given a diet of 2% cholesterol and 15% butter for
six months, and injected with bovine serum albumin, aortic ACAT
was increased but there was no change in cholesteryl ester hydrolase so that the S/H was increased (21).
Rats given atherogenic diets containing cholesterol, cholic acid,
thiouracil and peanut oil, which produces aortic atherosclerosis
have reduced aortic cholesteryl ester hydrolase (20). There have
been no studies on ACAT. Out of these diverse and different results it is difficult to draw any firm conclusions as to the
Cholc.,ll'rol
[.,lera't
erum
Fig. 3: Changes in lipolytic enzymes in rabbits given 1 % cholesterol diet for 12 weeks; and effect of EPL
solution (70 mg EPL / kg i.v. ) given as a single dose (animals killed 15 min after injection)
193
Choicstcrol
Esterase
o
tJ
Control
Acyl Co A
Synthctas~
Dict
aline
EPL
Cholestcrol Acyl
Transrcrase
Cholesu:rol Ene r
synthcLaSCS/ hydrol;uc
oAtherogenic Diet
aline
EPL
Fig. 4: Effect of 1 % cholesterol diet and EPL solution on aortic cholesterol ester hydrolase and ACAT
activities. Rabbits given 1 % cholesterol diet for 12 weeks and effect of EPL solution (70 mg / kg i. v. )
given as a s ingle dose (animals killed 15 min after injection)
general role of cholesteryl ester hydrolase/synthetase system

in experimental models. Where the S/H ratio is increased (as in
the baboon model) one would expect the preferential deposition
of cholesteryl esters. Where cholesteryl ester hydrolase activity is decreased, this would pOint to a defect in the removal
mechanism for cholesteryl esters either synthesized in situ or
deposited directly as esterified cholesterol from LDL and VLDL.
4.
Effect of Single Injection of EPL Solution in Cholesterol - fed Rabbits
Rabbits were given 1% cholesterol for 12 weeks and then injected intravenously with a single dose of EPL solution and killed
15 minutes later (15). In serum and liver, phospholipase and lipase were increased, but there was no effect on cholesteryl
ester hydrolase. In the aorta, cholesteryl ester hydrolase was
increased slightly and ACAT decreased slightly to give a significant decrease in the S/H ratio.
The acute effect of EPL solution on serum and liver lipase in
cholesterol-fed rabbits was similar to that seen in normal rabbits (Fig.3). However, the most interesting finding was the
changed S/H ratio (Fig.4) which would indicate a tendency for
decreased cholesteryl ester deposition, and may partially explain the beneficial effect of EPL solution in decreasing the
severity of atherosclerosis in cholesterol-fed rabbits.
194
5,
Effect of Intravenous EPL Solution in Experimental Atherosclerosis
Since EPL
study was
this drug
synthetic
They were
with 1 ml
solution is known to affect the arterial enzymes, a

made of rabbits fed atherogenic diets, injected with
(22). The experimental animals were all fed a semidiet which produced hyperlipemia and atherosclerosis.
injected every second day via the marginal ear vein
EPL solution (100 mg EPL/kg body weight).
The specific activities of the lipolytic enzymes after 18 weeks

treatment are presented in Fig. 5. Animals fed atherogenic diets
alone and injected with saline showed increased phospholipase
and lipase activities and decreased cholesterol esterase activities compared with normal. The arterial lipolytic enzyme activities of animals fed the atherogenic diet and injected with
EPL solution were not significantly different from normal.
Atherosclerotic lesions could be seen macroscopically in experimental animals fed for 18 weeks but not in those fed for 10 weeks.
As shown in Table 3, plasma cholesterol was elevated four-fold
in animals fed the semi-synthetic diet, but there was no significant difference between those injected with saline or EPL solution. The extent and severity of the atherosclerotic lesions
was reduced. The decrease in aortic atherosclerosis observed
with EPL solution could not be explained on the basis of decreased hypercholesterolemia, but was more likely due to the
changes in aortic enzyme activities.
60
50
Phospho lipase
ipase
Cholesterol
esteras
nequ i v /
mg/m'n
~ Control
o Salin
Ltpos abi I
30
20
Fig, 5: Changes in aortic enzymes and effect of EPL solution in atherosclerotic rabbits (control group given
normal diet; saline and EPL solution (Lipostabil), 25 mg EPL / kg thrice weekly, injected into rabbits
given atherogen ic diet)
195
Table 3:
Severity of lesions and lipid composition of aortas
Diet
Time
on
diet
Injectiona
-wks.
Control
18
Atherogenic
18
semi -synthetic
diet
containing
18
20%
beef
tallow
Aortic atherosclerosis
Aorta
Total No. No. of animals

of
at each grade
animals
of lesion b
Cholesterol
mg/100 ml
Ester
Free
mg/100 ml
None
114
11.7
0.4
0.26 c
2.4
0.91 .
Saline
485
217
(**)
4.8
1. 33
(***)
6.6
2.40
(***)
EPL
solution
578
139
(***)
3.9
2.41
(* )
5.6
1. 92
(* )
aInjected thrice weekly with

bGrade 0
Plasma
cholesterol
= no lesions; grade
ml saline or EPL solution (100 mg EPL)
= 1%; grade 2 = 1-5%; grade 3 = 5% diseased aorta
cMeans standard deviation

*p < 0.05; **p < 0.01; ***p < 0.001
A similar study was also made in hypercholesterolemic baboons

given injections of bovine serum albumin (23). Groups of 5 - 8
baboons were given either a control or hypercholesterolemic diet
for 6 months. During the last 90 days, each group was given
5 i.v. injections of bovine serum albumin (BSA) at 16 day intervals or control injections of saline. Only those animals which
were both hypercholesterolemic and injected with BSA developed
aortic atherosclerosis (Table 4).
An i.v. injection of 1 g EPL solution thrice weekly into animals receiving the atherogenic diet and BSA, reduced the incidence and severity of aortic atherosclerosis, but had no effect
on plasma cholesterol, or plasma phospholipids or on the fatty
acid composition of the cholesteryl esters and of lecithin.
In the hypercholesterolemic baboon, aortic lipase was found to

be elevated, but there was no change in cholesteryl esterase
compared with normal animals (Table 4). Species differences may
be an explanation of the conflicting result, but it is noted that
in the rabbit experiments, the plasma cholesterol in the experimental group was almost double that in the hypercholesterolemic
baboons and this may be of importance.
Injections of EPL solution in the hypercholesterolemic rabbit
caused a decrease in aortic atheroclerosis and normalization of
the aortic enzymes. In the baboon experiments, a similar result
has been achieved with respect to lipase, but in contrast the
activity of cholesterol esterase was increased by over 50% above
196
Table 4:
Mean plasma lipids, aortic atherosclerosis, and lipolytic enzymes in baboons
Group n Treatment(+) Plasma

cholesterol
Diet BSA EPL
1
2
3
4
5
8
8
5
5
5
A
A
A
+
+
(+) A = atherogenic, C
EPL = EPL solution
Aortic
atherosclerosis
mg%
% area
245
226
286
118
116
46.3
9.5
0
0
0
= control,
BSA
Aortic
Lipase
Aortic
cholesteryl
esterase
neq/min/mg
67
54
19
36
38
22
= bovine
serum albumin,
normal. The more pronounced effect in the baboon compared with

the rabbit may be due to the use of a much higher dose of EPL
solution (5 times greater/kg body weight). As for the rabbit
given an atherogenic semi-synthetic diet, plasma cholesterol in
baboons was unaffected by the administration of EPL solution and
its effect on atheroclerosis was not mediated by a lowering of
plasma cholesterol.
6. Regression of Lesions
Recent experiments (21) have been concerned with the possible

effect of EPL solution on the regression of established atherosclerotic lesions in the baboon. Animals were made to develop
aortic atherosclerosis using a cholesterol containing diet and
then injected with BSA as described above. They were then changed
to a control diet and treated with saline or EPL solution (250
mg EPL/kg thrice weekly) for 16 weeks. As shown in Table 5, EPL
solution had no demonstrable effect.
Similar experiments in rabbits made atherosclerotic by feeding
1% cholesterol for 12 weeks, and then injected with 2 ml EPL solution (i.e. 100 mg EPL/kg) daily for 16 weeks, whilst changed
to a normal diet, have also given negative results (19).
These results contrast with a positive effect on EPL solution
reported for regression in the Japanese quail (24), rat (25),
and minipig (26), by other workers. Thus the demonstration of
regression may depend on the species, the type of experimental
model, the dosage and duration of EPL solution treatment. A study
of the conditions necessary to obtain a positive response may
eventually prove useful with respect to the regression of atherosclerotic lesions in man.
197
Table 5:
Serum cholesterol, aortic atherosclerosis, and ACAT in baboons
Aortic
Serum cholesterol
Control
diet
Initial
diet
Group Injection
atherosclerosis
% disease
ACAT activity
neq/min/mg
Saline
383
115
26.9
1.6
EPL soln. A
C
None
388
104
114
26.8
0
0.1
112
atherogenic
0.6
control
However, one interesting result came to light in these studies:

EPL solution had a profound effect on ACAT in baboon aorta
(Table 5). Compared with control animals, enzyme activity was
increased in atherosclerotic aorta from saline injected animals.
In atherosclerotic animals injected with EPL solution the enzyme
could not be demonstrated. It is therefore concluded that the
drug strongly inhibits this enzyme.
Previous work in atherosclerotic animals had not included a
study of ACAT since the optimal parameters for its estimation
had not been sufficiently characterized at that time.
VASCULAR
LUMEN
INTIMA
LIPOPROTEINS~----+--~
CHOLESTEROL
ESTERS
,. "',
} deposited in
athe rosderosis
cholesterol I
c
ester
I
r
synthetase I
(' cholesterol
inhibited It esterase
I
('
II
trunsport into
Fig.
6:
Lumen
Anti-atherogenic action of EPL solution
CHOLESTEROL
198
CONCLUSION: Mechanism of Action of EPL Solution
The detailed mechanism by which cholesteryl esters accumulate

in the arterial wall has not so far been elucidated. But, as
discussed earlier, several factors are involved and of these
the increased esterification of cholesterol and the decreased
hydrolysis of esters appear to play an important role. The view
has been presented that an increase in cholesteryl ester hydrolase and a decrease in cholesteryl ester synthetase activity
would lead to less arterial lipid deposition. EPL solution when
given intravenously has these two properties and it is suggested
that they explain its observed anti-atherogenic effect (Fig.6).
Which of these two effects is more important is difficult to
establish at present. The positive prophylactive effect would
point to the inhibition of ACAT as being of major importance.
The lack of effect of EPL solution on regression would suggest
that the stimulation of cholesteryl ester hydrolase is of lesser
importance.
Whether both components of EPL solution (either EPL and sodium
desoxycholate) are responsible for the above mentioned enzyme
effects, is currently unknown and there is need for further
experimentation using EPL hydrosol. Sodium taurocholate stimulates aortic cholesteryl ester hydrolase (24) and inhibits ACAT
in vitro at certain concentrations (19). If this is a general
property of all bile acids then part of the activity of EPL
solution could be explained by the presence of bile salt. Experiments to investigate this point are currently in progress.
REFERENCES
- HOWARD A.N.: Experimental models for atherosclerosis and

the study of arterial metabolism. In: Proceedings of the
1968 Deuel Conference on the Turnover of Lipids and Lipoproteins, edited by C. COWGILL, D.L. ESTRICH and P.O. WOOD.
U.S. Department of Health, Education and Welfare, Washington,
1968, p. 171
2
- NELSON W.R., WERTHESSEN N.T., HOLMAN R.T., HADAWAY H. and

JAMES A.T.: Changes in fatty acid composition of human aorta
associated with fatty streaking. Lancet li, 86 (1961)
- SMITH E.B., EVANS P.H. and DOWNHAM M.D.: Lipid in the aortic
intima. The correlation of morphological and chemical characteristics. J.Atheroscler.Res. 2, 171 (1967)
- PATELSKI J., WALIGORA Z. and SZULC S.: Demonstration and

some properties of phospholipase A, lipase and cholesterol
esterase from the aortic wall. J.Atheroscler.Res. 7, 453
(1967)
-
199
- ROTHBLAT G.H., HARTZELL R., MIALHE R. and KRITCHEVSKY D.:

In: Lipid metabolism in tissue culture cells, edited by:
ROTHBLAT G., KRITCHEVSKY D., Wistar Institute Press,
Philadelphia, Pa. 1967, p. 129
- MURPHY J.: Erythrocyte metabolism. III. Relationship of

energy metabolism and serum factors to osmotic fragility
following incubation. J.Lab.clin.Med. 60, 86 (1962)
- PATELSKI J., PNIEWSKA B., PIERUNSKA B. and OBREBSKA M.:

The arterial acyl CoA: cholesterol ester hydrolase activities: Effect of substrates with fatty acid of different
chain lengths and saturation. Atherosclerosis ~, 287 (1975)
- KOTHARI H.V. and KRITCHEVSKY D.: Purification and Properties

of aortic cholesteryl ester hydrolase. Lipids 12, 322 (1975)
- PORTMAN 0.: Advances in lipid research. Edited by PAOLETTI R.

and KRITCHEVSKY D., Academic Press, New York, N.Y., 1970
p. 41
10 - BmoJYER D.E., HOWARD A.N., GRESHAM G.A., BATES D. and

PALMER B.V.: Aortic perfusion in experimental animals.
A system for the study of lipid synthesis and accumulation.
Biochem. Pharmacol. !, 235 (1968)
11 - MILLER G.J. and MILLER N.E.: Plasma-high-density-lipoprotein
concentration and development of ischaemic heart disease.
Lancet 1, 16 (1975)
12 - GLOMSET J.A.: Physiological role of lecithin-cholesterol
acyl transferase. Am. J. clin. Nutr. ~, 1129 (1970)
13 - STEIN O. and STEIN Y.: The removal of cholesterol from
Landschlitz ascites cells by high-density lipoproteins.
Biochem. Biophys. Acta 326, 232 (1973)
14 - SCHRADE W., BOHLE E. and BIEGLER R.: In: Drugs affecting
lipid metabolism; edited by GARATTINI S. and PAOLETTI R.,
Amsterdam, Elsevier Publ., 1961 p. 454
15 - WALIGORA Z., PATELSKI J. BROWN B.D. and HOWARD A.N.: Effect
of a hypercholesterolemic diet and a single injection of
polyunsaturated phosphatidylcholine solution on the activities of lipolytic enzymes, acyl-CoA synthetase and acylCoA cholesterol acyl-transferase in rabbit tissues. Biochemical Pharmacology ~ (1975) in press
16 - PATELSKI J., WALIGORA Z. and HOWARD A.N.: Effects of hepari~ on lipolytic enzyme activities "in vivo" and "in vitro".
Enzyme (1975) in press
17 - GRETEN H. and WALTER B.: purification of rat adipose tissue
lipoprotein lipase. FEBS Letters ~, 36 (1973)
200
18 - EHNHOLM CH., SHAW W., GRETEN H. and BROWN W.V.: Purification
from human plasma of a heparin-released lipase with activity against triglycerides and phospholipids. J.Biol.Chem.
250, 6756 (1975)
19 - HOWARD A.N., PATELSKI J., BROWN B.D. and WALIGORA Z.:
(unpublished data)
(1975)
20 - PATELSKI J., BOWYER D.E., HOWARD A.N. and GRESHAM G.A.:

