0021-972X/92/7404-0766$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 4 Printed in U.S.A.
Properties of Spontaneous Growth Hormone Bursts and Half-Life of Endogenous Growth Boys with Idiopathic Short Stature*
Secretory Hormone
in
JOHANNES D. VELDHUIS, ROBERT M. BLIZZARD, ALAN D. ROGOL, PAUL M. MARTHA, JR:~, JOHN L. KIRKLAND, BARRY M. SHERMAN, AND GENENTECH COLLABORATIVE GROUP Division of Endocrinology and Metabolism, Departments of Internal Medicine (J.D. V.), Pharmacology (A.D.R.), and Pediatrics (R.M.B., A.D.R., P.M.M.), National Science Foundation Science Center in Biological Timing (J.D. V.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; Department of Pediatrics, Baylor College of Medicine (J.L.K.), Houston, Texas 77030; and the Clinical Research Division, Genentech, Inc. (B.M.S.), South San Francisco, California 94080
ABSTRACT.
We analyzed endogenous GH secretory dynamics and MCRs by a novel quantitative deconvolution technique in 20 boys with idiopathic short stature (ISS) and 35 boys of normal stature in Tanner stage I of puberty. We tested the null hypotheses that 1) ISS is not associated with any alterations in the frequency, mass, amplitude, or duration of spontaneous GH secretory bursts and/or the 24-h GH production rate; and 2) the half-life of endogenous GH is not altered in ISS. The boys with ISS had a mean (+SEM) bone age of 8.0 + 0.42 yr and a chronological age of 10 f 0.50 yr. The latter was similar to the chronological (and bone) age of the normal boys of 9.8 f 0.23 (and 9.3 + 0.34) yr. Mean height SD scores were significantly lower in ISS boys, uiz. -2.7 + 0.15 in ISS us. +0.34 + 0.13 in normal boys (P < 0.001). Plasma insulin-like growth factor-I concentrations were similar in the two groups, as were (24-h) mean serum GH concentrations, uiz. 3.5 + 0.29 rg/L in ISS and 4.15 0.49 rg/L in normal boys (P = NS). Deconvolution analysis revealed that the mean number of GH secretory events per 24 h was similar in normal and ISS boys, uiz. 9.6 + 0.76 (normal) us. 8.4 + 0.55 (ISS), and that there was no significant difference in mean GH interburst intervals. The amplitude, mass, and dura-
T
HE PATHOPHYSIOLOGY of impaired linear growth in children with short stature has been the object of intensive clinical investigation (l-4). Many Received March 18, 1991. Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. * This work was supported in part by NIH Grant RR-00847 (to the Clinical Research Center of the University of Virginia), Research Career Development Award l-K04-HD-00634 (to J.D.V.), Diabetes and Endocrinology Research Center Grant 5-P60-AM-22125-05, NIHsupported Clinfo Data Reduction Systems, the Pratt Foundation, the Academic Enhancement Fund, and the NSF Science Center in Biological Timing. The members of the Genentech Collaborative Study Group are: Ann Johanson (Genentech), Robert Blizzard (University of Virginia Medical Center), Jose Cara (University of Chicago Hospital), Steven Chernausak (Children’s Hospital Medical Center, Cincin
tion of computer-resolved GH secretory bursts also did not differ in normal and ISS boys. The half-lives of endogenous GH were estimated to be 16 f 0.77 min in the ISS and 18 + 0.93 min in the control boys (P = NS). The calculated daily GH secretion rate per unit distribution volume was not significantly reduced in ISS, i.e. 194 + 19 pg/L.day in ISS us. 177 + 19 pg/L.day in control boys. Moreover, daily GH secretion rates corrected for body mass index (weight/height’) in the twp groups were not significantly different. In summary, the present cohort of boys with ISS manifested no significant alterations in GH secretory burst frequency, duration, mass, or amplitude or in the half-life of endogenous GH compared to normal boys in Tanner stage I of pubertal development. Indeed, whether daily GH secretion rates are expressed per unit distribution volume or per unit body mass index, groups of boys with ISS and normal height controls secrete similar total amounts of GH. We conclude that the overall dynamics of GH secretion and clearance in boys with ISS considered as a whole cannot be distinguished readily from physiological patterns observed in prepubertal boys of normal height. (J Clin Endocrinol
Metub 74: 766-773,1992)
studies have focused on possible disturbances in the neuroendocrine regulation of episodic GH release (5-7). Evaluations of pulsatile GH secretion are based on the premise that bursts of GH release are important in the physiological activation of relevant target tissues, including, for example, the stimulation of linear bone growth nati, OH), James Frane (Genentech), Joseph Gertner (New York Hospital, Cornell Medical Center), Raymond Hintz (Stanford University Medical Center), Nancy Hopwood (University of Michigan Medical Center), Selna Kaplan (University of California School of Medicine), John Kirkland (Baylor College of Medicine, Houston, TX), Barbara Lippe (UCLA Hospital, Los Angeles), Leslie Plotnick (Johns Hopkins Hospital, Baltimore, MD), Paul Saenger (Montefiore Hospital, New York, NY), and Barry Sherman (Genentech). t%urrent address: Baystate Medical Center, 759 Chestnut Street, Springfield, Massachusetts 01199.
766
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GH SECRETION
and the induction of specific hepatic and muscle proteins and genes in the rat (8, 9). The pathophysiological regulation of pulsatile GH signaling to target tissues is achieved by two major hypothalamic effector systems, which comprise an inhibitory (somatostatin) and stimulatory (GH-releasing hormone) limb (10, 11). Despite extensive studies of putative neuroendocrine disturbances in children with idiopathic short stature (ISS), there is no published information regarding the underlying GH secretory dynamics and subject-specific metabolic clearance of endogenous GH in boys with this clinical diagnosis. In general, detailed assessment of endogenous GH secretion and clearance in man has been limited by the previous difficulties inherent in quantitating hormone secretion and disposal rates in uiuo (12). Such difficulties arise in part because most methods to estimate endogenous hormone secretion rates depend upon the infusion of radiolabeled or “cold” hormone under equilibrium or nonequilibrium conditions. In such circumstances, the MCR of the infused hormone species can be calculated and the production rate of endogenous hormone estimated. To our knowledge, such studies have not been carried out to compare large groups of normally growing boys and individuals with ISS. The development of specialized analytical tools, such as deconvolution analysis, permits the estimation of endogenous hormone secretory rates and subject-specific half-lives from pulsatile serum hormone concentration profiles without the infusion of radiolabeled or unlabeled hormone (13). For example, one technique, termed multiple parameter deconvolution analysis, allows a statistically based appraisal of the number, amplitude, mass, and duration of underlying hormone secretory bursts and a simultaneous estimate of the endogenous half-life of the hormone of interest based on all measured serum hormone concentrations and their associated dose-dependent intrasample variances (13-15). Accordingly, we have used this new analytical technique to quantitatively evaluate the episodic mode of GH secretion and clearance in 35 individual prepubertal (Tanner stage I) boys of normal height and 20 chronological age-matched boys with ISS. We have been able to test the null hypothesis that the mean number, amplitude, mass, and duration of spontaneous GH secretory bursts and the mean half-life of endogenous GH do not differ between groups of normally growing boys and their counterparts with ISS. Materials
and Methods
Clinical protocol Pulsatile GH secretion and endogenous GH half-lives were studied in 35 healthy prepubertal boys of normal height and 20 boys with ISS. Control boys had heights and weights within 1.96 SD of age-derived normal measures. Boys with ISS were
IN SHORT BOYS
