Radioimmunoassay: Tool for Biomedical Investigation and Clinical Medicine
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Transkript: Englisch(automatisch erzeugt)
00:11
honored laureates, students, other guests. As scientists, we must appreciate that it is because of science and the achievements associated
00:24
with its advances that there are now four billion people in the world better fed, better housed, and in better health than ever before in history. If the problems to which Dr. Wald referred this morning
00:43
are to be solved, it is by an appreciation that the population of the world should not increase because this will be the only solution to our problem. But who among us has the wisdom to know who shall live
01:03
and whose lives should not be maintained? Who shall reproduce and who shall not? I am not that wise, and so I will restrict my remarks to fields in which I can offer scientific proof that you all
01:22
can accept. That is, I shall discuss only the science to which I have contributed sufficiently to find my place among these honored speakers. To primitive man, the sky was wonderful, mysterious,
01:41
and awesome. But he could not even dream of what was within the old golden disk, a silver point of light so far beyond his reach. The telescope, the spectroscope, the radio telescope, all the tools and paraphernalia of modern science
02:02
have acted as detailed probes to enable man to discover, to analyze, and hence better to understand the inner contents and fine structure of these celestial objects. Man himself is a mysterious object,
02:21
and the tools to probe his physiologic nature and function have developed only slowly through the millennia. Becquerel, the curies, the Joliot curies with a discovery of natural and artificial radioactivity, and Hevesy, who pioneered in the application of radio
02:43
isotopes to the study of chemical processes with the scientific progenitors of my career. For the past 30 years, I have been committed to the development and application of radio isotopic methodology to analyze the fine structure
03:02
of biologic systems. From 1950 until his untimely death in 1972, Dr. Solomon Berson was joined with me in this scientific adventure. And together, we gave birth to and nurtured through its infancy radioimmunoassay,
03:22
a powerful tool for determination of virtually any substance of biologic interest, essentially an amalgamation of physics and medicine, a physicist and physician. How did radioimmunoassay begin? Were we setting out to measure anything?
03:42
Let me share with you today its history, its past, and something of its potential. Radioimmunoassay came into being not by directed design, but more as a fallout from our investigations
04:01
into what might be considered an unrelated study. Dr. I. Arthur Mirsky postulated some 25 years ago that the diabetes of the adult, maturity-onset diabetes, might not be due to a deficiency of insulin
04:21
secretion, but rather to abnormally rapid degradation of insulin by a liver enzyme, which he deemed hepatic insulinase. Why did he make this suggestion? It was already known that although the pancreas
04:43
of the juvenile diabetic, the child with onset of diabetes has practically no insulin in his pancreas. However, it was known from post-mortem studies that the maturity-onset diabetic generally
05:02
had almost normal, normal, and even supranormal amounts of insulin in his pancreas. And yet 25 years ago, before the days of the oral hypoglycemic agents, virtually all diabetics were treated with insulin.
05:23
If the pancreas has enough insulin and it is presumed that the circulation does not have enough insulin, then the idea of Dr. Mirsky that insulin was being destroyed abnormally rapidly
05:41
in the diabetic seemed very reasonable. At the time, Dr. Berson and I were working in studies concerned with the turnover, the distribution and disappearance from the plasma of serum proteins. It therefore appeared not unreasonable to perform
06:04
the same type of studies to determine how rapidly was insulin disappearing from the plasma of the diabetic subject as compared to the plasma of the non-diabetic subject. If the Mirsky hypothesis were correct,
06:22
then we would have expected insulin to disappear more rapidly from the plasma of the diabetic than from the plasma of the non-diabetic subject. May I have the first slide, please? We therefore administered labeled insulin,
06:42
insulin labeled with radioisotope of iodine, I-131, intravenously to diabetic and non-diabetic subjects. If the Mirsky hypothesis were right, the insulin would have disappeared more rapidly
07:02
from the diabetic. What we found, to our surprise, is that the radioactive insulin disappeared more slowly from the plasma of the diabetic subjects than from the non-diabetic subjects shown in the lighter curves at the bottom.
