Protides of the biological fluids : proceedings of the thirty-second Colloquium, 1984

376 Pages·1985·36.849 MB·English

by  H Peeters

LIST OF COMMITTEE MEMBERS
R. BALLIEUX T. HUISMAN
Academisch Ziekenhuis Medical College of Georgia
Immunologisch Laboratorium Sickle Cell Center
UTRECHT AUGUSTA (GEORGIA)
THE NETHERLANDS U.S.A.
P. BURTIN E. LÜSCHER
Institut de Recherches Theodor Kocher Institute
Scientifiques sur le Cancer University of Bern
VILLEJUIF BERN
FRANCE SWITZERLAND
A. CARBONARA P. MASSON
Istituto di Genetica Medica Université de Louvain
deU'Universita di Torino Dept. de Méd. Expérimentale
TORINO, ITALY BRUSSELS
BELGIUM
H. CLEVE
Institut für Anthropologie V. OREKHOVICH
und Human Genetik Inst, of Medical Chemistry
MUNICH Academy of Medical Sciences
WEST GERMANY MOSCOW
U.S.S.R.
Z. DISCHE
College of Physicians and H. PEETERS
Surgeons of Columbia University Director
NEW YORK, U.S.A. Institute for Medical Biology
BRUSSELS
P. GRABAR
BELGIUM
Institut Pasteur
PARIS, FRANCE D. POULIK
William Beaumont Hospital
H. HARBOE
ROYAL OAK (MICHIGAN)
Harboes Laboratorium
U.S.A.
COPENHAGEN
DENMARK F. PUTNAM
Div. of Biological Sciences
H. HIRAI
Indiana University
Tumour Laboratory
BLOOMINGTON (INDIANA)
TOKYO
U.S.A.
JAPAN
A. SCHADE
S. HJERTEN 3901 Indian School Road
Institute of Biochemistry ALBUQUERQUE
University of Uppsala (NEW MEXICO)
UPPSALA U.S.A.
SWEDEN
H. SCHULTZE
J. HOBBS La Laguna
Westminster Medical School Apartado 32
University of London TENERIFE
LONDON, GREAT BRITAIN ISLAS CANARIAS
PROTIDES OF THE
BIOLOGICAL FLUIDS
PROCEEDINGS OF THE THIRTY-SECOND
COLLOQUIUM, 1984
Edited by
H. PEETERS
Director
Institute for Medical Biology
Brussels, Belgium
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PREFACE
1
This years Colloquium was introduced by a lecture on LOW MOLECULAR WEIGHT PROTEINS OF BIOLO-
GICAL FLUIDS held by D. Poulik. In this talk he summarized our knowledge in the field
and opened up vistas for the future.
The first section of the meeting was concerned with PROTEINS IN SECRETIONS. Immunoglobu-
lins were a great first in this part of the program and a description of the defense mechanism
was completed by several studies on IgA. Enzymes such as lactoperoxidase were considered
next. Lactoferrin received fair attention. After these individual proteins several secre-
tions were considered as such namely CSF, colostrum, milk, female and male genital secretions,
saliva, gastrointestinal juice, nasal and bronchoalveolar secretions, tears and synovial
fluid.
The second topic was concerned with TUMOR MARKERS AND TARGETING. Immunotoxins received
attention and tumor imaging rounded off this in vivo part of the program. Immunohistoche-
mistry was extensively reviewed in over 20 papers. Next came tumor markers : new markers,
ferritin, CEA, TPA and various other markers were seen in succession. A last set of papers
was grouped around organs such as the breast, lymphoid malignancies, and other various
tumors.
A third section of the meeting was devoted to ADVANCES IN THE SEPARATION OF PROTIDES. A
new staining procedure for protein detection received first attention. Two dimensional
electrophoresis and other electrophoretic techniques and methodologies were covered next.
The HPLC and affinity chromatography were duly considered and this closes the rich harvest
of papers for this Colloquium.
All in all these proceedings cover a series of advances in the protein field and contain a
mountain of information on recent problems. As showroom of international cooperation the
meetings have established their reputation as an open forum and a beacon for further research.
PBF-B
xxxiii
ACKNOWLEDGEMENTS
We, the members of the Scientific Committee of the XXXIInd Colloquium, wish to thank the
Burgomaster and the Aldermen of the city of Brussels for the reception at the Town Hall.