defined by 1) serum GH concentrations exceeding 10 pg/L after provocative testing (L-dopa, clonidine, arginine, sleep, or exercise), 2) height at least 2 SD below age- and sex-matched normative values (absolute range, -2.0 to -4.6), and 3) the absence of any history of intrauterine growth retardation, underlying systemic, metabolic, or known endocrine disease, dysmorphic traits, or central nervous system irradiation. We did not attempt to quantitate the relative contributions of genetic vs. constitutional delay of growth in ISS boys. The bone age of the ISS boys determined radiographically at the wrist (16) averaged 8.0 f 0.42 yr, and 13 of 20 boys had a bone age delay of at least 2 yr. The mean chronological ages (+SEM) of the normal and short subjects were, respectively, 9.8 * 0.23 and 10.0 f 0.50 yr (P = NS). ISS boys had a mean growth rate of 4.3 + 0.29 cm/yr. Other clinical comparisons are given in Table 1. All subjects were in Tanner genital and pubic hair stage I (17) and were studied after a guardian provided written informed consent, approved by the Institutional Human Investigations Review Board. Each guardian provided a detailed clinical history. The boys each underwent a complete physical examination and had normal fasting plasma glucose concentrations, unremarkable biochemical tests of renal, hepatic, and hematological function, and normal serum concentrations of Tq. Boys were admitted to the study center the evening or morning before blood sampling, which was carried out at 20min intervals for 24 h, beginning at least 1 h after venipuncture. Blood was withdrawn through an iv catheter placed in a forearm vein. Subjects were permitted to ambulate and were given three TABLE
1. Clinical characteristics
of boys with normal stature and ISS Normal stature (n = 35)
Chronological
age (yr)
9.8 + 0.23 (6-12)
Bone age (yr)
Wt 04 Ht age (yr) Height SD score 24-h mean serum GH cont. WJJ Plasma IGF-I (U/L) Body mass index (kg/m’)
9.3 + 0.34 (6.0-12.5) 33 + 1.2 (21-50) 10.0 f 0.28 (5-12) 0.31 + 0.14 (-1.5 to 2.0) 4.1 + 0.49 (1.0-15)
0.93 + 0.10 (0.29-2.1) 17.2 + 0.42 (11-23)
ISS (n = 20) 10.0 t 0.50" (6-14) 8.0 + 0.42* (3.6-10.6) 23 + l.O* (14-33) 7.1 * 0.40* (3-10) -2.7 + 0.15' (-4.6 to -2.0) 3.5 + 0.29" (1.5-6.2) 0.76 + 0.08" (0.20-1.5) 15.6 f 0.31' (12-20) -0.70 + 0.25 (-3to 1.5)'
Body mass index SD score for +0.33 + 0.25 (-4.6 to 3.8)” age and sexd Data are the mean + SEM. Parentheses contain ranges. Bone age was determined in only 24 of the 35 boys of normal stature. “P=NS. * P < 0.001 us. normal stature. ‘P = 0.004. d Calculated as the difference between the logarithms of the observed and normative (age- and sex-specific) body mass indices divided by the corresponding soteference value (Table 5 in Ref. 21). ‘P = 0.007.
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VELDHUIS
768
meals per day (at 0800, 1200, and 1700 h). They were not allowed to nap or sleep until 2200 h. After the blood clotted at room temperature for 1 h, the sera were separated and frozen for later immunoradiometric assay (IRMA) of GH concentrations. Assays Serum GH concentrations were determined in replicate (duplicate) by IRMA, in which the mean (*SD) intraassay coefficient of variation determined from all 73 replicated samples was 5.1 f 1.2% in the 35 control subjects and 4.4 f 1.1% in the ISS boys. All data were extrapolated from and/or expressed in relation to human GH standards (Nichols Institute, San Juan Capistrano, CA) diluted with horse serum matrix (18, 19). The assay sensitivity was 0.50 Kg/L, and the interassay coefficient of variation was less than 9.5%. The plasma somatomedin-C [insulin-like growth factor-I (IGF-I)] content was determined by the Nichols Institute direct assay. Convolution modeling Deconvolution analysis permits quantitative estimates of individual hormone secretory parameters and the calculation of subject-specific hormone half-life from all serum hormone concentrations and their dose-dependent intrasample variances considered simultaneously (13, 15). Calculations assume that the serum GH concentration at any given instant reflects the simultaneous operation of four determinable secretory and clearance features of interest: 1) the number and locations, 2) the amplitudes, and 3) the durations of all significant GH secretory bursts, which are acted upon by 4) endogenous subject-specific clearance kinetics. The GH disappearance function was defined by a subject-specific monoexponential half-life fitted simultaneously with the various secretion parameters. GH concentrations were assumed to decay to the sensitivity of the GH IRMA. We calculated the following features of GH secretion and clearance: the half-duration (duration at half-maximal amplitude) of GH secretory bursts, amplitudes (maximal values) and temporal positions of all significant GH secretory bursts, and GH half-life. The mass of GH secreted per pulse is the analytical integral (area) of the resolved secretory burst, and the 24h production rate is the product of the total number of secretory bursts and the mean mass of GH released per burst (13-15). Although an interpeak basal secretory rate can be fitted in principle, no tonic secretion function was required to model the present data.
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JCE & M .1992 Vol74.No4
on the logarithm of the expected normative value (21). P < 0.05 was construed as statistically significant.
Results The clinical features of the 35 boys of normal stature and the 20 subjects with ISS are summarized in Table 1. The chronological ages of the ISS and control boys were similar, as were mean (24-h) serum GH concentrations. Individual values of mean 24-h serum GH concentrations are shown in Fig. 1. Plasma IGF-I concentrations also were not significantly different, but boys with ISS had significantly reduced body mass index, whether expressed as an absolute value (kilograms per m”) or as an age- and sex-adjusted SD score (21). As illustrated in Fig. 2, both normal boys and those with ISS exhibited an exclusively episodic or pulsatile mode of GH release over the 24 h of blood sampling. We have depicted the observed serial serum GH concentrations over 24 h in two normal boys and two subjects with ISS. The fitted continuous curves through the GH concentration data (Fig. 2, upper subpanels) represent the predictions of the multiple parameter convolution model. The lower subpanels of Fig. 2 show the deconvolutionresolved GH secretory bursts and give their amplitudes (maximal rate of GH secretion attained per burst), halfdurations (duration of the GH secretory burst at halfmaximal amplitude), and locations in time. The mean quantitative characteristics of episodic GH secretion and clearance are given in Table 2. The frequency of spontaneous GH secretory bursts was similar statistically in boys with ISS and their controls. The mean interburst intervals, half-lives of endogenous GH, and half-durations of GH secretory bursts (duration of 3.5*0.29
Statistical analyses Unpaired two-tailed Student’s t testing and the Wilcoxon rank sum (nonparametric) test were used respectively for normal and nonGaussian distributed variables, as defined by the Wilk-Shapiro statistic (20). Data are given as the mean f SEM. Linear regression analysis was used to assess relationships between specific measures of GH secretion and clearance and bone age or body mass index (weight/height’). The latter was transformed to an age-dependent male-specific SD score based
01
/
Control
I
I
Short
,
Stature
1. Individual mean (24-h) serum immunoradiometric GH concentrations in 20 boys with ISS and 35 normal prepubertal (Tanner stage I) boys. Etch value is the mean of 72 sample estimates derived by withdrawing blood at 20-min intervals for 24 h.
FIG.
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GH
SECRETION
IN
A
SHORT
BOYS
769
NORMAL 12.5
h
B
10.0
5.0 2.5
. !
1.51
FIG. 2. Profiles of serum GH concentration peaks and deconvolution-resolved GH secretory bursts in two boys with normal stature (A) and two boys with 1% (B). Each upper subpanel gives the measured serum GH concentrations in blood collected serially at 20-min intervals over 24 h. The continuous curves through the observed serum GH concentration data were calculated by the multiple parameter convolution model. The lower subpanels depict the deconvolution-resolved GH secretory rates as a function of time. Quantitative secretion profiles were determined in each boy by simultaneously estimating values of relevant secretion parameters and the subject-specific GH half-life. The mean results for all 35 boys with normal stature and the 20 boys with ISS are summarized in Table 2. GH concentration units in nanograms per mL = micrograms per L. Note the different absolute values of the vertical axes.
