Protides of the Biological Fluids. Proceedings of the Nineteenth Colloquium, Bruges, 1971

532 Pages·1972·38.034 MB·English

by  H. Peeters (Eds.)

List of Committee Members
Monsieur le Docteur P. BURTIN Professor V. N. OREKHOVICH
Institut de Recherches Scientifiques Institute of Biological and Medical
sur le Cancer Chemistry USSR
B.P. No 8 Academy of Medical Sciences
F-94 Villejuif, France Pogodinka 10
Moscow, USSR
Professor Z. DISCHE
College of Physicians and Surgeons
Dr. H. PEETERS
Department of Biochemistry Simon Stevin Instituut voor
Corneal Center Wetenschappelijk Onderzoek
630 West 168th Street Jerusalemstraat 34
New York, N.Y., USA B-8000 Brugge, Belgium
Dr. T. FREEMAN
Dr. F. W. PUTNAM
National Institute for Medical Research
Division of Biological Sciences
The Ridgeway
Indiana University
Mill Hill
Bloomington, Indiana 47401, USA
London, England
Dr. A. L. SCHADE
Monsieur le Professeur P. GRABAR
N.I.H.
6 avenue Marcel Doret
Bldg5,Room215
F-75 Paris XVI, France
Bethesda, Md. 20014, USA
Monsieur le Professeur J. HEREMANS
Universite de Louvain Professor H. E. SCHULTZE
Departement de Medicine Experimentale La Laguna
Avenue Chapelle-aux-Champs 4 Apartado 32
B-1200 Bruxelles, Belgium Teneriffe, Islas Canarias
Spain
Dr. H. HIRAI
Hokkaido University Dr. C. P. STEWART
School of Medicine 17 Orchard Road South
Sapporo, Japan Edinburgh, Scotland
Professor T. H. J. HUISMAN
Professor A. TISELIUS
Medical College of Georgia
Institute of Biochemistry
Department of Biochemistry
Uppsala, Sweden
Augusta, Georgia 30902, USA
Professor E. E. LUSCHER
Theodor Kocher Institut
Bern, Switzerland
PROTIDES OF THE
BIOLOGICAL FLUIDS
PROCEEDINGS OF THE NINETEENTH COLLOQUIUM
BRUGES, 1971
Edited by
H. PEETERS
Director of the Simon Stevin Institute and of the
Laboratory of St. Jan's Hospital, Brugge {Belgium)
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Preface
THE Proceedings of the XlXth Colloquium are devoted to three main topics selected from
the general field of protein chemistry. The main topic is devoted to lipoproteins, the second
is concerned with hydrodynamic properties of proteins and the third topic is related to
protein catabolism, and to new techniques.
The main section of the Colloquium on lipoproteins groups more than 60 papers,
extensively describing the composition and structure of lipoproteins, their synthesis and
genetics and their function. The pathology of these proteins in atherosclerosis together with
dietary measures and drug therapy is also discussed.
Consideration of the hydrodynamic properties constitutes an apart section where the
recent developments and applications of dielectric relaxation and fluorescence depolariza­
tion to the determination of protein structure are described.
The third topic is concerned with the catabolism of proteins, specially of albumin and
gammaglobulins, both in health and disease.
The newest techniques and applications of immunoelectrophoresis to protein quantita-
tion are extensively described in the technical section. Microcalorimetry and isotachophore-
sis are also introduced.
The Academic Lecture presented by P. Burtin—one of the leading scientists in this
fascinating field—is a critical survey about the significance and application of Immuno-
chemical studies on human cancers.
Again, as in the years past, a numerous attendance followed the meeting, exchanging
information and enquiring about techniques, thus setting the pace and drawing the shape of
new work. To the best of its ability this Colloquium intends to remain an open window on
the moving landscape of Protein Chemistry.
xiii
Acknowledgements
WE, the Members of the Scientific Committee of the XlXth Colloquium, are glad to have
the opportunity of expressing our sincere appreciation for the support given by the Provin­
cial Government of West-Flanders.
The personal interest shown by the Governor, P. van Outryve d'Ydewalle and by Mr.
L. Gillon and Mr. J. Storme, members of the Council, was of great help in our work.
It is a pleasure to thank the authorities of the city of Bruges, especially Mr. P. Vandamme,
Burgomaster, as well as the President, Mr. R. Waes, and the members of the Public Assis­
tance Committee, for their collaboration in the general organization of the congress.