Changes in phospholipase A, lipase and cholesterol esterase
activity in the aorta in experimental atherosclerosis in
the rabbit and rat. J. Atheroscler. Res. ~, 221 (1968)
21 - HOWARD A.N. and PATELSKI J.: Hydrolysis and synthesis of
aortic cholesterol esters in atherosclerotic baboons.
Atherosclerosis 20, 225 (1974)
22 - PATELSKI J., BOWYER D.E., HOWARD A.N., JENNINGS I.W.,
THORNE C.J.R. and GRESHAM G.A.: Modification of enzyme
activities in experimental atherosclerosis in the rabbit.
Atherosclerosis .ll, 41 (1970)
23 - HOWARD A.N., PATELSKI J., BOWYER D.E. and GRESHAM G.A.:
Atherosclerosis induced in hypercholesterolemic baboons
by immunological injury; and the effects of intravenous
polyunsaturated phosphatidyl choline. Atherosclerosis 14,
17 (1971)
24 - STAFFORD W.W. and DAY C.E.: Regression of atherosclerosis
effected by intravenous phospholipid. Artery 1, 106-114
(1975)
25 - SAMOCHOWIEC L., KADLUBOWSKA D. and RCZEWICKA L.:
Investigations in experimental atherosclerosis. Part 1:
The effects of phosphatidylcholine (EPL) on experimental
atherosclerosis in white rats. Atherosclerosis 23, 305-317
(1976)
26 - SAMOCHOWIEC L., KADLUBOWSKA D., RCZEWICKA L., KUZNA W. and
SZYSKA K.: Investigations in experimental atherosclerosis.
Part 2: The effect of phosphatidylcholine (EPL) on experimental atherosclerotic changes in miniature pigs.
Atherosclerosis~, 319-331 (1976)
27 - PATELSKI J., WALIGORA Z. and SZULC S.: Investigations of
the Aortic Phospholipase A, Lipase and Cholesterol Esterase.
Enzyme .ll, 299 (1971)
Arterial Metabolism of
Cholesteryl Esters
Mechanism of Action of Polyunsaturated Phosphatidylcholine
J. Patelski
Lipid Metabolism Laboratory, Department of Biochemistry, Medical Academy,
Poznan, Poland
Abstract: Injections of polyunsaturated phosphatidylcholine

solution in sodium desoxycholate are known to alter the activity of a number of enzymes of the aortic wall. that are involved
in the metabolism of phospholipids. triglycerides and cholesteryl esters and to reduce aortic atherosclerosis.
This overview of investigations concerned with the aortic enzymes demonstrates the types of reactions in the metabolism of
cholesterol and cholesteryl esters in the arterial wall. characterizes the enzymes (reaction rates. pH optima). and reveals
the importance of saturation and chain length of the acyl
moieties in the substrate molecules. The acyl exchange in cholesteryl esters as mediated by polyunsaturated phosphatidylcholine is discussed with regard to its importance for the degradation of the cholesteryl esters in the arterial wall.
INTRODUCTION
The relationship betv1een the metabolisr:l and accumulation of

cholesterol, cholesteryl esters, glycerides and phospholipids
in the arterial wall has become a subject of increasing interest.
Interactions between the enzyme-catalyzed processes of synthesis
and decomposition of cholesteryl esters are considered to be iMportant for the development, prevention and treatment of atherosclerosis.
In different experimental animals, the following types of dietdependent changes of the reaction rates in the aorta were found:
1) a decreased hydrolysis and increased synthesis of cholesteryl
oleate concomitant with an increased hydrolysis of both glyceryl
trioleate and lecithin (1,2); 2) a decreased hydrolysis of cholesteryl oleate and decreased synthesis of cholesteryl palmitate
concomitant with an unchanged hydrolysis of both glyceryl trioleate and lecithin (3); 3) an unchanged hydrolysis of cholesteryl
oleate concomitant with an increased hydrolysis of glyceryl trioleate (4); and 4) an unchanged hydrolysis and increased synthesis
of cholesteryl oleate concomitant with an unchanged hydrolysis
of both glyceryl trioleate and lecithin (5).
202
Long-term intravenous injections of essential phospholipids

(EPL, NATTEru1ANN), a drug containing a polyunsaturated phosphatidylcholine solution in sodium desoxycholate, resulted in:
1) an increased hydrolysis (4) and decreased synthesis (5) of
cholesteryl oleate concomitant with a normalized hydrolysis of
glyceryl trioleate (4), and 2) a normalization of the rates
of other above mentioned enzyme-catalyzed reactions in the aorta
(2)
A single intravenous injection of EPL lowered the ratio of decreased rates of cholesteryl palmitate synthesis to cholesteryl
oleate hydrolysis and also the ratio of decreased hydrolysis of
glyceryl trioleate to increased hydrolysis of lecithin in experimental animals, but it elevated the ratio of synthesis to
decreased rates of hydrolysis of cholesteryl esters and of glyceryl trioleate concomitant with an unchanged hydrolysis of lecithin in control animals (3).
Both the polyunsaturated phosphatidylcholine and the bile salt
may be important for the response of the enzyme-catalyzed reactions to the injections of the drug. A mechanism of action
was proposed taking into account the importance of phospholipiddependent reactions of cholesteryl esters in the arterial wall
(6)
The present overview is concerned with our investigations on

the mechanisms of decomposition and synthesis of arterial cholesteryl esters, on the synthesis and decomposition of cholesteryl oleate and on the effect of substrates with different fatty
acids in relation to the physiological role of phospholipid-dependent reactions in the metabolism of cholesteryl esters.
METHODS,
Details of the experimental procedures are described in the references cited in this overview. Abbreviations and enzyme catalogue numbers of the enzymes which were prepared from arterial
tissue or used in the experiments are the following: ACAT = acylCoA:cholesterol acyltransferase (=cholesterol:acyltransferase),
(EC 2.3.1.26), LCAT = lecithin:cholesterol acyltransferase
(EC 2.3.1.43), LLAT = lysolecithin:lysolecithin acyltransferase
(EC 2.3.1.), phospholipase A = phosphatide acyl-hydrolase
(EC 3.1.1.4.), phospholipase B = lysolecithin acyl-hydrolase
(EC 3.1.1.5), CEH = cholesteryl ester hydrolase = steryl ester hydrolase = cholesteryl esterase (EC 3.1.1.13), Acid:CoA ligase
(AHP) (EC 6. 2 1 3)
203
RESULTS
I.
Mechanism of Decomposition and Synthesis of Cholesteryl Ester in the Arterial Wall
Cholesteryl esters are hydrolyzed by a cholesteryl esterase

present in protein extracts from acetone-butanol powders of the
aorta. The enzyme (7) differs in its physico-chemical properties
from other lipolytic enzymes (8,9).
Cholesteryl esters are synthesized in vitro from cholesterol and
free fatty acids in the presence of a lipid-free arterial enzyme
preparation, with (10) and without the addition of ATP and CoA
(7, 10). In the presence of the cofactors, the reaction rate is
much higher (10) which might indicate the relative importance
of the ATP- and CoA-dependent enzyme (acyl-CoA:cholesterol acyltransferase) for the acylation of cholesterol.
Another (ATP- and CoA-independent) esterification of cholesterol with fatty acids in the arterial wall by the lecithin:cholesterol acyltransferase (LCAT), was established (11, 12, 13).
The possibility of the decomposition of cholesterol esters in
the reverse reaction, i.e. by the acyl transfer from cholesteryl
esters to lysolecithin (LCAT-) has recently been demonstrated
in vitro (13, 14).
The enzyme-catalyzed reactions of synthesis and decomposition
of cholesteryl esters are summarized in Fig. 1.
e.A
ACYL-C.A
PPi; AMP
CHOLESTEROL ~
,-r '-" '"
LECITHIN
CHOLESTEROL
ESTE R
lYSOlEtlTHIN
AT?; e.A
FATTy
ACID
H,D
Fig. 1: Scheme of pathways of synthesis and decomposition of cholesteryl esters in the arterial wall:
CD= ACAT, 0= LCAT, @= CEH,@= Acid: CoA ligase (for abbreviations see the METHODS
section)
204
2.
Synthesis and Decomposition of Cholesteryl Oleate
Fig. 2 shows the activities vs. pH of the following enzymes:

Oleyol-CoA:cholesterol acyltransferase (ACAT), lecithin:cholesterol acyltransferase (LCAT), and cholesteryl oleate:lysolecithin acyltransferase (LCAT-), and cholest~ryl oleate hydrolase, according to (7, 10, 13-16). The double and triple pH optima for LCAT+ and CEH, respectively, may arise from various causes. According to the theory of double pH optima (17) the presence of isoenzymes or ampholyte inhibitors and conformational
changes have to be considered. Moreover, multiple pH optima
could arise from the presence of more than one enzyme in different subcellular fractions of the aorta catalyzing the same
reaction. The intracellular distribution of LCAT is not known,
but the distribution of CEH was found to be clearly cytoplasmic
(18). For LCAT+, the complex fatty acid composition of lecithin
and possible differences in the enzyme-substrate affinities have
also to be taken into account.
ACTIVITY
IS
[mU/mg]
10
~~--r-------~i~-------,r--------,----pH
;z
Fig.
2:
In vitro activities vs. pH of various enzymes prepared from the aorta of the pig as determined under
optimum experimental conditions:

II
.9
= cholesteryl oleate:
I = oleyl CoA : cholesterol acyltransferase (ACAT).
lysolecithin acyltransferase (LCAT).
III
II + = LCAT +.
= cholesteryl oleate hydrolase
(CEH)
The specific activities of the enzymes are listed in Table 1.

The highest activity values of ACAT, CEH, LCAT+, and LCAT- are
significantly different from each other, and their proportion
amounts to about 10:5:2:2, respectively. This data may reflect
the relative contribution of the enzymes to synthesis and decomposition of cholesteryl oleate in the arterial wall. A considerably higher ACAT-catalyzed synthesis than hydrolysis of
cholesteryl oleate may favor accumulation of the ester in the
arterial wall.
205
Table 1:
In vitro activities (means.. S.D.) of enzymes prepared from the aorta of the pig. The maximum
average values for ACAT,
LCAT +,
LCAT and CEH, respectively, are statistically different from each
other as evaluated by Student's paired t test. CE

Abbreviations for enzymes as in Fig. 2
= cholesteryl ester,
Enzyme
pH
optimum
Activity
(mU/mg
ACAT
6.5
14.0 + 1.6
LCAT+
7.0
2.7 + 0.7 (CE)
8.0
2.0 + 0.5 (CE)
LCAT-
8.0
2.5 + 0.6 (C )
CEH
8.6
6.9 + 1.0
8.0
3.3 + 1.0
7.0
4.7 + 0.9
= free cholesterol.
SYNTHESIS /HYDROLYSIS
2.0
1.0
0.5"
0.25
CONTENT
~--------~--~----~--~~--~
6.2S
12.5
aD
50,0
['.J
Fig. 3: Correlation between the ratios of the rates of aortic synthesis to hydrolysis of cholesteryl esters
( ACAT / CEH . activities, mU I mg) and the contents of the cholesteryl esters in fatty aortic intima and
media: r > 0.96 (p < 0.05). C subscript = number of Carbon atoms in the fatty acid and number
of double bonds
206
3. Effect of Substrates Containing Different Fatty Acids on the Synthesis and Decomposition of
Cholesterol Esters
The ACAT- and CEH activities were examined in the presence of

substrates containing fatty acids of different chain length and
saturation. The ratios of the rates of synthesis to hydrolysis
were calculated. The values obtained result in the following
numerical order: cholesteryl oleate > palmitate ~ linoleate >
linolenate> stearate, amounting to approximately 8:4:4:2:1 (15).
The ratios of the rates of synthesis to hydrolysis, but not the
absolute values of either synthesis or hydrolysis, are positively
correlated with the percental, as well as with the absolute contents of corresponding cholesteryl esters in the fatty aortic
intima (19, 20) and media (19) see Fig. 3.
Therefore, if free, but not esterified cholesterol is easily
transported across cell membranes (21), the differences in both
hydrolysis and the ACAT-catalyzed synthesis of cholesteryl esters
could determine the different intracellular accumulation of the
esters in the arterial wall.
Substrates and products of the phospholipid-de?endent reactions
are presented in Fig. 4.
The activity of the LCAT+ enzyme with respect to lecithins of
different fatty acid composition was measured. The phosphatidyl
choline from egg yolk (SIG!-1A) and the polyunsaturated phospha-
acD<
cD<
H0
FATTY ACID
LECITHIN
LYSOLECITHIN
CHOLESTEROL
CHOLESTEROL
CHOLE srE ROL
CHOLESTEROL
ESTER
LYSOLECITHIN
LECITHIN
LYSOLECITHIN
GlYC ERYlPHO$PHORYlCHOllNE
HaO
FATTY ACID
cD<
cD<
cD<
ESTE R
FATTY ACID
Fig.
4:
Phospholipid and cholesterol substrates and products of reactions catalyzed by arterial enzymes,
<2)= phOSpholipase A, ~ LLAT, 0~ phospholipase B. For abbreviations
see the METHODS section
~ LCAT, ~ CEH.
207
tidyl choline from soya (NATTERMANN) were used (12, 22). The
enzyme activities amounted to 1.8 + 0.4 and 1.2 + 0.4 of cholesterol esterified per minute and mg at pH 7.3 (means + S.D.,
n=5) (12). The difference of the means, 0.6, (S.E.M. =-0.12)
is statistically significant according to Student's paired ttest (p<0.01). The LCAT-dependent release of lysolecithin
(mU/mg) in this reaction is higher and almost equal for the two
lecithins (12,22). This may be attributed to subsequent hydrolysis of cholesteryl esters by CEH, with preference for those
formed in the presence of polyunsaturated lecithin. This supported by the different composition of cholesteryl esters found in
the two reaction mixtures (22) and is consistent with different
rates of hydrolysis of'the esters. At optimum experimental conditions (15,16), the rates of hydrolysis of cholesteryl palmitate, stearate, oleate linoleate, and linolenate amounted to:
8.4 + 1.0, 7.9 + 1.0, 6.9 + 1.2, 11.3 + 1.2 and 9. 7 + 1.1 mU/mg,
respectively, the means beIng statistically different from each
other according to an analysis of variance (p < 0.01, n=6).
The LCAT- activities were assayed in the presence of different
cholesterol esters at optimum conditions for acyl transfer from
cholesteryl oleate to lysolecithin and at optimum concentration.
for hydrolysis of the former substrate. The activity values
(expressed as free cholesterol) for cholesteryl palmitate, stearate, oleate, linoleate and linolenate amounted to: 1.1 + 0.56,
0.8 + 0.2, 2.6 + 0.43, 1.3 + 0.33, and 1.6 + 0.67 mU/mg,-respectively, the-means being-statistically dIfferent from each
other according to an analysis of variance (p <0.01, n=4). A
high and significant correlation was obtained between the activity values of LCAT- and the contents of appropriate cholesteryl esters found in fatty aortic intima (19,20) and media (19)
(Fig. 5).
ACTIVITY
[mU/m9]
C18:1
C18 : 2
.
(16;0
C18 !O
CONTENT
2)
SO
[r.]
Fig. 5: Correlation between activity of LCAT - in the presence of different cholesteryl esters and the
contents of the cholesteryl esters in the fatty aortic intima and media: r :;;.. 96 (p "";:0.05). C subscript:
see Fig. 3
208
Thus, the decomposition by transacylation with lysolecithin