0
1000
500
1500
0
TIME (MINI
6 12
A
SHORT
STATURE
25
yB
-4
/\I A AA 0
500
1000
1500
TIME the secretion event at half-maximal amplitude) were similar in both study groups, as were the mean amplitudes of GH secretory bursts (maximal rate of GH secretion attained within a release episode). The mass of GH released per secretory event (calculated as the area under the computer-resolved GH secretory pulse) also was not significantly different between normal and ISS boys (Table 2). Since the daily secretion or production rate is the product of the mean mass of
0
500
A
br
! ,L 1000
, , ,I 1500
(MINI
hormone secreted per pulse and the number of pulses per 24 h, we estimated a mean production rate of endogenous GH of 177 f 19 pg/L.day in the normal boys and 194 f 19 pg/L. day in those with ISS (P = NS; Fig. 3). Because of significant weight differences between the two groups, GH production rates were also expressed per kg body mass. According to such calculations (Table 2), daily GH secretion rates were statistically higher in the ISS group; uiz. 8.9 f 0.98 us. 5.9 +- 0.69 Kg/L. kg in ISS and control
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770
VELDHUIS
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JCE & M .1992 Vol74.No4
TABLE 2. Dynamics mal stature and ISS
GH half-life
of GH
(min)
Secretory burst half-duration (min) No. of GH secretory pulses/ 24 h Interpulse interval (min) Secretory burst amplitude (pg/L. min) * GH mass secreted/pulse km * Daily production pg/L. 24 hb
kg/m’.
and clearance
in boys
with
Normal stature (n = 3.5)
ISS (n = 20)
18 f 0.93
16 + 0.77”
(8-35) 24 + 1.6
(8-23) 25 + 1.4“
9.6
(9-44) + 0.74 (2-21)
nor-
A
9.6kO.7
8.410.6
(17-40) 8.4 + 0.55"
168k 14 (66-467) 0.74 f 0.06
(5-15) 164 + 11" (98-251) 0.92 + 0.11"
(0.31-1.9) 21k2.9 (3.7-97)
(0.22-1.8) 25 + 2.4" (7-39)
rate 177*
a/L~k/24 h rg/L.
secretion
24 h
19
194
(14-423) 5.9 + 0.69 (0.36-13)
(48-353) 8.9 + 0.98" (1.5-16)
11 f (0.71-25)
13 + 1.3" (2.4-22)
1.2
Short
Control
+ 19”
164+10.8
168+14.2
500
B
-I-
In
i
5
Stature
0
m
Data are the means + SEM. Ranges are given in parentheses. “P=NS. * The volume term (L) refers to unit distribution value for GH. ‘P = 0.017 US. boys of normal stature.
boys, respectively (P = 0.017). However, there was substantial overlap between values in the two groups. Moreover, when daily GH secretion rates were expressed further per unit body mass index (weight/height’; Table 2) or the corresponding SD scores (not shown), there were no significant differences between ISS boys and normal stature controls. Linear regression analysis revealed that in normal boys the body mass index SD score tended to correlate negatively with the daily GH secretory rate (r = -0.287; P = 0.09). The corresponding correlation in boys with ISS was r = -0.435 (P = 0.05). Of interest, in boys with ISS, this measure of sex- and age-adjusted body mass index also correlated negatively with GH secretory burst amplitude (r = -0.590; P = 0.006) and mass (r = -0.440; P = 0.05). Bone age correlated negatively with several quantitative features of GH secretion in normal boys, viz. GH secretory burst half-duration (r = -0.572; P = 0.003), mass of GH secreted per burst (r = -0.415; P = 0.04), and daily GH secretion rate (r = -0.440; P = 0.03). Discussion We have tested the null hypothesis that as a group young boys with ISS exhibit no measurable alterations in specific quantitative features of GH secretion and/or clearance compared to prepubertal boys of normal stature. Using deconvolution analysis, we observed statistically similar daily GH production rates in 20 prepubertal boys with ISS and 35 Tanner stage I counterparts of
01
I
Control
FIG. 3. Number of GH secretory (B) in individual normal (control) may represent 1 or more subjects.
/
I
Short
1
Stature
bursts (A) and interburst and short boys. An individual
intervals symbol
normal stature. The 2 groups also manifested indistinguishable mean GH secretory burst mass, frequencies (and, correspondingly, interburst intervals), durations, and amplitudes and endogenous GH half-lives. Therefore, we infer that the overall dynamics of GH secretion and clearance are not statistically distinguishable in this group of boys with ISS considered as a whole and their Tanner stage I (prepubertal) male counterparts of normal stature. These results contrast with some previous studies using various empirically based methods to identify numbers of plasma GH concentration peaks, which suggested either similar or reduced numbers and/or amplitudes of GH concentration peaks (l-6). Most of these studies involved fewer subjects than evaluated here (35 controls and 20 boys with ISS), and none quantitated GH secretory rates and subject-specific GH half-lives. In addition, our study may differ in the selection and/ or ascertainment of study subjects with normal growth
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GH SECRETION
or ISS. Plasma IGF-I concentrations were similar in our boys of normal stature and those with ISS. This suggests that GH action, at least on target tissues that contribute substantially to circulating total IGF-I levels (e.g. liver), is normal in ISS boys. However, normal GH action cannot necessarily be inferred for other sites of locally synthesized IGF-I, if such sites contribute only sparingly to the large plasma IGF-I pool, e.g. GH target cells in the skeleton, pituitary gland, and hypothalamus (22, 23). Furthermore, similar plasma total IGF-I concentrations in boys with ISS and controls do not rule out differences in sensitivity to the negative feedback actions of IGF-I on the somatotropic axis and/or differences in the specific association of IGF-I with one or more circulating IGF-I-binding proteins. Recent studies of serum GH concentration peaks (as distinguished from computer-resolved GH secretory bursts, described here) have suggested that the amplitude (height) and/or area, rather than the number of serum GH concentration peaks correlates positively with linear growth velocity in healthy pubertal children (24-26) and in boys (26) and girls (27) treated with sex steroid.hormones. Mathematical modeling of pulsatile endocrine signals indicates that an increase in the incremental height or area of plasma hormone concentration peaks can result from either an augmented secretory burst amplitude (maximal secretory rate achieved within each pulse) and/or an increased duration of each underlying secretory burst (28)l. In this regard, recent deconvolution studies have disclosed that GH production rates rise because GH secretory burst amplitude and mass specifically increase at the time of puberty, whereas GH secretory burst frequency and duration, and the endogenous GH half-life do not vary in puberty (29). We observed that boys with ISS have statistically similar mean GH secretory burst amplitudes, mass, and daily GH secretion rates when secretory data are expressed as micrograms of GH secreted per L distribution volume. However, when daily GH secretion is computed as a volume- and weight-adjusted value (micrograms per L/kg. day), ISS boys secrete slightly more GH than boys of normal stature, which presumably reflects the former group’s significantly reduced body mass. When GH secretion is evaluated further per unit body mass index, wherein body weight is expressed either in absolute terms or as a SD score in relation to the square of the child’s height (al), equivalent daily GH secretion rates are estimated in boys of normal stature and those with ISS. Consequently, we infer that as a whole, groups of idiopathically short and normal stature boys do not secrete 1 Increased tonic (interburst) GH secretion, diminished GH distribution volume, and/or a prolongation of GH half-life in principle also could increase maximal serum GH concentration peak heights (26).