Prof. R. Ballieux, Prof. A. Carbonara, Prof. H. Hirai, Prof. J. Hobbs, Prof. D. Poulik,
Prof. F. Putnam and Prof. A. Schade have been responsable for the concept and establishment
of the different parts of the program.
We thank Mrs. A.M. Lerou, Miss G. Peeters and Miss M. Declerck for their help in the practical
organization of the meeting.
The proceedings are being published with a subsidy of the Belgian Ministry of Education.
LOW MOLECULAR WEIGHT (LMW)
URINARY PROTEINS
M. D. POULIK* and T. SERINE**
*Div. Immunopathology, Dept. Clin. Path., Wm. Beaumont Hosp., Royal Oak, Ml
48072 USA and Dept. of Immunol. & Microbiol., Wayne State Univ. School of
Medicine, Detroit, MI 4820J, USA
**High Risk Study Div., Natl. Cancer Center Res. Inst., Tsukiji 5-Chome, Chou-ku,
Tokyo, Japan
INTRODUCTION
The influence of Arne Tiselius' work can be felt in every branch of biological and medical
science. The introduction of an electrophoretic method of protein separation opened new
possibilities not only for basic research in protein chemistry but also for new approaches in
diagnosis of a variety of pathological conditions (Tiselius, 1937). This work brought Prof.
Dr. Arne Tiselius the ultimate reward, the Nobel Prize for Chemistry, which was awarded to him
in 1948.
One of the authors (M. Poulik) was profoundly influenced by his work and especially by a paper
published by him and the late Henry G. Kunkel. Kunkel and Tiselius (1951) described a new
method of electrophoresis in which the supporting medium for the electrophoresis separation
was a filter paper soaked in an appropriate buffer and sandwiched between two glass plates.
The electrophoretic separation of the proteins present in human serum was remarkable. An
earlier paper by Tiselius and Kabat (1939) on separation of immune sera gave one of the authors
the idea to combine two methods (electrophoresis and immunodiffusion) and develop a new type
electrophoresis: a prototype of Immunoelectrophoresis (Poulik, 1952).
The authors are honored to contribute this paper to the memory of Prof. Dr. Arne Tiselius. We
have chosen to discuss a group of proteins, the detection of which were achieved by electro-
phoretic methods. Richard Bright (1836) showed that presence of albuminous substances in
urine herald serious renal disease. Bence Jones detected (Bence Jones, 1847) "deutoxide of
albumen" which betrayed another serious illness known at that time as mollities ossium (myelo-
matosis or myeloma). Edelman and Poulik (1961) and Poulik and Edelman (1961) have proved that
this rather mysterious substance (which became known as Bence-Jones protein) is the light chain
of the "myeloma protein" usually present in high concentration in such patients. A marked pro-
teinuria due to severe exercise was observed by Leube (1878) who named this type of proteinuria
"march proteinuria" since it was frequently observed in troops who had to cover on foot long
distances in a short time. Other types of proteinuria were also described, e.g., proteinuria
of pregnancy by Lever (1843), postural proteinuria by Moxon (1878). Other types of protein-
uria were also subsequently described. In 1966 a Swedish group of scientists, led by the late
Ingemar Berggard, began to investigate systematically the role of low molecular weight proteins
(LMW) in clinical medicine. They were stimulated by the work of Piscator (1962) who studied
extensively the effects of cadmium dust on workers employed in cadmium mines in Sweden.
Demonstration of $m in urine of workers exposed to cadmium by Piscator confirmed the observa-
2
tion of Fridberg (1950) that the proteinuria observed in these patients was different from
that due to increased glomerular permeability. The new type of proteinuria was subsequently
observed also in patients with a variety of pathological conditions, e.g., Fanconi syndrome,
galactosemia, Wilson's disease, acute tubular necrosis and in kidney transplant patients, to
name a few. Since the underlying pathological condition pointed to tubular dysfunction this
proteinuria became known as tubular proteinuria. Low molecular weight proteins are normally
reabsorbed in proximal tubules after passing through the glomerular membrane. Damage to these
cells due to whatever cause (drugs, heavy metals, anoxia, antibiotics, etc.) leads to poor
3
4 THIRTEENTH ARNE TISELIUS MEMORIAL LECTURE
reabsorption of the LMW proteins and, consequently, their excretion and thus increased concen-
tration in the urine. A concomitant rise of their concentration in serum is also observed.
