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CLIN. CHEM. 36/3, 415-420 (1990)
Growth-Hormone-Releasing
Mary Lee
Hormone
Vance
Growth-hormone-releasing
hormone (GHRH, somatoliberin)
is the hypothalamic peptide hormone that specifically stimulates synthesis and release of growth hormone (GH, somatotropin) by somatotrope cells of the anterior pituitary gland.
GHRH is the last of the classically postulated hypothalamic
hormones to be characterized,synthesized,and used in
clinical medicine. In this review of GHRH, I discuss the
discovery and characterization of the peptide, its role in the
regulation of GH secretion, and its clinical use in pathological
states of GH excess and GH deficiency. The two most
clinically useful aspects of GHRH are to establish the etiology
of GH deficiency, most commonly the result of a hypothalamic GHRH deficiency, and to treat GH-deficient children.
Use of GHRH as therapy for GH deficiency currently is
experimental
and, to date, results encourage the idea of a
therapeuticrolefor this peptidein promotingendogenous GH
secretionwith resultingaccelerationof lineargrowth.
AddItIonal
Keyphrases:
hormone deficiency
somatoliberin . somatotropin . growthregulation of growth hormone secretion
Growth-hormone-releasing
hormone
(GHRH,
somatolibenn)
is the last of the classical hypothalamic
hormones
to be identified
and characterized.1
The existence
of such
substances
was postulated by Green and Harris in 1947 (1).
The other known
hypothalamic
regulatory
hormones in.
dude
several
peptides-thyrotropin-releasing
hormone
(thyroliberin),
gonadotropin-releasing
hormone (GnRH, gonadoliberin),
corticotropin-releasing
hormone
(corticoliberin),
and somatostatin
(SRIF)-and
the, monoamine,
dopamine.
Except for somatostatin
and dopamine,
these
compounds
specifically
stimulate
the release
of specific
pituitary
hormones.
The characterization
and synthesis
of
these hypothalamic
hormones
and of their agonists
and
antagonists
have greatly aided the study of both normal
and abnormal
neuroendocrine
physiology,
resulting
in the
detection and treatment
of several hypothalamic-pituitary
disorders. This discussion
is directed to GHRH, its discovery, biological properties,
and clinical usefulness.
Growth hormone
(GH, somatotropin)
secretion
by the
anterior pituitary
gland is regulated
by two hypothalamic
hormones: GHRH, which stimulates
its release, and somatostatin,
which inhibits
its release.
GHRH is secreted
episodically
in bursts that can be detected
by frequent
measurement
of GH concentrations
in normal subjects and
by measurement
of GHRH concentrations
in patients
with
Department of Internal Medicine, University of Virginia Health
Sciences Center, Box 511, Charlottesville,
VA 22908.
1 Nonstandard
abbreviations:
GH, growth hormone; GHRH,
growth-hormone-releasing
hormone; and GnRH, gonadotropinreleasing hormone.
Received October 13, 1989; accepted November 21, 1989.
ectopic
tumors.
GHRH-producing
as often as every 20
determine
total
mm or
secretion
When blood is sampled
every 5 mm during 24 h, one can
and characterize
secretory
pro-
ifies. Studies of normal men and women have demonstrated
that most GH secretion occurs in bursts at night (2). The
burst of GH probably is a result of the concomitant
secretion of GHRH and withdrawal
of somatostatin
release by
the hypothalamus
(3). The interaction
of these hormones
and other modulators
of GH release is being studied by
numerous
groups.
HistoricalPerspective
GHRH was isolated, sequenced, and synthesized in 1982
by two independent groups, one led by Vale (4), the other by
Guillemin
(5). The method
of discovery
of GHRH was
unique in that all work was carried out with human
pancreatic tumors that secreted GHRH and caused acromegaly. In contrast, the other hypothalamic
hormones had
been characterized
by using thousands
of hypothalami
from animals. The GHRH peptide in the tumor used by the
Vale group was found to consist of 40 amino acids (4); the
tumor used by the Guillemin group contained three peptides with 37, 40, and 44 amino acid residues (5). The 1-37
and 1-40 amino acids of the GHRH isolated by Vale and
colleagues were homologous with the first 37 and 40 amino
acids of the 1-44 GHRH isolated by Guillemm and colleagues. Additionally, 64% of the GHRH in the tumor used
by the Guillemin group was 1-40, 12% was 1-37, and 22%
was 1-44, which makes it unlikely that the 1-40 form is a
degradation
product of the 1-44 form (6). In vivo studies
have
demonstrated
that
the
37-, 40-,
and 44.amino-acid
peptides are equipotent
in stimulating
GH release (7).