07:22
There were included in the lower group some diabetic subjects. In fact, one MN, when first seen as a fresh diabetic, the disappearance curve was normally rapid. However, following several months of insulin therapy,
07:42
he joined the more slowly disappearing group. In addition, there was an occasional non-diabetic subject shown in this heavy dotted line who also had a slow rate of disappearance. And this was a schizophrenic subject
08:00
who had received insulin shock therapy. The difference between the slowly and the rapidly disappearing group, therefore, was not a history of diabetes per se, but rather a history of previous therapy with insulin.
08:22
We therefore suspected that the retarded rate of insulin disappearance was due to binding of labeled insulin to antibodies, which developed in response to administration of exogenous insulin obtained from cows or pigs.
08:42
However, classic immunologic techniques were not satisfactory for the detection of antibodies, which we presume were likely to be of such low concentration as to be non-precipitating. We were confronted with the idea
09:01
that insulin was not antigenic or its antigenicity would have been observed in the 25 year previous history of insulin therapy. We therefore felt that we must introduce new techniques of high sensitivity for detection
09:21
of soluble antigen antibody complexes, and these techniques depended upon the use of labeled insulin, insulin labeled with radioactive iodine. In the next slide, next slide please, we see some of the techniques.
09:41
The first of the techniques we used were called electrophoresis. In these techniques, essentially we are separating proteins on the basis of their differences in charge and their response to the superposition of an electric field.
10:03
Shown here in the center are the electrophoresis patterns of labeled insulin in the plasma of a patient never treated with insulin, what we are calling our non-immune plasma, and in the plasma of patients treated with insulin, we are calling it immune plasma.
10:22
In the plasma of the patients never treated with insulin, the labeled insulin binds to paper as the rest of the serum proteins migrate. However, in the plasma of the insulin treated subject, the insulin migrates in whole or part with the serum proteins,
10:42
here shown as an inter-beta gamma globulin. Saul Berson and I were always in a great hurry, and therefore we designed a new, very simple technique called paper electrophoresis, in which we left the top of the electrophoresis box open,
11:04
we encouraged water flow chromatography, and therefore could affect separation between the free insulin, which remains at the site of application, and the protein-bound insulin, which migrates in about 15 minutes
11:22
as compared to the overnight or 16 hours required for standard electrophoresis. Here we see starch block electrophoresis, again a system that separates only on the basis of charge, where free insulin migrates almost in the region
11:41
of albumin, and the labeled insulin bound to a gamma globulin remains quite close to the site of application. Using a variety of such systems, we were able to demonstrate the ubiquitous presence of insulin-binding antibodies
12:02
in virtually all subjects treated with insulin for a period of a month or more. This concept was not acceptable to the immunologists of the mid-1950s. Heidelberger had just stated it in his book that peptides less than 10,000 in molecular weight
12:23
could not be antigenic. Next slide please. The original paper describing these findings was rejected by science, and initially rejected by the leading American Journal of Clinical Investigation.
12:42
A compromise with the editors eventually resulted in acceptance of the paper, but only after we omitted the word antibody from the title, because they were unable to accept our conclusion that the globulin responsible for insulin binding was,
13:02
in fact, an acquired antibody. Here we see the bound insulin migrating with the gamma globulins, the free insulin remaining at the site of application. Note that as we increase the insulin concentration from less than one milliunit of insulin per milliliter
13:23
up to a value 10-fold greater, we get a reduction in the ratio of antibody bound to free insulin, some 67% being bound in the first case, 64, 57, less than half, and 31% being bound
13:42
as the insulin concentration has gone up 10-fold. This observation provided the basis for the radioimmunoassay of plasma insulin. However, investigations and analyses which lasted for several years, and which included studies on the quantitative aspects
14:03
of the reaction between insulin and antibody, and the species specificity of the available antisera were required to translate the theoretical concepts of radioimmunoassay into its practical application
14:20
almost 20 years ago to the measurement of insulin in unextracted plasma. Next slide, please. Radioimmunoassay is simple in principle. It is shown in the competing reactions in this slide. The concentration of antigen in the unknown sample
14:43
is determined by comparing its behavior in inhibiting the binding of labeled antigen to antibody with a behavior of known standard solutions. Radioimmunoassay is not an isotope dilution technique,
15:03
as originally described by Hevesy. Since there is no requirement for identical immunologic activity of the unlabeled antigen with that of the labeled antigen. The validity of radioimmunoassay
15:20
is dependent only on identical immunologic behavior of the unknown samples and of the known standards. The specificity of immunologic reactions can permit ready distinction, for instance, between corticosterone and cortisol,
15:43
two steroids which differ only in the presence of a single hydroxyl residue. There is no requirement in radioimmunoassay for standards and unknowns to be identical chemically
16:01
or to have identical biologic behavior. It is therefore necessary if one is concerned with the biologic behavior of quantities measured by radioimmunoassay to have some additional proof as to the validity of the biologic comparison.
