Introduction Many honnones, growth factors and other biologically active agents are extremely

potent, eliciting their effects at exceedingly low concentrations. For instance, the
honnone glucagon (Fig. 12.1) , the subject of this problem，的 normally present in
plasma at about 3 X 10- 11 M or 100 pg/ml (1 pg = 10一 12 g). The concentrations in
plasma or serum of some honnones are given in Appendix 4.
Clinical investigation frequently requires the rapid and accurate measurement of the
concentrations of these honnones. Bioassays , which measure the honnone's effects in
animals or organ systems, are expensive, slow and insensitive. Often they fa過 to
distinguish between honnones whose biological effects are rather similar; for instance ,
there are several honnones that will increase blood glucose or cause bone growth.
Antibodies provide the basis for a large variety of modern assay kits for compounds of
clinical importance. They are quick and easy to use , extremely accurate , sensitive and
specific.
Confinnation of human pregnancy is based on the detection of human chorionic
gonadotropin (hCG) in the urine of a pregnant woman. This used to involve a
bioassay - the gonadotropic effects of hCG were detected in toads - conducted by
specialist laboratories. Not only was this expensive, but its insensitivity, lack of
reproducibility and the time taken to obtain the results meant that pregnancy could
only be confinned once it was fairly well established. By using antibodies to hCG,
highly accurate and rapid pregnancy testing can be done now in the privacy of the
home using simple and inexpensive ‘over-the-counter' kits.

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 Getting the Measure of Hormones

The Problem A clinical biochemistry laboratory established a radioimmunoassay (RIA) for human
g1ucagon. A series of incubation tubes was set up , each tube containing a constant but
limiting amount of an antiserum against human glucagon and a fIxed amount of
radiolabelled glucagon. Various amounts of unlabelled g1ucagon were also added
After incubation , the radioactive immune complex formed was assayed (Figure 12.2).
The RIA was then used to assay plasma from three individuals (Table 12.1). Alan G.
was a young child with a history of repeated convulsive limb movements. His parents
repo此ed that these seizures could be lessened by frequent feeding though this was
becoming less e位 ctive. Barbara W. was a somewhat overweight post-menopausal
woman who had been referred by a derrnatology clinic because of her characteristic
skin lesions. Fred C. was asymptomatic and had been screened as an apparently
norrnal member of the general publi c. All three provided fasting blood samples for
analysis (Table 12.2). Both Alan G. and Fred C. had a norrnal blood glucose response
after 峙的ed glucagon 但 arbara W. was not tested).

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Glucagon (pg)

Patient Volume of plasma Immune complex

assayed (rnl) fonned (dpm)

Alan G. 0.10 3700

Barbara W. 0.01 2600
Fred C 0.01 6400

Patient Glucose (mM) Ins叫血 (μU/叫)

Al an G. 2.4 9.6
Barbara W 6.5 30.1
Fred C 4.1 12.8
Norrnal subjects (range) 3.5-5 .5 9.0-18.0

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 Getting the Measure of Hormones

1. What are the plasma glucagon concentrations for each subject? The range for a group
of normal subjects was found to be 85-150 pg/rnl.
2. Why was it necessary to use a lirniting amount of antiserum in the RIA?

3. Why was a group of individuals used for the control data?

4. What is the reason for Alan G.'s convulsive seizures and why were they relieved by
feeding?
5. What is the metabolic defect in Alan G.?

6. What is the likely explanation for Barbara W.'s condition?

7. Fred c. 's plasma glucagon was tested again with the same result. Bearing in mind
which prope前y of glucagon is being measured in the RIA, how can his asymptomatic
condition be reconciled with his plasma glucagon?