This contrasts with a glomerular type of proteinuria where the larger molecular weight proteins,
usually present in greater abundance in serum, are excreted. Unfortunately, both types of
proteinuria may be sometimes simultaneously present in lesser or greater degree and thus
obscure a clear-cut identification of the patho-physiologic condition (Peterson, et al, 1969).
Thus analysis of proteins excreted in various forms of renal diseases led to the notion that
the type(s) of protein excreted may depend on the specific nature of the disease. Electrophor-
esis and immunological methods were instrumental in the formulation of concepts of looking at
the glomerular membrane as a molecular sieve which does not exactly obey all the rules of simple
filtration. Other processes are also involved and thus the kidney emerged as an organ of the
site of catabolism. The kidney is involved in many functions: it maintains protein homeosta-
sis with a molecular barrier which due to the size of its pores prevents the loss of large
molecular weight proteins. The LMW proteins are processed with great care being allowed to be
filtered, reabsorbed and those of special "interest" catabolized. The final product found in
the urine is the toil of all these functions. The work presented here will discuss a selected
group of proteins which are of such a special "interest" to the kidney, clinicians and research
workers. The proteins to be discussed are 3 -microglobulin (32m), a -microglobulin (a m),
2 2 2
cq-microglobulin (c^m) and post-gamma globulin (ρ-γ).
^-MICROGLOBULIN (3 m)
2
Many research groups have been engaged for the last decade in search for the function of an
ubiquitous low molecular weight protein first described by Berggard (1964). This protein is
beta-2-microglobulin (3 m). The first detailed physico-chemical characterization of the
2
protein was given by Berggard and Beam (1968). However, no indication of its function was
suggested at that time. By a brief historical introduction the span of these investigations
will be outlined. Since 3 m was isolated from urine its excretion was studied early and the
2
kidney was identified as the site of its catabolism (Bernier and Conrad, 1969). This protein
was "rediscovered" by Poulik in 1971 and partially sequenced (Smithies and Poulik, 1972).
The authors determined that 3 m is structurally related to immunoglobulins. This notion was
2
corroborated by Peterson et al. (1972). Smithies and Poulik (1972) subsequently isolated dog
3 m and demonstrated interspecies amino acid sequence homology of human 3 m and 3 m of other
2 2 2
species. Presence of 3 m on cell surfaces was shown with the aid of antisera specific for ß m
2 2
(Poulik and Motwani, 1972) and the first report on the levels of 3 m in various biological
2
fluids and in diseases was reported (Evrin and Wibell, 1972, Poulik, et al, 1972). Inter-
action of antisera against 3 m with lymphocytes was studied by Lightbody, et al (1974) and
2
Bach, et al (1973). This work stimulated research of the biological effects of such antisera
on cell-mediated cytotoxicity, cell migration, allograft rejection, etc. The discovery that
3 m is an integral part of the histocompatibility antigens (HLA) (Grey, et al, 1973, Nakamuro,
2
et al, 1973) opened a new era of understanding the structures of a number of cell membrane
antigens. Faber, et al, (1976) as well as Goodfellow, et al, (1975) demonstrated that the
human 3 m gene is located on chromosome 15. 3 m was found to contain a leader sequence when
2 2
synthesized in cell-free medium (Lingappa, et al, 1979). Suggs, et al, (1981) isolated cDNA
for human and mouse 3 m. Structure of the mouse 3 m gene was partially determined (Parnes and
2 2
Seidman, 1982). Through all these years 3 m has been a subject of numerous clinical investi-
2
gations of every possible disease condition using every available body fluid. In spite of all
of these basic and clinical studies the true function of 3 m remains elusive.