However, the full biological activity of GHRH peptides
isolated from both tumors resides in the 29 residues ending
at the amino terminus
(4, 5). The stimulatory
effect of
GHRH on GH release occurs via stimulation
of cyclic AMP
production (8, 9).
Isolation of messenger RNA from both tumors led to the
development of cDNA probes (10, 11). Subsequently,
Mayo
et al. (12) sequenced the human GHRH gene, located on
chromosome 20, and determined that it spans 10 kilobases
and contains five exons.
The sequence of GHRH from other animals has also been
determined.
Rat hypothalamic
GHRH consists of 43 amino
acids and has a free carboxyl terminus (13). This peptide
differs from the human 1-44 in 15 amino acid substitutions
or deletions. Porcine (14) and bovine (15) hypothalamic
GHRH are 44 amino acids long and are amidated at the
carboxyl
terminus.
Although GHRH was originally isolated from an extrahypothalamic
site, subsequent
studies demonstrated
its
presence in the hypothalamic
nuclei of human and primate
brains. GI{RH-containing
neurons have been identified by
immunostaining
in the infundibular
and ventromedial
CLINICAL
CHEMISTRY,
Vol. 36, No. 3, 1990
415
nuclei (16-18). GHRH
detected in the human
immunoreactivity
also has been
pancreas,
placenta, brain (other
than hypothalamus),
and gut (19-21). The presence
of
GHRH in the gut may be of clinical importance
because
radioimmunoassayable
GHRH is present in the serum of
patients with ectopic GHRH secretion, in normal subjects,
and in GH-deficient
patients who are presumably
deficient
in hypothalamic
GHRH (22-25). Thus, detection of a 100to 1000-fold increase
in GHRH concentrations
(i.e., microgram per liter concentrations
vs the normal nanograms per
liter) in a patient with acromegaly is useful to determine
whether the patient has an ectopic GHRH-secreting
tumor.
However, measurable GHRH in normal subjects and in
GH-deficient patients probably reflects gastrointestinal
secretion rather than hypothalamic
secretion. GHRH concentrations in the plasma of normal subjects may range from
<10 to 300 ng/L, depending upon the specific radioimmunoassay and antibody; commercial GHRH assays are not
yet available. Additionally,
GHRH has been detected by
immunocytochemical
staining in several types of neuroendocrine tumors, including pancreatic,
bronchial carcinoid,
gut carcinoid, thymic carcinoid, medullary
carcinoma
of
the thyroid, pheochromocytoma,
and small-cell carcinoma
of the lung (26). However, only two of the 24 patients
studied had clinical features
of acromegaly,
suggesting
that the peptide was not biologically
active. Similarly,
iminunoreactive
GHRH has been detected in pheochromocytomas, ganglioneuroblastomas,
medullary carcinoma of
the thyroid,
insulinomas,
gastrinomas,
glucagonomas,
small-cell carcinomas,
and vasoactive
intestinal
peptidesecreting tumors (19). Although several types of neuroendocrine tumors may synthesize GHRH, the associated clinical syndrome of acromegaly is rare.
40
A
30
20
0600
000
200
400
600
TuIE
(HOURS)
Fig. 1. Serum OH, ng/mL (y.axis), before and after (A) placebo and
( GHRH, 1 pg/kg, in six normal men
Note variability in the degree of OH responsiveness
printed, with permission, from ref. 27
among subiects. Re-
20
n’6
Growth
Hormone 10
GHRH Effects on GH Secretion
Initial studies
with
its ability to stimulate
ng/mL.
GHRH were
designed
to determine
GH release, its specificity, its clinical effects and safety, the likeithood of a dose-response
relationship, and its indirect effects on the generation
of
insulin-like growth factor I (somatomedin C). GHRH, as
GHRH-40, was first administered
to healthy male volunteers in December 1982 (27). Six young men were given
GHRH-40
(1 zg per kilogram body weight) intravenously,
and the concentrations
of GH and other pituitary hormones
were measured and compared with the concentrations
after
placebo
administration.
subjects
GHRH,
had small spontaneous
serum.