16:24
In addition, radioimmunoassay can even be clinically useful in some assays which cannot be properly validated due to a lack of immunologic identity between standards and the sample
16:41
whose concentration is to be determined. The use of radioimmunoassay for the measurement of parathyroid hormone, the hormone which controls the body's handling of calcium is typical of this. Radioimmunoassay is a test tube method.
17:01
To perform a radioimmunoassay, we use one or another variation of the following. We mix in the test tube a fixed amount of labeled antigen, a fixed amount of antibody, and in some tubes, the known standards or in other tubes, the unknown samples. At the end of a period of time
17:22
which may be from minutes to hours to days, we provide some way of separating the antibody bound labeled antigen from that which is free because as I have described earlier, the antibody bound antigen is in the form of soluble complexes.
17:42
Tens of techniques have been used to affect this separation. Next slide, please. We then plot a standard curve which consists of the ratio of antibody bound to free labeled antigen as a function of the concentration
18:00
of the unlabeled antigen. We then measure the percent binding or the B over F ratio in the unknown tube and from this calibration curve can measure directly the concentration of the unlabeled antigen. As shown here, the sensitivity
18:21
of radioimmunoassay is quite remarkable. As little as a 10th picogram per milliliter or five times 10 to the minus 14th molar gastrin is readily measurable. Next slide, please. The radioimmunoassay principle
18:41
is not limited to immune substances but can be extended to other systems in which in place of the specific antibody, there is now a specific reactor. The specific reactor can be any type of binding substance. This might be a binding protein in plasma,
19:02
an enzyme or a tissue receptor site. Furthermore, it is not necessary that a radioactive atom be used as the marker. Recently, there has been considerable interest in employing as markers enzymes
19:21
which are covalently bound to the antigen. Although many variations of competitive assay, the more general name for radioimmunoassay have been described, radioimmunoassay has remained the method of choice and is likely to remain so at least in those assays
19:42
which require high sensitivity. The receptor site assays for the peptide hormones have the advantage of measuring biologic activity but are generally at least 10 to 100 fold less sensitive than radioimmunoassay.
20:03
Enzyme marker assays which have been used rather extensively the last few years do have several disadvantages. The most important is that the steric hindrance introduced into the antigen antibody reaction because of the presence of the enzyme molecule
20:23
almost inevitably decreases the sensitivity of the assay. Two decades ago when bioassay procedures were in the forefront, the first presentation on the potential of hormonal measurements by radioimmunoassay went virtually unnoticed.
20:42
Somewhat more interest was generated by our demonstration in 1959 of the practical application of radioimmunoassay to the measurements of plasma insulin in man. Next slide please. Nonetheless, in the early 60s, the rate of growth of radioimmunoassay was quite slow.
21:01
Only an occasional paper other than those from our laboratory being found in the leading American journals. Not only did we describe radioimmunoassay but in the early 60s, we conducted training courses in our laboratory over a three year period in which we trained more than 100 American investigators
21:23
in the use of its techniques. And as you can see at the end of this training period, the procedure took off and there was in fact an exponential and continuing exponential growth of radioimmunoassay. Like most scientists, we did not patent our discoveries.