2
ß.-microglobulin, when isolated from urine, is a low molecular weight protein (11,800 daltons)
which is devoid of carbohydrate and free sulphydryl groups. Detailed physico-chemical charac-
teristics were established (Karlsson, 1974) and the first indication that 3 m is not exclus-
2
ively a urinary protein was provided (Smithies and Poulik, 1972). The authors determined the
sequence of the first 44 amino acid residues and showed that a significant homology exists
with the amino acid sequence of human gamma globulin heavy chain. By statistical analysis
they proved that such a structural relationship is not due to change alone and postulated that
the 3 m gene may have evolved from an ancestor of the present-day immunoglobulin gene. Thus,
2
indirectly, the role of 3 m in immunological phenomena was suggested. Peterson, et al (1972)
2
completed the sequence of 3 m and demonstrated that 3 m has a tertiary structure similiar to a
2 2
constant "domain" of gamma globulins. They have established that about 30% of the one hundred
amino acid residues are homologous with the constant C3 immunoglobulin domain, as well as the
other constant domains of the immunoglobulins. They called 3 m the "free" immunoglobulin
2
domain" and corroborated the postulate of Smithies and Poulik (1972) that 3 m evolved from an
2
ancestral gene of immunoglobulins. Parker and Strominger (1982) provided the definitive amino
acid sequence for human 3 m and proved that 3 m has only 99 amino acid residues. Subsequently,
2 2
3 m was isolated from mouse (Gates, et al, 1981), rat (Poulik and Smithies, 1979), cow
2
LOW MOLECULAR WEIGHT URINARY PROTEINS 5
(Groves and Greenberg, 1982), guinea pig (Cigen, et al, 1978), rabbit (Wolfe and Cebra, 1980)
and, most recently, chicken and turkey (Winkler and Sanders, 1977; Lillehoj, et al, 1982).
All of the above 3 m proteins were partially or completely sequenced and the seauences showed
2
high degree of interspecies homologies. Comparison of complete sequence data of mouse (Gates,
et al, 1981) and human 3 m reveals that most of the amino acid variations throughout the entire
2
molecule can be attributed to single base pair alterations. The primary structural similarity
among various 3 ms suggest that this protein has been well conserved through evolution,
2 n ae vnt o D ene
although non-mammalian 3 ms extensively studied in this respect. The amino acid
2
sequence homology between 3 m and immunoglobulins is well recognized, however controversy
2
exists concerning immunological cross-reaction between them. Poulik and Bloom (1973) demon-
strated that antisera prepared against human 3 m will detect ß m of chimpanzee and Poulik and
2 2
Weiss have shown (unpublished) that the same antisera also cross-reacted with the 3 m of
2
gorilla, but not with gibbon, rabbit or rat 3 m. Antiserum to rat 3 m produced in sheep
2 2
readily recognized both mouse and rat 3 m and became indispensible for subsequent work on
2
identification of histocompatibility antigens of the mouse lymphocytes (Vitetta, et al, 1976).
Detection of the presence of 3 m on the cell surface of lymphocytes by Poulik (1973) paved the
2
way for the subsequent discovery that 3 m is an integral part of histocompatibility antigens
2
of man and other mammalian species. At least three classes of the major histocompatibility
(MHC) antigens have so far been recognized. Classical transplantation antigens or serologic-
ally defined antigens (HLA-A, Β and C in man and H-2K, D, L, R in mouse) are called Class I
antigens. Products of the Class I MHC gene consist of two polypeptide chains in a noncovalent
association: a 44-46,000 dal ton transmembranal glycoprotein expressing alloantigenie deter-
minants of the MHC (heavy chain) and a 12,000 dal ton chain (light chain) which is 3 -microglo-
2
bulin. It was proposed that the heavy chain of the MHC complex may also have a structural
relationship to gamma globulins as 3 m does. This suggestion proved to be correct. After the
2
demonstration that 3 m is a part of the HLA molecule (Nakamuro, et al, 1973; Grey, et al, 1973),
2
the heavy chain of the HLA was sequenced and was shown to consist also of domains. One of
the domains is characterized by a sequence which is reasonably homologous with the immunoglo-
bulin constant domain. Furthermore, even Class II MHC proteins (HLA-DR of main) are glyco-
proteins and also composed of two non-covalently associated chains (alpha and beta) which
contain sequences homologous with gamma globulins. It was also proposed that some of the
tumor associated antigens may have a similar structure and contain 3 m (Thomson, et al, 1976).
2
Except for mouse thymus leukemia antigen (TL antigen) and human T6 and M241 antigens there
is no definite evidence of the association of 3 m with tumor antigens (van Rijn, et al, 1983).
2
The T6 and M241 antigens are not structurally related to the TL antigen as recently shown by
van Rijn (1983). They may however represent a novel Class I of the MHC antigen (van Rijn,
et al, 1983). The relationship of 3 m to tumor associated antigen thus remains an open
2
question.