5
hpGRF-40
day (placebo), two
GH peaks; after receiving
On the control
all had increased concentrations
of GH in their
This increase
varied
among
the subjects, which
probably reflects variable
release
of endogenous
hypothato diminish
GH secretion (Figure 1)
(27). GHRH was specific in stimulating
GH release; there
was no effect on other anterior pituitary hormones Eprolactin, thyrotropin,
or luteinizing
hormone (lutropin)]
or on
plasma
cortisol [a reflection
of adrenocorticotropin
hormone (corticotropin)
secretion], or on eight enteropancreatic hormones.
A full dose-response
study was carried out
by administering
eight single intravenous
bolus doses of
lamic
somatostatin
GHRH to normal men (28, 29). Doses of 0.1 to 10 igfkg
significantly
stimulated
GH release: the lower doses of 0.1,
0.3, and 1.0 pg/kg caused a monophasic
response;
the
higher doses of 3.3 and 10 pg/kg resulted in more prolonged
stimulation
of GH release and a second “peak,” indicative
of a biphasic
response
(Figure 2). Serum insulin-like
growth factor I concentrations
also increased. The only
416
CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990
Time
Fig. 2. Mean
increasein serum OH after placebo (vehicle) and five
doses of GHRH in normal
men
Note that the highest doses of 3.3 and 10 pg/kg resulted in a biphasic
response. Repnnted, with permission, from ref. 28
notable side effect was transient
(<5 mm) flushing when
the highest dose (10 tug/kg) was administered;
there was no
evidence of toxicity (28). Similar results were obtained by
Rosenthal et al. (30), who used the 44-amino-acid
peptide.
In addition, GHRH given intravenously every 3 h for 108 h
to GH-deficient
adults stimulated
GH release; this suggested that the GH deficiency was a result of a defect in
hypothalamic
abnormality
GHRH
(31).
secretion
and
not
a somatotroph
Pharmacokinetic
studies included determination
of the
serum half-life (t112) and metabolic
clearance
rate of GHRH
after a single intravenous injection and after a continuous
GHRH infusion. Immunoreactive
GHRH was measured by
a radioimmunoassay
developed
by Frohman et al. (32). In
their study, single intravenous injections were used, three
different
doses of GHEH being administered
on separate
occasions to normal volunteers, and GHRH concentrations
were measured over the subsequent
180 min. Bi-exponential analysis revealed that the rate of disappearance from
plasma was divided into two linear phases: an initial
equilibration
phase with t112 of 7.6 (SE 1.2) mm and an
elimination phase with t1,2 of 51.8 (SE 5.4) min. The latter
was similar to the disappearance
rate of 41.3 (SE 3.0) min
observed after cessation
of the constant
infusion.
The
calculated
metabolic
clearance
rate (L per m2 of body
surface per day) was 194 (SE 17.5) in the intravenous
single bolus study and 202 (SE 16) in the constant-infusion
study; these results were not significantly
different from
one another (32). Subsequent
“high-performance”
liquid
chromatography
studies by Frohman
et al. (33) demonstrated that the t112 was 6.8 mm. They determined
that
rapid degradation
and modification of the amino terminus
results in cleavage of the first two amino acids, with the
remaining
fragment
being immunologically
active. This
accounts for the findings in their previous study (32).
Because GHRH is a potential therapeutic agent, studies
have been undertaken
to determine its effectiveness
when
administered
subcutaneously
or mntranasally. Normal male
volunteers
were given different subcutaneous
and intranasal doses of GHRH-40, and the results were compared
with the response to intravenous
GHRH. Both subcutaneous and intranasal
administration
of GHRH stimulated
GH release, but a 30-fold higher subcutaneous
dose and a
300-fold higher intranasal
dose were required to stimulate
an amount
of GH release comparable
with that after
intravenous
administration
of GHRH (29). The concentrations of immunoreactive
GHRH in plasma were 60- and
500-fold higher after intravenous
administration
than after subcutaneous
and intranasal
administration,
respectively (29). Thus, although the subcutaneous
and intranasal routes of GHRII administration
stimulate
GH release, much larger doses of peptide are required. Because
synthesis of GHRH-40 is time-consuming
and expensive,
it
is not currently being administered
therapeutically
by the
intranasal
route. The 44-amino-acid
form is similarly difficult to synthesize.