seems to be more important for cholesteryl oleate than for
other cholesteryl esters: the greater the capability of ester
accumulation the greater is the importance of the reaction.
DISCUSSION
The importance of LCAT-catalyzed synthesis and decomposition

of cholesteryl esters in the arterial wall and the role of
polyunsaturated phosphatidyl choline are to be considereu from
a qualitative rather than a quantitative point of view. This is
suggested by the markedly lower LCAT than both ACAT and CEH activities along with the differences observed in the enzyme-substrate affinities.
In the reaction catalyzed by LCAT, different cholesteryl esters
are formed and the same amounts of lysolecithin are released.
Lysolecithin, in turn, can be utilized for transacylation with
cholesteryl esters, to a higher degree with cholesteryl oleate.
The acyl exchange between lecithin and cholesterol esters favors
the decomposition of cholesteryl oleate and the formation of
other cholesteryl esters which are less resistant to a cleavage
by hydrolysis.
The phospholipid-mediated acyl exchange in cholesteryl esters,
i.e. the substitution of oleyl residue by other fatty acyls,
is highly effective with polyunsaturated phosphatidyl choline
rendering the cholesteryl esters more susceptible to hydrolysis.
This is mainly due to the predominant content of linoleyl residue and to the preferential hydrolysis of cholesteryl linoleate
as compared with other cholesteryl esters.
Also other enzyme-catalyzed reactions, such as formation of lecithin by lysolecithin:lysolecithin acyltransferase and hydrolysis of lecithin and lysolecithin by phospholipase A and B respectively (see Fig. 4), are relevant to the arterial synthesis
and degradation of cholesteryl esters. However, the relative
importance of these three reactions is still unknown.
REFERENCES
- PATELSKI J., BOWYER D.E., HOWARD A.N. and GRESHAM G.A.:

Changes in phospholipase A, lipase and cholesterol esterase
activities in the aorta in experimental atherosclerosis in
the rabbit and rat. J.Atheroscler.Res.~ 221 (1968)
2
- PATELSKI J., BOWYER D. E., HOWARD A. N., JENNINGS cT. v.J. ,

THORNE C.J.R. and GRESHAM G.A.: r.1.odification of enzyme
activities in. experimental atherosclerosis in the rabbit;
Atherosclerosis 1l 41 (1970)
209
- WALIGORA Z., PATELSKI J., BROWN B.D. and HOWARD A.N.:

Effect of a hypercholesterolaemic diet and a single injection of polyunsaturated phophatidyl choline solution
on the activities of lipolytic enzymes, acyl-CoA synthetase
and acyl-CoA:cholesterol acyltransferase in rabbit tissues;
Biochem. Pharmacol. ~ (1975)
- HOWARD A.N., PATELSKI J., Bm~'YER D.E. and GRESHAM G.A.:

Atherosclerosis induced in hypercholesterolaemic baboons
polyunsaturated phosphatidyl choline; Atherosclerosis 14
17 (1971)
- HOWARD A.N. and PATELSKI J.: Hydrolysis and synthesis of

aortic cholesterol esters in atherosclerotic baboons. Effect
of polyunsaturated phosphatidyl choline on enzyme activities; Atherosclerosis 20 225 (1974)
PATELSKI J.,: Participation of phospholipids in arterial

metabolism of cholesterol esters. Proceedings of the International Workshop Conference on Atherosclerosis,
London, Ontario 1975, in press
- PATELSKI J.: Esteraza cholesterolowa Tetnicy Grownej

(Cholesterol esterase of the aorta) Panstwowy Zaklad
Wydawnictw Lekarskich, Warszawa, 1964
- PATELSKI J., WALIGORA Z. and SZULC S.: Demonstration and

some properties of the phospholipase A, lipase and cholesterol esterase from the aortic wall; J.Atheroscler.Res.
2 453 (1967)
- PATELSKI J., WALIGORA Z. and SZULC S.: Investigations of

the aortic phospholipase A, lipase and cholesterol esterase;
Enzyme ~ 299 (1971)
10 - KLIMINSKA-PNIEWSKA B.: Enzymatyczna estryfikacja cholesterolu w tetnicy grownej (Enzymatic esterification of

cholesterol in the aorta); Poznanskie Towarzystwo Przyjacior
Nauk, Prace Komisji Medycyny Doswiadczalnej 42 167 (1970)
11 - ABDULLA Y.H., ORTON C.C. and ADAMS C.W.H.: Cholesterol
esterification by transacylatioQ in human and experimental
atheromatous lesions; J.Atheroscler.Res. ~ 967 (1968)
12 - PATELSKI J. and TORLINSKA T.: Lecithin: cholesterol acyltransferase and phospholipase A activities of the aortic
wall. in: SA110CHOHIEC L. and WOJCICKI J. (Eds.) Proceedings
of the 1972 International Symposium on Phospholipids; International Soc. Biochem.Pharmacol. and Polish Pharmacol.Soc.,
Szczecin, 1973, p.91
13 - PATELSKI J. and PIORUNSKA M.: Cholesterol ester:lysolecithin
acyltransferase activity of the aorta; Proceedings of the'
International Workshop Conference on Atherosclerosis; London,
Ontario, 1975, in press
210
14 - PATELSKI J. and PIORUNSKA M.: Cholesterol ester degradation

by trans acylation with lysolecithin and hydrolysis in the
arterial wall. To be published
15 - PATELSKI J., PNIEWSKA B., PIORUNSKA M. and OBREBSKA ~1. :
The arterial acyl-CoA:cholesterol acyltransferase and
cholesterol ester hydrolase activities. In vitro effect
of substrates with fatty acid od different chain length
and saturation; Atherosclerosis ~ 287 (1975)
16 - PATELSKI J., PIORUNSKA A. and PIORUNSKA M.: Effect of substrate composition and concentration on the aortic cholesterol ester hydrolase activity. To be published
17 - SCHWIMMER S.: Theory of double pH optima enzymes; J.Theoret.
Biol. 3 102 (1962)
18 - PATELSKI J. and TIPTON K.F.: The subcellular localization
of lipolytic enzymes in pig aorta; Atherosclerosis 13 288
(1971)
19 - NELSON W.R., WERTHESSEN N.T., HOLMAN R.T., HADAWAY H. and
JAMES A.T.: Changes in fatty acid composition of human
aorta associated in fatty streaking; Lancet 1 86 (1961)
20 - SMITH E.B., EVANS P.H. and DOWNHAM M.D.: Lipid in the aortic
intima. The correlation of morphological and chemical
characteristics; J.Atheroscler.Res. 2 171 (1967)
21 - ROTHBLAT G.H., HARTZELL R., MIALHE R. and KRITCHEWSKY D.
In : ROTHBLAT G.H. and KRITCHEWSKY D. (Eds.), Lipid r-ietabolism in Tissue Culture Cells, Wistar Institute Press,
Philadelphia, Pa. 1967, p. 129
22 - PATELSKI J., FILIPEK-WENDER H. and WALISZE~vSKA A.: Fatty
acid composition of cholesterol esters formed by the
aortic lecithin:cholesterol acyltransferase. To be
published
On the Action of Essential

Phospholipids in Experimental
Atherosclerosis
l. Samochowiec
Department of Pharmacology, Pomeranian Medical Academy, Szczecin, Poland
Abstract: 108 male and female WISTAR rats and 39 vietnamese

miniature pigs were used to test the preventive and curative
potency of orally applied essential phospholipids (EPL)J i.e.
polyunsaturated phosphatidylcholine J against experimentally induced atherosclerosis. The animals were fed an atherogenic diet
for 60 daysJ one group received then a basic diet (atherosclerotic control)J 3 groups received basic diet plus EPL (280 J 900 J
2800 mg/kg/d for rats and 28 J 90 J 280 mg/kg/d for pigs) (curative therapy) for further 60 days. Another 3 groups of rats received 3 different doses of EPL in addition to the atherogenic
diet (preventive therapy) and one group of r<ats and pigs) respectivelYJ received basic diet for 120 days (zero control).
EPL given orally (900 mg/kg/d in rats and 90 mg/kg/d in pigs)
almost prevented and cured the experimentally induced atherosclerosis in rats as well as in pigs as evidenced by macroscopic
and microscopic histopathologic investigation. The total serum
lipids in pigs were lowered within 60 days of EPL-treatment (280
mg/kg/d) from 300 per cent elevated to normal values J while they
decreased in the zero control group by only one third. The fatty
acid composition of the phospholipids) triglycerides and cholesteryl esters of the serum J the liver J and the aorta was significantly changed in favor of the polyunsaturated fatty acids depending on dose of EPL J while the atherogenic diet had changed
it beforehand in favor of the saturated ones. In addition J ophtalmologic planimetry of retinal vessels and electroretinography
were tested for determination of the grade of atherosclerosis
in vivo. The results are in agreement with the fundamental finding of this study, that EPL is capable of preventing and curing
experimental atherosclerosis in rats and in pigs as well.
INTRODUCTION
There have been several reports in recent years that polyunsaturated phosphatidylcholine or essential phospholipid (EPL) exerts
a protective and curative action in experimentally induced atherosclerosis. BYERS and FRIEm1AN (1) observed already in 1960 that
sustained infusion of phosphatides of soybean origin reduced
atherosclerosis in rabbits being on an atherogenic diet. They
212
concluded from their experiments that mobilization of cholesteryl esters deposited in the aortic wall rather than reduction of
endogenous cholesterol synthesis caused the observed antiatherosclerotic effects. AD~lS et al. (2) observed a significant decrease of aortic atheroma grade in cholesterol-fed rabbits treated
with 2 g of LIPOSTABIL i.v. weekly for 4 weeks. They showed that
saturated phosphatidylcholine (egg lecithin) in contrast to EPL
even accentuated the formation of atherosclerotic lesons. HOWARD
and PATELSKI (3) reported a reduction of the extent and severity
of aortic atherosclerotic lesons in rabbits fed a semisynthetic
atherogenic diet and injected with EPL thrice weekly. An i.v. injection of 1 g EPL thrice weekly into baboons receiving a hypercholesterolemic diet plus bovine serum albumin reduced the incidence and severity of aortic atherosclerosis also in these
animals (4).
Similar results were obtained after i.v. injection of EPL (2 g
LIPOSTABIL weekly for 4 weeks) in rabbits by ADM1S and ABDULLA
(5). STAFFORD and DAY (6) recently reported a significant regression of experimentally induced atherosclerosis in Japanese
quails receiving once weekly an i.v. injection of 400 mg/kg EPL
(0,8 ml LIPOSTABIL) while being on an atherogenic diet for 6
months. A preventive effect of EPL on coronary atherosclerosis
was measured by LEUSCHNER et al. (7) in chicken fed an atherogenic diet. This beneficial effect of the orally applied therapeutic agent was visible in the coronary vessels already at a
dose of 50 mg/kg/d and was significant (p<0.o1) at 450 mg/kg/d.
At the high dose of EPL the severity of atherosclerotic changes
in the chicken aorta was reduced.
In the light of these results, the objective of the present investigation was 1) to extend these studies to further experimental animals species; 2) to inv~stigate the therapeutic potency
of orally applied essential phospholipids; and 3) to atte~pt to
correlate the autoptically found atherosclerotic chanyes with
vascular alterations measured in vivo using planimetry of the
retinal vessels and electroretinography.
METHODS
Male and female WISTAR rats (100-150 g body weight) and vietnamese
miniature pigs (20-23 kg body weight) were used in the experiments. The animals received a basic diet according to LEHBECK
(8) and, in addition, an atherogenic diet for rats according to
HOOGERWERF (9) and HARTROFT & THOMAS (10), and for pigs of the
following composition: cholesterol (2 %), rape see oil (22 %),
cholic acid (0,5 %), salt mixture (4 %), hydrogenated fat (40 %),
methyluracil (0,01 %), and wheat flour (31,5 %). Essential phospholipid (EPL-NATTERMANN) was administered by a gastric tube once
daily.
213
1.
Prevention and Regression of Experimentally Induced Atherosclerosis
The dietary and therapeutic regimen was as follows:

