The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 7 2569-2572
Copyright © 1998 by The Endocrine Society
Preserved Growth Hormone (GH) Secretion in Aged and Very Old Subjects after Testing with the Combined Stimulus GH-Releasing Hormone plus GH-Releasing Hexapeptide-61
Dragan Micic,
Vera Popovic,
Mirjana Doknic,
Djuro Macut,
Carlos Dieguez and
Felipe F. Casanueva
Institute of Endocrinology, University Clinical Centre (D.M., V.P.,
M.D., D.M.), Belgrade Yugoslavia Y-11000; and Department of Physiology
(C.D.) and Medicine (F.F.C.), School of Medicine, Santiago de
Compostela University, Santiago de Compostela, Spain E-15780
Address all correspondence and requests for reprints to: F. F. Casanueva, P.O. Box 563, Santiago de Compostela, Spain E-15780. E-mail:
meffcasa{at}uscmail.usc.es
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Abstract
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Either spontaneous or pharmacological stimulated GH secretion is
reduced with advanced age. This observation is an added difficulty for
the biochemical diagnosis of GH deficiency in adults. Furthermore, the
combined administration of saturating doses of GH-releasing hormone
(GHRH) plus GH-releasing hexapeptide (GHRP)-6 is nowadays the most
effective GH-releasing stimulus tested in a variety of settings related
to altered somatotroph function. To understand whether the GH discharge
elicited by the combined stimulus declines with age, 26 normal subjects
of both sexes, divided into 3 age groups [adults 19–40 yr; aged
46–65 yr; and very old (75–96 yr) subjects] were studied. They were
administered iv, as bolus and in combination, 90 µg GHRH plus 90 µg
GHRP-6.
In the three groups, the combined administration of GHRH plus GHRP-6
elicited a GH area under the curve (µg/L per 120 min) of 3,127
± 262, 3,409 ± 573, and 4,655 ± 737 for adults, aged, and
very old subjects, respectively (nonsignificant differences). The mean
GH peak was 47.5 ± 4.5 µg/L for adults, 52.9 ± 8.4 µg/L
for aged subjects, and 76.0 ± 11.7 for very old subjects
(nonsignificant differences). Individually examined, there were no
nonresponders to the combined stimulus, and all subjects (independently
of age) showed a GH peak over 25 µg/L (the lowest peak was 27.3
µg/L, and the highest peak was 119.2 µg/L).
In conclusion, the GHRH plus GHRP-6-induced GH release is well
preserved in aged and very old subjects, which suggests that the GH
secretory capability of the combined test is not reduced by age. This
combined test may be useful for the diagnosis of GH-deficient states in
adults.
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Introduction
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THE BIOCHEMICAL diagnosis of GH
deficiency in the adult patient is an awkward task (1, 2, 3), and it has
even been questioned whether such a diagnosis is viable (4). Because
the random measure of serum concentrations is worthless, because of the
pulsatile nature of GH secretion, and because 24-h spontaneous GH
secretion gives conflicting results, clinicians must rely on the
pharmacological stimulation of the pituitary somatotroph to establish
the diagnosis of a suspected deficient secretion of GH. Nowadays, the
insulin tolerance test (ITT) may be considered the gold standard test
for assessing GH deficiency in adults (5, 6) being the combination of
arginine-GHRH (7), an excellent alternative when hypoglycemia is
contraindicated (6). However, the side effects of ITT made compulsory
the search for alternative provocative stimulus of GH secretion,
reproducible, without side effects and with cut-off points that do not
overlap between normal and pathological subjects. Furthermore, because,
in aging and in obesity, there is a progressive decline in stimulated
GH secretion (8, 9, 10), and because most suspected GH-deficient adults
present an advanced age and some degree of adiposity, a test that is
nonaffected by age or adiposity would be very useful.
GH-releasing hexapeptide (GHRP)-6, hexarelin, and other nonclassical
GH-releasing peptides (11, 12, 13, 14) seem to be a new and promising tool for
exploring GH secretory mechanisms in patients with suspected GH
deficiency. In fact, GHRP-6, when administered alone, is a potent
stimulus of somatotroph discharge in normal subjects. Moreover, the
combined administration of GHRH plus GHRP-6 elicits a powerful GH
discharge of potentiating characteristics, being probably the most
potent GH stimulus known to date (15). The GH secretion elicited by the
combined stimulus is similar for men and women, or normal children, and
it is not affected by obesity (16, 17, 18).