As noted above, the active portion of the GHR}I molecule
resides in the first 29 amino
acid residues. Thus, synthesis
of shorter analogs is easier and less expensive. Two 29amino-acid
residue GHRH analogs have been administered
to humans, and both preparations stimulate GH release. A
full dose-response
study of [N1e27]GHRH(1-29)-NH2
was
performed
by administering
the analog intravenously, subcutaneously, and intranasally
to normal men. This analog
has one amino acid substitution at position 27 and stimulates GH release in a dose-dependent fashion. Similar to
the studies with GRRH-40, a 10-fold higher subcutaneous
dose than intravenous
dose was required to stimulate
a
comparable amount of GH secretion, and a 30-fold higher
intranasal
dose was required to stimulate
approximately
one-fifth
the amount
of GH release. This analog was not
longer acting than native GHRH (34), and, although it
effectively stimulated
GH secretion,
it is not currently
being used for therapy.
Another important
physiological
question addressed by
the early GHRH studies in normal men concerned the
similarity
of the somatotroph
to the gonadotroph
in its
responsiveness
to prolonged
stimulation
by its hypothalamic-releasing
hormone. When GnRH is administered
intermittently,
luteinizing
hormone
and
folliclestimulating
hormone
(follitropin)
are released;
when
GnRH is administered
continuously,
however, secretion of
luteinizing
hormone and follicle-stimulating
hormone diminish and cease, suggesting
a “down regulation”
of the
gonadotroph receptor. This observation is the basis for the
treatment
of precocious puberty or carcinoma of the prostate with long-acting GnRH analogs or agonists, because
they provide a reversible “medical gonadectomy” (35-37).
Thus, before a suitable treatment
regimen could be designed for GH-deficient children, it was necessary to determine whether the somatotroph
was responsive
to both
intermittent
and continuous stimulation
by GHRH. Normal men were given continuous intravenous
infusions of
GHRH for 6 or 24 h, and their response to a supramaximal
bolus injection at the end of the infusions was measured.
GH secretion was preserved during continuous infusion of
GHRH, as was the response to the bolus injection of GHRH
(Figure 3). These findings suggest that the somatotroph
can
respond to prolonged stimulation and that treatment
with
a long-acting GHRH analog may be feasible (38,39). These
findings were confirmed by administering
a continuous
GHRH infusion to normal men for 14 days. On the 14th
infusion day, GH secretion had increased to double the
amount measured on the pretreatment day (40).
GHRH as a Diagnostic Agent
In an attempt to define better the abnormality, hypothalamic peptides are frequently administered
to patients with
suspected hypothalamic-pituitary
dysfunction.
Administration of thyrotropin-releasing
hormone with subsequent
measurement of the thyrotropin response, probably the
most widely used of these tests, is used to diagnose subclinical hypothyroidism or hyperthyroidism.
Repeated administration of GnRH usually is necessary
to determine
whether functional gonadotrophs are present. Similarly,
GHRH has been administered
to GH-deficient patients to
Vehicle or hpGRF-40
hpGRF-40
3.3,ig/kg
#{149}
.V4jcl6
40
IV
#{149}.
6eGRF-40
‘6
35
30
I
2ng/kg/min
45
-
25
20
15
tO
5
SI
800
1100
400
700
2000
2300
0200
0500
0800
II0d’
Time
Fig. 3. Mean serum GH, ng/mL (y-axis), during a 24-h placebo
(vehicle) and GHRH-40 (hpGRF-40) infusion in normal men
During continuous GHRH stimulationOH secretion is preserved, and there is
augmentation of naturally occurring OH pulses.Reprinted, with permission,
from ref. 38
CLINICAL CHEMISTRY, Vol. 36, No. 3, 1990
417
determine if functional
somatotrophs
are present. Although a single intravenous dose of GHRH may stimulate
GH release in some patients with idiopathic GH deficiency,
no longer available in the United States. Fortunately,
repetitive intermittent
administration may be necessary to
“prime” the somatotroph
before significant GH stimulation
occurs (31).
Children
with short stature
of various etiologies have
been given GHR}I. Children with short stature secondary
to intra-uterine
growth retardation or with familial short
stature had a normal GH response, whereas the majority of
those with organic hypopituitarism or idiopathic GH deficiency had a smaller GH response than did children in the
other groups; nonetheless,
an increase in GH indicated the
presence of functional somatotrophs (41). Similarly, four
An alternative
caveat is that the
degree of GH responsiveness varies in normal subjects and
in GH-deficient patients alike. Thus, although most normal
subjects have an increase in serum GH of>10 pg/L after an
intravenous GHRH dose of 1 pg/kg, a less dramatic increase may not be abnormal (28,30).