Group I
(atherosclerotic control): atherogenic diet for

60 d, then basic diet for 60 d
Group II
(preventive therapy): atherogenic diet + EPL

for 60 days
Group III (curative therapy): atherogenic diet for 60 d

then basic diet + EPL for 60 d
Group IV
(zero control): basic diet 120 d
Male and female animals were separately investigated in each

group. Group II (preventive therapy) was only investigated in
rats. The therapy groups were divided into 3 subgroups receiving
280, 900 and 2800 mg/kg/d EPL (rats) and 28, 90 and 280 mg/kg/d
EPL (pigs), respectively.
At the end of the experimental periods the animals were killed,
the internal organs were examined at autopsy and the degree of
atherosclerosis in the blood vessels was macroscopically determined (see Figures 1 and 3). The aorta and the coronary vessels
were subjected to further microscopic histopathologic investigation.
The lipids were extracted from the blood serum, from the aortic
wall and from the liver by the method of FOLCH (11) and the fatty
acid composition in the three fractions of phospholipids, triglycerides and cholesteryl esters was measured using gas-liquid
chromatography according to HAWKE (12).
2.
Investigation of Experimentally Induced Atherosclerotic Retinopathy
For this study 9 miniature pigs were used. The dietary and therapeutic regimen was according to Group I (n = 4) and Group III
(n
5). 'l'he EPL-doses were 90 mg/kg/d (n = 3) and 28 mg/kg/d
(n
2).
Pictures of the eye fundus were taken with the aid of a ZeissJena Retinophote. The area of the vessels was measured using a
pole planimeter type PL (17). The statistical analysis followed
the test of small random samples.
The electroretinography was performed using a Karpe-electrode
on a Keiser's 8-channel encephalograph. Light stimuli of 20 ms
duration were used during mesoptic and dark adaptation of 10 min,
respectively. The pigs were anesthetized with nembutal (30 mg/kg
body weight).
214
RESULTS
1.
Prevention and Regression of Experimentally Induced Atherosclerosis
The atherogenic diet or EPL did not cause any changes in the
behavior of the animals and no mortality was induced. The body
weight increased steadily, to a higher extent in animals under
atherogenic diet than under control conditions.
Fig. 1 depicts schematicly the extent and severity of the experimentally induced atherosclerotic changes in rat aortas and
the effect of the EPL-therapy. After 60 days of atherogenic diet
(Group ~) severe infiltrations and lesions of the endothelial
surface and numerous nodules of various sizes were observed as
G,oup
(b
go
fib
~c;
m;''''1~~,,1
Iw~'I"~''11
1~'4\"iI4\1 ~""!f\)l\1
1Cf\\"4\4\4\h~~4\4\1
1i1\4\'l~~OO4\4\4\4\1
I !lo A'1'1'1'1f1IQqCft4\,
I !lib 19f1~4\~1 '1f1'lfffl
I - ICft'\'1ft~1 ~
Iv
I ~qqqq'iJ '1\'1)1\'1\'1'11
nk
Fig. 1: Schematic depiction of the effect of preventive (II a . c) and curative (III a - c) EPLtherapy in atherosclerotic male and female rats. The diagrams show the single aortas with the semilunar
valves of each animal. Shaded areas = infiltrations and lesions of the endothelial surface. points = nodules.
Atherosclerotic control after 60 d (~) and after 120 d ( I ). zero control ( IV ) after 120 d
215
Fig. 2 a : Male rat, atherosclerotic control (fJ) . The semilunar valves, the ascend ing and descending parts
of the aorta show numerous nodules and lesions
Fig. 2 b: Male rat , curative therapy (" I c) with 2800 mg / kg / d EPL. The semilunar valves and the
intima of the aorta do not exhibit any change
216
well on the aortic valves as on the intima (see also Fig. 2a).
These alterations of the aortic wall were reduced during the
successive 60 days period of basic diet (Group I, atherosclerotic
control), but were still severe and extended. EPL prevented as
well as cured, dependent on dose, these atherosclerotic changes.
The low curative doses were more potent than the preventive ones.
At doses of 2800 mg/kg EPL per day practically no effect of the
atherogenic diet was visible in both cases. The intima of the
aorta and the semilunar valves were smooth and showed no signs
of lipid infiltrations (see also Fig. 2b), as it was also the
case in the zero controls (Group IV). These macroscopic results
were confirmed by extensive histopathologic investigations of
the aortic wall and the coronary vessels (13, 14).
Grou.p
~ ~ 0 ~ UB
~-
~ ~:~.
:.
'.
--
-....
..
I rna ITIlolD11 81 ill TIl

I rnb IDIQIQII UIDID I
D Q D DD
(l
lIIe
IV
DDD UDD
Fig. 3: Schematic depiction of the effect of curative therapy in atherosclerotic male and female miniature
pigs. corresponding to Fig. 1
217
Fig. 3 gives a schematic description (similar to Fig. 1) of the

results obtained with miniature pigs. Gross infiltrations and
lesions of the endothelial surface and numerous nodules were
found in the atherosclerotic control (Group I), sporadic nodules
were seen in the low dose therapy group (28 mg/kg/d EPL) , but no
signs of atherosclerosis were seen after medium and high doses
of EPL (90 and 280 mg/kg/d) and no changes in the zero controls
(Group IV) either.
The biochemical investigations of the lipids of the serum, the
aorta, and the liver correlated strongly with the histopathologic
findings.
Table 1 shows the serum lipids measured in miniature pigs after
120 days of treatment. The total lipids, the triglycerides, and
the B-lipoproteins were highly significantly increased (Group I,
atherosclerotic control) above the normal values (Group IV, zero
control), whereas the phospholipids were somewhat decreased. All
values were normalized or even decreased below the normal values
due to the EPL therapy, and the extent of the curative action
depended on the applied dose. Similar observations were made in
rats (13).
Table 1: Effect of orally applied EPL (28, 90, 280 mg / kg / d) on the levels of total lipids, lipid
phosphorus, triglycerides and betalipoproteins in the serum of atherosclerotic miniature pigs. Means (mg /
100 ml) of n = 3 each. I, IV = atherosclerotic and zero control, III = curative therapy (see METHODS)
Total
Lipids
Lipid
Phosphorus
Triglycerides
Betalipoproteins
255 50
260 + 33
5.5 + 0.4
5.4 + 0.6
120 + 17
112 + 19
250 + 27
270 + 21
28
180 + 17
200 + 17
6.8 + 0.6
6.2 + 0.3
80 + 9
90 + 11
150 + 14
190 + 18
90
150 + 18
180 + 24
-
6.2 + 0.5
6.9 + 0.7
75 +
97 +
120 + 15
115 + 11
93 + 15
75 + 10
5.9 + 0.2
7.9 + 0.6
33 +
19 +
74 + 8
97 + 12
130 + 12
145 + 10
6.9 + 0.5
7.2 + 0.3
77 + 10
72 + 8
82 + 12
69 + 11
Dose
Group
Sex
I
&
~
III
c1
~
c1
~
r1
280
IV
r1
~
9
4
190 + 19
120 + 30
Zero
control
IV
Curative
therapy
III
&
180 67
50
250 21
110 + 10
120 + 15
90 + 12
110 + 12
95 10
520 + 80
200 + 15
290 + 25
250 22
40
atherogenic phase
11012
83 +
150 + 12
90 +
114
Atherosclerotic
control
20
Sex
Group
in comparison with a 120 days period of basic diet

(IV).
136 + 18
130 + 15
443 + 50
33
15
17
140 12
130
250 19
220 17
145 10
130 12
75 10
93
260
72
260
255 50
120
(I)
230 52
therapeutic phase
130 + 12
135 14
380 + 28
300 14
330 70
370 + 35
350 25
280 27
40
300
80
60
100
Means of n = 3 each
Time after beginning of the experiments (days)
(III)
Changes with time of the total serum lipids in miniature pigs during a 60 days period of atherogenic diet followed by 60 days of basic diet
EPL
2:
basic diet + 280 mg / kg / d
Table
and
I
I
co
219
Table 3: Effect of orally applied EPL (280 mg / kg / d) on the ratio of unsaturated to saturated fatty
acids, U / 5, in the lipid fractions of the serum, the liver and the aorta in atherosclerotic rats. % of IV =
ratio U / 5 relative to the zero controls
Group
Phospholipids
U/S
Atherosclerotic
control: I
% of IV
114
1.22
71
2.32
60
1. 52
142
2.15
124
5.92
153
1. 48
138
2.07
120
7.60
196
1.07
100
--
1.73
100
--
3.~8
100
0.65
63
0.95
58
0.92
65
Preventive
therapy: II
1.03
99
1. 87
115
1. 43
101
Curative
therapy: III
1. 35
130
2.08
128
1. 63
116
Zero
control:IV
1.04
100
--
1. 63
100
--
1. 41
100
--
0.97
90
0.96
58
1.10
99
Preventive
therapy: II
1. 43
132
2.12
129
1.30
117
Curative
therapy: III
1. 34
124
2.06
125
1. 49
134
Zero
control:IV
1.08
100
--
1.65
100
--
1. 11
100
--
blood
serum Curative
therapy: III
Zero
control: IV
Atherosclerotic
control:I
Atherosclerotic
control: I
aorta
U/S
Cholesterylesters
U/S % of IV
1.22
Preventive
therapy: II
liver
% of IV
Triglycerides
220
Table 4: Effect of orally applied EPL (280 mgjkgjd) on the ratio of unsaturated to saturated fatty acids, UjS,
in the lipid fractions of the serum, the liver and the aorta in atherosclerotic miniature pigs. % of IV = ratio UjS
relative to the zero controls
Group
Phospholipids
Triglycerides
U/S
% of IV
U/S
0.54
51
1. 44
83
5.27
82
1.03
97
2.03
117
7.61
118
1.06
100
1. 73
100
--
6.44
100
--
0.77
84
1. 63
99
0.90
70
Curative
therapy: III
1.80
196
3.22
196
2.06
161
Zero
control IV
0.92
100
--
1. 64
100
--
1. 28
100
0.77
81
1. 42
112
0.94
94
1. 32
135
1. 82
143
1. 63
163
0.95
100
--
1. 27
100
--
1.00
100
--
Atherosclerotic
control: I
blood Curative
serum therapy: III
Zero
control:IV
Atherosclerotic
control:I
liver
Atherosclerotic
control: I
aorta Curative
therapy: III
Zero
control: IV
% of IV
Cholesterylesters
U/S % of IY
The temporal changes of the total serum lipids during the atherogenic and the therapeutic periods are summarized in Table 2. The
serum lipids showed only a slight increase in the control animals
being on a basic diet over the whole period of 120 days (Group
IV). The serum lipids increased under the atherogenic diet steadily during 60 days reaching values 3 times higher than the normal
level. They decreased within the following period of basic diet
to values two times higher than the normal level (Group I), whereas EPL (280 mg/kg/d) reduced the total lipids to the initial normal level (Group III). This therapeutic effect was a slow steady
221
process over 60 days. Similar observations were made for the individual lipid fractions (14).
Concomitantly, there was a marked difference in the fatty acid
composition of the lipid fractions in the serum, the liver, and
the aortic wall with and without EPL-therapy (Tables 3 and 4).
EPL caused an increase of the ratio of unsaturated to saturated
fatty acids beyond the normal values (Group IV, zero control),
while this ratio was lower than the control values in practically
all atherosclerotic controls (Group I). The atherogenic diet obviously increased the portion of saturated fatty acids, which
were exchanged for polyunsaturated ones due to the action of
essential phospholipids.
These changes in favor of the unsaturated fatty acids due to the
oral EPL-therapy depended strongly on dose (13, 14); they were
less pronounced at low doses, but were statistically significant
at high doses in most cases both in rats (Table 3) and in pigs
(Table 4). There were no significant differences between male
and female animals (13, 14), therefore the data of both sexes
were averaged in Table 3 and 4.
2. Experimentally Induced Atherosclerotic Retinopathy
The results of the ophthalmoscopic investigations of the arterial

vessels of the retina are summarized in Table 5. The area of the
vessels was determined before and after an atherogenic diet of 60
days and after EPL-therapy. A narrowing of the arterial vessels
due to the effects of the atherogenic diet was observed, which
was partly restored after the oral application of EPL.
Table 5: Changes of the area of retinal blood vessels in 6 miniature pigs fed an atherogenic diet of 60 d (0).
followed by additional 60 d of basic diet (I), or basic diet plus EPL (28 and 90 mg/kg/d) (III). Data given in
planimetric units
Before treatment
28
29
16
12
Atherogenic diet
23
24
10
10
Atheroscl. control
III a
Curative therapy 28
11
10
III b
Curative therapy 90
27
27
16
Animal No.
red
li g ht
II.
-oj.
1,. 1'
, ,;'H
.':'
"
- ..
.,
bl ue
tl.
OA
OA
blue
light
fl.
fl.
::&II
MA
Ii ght
.'
rf :
~ ~
.-++,.
red
Ii ght
fl.
light
white
..;..
. ".
"',)"
' .
iii
.'"
white
light
11.
MA
"
11.
"
,~l
I..,. . 1 . _ .
ft .
Err
1l.
blue
light
I
blue
fl.
lig ht
"'t-t-~~
OA
OA
! III ,I I H-t-i !.I .' I
.-W-I+-~ ! II L ~+:-f......:.-
MA
liF.,E~J
l:1:tt! . LIT'1"''''
.,7;;;,.
17;~~ HJIIH
lig ht
red
light
fl.
red
11.
~hite
lIght
MA
'"
N
N
223
MA
white
light
fl .
MA
red
lighi
blue
Iig hi
t t.
fl.
white
Iig h I
fSJ
DA
~~ I=~~
1... 11
l't
.J
,~
. ~~
fl.
DA
i ~"
red
lig hi
fl .
blue
lig hi
tl.
[I
fRS
!Iii
~:
-'-
i""
"t
,~
,1
.",
v
",.~
~ ~~~~
i:l_,-"
Fig. 4: Electroretinographic records in the pig under mesoptic (MA) and dark adaptation (DA) before (a)
and after (b) atherogenic diet of 60 days and after another 60 days of EPL-treatment (c)
Examples of the electroretinograms (ERG) determined after mesoptic

(I1A) and dark adaptation (DA) before and after atherogenic diet
of 60 days and after EPL-therapy of the induced atherosclerosis
(60 days) are shown in Fig. 4a-c. White, red and blue light flashes
were used for eliciting. The normal ERG showed a negative a-wave
followed by a positive b-wave under white stimuli, a negative
a-wave under red, and a positive b-wave under blue stimuli (Fig.4a).
After 60 days of atherogenic diet the a-wave often was missing
or was reduced in amplitude, and both waves seemed to be slowed
down (Fig. 4b). These changes were observed in 7 of 9 animals,
but they were no longer visible after another 60 days treatment
of the induced atherosclerosis by EPL in 4 of 5 animals (Fig. 4c).
224
DISCUSSION
The results of this investigation give evidence that orally

applied essential phospholipids (EPL) are capable of preventing
and curing experimentally induced atherosclerosis in rats and
in miniature pigs. The prevention and the curative effects both
strongly depend on the applied dose of EPL, suggesting a pharmacodynamic effect. The doses of EPL necessary to exert the therapeutic effects are high in rats, which are known to be fairly
resistant to induction of atherosclerosis and therefore require
great amounts of the drug for curing. Miniature pigs, more com~
parable in their metabolism to humans, need far less amount of
the therapeutic agent. The daily effective curative or preventive dose (90 mg/kg) in pigs being on a strong atherogenic diet
is only thrice ~s high as the one known to be effective in human
hyperlipidemia.
As experienced in human hyperlipidemia, the rate of lipid lowering with time due to the EPL-treatment was rather slow. It took
60 days of EPL-treatment to reduce the total serum lipids from
300 per cent after atherogenesis to normal values, while in the
untreated group the total lipids spontaneously decreased by only
one third. In contrast to this slow but effective lipid lowering, the change the fatty acid composition in favor of the unsaturated fatty acids in man occurs within a few days, see
DITSCHUNEIT et al. (15). This might, therefore, be the more important antiatherosclerotic mechanism.
The effectiveness of orally applied essential phospholipids,
which had been questioned occasionally, is now well understood,
since detailed data on the pharmacokinetics of orally applied
essential phospholipids are available (eg. 16).
The use of ophtalmologic planimetry of retinal vessels and of
electroretinography for in vivo determination of the grade of
atherosclerotic changes appears to be promising, however, the
relevance of these techniques and their reproducibility have to
be evidenced by future work. On the other hand the data obtained
so far indicate a confirmation of the histopathologic results
that orally applied EPL is capable of curing and preventing experimentally induced atherosclerosis in rats and pigs.
REFERENCES
- BYERS S.O. and FRIEDMAN M.: Effect of infusions of phosphatides upon the atherosclerotic aorta in situ and as an
veular aortic implant. J. Lipid Res. 1, 343-348 (1960)
2
- ADAMS C.W., ABDULLA Y.H., BAYLISS O.B. and MORGAN R.S.:

Modifikation des Aorta-Atheroms und der Fettleber bei Cholesterin-geflitterten Kaninchen durch intravenose Injektionen
von gesattigten und mehrfach ungesattigten Lecithinen. J.
Pathol. Bacteriol 94, 77-87 (1967)
225
3
- HOWARD A.N. and PATELSKI J.: Effect of Intravenous Polyunsaturated Phosphatidyl Choline in Experimental Atherosclerosis. Verh. Dtsch. Ges. Inn. Med. 78, 1245-1248 (1972)
- HOWARD A. N., PATELSKI J., BOWYER D.E. and GRESHM1 G.A.:

Atherosclerosis induced in hypercholesterolemic baboons
polyunsaturated phosphatidyl choline. Atherosclerosis li,
17 (1971)
- ADAMS C.W.H. and ABDULLA Y.H. in: G. SCHETTLER (ed.)