It has to be stated again that in spite that 3 m was found to be associated with a multitude
2
of immunological phenomena, its true function is still unknown. 3 m is present on membranes
2
of all nucleated cells regardless of origin (Evrin and Nilsson, 1974). Notable exceptions
are erythrocytes, trophoblastic cells (Faulk and Temple, 1976), embryonal carcinoma cells,
gestational human choriocarcinoma (Tanaka, et al, 1981) and Daudi cells (Nilsson, et al, 1979).
In vitro, both normal and neoplastic cells of mesenchymal, epithelial, and hematopoetic origin
are endowed with the capacity to synthesize 3 m and consequently also HLA. The Daudi cell line
2
is an exception to this rule since it does not express 3 m on the surface and also lacks HLA
2
antigens. However, the Daudi cell is able to express a complete molecule of HLA including 3 m
2
when hybridized with tissue culture cell lines which bear histocompatibility antigens (Sege,
et al, 1981). Presence of 3 m is a sine qua non-necessity to express the HLA molecules. Con-
2
sequently it is important for translational modification and transport of the MHC heavy chain.
The absence of HLA in Daudi cell is due to an aberrant form of mRNA for 32^ (dePreval and
Mach, 1983; Rosa, et al, 1983). It was postulated that one of the functions of 3 m is to
2
stabilize the conformation of the heavy chain of the HLA molecule, especially the so-called
alpha-2 and alpha-3 domains (Lancet, et al, 1979). In addition to these possible functions,
3 m has been implicated in numerous immunological effects, e.g., inhibition of leucocyte
2
migration, stimulation of monocyte migration, etc. (Conway and Poulik, 1982). 3 m seems also
2
to share some functional properties with the domains of the immunoglobulins, especially those
of the Fc fragments. Thus macrophage rosetting of sheep red blood cells is promoted by 3 m
2
and 3 m seems to fix complement (Painter, et al, 1974).
2
As far as we know, 3 m is not an etiological factor in any disease. It can serve as a good
2
indicator of certain disease processes, e.g., renal diseases, failure of kidney transplanta-
tion, inflammation and possibly leukemias. Consequently, it can be used to monitor a disease
process. The clinical diagnostic potential of 3 m becomes evident when the metabolism of the
2
protein is understood and appreciated. Being a low molecular weight protein obtainable in
PBF-B*
6 THIRTEENTH ARNE TISELIUS MEMORIAL LECTURE
high purity and sufficient quantities, it became a model for studies of the physiological
role of proteins of this class. ß -microglobulin metabolism was studied in great detail and
2
the mechanism of the renal handling of all LWM proteins is well established. Consequently
ß m became a model for investigation of renal handling of all LMW proteins. The process will
2
be briefly described. ß m passes freely through the glomerular barrier into primary filtrate
2
and is reabsorbed by the proximal tubules and then transferred to lysozymes by pinocytosis.
Subsequently, it is completely degraded to amino acids which are used in the amino acid pool
for synthesis of new proteins. The reabsorption efficiency is 99% as shown for rat ß m and
2
human ß m (Conway and Poulik, 1977; Karlsson, et al, 1978). The source of ß m in the urine
2 2
and other biological fluids can be traced to nucleated cells. Bernier and Fanger (1972) and
Poulik and Bloom (1973) have shown that ß m is secreted into tissue culture fluid when lympho-
2
cytes are stimulated by mitogen(s). Studies of synthetic rates in large numbers of tissue
culture cell lines have demonstrated that the synthetic rate of ß m is essentially constant
2
(Hutteroth, et al, 1973). The turnover studies performed by Karlsson, et al (1978) estab-
lished that 0.13 mg/hr/kg of ß m is synthesized by a normal adult (a mean 24 hr production of
2 m
about 200 mg of ß m in a 70 kg man). The HLA molecules containing ß are shed continually
2 2
from the surface of the cells into the body fluids or in vitro into the supernatant fluids.