Acromegaly, a disease of excess GH secretion, is most
commonly caused by a pituitary
adenoma. Rare cases of
acromegaly
result from GHRH secretion by an ectopic
source, such as a pancreatic tumor or carcinoid tumor that
stimulates
pituitary
GH secretion. The prevalence of ectopic GHRH secretion is low. In one survey of 177 unselected acromegalics (25), none had an increase in plasma
GHRH, but three other patients with known ectopic
GHRH-secreting
tumors all had increased plasma GHRH
concentrations (25). Thus, the prevalence of ectopic GHRH
secretion as the cause of acromegaly is <1%. Measuring
GHRH in plasma before any therapeutic intervention can
identifS’ ectopic GHRH secretion as the etiology of the
acromegaly, and proper attention can be directed to identil5ring the source. Measurement of GHRH in plasma
should be an essential part of the evaluation of a patient
with acromegaly.
Acromegalic patients also have been administered
GHRH to determine
pituitary GH responsiveness.
Gelato
et al. (43) administered
GHRH to 29 acromegalic patients,
and
total treatment
duration
among studies, a substantial
proportion of children showed increased growth velocity
with GH deficiency secondary to hypothalamic
to GHRH (42). These studies
indicate that GH deficiency most commonly results from a
hypothalamic
GHRH deficiency.
Thus, children with suspected GH deficiency may now be evaluated with indirect
of therapy with GHRH. One important
for GH deficiency is adminis-
tions, total daily dose, frequency of administration,
had a GH response
stimuli, such as the classic insulin-induced
hypoglycemia
or oral 1.-dopa and intravenous arginine, and with direct
pituitary stimulation
(i.e., by the GHRH test). A GH
response to GHRH may also be used to assess the feasibility
treatment
tration of GHRH to stimulate GH release. GH-deficient
children have been treated with GHRH-40, GHRH-44, and
a 29-amino-acid GHRH analog, with somewhat inconsistent results (47-52). Despite variations in GHRH prepara-
patients
tumors
the
development of techniques for biosynthesis of GH by recombinant DNA technology has provided an ample supply of
human GH for treatment of these children. Clinical trials
of a methionyl-GH
and pure GH, currently under way,
indicate that these preparations are effective in promoting
linear growth and are not associated with toxicity (45,46).
during therapy with GHRH.
In one study of 24 GH-deficient children treated for six
months or longer with GHRH-40, 21 had acceleration
of
growth. The peptide was administered
either every 3 h for
24 h, every 3 h overnight only, or by twice-daily subcutaneous injections. Among these three subgroups, regression
analysis revealed a correlation between average daily dose
and growth velocity; i.e., those children who received the
higher total daily dose had the most pronounced acceleration of growth (Figure 4) (48). In two studies with GHRH44, five of six and five of seven children had an increase in
growth rate (50, 51). However, the pretreatment response
of serum GH to intravenous GHRH, the concentrations of
insulin-like growth factor I in serum, and the maximal GH
response during subcutaneous
GHBH therapy
were not
predictors of clinical response (51). An amidated 29-aminoacid GHRH analog was administered
to 18 GH-deficient
children for at least six months. Twice-daily subcutaneous
administration resulted in an increase in height in 12, and
eight were classified as “responders”
as judged
by an
increase in height velocity of >2 cm/year (52). The bioavailability of this 29-amino-acid analog is not known. Antibodies to GHRH developed in children receiving
all three
GHRH preparations;
antibodies disappeared
in some, decreased in titer in some, and remained unchanged in others
during continued treatment or after discontinuation
(48,
50,52). In all studies, the presence of anti-GHRH antibodies did not appear to interfere with the increase in growth
velocity. Because there are no parallel studies comparing
the growth-promoting
effects of GHRH vs recombinant
DNA-produced human GH, the possible superiority of one
3
imur,
lice
duih
IImp
injeciio.n
even
3 houn:
,nernight
all but four of whom showed a GH response; the unresponsive patients had received prior pituitary irradiation. Although it is not known whether testing with GHRH is
useful in determining the pathophysiology of acromegaly,
this test may be useful in assessing the effectiveness of
therapies, including pituitary irradiation and surgery.
Studies are in progress to determine whether or not the
absence of a GH response to GHRH after pituitary surgery
is predictive of “cure.”
-
a
l
Therapy with GHRH
The traditional treatment of GH-deficient children is
administration of growth hormone. Because of the development of Creutzfeldt-Jakob
disease in a few young adults
who received GH extracted from virus-contaminated
human pituitary glands (44), these biological preparations are
418 CLINICAL CHEMISTRY, Vol. 36, No. 3,
IL
1990
/
I
/
BEFORE
.i.
