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Clinical Research Center Studies |
Department of Endocrinology, Christie Hospital (A.A.T., S.M.S.), Withington, Manchester, United Kingdom M20 4BX; the Department of Medicine, University of Virginia Health Science Center (R.M.N., S.S.P., M.O.T.), Charlottesville, Virginia 22908; and the University Department of Geriatric Medicine, South Manchester University Hospital Trust (P.A.O.), Manchester, United Kingdom M20 2LR
Address all correspondence and requests for reprints to: Dr. Stephen M. Shalet, Department of Endocrinology, Christie Hospital, Withington, Manchester, United Kingdom M20 4BX.
Abstract
Detailed assessment of physiological and pathophysiological GH secretion has, until recently, been limited by the poor sensitivity of the available assays. We have used an ultrasensitive chemiluminescence GH assay (sensitivity, 0.002 µg/L) to study 24-h GH profiles (20-min sampling) from 24 patients who had been treated for hypothalamic-pituitary disease with surgery and irradiation and from 24 healthy control subjects matched for age, sex, and body mass index.
Twenty-three of the 24 patients demonstrated pulsatile GH secretion, determined by Cluster. The median (range) area under the curve for GH, mean pulse area, mean pulse height, average valley mean level, and mean interpeak nadir were lower in the patients than in the controls [119.25 (7.273–843.600) vs. 968.539 (227.200–4625.000) min/µg·L (P < 0.00001); 3.777 (0.288–30.850) vs. 61.390 (12.880–224.210) min/µg·L (P < 0.00001), 0.107 (0.010–0.958) vs. 1.408 (0.368–5.050) µg/L (P < 0.00001), 0.074 (0.006–0.415) vs. 0.348 (0.048–2.350) µg/L (P < 0.00001), and 0.066 (0.003–0.270) vs. 0.205 (0.021–1.838) µg/L (P = 0.0004), respectively]. The median (range) number of pulses, mean pulse duration, and mean interval between pulses did not differ between the patients and controls [10 (4–15) vs. 10 (7–15; P = 0.36), 96.4 (68.0–220.0) vs. 104.0 (72.0–151.4) min (P = 0.65) and 128.0 (92.8–255.0) vs. 126.2 (90.0–180.0) min (P = 0.73), respectively]. The diurnal rhythm of GH secretion was present in the controls, but there was only limited evidence of residual diurnal rhythm in the patients.
This study has demonstrated that GH secretion remains pulsatile in GH-deficient patients despite the mass effect of hypothalamic-pituitary pathology, pituitary surgery, and radiotherapy. With the development of potent GH secretagogues that are active orally, our findings may have important implications for the future management of GH-deficient subjects.
GH IS SECRETED in pulses that occur approximately every 3 h. There is a diurnal variation, with more GH secreted at night than during the day. Between pulses, the GH nadir values are frequently below the limit of detection of conventional RIAs. Under circumstances in which GH secretion is known to be reduced, e.g. hypopituitarism (1, 2, 3) and aging (4, 5, 6, 7, 8, 9), most of the 24-h GH values cannot be estimated. In our recent study of GH secretion in elderly patients with pituitary disease, 93% and 61% of the GH estimations in the 24-h profiles of patients and controls (72 samples) fell below the sensitivity of the conventional assay (1). Thus, we were unable to comment on the true nature of GH secretion in either population.
Recent advances in assay techniques have increased the sensitivity of the GH assay and allowed detection of GH concentrations up to 50–200 times lower than previously possible using radioimmunometric techniques (10, 11). As a result of these advances, it has been possible to quantify GH concentrations at all time points of a GH profile, thereby allowing GH secretion to be studied more meaningfully in healthy adults and those with pathophysiology. Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA), GH profiles in adults with hypothalamic-pituitary disease have been described, demonstrating that, although greatly reduced, GH remains detectable throughout the 24 h (12). We have now used an ultrasensitive chemiluminescence assay (10) to reexamine the GH profiles of 24 patients with hypothalamic-pituitary disease and 24 controls with normal hypothalamic-pituitary function (all >60 yr of age) and report here the true nature of GH secretion in these 2 groups.
