Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
Festschrift for Sir John McMichael
53
Standard procedures for assessing hypersecretion or secretory
capacity for human growth hormone, using the radioimmunoassay
RUSSELL FRASER
A. D. WRIGHT
Royal Postgraduate Medical School, London
THERE are no easily measured clinical signs of
growth hormone (GH) activity though it is
closely reflected by growth in children, and
by skin thickness, hair growth and skin secretory
activity in adults. Yet there is need to assess
GH activity, not only for the diagnosis and
management of various pituitary disease, but
also in the study of disease such as diabetes
mellitus and its complications. A radioimmunoassay sufficiently sensitive to measure GH levels
in body fluids (Glick et al., 1963; Hunter &
Greenwood, 1964) has recently offered hopeful
prospects for doing this. But the initial studies
soon revealed the variability of normal GH levels
(Knobil, 1966; Copinschi et al., 1967) and their
rapid fluctuation with various stimuli (Glick et
al., 1965). However, suitable diagnostic procedures for assessing GH activity in relation to
various clinical problems have now evolved and
here we shall appraise the use of some of these
procedures, and define the normal responses.
The radioimmunoassay of human growth
hormone (HGH)
While only one bioassay (Daughaday, Salmon
& Alexander, 1959; Daughaday & Parker, 1963)
is sufficiently sensitive to measure GH activity
in serum, the radioimmunoassay is more sensitive and more specific. In this method radioactive HGH (labelled with 1311 or 125I) and a
known amount of anti-HGH antibody is added
to a series of tubes containing either a standard
amount of unlabelled pure HGH or a diluted
serum sample. Subsequent measurement of the
percentage 131I or 1251 bound to the antibody
is the basis of the assay, for this clearly should
index the amount of unlabelled hormone added.
When an effect is obtained from an unknown
sample, its specificity can be assessed by comparing its dose response effect with that of the
standard HGH preparation. Some substances,
such as human placental lactogen, are partly
identical with HGH (Greenwood, Hunter &
Klopper, 1964), while GH from non-primate
species are non-identical. Further, the radioimmunoassay can be affected non-specifically by
such factors as plasma proteins, or changes of
pH or ionic strength; effects which must be
covered by the standardization of the assay procedure and adequate dilution of serum samples.
Different methods are used to separate the
antibody-bound and free fraction of labelled
HGH. In our Unit we have developed this assay
using the double-antibody method (Hartog
et al., 1964b), using anti-y-globulin antiserum
to precipitate the antibody-bound fraction. With suitable precautions (Welborn &
Fraser, 1966), this method has certain advantages
over the original chromatoelectrophoresis separation of Glick et al. (1963). It is particularly
important to ensure that complete precipitation
will occur under all the conditions possible in
the assay. 131I-labelled -y-globulin in trace
amounts provides a useful check for this precipitation, and the optimal concentrations of
anti-y-globulin antiserum can be determined for
the chosen concentration of y-globulin. Recent
developments are evolving simpler separation
methods, still needing careful appraisal of their
sensitivity and specificity-such as dextran-coated
charcoal (Lau, Gottlieb & Herbert, 1966) and
silica powders (Rosselin et al., 1966) and solid
synthetic protein-binding discs (Catt, Niall &
Tregear, 1966). A good clinical assay should be
adaptable to semi- or complete automation which
is true of the double-antibody procedure.
An immunologically pure, radioactively labelled
hormone is essential for the assay. The hormone
becomes damaged to some extent during the
labelling, during subsequent storage and during
the 4-7 days incubation for the assay. Fractionation of the prepared 1311-HGH by gel filtration as described by Touber & Maingay (1963)
is a simple purification step which we use
routinely. A fraction, up to 40% of which will
bind to protein in the absence of anti-HGH
Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
54
Postgraduate Medical Journal
antiserum, can be separated from the immunologically pure hormone (Fig. 1). Incubation
damage of the labelled hormone can be avoided
by limiting the specific radioactivity to 200 uCi/
,tg HGH and also by assaying the serum diluted
to at least 1: 5 in the final incubate.
W
steroids (Hartog et al., 1964a) and some drugs
acting on the central nervous system (Muller et al.,
1967), the response to insulin hypoglycaemia is
suppressed. It is therefore important to standardize the conditions under which samples are
collected.