The staff of the Simon Stevin Institute for Scientific Research have been the backbone
of all the practical work throughout the preparation of the meeting and the editing of
this book.
We are also indebted to many others too numerous to mention who gave unstintingly of
their time and effort to make this Colloquium a success.
The publication of these proceedings is supported by a grant from the Ministry of
National Education.
xv
Academic Lecture
Immunochemical Studies on Human Gastro-intestinal Tumors
P. BURTIN
Laboratoire (Tlmmunochimie,
Institut de Recherches Scientifiques sur le Cancer, B.P. 8, 94 Villejuif, France
THERE is now an important problem for those immunologists who are interested in cancer.
That is the possible existence of cancer-specific antigens. When dealing with cancer of the
gastro-intestinal tract, the problem can be formulated as follows: Do the malignant tumors
of the GI tract contain specific antigens ? Have these antigens an individual specificity or
are they common either to all the GI tumors, or at least to the tumors of one organ ?
In this sense, there could be an analogy between the antigens of human spontaneous
tumors and those of the experimental tumors induced in laboratory animals.
The tumors of viral origin share common neoantigens whose specificity is strictly
related to the causative virus.
The chemically induced tumors carry at least theoretically antigens having an individual
specificity.
All these antigens, either of common or individual specificity, are located on the
membranes of cancerous cells. Do such antigens exist in GI tract human cancers? It is
likely so, as there seems to be a certain level of immunity, or at least of immunological
defence, against these tumors. This is proved by the Hellstroms, with their colony
inhibition test.(1) These authors put in culture cells dissociated from surgical tumors,
namely colonic tumors, and count the number of colonies growing from those cells. This
number is reduced by 50 % in the presence of lymphocytes obtained from patients having a
cancer of the colon. This reduction is specific in the sense that the lymphocytes of colonic
cancer patients do not inhibit the growth of fibroblasts, or of cells of tumors of other
organs. But these lymphocytes react as well with the tumor of the patient, or with other
tumors of the same organ. The antigen, or antigen(s), to which the lymphocytes react is
thus a group antigen, probably common to all tumors of the same organ. Let me remind
you that similar findings were obtained for many other human tumors, neuroblastomas,
nephroblastomas, cancers of the breast, the bladder, etc.
Moreover, the Hellstroms demonstrated that the lymphocytes of the cancer patients
inhibited also the growth of cells of the fetal intestine. It is thus likely that the tumor
antigen(s) is also present in this fetal organ.
Did the immunochemical studies on GI tumors obtain similar results ? Did they show
the existence of such an antigen(s) postulated by the results of the Hellstroms, that is cancer-
specific antigens found also in fetal intestine. The present answer is: the immunochemists
for a time thought they had found such an antigen, the carcinoembryonic antigen (CEA).
In gastric cancer, the sulphoglycoprotein described by Hakkinen is also of fetal type,
but is not strictly cancer-specific.(2)
It is time now to synthesize the information obtained on the CEA, and to determine
whether it could be the antigen or one of the antigens involved in the defence mechanisms
against cancer.
3
4 ACADEMIC LECTURE
The CEA was described first by Gold and Freedman,(3) as an antigen present in cancers
of the colon, absent from the normal digestive mucosa, and present also in fetal intestine.
Its reappearance in cancerous glands was thought to be due to a derepression. It was found
independently by Mrs von Kleist and myself,(4) and Mrs. von Kleist presented here last
year our results on this antigen. The CEA is a perchlorosoluble glycoprotein of /?
mobility. It is revealed by specific antisera. It is present in all the glandular tumors of the
colon and the rectum, of the stomach and the pancreas. It is found also in traces in some
hepatomas. Recently we detected it in a liberkuhnian carcinoma of the small intestine. The
CEA is also present in the metastasis of these tumors, especially in the hepatic metastasis
which due to their size provide a very good material for the purification of the antigen. As
shown by immunofluorescence, the CEA has a typical localization at the apical pole of the
glandular cells.(5) We thus observe a fluorescent line bordering the lumen of the gland.
This localization is identical in cancerous tissues and in the fetal intestine, but the intensity
of the fluorescence is weaker in the latter organ, due to a low amount of CEA in fetal tissues.
When the tumor loses its glandular organization, CEA is found in deposits, and surrounding
at least a part of the invasive cells: it behaves sometimes as a membrane associated antigen.