Subjects and Methods
Patients and controls
Twenty-four patients with nonacromegalic pituitary disease were
studied (16 men and 8 women, aged 61–85 yr) (1). All patients were
recruited from our out-patient population. We surveyed all of our
patients with known hypothalamic-pituitary disease who were over 60 yr
of age and approached all those who had previously had a peak GH
response to a provocative test less than 7.5 µg/L. This GH peak was
chosen arbitrarily in the absence of a clearly defined threshold for
the diagnosis of GH deficiency in this age group. All patients had
hypothalamic-pituitary disease that developed in adult life. Twenty-one
patients had been treated with cranial irradiation administered using
linear accelerator equipment, using a 3-field technique (13). Eighteen
patients received 37.5–42.5 Gy in 15 fractions over 3 weeks, a lower
total dose than that used in other centers (40–50 Gy in 20–25
fractions over 4–5 weeks); however, the larger fraction size and
shorter treatment period resulted in a slightly greater radiobiological
equivalent dose. The remaining 3 patients treated with radiotherapy
received 20 Gy in 8 fractions. Eighteen patients had undergone surgery
before receiving radiotherapy, 3 patients were treated with
radiotherapy alone, and 1 patient was treated with surgery alone (Table 1
).
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. Twenty-one patients had additional anterior pituitary hormone
deficiencies. All had gonadotropin deficiency, 16 had ACTH deficiency,
and 13 had TSH deficiency. Eleven of the 15 men with gonadotropin
deficiency were receiving testosterone replacement. Two had received
the most recent testosterone implant 3 months before entering the
study, the remaining 9 had received an im injection of testosterone
esters within the 2 weeks preceding the study. None of the women had
received sex steroid replacement therapy during the 5-yr period
preceding the study. All patients with ACTH deficiency were receiving
replacement therapy with hydrocortisone (n = 9), cortisone acetate
(n = 5), or prednisolone (n = 2), and those with TSH
deficiency were receiving T4. Twenty-four control subjects (17 men and 7 women, aged 60–87 yr) were recruited from a panel of normal volunteers. Exclusion criteria consisted of a history of diabetes mellitus, current treatment with psychotropic medication, treatment for thyroid disease, or abnormal thyroid function tests. The controls were matched with the patients for age and body mass index (BMI). None of the women had received estrogen replacement therapy within 1 yr of entering the study.
Study protocol
The study was approved by the South Manchester Area Health Authority ethics committee. All subjects gave written consent before entering the study.
Patients and controls were admitted to the ward at 0800 h, having eaten a normal breakfast. A cannula was inserted into a forearm vein and kept patent with heparinized saline. Blood samples were drawn every 20 min for 24 h. There was no restriction on activity within the ward. A standard hospital diet was consumed. Meal times were 1200, 1730, and 2200 h. The subjects retired to bed at 2230 h when the lights on the ward were turned out and were awakened at 0630 h. At 0900 h the following day, blood was drawn for serum insulin-like growth factor I (IGF-I) estimation. All samples were separated, and sera were kept at -80 C until the assays were performed.
Serum GH assay
GH concentrations were determined in duplicate using the Nichols Luma Tag hGH chemiluminescence immunometric assay (Nichols Institute, San Juan Capistrano, CA). The sensitivity of this assay was 0.002 µg/L. The intraassay coefficients of variation were 11.3%, 9.8%, and 11.7% for GH concentrations of 8.22, 0.293, and 0.027 µg/L, respectively. The interassay coefficients of variation at the same GH concentrations were 6.6%, 7.7%, and 10.4%, respectively.