In vivo the HLA-heavy chain is rapidly degraded and the low molecular weight ß m is liberated
2
into the tissue fluids where it can be quantitated by radioimmunoassay (Evrin, et al, 1971)
or other convenient method. ß m is a stable molecule when present in serum, however in urine
2
a degradation process may set in whenever the pH of the urine drops below 5.5. Time and
temperature play a role in the degradation process and substances may be encountered which
may interfere with the quantitation procedure. The level of ß m was determined in practically
2
all biological fluids and for all ages. Levels of ß m in serum does not show any appreciable
2
diurnal or day to day variation and there is also no significant difference in the amount
among the sexes. The serum of a normal adult contains 0.6-2.6 mg/1, cerebrospinal fluid
0.7-2.4 mg/1 and saliva 0.8-2.4 mg/1 of ß m. Mean urinary excretion is 33-360 pg/24 hr. At
2
birth about 3 mg/1 (Jonasson et al, 1974) of ß m are present and this level decreases gradu-
2
ally until puberty is reached. Subsequently, a gradual rise from 2-3 mg/1 to slightly higher
level is the usual finding (Evrin and Wibell, 1972). This increase is probably due to the
decreasing number of glomeruli and thus less efficient glomerular filtration. The serum level
is dependent on (a) rate of synthesis and (b) rate of degradation. As mentioned above, the
rate of synthesis is remarkably constant. The rate of degradation is primarily a renal pro-
cess and only a very small amount of ß m is cleared extrarenally (0.1%). When renal absorp-
2
tion is impeded by whatever mechanism, levels of ß m in serum and urine will change. In
2
healthy subjects the excretion rate of ß m is fairly independent of the urinary flow rate and
2
the simultaneous excretion of sodium or osmomoles (Wibell and Karlsson, 1976). Estimation of
glomerular filtrate rate (GFR) is of paramount importance when renal disease is suspected. It
should be stressed at the onset that unless the renal status of the patient is carefully
determined, the quantitation of ß m may be of limited diagnostic and/or prognostic signifi-
2
cance. A disturbance of the proximal tubular function has a great influence on the urinary
ß-microglobulin output. The most notable examples of such a disturbance are aminoglycosides
2
nephrotoxicity (Sethi and Diamond, 1981), Balkan nephropathy (Hall, et al, 1972), Fanconi
syndrome (Butler and Flynn, 1958) and other causes such as cadmium and Itai-Itai disease
(Shiroishi, et al, 1977) and gold (Latt, et al, 1981), to mention the most common ones.
Retrospective studies have shown that 2-36% of patients treated with aminoglycosides
(gentamicin) develop signs of drug induced renal tubular dysfunction. Screening for ß m
2
levels may be extremely useful to assess the renal damage. Monitoring of the outcome of a
kidney transplant also falls into this category (Ravnskov, 1981). The authors' experience
in this area is that ß m is an inadequate indicator of acute rejection. This is mainly due
2
to the time factor of obtaining results by the RIA method of quantification.
In non-renal diseases the GFR is usually normal. However, in cases where reduction of GFR
has been taken into account, the serum levels of ß m were found to be disproportionately
2
elevated. Clinical entities which fall into this category are (a) some chronic inflammatory
diseases and (b) a wide range of neoplastic disorders. In chronic inflammatory diseases such
as systemic lupus erythematosus (SLE) (Parving, et al, 1980), rheumatoid arthritis (Manicourt,
et al, 1978), Sjogren's Syndrome (Michalski, et al, 1975), Crohn's disease (Descos, et al,
1979), etc., may exhibit high serum levels of ß m. However, the role of ß m determination
2 2
to monitor the effect of therapy of these patients requires further studies. Many workers
find relevance in changes of serum concentration of ß m in hematological malignancies (Kithier,
2
et al, 1974; Spatti, et al, 1980). In chronic lymphocytic leukemia the level of ß m increases
2
with the stage of the disease but it is not correlated with the lymphocyte count. In T-cell
lymphoma or acute leukemia the level is often unchanged. Cooper and Child (1981) have
reviewed the problem of serum ß m in lymphoid neoplasia and the authors warn that "the level
2
of ß m has no part in the diagnosis of lymphoid neoplasia." Furthermore, measurement of ß m
2 2
does not help in the differential diagnosis.
LOW MOLECULAR WEIGHT URINARY PROTEINS 7
as 3
Solid tumors represent yet another category where strides have been made to use ß m tumor
2
marker (Shuster, et al, 1976; Bunning, et al, 1979; Daver, et al, 1978). The levels of ß m
2
are usually only moderately elevated (Teasdale, et al, 1977). Patients with bronchial
(Schweiger and Tomczany, 1978), breast (Teasdale, et al, 1977), gastrointestinal (Satake,
et al, 1981), oral (Scully, 1981), and liver (Rashid, et al, 1981) malignancies fall into
this group. Detailed statistical analysis of 3 m levels in serum of lung and gastrointestinal
2
tract (GL) cancer patients was undertaken by Poulik, et al. (1980). However, their results
cast doubt on the potential of 3 m screening at least for the two sites. This study also
2
underlined the need for careful evaluation of parameters that determine the serum levels not
only of ß m but also any other potential tumor marker.