From the viewpoint of molecular biology, we recently noticed that the derepression of
the synthesis of the CEA is associated with the disappearance of a normal membrane
antigen of the colonic mucosa.(6) This antigen, called CMA, is revealed by some antisera
against normal colonic mucosa (NCM). It is pronase sensitive and thus of protein nature.
There seems to be a correlation between the synthesis of CMA and that of CEA. In cancerous
glands and in pericancerous areas the CMA decreases or is apparently lacking. Other
membrane antigens are now studied which are present in both gastric and intestinal mucosa.
They seem to be also lacking in cancerous glands.
An important finding obtained by Gold was the autoantigenicity of the CEA.(7) He
found antibodies against CEA, by passive hemagglutination, in about 50% of the patients
having a cancer of the colon. Studies were made also on a possible cellular immunologic
reaction against CEA, by the group of Herberman:(8) results were not conclusive.
Thus the CEA was thought as very likely involved in the immunological defences against
colonic tumors.
Recently, however, this conclusion suffered some criticism. First of all the CEA is by
no means a cancer-specific antigen. We found it by immunofluorescence in NCM of infants,
and also in all the polyps we studied.(9) Polyps of the colon are benign tumors, often
associated with a malignancy or able to transform themselves in cancers. But sometimes
they remain benign. In twenty-five polyps CEA was always found, even in those which were
considered as certainly benign. A perchloric extract of these benign polyps precipitated with
a monospecific antiserum CEA. We detected it also in some samples of mucosa taken for
non-cancerous diseases, as sigmoiditis. It could even be present in traces in almost every
normal colon, according to Martin, who used a radioimmunological method/10)
At the present time CEA must be considered as an antigen increased in GI cancers, but
not as cancer-specific. Actually its derepression seems to be easy. It could be linked to a
special localization of cell proliferation, observed by Deschner(11) in polyps and some
samples of normal colonic mucosa. We plan to study now this possible correlation.
Second, the CEA is not autoantigenic. At least, it does not give a production of auto-
antibodies. In our laboratory, antibodies present in the sera of colonic cancer patients, and
able to agglutinate red cells sensitized by extracts of colonic tumors, were carefully studied.
Such antibodies were found in many cancer sera, but also in non-cancer sera. Their titer
IMMUNOCHEMICAL STUDIES ON HUMAN GASTROINTESTINAL TUMORS 5
was not higher when using for sensitization of the red cells perchloric instead of saline
extracts of tumor. Moreover, these antibodies were absorbable by extracts of NCM. They
were not cancer-specific. In order to control this result, E. Collatz and Mrs von Kleist(I2)
tried to isolate the antigen(s) present in colonic tumor extracts and able to react with the
autoantibodies. They used the immunoadsorption method. They prepared an insoluble
immunoadsorbant by polymerizing antibody-rich sera. They made their extracts to react
with this immunoadsorbant. After careful washings, they recovered by acid elution several
antigens, which they studied by immunochemical methods. Three precipitin lines were
obtained, one of which joined that of purified or perchlorosoluble a-globulin. No CEA line
was found. This is strong evidence against the autoantigenicity of the CEA.
The present conclusions of the immunochemists are thus.
1. There is in the tumors a lack of normal antigens, specially of membrane antigens.
2. We have no evidence for a true cancer-specific antigen, but we can demonstrate the
considerable increase in tumors of some antigens, which are also present in fetal intestine.
It is conceivable that an immunological reaction may be induced by some of these antigens,
perhaps as they are more easily accessible. It could be imagined that antigens normally
confirmed to the cytoplasm may reach the plasmic membranes in pathological conditions
and thus be able to induce immunological reactions. This is yet to be proven.
There is still a chapter to be shortly developed. This is the temptative dosage of the CEA
by radioimmunoassay in the serum of the patients, as described by Thompson, in Gold's
group.(13) This group published in 1969 very promising results: detectable levels of CEA
were found only in cases of gastro-intestinal cancer. All the non-cancer sera and the sera
of patients with cancer of another organ were negative. These findings are now controlled
by several laboratories, especially in the USA. Very recently one group published less
decisive results, as some sera of patients having a non-digestive cancer were also positive/14)
In France, Martin(15) obtained similar results, that is, positive levels in some controls. I
must say that this dosage is made difficult by technical problems, such as non-specific
inhibition of the precipitation of the radioactive antigen by some normal sera. We have
thus to wait a little before being able to establish the value of the dosage of the CEA in
patients' sera. If it is valid, it could be of great help in the diagnosis of intestinal cancers.