21:42
By the late 1960s, radioimmunoassay had become a major tool in endocrine laboratories. More recently, it has expanded beyond the research laboratory into the nuclear medicine and clinical laboratories. It has been estimated that in 1975,
22:00
in the United States alone, over 4,000 hospital and non-hospital clinical laboratories performed radioimmunoassays of all kinds, almost double the number of a year or two earlier. And the rate of increase appears not to have diminished over the past two or three years.
22:20
The technical simplicity of radioimmunoassay and the ease with which the reagents may be obtained have enabled its extensive use even in scientifically underdeveloped nations. In fact, I would say the current problem in radioimmunoassay is its overuse as is the problem with many of the tools
22:42
that modern medicine has given us. Next slide please. The explosive growth of radioimmunoassay has derived from its general applicability to many diverse areas in biomedical investigation and clinical diagnosis. A representative, illegible and incomplete listing
23:02
of substances measured by radioimmunoassay is shown here. This slide is meant more to impress than to be read so I'll go on to the next slide which has only the listing of the substances shown. On the left is a listing of the peptidyl hormones
23:21
in the center, the non-peptidyl hormones and in the right the non-hormonal substances which have been measured by radioimmunoassay. And I would first like to describe some very typical uses to let us know how with radioimmunoassay we have gained new insight into physiology and pathophysiology.
23:43
I started this lecture by indicating in the 1950s it was thought that all diabetics had an absolute deficiency of insulin. This is the reason for the Mersky hypothesis with an adequate pancreas
24:03
and with the thought that the circulating insulin was too low, he therefore thought insulin was being degraded abnormally. In fact, the first discovery made with radioimmunoassay was the recognition that the maturity onset diabetic subject
24:24
did not have an absolute deficiency of insulin but in fact his insulin levels generally exceeded that of the non-diabetic patient because there was something wrong in diabetes which inhibits the body from properly using the insulin
24:44
so that the first discovery made with radioimmunoassay was the recognition that in diabetes the elevated blood sugar was due in the adult not to an absolute deficiency of insulin
25:00
but to something in the disease state that accounts for the barrier of the diabetic subject to use his insulin as effectively as does the non-diabetic subject. The second discovery with radioimmunoassay was the ability to measure one of the pituitary hormones,
25:22
growth hormone, the hormone concerned with the growth of small children. If these children are treated in time with growth hormone, then they can achieve almost normal growth. Are all children of small stature
25:43
due to a deficiency of growth hormone? The answer is no. There are many other causes for the short stature of some children. Malnutrition, genetic constitution, even lack of love
26:01
can prevent some small children from growing. At present, we have no way of obtaining human growth hormone except as autopsy material from human subjects. Perhaps someday E. coli will grow it but at the present time,
26:21
our only source is from autopsy material. We cannot use animal growth hormones to treat growth deficient human children. The importance of radioimmunoassay is it provides us with ways of determining which small children are small
26:41
due to an absence of growth hormone and which small children are small due to other causes. And why is it important to make this distinction? Because we only have enough growth hormone to treat less than half the children who really need it. And if we wasted it by treating those children
27:02
who do not need it, we would not have enough of it to treat those who do. Second role for radioimmunoassay. With radioimmunoassay, we are able to measure the hormones which control calcium metabolism, the calcitropic hormones.
27:21
And we have understood something about the secondary hyperparathyroidism, the bone disease of patients with renal disease. We've been able to make the diagnosis of calcium secreting tumors, of excess parathyroid hormone
27:43
from tumors of the parathyroid gland and so on. We have learned a good deal more about sterility and fertility with assays for the gonadotropins. To me, perhaps the most exciting new development
28:02
of radioimmunoassay is one that has not come from my laboratory because I work at a veterans administration hospital and our patients are adult males for the most part, but the use of radioimmunoassay for the screening of hypothyroidism of the newborn.