IN SHORT
BOYS
771
different amounts of GH over 24 h given their differences in both weight and height. In the 35 prepubertal boys of normal stature, we found that age was a significantly negative predictor of GH secretory burst mass and duration and daily GH secretion rate. These relationships have not been recognized previously in this interval just before puberty, but they suggest that there may be regulation of GH secretion by as yet unspecified nutrient and/or metabolic signals that covary with prepubertal age. Alternatively, if the narrow time window of relatively decreased linear growth velocity before the adolescent growth spurt (17) is associated with decreased GH release, and if only children in prepuberty are studied (as here), then a negative correlation might be expected between age and GH release within this particular time span. Recent studies in men and pubertal boys have revealed an inverse relationship between body mass index, a measure of relative weight per unit surface area, and daily GH secretory rates (29,30). Similarly, the normal cohort of prepubertal boys studied here manifested a significantly negative correlation between body mass index and total GH production. Within the group of ISS boys, we found significantly negative associations between the SD score for sex- and age-adjusted body mass index and GH secretory burst amplitude and mass as well as daily GH secretion rates. Of interest in this regard is the recent finding that body mass index is a positive statistical correlate of plasma GH-binding protein concentrations in children (31). The high affinity plasma GH-binding protein closely resembles, and in some measure may reflect activity of, the extracellular domain of tissue GH receptors (32). Accordingly, it is possible that the association of low plasma GH-binding protein levels with a low body mass index might account for poor skeletal growth responses in some short patients with apparently normal GH secretion. This speculation also suggests the possibility that local generation of IGF-I and/or trophic actions of IGF-I on growth-related target tissues may be attenuated in some ISS patients. Larger amounts of GH might be expected to overcome such putative tissue resistance. The latter consideration is consistent with the demonstration that GH administration can stimulate significantly increased linear growth in some boys with ISS (33, 34). In normally growing prepubertal boys, deconvolution analysis revealed approximately 10 secretory episodes/ 24 h, which corresponds to an interpulse interval of 168 min. The average half-duration (duration of the calculated secretory event at half-maximal amplitude) of the GH secretory burst was 24 min, which indicates that sustained GH secretion occurs within any given release episode. This secretory event duration is approximately 2-fold longer than that reported for immunoactive LH
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772
VELDHUIS
or FSH or biologically active LH (13, 14), but similar to that estimated recently for cortisol in healthy men (15). The mass of GH discharged per burst averaged 21 pg/L distribution volume. Assuming a nominal GH distribution volume of 8% body weight (35), we can calculate that approximately 56 pg GH are secreted per spontaneous pulse in the normal Tanner stage I boys studied here, who weighed an average of 33 kg. The corresponding daily GH secretion rate is approximately 0.56 mg according to the assay, standards, and reagents used here. Of course, the absolute value of this estimate depends on the specific GH assay, reagents, matrix, and standards employed (18, 19). Direct estimates of subject-specific GH MCRs are limited in normal prepubertal (Tanner stage I) boys. However, our calculated endogenous GH half-lives are similar to many adult values inferred by others using exogenous GH. We estimated a mean half-life of endogenous GH of 18 f 0.93 min in normal boys, which falls within the range of values determined after the injection of purified pituitary or recombinant GH in adults and children (35-45). The present values also agree with recent estimates of endogenous GH half-lives in normal men, in whom pituitary secretion of GH was stimulated transiently by a bolus injection of GH-releasing hormone, after which further GH secretion was blocked by infusion of somatostatin (46). We observed a large range of GH secretion and clearance values in boys with ISS, some of which considered alone would differ considerably from the normal mean. However, prepubertal boys of normal stature also exhibited a wide dispersion of GH secretory and clearance values, thus emphasizing the broad spectrum of normal physiological activity of the somatotropic axis before puberty. Other data indicate similar variability among individuals during and after puberty (24-27, 29, 30, 3548). Of interest, some of the differences identified in GH secretory behavior among otherwise comparable individuals within a given diagnostic category can be related to specific variables, such as body mass index, bone age, or sex steroid hormone concentrations (29, 30, 36, 45, 4850). Acknowledgments We thank Patsy Craig for her skillful preparation manuscript, and Paula P. Azimi for the artwork.
of the
References 1. Costin G, Kaufman FR. Growth hormone secretory patterns in children with short stature. J Pediatr. 1987;110:362-8. 2. Spiliotis BF, August GP, Hung W, Sonis W, Mendelson W, Bercu BB. Growth hormone neurosecretory dysfunction: a treatable cause of short stature. JAMA. 1984;251:2223-30. 3. Adlard P, Buzi F, Jones J, Stanhope R, Preece MA. Physiological growth hormone secretion during slow-wave sleep in short prepu-
ET AL.
JCE & M -1992 Voll4.No4
bertal children. Clin Endocrinol (Oxf). 1987;27:355-61. hormone secretion in patients with constitutional 4. Lanes R. Growth delay of growth and pubertal development. J Pediatr. 1986;109:781n 5. goose SR Ross JL Uriarte M Barnes KM Casorla FG Cutler Jr GB. The advantage of measuring stimulated as compared with spontaneous growth hormone levels in the diagnosis of growth hormone deficiency. N Engl J Med. 1988;319:201-7. 6. Bercu BB, Shulman D, Root AW, Spiliotis BE. Growth hormone (GH) provocative testing frequently does not reflect endogenous GH secretion. J Clin Endocrinol Metab. 1986:63:709-16. 7. Hindmarsh PC, Matthews DR, Brook CG. Growth hormone secretion in children determined by time series analysis. Clin Endocrinol (Oxf). 1988;29:35-55. 8. Isgaard J, Carlsson L, Isaksson OGP, Jansson JO. Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases insulin-like growth factor I messenger RNA in skeletal tissues more effectively than continuous GH infusion. Endocrinology. 1983;123:2605-10: 9. Maiter D. Underwood LE. Maes M. Davenuort ML. Ketelslegers JM. Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedin-C/insulin-like growth factor I and liver GH receptors in hypophysectomized rats. Endocrinology. 1988;123:1053-9. 10. TannenbaumGS. Somatostatin as a physiological regulator of nulsatile erowth hormone secretion. Horm Res. 1988;29:70-4. 11. Plotsky PM, Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophyseal-portal circulation of the rat. Science. 1985;230:461. 12. Urban RJ, Evans WS, Rogol AD, Kaiser DL, Johnson ML, Veldhuis JD. Contemporary aspects of discrete peak detection algorithms. I. The paradigm of the luteinizing hormone pulse signal in men. Endocr Rev. 1988;9:3-37. 13. Veldhuis JD, Carlson ML, Johnson ML. The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proc Nat1 Acad Sci USA. 1987;84:7686-90. 14. Veldhuis JD, Johnson ML, Dufau ML. Physiological attributes of endogenous bioactive luteinizing hormone secretory bursts in man: assessment by deconvolution analysis and in vitro bioassay of LH. Am J Physiol. 1989;256:E199-207. 15. Veldhuis JD, Iranmanesh A, Lizarralde G, Johnson ML. Amplitude modulation of a burst-like mode of cortisol secretion gives rise to the nyctohemeral glucocorticoid rhythm in man. Am J Physiol. 1989;257:E6-14. 16. Roche AF, Chumlea WC, Thissen D. Assessing the skeletal maturity of the hand wrist. Springfield: Thomas; 1988;1-339. 17. Marshall WA, Tanner JM. Variation in the pattern of pubertal changes in boys. Arch Dis Child. 1970;45:13-23. AC, Chen AB, Wert RM, Sherman BM. Variability in the 18. Celniker quantitation of circulating growth hormone using commercial assays. J Clin Endocrinol Metab. 1989;68:469-76. 19. Felder RA, Ho11 RW, Martha Jr P, et al. Influence of matrix on concentrations of somatotropin measured in serum with commercial immunoradiometric assay. Clin Chem. 1989;35:1423-6. principles in experimental design. New York: 20. Winer BJ. Statistical McGraw-Hill: 1971;300-50. 21. Rolland-Cachera M, Sempe M, Guilloud-Bataille M, Patois E, Peauienot-Gueeenbuhl F. Fautrad V. Adiuositv indices in children. Am J Clin Nt&. 1982;36:178-84. _ ” ME, Van Wyk JJ, Underwood LE. Human growth 22. Abe H, Molitch hormone and somatomedin-C suppress the spontaneous release of growth hormone in unanesthetized rats. Endocrinology. 1983; 113:1319-22. 23. Berelowitz M, Szabo M, Frohman LA, et al. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and pituitary. Science. 1981;212:1279-81. 24. Hindmarsh P. Smith PJ. Brook CG. Matthews DR. The relationship between height velocity and growth hormone secretion in short prepubertal children. Clin Endocrinol (Oxf). 1987;27:581-91. 25. Martha Jr PM, Rogol AD, Veldhuis JD, Kerrigan JR, Goodman
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GH SECRETION
26.
21.
28. 29.
30.
31.
32. 33. 34. 35.
36.
37.