In the present work, the GH secretion elicited by the administration of
GHRH plus GHRP-6 was assessed among a group of aged and very old normal
subjects and was compared with that elicited in normal adults as
internal controls. The target of this study was to evaluate whether
this combined stimulus would be affected by the age of the tested
subjects.
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Subjects and Methods
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Twenty-six normal healthy volunteers (16 women and 10 men),
53.5 ± 4.5 yr old (range 19–96 yr), participated in this study
after providing informed consent. All of them had normal life styles,
were taking no medication, and were within 10% of their ideal body
weight. The study was approved by the Hospital Bioethical Committee.
Testing subjects had been previously divided, according to age, into 3
groups: 1) adults, n = 10 (6 women, 4 men), 29.0 ± 1.9 yr
old (range 19–40 yr), body mass index (BMI) = 25.3 ± 1.0; 2)
aged subjects, n = 8 (4 women, 4 men), 56.5 ± 2.1 yr old
(range 46–65 yr), BMI = 22.2 ± 0.9; and 3) very old
subjects, n = 8 (5 women, 3 men), 81.3 ± 2.9 yr old (range
75–96 yr), BMI = 21.0 ± 1.5.
Tests started at 0800 h, after an overnight fast, with the
subjects recumbent. An indwelling catheter was placed in a forearm vein
and kept permeable with a slow infusion of 150 mmol/L NaCl. The first
blood sample was obtained at -30 min; the GH stimulus was administered
at 0 min, and additional blood samples were obtained at appropriate
intervals. Women were tested in the follicular phase of the menstrual
cycle. As GH stimulant, it was administered the combined test of an iv
bolus injection of 90 µg GHRH (GRF 1–29 NH2, Geref Serono
Laboratories, Madrid, Spain), immediately followed by an iv bolus
injection of GHRP-6 (His-DTrp-Ala-Trp-DPhe-Lys-NH2; Peninsula
Laboratories, Madrid, Spain), prepared as previously described (see
Ref. 25).
Serum GH concentrations were determined by using a time-resolved
fluoroimmunoassay (Delfia, Wallac Oy, Turku, Finland) with a GH
sensitivity of 0.02 µg/L and coefficients of variation of 6.3% (0.4
µg/L), 5.3% (10.2 µg/L), and 4.2% (43.4 µg/L). Hormone levels
are presented and analyzed as absolute values or as the mean GH peak.
The areas under the secretory curve (AUC) were calculated by a
trapezoidal method and were compared between groups by the Wilcoxon
rank test. The statistical level of significance was set at
P < 0.05.
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Results
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As expected, the combined administration of GHRH, immediately
followed by GHRP-6 (both at saturating doses), induced a clear-cut GH
secretion in the three studied groups of subjects (Fig. 1
), with a quite synchronized pattern of
GH discharge. No differences were observed between men and women;
therefore, values were pooled. The GHRH+GHRP-6-mediated mean GH peak
was 47.5 ± 4.5 µg/L for adults, 52.9 ± 8.4 µg/L for
aged subjects, and 76.0 ± 11.7 for very old subjects (Fig. 2). Compared with the adult group, no
statistically significant differences were observed in either the aged
or the very old group. Analyzed as area under the curve, the values in
µg/L per 120 min were: 3,127 ± 262, 3,409 ± 573, and
4,655 ± 737 for adults, aged, and very old subjects,
respectively, without statistical differences among the groups.
Individually examined (Fig. 2), there were no nonresponders to the
combined stimulus; and all subjects (independently of the age) showed a
GH peak over 25 µg/L (the lowest peak being 27.3 µg/L; and the
highest peak, 119.2 µg/L). The four higher GH peaks, in decreasing
order, were observed in subjects who were 96, 77, 79, and 53 yr old.
There was a positive correlation between the GHRH-GHRP-6-mediated
GH response and age (r = 0.45, P < 0.01);
how-ever, the r value was too low to consider the correlation of
biological relevance.
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Figure 1. Mean ± SE of plasma GH
values after the administration iv of GHRH (90 µg) plus GHRP-6 (90
µg) at 0 min in three groups of healthy subjects with different age
intervals: adults (19–40 yr), aged (46–65 yr), and very old subjects
(75–96 yr).