28:23
In our country, we have been familiar for a number of years with a screening for newborns with phenylketonuria, the inability to properly metabolize phenylalanine. These children, if not given a special diet
28:43
in the first year of their life, develop irreversible mental retardation. And as a result, in our country, we've had a screening program in effect throughout the 48 or 50 states. With the development of radioimmunoassay,
29:03
we are able to measure the concentration of thyroid hormones also in a drop of blood on filter paper taken from the newborn. Phenylketonuria in Northern Europe in the United States occurs one in 25,000 births,
29:25
and actually the treatment is a very difficult one. Hypothyroidism of the newborn, inactive thyroid of the newborn, occurs in about one to 5,000 to one to 8,000 births, three to fivefold greater incidence.
29:44
If not detected by three months, mental retardation is inevitable, a lowering of the IQ, the intelligent quotient, by more than 30 to 40% below that of their siblings.
30:01
The cost of treatment is a dollar a year. And in many states now, screening of the newborn for neonatal hypothyroidism is being required. And we can look to the end in one in 5,000 births of this terrible tragedy for the family,
30:23
for the child, and for the community. More recently, I have returned from India where I have noted that in India infectious diseases are second in the cause of death as opposed in Europe and in the United States,
30:41
15 among the causes of death. Radioimmunoassay will have an important role in the early detection of carriers of disease. Some eight years ago, we described from our laboratory the first application of radioimmunoassay
31:01
to a viral antigen, the measurement of hepatitis B antigen and the antibody using radioisotopic techniques, using radioimmunoassay. It is now the method of choice in our country for the detection of blood which has been infected with hepatitis B antigen.
31:22
We have more recently described from our laboratory the application of radioimmunoassay for the measurement of purified protein derivative. This is a protein that is derived from the cell wall of the mycobacterium tuberculosis.
31:44
We have described the ability to detect this antigen in situations of miliary tuberculosis. We hope to be able to apply it to other very severe problems such as tubercular meningitis,
32:02
where an early diagnosis is very important if treatment is to be affected in time and where the usual biologic culture techniques frequently will take six weeks or so before the diagnosis can be made. This might be less exciting in the States. In India, where the differential diagnosis on brain scan
32:25
is not between stroke and tumor, but between stroke and tuberculous granuloma, it is in fact a very important problem. Other uses which I can envision would be, for instance, in the case of leprosy,
32:41
a disease in which there is a very long incubation period and which could be treated in time if we could identify the carriers. I believe that in the 1980s, radioimmunoassay will find as wide applicability in the study of infectious diseases
33:01
as it has proven its role in the 60s in the study of endocrinology. Rather than go on with this very generalized description, I would like to consider a few specific examples of radioimmunoassay to illustrate how it can and should be used.
33:23
Proper interpretation of plasma hormone levels, particularly of the peptide hormone concentrations in clinical diagnosis, requires a clear understanding of the factors involved in the regulation of hormonal secretion.
33:40
Next slide. Generally, such secretion is stimulated by some departure from the state of biologic homeostasis that the hormone is designed to modulate. A representative model for one such system is shown in this slide. Regulation is affected through the operation
34:03
of a feedback control loop which contains the hormone at one terminus and at the other, the substance which it regulates. Gastrin is a hormone which is secreted in the stomach and which controls gastric acidity.
34:21
Gastrin secretion increases gastric acidity which then suppresses the secretion of antral gastrin. Modulation of this system can be affected by a number of factors, perhaps the most important of which is feeding. Feeding promotes gastrin release
34:42
directly by its chemical action on the antrum by distension of the stomach or by the buffering action of food which reduces gastric acidity and through this mechanism promotes gastrin release. Next slide. The normal fasting gastrin concentration
35:03
in most types of ulcer patients and normal patients is less than a 10th nanogram per milliliter. Here we see three different clinical conditions and I will describe them in which the gastrin is abnormally elevated.
35:22
The first group is a group of patients with pernicious anemia. These patients who develop, if untreated, the fatal form of anemia, the inability to make blood, are patients whose stomachs are characterized by marked hypoacidity.