REFERENCES
1. HELLSTROM, I., HELLSTROM, K. and SHEPARD, T., Int. J. Cancer 66, 346-351 (1970).
2. HAKKINEN, I. and YIIKART, S., Ann. Surgery 169, 277-281 (1969).
3. GOLD, P. and FREEDMAN, S., /. Exp. Med. 122, 467-481 (1965).
4. VON KLEIST, S. and BURTIN, P., Cancer Res. 29, 1961-1964 (1969).
5. VON KLEIST, S. and BURTIN, P., int. J. Cancer 4, 874-879 (1969).
6. BURTIN, P., VON KLEIST, S. and SABINE, M. C, Cancer Res. (1971) in press.
7. GOLD, P., Cancer 20, 1663 (1967).
8. HOLLINSHEAD, A., GLEW, D., BUNNAG, B., GOLD, P. and HERBERMAN, R., Lancet 1 1191-1195 (1970).
9. BURTIN, P., /. Nat. Cancer Inst., in preparation.
10. MARTIN, F. and MARTIN, M. S., Int. J. Cancer 6, 352-360 (1970).
11. DESCHNER, E., Autoradiographic studies of synthesis DNA, RNA and protein in normal and diseased
colonic mucosa in carcinoma of the colon and antecedent epithelium. In W. Burdette (Ed.), Ch.C.
Thomas Pub., Springfield, 1970, pp. 222-229.
12. COLLATZ, E., VON KLEIST, S. and BURTIN, P., Int. J. Cancer, in preparation.
13. THOMSON, D., KRUPEY, J., FREEDMAN, S. and GOLD, P., Proc. Nat. Acad. Sci. 64, 161-167 (1969).
14. MOORE, T., ZUPCHIK, H., MARCON, N. and ZAMCHEK, N., Am. J. Dig. Dis. 16, 1-7, 1971.
15. MARTIN, F., Unpublished results.
A. Introduction
Conceptual Development of the Classification Systems of Plasma
Lipoproteins
P. ALAUPOVIC
Cardiovascular Section, Oklahoma Medical Research Foundation and Department of Biochemistry,
University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73104, U.S.A.
OPERATIONAL CLASSIFICATION SYSTEMS OF PLASMA LIPOPROTEINS
Nerking's postulate, at the beginning of this century, that plasma lipids are chemically
bound to proteins(1) was confirmed almost thirty years later by MacheboeuPs successful
isolation from horse plasma of a lipoprotein of constant composition.(2) After precipitating
globulins by half saturation of plasma with ammonium sulfate, acidification of the supernate
to pH 3.9 resulted in precipitation of a fraction containing 59.1 % protein, 22.7% phospho-
lipid and 17.9% cholesterol esters. The lipid and protein composition of this lipid-protein
complex soluble at neutral or alkaline pH remained constant after nine consecutive
precipitations. Subsequently, Macheboeuf and his coworkers showed(3) that the lipoprotein
exhibited a single sedimenting boundary in the ultracentrifuge and migrated as a single
band with a-globulin mobility in free electrophoresis. Another important result of these
studies was the finding that the protein moiety obtained by ethanol-diethyl ether delipidiza-
tion of lipoprotein differed from albumin and any other known globulin. These historic
accomplishments provided the necessary experimental background for the first formulation
of a lipoprotein concept based on the existence of a specific protein capable of binding
various lipid classes in a constant ratio. Systematic studies on the extraction of serum lipids
by diethyl ether and ethanol showed that lipids in the "albumin" fraction were more resistant
to extraction than those in the "globulin" fraction.(4) The possibility that this phenomenon
was due to the presence of several specific proteins differing in lipid binding properties was
fully substantiated during subsequent years when the accumulated evidence indicated that
all plasma lipids were combined with specific proteins into two major groups of lipoproteins
migrating in the electrical field either with a - or /^-globulin fractions.(5) The concept that
x
plasma lipoproteins exist as two major distinct groups culminated with the studies by Cohn
and his associates(6) who were the first to develop a practical preparative procedure for
isolation of lipoproteins. Fractionation of human plasma in ethanol-water mixtures at
low ionic strength and low temperatures resulted in precipitation of two types of lipid-
protein complexes which differed in their physical and chemical properties. Due to their
characteristic mobilities in the electrical field, these two lipoproteins were designated as
a (c^-LP) and ^-lipoproteins (/?-LP).* Although acknowledging the heterogeneity of plasma
r
lipoproteins, this classification system conveyed the impression that all plasma lipids were
bound only to two specific types of proteins. With some refinements, this simplistic view of
plasma lipoproteins has survived until the present time.