2
The most recently application of e m screening is in acquired immunodeficiency syndrome (AIDS).
2
Elevated levels of serum ß m, which occur in this syndrome, should aid in diagnosis (Fracioli
2
and Clemens, 1982; Bhalla, et al, 1983).
^-MICROGLOBULIN (a m)
2
Understanding all the functions of vitamin A remains an enigma in spite that this vitamin
was discovered as early as in 1915. It plays a role in a multitude of functions, e.g.,
differentiation, reproduction and in the visual process. Utilization of vitamin A in the body
is a tightly regulated process and there are several sites of control, e.g., transport from
its storage site in liver to tissues by a special carrier protein - retinol binding protein
(RBP) - isolated by Kanai(Kanai, et al, 1968). The alcohol form (retinoic acid) is
apparently transported by serum albumin (Smith, et al, 1973). There is also control at the
level of entry into the cell and at the cellular level where vitamin A appears to be bound to
a cellular receptor protein(s) in the cytosol. Evidence is accumulating with regard to the
anticancer effects of vitamin A and its precursors. A vast body of literature exists which
stimulates the interest not only in vitamin A but also its carriers, in biological fluids and
cytosol alike (Peto, et al, 1981).
In 1961 Berggard observed in urine a "long a -protein" (Berggard, 1961), isolated the protein,
2
and gave it a name, a -microglobulin, in analogy to $ m (Peterson and Berggard, 1971; Peterson,
2 2
1971). Two groups were involved in the study of this protein at that time. The American
group pursuing mainly research on the metabolism of vitamin A and the Swedish group pursuing
research on low molecular weight protein in the urine including retinol-binding protein. In
plasma the RBP (isolated by Goodman's group) was found to be bound to yet another protein,
thyroxin-binding prealbumin. It became obvious to Peterson and Berggard (1971) that the
a-microglobulin is similar, if not identical, to Kanai's plasma RBP. Subsequently, Cejka
2
and Poulik isolated the same protein from urine of kidney transplant patients (Cejka and
Poulik, 1971).
RBP is a minor component of human serum and it is not too difficult to isolate. The detection
during the different phases of purification is facilitated by the fluorescence properties of
vitamin A. Ion exchange chromatography is the method of choice and in a single step more than
200-fold purification can be achieved and the pure protein can be crystalized (Haupt and Heide,
1972). Isolation of RBP from other species than human is more difficult due to the charge
characteristics of the RBP-prealbumin complex. The interaction of RBP with prealbumin can be
abolished by buffers of low ionic strength or by buffers containing 6 M urea. The two proteins
can be separated electrophoretically by preparative Polyacrylamide gel electrophoresis in
sodium dodecyl sulfate (SDS) or in non-denaturing buffers (Kanai, et al, 1968). Highly
purified RBP which migrates with a -globulin in low resolution media, e.g., agarose, when
2
subjected to electrophoresis in Polyacrylamide gel or starch gel, 5-6 protein bands can be
detected. This heterogenity is probably due to the loss of amide groups and there seems to
be no evidence for genetic polymorphism. In spite of the electrophoretic heterogeneity the
protein is homogeneous with regard to size and molecular weight. The molecular weights were
established for RBP (21,000 daltons), pre-albumin (55,000) and RBP-prealbumin (76,000 daltons).
[Ultraviolet spectrum of RBP containing retinol (holo-RBP) has two spectra (a) 280 nm (aromatic
amino acids of the polypeptides) and (b) 330 (retinol molecule). The protein is devoid of
carbohydrates and fatty acids. Partial amino acid sequence was obtained originally by two
groups. Morgan et al. (1976) sequenced the first 15 amino acids and Poulik et al. (1975)
sequenced the first 50 amino acid residues of human and dog RBP isolated from urine. Results
of both groups agreed. The complete amino acid sequence was provided in 1979 (Rask, et al,
1979). The protein has 182 amino acid residues. Glutamine is the N-terminal and leucine is
the C-terminal amino acid residue.
The RBP-prealbumin complex is involved in two protein-1igand interactions in addition to the
protein-protein interaction. Thyroxin can bind to prealbumin without its binding to RBP.