35:44
Since gastric acid normally suppresses gastrin secretion, the continued absence of acid and the repeated stimulation by feeding eventually produces secondary hyperplasia of the gastrin producing cells.
36:03
The high level of gastrin in these patients is then considered very appropriate because of the absence of the inhibitory effect of hydrochloric acid on the secretion of antral gastrin. The second group is a group
36:21
we call them Zollinger-Ellison syndrome. Essentially these are patients with a tumor that secretes gastrin. This tumor is frequently a malignant tumor. It is a form of cancer. And in this case, secretion of gastrin from the tumor
36:41
is not being appropriately regulated. The patients have marked hyperacidity, developed ulcers in the duodenum due to the marked hyperacidity. It is important to be able to distinguish
37:00
between the patients who have a gastrin-secreting tumor and patients with elevation of gastrin because of hypoacidity. And so it is evident that we must measure both the hormone and also the level of acid. In application of radioimmunoassay
37:22
in the case of the peptide hormones, it is not sufficient to measure the peptide hormone concentration per se. We must measure the hormone and the substrate which is designed to regulate. Interesting enough, we can also get marked hyperacidity
37:43
not because of a malignant condition such as a gastrin-secreting tumor but simply because of hyperactivity of the antrum, hyperactivity of the gastrin-secreting cells of the stomach.
38:03
How do we distinguish between those patients with marked hyperacidity and excess gastrin secretion in the region of overlap? Certainly the treatment of a tumor is likely to be quite different from the treatment of simple hyperactivity
38:22
of the gastrin-secreting cells. With radioimmunoassay, we can do dynamic studies. Before radioimmunoassay, it took a cup of blood to measure the concentration of insulin in the blood. With radioimmunoassay, we can make
38:42
the same measurement with a finger stick. As a result, we can apply dynamic studies. We can perform five, 10, 20 successive radioimmunoassays to determine how the particular substance concentration is changing in plasma.
39:02
Next slide, please. And in this slide, we see how we make the separation between gastrin hypersecreters due to the tumor as shown on the left and due to hyperactivity of the antral gastrin-secreting cells as shown on the right.
39:21
Patients with hyperactivity of the gastrointestinal tract respond dramatically to feeding and not to other secretagogues, such as calcium and secretin, because they are characterized by overactivity of the gastrin-secreting cells, which respond to feeding.
39:42
Patients with a tumor do not respond to feeding down here as opposed to up there, whereas they do respond to other secretagogues. So that with appropriate choice of secretagogues, we are able to make diagnostic differentiation
40:01
between two groups that clinically would resemble each other. They both have high levels of gastrin, both have high levels of gastric acid, but with appropriate stimulatory tests, we are able to make the distinction. Thus, in the application of radioimmunoassays
40:21
to problems of hypo or hypersecretion, we seldom rely on a single determination of the plasma hormone. Generally, to test for deficiency states, we measure concentrations not only in the basal state, but in response to administration of appropriate physiologic or pharmacologic stimuli.
40:44
When hypersecretion is suspected, sometimes we use suppressive tests. Studies such as these are common now in endocrinology and would not have been possible without radioimmunoassay. The study of the peptide hormones
41:02
has been further complicated by a change in our concepts of the chemical nature of the peptide hormones. We now know that the peptide hormones are found in plasma in more than one form and in the glandular tissues from which they come.
41:23
These forms may or may not have biologic activity and may represent either precursors or metabolic products of the well-known, well-characterized biologically active hormone. Their existence has certainly introduced complications
41:41
into the interpretation of hormonal concentrations as measured by radioimmunoassay and as measured by bioassay as well. A typical example of work in this area is the current interest in the heterogeneity of gastrin.
42:01
Next slide, please. Several analytical methods were used to elucidate the nature of plasma gastrin. The technique shown here is called Sephadex gel filtration and it separates molecules on the basis of their molecular radius,
42:22
essentially on molecular weight and configuration of the molecule. This is done in a column in our hands about a half a meter long. We have marker molecules which mark the void volume and marker molecules which mark the salt peak.