DW, Blizzard RM. Alterations in the pulsatile properties of circulating growth hormone concentrations during puberty in boys. J Clin Endocrinol Metab. 1989;69:563-70. Mauras N. Blizzard RM. Link K. Johnson ML. Roe01 AD. Veldhuis JD. Augmentation of growth hormone secretion-during puberty: evidence for a pulse amplitude-modulated phenomenon. J Clin Endocrinol Metab. 1987;64:596-601. Mauras N. Roe01 AD. Veldhuis JD. Snecific time denendent actions of low-dose e&adiol’administration bn the episodic release of GH, FSH, and LH in prepubertal girls with Turner’s syndrome. J Clin Endocrinol Metab. 1989;69:1053-8. Veldhuis JD, Johnson ML. A novel general biophysical model for simulating episodic endocrine gland signaling. Am J Physiol. 1988;255%749-59. Martha Jr PM. Rosol AD. Blizzard RM. Veldhuis JD. The nature of pituitary growth hormone secretory events and endogenous production rates in pubertal boys and young men. Pediatr Res. 1990;27:81A. Veldhuis JD, Iranmanesh A, Ho KKY, Lizarralde G, Waters MJ, Johnson ML. Dual defects in pulsatile growth hormone secretion and clearance subserve the hyposomatotropism of obesity in man. J Clin Endocrinol Metab. 1991;72:51-9. Martha Jr PM. Roe01 AD. Blizzard RM. Shaw MA. Baumann G. Growth hormone-bmding protein activity is inversely related to 24 hour growth hormone release in normal boys. J Clin Endocrinol Metab. 1991;73:175-81. Daughaday WH, Trivedi B, Andrews BA. The ontogeny of serum GH binding protein in man: a possible indicator of hepatic GH receptor development. J Clin Endocrinol Metab. 1987;65:1072-4. Undate on the Kabi International Growth StudvI Aoril _ 1989. Acta P’aed Stand. 1989;356:173-7. Genentech Collaborative Study Group. Idiopathic short stature. Results of a one-year controlled study of human growth hormone treatment. J Pediatr. 1989;115:713-9. Owens D, Srivastava MC, Tompkins CV, Nabarro JDN, Sonksen PH. Studies on the metabolic clearance rate, apparent distribution space, and plasma half-disappearance time of unlabeled human growth hormone in normal subjects and in patients with liver disease, renal disease, thyroid disease, and diabetes mellitus. Eur J Clin Invest. 1973;3:284-93. Kowarski A, Thompson RG, Migeon CJ, Blizzard RM. Determination of integrated plasma concentrations and true secretion rates of human growth hormone. J Clin Endocrinol Metab. 1971;32:35660. Rosenbaum M, Gertner JM. Metabolic clearance rates of synthetic human growth hormone in children, adult women, and adult men. J Clin Endocrinol Metab. 1989;69:821-4. ,”
I
IN SHORT BOYS 38. Parker ML, Utiger RD, Daughaday WH. Studies on human growth hormone. II. The physiological disposition and metabolic fate of human growth hormone in man. J Clin Invest. 1962;41:262-7. 39. Glick SM, Roth J, Lonergan ET. Survival of endogenous human growth hormone in plasma. J Clin Endocrinol Metab. 1964;24: 501-6. human growth 40. Boucher BJ. Disappearance of iodine-131-labelled hormone from the plasma of diabetic, non-diabetic, and acromegalic subjects. Nature. 1966;210:1288-9. 41. Refetoff S, Sonksen PH. Disappearance rate of endogenous and exogenous human growth hormone in man. J Clin Endocrinol Metab. 1970;30:386-92. 42. Hendricks CM, Eastman RC, Takeda S, Asakawa K, Gorden P. Plasma clearance of intravenously administered pituitary human growth hormone: gel filtration studies of heterogeneous components. J Clin Endocrinol Metab. 1985;60:864-71. 43. Baumann G, Stolar MW, Buchanan TA. The metabolic clearance, distribution, and degradation of dimeric and monomeric growth hormone (GH): implications for the pattern of circulating GH forms. Endocrinology. 1986;119:497-501. 44. Hindmarsh PC, Matthews DR, Brain CE, et al. The half-life of exogenous growth hormone after suppression of endogenous growth hormone secretion with somatostatin. Clin Endocrinol (Oxf). 1989;30:443-50. K. Rosbers S. Libre E. Lundberg L-O. Groth 45. Albertsson-Wikland T. Growth hormone skcretory”rates in children as estimated by deconvolution analysis of 24-hr plasma concentration profiles. Am J Physiol. 1989;257:E809-14. 46. Faria ACS. Veldhuis JD. Thorner MO. Vance ML. Half-time of endogenous growth hormone disappearance in normal man after stimulation of GH secretion by GHRH and suppression with somatostatin. J Clin Endocrinol Metab. 1989;68:535-41. 47. Veldhuis JD, Faria A, Vance ML, Evans WS, Thorner MO, Johnson ML. Contemporary tools for the analysis of episodic growth hormone secretion and clearance in uiuo. Acta Paediatr Stand. 1988;347:63-82. 48. Hartman ML, Faria ACS, Vance ML, Johnson ML, Thorner MO, Veldhuis JD. Temporal structure of in uivo growth hormone secretory events in man. Am J Physiol. 1991;260:ElOl-10. 49. Kerriaan JR. Martha Jr PM. Blizzard RM. Christie CM, Rogol AD. Variations of pulsatile growth hormone release in healt?ly short prepubertal boys. Pediatr Res. 1990;28:11-4. 50. Iranmanesh A, Lizarralde G, Veldhuis JD. Age and relative adiposity are specific negative determinants of the frequency and amplitude of growth hormone (GH) secretory bursts and the halflife of endogenous GH in healthy men, J Clin Endocrinol Metab. 1991;73:1081-8.
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Vol. 74, No. 4 Printed in U.S.A.
Properties of Spontaneous Growth Hormone Bursts and Half-Life of Endogenous Growth Boys with Idiopathic Short Stature*
Secretory Hormone
in
JOHANNES D. VELDHUIS, ROBERT M. BLIZZARD, ALAN D. ROGOL, PAUL M. MARTHA, JR:~, JOHN L. KIRKLAND, BARRY M. SHERMAN, AND GENENTECH COLLABORATIVE GROUP Division of Endocrinology and Metabolism, Departments of Internal Medicine (J.D. V.), Pharmacology (A.D.R.), and Pediatrics (R.M.B., A.D.R., P.M.M.), National Science Foundation Science Center in Biological Timing (J.D. V.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; Department of Pediatrics, Baylor College of Medicine (J.L.K.), Houston, Texas 77030; and the Clinical Research Division, Genentech, Inc. (B.M.S.), South San Francisco, California 94080
ABSTRACT.