Analysis of pulsatile GH release
Estimates of the uncertainty associated with each serum GH concentration measurement were derived using a recently reported data reduction method (10). Attributes of pulsatile GH release were analyzed using the Cluster algorithm (14). The threshold parameters used (test peak = 1, test nadir = 1, t statistic = 1) had a sensitivity of 75% for detection of GH concentration pulses and a positive predictive accuracy of 93% in a validation study employing computer simulations of GH pulse series at 20-min intervals. Specifically identified properties of pulsatile GH release included pulse frequency (number of significant GH peaks per 24 h), mean interpulse interval (time in minutes separating consecutive peak maxima), mean pulse duration, mean pulse height (maximal GH concentration in a peak), mean pulse area (integrated concentration under a peak in excess of the mean pre- and postpeak nadirs), mean interpeak nadir concentrations, interpeak valley mean (a valley was defined as a region embracing nadirs without significant intervening peaks), and integrated GH concentrations for the entire sampling period [area under the curve (AUCGH)].
Serum IGF-I assay
Serum IGF-I was measured, after acid-alcohol extraction, using an in-house RIA. The samples were assayed in duplicate as a single batch. The reference preparation used was National Institute of Biological Standards and Control 87/518. The intraassay coefficients of variation for mean IGF-I concentrations of 46, 246, and 706 µg/L were 11.3%, 6.5%, and 4.7%, respectively. The sensitivity of this assay was 14 µg/L.
Statistical analysis
Results are expressed as the median (range). Comparison between groups was made using the Mann-Whitney U test. Relationships between parameters were determined by Pearson’s correlation. To establish the presence of a downward trend in the characteristics of GH release with increasing severity of hypopituitarism in the patients, the Jonckheere test (15) was employed. This is a nonparametric test that looks for an ordered trend in medians. A positive J value indicates an increasing trend; a negative J value indicates a decreasing trend. To determine whether a diurnal rhythm was present, parameters of GH secretion during the night (2230–0630 h) were compared with those during the day (1030–1830 h) using a paired t test. P < 0.05 was considered statistically significant.
Results
BMI [median (range)] did not differ between the two groups \[28.2 (22.6–37.3) kg/m2 in the patients vs. 26.1 (20.1–37.0) kg/m2 in the controls\]. The controls were slightly, but significantly, older than the patients \[70.6 (60.8–87.5) vs. 66.0 (61.0–85.7) yr; P = 0.04\].
GH profiles
Pulsatile GH release was observed in all subjects except patient
2. Representative profiles of four patients and four controls are shown
in Figs. 1 and 2. The median AUCGH in the
patients was reduced by 88% of the control value. The
mean pulse area, mean pulse height, interpeak mean level, and mean
interpulse nadir were also significantly lower in the patients than in
the controls. The number of pulses, mean pulse duration,
and mean interpulse interval, however, did not differ between the
patients and controls (Table 2).
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).
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Age, BMI, and sex. Age did not have an effect on GH secretion in either the patients or the controls. There was a negative correlation between BMI and AUCGH in the patients (r = -0.40; P = 0.05) and the controls (r = -0.514; P = 0.01). BMI was correlated significantly with GH pulse height in the controls (r = -0.60; P < 0.002) and to a lesser extent in the patients (r = -0.44; P = 0.037).
Female controls had a greater AUCGH \[1462.1 (978.18–4624.990) vs. 788.5 (227.172–2923.600) min/µg·L; P = 0.003\], mean pulse height \[2.76 (1.388–5.050) vs. 0.973 (0.368–3.822) µg/L; P = 0.007\], average valley mean level \[0.498 (0.190–2.350) vs. 0.191 (0.048–1.191) µg/L; P = 0.01\], and mean interpeak nadir \[0.398 (0.150–1.838) vs. 0.123 (0.021–0.825) µg/L; P = 0.008\] than their male counterparts, who were matched for age and BMI. These sex-related differences were not observed in the patients.
Diurnal variation.