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Figure 2. A, Mean ± SE of the AUC
(in µg/L per 120 min) and mean GH peak in three different groups of
healthy subjects with different age intervals, after the administration
of GHRH plus GHRP-6 at saturating doses; B, individual peaks after the
same combined stimulus.
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No side effects were reported in any of the tests.
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Discussion
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The signs and symptoms of GH deficiency in adults are similar to
those of normal aging, making the diagnosis of GH deficiency a
cumbersome and uncertain task in adult patients (1). The diagnosis of
GH deficiency must rely on biochemical determinations, and provocative
or pharmacological tests have been the only validated means for such
diagnosis (6). Furthermore, because either spontaneous or stimulated GH
secretion is reduced with the advancing of age, a new degree of
uncertainty is shown in the diagnostic process. In fact, all
provocative stimuli of GH secretion (including hypoglycemia, GHRH and
GHRP-6 or hexarelin) show a lower effectiveness when aging progresses
(19, 20, 21, 22, 23). So, the availability of a provocative test not affected by
aging should be clinically useful.
The new GH secretagogues, among which GHRP-6 is the most widely
studied, may represent a new physiological system that participates in
the regulation of GH secretion in man (11, 12, 13, 14), and these nonclassical
secretagogues have been widely used in the last few years in the
testing of the GH reserve in man (24, 25, 26). Although the mechanism of
action is not fully understood, the combined administration of GHRH
plus GHRP-6 (both at saturating dose) is nowadays considered the most
potent stimulus of GH secretion in man (15), being able to restore the
GH secretion in states associated with chronic blockade of somatotroph
activity (as in obesity) (18). In the present work, the combined
stimulus of GHRH-GHRP-6 has been studied in a group of very old
subjects (age higher than 75 yr) showing no decline in the amount of GH
secretion, as compared with both normal adults (less than 40 yr) and
aged subjects (age 46–65 yr). A similar lack of age-related decline
has been reported for normal subjects in their late adulthood (21). The
GHRH-GHRP-6-mediated GH discharge was similar for the three groups of
age, with similar mean GH peaks, AUC, and secretory pattern, which
suggests a very synchronized type of secretion. However, the most
interesting information came from the analysis of individual GH peaks.
In fact, as Fig. 2
shows, all the subjects had a positive response to
the stimulus, an unusual fact when facing a GH provocative test.
Moreover, despite the dispersion in GH peaks in any of the age groups,
no subject responded with a GH peak under 25 µg/L, a cut-off far from
the highest level of GH secretion elicited in patients with GH
deficiency, studied with similar tests (26).
When considering the suitability of GHRH plus GHRP-6 as a first-choice
stimulus for assessing GH reserve, it is worthy to consider that it
elicits a near-normal GH discharge in obesity (18) in patients with
hypothyroidism (27) and in patients with type 2 diabetes mellitus (28).
All of them are confusing situations when facing an individual
diagnosis of GH deficiency in adults. Another positive fact is that it
is a safe stimulus, without side effects, and that it does not require
medical supervision under its realization. The lack of age-related
decline in the GHRH-GHRP-6-mediated GH release is an added value to
this promising test, and this opens the possibility of using it as a
therapeutical tool to revert some deleterious manifestations of aging
in man, as has been showed with the nonpeptidil GH secretagogues (29).
It seems then that the combined test may be used for the diagnosis of
GH-deficient states in adults, because it is scarcely affected by age,
obesity, sex steroid levels, and so on. Because the maximal amount of
GH that a given individual is able to release upon stimulation must
have relationship with the pituitary reserve of the hormone, and
because GHRH plus a GHS is the most powerful stimulus, it may have a
relevant diagnostic role in the future.
Considerable work with larger groups of either normal or GH-deficient
subjects is needed before the diagnostic advantages of this test, in
confrontation with the ITT and the arginine-GHRH, may be
established.
In conclusion, the GHRH plus GHRP-6-induced GH release is preserved in
aged and very old subjects, suggesting that the GH secretory capability
of the combined test is not reduced by age. This combined test may be
useful for the diagnosis of GH-deficient states in adults.
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Acknowledgments
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The expert technical help of Ms. Mary Lage is gratefully
acknowledged.
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Footnotes
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1 This work was supported by research grants from: Fondo de
Investigacion Sanitaria, Spanish Ministry of Health and Conselleria de
Educacion, Xunta de Galicia. 
Received December 3, 1997.
Revised January 27, 1998.
Accepted February 3, 1998.
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Abstract
Introduction