Standard clinical tests
The HGH level in routine fasting serum
samples varies considerably unless the status at
collection is basal (Copinschi et al., 1967). Basal
samples should only be taken after ensuring rest
and calm for +-1 hr. Since, however, a basal
status cannot be ensured, a standard suppression
test, such as oral glucose (GTT), is useful. Further truly basal levels are often too low to be
measured in normals and a standard stimulation
such as intravenous insulin (ITT) is also important for suspected loss of secretory capacity.
0
0
0
4-
4
8
Sample No.
12-->23/6/65
[5ml sampl
FIG. 1. Fractionation of 1311-HGH on Sephadex G-200
(15 x 1 *2 cm).
The results of the assays in this paper are
expressed in terms of the M.R.C. reference standard 'A', kindly supplied by Dr D. R. Bangham.
The precision of our assay has been assessed
by reviewing the results of the standard sera
assayed each day. For samples above 10 ng/ml,
the coefficient of variation of the mean value
has ranged between 14 and 17% ; for those with
lower values this has varied from 23 to 25%. In
normal urine without concentration HGH is
barely demonstrable as have found Girard &
Greenwood (1967) and Roth et al. (1967), contrary to the reports of Geller & Loh (1963)
based on the less specific haemagglutination
separation of bound and free. A suitable urinary
concentration procedure has yet to be evolved.
(1) GH levels at 1 and 2 hr after oral glucose: a
test for acromegaly
The serum GH levels observed in nineteen
normal adults taken while at rest in bed following an oral glucose load of 40 g glucose per m2
body surface area are shown in Fig. 2. Clearly
the lowest values are at 1-2 hr; the secondary
postprandial rise may sometimes appear at 21 hr
but not before. Evidently, if the subject has
been at rest during the test, though the fasting
Ilustrative cases
Mean and 2 hr values
Acromegaly
subjects
Normal
01
0lo
08
E
88
Stimuli which can increase or suppress GH
secretion
In normal subjects serum levels of GH increase following insulin-induced hypoglycaemia
or 2-desoxyglucose, intravenous tolbutamide,
stress, surgical operations, exercise, pyrogen, prolonged fasting and intravenous arginine or other
amino acids. Conversely serum levels are suppressed to a minimum for the first 2 hr following
a meal or a glucose load. Also, oestrogens
potentiate the GH response to exercise (Frantz
& Rabkin, 1965) and to arginine (Merimee, Burgess & Rabinowitz, 1966) while after cortico-
a
I/c_
*8
nr
I
I
0_
2
Hours after oral glucose
FIG. 2. Serum HGH levels during oral GTTs in normal
subjects (nineteen) and acromegaly (ninety). 0, Normal
subjects; 0, acromegalic patients.
Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
Festschrift for Sir John McMichael
level may be variable, the mean 1-2-hr levels
should rarely be as high as 5 and never exceed
10 ng/ml serum. Fig. 2 also shows our findings during this test in ninety acromegalic
patients. Firstly the GH levels usually did not
alter significantly during the oral GTT and
secondly all but one of the mean 1-2-hr levels
exceeded 10 ng/ml serum. These levels varied
widely within this group of acromegalics, twenty
"I
of whom had been given external irradiation to
the pituitary; but the level did not appear to
correlate with the severity or duration of the
clinical syndrome, or with the age of the patient.
These levels offer the best measure for assessing
any treatment. After pituitary implantation with
90yttrium, serum levels are usually restored towards normal (Fig. 3).
Treated
(Before ond 3monthssofter-with clin. remission)
Satisfactory
400
ry
70
W'
0~~~~~~~~~~~~~~
~~I
I
LI:
L
r~~~~L~
10
2
0
Hours (after i.v. insulin 0C1 u/kg)
FIG. 4. Serum HGH during intravenous insulin tolerance test for GH reserve; in twelve normal subjects.
-, Mean. (Insulin 0-1 u/kg causing hypoglycaemia under
50 mg/100 ml in each subject.)
CIQ
0
50
0
0'
0
0'
E
~~~~~0
~~~~~~
c
o
serum (Fig. 4). The peak GH levels were closely
reproducible in one normal subject (DW) on
six separate tests (mean 67 ng/ml, range 53-88).
NiI
Partial
_200-
E
55
Upper
onormal
jimit
3-
.01
(6)
(12)
(10) (15)
No. of subjects
(2)
(3)
FIG. 3. Serum HGH before and after 90Y treatment of
acromegaly: mean 1- and 2-hr values of oral GTT, in
groupings by clinical response. * Mean values at 60-120
min of oral GTT.