8. What treatments could be offered to Al an G. and to Barbara W.?

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Getting the Measure of Hormones

Commentary The RIA is based on the ability of antibodies in the antiserum to recognise human
glucagon with high specificity. These antibodies (Ab) form an immune complex with
the radiolabelled glucagon (G*) according to the following equ山brium :

The radiolabelled complex (Ab-G*) can be assayed by separating it from the

remammg uncomplexed radiolabelled glucagon. Provided the Ab is present in
limiting amounts , the presence of additional unlabelled glucagon (G) will result in
competition between G and G* for Ab:

The amount of radiolabelled complex will therefore decrease in proportion to the

amount of unlabelled glucagon added as seen in Figure 12.2 . Using this standard curve
the plasma glucagon concentrations can be calculated as shown in Table 12.3.
Thus , compared to the normal range of85- 150 pg/ rnl , Al an G. has a substantially
decreased plasma glucagon while the others have high plasma glucagon , Barbara W.'s
being particularly elevated. A group of normal individuals should always be used to
generate reference data rather than a single normal control since there may be
considerable variation even in the normal population. In addition, it may be necessary
to choose normal controls of the same sex, age, etc. to have meaningful data for
comparison. Control data are normally given as a range (as in this problem) or as the
mean 土 SD.
The central nervous system is particularly dependent on an adequate supply of
glucose. Under conditions of severe hypoglycaernia , as experienced by Alan G., the
brain functions abnormally and limb seizures result. By feeding him frequently , an
adequate plasma glucose concentration can be maintained to relieve his symptoms.
In the fasting state , hypoglycaemia signals the pancreatic A cells to secrete glucagon
whose target , the liver, releases glucose from mobilised glycogen stores. Al an G.
appears to be unable to produce enough glucagon to counteract his hypoglycaem尬，
probably because of a pancreatic A-cell deficiency. However, he is able to respond
normally to injected glucagon and so glucagon-replacement therapy (analogous to
insulin treatment of diabetic pati凹的) might provide a basis for his treatment. Al an's
plasma insulin should be very low because of his hypoglycae口凶. The value actually

Patient Plasma glucagon (p g/ml)

Al an G. 55
Barbara W. 800
Fred C. 240

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 Getting the Measure of Hormones

reported in the case (Table 12.1) is not quite as low as might have been expected but
the reasons for this are not evident.
Barbara W . on the other hand has a massively elevated plasma glucagon
concentration which causes the liver to maintain a hyperglycaemic state. Insulin
levels rise secondarily in an attempt to counteract this hyperglycaemia. Here the likely
cause is a malignant tumour of the pancreatic A cel1s (glucagonoma). This would have
to be treated with chemotherapy , but the prospects for such patients are not good.
The skin lesions seen in Barbara W. , necrolytic migratory erythema , are a
characteristic dermatological feature of hyperglucagonaemia. As a result , this
condition, though relatively rare, is often fìrst diagnosed by dermatologists.
Fred C. is asymptomatic. His plasma glucose and insulin are both normal and he
also responds normally to 月的ed glucagon. Yet the RIA suggests a signifìcantly
elevated plasma glucagon concentration. This conundmm can be explained by
considering how the RIA works. It estimates any material that will cross-react with
the antiglucagon antibody whether or not it has glucagon activity. Besides normal
glucagon, Fred c. 's plasma may contain other proteins that cross-react with the
antibody but are devoid of glucagon activity. Several types of such cross-reacting
material have been described in asymptomatic individuals , including proglucagon
secreted prematurely from the A cel1s. An altemative explanation is that Fred's
glucagon has an inherently low intrinsic activity and so is synthesised in larger
quantities to compensate. Fred is partic叫arly illustrative of the dangers inherent in
relying on a single 'normal' or asymptomatic control with which to compare patient
data.
In an average clinical biochemistry laboratory , the bulk of RIAs are for assay of
TSH , human chorionic gonadotropin and luteinising hormone. The assay of 200
samples per week for TSH is typical whereas only one insulin assay per month might
be required. Glucagon assays are performed extremely rarely.