The results of Cluster analysis of the
daytime period (1030–1830 h) compared to that of the lights out
(2230–0630 h) period are shown in Table 3. The
mean \ SEM nocturnal GH concentration was
significantly higher than the mean daytime GH concentration in the
controls (1.01 \ 0.18 vs. 0.59 \ 0.10 µg/L;
P = 0.005), but not in the patients (0.15 \ 0.03
vs. 0.11 \ 0.02 µg/L; P = 0.09). The
mean interpeak valley concentration was significantly higher during the
night than during the day in the controls (0.633 \ 0.153
vs. 0.285 \ 0.072 µg/L; P = 0.004)
and in the patients (0.128 \ 0.026 vs. 0.083
\ 0.015; P = 0.014).
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). There was a
tendency for the pulse frequency to fall as the number of hormone
deficits increased, but this did not quite reach significance
(P = 0.08; Table 4).
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The median serum IGF-I concentration was 102 (<14–162) and 147 (65–255) µg/L in the patients and controls, respectively (P = 0.0002). The serum IGF-I concentration did not correlate with AUCGH or the absolute GH peak in the patients or controls. Serum IGF-I did, however, correlate with the absolute GH nadir in the patients (r = 0.52; P = 0.01), but not in the controls.
Discussion
In the past, determination of pulsatile GH secretion in healthy subjects and patients with hypothalamic-pituitary disease has been limited by the lack of sensitivity of the assays used to measure GH. Studies using standard RIA for GH determination suggested that GH was secreted in bursts followed by episodes of apparent secretory quiescence. With the development of more sensitive assays, the description of pulsatile GH secretion has required modification; in particular, an increase in pulse frequency has been appreciated. Information about trough and nadir GH concentrations, however, has remained undefined (9). With the advent of ultrasensitive GH assays, basal GH secretion has been unmasked. GH secretion is continuous and consists of two distinct modalities: secretory bursts that occur intermittently overlying a low level, continuous tonic production of GH (12, 16). We have previously estimated the GH concentrations in the samples from these profiles using an in-house immunoradiometric assay with a sensitivity of 0.4 µg/L. GH concentrations were measurable in 39% of the 24-h samples (n = 72) from the controls, but in only 7% of the samples from the patients; 16 of the patients had no detectable GH secretion during the 24-h period (1). Now, using the ultrasensitive assay, we have been able to determine the GH concentration in all of the samples, allowing us to examine the true nature of GH secretion in healthy elderly subjects and in those with organic disease of the hypothalamic-pituitary axis.
It is well recognized that spontaneous GH secretion declines with increasing age (6, 8, 17); it was even suggested in one early study that GH secretion may cease in the elderly (4). The assay used in that study, however, lacked sensitivity (1.0 µg/L), and three of five subjects had no measurable GH concentrations. In our present study we have confirmed the findings of Iranmanesh et al. (16), showing that, using an ultrasensitive GH assay in elderly subjects, GH concentrations remain detectable throughout the 24-h period. In addition, we have demonstrated that GH release remains pulsatile in healthy individuals well into the ninth decade of life.
GH secretion is markedly reduced in adults who have organic pituitary disease compared with that in healthy controls. Recently, Reutens et al. used an ultrasensitive GH ELISA and deconvolution analysis to demonstrate that patients with organic disease of the hypothalamic-pituitary axis continue to secrete GH throughout the 24-h period (12). GH secretion in their GH-deficient subjects was reduced to 5% of that in the controls who were matched for age, sex, and BMI. The patients and controls were separated in terms of the absolute peak value during the profile, but there was overlap of the values for absolute nadir between the two groups. In our study GH release in the patients was 12% of that in the controls, and the two groups could not be clearly separated in terms of either absolute nadir or absolute peak. There are two possible explanations for these observations. Firstly, all of the subjects studied by Reutens et al. (12) had two or more additional pituitary hormone deficits. We have previously reported that the severity of GH deficiency, determined by the peak GH response to an insulin tolerance test, increases in the presence of additional pituitary hormone deficiencies (18). In this study we have shown that spontaneous GH release also falls as the degree of hypopituitarism increases. In the GH-deficient subjects with two or more additional pituitary hormone deficiencies, GH release was reduced to 8% of that in the controls, a decrease similar to that reported by Reutens et al. (12). This subgroup of patients was also distinct from the control group in terms of absolute peak GH concentration during the 24-h profile. Secondly, the control subjects in our study were, on the average, almost 20 yr older than those studied by Reutens et al. (12) and, therefore, would have reduced GH secretion because of their increased age. With increasing age, the distinction between GH deficiency and normality is quantitatively reduced.