We analyzed endogenous GH secretory dynamics and MCRs by a novel quantitative deconvolution technique in 20 boys with idiopathic short stature (ISS) and 35 boys of normal stature in Tanner stage I of puberty. We tested the null hypotheses that 1) ISS is not associated with any alterations in the frequency, mass, amplitude, or duration of spontaneous GH secretory bursts and/or the 24-h GH production rate; and 2) the half-life of endogenous GH is not altered in ISS. The boys with ISS had a mean (+SEM) bone age of 8.0 + 0.42 yr and a chronological age of 10 f 0.50 yr. The latter was similar to the chronological (and bone) age of the normal boys of 9.8 f 0.23 (and 9.3 + 0.34) yr. Mean height SD scores were significantly lower in ISS boys, uiz. -2.7 + 0.15 in ISS us. +0.34 + 0.13 in normal boys (P < 0.001). Plasma insulin-like growth factor-I concentrations were similar in the two groups, as were (24-h) mean serum GH concentrations, uiz. 3.5 + 0.29 rg/L in ISS and 4.15 0.49 rg/L in normal boys (P = NS). Deconvolution analysis revealed that the mean number of GH secretory events per 24 h was similar in normal and ISS boys, uiz. 9.6 + 0.76 (normal) us. 8.4 + 0.55 (ISS), and that there was no significant difference in mean GH interburst intervals. The amplitude, mass, and dura-
T
HE PATHOPHYSIOLOGY of impaired linear growth in children with short stature has been the object of intensive clinical investigation (l-4). Many Received March 18, 1991. Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. * This work was supported in part by NIH Grant RR-00847 (to the Clinical Research Center of the University of Virginia), Research Career Development Award l-K04-HD-00634 (to J.D.V.), Diabetes and Endocrinology Research Center Grant 5-P60-AM-22125-05, NIHsupported Clinfo Data Reduction Systems, the Pratt Foundation, the Academic Enhancement Fund, and the NSF Science Center in Biological Timing. The members of the Genentech Collaborative Study Group are: Ann Johanson (Genentech), Robert Blizzard (University of Virginia Medical Center), Jose Cara (University of Chicago Hospital), Steven Chernausak (Children’s Hospital Medical Center, Cincin
tion of computer-resolved GH secretory bursts also did not differ in normal and ISS boys. The half-lives of endogenous GH were estimated to be 16 f 0.77 min in the ISS and 18 + 0.93 min in the control boys (P = NS). The calculated daily GH secretion rate per unit distribution volume was not significantly reduced in ISS, i.e. 194 + 19 pg/L.day in ISS us. 177 + 19 pg/L.day in control boys. Moreover, daily GH secretion rates corrected for body mass index (weight/height’) in the twp groups were not significantly different. In summary, the present cohort of boys with ISS manifested no significant alterations in GH secretory burst frequency, duration, mass, or amplitude or in the half-life of endogenous GH compared to normal boys in Tanner stage I of pubertal development. Indeed, whether daily GH secretion rates are expressed per unit distribution volume or per unit body mass index, groups of boys with ISS and normal height controls secrete similar total amounts of GH. We conclude that the overall dynamics of GH secretion and clearance in boys with ISS considered as a whole cannot be distinguished readily from physiological patterns observed in prepubertal boys of normal height. (J Clin Endocrinol
Metub 74: 766-773,1992)
studies have focused on possible disturbances in the neuroendocrine regulation of episodic GH release (5-7). Evaluations of pulsatile GH secretion are based on the premise that bursts of GH release are important in the physiological activation of relevant target tissues, including, for example, the stimulation of linear bone growth nati, OH), James Frane (Genentech), Joseph Gertner (New York Hospital, Cornell Medical Center), Raymond Hintz (Stanford University Medical Center), Nancy Hopwood (University of Michigan Medical Center), Selna Kaplan (University of California School of Medicine), John Kirkland (Baylor College of Medicine, Houston, TX), Barbara Lippe (UCLA Hospital, Los Angeles), Leslie Plotnick (Johns Hopkins Hospital, Baltimore, MD), Paul Saenger (Montefiore Hospital, New York, NY), and Barry Sherman (Genentech). t%urrent address: Baystate Medical Center, 759 Chestnut Street, Springfield, Massachusetts 01199.
766
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GH SECRETION
and the induction of specific hepatic and muscle proteins and genes in the rat (8, 9). The pathophysiological regulation of pulsatile GH signaling to target tissues is achieved by two major hypothalamic effector systems, which comprise an inhibitory (somatostatin) and stimulatory (GH-releasing hormone) limb (10, 11). Despite extensive studies of putative neuroendocrine disturbances in children with idiopathic short stature (ISS), there is no published information regarding the underlying GH secretory dynamics and subject-specific metabolic clearance of endogenous GH in boys with this clinical diagnosis. In general, detailed assessment of endogenous GH secretion and clearance in man has been limited by the previous difficulties inherent in quantitating hormone secretion and disposal rates in uiuo (12). Such difficulties arise in part because most methods to estimate endogenous hormone secretion rates depend upon the infusion of radiolabeled or “cold” hormone under equilibrium or nonequilibrium conditions. In such circumstances, the MCR of the infused hormone species can be calculated and the production rate of endogenous hormone estimated. To our knowledge, such studies have not been carried out to compare large groups of normally growing boys and individuals with ISS. The development of specialized analytical tools, such as deconvolution analysis, permits the estimation of endogenous hormone secretory rates and subject-specific half-lives from pulsatile serum hormone concentration profiles without the infusion of radiolabeled or unlabeled hormone (13). For example, one technique, termed multiple parameter deconvolution analysis, allows a statistically based appraisal of the number, amplitude, mass, and duration of underlying hormone secretory bursts and a simultaneous estimate of the endogenous half-life of the hormone of interest based on all measured serum hormone concentrations and their associated dose-dependent intrasample variances (13-15). Accordingly, we have used this new analytical technique to quantitatively evaluate the episodic mode of GH secretion and clearance in 35 individual prepubertal (Tanner stage I) boys of normal height and 20 chronological age-matched boys with ISS. We have been able to test the null hypothesis that the mean number, amplitude, mass, and duration of spontaneous GH secretory bursts and the mean half-life of endogenous GH do not differ between groups of normally growing boys and their counterparts with ISS. Materials
and Methods
Clinical protocol Pulsatile GH secretion and endogenous GH half-lives were studied in 35 healthy prepubertal boys of normal height and 20 boys with ISS. Control boys had heights and weights within 1.96 SD of age-derived normal measures. Boys with ISS were
IN SHORT BOYS
defined by 1) serum GH concentrations exceeding 10 pg/L after provocative testing (L-dopa, clonidine, arginine, sleep, or exercise), 2) height at least 2 SD below age- and sex-matched normative values (absolute range, -2.0 to -4.6), and 3) the absence of any history of intrauterine growth retardation, underlying systemic, metabolic, or known endocrine disease, dysmorphic traits, or central nervous system irradiation. We did not attempt to quantitate the relative contributions of genetic vs. constitutional delay of growth in ISS boys. The bone age of the ISS boys determined radiographically at the wrist (16) averaged 8.0 f 0.42 yr, and 13 of 20 boys had a bone age delay of at least 2 yr. The mean chronological ages (+SEM) of the normal and short subjects were, respectively, 9.8 * 0.23 and 10.0 f 0.50 yr (P = NS). ISS boys had a mean growth rate of 4.3 + 0.29 cm/yr. Other clinical comparisons are given in Table 1. All subjects were in Tanner genital and pubic hair stage I (17) and were studied after a guardian provided written informed consent, approved by the Institutional Human Investigations Review Board. Each guardian provided a detailed clinical history. The boys each underwent a complete physical examination and had normal fasting plasma glucose concentrations, unremarkable biochemical tests of renal, hepatic, and hematological function, and normal serum concentrations of Tq. Boys were admitted to the study center the evening or morning before blood sampling, which was carried out at 20min intervals for 24 h, beginning at least 1 h after venipuncture. Blood was withdrawn through an iv catheter placed in a forearm vein. Subjects were permitted to ambulate and were given three TABLE
1. Clinical characteristics
of boys with normal stature and ISS Normal stature (n = 35)
Chronological
age (yr)
9.8 + 0.23 (6-12)
Bone age (yr)
Wt 04 Ht age (yr) Height SD score 24-h mean serum GH cont. WJJ Plasma IGF-I (U/L) Body mass index (kg/m’)
9.3 + 0.34 (6.0-12.5) 33 + 1.2 (21-50) 10.0 f 0.28 (5-12) 0.31 + 0.14 (-1.5 to 2.0) 4.1 + 0.49 (1.0-15)
0.93 + 0.10 (0.29-2.1) 17.2 + 0.42 (11-23)
ISS (n = 20) 10.0 t 0.50" (6-14) 8.0 + 0.42* (3.6-10.6) 23 + l.O* (14-33) 7.1 * 0.40* (3-10) -2.7 + 0.15' (-4.6 to -2.0) 3.5 + 0.29" (1.5-6.2) 0.76 + 0.08" (0.20-1.5) 15.6 f 0.31' (12-20) -0.70 + 0.25 (-3to 1.5)'
Body mass index SD score for +0.33 + 0.25 (-4.6 to 3.8)” age and sexd Data are the mean + SEM. Parentheses contain ranges. Bone age was determined in only 24 of the 35 boys of normal stature. “P=NS. * P < 0.001 us. normal stature. ‘P = 0.004. d Calculated as the difference between the logarithms of the observed and normative (age- and sex-specific) body mass indices divided by the corresponding soteference value (Table 5 in Ref. 21). ‘P = 0.007.