Phospholipide in Biochemie Experiment und Praxis. Thieme
Stuttgart 1973, p.p. 49-53
- STAFFORD W.W. and DAY CH.E.: Regression of Atherosclerosis

Effected By Intravenous Phospholipid. Atery 1, (2) 106-114
(1975)
-
- LEUSCHNER F., WAGENER H.H. und NEUMANN B.: Antihyperlipamische und antiatherogene Wirksamkeit der EPL-Substanz im
pharmakologischen Versuch. Arzneimittelforsch. (Drug.Res.)
1976 in press
- LEMBECK F.: Die Wartung und Flitterung von Ratten im Laboratorium. Arzneimittel-Forsch. 1, 50 (1953)
- HOOGERWERF S.: Der EinfluB von Vasolastine auf klinstliche

Sklerose bei Ratten und Arteriosklerose des Menschen. Arztl.
Forsch. ~, 540 (1955)
10 - HARTROFT W.J. and THOMAS W.A.: Pathological lesions related

to disturbances of fat and cholesterol metabolism in man.
J. Am. Med. Ass., 164, 1899 (1957)
11 - FOLCH J., ASCOLY J., LESS M., MEATH J.A. and LE BARON F.N.:
Preparation of lipid extracts from brain tissue. J.Biol.
Chern. 111, 833 (1951)
12 - HAWKE J.C., HANSEN R.P. and SHORLAND F.B.: Gas-liquid chromatography, J. Chromatogr. ~, 547 (1959)
13 - SAMOCHOWIEC L., KADLUBOWSKA D. and ROZEWICKA L.: Investigations in experimental atherosclerosis. Part 1: The effects
of phosphatidylcholine (EPL) on experimental atherosclerosis
in white rats. Atherosclerosis ~, 305-317 (1976)
14 - SAMOCHOWIEC L., KADLUBOWSKA D., ROZEWICKA L., KUZNA W. and
SZYSZKA K.: Investigations in experimental atherosclerosis.
Part 2: The effect of phosphatidylcholine (EPL) on experimental atherosclerotic changes in miniature pigs. Atherosclerosis ll, 319-331 (1976)
15 - DITSCHUNEIT H., KLOR H.-U. and DITSCHUNEIT H.H.: Effect of
Essential Phospholipids on the Carbohydrate-Induced Hypertriglyceridemia. In: Phosphatidylcholine - Biochemical and
clinical aspects of essential phospholipids; edited by H.
PEETERS, Springer, Berlin-Heidelberg-New York, (1976)
226
16 - LEKIM D. and GRAF E.: Tierexperimentelle Studien zur Pharmakokinetik der EPL-Substanz. Arzneimittelforschung (Drug.
Res.) 1976, in press
17 - KARCZEWICZ D.: Ocena Zachowania sie naczyc siatkowki swin
wietnamskich po dozylnym podaniu witnaminy. PP. Klin.
Oczna i2, 433-436 (1975)
Essential Phospholipids in Red Cells

and Thrombocytes

on the Flow Properties of the Blood
A. M. Ehrly and R. Blendin
Klinikum der Johann-Wolfgang-Goethe-Universitat, Zentrum der Inneren Medizin,
Abteilung fUr Angiologie, D-6000 Frankfurt a. M. 70, Germany
Abstract: In 11 healthy test subjects and 8 patients with chronic

arterial occlusive diseases, the blood flow properties were determined before and 15, 30 and 45 min. after the intravenous
injection of 750 mg essential phospholipids (LIPOSTABIL, NATTERMANN). LIPOSTABIL did not change the "macro-rheological" parameters, such as hematocrit, blood viscosity and plasma viscosity. However, a statistically significant increase of the passage
of erythrocytes did occur through a capillary filter (with
capillaries of 8 um in diameter), which simulates the conditions
prevailing in the microcirculation. In the patients with peripheral arterial occlusive diseases, an increase of the capillary
filtrate could similarly be found, but these differences Were
just below the borderline of statistical significance. The present findings indicate that, in some way, the flow properties of
the erythrocytes, and thus their deformability, are influenced
in a manner such that their passage through narrow capillaries
is facilitated, and the capillary flow-rate is thereby increased.
INTRODUCTION
The knowledge about the influence of the phospholipids (especially the choline phosphatides) on the flow properties of the
blood is still fragmentary. The first indication that the essential phospholipids (EPL) might exert a disgregating action were
provided by previous results showing that the choline phosphatides can influence the erythrocyte sedimentation rate (1, 2, 3,
4). It is known also that surface-active substances can affect
the blood viscosity (5). EIKERMANN (6) found that the EPL substance is surface-active. Earlier experiments showed that the
addition of a soluble preparation of essential phospholipids
(LIPOSTABIL, NATTERMANN), in concentrations around 100 mg per
100 ml blood, produces an appreciable disgregation of erythrocyte
aggregates in vitro (1, 3, 4). These high concentrations do not,
of course, correspond to those that can be expected in the blood
when this agent is injected into patients. However in further in
vitro experiments (7), where most of the solubilizing agent had
been dialyzed off from the LIPOSTABIL-preparation, it was found
that the phospholipids could then disgregate erythrocyte aggre-
229
gates, even in low concentrations, and could reduce the structure viscosity of the blood in vitro. It was concluded that the
bile acid, which acts as the solubilizing agent in the phospholipid product, inhibits the inherent disgregating action of the
phospholipids in vitro. Comparative tests undertaken with highly
water-soluble lysolecithins showed (2, 3) that a disgregating
action can be exerted by these compounds at concentrations
around 5 mg/100 ml blood. Since it is known that, after injection of LIPOSTABIL, the bile acids are rapidly excreted by the
liver, it can be calculated theoretically that an essential phospholipid concentration of some 5 mg/100 ml blood would be sufficient to exert a disgregating effect in vivo.
And in fact, after injection of LIPOSTABIL into 10 healthy test
subjects, a slight inhibition of the erythrocyte sedimentation
values, together with comparable hematocrit values, has been
found, and has been interpreted as a manifestation of a slight
disgregation of erythrocyte aggregates (2, 3). In some instances,
the blood viscosity at low shear rates (structure viscosity) was
found to be diminished.
The purpose of this present in vivo study is to obtain further
information about the effect of phospholipids on the flow properties of the blood, particularly in the region of the microcirculation.
METHODS
In 11 healthy test subjects, two control samples of venous blood

was taken through a Braunlile at a 10-minute interval, and heparin was added to prevent blood clotting. Within 10 minutes,
15 ml LIPOSTABIL (corresponding to 750 mg EPL) was administered
intravenously. Further blood samples were taken 15, 30 and 45
minutes after the injection of LIPOSTABIL.
In 8 patients with chronic occlusive diseases of the peripheral
arteries in stage II, the same procedure was adopted; blood
samples were taken 15, 60 and 90 minutes after the injection of
750 mg essential phospholipids had been completed.
The erythrocyte sedimentation rate was determined in the normal
way using WESTERGREN pipettes. The number of erythrocytes per
mm 3 blood was counted with an electronic counter (Picoscale,
HELLIGE Company, Freiburg). The hematocrit was determined with
a microhematocrit centrifuge (A. HATTICH Company, Tuttlingen).
The blood and the plasma viscosity were determined by means of
an OSTWALD capillary viscosimeter, and expressed as a relative
viscosity in comparison to water (H20 = 1). The ca~illaries for
blood had a diameter of 0.6 rnm, and for plasma, 0.5 rom; the
length of capillary amounted to 100 mm. The range of error of
this method is ~ 0.5% (8).
230
For the measurement of the blood viscosity at various shear rates,
the LVT-micro-cone-plate-viscometer (BROOKFIELD Company, Houston,
Texas) was used. This apparatus has been described in detail by
WELLS (9). It was used at shear rates of 5.6; 11.5; 23.0 and
46.0 s-l. The filtration rate of the blood was measured using
the procedure of EHRLY and ROSSBACH (10, 11). Erythrocyte suspensions with a hematocrit of 10% were passed through Milliporefilters Type SCWP 02500, the driving force being produced by
the hydrostatic pressure of the blood column (12.9 mm). The mean
pore diameter of the filters is 8 + 1.4 ~m; all the filters belonged to one batch, because differences may occur between different batches. The parameters measured were the filtrate volume
(ml) passing through the filter in 5 minutes, the number of
erythrocytes/mm 3 contained in this filtrate, and the total number
of erythrocytes calculated therefrom. This total number of erythrocytes in the filtrate is taken as a measure of the deformability of the erythrocytes (10, 11).
RESULTS
After injection of 750 mg essential phospholipids (EPL) into

11 healthy test subjects, slight decreases occur in the erythrocyte sedimentation rate and also in the number of erythrocytes
in the whole blood (see Table 1). The hematocrit value remains
unaltered during the experiment. The blood and plasma viscosities, measured with the Ostwald viscometer, also remain unaltered after injection of LIPOSTABIL.
The viscosity values measured with the BROOKFIELD viscometer
similarly show no change after the injection of LIPOSTABIL.
In regard to the erythrocyte filtration through the 8- ~m filters,
both the filtrate volume and the number of erythrocytes per mm 3
of filtered blood are increased after the injection of LIPOSTABIL
in comparison with the two control values (Table 2). The total
number of filtrated erythrocytes is correspondingly increased.
This is shown in more detail in Table 3: The changes for the
values measured 45 minutes after the injection as compared with
the second control values are statistically significant and there
is a corresponding trend for the other values. These tests were
performed at 23C.
Table 4 shows the influence of 750 mg essential phospholipids
on the flow properties of the blood of 8 patients with chronic
arterial occlusive disease. Here, only the blood viscosity, the
plasma viscosity and the erythrocyte filtration were measured.
These investigations were performed at 37C. It is found that,
in patients also, the blood and plasma viscosities do not appreciably change during the experiment. In the erythrocyte filtration test, a slight increase occurs in the number of erythrocytes passing through the capillary filter per unit time, the
maximum value being attained 60 minutes after the injection is
completed; after 90 minutes, this value returns to the initial
231
Table 1: Effect of essential phospholipids (750 mg EPL intravenously injected as 15 ml LIPOSTABIL) on rheological
parameters of the blood. Average values as obtained from 11 healthy test subjects. The blood samples were taken 20 ( I )
and 10 ( II ) minutes before and 15 ( III ), 30 (IV
l.
and 45 (V) minutes after the injection of EPL was completed.
The relative viscosity was determined by the method according to OSTWALD, the absolute blood viscosity by the method
according to BROOKFIELD
Sample
II
III
IV
10.2/
25.2
9.6/
25.1
Ery. sedimentation
rate (rom)
11 .3/
26.0
10.9/
26.0
10.f:i/
Number of Ery.
UJIio/rom 3 )
4.23
4.12
4.12
4.14
4.16
Hematocrit (%)
33.8
33.8
33.8
33.8
33.8
Relative viscosity
of blood (H20 = 1)
5.39
5.38
5.38
5.36
5.42
Relative viscosity
of plasma (H20 = 1)
1. 69
1. 69
1. 69
1.68
1. 70
14.7
12.7
10.6
8.1
14.3
12.5
10.8
8.1
14.3
12.8
10.7
8.0
15.0
13.0
10.4
8.2
Blood viscosity (cp)

-1
5.6 5 ~1
11. 5 5 -1
23.0 5 -1
46.0 5
24.6
16.2
12.1
11.4
8.1
Table 2: Effect of essential phospholipids on the flow properties of the blood of 11 healthy test subjects. The average
values from the erythrocyte filtration through a,.um Millipore filter are given. Nomenclature of the blood samples as
in Table 1
II
III
IV
IV
Time after EPLinjection
-20
-10
15
30
45
Filtrate volume
(Ill)
258
272
296
307
311
Number of ery.
in the filtrate
(X 10 3 / mm 3 )
377
372
405
431
441
1078
1128
1237
1368
1440
Sample
Total erythrocyte
count (X 10 5 )
232
Table 3: Effect of essential phospholipids injected into 11 healthy test subjects on the total erythrocyte count (x 10 5 )
in the erythrocyte filtration experiment (see Table 2 )
Sample
II
III
IV
-20
-10
15
30
45
1
2
3
4
5
1436
701
627
919
1862
1233
725
715
1522
1571
1303
707
1104
1441
2150
1984
784
1571
865
2321
1345
610
1314
1111
3171
6
7
8
9
10
11
1950
1843
767
600
705
450
1949
1843
894
851
505
605
1918
1535
669
831
813
1133
1897
1847
1008
909
726
1139
1924
2174
874
1377
928
1010
mean
S.D.
S.E.M.
1078
576
174
1128
517
156
1237
491
148
1368
569
172
1440
733
221
Time after EPLinjection (min)