1v
r
I
2
6M0
/
4
#{149}
1.2
/
BEFORE
p,
#{149}
6 MO
Fig. 4. Effect of six monthsof GHRH-40
BEFORE
treatment
6 MO
on linear growth
of GH.deflcient children
Note that there were three regimensof GHRH therapy. Reprinted, with
permission, from ref. 48
treatment
over the other is not known. However, in seven
children who were treated with Gil before GHRH treatment, the rates of height increase were similar during both
treatment regimens (52). Comparative studies of these two
treatments are in progress.
Summary
Study of the physiology of GH secretion entered a new
era with the discovery of GHRH. Less than four decades
elapsed between the original hypothesis of the existence of
hypothalamic-releasing
hormones and the characterization
of the last of these hormones. The collaboration of many
investigators
has produced a large volume of information
on the regulation
of GH secretion by GHRH and other
factors
and ultimately
has led to the therapeutic
use of
GHRH in the treatment of GH-deficient children.
I thank Ms. Sandra W. Jackson
and the nurses and staff of the
Research Center for the
excellent care of the patients and study subjects and Ms. Colleen
Lanigan for preparation of the manuscript.
University
of Virginia
General
Clinical
References
1. Green JD, Harris GW.The neuroendocnne link between the
neurohypophysis. J Endocrinol 1947;5:136-46.
2. Ho KY, Evans WS, Blizzard RM, et al. Effects of sex and age on
the 24 hour secretory profile of GH secretion in man: importance of
endogenous estradiol concentrations. J Clin Endocrinol Metab
1987;64:51-8.
OS, Ling N. The interrelationship
of growth
hormone (GH)-releasing factor and somatostatin in generation of
the ultradian rhythm of GH secretion. Endocrinology 1984;115:
3. Tannenbaum
1952-7.
Rivier J, Spiess J, Thorner M, Vale W. Characterization of a
growth hormone releasing factor from a human pancreatic islet
tumor. Nature (London) 1982;300:276-8.
5. Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg
WB. Growth hormone releasing factor from a pancreatic tumor
4.
that caused acromegaly. Science 1982;218:585-7.
6. Thorner MO, Vance ML, Evans WS, et al. Physiological and
clinical studies of GRF and GH. Recent Prog Horm Res
1986;42:589-640.
WB, Ling N, Brazeau P, et a!. Somatocrinin,
growth hormone releasing factor, stimulates secretion of growth
hormone in anesthetized rats. Biochem Biophys Res Commun
7. Wehrenberg
1982;109:382-7.
8. Cronin MJ, Rogol AD, MacLeod RM, et al. Biological activity of
a growth hormone-releasing factor secreted by a human tumor.
Am J Physiol 1983;244:(Endocrinol Metab)E346-3.
9. Belezikjian LM, Vale WW. Stimulation of adenosine 3’,5’.
monophosphate production by growth hormone-releasing factor
and its inhibition by somatostatun in anterior pituitary cells in
vitro. Endocrinology
1983;113:1726-31.
10. Gubler U, Monahan JJ, Lomedico PT, et al. Cloning and
sequence analysis of cDNA for the precursor of human growth
hormone-releasing factor, somatocrunin. Proc Natl Acad Sci USA
1983;80:4311-4.
11. Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM. Characterization of cDNA and genomic clones encoding the precursor to rat
hypothalamic growth hormone-releasing factor. Nature (London)
1985;314:464-7.
12. Mayo KE,
Vale W, Rivier J, Rosenfeld MG, Evans RM. Gene
encoding human growth hormone-releasing factor precursor:
structure, sequence, and chromosomal assignment. Proc Natl Acad
Sci USA 1985;82:63-7.
13. Spiess J, Rivier J, Vale W. Characterization
of rat hypothalamic growth hormone-releasing
factor. Nature (London) 1983;
303:532-5.
14. Bohlen P. Brazeau P, Bloch B, Ling N, Gaillard R, Guillemin
R. Human hypothalamic growth hormone releasing factor (GRF):
evidence for two forms identical to tumor derived GRF-44-NH2
and GRF-40. Biochem Biophys Res Commun 1983;114:930-6.
15. Esch FS, Bohlen P, Ling N, et a!. Characterization of a 40
residue peptide from a human pancreatic tumor with growth
hormone releasing activity. Biochem Biophys Res Commun 1982;
109:152-8.