1. To what extent would the symptoms of glucose 6-phosphatase defìciency (von

Gierke's glycogen storage disorder) and insulinoma resemble those of Al an G.? Devise
a protocol for their differential diagnosis

2. Apart from liver glycogen , what other reserves can the body draw on to maintain the
blood glucose concentration? Wh at mechanisms are involved?

3. Glucagon action on hepatocytes does not involve its entry into the cel1. Predict the
molecular mechanism whereby the hormonal signal is transduced across the plasma
membrane. How is the hormonal specifìcity confe叮ed?

4. What other hormones are produced by the endocrine pancreas? What else does the
pancreas produce and how does this explain why early attempts to purifY insulin were
unsuccessful before Banting and Best showed the way in 1921?

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Getting the Measure of Hormones

5. What are the advantages and disadvantages of immunological methods in medical

diagnosis? Can you distinguish between RIA and E LlSA methods?
6. What are the biochernical and physiological consequences of sustained hyperglycae-
rnia?

7. Apart 企om nervous tissues, what other tissues or cells have an absolute requirement
for glucose?
8. e
One of the ways in which proteins may be radiolabelled is with 25 I]iodide. Which
residues in the protein would be labelled? What parallel does this have in normal
physiology?

Connections • Use this problem also to review the immune response and the structure of
antibody molecules. Distinguish between 'polyclonal' and ‘monoclonal' antibodies
and describe how they are produced.
• Use this problem to consider the use of radioisotopes in biochernistry and
medicine. Think what altemative methods there are to the use of radioisotopes in
assay procedures and what their relative advantages are.
. This problem should lead you to consider how the blood glucose level is
controlled. Remember the central role of the liver in energy metabolism ,
especially glycogenolysis and gluconeogenesis, and the antagonistic effects of
insulin (generally anabolic) and glucagon , adrenali肘， growth hormone and
corticosteroids (catabolic). (See also Problems 2 and 17.)
. Outline the synthesis and secretion of the pancreatic peptide hormones. Y ou
should know something of the proteolytic processing of prohormones, the role of
signal peptides , rough endoplasrnic reticulum and the Golgi apparatus. Remember
the anatomy of the pancreas and both its endocrine and exocrine functions. (See
also Problems 8, 9, 16 and 18.)
• Recall what metabolic fuels are used by the untreated diabetic individual and what
the consequences are. Compare the metabolism of a diabetic with that of someone
who is starving. Y ou should understand what ketoacidosis is , how it arises , its
consequences and how they would be treated. (See also Problems 1, 2 and 10.)
• The action of peptide hormones such as glucagon requires transrnission of a signal
across the plasma membrane. Review the various components involved in this
signalling process (receptors , G protei郎， second messengers , protein kinases and
phosphatases) . (See also Problem 15.)
• Ensure that you understand the statistical terms ‘mean' ,‘ standard deviation' and
‘ no口nal distribution' and how statistical methods can be used to compare sets of
data (for instance patients versus controls).

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 Getting the Measure of Hormones

References Edwards R (1985) Immu仰的5句 : An Introduction. Heinemann , London.

Gosling J P (1990) A decade of development in immunoassay methodology. Clinical
Chemistry 36, 1408-27.
Gow 1 F and Williams B C (1989) Immunoassays for antigens. Current Opinion in
Immunology 1, 940-7.
Kemeny D M (1991)λ Practical Guide to ELISA , pp 1-115. Pergamon Press , Oxford.
Montague W (1983) Glucagon , Chapter 5 , pp 86-102. In Dia仰的 and the Endocrine
Pancreas: A Biochemical Approach. Croom Helm , London and Canberra
Vidnes J and 臼yasæter S (1977) Glucagon deficiency causing severe neonatal
hypoglycaemia in a patient with normal insulin secretion. Pediatric Research 11 ,
943-9. Original report.

Many of the ‘ standard' biochemistry and clinical chemistry textbooks have short
sections on the principles and applications of immunological methods.