Test subject no.
t-test:
II/III:
II/ IV:
II/ V:
(8)
(8)
(S)
2.983
4.393
5.270
t
t
t
=
=
1.201
1.527
1.960
<
0.15
<
0.05
p < 0.1
value. This increase of the total number of erythrocytes passing

through the filter found 60 minutes after the injection was not
statistically significant as compared with the control however,
a statistical significance (p < 0.05) is found bebleen the maximum value (after 60 minutes) and the value found 90 minutes
after the injection (see Table 4).
233
Table 4: Effect of essential phospholipids on rheological parameters and blood flow properties as determined from the
erythrocyte filtration experiment (temperature 37 0 C I. Mean values from 8 patients with chronic arterial occlusive
diseases (for details see Table 1 1
II
III
IV
Time after EPLinjection (min)
-20
-10
15
60
90
Relative viscosity
of blood (H 20 = 1)
5.44
5.36
5.34
5.46
5.44
Relative viscosity
of plasma (H 20 = 1)
1. 85
1.84
1.85
1. 84
1.82
Total count of
filtrated ery.
(X 10 5 )
420
450
590
600
460
S.D.
350
350
670
420
400
Sample
t-test:
II/IV:
IV/ V:
p
p
<
<
0.05
0.05
DISCUSSION
The peripheral circulation, especially in the region of the

microcirculation, depends largely on the f~w properties of the
blood. A new concept of the pathophysiology of chronic arterial
vascular diseases, and the therapy derived from it has, therefore, been developed (12, 13, 14), according to which an improved capillary perfusion should be based on improved flow properties of the blood. When essential phospholipids are given intravenously, a slight disgregation of erythrocytes can be deduced from the change of the erythrocyte sedimentation rate,
whereas the "macro-rheological" parameters, such as blood and
plasma viscosities and structure viscosity, show no significant
changes. These macrorheological methods, however, cannot detect
alterations in the microcirculation, and therefore the erythrocyte filtration through very narrow, capillary-like systems were
tested in vitro. In healthy test subjects, 45 minutes after the
end of the injection, the total number of erythrocytes that pass
through the filter system in vitro per unit time is increased by
more than 25%. These variations could be confirmed statistically.
In patients with chronic arterial occlusive diseases also a
marked increase of the total perfusion of blood through narrow
234
capillaries could be detected in vitro; this finding could not,

however, be secured statistically, probably on account of the
relatively small number of cases. Here again, though, the trend
is comparable with the findings obtained in healthy test subjects.
The improvement of the blood flow properties produced by an intravenous injection of LIPOSTABIL in man cannot at present be
definitively explained. The results of the erythrocyte filtration simply show that, without any increase of the pressure
gradients and without any vasodilatation or any hemodilution,
the flow-rate of blood in these narrow, capillary-like cavities
or the filters has been increased. The plasma viscosity itself
probably has no effect, since it remained unchanged throughout
the investigation. No spontaneous alteration of the erythrocyte
filtration can be assumed to have occurred, because the experiments were performed at a time when spontaneous alteration of
the blood flow properties are minimal (1S, 16). The most plausible explanation would be that the deformability of the erythrocytes has increased, and that their passage through the
narrow and highly ramified capillaries of the test-filters might
thus be facilitated. This might be caused, for example, by
changes of the lipid composition of the erythrocyte membrane
produced by the exchange of membrane phosphatides with saturated
fatty acids against phosphatides with unsaturated fatty acids
(EPL). It may also be discussed whether a slight inhibition of
the aggregation of the erythrocytes might similarly exert an
influence on the filtrability of the blood after LIPOSTABIL. As
a further possibility, the injection of LIPOSTABIL might modify
the lipids of the blood and thus also the blood flow properties.
An accurate analysis of these various factors would be required,
in order to weigh these theoretical possibilities against each
other, and to identify the true mechanism.
These present studies comprise a combination of in vivo and in
vitro tests. The preparation LIPOSTABIL was injected intravenously into healthy subjects and into patients with chronic
arterial occlusive diseases; the effect on the blood flow properties or the microcirculatory properties, however, were measured on in vitro test systems. Other methods would have to be
adopted, in order to solve the question whether the in vitro
alteration of the blood flow properties can also be detected
in the microcirculation of human tissue, and might exert there
a therapeutic effect.
Such a method has recently been provided by the measurement of
the oxygen tension in the ischemic muscle of patients with
chronic arterial occlusive diseases (17). An increase of the
oxygen tension in the tissue, taken in conjunction with these
present results, might then be interpretable as an improvement
of the microcirculation in the human tissue.
235
REFERENCES
1 -
BARTELS, U.: tiber den EinfluB der Cholinphosphatide auf

die Aggregation der Erythrozyten und die Viskosit~t des
Blutes. M.D. TheSiis, r1ed. Univ. Klinik, Frankfurt (1971).
2 -
EHRLY, A.M.: EPL and blood viscosity. In: Phospholipids.

Ed. by G. SCHETTLER, Thieme, Stuttgart 1972, S. 97
3 -
EHRLY, A.M.: Desaggregation von Erythrozytenaggregaten

durch "essentielle" Phospholipide. In: Gef~Bwand und Blutplasma IV. Ed. R. E~mRICH, VEB Gustav Fischer Verlag, Jena,
1974, S. 165
4 -
EHRLY, A.M.: Influence of phospholipids on blood viscosity.

- First International Symposium of Chemistry, Metabolism,
Function and Pharmacology of Phospholipids Scecin Sept. 1972.
Ed. by L. SAMOCHOWIEC and J. WOJCICKI 1973, p. 45
5 -
EHRLY, A.r1.: Reduction in blood viscosity at low rates of

shear by surface active substances: A new hemorheologic
phenomenon. - Biorheology 2' 209 (1968)
6 -
EIKERMANN, H.: Sonderstellung und Therapie der Atherosklerose und arteriosklerotischen Krankheitsgeschehen. - Fortschr. Med. 74, 381 (1956)
7 -
EURLY, A.M.: H~morheologische Probleme bei Venenerkrankungen. - Zentralbl. Phlebologie ~, 338 (1967)
8 -
KtlHJ.ER, S.: Untersuchungen iiber den EinfluB von Diuretika

auf die FlieBeigenschaften des Blutes. - Statistischer Anhang S. 85. M.D. Thesis, Med. Univ. Klinik, Frankfurt (1973)
9 -
WELLS, R.E., et.al.: Measurement of viscosity of biologic

fluids by cone plate viscosimeter. - J. Lab, Clin. Med. 57,
646 (1961)
10 - EHRLY, A.M. and ROSSBACH P.: The detection of changes in

erythrocyte shape by a filtration method using 8 ~m filters.
- II. International Symposium on metabolism and membrane
permeability of erythrocytes, thrombocytes and leucocytes,
Wien 1972. In: Erythrocytes, Thrombocytes and Leucocytes.
Ed. by GERLACH, MOSER, DEUTSCH, WILr1A.~S, Thieme, Stuttgart
(1973), S. 52
11 - EHRLY, A.M. and ROSSBACH P.: Studies with an 8 ~m filter
system. - VII. Europ. Soc. Microcirculation, Aberdeen 1972,
Pt. I. Bibl. anat. 11, 55 (1973)
12 - EHRLY, A.M.: Untersuchungen iiber die Senkung der Strukturviskositat menschlichen Blutes im Hinblick auf die Therapie
angiologischer Erkrankungen unter besonderer Beriicksichtigung der Blutlipide. - Habilitationsschrift Med. Univ.
Klinik, Frankfurt, 1969
236
13 - EHRLY, A.M.: Increased blood flow by improvement of the

flow properties of blood: A new concept in the treatment of
vascular diseases. - VIII. Intern. Congr. of Angiology, Rio
de Janeiro 1972. In: Progress on Angiology: Prodeedings
VIII. Intern. Congress of Angiology 1972. Ed.: G.C. de LEMOS
CORDEIRO and R.C. MAYALL. Vol. III, 987 (1974)
14 - EHRLY, A.llf.: Verbesserung der FlieBeigenschaften des Blutes:
Ein neues Prinzip zur medikamentosen Therapie chronischer
peripherer arterieller Durchblutungsstorungen. - VASA 2,
Suppl. 1 (1973)
15 - EHRLY, A.M. and JUNG G.: Circadian rhythm of human blood
viscosity. - I. Internat. Kongr. tiber Biorheologie Lyon
1972, Biorheologie 1, 577 (1973)
16 - WARSTAT, TH.: Untersuchungen,tiber den EinfluB von niedermolekularem Dextran auf die Filtrabilitat des Blutes in
8 urn Filtern. - M.D. Thesis, Med. Univ. Klinik, Frankfurt
(1974)
17 - EHRLY, A.M., K~HLER H.-J., SCHROEDER W. und MULLER R.:
Sauerstoffdruckwerte im ischamischen Muskelgewebe von Patienten mit chronischen peripheren arteriellen VerschluBkrankheiten. Klin. Wschr. 21, 687 (1975)
The Treatment of Arterial andVenous

CirculatoryDisturbances with EPL
J. Klemm
Rontgen-Abteilung der II. Medizinischen Universitatsklinik,
0-8000 Munchen, Germany
Abstract: The therapeutic effect of essential phospholipids

(EPL) on the circulatory parameters 1) of blood flow in the muscle at rest and during reactive hyperemia, 2) of transport speed
in the lower extremities, 3) of cerebral blood flow, and 4) cf
relative change of blood viscosity were investigated. 26 patients
suffering from circulatory disturbances received 3 x 600 mg
EPL/d per os for 30 days. The data of 20 patients were evaluated
The circulatory parameters as determined by the 13 3 Xe-clearance
method were improved on a high level of statistical significance
(0.01 ~ p ~ 0.001). The amount of improvemen't was inbetween 7 and
19 per cent as compared to the control. The blood viscosity showed
a decrease by 2.7 per cent relative to the vaZue found in normals.
It is concluded that EPL causes a marked improvement of the blood
flow properties, since also in normal persons an increase of the
blood flow after the treatment was observed. No side effects were
seen during the whole t~ial of EPL.
INTRODUCTION
In a series of long-term trials (from 1972 to 1975) eleven different therapeutic preparations -claiming an improvement of
blood circulation in the brain and in the lirnbs- were tested on
237 patients suffering from circulatory disturbances. One of the
agents tested was EPL and the present contribution will report
the results of this part of the investigations. It will be shown
that EPL causes a significant improvement of the cerebral and
peripheral blood circulation as determined by the 133Xe-clearance
method.
METHODS
Before starting the therapy the patients were subjected to a

general clinical examination and to angiography of the brain and
of the lower extremities. The circulatory disturbances were
classified according to FONTAINE. In case a cardiotherapy was
238
Left
Right
Fig.1 1:
Scheme of the technique of muscle blood flow measurement
Reactive hyperaemia
Arterial obstruction (Z5U mmHgl

r---defined --,
Resting
foot-lifting
blood flow
work
~VI~
Distn bullon lime
----
il~~~______j~__~~~~--~
A"1
IMBF= 0' 1611
:~
-j1 Min_i+
:
I
DH
I
II
II
II
0.: I Min-Ii
I
I
I
II
:
:I
Duration of
~ reaclhyperaemia
+I
3Min.5Min.
....-t"I"'" 2 Min.
4 Min.
Fig. 2: Pattern of blood flow determination in leg (arm) using the 133Xe clearance method: Activity vs.
time curve (read from right to left )
239
necessary, it was kept unchanged during the whole period of the

trial. The patients received 3 x 600 mg/d of essential phospholipids (EPL) (NATTERMANN) per os for a period of 30 days. The
therapeutic effect was determined by measuring the following
parameters before and after the treatment:
1.
2.
3.
4.
133Xe
13 3Xe
13 3Xe
Change
muscle clearance (ml/100 g muscle/min)

cerebral blood flow (ml/100 g brain/min)
transport velocity (cm/sec)
of blood viscosity
For measuring the muscle clearance a depot of 100 ~Ci of 133Xe

dissolved in 0.5 ml of a physiological saline was injected into
the musculus tibialis of both of the lower extremities of the
reclining patients (Fig. 1). The velocity of Xenon depletion was
measured by scintillation tubes. The pattern of the blood flow
measurements is illustrated in Fig. 2 (read from left to right).
After a period of Xenon distribution the blood flow in the resting
state was determined. Then the arteries in the thighs were obstructed for five minutes and defined foot lifting work was performed under bloodless conditions for-3 minutes. In type III and
IV patients the footlifting work was strictly controlled and limited to the still tolerable pain, i.e. the working time was reduced accordingly. The resulting metabolic deficit induces a
maximum reactive hyperemia and a correspondingly high rate of
Xenon depletion after removal of the arterial obstruction_
The technique of measuring the transport velocity is illustrated
in Fig. 3. Scintillation tubes were adjusted above the epimalleo-
~----cm
Fig.
3:
.. I
Scheme of the technique of transport velocity measurement. The time of transport of injected
133Xe from the epimalleolar to the inguinal region is determined
240
jugular v.
excreted
in lungs
injection
completed
excreted
in lungs
Fig. 4: Scheme of the technique of determination of the cerebral blood flow. Immediately after the
injection of 133Xe the radioactivity is distributed over the brain (left). Successively, the activity
is washed out (right)
lar and the inguinal regions and their distance was measured.
200 pCi 133Xe were injected into a vein of the instep and the
time of transport of radioactivity between the two measuring
points was determined. The resulting transport velocity is a
measure of the flow properties of the blood provided the blood
pressure remains unchanged.
The cerebral blood flow was measured accordingly. 400 pCi 133Xe
dissolved in 1 ml saline were injected in the arteria carotis
interna (Fig. 4). The radioactivity is distributed in the brain
along the concentration gradients, since diffusion of inert gases
is not restricted by the blood-tissue barriers. The wash-out of
133Xe by blood free of activity after the bolus injection was
recorded by outside located scintillation counters. From the
wash-out curve the average blood flow volume (CBF m) was evaluated.
The methods used to determine the blood viscosity have been previously described in detail.
241
Table 1: Effect of essential phospholipids on the muscle blood flow in the lower extremities. Mean values
before and after EPL - therapy ( 1.8 g / d per os for 30 days) and their differences are presented.
Classification according to FONTAINE
Resting blood flow
Group of
after
disease N before
](1
t::.
x2
X2-X1
ml
~n
ml
ml
normal
2.3
I II
10
1.5
reactive hyperaemie
t::. in% before after

of before X1
X2
t::.
X2- X1
statist.
t::. in% rest. bL fl. reactHyp

of before
p
p
ml
1.1
3.4
0.01
0.005
21.7 24.2 2.5
11.5
0.02
0.005
ml
ml
2.6 0.3
13
32.4 33.5
1.75 0.25
16.7
"-
III IV 10 0.9
1.12 0.22 [ 23.2
13.8 15.7
2.9
21.0
0.001
0.001
RESULTS
The effects of the EPL-treatment on the muscle blood flow in

the lower extremities are summarized in Table 1. In normal test
persons, the flow rate in the resting state was found in the
range of 1.8-2.5 ml/100 g muscle/min and for maximum reactive
hyperemia it varied from 30 to 36 ml/loo g muscle/min. In normals
as well as in type I + II and type III + IV patients there was a
statistically significant increase of the blood flow after the
EPL treatment as compared with the values measured before. The
fact that this improvement of the blood flow was observed not
only for reactive hyperemia but also in the resting state indicates an effect on the blood flow properties rather than on the
blood vessels.
Table 2 shows the influence of EPL on the velocity of transport
in the lower extremities. The velocity of transport was increased
significantly in normals as well as in type I + II and type III
+ I patients due to the treatment. This result again favors an
improvement of the blood flow properties. The lower therapeutic
effect observed in the type III + IV patients possibly can be
explained by the presence of longer collateral pathways.
An example of a wash-out curve for 13 3 Xe from the brain is shown
in Fig. 5. The normal value of cerebral blood flow volume as derived from these curves depends on age and is CBFm = 55 6 ml/
100 g brain/min for normals of 30 years and younger. CBFm decreases with age and was on the average 43 4 ml/loo g brain/
min for the group tested. This value was increased by the treat-
242
Table 2: Effect of EPL - therapy (3 x 600 mg EPL / d per as for 14 days) on the transport velocity
in the lower extremities measured with the aid of two scintillation counters after the injection of 133Xe
into a vein of the instep. Notation as in Table 1
Group of vessels
disease
------------
Velocity
II ino,o
before after II
em/sec
.,.
8.8
0.01
x,
x2
X2- X1 of x1
em/sec em/sec
normal
6.72 7.31 0.59
II
4.12
4.47 0.35
8.5 0.001
III
IV
2.39
2.51 0.12
5.02 0.05
~.,36 8.K. 83a