16. Bloch B, Brazeau P, Bloom F, et al. Immunohistochemical
detection of growth hormone-releasing factor in brain [Letter].
Nature (London) 1983;301:607-8.
17. Bloch B, Gaillard RC, Brazeau P, Ling HD, Ling N. Topo.
graphical and ontogenetic study of the neurons producing growth
hormone-releasing factor in human hypothalamus. Regul Pept
1984;8:21-.31.
18. Bresson JL, Clavequin MC, Fellman D, Bugnon C. Ontogeny
of the neuroglandular system revealed with HPGRF-44 antibodies
in human hypothalamus. Neuroendocrinology 1984;39:68-73.
19. Christ.ofides ND, Stephanou A, Suzuki H, Yiangou Y, Bloom
SR. Distribution of immunoreactive growth hormone-releasing
hormone in the human brain and intestine and its production by
tumors. J Cliii Endocrinol Metab 1984;59:747-51.
20. Shibasaki T, Kiyosawa Y, Masuda A, et al. Distribution of
growth hormone-releasing
hormone-like immunoreactivity
in human tissue extracts. J Clin Endocrinol Metab 1984;59:263-8.
21. Tanako K, Honda N, Shizume K, Hizuka N, Ling NC. Plasma
growth hormone (GH) responses to single and repetitive subcutaneous administration
of GH releasing factor (hpGRF1-44) in normal and GH deficient children. Acts Endocrunol 1985;108:11-9.
22. Saito H, Saito 5, Yamazaki R, Hosoi E. Clinical value of
radioimmunoassay of plasma growth-hormone-releasing
factor.
Lancet 1984;i:401-2.
23. Sopwith AM, Penny ES, Besser GM, Roes LH. Stimulation by
food of peripheral plasma immunoreactive growth hormone releasing factor. Cliii Endocrunol 1985;22:337-40.
24. Sopwith AM, Penny ES, Grossman A, Savage MO, Besser GM,
Rees LH. Normal circulating
immunoreactive
growth hormone
releasing factor (hGRF) concentrations in patients with functional
hypothalamic hGRF deficiency. Clin Endocrinol 1986;24:395-400.
25. Thorner MO, Frohman LA, Leong DA, et al. Extra-hypothalamic growth-hormone-releasing factor (GRF) secretion is a rare
cause of acromegaly; plasma GRF levels in 177 acromegalic
patients. J Clin Endocrinol Metab 1984;59:846-.9.
26. Asa SL, Kovacs K, Thorner MO, Leong DA, Rivier J, Vale W.
Immunohistological
localization of growth-hormone releasing hormone in human tumors. J Cliii Endocrunol Metab 1985;60:423-7.
27. Thorner MO, Rivier J, Spiess J, et a!. Human pancreatic
growth-hormone-releasing
factor selectively stimulates
growthhormone secretion in man. Lancet 1983;i:24-8.
28. Vance ML, Borges JLC, Kaiser DL, et a!. Human pancreatic
tumor growth hormone-releasing
factor dose-response relationships in normal man. J Clin Endocrino! Metab 1984;58:838-44.
29. Evans WS, Vance ML, Kaiser DL, eta!. Effects of intravenous,
subcutaneous, and intranasal administration of growth hormone
(GH)-releasing hormone-40 on serum GH concentrations in normal men. J Clin Endocrinol Metab 1985;61:846-50.
30. Rosenthal SM, Schriock EA, Kaplan SL, Guillemin R, Grumbach MM. Synthetic human pancreas growth hormone-releasing
factor (hpGRFl-44-NH2) stimulates growth hormone secretion in
normal men. J Clin Endocrmol Metab 1983;57:667-9.
31. Borges JLC, Blizzard RM, Evans WS, et a!. Stimulation of
growth hormone (OH) and somatomedin C in idiopathic OHdeficient subjects by intermittent pulsatile administration of synthetic human pancreatic tumor GH-releasing factor. J Clin Endocrinol Metab 1984;59:1-6.
32. Frohman LA, Taminet
JL, Webb CB, et a!. Metabolic clearance and plasma disappearance rates of human pancreatic tumor
growth hormone releasing factor in man. J Clin Invest 1984;73:
1304-11.
33. Frobman LA, Downs TR, Williams TC, Heimer EP, Pan YE,
Felix AM. Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically
inactive product cleaved at the NH2 terminus. J Clin Invest
1986;78:906-13.