42:41
When we add gastrin, it's heptadecopeptide gastrin. The gastrin purified from the antrum is a 17 amino acid peptide. We add this to plasma. We note that the gastrin elutes after insulin which weighs only 6,000,
43:03
pro-insulin weighing 9,000. If we examine the nature of the gastrin in plasma, we see that it elutes between insulin and pro-insulin, clearly behaving different chemically from the gastrin which has been purified from the antrum.
43:23
Next slide, please. We can use other physical chemical methods to affect that separation. You can leave it that way. The anode is shown up. The material is applied here at the origin. We note that the gastrin in plasma
43:40
differs in charge also from the 17 amino acid peptide which had been separated from the antrum. We have called this new form of gastrin big basic gastrin. Next slide, please. Again, it's not lined up right.
44:01
We note here's the big gastrin eluting between the void volume and the insulin. If we now use tryptic digestion, we can convert the big gastrin into the heptadecopeptide gastrin. We therefore predicted that big gastrin
44:21
was a precursor molecule for the 17 amino acid peptide and was linked to the 17 amino acid peptide through a lysine or arginine residue. Next slide, please. Soon thereafter, Gregory and Tracy were able to show that the 17 amino acid heptadecopeptide gastrin
44:45
was incorporated in big gastrin through two lysine residues. Thus, our prediction based on the measurement of picogram to nanogram amounts of immunoreactive gastrin
45:00
in the presence of billion-fold higher concentrations of other proteins was justified by the work of Gregory and Tracy purifying and chemically characterizing this material. Next slide, please. Unlike proinsulin,
45:21
which is virtually devoid of biologic activity, the in vivo administration of immunochemically equivalent amounts of big gastrin and heptadecopeptide gastrin results in the same physiologic response, the same acid output in a dog.
45:42
On this basis, we would say that big gastrin and heptadecopeptide gastrin have equal biologic activity. You administer the same amount, you get the same biologic response. Defined in this way, the classic physiologic description,
46:02
big and heptadecopeptide gastrins have the same biologic activity. However, the turnover time for big gastrin is five times as long as that of heptadecopeptide gastrin so that when you administer it intravenously, it disappears more slowly.
46:21
Therefore, under the conditions of a continuous infusion, the plasma concentration of the big gastrin is five times as high as that of the heptadecopeptide gastrin. Therefore, if we define biologic responsiveness, not as dose-administered biologic response,
46:43
but plasma level versus biologic response, we would say under those conditions, big gastrin has only a fifth the biologic activity as does the heptadecopeptide gastrin. So the concept of heterogeneity
47:00
has introduced complications not only into immunoassay but how we define things in terms of bioassay as well. At present, a decade after the concept of heterogeneity was developed, and in spite of an enormous body of descriptive data in this field,
47:20
we still do not know very much about the rules or reasons for this precursor product synthetic scheme. Is the synthesis of the peptide hormones in the form in which they are linked to another peptide essential only for the method of synthesis? What are the enzymes involved in the conversion process?
47:42
10 years later, we don't know if the enzyme converting enzymes are hormone-specific or species-specific. There are still arguments as to whether the conversion is affected only in the secreting tissue or whether there is peripheral conversion from inactive to active form.
48:00
What is the role of the part of the precursor molecule which is discarded after biosynthesis? In fact, is it discarded? There is now evidence to suggest that ACTH, lipotropin, and others are part of the same molecule, and each part has different physiologic function. Finding the answers to these and related questions
48:22
will keep many of us busy for quite a while. In the few minutes left, I would just like to discuss with you what is, to me, a very new and exciting development, again, making use of radioimmunoassay. The findings by Vanderhagen et al.