A. CAR.INT. L1.
PARI ETAl
vp~ 1.5 inch/min

FRONTAl
rc!:10
RANGE : 100 K x 2
I,,!J. MENGE: 400,ci 1331Ce
CBFe = 34.7
CBFw ='16.3
CBFm ='27.3
~~ = 104.3
IllFw= 18.6
CBFm=34.0
Fig.
5:
Wash - out of
133Xe from the brain
(Original record, read from right to left )
243
Table 3: Effect of EPL therapy (3 x 600 mg EPL / d per as for 30 days) on the cerebral blood
flow as determined by the 133Xe clearance method. Mean values and differences in ml / 100 9 brain / min
are presented
CBFm
Group of
N before after
disease
~n
l:l
l:l in%
Xl
x2
x2- xl
of before
ml
ml
ml
Cerebral
vascular 15 43.2 46.1
sclerosis
2.9
6.7
0.05
ment with EPL by 6.7 per cent (Table 3). Since the blood vessels
of the brain are known not to be influenced, this result suggests
an effect of EPL on the blood flow properties.
The value of blood viscosity of 18 patients was found to be reduced by 2.7 per cent due to EPL-treatment as compared with the
absolute value found in normals. In this case the duration of
therapy was only 18 instead of 30 days.
CONCLUSION
EPL orally applied (3 x 600 mg/d) for 30 days increased significantly the muscle blood flow, the velocity of transport in the
lower extremities, and the cerebral blood flow, and decreased
the viscosity of blood in patients suffering from circulatory
disturbances. The results indicate that the therapeutic effect
is rather due to an improvement of the blood flow properties
than to an effect on the blood vessels.

on Human Platelet Aggregability
J. Schneider. G. Fuchs and H. Kaffarnik
Medizinische Poliklinik der Universitat, D-3550 Marburg, Germany
Abstract:
The platelet aggregability in 31 patients suffering
from a hyperlipoproteinemia of either type IIa, or lIb, or IV
was measured using BREDDINs tests before and after a therapy with
essen tial phospho lipids (EPL) (3g Idie per os). The total period
of the therapy was 3.5 months. Already after 8 weeks of treatment
a significant decrease of platelet aggregability was observed,
whereas the number of platelets remained unchanged and no alteration of coagulation parameters of fibrinolysis was found. These
results are discussed in the light of the role of hyperlipoproteinemia as a risk factor in atherosclerosis.
INTRODUCTION
The occurrence of an enhanced platelet adhesivity after fatty

meals has been demonstrated by HOOLTEN et al. (1) and also by
MUSTARD et al. (2). SCHULZ and WEDELL (3) reported phagocytosis
of fat into platelets. The incorporation of lipids into platelets
has been observed in vivo and in vitro (4,5). Different effects
of lipids or of fatty acids on platelets have been reported: an
increased adhesivity, an enhanced aggregability, or a disturbed
reactive release of ADP on physiological agents like ADP, arterenol, or collagen. RENAUD et al. (6) observed a prolongation
of the coagulation time in relation to the ratio of stearic:
linolenic acids in the ingested fats. GROTTUM et al. (7) were
able to show that "intralipid" infusions (of 46% linoleic acid)
led to a decrease of the release of platelet factor III by ADP or
by caolin, and to a decrease of platelet adhesivity, but without
changing the number, the life time or the aggregability of the
thrombocytes. Under these circumstances a study of the platelet
adhesivity in hyperlipoproteinemic patients and of the effect of
the therapy was of great interest.
METHODS
31 patients (14 male, 17 female; average age: 49 years) suffering

from a hyperlipoproteinemia of either type IIa, or type lIb, or
245
type IV received a therapeutic dose of 3 g/die of liquid EPL

(NATTERMANN) for 3.5 months. The platelet aggregability was determined before and during the therapy using the test according to
BREDDIN (8). After a period of 10 minutes of rotation in glass
tubes the platelet aggregation was classified in five grades independently by several different investigators from slides labelled with a number only.
RESULTS
Already after 8 weeks of therapy a significant decrease of the

aggregability of the platelets was measured. The most impressive
case is illustrated in Figs. 1 and 2 showing the platelets after
10 minutes of rotation in glass tubes before and after the treatment with EPL. A grade IV aggregability was turned down to a grade
I by the therapy; this patient suffered from a hyperlipoproteinemia of type lIb. The results from the investigated 31 cases are
summarized in Table 1. The significance of the changes relative
to the values measured before treatment was determined using the
Mann-Whitney test (significance for ex ~ 5).
Table 1: Platelet aggregation in hyperlipemic patients (type II a, II b, IV) before and after oral therapy with 3 g/d of
essential phospholipids (EPL), Changes are significant for
ex ..
5 (MANNWHITNEY test!.
PAT + S.E.M.
before
after 4 weeks
after 8 weeks
end (mean 15 weeks)
12
11
11
10
2.79
3.32
1.68
1. 75
+ 0.45
+ 0.78
+ 0.40
+ 0.49
7.3
0.0
0.0
DISCUSSION
explanation of the effect of EPL on platelet aggregability may

be derived from the results of VERGROESEN (9). He demonstrated
that the adhesivity of platelets induced by collagen was increased
after feeding of different dietary fats. This activation was less
pronounced, if the diet contained a high portion of linoleic acid.
He concluded that the metabolism of linoleic to arachidonic acid
might favor the production of prostaglandin E 1 , which is known to
be a potent inhibitor of platelet aggregation and -adhesivity and
is -in vivo- one of the strongest antilipolytic compounds known
An
(9)
246
Flg . 1
Flg . 2
The oral dose of 3 g of the substance seems to be very small in

comparison to the amounts of polyunsaturated fatty acids, which
can be ingested by changing the diet. Because of this fact, one
could argue that EPL partly may be resorbed as a whole or may be
resynthesized in the mucosal cells of the intestine to an identical form of phosphatidylcholine.
247
PFLEIDERER, WEBER and MORGENSTERN (10) proposed that EPL may coat
the platelets and protect them against the activating forces of
triglycerides or of fatty acids in the medium.
Polyunsaturated lyso-phosphatidylcholine fractions affect platelet
aggregation in a very different way: BESTERMANN and GILLET (11)
observed an inhibition of irreversible aggregation by fully saturated lysolecithin, but no effect of a lysolecithin fraction containing a very high proportion of polyunsaturated fatty acids.
The speculation on the possible action of EPL could be confirmed,
if alterations in the pattern of platelet phospholipids, before
and after EPL treatment could be measured as it was observed after
dietary regimen by RENAUD (12).
The clinical relevance of our results is elucidated by the fact
that hyperlipoproteinemia is considered to be a risk factor in
atherosclerosis. One of the pathogenetic principles, which has
to be taken into account, is an increased tendency to arterial
thrombosis and microthrombosis, possibly on a preinjured endothelium. HORNSTRA (13) stated that arterial thrombosis may be promoted by an increased aggregability of platelets, and venous thrombosis by an enhanced release to the platelet factor III. Thus, EPLtherapy of hyperlipoproteinemia -with respect to platelet aggregability- also shows a curative effect on the risk of atherosclerosis.
REFERENCES
1 - MOOLTEN S.E., JENNINGS P.B. and SOLDEN A.: Amer. J. Cardiol.

ll, 290 (1963)
2 - MUSTARD J.F. and MURPHY E.A.: Brit. Med. J. !, 1651 (1962)
3 - SCHULZ H. and WEDELL J.: Klin. Wschr. 40 1114 (1962)
4 - WEBER E., MORGENSTERN E. and PFLEIDERER TH. In: Platelets
and the Vessel Wall-Fibrin Deposition. Ed: G. SCHETTLER,
G. Thieme Verlag, Stuttgart (1970), p. 71
5 - PFLEIDERER TH. and 10RGENSTERN E. In: Platelets and the
Vessel Wall-Fibrin Deposition. Ed.: G. SCHETTLER, G. Thieme
Verlag, Stuttgart (1970), p. 79
6 - RENAUD S. and GAUTHERON P.: Atherosclerosis
~,
115 (1975)
7 - GROTTUM K.A., NORDOY A. and HELLEM A.: Thrombos. Diathes.

haemorrh. (Stuttg.) 29, 701 (1973)
8 - BREDDIN U. In: Die Thrombozytenfunktion bei hamorrhagischen
Diathesen, Thrombosen und GefaBkrankheiten. Schattauer,
Stuttgart-New York (1968), p. 114
248
9 - VERGROESEN A.J.: Proc. Nutr. Soc.
11,
323 (1972)
10 - PFLEIDERER TH., WEBER E. and MORGENSTERN E. In: Phospholi-
pide. Ed.:
p. 106
G. SCHETTLER, G. Thieme Verlag, Stuttgart (1973),
11 - BESTERMANN E.M.I-1. and GILLETT M.P.T.: Atherosclerosis..!.,
89 (1972)
12 - RENAUD, S.: Thrombosis Res.

13 - HORNSTRA G.: Thrombosis Res.
!,
!,
25 (1974)
69 (1974)
Subj ect Index
For abbreviations see

(enzymes)
p.4 (lipids), p.5 (lipoproteins), p.202
absorption, intestinal 48-63,

66,77,78
ACAT-enzyme
187,188,192,197,
198,203
acetate 66,67,152,158,160,173
acetone-butanol powders
203
acyl CoA 141,161,180,188,207
acyl CoA carboxylases
acyl CoA transferases
180, 207
adaptation, eye 213, 223
adipose tissue
115
ADP 244
age differences 88,90-95
albumin 72,163,192,195
alpha-lipoproteins 6,16,20,41,
72,127,128
amino acid composition, apoproteins
22
AMP,
see cAMP
amphiphatic helices
37
amphiphilic molecules
133
anemia 41
angiography 237
aorta 28,89,160,163,187,191,
195
, see also arterial
apoA subfractions 21-27,36-41
apoC-I 36-41
apoD
37
apoE
38
apolopoproteins 5,6,11,20-24,
27,36,38
arachidonic acid 4,107,113,245
arginine-rich protein 38
arterenol 244
arterial wall 29,99,140-158,
160-183,187-198,201-208
c hoI est e ry 1 est e r s

1 12 , 19 4 ,
211-214,218,220
lip i d me tab 0 l i s m 15 8, 1 88 ,
189,201
atherogenic diet
191,192,194,
211-214,218,220
atheroma grade
141,146,147,
195
atherosclerosis, human
11,12,
20,28,34,98,99,125,141,
160,194,230,233,237-243,
247
-blood flow 228-234,237-243
, -risk factors
28,98,99,247
atherolsclerosis, experimental
11,20,30,49,123,140,141,
160,187,192-194,211,212
prevention 141,195,211-214
regression 30,34,41,196,
211-216
atherosclerotic lesions
28,30,
140,157,160-162, 182, 187,
195,196,214-217
baboon 21-23,27,187,192,195,
197,212
behaviour 214
betalipoproteins 6,16,20,29,
72,127,217
bile acids
72,189,192,212,
229
bile cannulation 63,78
binding, lipid-protein 20,
22-25
blood-brain-barrier 66,76
250
blood, flow properties 228234,237-243

-, viscosity 30,228-230,237,
243
brain 53,58,74-76,89
c
cAMP
1 15 - 1 1 7 , 12 0
caolin 244
capillaries 228,229,234
carbohydrate 17,18,98-100,
107,112
cerebral blood flow 237,239241
cephalins, see PE
chain length of FA in lipids
24,28,87,205
chain length elongation 141,
171
chicken 212
chimpanzee
17-19,21-23
choles tasis 41
cholesterol
17,34,35,87,88,
127,187,189,197,201,203
212
arterial wall
141,161,168,
169
es terification 40,131,141,
162,194,198,203
lipoproteins
35,36,38,133,
membranes 91
perifibrous lipid 161
plasma 11,13,28,39,99,100,
128,141,144,195,197
- / PL ratio
13,15,17,127,128
cholesteryl esters 4,27,28,35,
41,100,107,127,133,135,144,
187,201,203
arterial synthesis
152,161,
162,201,204,206
arterial turnover 39,40,148152,156,160,161,179,183,
188,193,201,203,206,212
lipoproteins
35,38
synthesis
37,38,151
Tangier disease 39
transesterification 39,73,
136,208
cholesterylesterase, see ChEhydrolase
cholesterylesterhydrolase 28,
131,187,188,189,192,198,
202
cholesterylester synthetase 28,

131, 188,189,192, 198
ChE synthetase / hydrolase ratio
192,193
cholesteryllinoleate
128,131,
134,161,206,207
cholesteryloleate
160,188,201,
202,204,206,207
cholesteryl palmitate
131,201,
202,206,207
cholic acid
192,212
choline 37,55,66,67
chromatography 11,21,27,41,42,
49,58,59,69,81,98,100,133,
135,190,213
chylomicronemia 39
chylomicrons 6,35,38,42,56,62,
72, 189
coagulationtime 244
collagen 99,244,245
cornea 39,41
coronary vessels 212,213
cylic AMP,
see cAMP
o
desoxycholate 72
diet
17,19,28,100,112,113,
, see also atherogenic diet
dilinoleoyl PC 3,48,49,67
dimer forms of apoA-II 22,26,
27
dimyristoyl PC 25,26
dipalmitoyl PC 39,87,94,95,
distearyl lyso PI 75
distearyl PC 75
distearyl PI 75
dose effects 213,217
dyslipidemias 49,97-137
differential diagnosis
16
E
egg yolk lecithin 40,191,206,