(2) GH levels during hypoglycaemia of an ITT:
a test for GH secretory capacity
The GH response to the hypoglycaemia induced by insulin is a useful test for abnormalities
of the hypothalamo-pituitary axis. There is often
a slight initial fall in serum GH, but then a
sharp rise to a peak at 60 or 90 min, whose
value is usually 30-70 and always over 20 ng/ml
In this test the maximal GH secretion must
be stimulated, though it is less easy to define
adequate hypoglycaemia for this purpose. A
rapid fall of blood sugar is more effective, which
an intravenous dose achieves while also offering the least hazard of prolonged hypoglycaemia.
In normal subjects (Fig. 4) a standard intravenous ITT (0-1 units soluble insulin/kg body
weight or 3 7 u/M2 body surface area) lowers
at least one of the '-hourly blood sugar values
under 50 mg/100 ml and usually induces symptoms of hypoglycaemia; we have felt that
equivalent hypoglycaemia should be obtained in
this test-i.e. after intravenous administration of
the insulin in a dosage suited to the patient's
sensitivity, at least one blood sugar should fall
below 50 mg/100 ml. In subjects with either
increased or decreased insulin sensitivity, the insulin dosage is varied. Subjects who are hypersensitive to insulin, for example hypopituitary
subjects, are tested with half the standard dose,
or those expected to be insulin resistant, for
example the obese or patients with Cushing's
disease or acromegaly and most diabetics, are
given three times the standard dose.
The adaptation of this test to diabetics (Oakley
et al., 1967) has helped confirm these criteria of
adequate hypoglycaemia for maximum GH stimulation. In a group of twenty-three diabetic
patients with retinopathy the preliminary diabetic
control was carefully adjusted to attain a near
Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
Postgraduate Medical Journal
56
normal fasting blood sugar (that is under 150 if
possible, or at least under 200 mg/ 100 ml), a
'Dextrostix' check of this being done before
giving the intravenous insulin, usually at a
dose of 03 u/kg body weight except when the
blood sugar was already well under 150 mg/
100 ml. Fig. 5(a) shows thirteen such tests on
diabetics whose blood sugar fell to under
50 mg/ 100 ml. It will be seen that all these
showed normal GH peaks, that is over 32 ng/ml;
while in Fig. 5(b) will be seen the test results
in the other ten diabetics tested whose blood
sugar did not fall below 50 mg/ 100 ml, only
four of whom showed normal GH peaks. It will
be noted that nine of these ten had initial fasting blood sugar levels of over 200 mg/100 ml
indicating the probability of inadequate hypoglycaemia within the test period. In several
Blood sugar
Serum HGH
200
Late GH
(a)
.
.
-
'
peaks
32+--i-i'\
-IN~~~~I
0
z
20
-
---
-
-
-
1I00
0
~~~~~~~E
/
10
501
-~ ~
~
-t
0~~~~~~~~~~~~~~~~~~~~~B
0
0
2
0
1
2
Hoursb(afr i..i Normal
:GH peak-,
32+
L---
.--
20
T
200
'~~~
0
-
I
0
afe iv nui---01~~Hur
/g
FIG. 5. Serum HGH during intravenous insulin tolerance test for GH reserve. (a) In
thirteen diabetics, whose blood sugar fell under 50 mg/100 ml. (b) In ten diabetics,
whose blood sugar did not fall under 50 mg/100 ml.
Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
57
Festschrift for Sir John McMichael
x
%
(Associated blood sugar)
I
0
0
c
E
2
0
2
0
Hours
Hours
FInsulin dose 0.3 u/kg pre 1
IO01 u/kg post
L
FIG. 6. Serum HGH during intravenous insulin tolerance test in Cushing's syndrome
before and after 90Y pituitary implantation.
patients considerable percentage falls in blood
(exceeding 50%) occurred without inducing maximal GH secretion, though occasionally
steep falls from high levels did attain this. Others
(Powell et al., 1966) have suggested that a 50%
blood sugar fall is a sufficient stimulus in this
sugar
test.
We have found the test useful in assessing
pituitary ablation performed to arrest diabetic
retinopathy (Joplin et al., 1967); a response to a
level of 5 or more ng/ml serum was found to
indicate an inadequate degree of pituitary ablation. The ITT response may be abnormal in
Cushing's disease (Fig. 6) indicating a subnormal
secretory capacity in this disease, but not necessarily primary pituitary deficiency. Similarly in
very severe obesity we have found that even
with adequate hypoglycaemia from 11-1 u soluble insulin/M2, the GH peak may be subnormal,
though there is usually some response. In malnutrition we find that the levels may be raised
and do not tend to be lowered.