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VELDHUIS
768
meals per day (at 0800, 1200, and 1700 h). They were not allowed to nap or sleep until 2200 h. After the blood clotted at room temperature for 1 h, the sera were separated and frozen for later immunoradiometric assay (IRMA) of GH concentrations. Assays Serum GH concentrations were determined in replicate (duplicate) by IRMA, in which the mean (*SD) intraassay coefficient of variation determined from all 73 replicated samples was 5.1 f 1.2% in the 35 control subjects and 4.4 f 1.1% in the ISS boys. All data were extrapolated from and/or expressed in relation to human GH standards (Nichols Institute, San Juan Capistrano, CA) diluted with horse serum matrix (18, 19). The assay sensitivity was 0.50 Kg/L, and the interassay coefficient of variation was less than 9.5%. The plasma somatomedin-C [insulin-like growth factor-I (IGF-I)] content was determined by the Nichols Institute direct assay. Convolution modeling Deconvolution analysis permits quantitative estimates of individual hormone secretory parameters and the calculation of subject-specific hormone half-life from all serum hormone concentrations and their dose-dependent intrasample variances considered simultaneously (13, 15). Calculations assume that the serum GH concentration at any given instant reflects the simultaneous operation of four determinable secretory and clearance features of interest: 1) the number and locations, 2) the amplitudes, and 3) the durations of all significant GH secretory bursts, which are acted upon by 4) endogenous subject-specific clearance kinetics. The GH disappearance function was defined by a subject-specific monoexponential half-life fitted simultaneously with the various secretion parameters. GH concentrations were assumed to decay to the sensitivity of the GH IRMA. We calculated the following features of GH secretion and clearance: the half-duration (duration at half-maximal amplitude) of GH secretory bursts, amplitudes (maximal values) and temporal positions of all significant GH secretory bursts, and GH half-life. The mass of GH secreted per pulse is the analytical integral (area) of the resolved secretory burst, and the 24h production rate is the product of the total number of secretory bursts and the mean mass of GH released per burst (13-15). Although an interpeak basal secretory rate can be fitted in principle, no tonic secretion function was required to model the present data.
ET AL.
JCE & M .1992 Vol74.No4
on the logarithm of the expected normative value (21). P < 0.05 was construed as statistically significant.
Results The clinical features of the 35 boys of normal stature and the 20 subjects with ISS are summarized in Table 1. The chronological ages of the ISS and control boys were similar, as were mean (24-h) serum GH concentrations. Individual values of mean 24-h serum GH concentrations are shown in Fig. 1. Plasma IGF-I concentrations also were not significantly different, but boys with ISS had significantly reduced body mass index, whether expressed as an absolute value (kilograms per m”) or as an age- and sex-adjusted SD score (21). As illustrated in Fig. 2, both normal boys and those with ISS exhibited an exclusively episodic or pulsatile mode of GH release over the 24 h of blood sampling. We have depicted the observed serial serum GH concentrations over 24 h in two normal boys and two subjects with ISS. The fitted continuous curves through the GH concentration data (Fig. 2, upper subpanels) represent the predictions of the multiple parameter convolution model. The lower subpanels of Fig. 2 show the deconvolutionresolved GH secretory bursts and give their amplitudes (maximal rate of GH secretion attained per burst), halfdurations (duration of the GH secretory burst at halfmaximal amplitude), and locations in time. The mean quantitative characteristics of episodic GH secretion and clearance are given in Table 2. The frequency of spontaneous GH secretory bursts was similar statistically in boys with ISS and their controls. The mean interburst intervals, half-lives of endogenous GH, and half-durations of GH secretory bursts (duration of 3.5*0.29
Statistical analyses Unpaired two-tailed Student’s t testing and the Wilcoxon rank sum (nonparametric) test were used respectively for normal and nonGaussian distributed variables, as defined by the Wilk-Shapiro statistic (20). Data are given as the mean f SEM. Linear regression analysis was used to assess relationships between specific measures of GH secretion and clearance and bone age or body mass index (weight/height’). The latter was transformed to an age-dependent male-specific SD score based
01
/
Control
I
I
Short
,
Stature
1. Individual mean (24-h) serum immunoradiometric GH concentrations in 20 boys with ISS and 35 normal prepubertal (Tanner stage I) boys. Etch value is the mean of 72 sample estimates derived by withdrawing blood at 20-min intervals for 24 h.
FIG.
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GH
SECRETION
IN
A
SHORT
BOYS
769
NORMAL 12.5
h
B
10.0
5.0 2.5
. !
1.51
FIG. 2. Profiles of serum GH concentration peaks and deconvolution-resolved GH secretory bursts in two boys with normal stature (A) and two boys with 1% (B). Each upper subpanel gives the measured serum GH concentrations in blood collected serially at 20-min intervals over 24 h. The continuous curves through the observed serum GH concentration data were calculated by the multiple parameter convolution model. The lower subpanels depict the deconvolution-resolved GH secretory rates as a function of time. Quantitative secretion profiles were determined in each boy by simultaneously estimating values of relevant secretion parameters and the subject-specific GH half-life. The mean results for all 35 boys with normal stature and the 20 boys with ISS are summarized in Table 2. GH concentration units in nanograms per mL = micrograms per L. Note the different absolute values of the vertical axes.
0
1000
500
1500
0
TIME (MINI
6 12
A
SHORT
STATURE
25
yB
-4
/\I A AA 0
500
1000
1500
TIME the secretion event at half-maximal amplitude) were similar in both study groups, as were the mean amplitudes of GH secretory bursts (maximal rate of GH secretion attained within a release episode). The mass of GH released per secretory event (calculated as the area under the computer-resolved GH secretory pulse) also was not significantly different between normal and ISS boys (Table 2). Since the daily secretion or production rate is the product of the mean mass of
0
500
A
br
! ,L 1000
, , ,I 1500
(MINI
hormone secreted per pulse and the number of pulses per 24 h, we estimated a mean production rate of endogenous GH of 177 f 19 pg/L.day in the normal boys and 194 f 19 pg/L. day in those with ISS (P = NS; Fig. 3). Because of significant weight differences between the two groups, GH production rates were also expressed per kg body mass. According to such calculations (Table 2), daily GH secretion rates were statistically higher in the ISS group; uiz. 8.9 f 0.98 us. 5.9 +- 0.69 Kg/L. kg in ISS and control
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770
VELDHUIS
ET AL.
JCE & M .1992 Vol74.No4
TABLE 2. Dynamics mal stature and ISS
GH half-life
of GH
(min)
Secretory burst half-duration (min) No. of GH secretory pulses/ 24 h Interpulse interval (min) Secretory burst amplitude (pg/L. min) * GH mass secreted/pulse km * Daily production pg/L. 24 hb
kg/m’.
and clearance
in boys
with
Normal stature (n = 3.5)
ISS (n = 20)
18 f 0.93
16 + 0.77”
(8-35) 24 + 1.6
(8-23) 25 + 1.4“
9.6
(9-44) + 0.74 (2-21)
nor-
A
9.6kO.7
8.410.6
(17-40) 8.4 + 0.55"
168k 14 (66-467) 0.74 f 0.06
(5-15) 164 + 11" (98-251) 0.92 + 0.11"
(0.31-1.9) 21k2.9 (3.7-97)
(0.22-1.8) 25 + 2.4" (7-39)
rate 177*
a/L~k/24 h rg/L.
secretion
24 h
19
194
(14-423) 5.9 + 0.69 (0.36-13)
(48-353) 8.9 + 0.98" (1.5-16)
11 f (0.71-25)
13 + 1.3" (2.4-22)
1.2
Short
Control
+ 19”
164+10.8
168+14.2
500
B
-I-
In
i
5
Stature
0
m
Data are the means + SEM. Ranges are given in parentheses. “P=NS. * The volume term (L) refers to unit distribution value for GH. ‘P = 0.017 US. boys of normal stature.
boys, respectively (P = 0.017). However, there was substantial overlap between values in the two groups. Moreover, when daily GH secretion rates were expressed further per unit body mass index (weight/height’; Table 2) or the corresponding SD scores (not shown), there were no significant differences between ISS boys and normal stature controls. Linear regression analysis revealed that in normal boys the body mass index SD score tended to correlate negatively with the daily GH secretory rate (r = -0.287; P = 0.09). The corresponding correlation in boys with ISS was r = -0.435 (P = 0.05). Of interest, in boys with ISS, this measure of sex- and age-adjusted body mass index also correlated negatively with GH secretory burst amplitude (r = -0.590; P = 0.006) and mass (r = -0.440; P = 0.05). Bone age correlated negatively with several quantitative features of GH secretion in normal boys, viz. GH secretory burst half-duration (r = -0.572; P = 0.003), mass of GH secreted per burst (r = -0.415; P = 0.04), and daily GH secretion rate (r = -0.440; P = 0.03). Discussion We have tested the null hypothesis that as a group young boys with ISS exhibit no measurable alterations in specific quantitative features of GH secretion and/or clearance compared to prepubertal boys of normal stature. Using deconvolution analysis, we observed statistically similar daily GH production rates in 20 prepubertal boys with ISS and 35 Tanner stage I counterparts of
01
I
Control
FIG. 3. Number of GH secretory (B) in individual normal (control) may represent 1 or more subjects.