212
elaidic acid 162
elastic fibres 99
electron microscopy 35,41
electron spin resonance 87
electrophoresis 5,11,27,35,69
electroretinography 211,213,
221-223
electrostatic interaction 37
endothelium, arterial
163,171,
175,180,214,217,247
251
endocytos is
171
enthalpy 24-27
EPL,
see polyenyl PC
erythrocytes 228-234
essential fatty acids
3,4,
141,246,247
essential phospholipids,
see
polyenyl PC
excretion, biliary 77
fat embolism 30
fatty acids
35,62,87,90,1151 17, 1 82
see individual compounds
arterial wall
141,171,
173,175,179,182,203
, chain elongation 141,171
fatty acid pattern, arterial
lipids
147,148,171
PC 4,29,68,70,195
plasma lipids 98, 107112,127-131,144-146,
195,213,221
, platelet aggregation 247
fatty streak,
see atherosclerotic lesions
160-162,182
fibrous plaques, see atherosclerotic lesions
fluidity, membrane 39,87,9395,234
fluorescence spectroscopy 37
heart 54,58,89
hematocrit 228-231
hemodynamics 30
hemolysis 70
heparin 123,190
hepatocytes 35,87,88
hydrophilic interaction 37
hydrophobic interaction 37,38
hyperbetalipoproteinemia 127
hypercholesterinemia 99,134
136,162,188,194
hyperemia, reactive 237-239,
241
hyperlipoproteinemia 11,12,20,
99,127,189,194
experimental
17,63,98-114,
188,192
type IIa 13-18,28,128,
130,244,245
type IIb
13-18,28,128,
130,244-246
type IV 13-15,17,18,28,
127-130
hypertension 162
hypertriglyceridemia, experime n tal 98- 1 14
infiltrations
214,216
inositol 66,67
intestinal PC-pool 52,53,63
intestinal tract 53,78,89
gas-chromotography 69,98,100
gas-liquid chromatography 81,
133,135,213
germination 67
glycerol 66,67,75,115,117,173
glycerol phosphate 75
glycerophosphorylcholine 55,
62
Golgi apparatu 35
Kennedy pathway
kidney 58,89
HDL
20-25,28,35-38,40-42,
81,82,98,103-107,109,
110,112,187,189
HDL deficiency, hereditary 39
HDL -subfractions 35,37,38
head groups
36,37,87,94
61
LCAT-enzyme 29,34-42,85,131,
141,144,162,188,189,202,
203,207
activity 28,36,38,131,141,
144
substrate specifity 35,38
Tangier disease 40
LCAT deficiency, hereditary
34,38,41,133
LDL 23,29,35,38-40,81,82,98100,103-107,131,187,189,
193
lecithin, see PC
252
lecithin, see egg yolk lecithin

lipases 27,28,35,38,115,131,
187,189,190,195
linoleic acid, linoleate 3,4,
38,68,81,82,85,88,107,113,
130,143,155-158,244,245
linolenic acid 4,244
lipids, arterial metabolism
141,150, 158,160,163,173,
182,187,201
lipids, arterial synthesis
148,149,150,152,168,169
lipids, organs 28,58,63,89
lipids, plasma 11,18,28,107,
135
lipids, transport disorders 39
lipid-protein interaction
36,37,95
lipoproteins 5,6,11,16,20,27,
29,34-38,72,85,98,99,107,
1 13, 131 ,187, 1 89
see also apolipoproteins
see also VLDL, LDL, HDL
molecular structure 20,2325,37,38
lipoprotein lipase 27,38,42,
113,122,190
lipoprotein-X 41
liposomes
38,39,87,94,95
LIPOSTABIL
11,28,80,82,123,
126,134,190,194,212,228,
229,234
liver 35,39,41,53,54,58,59,
73,76-78,87-89,113,158,
189, 190, 191 , 193,213,219
LLAT 202
lungs 53,58,73,89
lymph 50,56,62
lymph cannulation 48,50,54,61
lymph nodes
39
lysolecithin, see lyso-PC
lyso-PI 66,69,73-76,78
lyso-PI, toxicity 70
lyso-PC 13-16,36,38,41,66,69,
73-77 , 1 7 1 , 173, 176 , I 80,
207,229
arterial wall
160,168,169,
183
platelet, aggregation 247
, toxicity 70
lysophosphorylcholine 90
M
membranes
234
membranes fluidity 93,234

methyluracil 212
mice 66,70
micelles
36,37
microsomes 88,89,180
microcirculation 228,229,
234
mitochondria 88,89,171
mobility, molecular 94
monkeys, see species
monomer form of apoA-II
22,26,27
muscle 89
mucopolysaccharides 99
myocardial infarction 99
myocardium 158
myristic acid
175
N
13
NMR spectroscopy ( C)
37,38
norepinephrine
115,116
o
oedema, sub-endothelial
161
oleic acid, oleate 4,38,107
113,130,143,158,162,173
-, arterial lipids
155-157
p
palmitic acid, palmitate 68,

107,113,128,173,175,193
perfusion technique, arterial
160,163,164,182
perifibrous lipid 161
pH-optima 204,205
phagocytosis 244
phosphate 66,67,75,154,173
phosphatidylcholine
13,14,16,
21,38,40,56,66,67,71,73
81,94,141,152-154,173,
201,202
see egg yolk lecithin
see also polyenyl-PC (EPL)
see diacyl-PCs
lipoproteins 36-38
molecular structure 3,4,11,
20,68,73
effect on lipases 27,28,
1 15, 162
34,87-96,133,179,
, effect on TG metabolism 28
phosphatidylethanolamine 13,
14,16,81,85
253
phosphatidylethanolymine,
arterial wall
160,168,
169
phosphatidylinositol
13,14,16
66,67,152,154,173,179
-, pharmakokinetics 66,67,7176,78
phosphatidylserine
13,14,16
-, arterial wall
160,168,169
phosphodiesterase
126
phospholipases 4,49,56,59
67,69 ,85, I 73, 1 80 , I 87 , 190,
191,194,202,206,208
phosholipids
20,24-27-35,37,
81,91,98,100,107,141,187,
20 I
lipoproteins 4,11,19,35
plasma 11-13,15,17,81,128,
2 I7
arterial wall
147,149-151,
161,162,168,169
phospholipid pattern, lipoproteins
11,16,18-21
phosphoryl choline 55,90
pig 196,211,212
pigeons
162
platelets
244-247
-, ADP release 244
platelet aggregation test (PAT)
245
platelet factor III 244,247
polyenyl phosphatidylcholine
(EPL)
3,4
absorption intestinal 4956,61,63,77,247
arterial wall 29,141,
146-149,151-158,160,163,
169,170,171,178,183,187,
195,197,202
brain incorporation 74-76
fatty acid composition 4,
29,68,70
intraduodenal injection
63,77,78
intraportal injection 58,
63,71,80
intravenous injection 63,
77,78,80,88,127,157,158,
202,212,229,234
lipoproteins 36-38,72
liver incorporation 54,
58,89,157,158
lymph 55,57,62
membrane incorporation
88-96,234
oral application 48,66,77,
87,98,127,211
pharmakokinetics 48-63,
66-79,80-86,89,158
plasma level 71,75,80,89
re-esterification (intestinal) 49,56,61,62,247
side effects 237
solution/suspension 71,
72,190
synthesis 49,67,75
therapy, long-time
127
effects on .
-ACAT-enzyme
187,197
-atherosclerosis 28,30,49,
147,157,194-198,223,224
-blood flow 30,230-233,237,
241,243
-cerebral blood flow
237,242,243
-cholesterol, plasma
195,197
-cholesterylester hydrolase
175,187,194,195
-cholesterylester metabolism 29,141,148,158,
173,179,183,194,195,207
-erythrocytes 228-234
-FA metabolism, arterial
wall
155,156,160,171,
173,178,183,202
-FA pattern, arterial
lipids
147-148
-FA pattern, plasma lipids
98,100,107-112,130,134,
144-146
fat embolism 30
hematocrit 229,230
-LCAT
144,148
-lipases 28,113,115,162,
187,189,193-195
-lipids, arterial wall
140,146-154,158,163,169171,183,202
-lipids, plasma 28-30,
52,63,76,100,113,127,128,
134,140,144,217-220,224
-lipoproteinlipase 113,162
-lipoprotein pattern 98,105,
106,127,131,217
-lyso-PC-turnover
179
-phospholipases
187,190,
191,194
-phospholipids, arterial
wall
152-154,158
-platelet aggregability
144-247
-proteins, arterial wall
147
254
-sterolester hydrolase
175
-transesterification of
arterial lipids
141,
148,149,158,207
polyunsaturated, see polyenyl
prebetalipoproteins 6
primates
20
prostaglandin E 245
protein-lipid interaction 87
protein-protein interaction
36
proteinuria 41
Q
quails
196,212
rabbits
123,140,141,160,162,
163,187,189,191,193,194,
196,211,212
radioimmunoassay 40
radiolabel exchange, body
water 54,56,62
radiolabeling 49,67
rats 48,50,66,70,87,88,115,
116,192,196,212
rheologic parameters, see
blood flow
rhesus monkey 21-23,62
reticuloendothelial system
39,41
retinopathy, atherosclerotic
221
s
saturation, acyls
36,87,107,
113,114,131,201,205,206,
219,221
scintillation counting 239,240
seasonal effects
13
sex differences 58,59,213,217,
218,221
side effects 237
soybeans
3,67,68
sphingomyelin 14,16,21,36-39,
152-154,177
-, arterial wall
161
sphingomyelin hydrolase
173
sphingomyelin / PC ratio
13,
15-18,23
spin label 87,93,94
stearic acid, stearate 88,107,
113,244
sterol ester hydrolase

161,
162,175
substrate specifity 35,40,204
succinic acid 37,88
sucrose
17,18,112
sunflower 50
surface-activity 228
T
Tangier disease 39
taurocholate
198
theophylline
115,116
therapy, long-time
12-15,125131,244
thermostability, ATPases 91
thin-layer chromatography
69,76,81,98
thin-line protein 38
thiouracil
192
thrombocytes,
see platelets
thrombosis 247
tissue culture 41,141-143,148
tonsills 39
transesterification, Ch E
38,39,73,133,136
transfer-reaction, lipoproteins
36,38,133
triglycerides, plama 11,13,27,
77,98-102,112,113,128,135,
136,144,189,217
arterial wall
148-152,177,
187
lipoproteins 38
lymph 56
metabolism 40,58,152
platelets 247
, Tangier disease 39
triglyceride hydrolase
162,
177
u
ultracentrifugation 5,11,23,
41,98,99
unsaturation,
see saturation
V
vascular diseases 30,228,230,

233,234,237-243
viscosity, blood 228-230-233
-, plasma 228,229,234
VLDL 35,38,40,42,81,82,98,99,
100,107,113,133,189,193
X
Xenon-clearance 237-243
X-ray scattering 37

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Phosphatidylcholine

Biochemical and Clinical Aspects

Dr. Huben Peelers

Proceeding s o f aSymposium held at the Simon Stevin Institute

e -IS BN-13: 978-3-642-66424-3

by Spr inger Verlag Berlin Heidelberg 1976

A Glossary of Essential Phospholipids, Lipids, and

The Biological Significance of the Plasma Phospholipids

PHARMACOKINETICS OF ESSENTIAL PHOSPHOLIPIDS

On the Pharmacokinetics of Orally Applied Essential

Pharmacokinetic Studies on Phosphatidylcholine and

Effect of Essential Phospholipids on the ATPases and

Effects of Essential Phospholipids on the CarbohydrateInduced Hypertriglyceridemia

The Human Plasma Lipids and Lipoproteins Under Influence

PHOSPHOLIPID METABOLISM IN THE ARTERIAL WALL

Influence of Essential Phospholipids on Serum Lipids

Effect of EPL on the Lipid Metabolism of the Arterial

ESSENTIAL PHOSPHOLIPIDS IN RED CELLS AND THROMBOCYTES

Influence of Essential Phospholipids on the Flow

subject Index . 249

MARES, P., 4th Chair of Internal Medicine, Faculty of Medicine,

A Glossary of Essential Phospholipids,

1,2-dilinoleoyl phosphatidyl choline (Fig.1) is the prototype

Since in these phospholipid molecules the acyl moieties consist

The fatty acid composition as determined by gas chromatography

Fatty acid composition (molar %) of soybean polyenyl phosphatidylcholine (EPL) as

determined after phospholipase A2 hydrolysis using gaschromatography on a 200 cm column of EGS

Animal fat contains three families of polyunsaturated fatty

All these fatty acids are incorporated into animal phospholipids,

3: Characteristic properties of serum lipoproteins in man

The Biological Significance of

I: The Phospholipids in Dyslipoproteinemia

Total plasma phospholipids

Experimental dietary-induced hyperlipoproteinemia in

Relationship between primary apoprotein structure and

Reassembly of apoprotein with phospholipid

PART III: Phospholipids and the Lipases

IV: The Therapeutic Potential of Phospholipids

This paper interrelates the work performed at this laboratory

PART I: THE PHOSPHOLIPIDS IN DYSLIPOPROTEINEMIA

1. Total plasma phospholipids

The plasma phospholipid concentration of 10 patients followed during 2 years

Plasma lipids and lipid ratios in normal and hyperlipoproteinemic patients

0.35 + 0.02 0.321 +

0.61 + 0.03 0.304 + 0.012

1.20 + 0.10 0.206 + 0.008

T. Chol . Total Cholesterol; TG Triglycerides ; PL .' Phospholipids

Cholesterol/PhoSpholipid ratio; TG/C Triglycerid/Cholesterol ratio

S/Pc Sphlngomyelin/Phosphatldylchollne ratio.

therefore it is mostly neglected. However if the cholesterol to

2 : The plasma phospholipid subclasses of 10 patients fOllowed during 1 year

The plasma phospholipid profiles in normal and hyperlipoproteinemic patients

ful parameter in the differential diagnosis between type lIb

The relation between S / PC in human plasma

3. The phospholipid pattern of individual lipoproteins

Percent phospholipids in human plasma lipoproteins obtained by electrochromatography

Phosphat idyl serine

In type II patients the sphingomyelin concentration is increased

Experimental Dietary Induced Hyperlipoproteinemia in the Chimpanzee

During the induction of a hyperlipoproteinemic state in the

and Fig. 6). This observation is in favour of the introduction

dietary induced atherosclerosis was

also positive. In type II