(3) Urinary GH levels: a possible measurement
of mean GH secretion
With the variability of serum levels of GH
found in all subjects except in acromegaly, it is
difficult to assess the daily GH production by
the pituitary. In such conditions as diabetes mellitus this may be an important factor and urinary
measurements might provide a useful estimate
of the mean rate of GH secretion. As yet a
satisfactory concentrating procedure has to be
evolved to enable the levels to be measured in
normal subjects. High levels have, however, been
observed in uraemia, which we have found partly
depend upon the higher clearance found in
uraemia.
Conclusion
Suitable tests are now available to reveal:
(1) GH hypersecretion, and so to index the primary disorder in acromegaly (GH levels at 1-2
hr of an oral GTT), and (2) the GH secretory
capacity and so potentially hypopituitary states
(GH levels during adequate hypoglycaemia during an ITT). Further evolution of the assay procedure is needed to enable measurement of the
usual daily GH secretory level, for which measurement of urinary levels combined with an
assessment of renal function may be most
helpful.
Acknowledgments
We are grateful to Dr R. M. Haslam and her colleagues in
the Department of Chemical Pathology for the blood sugar
measurements. The M.R.C. standard 'A' was kindly supplied
by Dr D. R. Bangham, Division of Biological Standards,
National Institute for Medical Research, London, N.W.7,
and the HGH for iodination by Dr Anne S. Hartree, Department of Biochemistry, University of Cambridge. This work
was supported by an M.R.C. technical grant. A.D.W. is in
receipt of an M.R.C. Junior Research Fellowship.
References
CATT, K., NIALL, H.D. & TREGEAR, G.W. (1966) Solid phase
radioimmunoassay of human growth hormone. Biochem. J.
100, 31c.
COPINSCHI, G., HARTOG, M., EARLL, J.M. & HAVELL, R.J.
(1967) Effect of various blood sampling procedures on
serum levels of immunoreactive human growth hormone.
Metabolism, 16, 402.
Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
58
Postgraduate Medical Journal
DAUGHADAY, W.H. & PARKER, M.L. (1963) Sulfation factor
measurement as an aid in the recognition of pituitary
dwarfism. J. clin. Endocr. 23, 638.
DAUGHADAY, W.H., SALMON, W.D. & ALEXANDER, J. (1959)
Sulfation factor activity of sera from patients with pituitary
disorders. J. clin. Endocr. 19, 743.
FRANTZ, A.G. & RABKIN, M.T. (1965) Effects of oestrogen
and sex differences on secretion of human growth hormone.
J. clin. Endocr. 25, 1470.
GELLER, J. & LOH, A. (1963) Identification and measurement
of growth hormone in extracts of human urine. J. clin.
Endocr. 23, 1107.
GIRARD, J. & GREENWOOD, F.C. (1967) The radioimmunoassay of urine for human growth hormone. J. Endocrin. 37,
xxxiv.
GLICK, S.M., ROTH, J., YALOW, R.S. & BERSON, S.A. (1963)
Immunoassay of human growth hormone in plasma.
Nature (Lond.), 199, 784.
GLICK, S.M., ROTH, J., YALOW, R.S. & BERSON, S.A. (1965)
The regulation of growth hormone secretion. Rec. Prog.
Hormone Res. 21, 241.
GREENWOOD, F.C., HUNTER, W.M. & KLOPPER, A. (1964)
Assay of human growth hormone in pregnancy at parturition and in lactation. Brit. med. J. i, 22.
HARTOG, M., GAAFAR, M.A. & FRASER, T.R. (1964a) Effect
of corticosteroids on serum growth hormone. Lancet, i,
376.
HARTOG, M., GAAFAR, M.A., MEISSER, B. & FRASER, T.R.
(1964b) Immunoassay of serum growth hormone in acromegalic patients. Brit. med. J. ii, 1229.
HUNTER, W.M. & GREENWOOD, F.C. (1964) A radioimmunoelectrophoretic assay for human growth hormone. Biochem.
J. 91, 43.
JOPLIN, G.F., OAKLEY, N.W., HILL, D.W., KOHNER, E.M. &
FRASER, T.R. (1967) Diabetic retinopathy, II. Comparison
of disease remission induced by various degrees of pituitary
ablation by Y90. Diabetologia (In press).