♦Abbreviations: VLDL, very low density lipoproteins, lipoproteins of d< 1.006 g/ml (S > 20);
f
LDL, low density lipoproteins, lipoproteins of d 1.006-1.063 g/ml (5/0-20); HDL, high density lipoproteins,
lipoproteins of d 1.063-1.21 g/ml; a -LP, a-lipoproteins, lipoproteins with an electrophoretic mobility of
x
«!-globulins; j3-LP, j8-lipoproteins, lipoproteins with an electrophoretic mobility of j3-globulins.
9
10 INTRODUCTION
Whereas solubility properties and electrophoretic behavior of lipoproteins are those of
typical proteins, their relatively low hydrated densities reflect the presence of various lipid
classes. It is, therefore, not surprising that, in addition to the electrophoretic behavior, this
very unique physical property of lipoproteins has been utilized very successfully not only
for the development of new isolation procedures but also for characterization of plasma
lipoproteins. Pederson's isolation, in 1945, of a lipid-protein complex ("X protein") with
^-globulin mobility by ultracentrifugal flotation(7) marked the beginning of a new develop­
mental phase in the analysis and preparative isolation of lipoproteins. Gofman and his
coworkers(8,9) established by fractional ultracentrifugal flotation of lipoproteins in salt
solutions of successively increased densities that plasma lipoproteins represented a wide
spectrum of particle sizes and hydrated densities. They observed that the lipoproteins
displayed a pattern of minimal and maximal concentrations along a density gradient from
0.92 g/ml to 1.20 g/ml and classified lipoproteins according to their density ranges into four
major classes designated as chylomicrons, very-low density (VLDL), low-density (LDL)
and high-density (HDL) lipoproteins/10) Macromolecular distributions showed that each
of these major density classes represented a polydisperse system, heterogeneous with respect
to both particle size and hydrated density/11,12) The relatively high degree of this hetero­
geneity, reflected in the spreading of otherwise symmetrical boundaries, prompted a
subdivision of VLDL,(1314) LDL,(1516) and HDL(1718) into density subclasses. Utilization
of hydrated densities as a major criterion for classification and characterization of lipo­
proteins has represented the single most important factor in shaping the current views on
chemistry and metabolism of plasma lipoproteins. In contrast to the simple electrophoretic
classification, it has revealed an unexpected heterogeneity of lipoproteins and underlined
the role and significance of lipid rather than protein components for this compositional and,
possibly, structural heterogeneity. The application of zonal electrophoresis to plasma lipo­
proteins disclosed lipid-stained bands not only in the a - and /8-globulin regions, but also
x
at the origin and in the a - or pre-j8-position/19'20) Although distribution studies indicated
2
clearly that each major density class contained a great variety of lipoproteins differing in
particle size and hydrated density, it was generally assumed that chylomicrons, VLDL,
LDL and HDL could be correlated with four narrow electrophoretic bands forming at the
origin, pre-j3-, /?- and c^-positions, respectively. In the later part of the fifties, studies on the
N-terminal amino acids(21,22) and immunological properties*23-24* indicated the existence
of two distinct proteins, one of which was considered to be characteristic for the low-density
and the other for the high-density lipoproteins. At the same time the recognized significance
of plasma lipids in the genesis and development of atherosclerotic lesions(25) focused the
attention of most investigators on the lipid components and contributed significantly to our
knowledge of the quantitative lipid composition of various lipoprotein classes. Results of
these studies indicated clearly that the heterogeneity of lipoprotein density classes was due
mainly to a great variation in the absolute and relative amounts of individual constituents
of the lipid complement. To reconcile this heterogeneity in the lipid composition of lipo­
proteins with the existence of only two specific proteins, one had to assume that the formation
of lipoproteins depended not only upon the capacity of each protein to bind directly various
combinations of lipids, characterized by a certain equivalency between the individual
representatives of major lipid classes, but also upon the relatively non-specific interactions
between the lipid moiety and additional lipid components/26*2"0 By the mid-sixties, the
soluble plasma lipoproteins could be defined as macromolecular complexes of neutral lipids,
phospholipids and at least two specific proteins bound through non-covalent interactions to