34. Vance ML, Evans WS, Kaiser DL, eta!. The effect of intravenous, subcutaneous and intranasal GHRH analog, N1e27GHRH29-N2, on growth hormone secretion in normal man: dose response
relationships. Clin Pharmacol Ther 1986;40:627-33.
35. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E.
Hypophysial responses to continuous and intermittent delivery of
CLINICALCHEMISTRY, Vol. 36, No. 3,
1990
419
hypothalamic
gonadotropin-releasing hormone. Science 1978;202:
631-3.
36. Mansfield MJ, Beardsworth
treatment
of central
precocious
DE, Loughlin JS, eta!. Long-term
puberty
with a long-acting
ana-
logue of luteinizing hormone-releasing hormone: effects on somatic
growth and skeletal maturation. N Engl J Med 1983;309:1286-90.
37. Leuprolide Study Group. Leuprolide versus diethylstilbestrol
for metastatic prostate cancer. N Engl J Med 1984;311:1281-6.
38. Vance ML, Kaiser DL, Evans WS,eta!. Pulsatile GH secretion
in normal man during a continuous 24 hour infusion of hpGRF-40:
evidence for intermittent somatostatin secretion. J Clin Invest
1985;75:1584-90.
39. Vance ML, Kaiser DL, Evans WS, eta!. Evidence for a !imited
growth hormone (GH)-releasing
(GHRH)-releasable
quantity of
GH: effects of 6 hour infusions of hpGRF-40 on growth hormone
secretion in normal man. J Clin Endocrinol Metab 1985;60:730.-5.
40. Vance ML, Kaiser DL, Martha Jr PM, et al. Lack of in vivo
somatotroph desensitization or depletion after 14 days of continuous growth hormone (GH)-releasing hormone administration in
normal men and a OH-deficient boy. J Clin Endocrinol Metab
1989;68:22-8.
41. Rogol AD, Blizzard RM, Johanson AJ, eta!. Growth hormone
release in response to human pancreatic tumor growth-hormonereleasing-hormone-40
in children with short stature. J Clin Endocrinol Metab 1984;59:580-6.
42. Grossman A, Lytras N, Savage MO, et a!. Growth hormone
releasing factor comparison of two analogues and demonstration
of hypothalamic defect in growth hormone release after radiotherapy. Br Med J 1984;288:1785-7.
43. Gelato MC, Merriam GR, Vance ML, et al. Effects of growth
hormone-releasing factor upon growth hormone secretion in acro-
420
CLINICAL
CHEMISTRY,
Vol. 36, No. 3, 1990
megaly. J Clin Endocrinol Metab 1985;60:251-7.
44. Brown P, et a!. Potential epidemic of Creutzfeldt-Jakob disease from human growth hormone therapy. N Engl J Med
1985;313:728-31.
45. Hints RL, Rosenfeld
RG, Wilson DM, et
al. Biosynthetic
methionyl human growth hormone is biologically active in adult
man. Lancet 1982;ii:1276-9.
46. Takano K, et a!. Short-term study of biosynthesized hGH in
man. Endocrunol Jpn 1983;20:79-84.
47. Thorner MO, Reschke J, Chitivone J, et a!. Acceleration of
growth in two children treated with human growth-hormone
releasing factor. N Engl J Med 1985;312:4-9.
48. Thorner MO, Rogo! AD, Blizzard RM, et a!. Acceleration of
growth rate in growth hormone-deficient
children treated with
human growth hormone-releasing hormone. Pediatr Rae 1988;
24:145-51.
49. Smith PJ, Brook CG, Rivier J, Vale W, Thorner MO. Nocturnal pulsatile growth hormone releasing hormone treatment in
growth hormone deficiency. Clin Endocrinol 1986;25:35-44.
50. Rochiccioli PE, Tauber MT, Coude FX, et a!. Results of 1-year
growth hormone (GH)-releasing
hormone-(1-44) treatment on
growth, somatomedin-C, and 24 hour GH secretion in six children
with partial OH deficiency. J ChinEndocrinol Metab 1987;65:26874.
51. Low LCK, Wang C, Cheung PT, et a!. Long term pu]satile
growth hormone (GH)-releasing
hormone therapy in children with
GH deficiency. J Clin Endocrunol Metab 1988;66:611-7.
52. Ross RJ, Rodda C, Tsagarakis S, at a!. Treatment of growthhormone deficiency with growth-hormone-releasing
hormone.
Lancet 1987;i:5-8.