48:42
of a new peptide in the vertebrate central nervous system that reacts with antibodies against gastrin was confirmed by Doc Ray, who suggested that the brain peptide resembled cholecystokinin-like more closely than it did gastrin-like peptides. These studies were based only upon
49:01
the differences in immunoreactivity of different antiserums. We extended these studies and demonstrated that the peptide in the brain was not gastrin, was not simply cholecystokinin-like, but was, in fact, intact cholecystokinin
49:21
and its C-terminal octapeptide, a gut hormone now being found in the brain. These observations depended on the use of two antisera with different immunochemical specificities. Next slide, please. One was prepared by immunization of a goat
49:41
with porcine cholecystokinin, the only species from which cholecystokinin has been purified, and this doesn't react with any of the other gut hormones, including gastrin or even the octopeptide, the C-terminal octopeptide of cholecystokinin. So it reacts with amino acids
50:02
in the N-terminal portion of the molecule. A second antiserum, next slide, was prepared by immunization with the four-terminal amino acids of gastrin. Gastrin and cholecystokinin share the same five C-terminal amino acids,
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and we see that the peptides cholecystokinin, its octopeptide, gastrin, the 39-amino acid cholecystokinin all behave about the same in this system. Using this antiserum, next slide, please,
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we observed that in all animal species studied, the immunoreactive content of cholecystokinin in the gut and in the brain were roughly comparable, so that in fact, if we consider man with a big brain,
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there is more of the gut peptide in the brain than there is in the gut. A very interesting observation. Note also that the concentrations among the different species are very constant within the range of five or six-fold. Furthermore, after trypsin digestion,
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which as you know breaks the lysine and arginine residues there is virtually no change in the amino reactivity because the cholecystokinin octopeptide does not contain lysine or arginine residues and therefore is not a substrate for trypsin.
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And in the next slide, we see the Sephadex gel filtration patterns. In yellow is shown the intact cholecystokinin. In white is shown the octopeptide, about half and half before tryptic digestion, all converted to the octopeptide after tryptic digestion
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with no loss in the amino reactivity. And essentially, the cholecystokinin in the gut of the pig is comparable to that in the cerebral cortex in the gut of the dog, comparable to that in the cerebral cortex. The gut of the monkey, comparable to that in the cerebral cortex.
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Next slide. In the same monkey and dog extracts in which the cholecystokinin-like material was present in about the same concentration in the pig extracts, we did not find it in the brain and gut extracts of the other species when we use the N-terminal antiserum.
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In other words, we found immunoreactivity with an antiserum directed to the C-terminal portion of the molecule, but not with an antiserum directed to the N-terminal portion of porcine cholecystokinin.
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We therefore predict on the basis of radioimmunoassay that there are major differences between pig and the other animal cholecystokinins in the amino terminal portion of the molecule. Since this portion of the molecule is not directly involved in its biologic action,
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it is not surprising that the amino acid sequences in this region have diverged during the course of evolution. In fact, the C-terminal octapeptide has about 10 times the biologic activity as intact cholecystokinin. We look forward to our predictions stimulating Victor Mutt
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to purify and chemically characterize the other animal cholecystokinins, and we are looking forward to what he finds. Next slide. Where in the brain is cholecystokinin found? Its concentration is highest in the cerebral cortex. Immunohistochemical studies as shown here
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indicates that the hormone appears to be concentrated in the cortical neurons. Lights, please. The finding of peptides resembling cholecystokinin and its octapeptide in the central nervous system raises intriguing questions
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about their physiologic function, particularly with respect to their potential roles as satiety factors. The observations of Gibbs et al. that injection of purified cholecystokinin or the octapeptide evokes satiety, although other gut hormones did not, has suggested a negative feedback mechanism
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from the gastrointestinal tract is the causative mechanism. The finding that cholecystokinin peptides appear to be endogenous in the brain suggests a more direct role for them as neuro regulators, and in fact, we are now examining the changes
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in the cholecystokinin content in configuration in obese animals as compared to that of normal or fasted animals. Where is radioimmunoassay going? I'm afraid I'm not the best predictor of where radioimmunoassay is going.
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I have now seen hundreds of different applications. I've seen whole new fields in medicine stimulated. I, like you, look forward to where radioimmunoassay goes from here. Thank you. Thank you. Thank you.