KNOBIL, E. (1966) The pituitary growth hormone: an adventure in physiology. Physiologist, 9, 25.
LAU, K.S., GOTTLIEB, C.W. & HERBERT, V. (1966) Preliminary
report on coated charcoal immunoassay of human
chorionic 'growth hormone-prolactin' and growth hormone. Proc. Soc. exp. Bio. (N. Y.), 123, 126.
MERIMEE, T.J., BURGESS, J.A. & RABINOWITZ, D. (1966) Sexdetermined variation in serum insulin and growth hormone
response to amino acid stimulation. J. clin. Endocr. 26, 79 1.
MULLER, E.E., SAITO, T., ARIMURA, A. & SCHALLY, A.V.
(1967) Hypoglycaemia, stress and growth hormone release:
blockade of growth hormone release by drugs acting on the
central nervous system. Endocrinology, 80, 109.
OAKLEY, N.W., WRIGHT, A.D., FRASER, T.R. & HASLAM,
R.M. (1967) Fasting blood-sugar and serum-growthhormone in hypopituitary diabetics. Lancet, i, 523.
POWELL, E.D.U., FRANTZ, A.G., RABKIN, M.T. & FIELD,
R.A. (1966) Growth hormone in relation to diabetic
retinopathy. New Engl. J. Med. 275, 922.
ROSSELIN, G., ASSAN, R., YALOW, R.S. & BERSON, S.A.
(1966) Separation of antibody-bound and unbound peptide
hormones labelled with iodine-131 by talcum powder and
precipitated silica. Nature (Lond.), 212, 355.
ROTH, J., GLICK, S.M., CUATRECASAS, P. & HOLLANDER, C.S.
(1967) Acromegaly and other disorders of growth hormone
secretion. Ann. intern. Med. 66, 760.
TOUBER, J.L. & MAINGAY, D. (1963) Heterogeneity of human
growth hormone. Its influence on a radioimmunoassay of
the hormone in serum. Lancet, i, 1403.
WELBORN, T.A. & FRASER, T.R. (1965) The double-antibody
immunoassay of insulin. Diabetologia, 1, 211.
Total fasting in the treatment of obesity
I. C. GILLILAND
Royal Postgraduate Medical School, and Prince of Wales'
General Hospital, London
IN AN affluent and labour-saving society energy
intake often exceeds output and consequently
obesity is the most frequently encountered metabolic disturbance. It is known to be associated
with an increased morbidity and mortality rate.
As Harrison & Harden (1966) point out, if this
increased mortality rate returns to normal on
losing weight (Metropolitan Life Assurance Co.,
1962) then measures to lose weight must necessarily be of major therapeutic importance. Current methods of sub-caloric diet weight reduction tend to be very unsatisfactory. Stunkard &
McLaren-Hume (1959) showed that out of 100
patients attending an ordinary weight-reduction
clinic only two had lost weight after 2 years.
Fasting was described by Folin & Denis in
1915 as 'a safe, harmless and effective method
for reducing the weight of those suffering from
obesity', and in the same year Benedict (1915)
reviewed many previous examples of lengthy
fasting as well as giving detailed observations
on his own patient. Bloom (1959) revived this
method of absolute starvation and much interest
has since been concentrated on it. Bloom's initial
periods of fasting were short, but very much
longer periods have since been reported by
Thomson, Runcie & Miller (1966) and Harrison
& Harden (1966). We are here recording our
experience with forty-six patients whose reducing regime started with a standard absolute fast
for 14 days.
Patients
Any patient with sufficient motivation to seek
Downloaded from http://pmj.bmj.com/ on March 4, 2016 - Published by group.bmj.com
Standard procedures for
assessing hypersecretion or
secretory capacity for human
growth hormone, using the
radioimmunoassay.
R. Fraser and A. D. Wright
Postgrad Med J 1968 44: 53-58
doi: 10.1136/pgmj.44.507.53
Updated information and services can be found at:
http://pmj.bmj.com/content/44/507/53.citation
These include:
Email alerting
service
Receive free email alerts when new articles cite this
article. Sign up in the box at the top right corner of
the online article.
Notes
To request permissions go to:
http://group.bmj.com/group/rights-licensing/permissions
To order reprints go to:
http://journals.bmj.com/cgi/reprintform
To subscribe to BMJ go to:
http://group.bmj.com/subscribe/