Twenty-two of 24 patients had a pituitary space-occupying lesion; 18 of them underwent pituitary surgery and irradiation. The findings that GH secretion remains pulsatile, the diurnal variation in GH secretion is reduced but is not absent, and the major alteration in physiological GH status is a reduction in the amplitude of the GH pulse are important observations. The GH pulse frequency and mean pulse duration were not significantly different in the patients compared with the controls. GH nadir characteristics were significantly reduced in the patients, but to a lesser extent than the alteration in pulsatile GH release.
Pulsatile GH secretion is controlled by two hypothalamic hormones, GHRH and somatostatin (19). There are several lines of evidence indicating that the hypothalamus is more radiosensitive than the anterior pituitary and that GH deficiency is indeed an early feature of radiation-induced damage to the hypothalamic-pituitary axis (20). Radiation-induced hypothalamic damage appears to be a result of direct injury to hypothalamic neurons rather than of vascular damage (21), and although endogenous somatostatin tone and GHRH secretion are reduced, they are not abolished by irradiation (22). The present finding that the qualitative pattern of GH secretion remains intact after a dose of hypothalamic-pituitary irradiation known to induce GH deficiency (23) is in agreement with these observations.
Evidence that the hypothalamic control of GH secretion remains intact suggests that it may be altered by exogenous factors. This has implications for the future management of patients with GH deficiency. The benefits of GH replacement therapy in GH-deficient adults are now recognized (24, 25). Currently, GH replacement is given as a single sc injection at night in an attempt to mimic the physiological diurnal rhythm. This is far from physiological and results in a nonpulsatile GH profile. The GH-releasing peptides and synthetic agents, some of which are active orally, cause the release of GH from the pituitary via a direct action on the pituitary somatotroph as well as an even greater action at the hypothalamus (26, 27, 28). The ability of GH-releasing peptides to release GH from the somatotroph is potentiated by GHRH, suggesting that an intact hypothalamic-pituitary axis is important to maximize the effect (29, 30). The vast majority of adults with adult-onset GH deficiency have acquired the deficiency as a consequence of the presence of a pituitary adenoma or treatment with pituitary surgery and/or irradiation, whereas a significant proportion of adults with childhood-onset GH deficiency have acquired the deficit as a consequence of radiation damage. We have demonstrated that GH release remains pulsatile in adults after hypothalamic-pituitary irradiation in addition to pituitary surgery and the mass effect of a pituitary lesion. Thus, the integrity of the hypothalamic-pituitary axis is preserved. In this group of patients, manipulation of GH secretion with GH secretagogues requires further investigation, but may provide a more appropriate means of replacing GH.
Acknowledgments
We thank the General Clinical Research Center Core Laboratory for performing the GH chemiluminescence assays, and Drs. Johannes D. Veldhuis, Michael Johnson, and Martin Straume for the computer programs for Cluster and the discrete variance data reduction method for hormone data. We also thank Mark L. Hartman for assistance with the pulse analysis and critical review of the manuscript. We are grateful to Jenny Jones at the Institute of Child Health (London, UK) who performed the serum IGF-I measurements.
Footnotes
1 This work was supported in part by NIH Grants DK-32632 (to M.O.T.)
and RR-00847 (to the General Clinical Research Center), the CDMAS
Laboratory at the University of Virginia, Pharmacia & Upjohn, Ltd. 
2 Supported by a grant from the Deutsche
Forschungsgemeinschaft.
Received October 30, 1996.
Revised January 15, 1997.
Accepted April 18, 1997.
References
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,
Patients with two or three additional hormone deficits; *, patients
with one additional hormone deficit; •, patients with isolated GH
deficiency; 
, controls.