/
I
Short
1
Stature
bursts (A) and interburst and short boys. An individual
intervals symbol
normal stature. The 2 groups also manifested indistinguishable mean GH secretory burst mass, frequencies (and, correspondingly, interburst intervals), durations, and amplitudes and endogenous GH half-lives. Therefore, we infer that the overall dynamics of GH secretion and clearance are not statistically distinguishable in this group of boys with ISS considered as a whole and their Tanner stage I (prepubertal) male counterparts of normal stature. These results contrast with some previous studies using various empirically based methods to identify numbers of plasma GH concentration peaks, which suggested either similar or reduced numbers and/or amplitudes of GH concentration peaks (l-6). Most of these studies involved fewer subjects than evaluated here (35 controls and 20 boys with ISS), and none quantitated GH secretory rates and subject-specific GH half-lives. In addition, our study may differ in the selection and/ or ascertainment of study subjects with normal growth
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GH SECRETION
or ISS. Plasma IGF-I concentrations were similar in our boys of normal stature and those with ISS. This suggests that GH action, at least on target tissues that contribute substantially to circulating total IGF-I levels (e.g. liver), is normal in ISS boys. However, normal GH action cannot necessarily be inferred for other sites of locally synthesized IGF-I, if such sites contribute only sparingly to the large plasma IGF-I pool, e.g. GH target cells in the skeleton, pituitary gland, and hypothalamus (22, 23). Furthermore, similar plasma total IGF-I concentrations in boys with ISS and controls do not rule out differences in sensitivity to the negative feedback actions of IGF-I on the somatotropic axis and/or differences in the specific association of IGF-I with one or more circulating IGF-I-binding proteins. Recent studies of serum GH concentration peaks (as distinguished from computer-resolved GH secretory bursts, described here) have suggested that the amplitude (height) and/or area, rather than the number of serum GH concentration peaks correlates positively with linear growth velocity in healthy pubertal children (24-26) and in boys (26) and girls (27) treated with sex steroid.hormones. Mathematical modeling of pulsatile endocrine signals indicates that an increase in the incremental height or area of plasma hormone concentration peaks can result from either an augmented secretory burst amplitude (maximal secretory rate achieved within each pulse) and/or an increased duration of each underlying secretory burst (28)l. In this regard, recent deconvolution studies have disclosed that GH production rates rise because GH secretory burst amplitude and mass specifically increase at the time of puberty, whereas GH secretory burst frequency and duration, and the endogenous GH half-life do not vary in puberty (29). We observed that boys with ISS have statistically similar mean GH secretory burst amplitudes, mass, and daily GH secretion rates when secretory data are expressed as micrograms of GH secreted per L distribution volume. However, when daily GH secretion is computed as a volume- and weight-adjusted value (micrograms per L/kg. day), ISS boys secrete slightly more GH than boys of normal stature, which presumably reflects the former group’s significantly reduced body mass. When GH secretion is evaluated further per unit body mass index, wherein body weight is expressed either in absolute terms or as a SD score in relation to the square of the child’s height (al), equivalent daily GH secretion rates are estimated in boys of normal stature and those with ISS. Consequently, we infer that as a whole, groups of idiopathically short and normal stature boys do not secrete 1 Increased tonic (interburst) GH secretion, diminished GH distribution volume, and/or a prolongation of GH half-life in principle also could increase maximal serum GH concentration peak heights (26).
IN SHORT
BOYS
771
different amounts of GH over 24 h given their differences in both weight and height. In the 35 prepubertal boys of normal stature, we found that age was a significantly negative predictor of GH secretory burst mass and duration and daily GH secretion rate. These relationships have not been recognized previously in this interval just before puberty, but they suggest that there may be regulation of GH secretion by as yet unspecified nutrient and/or metabolic signals that covary with prepubertal age. Alternatively, if the narrow time window of relatively decreased linear growth velocity before the adolescent growth spurt (17) is associated with decreased GH release, and if only children in prepuberty are studied (as here), then a negative correlation might be expected between age and GH release within this particular time span. Recent studies in men and pubertal boys have revealed an inverse relationship between body mass index, a measure of relative weight per unit surface area, and daily GH secretory rates (29,30). Similarly, the normal cohort of prepubertal boys studied here manifested a significantly negative correlation between body mass index and total GH production. Within the group of ISS boys, we found significantly negative associations between the SD score for sex- and age-adjusted body mass index and GH secretory burst amplitude and mass as well as daily GH secretion rates. Of interest in this regard is the recent finding that body mass index is a positive statistical correlate of plasma GH-binding protein concentrations in children (31). The high affinity plasma GH-binding protein closely resembles, and in some measure may reflect activity of, the extracellular domain of tissue GH receptors (32). Accordingly, it is possible that the association of low plasma GH-binding protein levels with a low body mass index might account for poor skeletal growth responses in some short patients with apparently normal GH secretion. This speculation also suggests the possibility that local generation of IGF-I and/or trophic actions of IGF-I on growth-related target tissues may be attenuated in some ISS patients. Larger amounts of GH might be expected to overcome such putative tissue resistance. The latter consideration is consistent with the demonstration that GH administration can stimulate significantly increased linear growth in some boys with ISS (33, 34). In normally growing prepubertal boys, deconvolution analysis revealed approximately 10 secretory episodes/ 24 h, which corresponds to an interpulse interval of 168 min. The average half-duration (duration of the calculated secretory event at half-maximal amplitude) of the GH secretory burst was 24 min, which indicates that sustained GH secretion occurs within any given release episode. This secretory event duration is approximately 2-fold longer than that reported for immunoactive LH
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772
VELDHUIS
or FSH or biologically active LH (13, 14), but similar to that estimated recently for cortisol in healthy men (15). The mass of GH discharged per burst averaged 21 pg/L distribution volume. Assuming a nominal GH distribution volume of 8% body weight (35), we can calculate that approximately 56 pg GH are secreted per spontaneous pulse in the normal Tanner stage I boys studied here, who weighed an average of 33 kg. The corresponding daily GH secretion rate is approximately 0.56 mg according to the assay, standards, and reagents used here. Of course, the absolute value of this estimate depends on the specific GH assay, reagents, matrix, and standards employed (18, 19). Direct estimates of subject-specific GH MCRs are limited in normal prepubertal (Tanner stage I) boys. However, our calculated endogenous GH half-lives are similar to many adult values inferred by others using exogenous GH. We estimated a mean half-life of endogenous GH of 18 f 0.93 min in normal boys, which falls within the range of values determined after the injection of purified pituitary or recombinant GH in adults and children (35-45). The present values also agree with recent estimates of endogenous GH half-lives in normal men, in whom pituitary secretion of GH was stimulated transiently by a bolus injection of GH-releasing hormone, after which further GH secretion was blocked by infusion of somatostatin (46). We observed a large range of GH secretion and clearance values in boys with ISS, some of which considered alone would differ considerably from the normal mean. However, prepubertal boys of normal stature also exhibited a wide dispersion of GH secretory and clearance values, thus emphasizing the broad spectrum of normal physiological activity of the somatotropic axis before puberty. Other data indicate similar variability among individuals during and after puberty (24-27, 29, 30, 3548). Of interest, some of the differences identified in GH secretory behavior among otherwise comparable individuals within a given diagnostic category can be related to specific variables, such as body mass index, bone age, or sex steroid hormone concentrations (29, 30, 36, 45, 4850). Acknowledgments We thank Patsy Craig for her skillful preparation manuscript, and Paula P. Azimi for the artwork.
of the
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GH SECRETION
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