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Clinical Research Center Studies |
Department of Pediatrics, Division of Endocrinology, and the Department of Internal Medicine (A.B.), University of Michigan Medical School, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Dr. Ayse Pinar Cemeroglu, D3252 Medical Professional Building, Box 0718, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718.
Abstract
Acceleration of linear growth during puberty is associated with increased GH secretion, although the relationship between growth and GH is complex. As GH exists as a family of isoforms, some of which may not be identified by immunoassay, there may be alterations in isoform secretion during pubertal maturation that result in increased growth. The changes in serum immunoreactive and bioactive GH concentrations across pubertal maturation were determined in 30 boys, aged 6.5–19.3 yr, with idiopathic short stature or constitutional delay of adolescence. Data were grouped as follows: 1) 6 prepubertal boys with bone age 7 yr or less; 2) 5 prepubertal boys with bone age of more than 7 yr; 3) 10 boys in early puberty; 4) 9 boys with mid- to late puberty. Blood was obtained every 20 min from 2000–0800 h. An equal aliquot of each serum sample was pooled for determination of GH by bio- and immunoassays. The mean serum immunoreactive GH concentration increased from 2.1 ± 0.3, 1.8 ± 0.3, and 2.9 ± 0.5 µg/L in groups 1, 2, and 3, respectively, to a peak of 4.6 ± 0.7 µg/L in group 4 (P < 0.05 vs. groups 1–3). The mean serum GH bioactivity was 48 ± 13 µg/L in group 1 and declined to 39 ± 8 and 31 ± 3 µg/L in groups 2 and 3, increasing to a maximum of 64 ± 15 µg/L in group 4 (P < 0.05 vs. group 3). The ratio of bioactive to immunoreactive GH suggests that the biopotencies of secreted isoforms do not increase during pubertal maturation. The role of E2 in increasing GH secretion was characterized in 8 additional early pubertal boys. Each boy received a saline infusion from 1000–0800 h, followed 1 week later by an infusion of E2 at 4.6 nmol/m2·h. Blood was obtained every 15 min from 2200–0800 h for GH and LH and every 60 min for E2 and testosterone. An equal aliquot of each overnight serum sample was pooled for insulin-like growth factor I (IGF-I) and GH by immuno- and bioassays. The mean serum LH concentration decreased from 5.0 ± 0.9 to 2.3 ± 0.6 IU/L (P < 0.01), and the E2 concentration increased from 22 ± 4 to 81 ± 26 pmol/L (P < 0.01) during saline and E2 infusions, respectively. Mean serum GH concentrations as measured by immunoassay were similar during both infusions (6.6 ± 1.4 vs. 9.7 ± 2.1 µg/L; saline vs. E2 infusion, respectively). In contrast, the mean serum GH concentration, as measured by bioassay, decreased from 48 ± 10 µg/L during saline infusion to 16 ± 3 µg/L during E2 infusion (P < 0.05). The mean serum IGF-I concentration also decreased significantly from 116 ± 17 to 93 ± 15 µg/L (saline vs. E2 infusion, respectively; P < 0.05). Thus, although mean overnight serum GH concentrations increase in late puberty, whether measured by immuno- or bioassay, an acute increase in E2 produces an acute decline in serum GH bioactivity and a lesser decline in the serum IGF-I concentration. These unexpected changes indicate that E2 may affect pubertal growth and GH secretion in a complex or biphasic manner depending on the context in which it is administered.
PUBERTAL growth in boys is associated with a marked acceleration of linear growth velocity, which peaks during late puberty. Although the rate of growth during puberty is associated with an increase in the serum GH concentration (1, 2, 3, 4, 5, 6, 7), the relationship is not linear (8, 9). This suggests that factors other than the GH concentration may influence the rate of growth during pubertal maturation. Alternatively, as GH is produced and secreted as a family of isoforms (10, 11, 12), and these isoforms may not be equally recognized by standard immunoassays, pubertal growth acceleration may be influenced by a change in the relative biopotency of secreted and circulating GH.
It has been difficult to assess GH bioactivity in serum because the available assays have either been relatively insensitive or have had nonphysiologic end points, limiting their utility (13, 14, 15, 16, 17). Recently, we developed an in vitro bioassay (18) that has enabled us to examine the changes in nocturnal serum bioactive as well as immunoreactive GH concentrations in a cross-sectional study of 30 boys in different stages of puberty. We hypothesized that the biopotencies of circulating GH isoforms increase more than the immunoreactive GH concentration during puberty, which may explain the nonlinear relationship between the serum immunoreactive GH concentration and pubertal growth.
Studies in patients with constitutional delay of growth and hypogonadism suggest that testosterone (T) replacement augments GH secretion and promotes the growth rate (19, 20). However, in pubertal boys, the estrogen concentration increases as well, mainly through peripheral aromatization of T (21). Thus, the relative importance of increased estrogen vs. T concentration on the pubertal increase in growth rate and GH secretion in boys is still not well understood (1). Estrogen receptor blockade with tamoxifen has been shown to diminish GH secretion in boys, providing indirect evidence for the role of estrogens in the male pubertal growth spurt (22). We have previously demonstrated that acute T infusion does not alter nocturnal GH secretion in pubertal boys, suggesting that T must be aromatized to estradiol (E2) to become effective (23). Thus, we designed another study to examined the effect of a 20-h E2 infusion on nocturnal serum immunoreactive and bioactive GH concentrations in early pubertal boys, testing the hypothesis that the pubertal increase in the serum GH concentration in boys may be related to an increased estrogen concentration.
Subjects and Methods
Subjects
The clinical characteristics of the 30 boys in the
cross-sectional study and those of the 8 boys in the E2
infusion study are shown in Tables 1
and 2 respectively. All of the boys studied had either
idiopathic short stature or delayed adolescence, but were otherwise
healthy and were not taking any medications. Thyroid function tests
were normal. For the purpose of comparison, the boys in the
cross-sectional study are divided into 4 groups as follows: group 1, 6
boys with stage I puberty with a bone age of 7 yr or less (young
prepubertal); group 2, 5 boys in stage I puberty with a bone age of
more than 7 yr (prepubertal); group 3, 10 boys in stage II puberty
(early pubertal); and group 4, 9 boys in stage III and IV puberty (mid-
to late pubertal). Subjects who had subnormal responses to GH
provocative tests (insulin and arginine tolerance tests) were excluded
from the study.
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The protocols were approved by the institutional review board of the University of Michigan. Informed consent was obtained from a parent and assent from the child before the study. All studies were carried out in the Kughn Clinical Research Center of the University of Michigan. All samples were immediately frozen after they were obtained and were thawed just before assay.
Cross-sectional study
An iv heparinized cannula was placed in a forearm vein 2 h before the study. Blood was obtained every 20 min from 2000–0800 h next morning. An equal aliquot of each serum sample was pooled from samples obtained between 2000–0800 h for determination of serum immunoreactive and bioactive GH, LH, FSH, E2, T, insulin-like growth factor I (IGF-I), and IGF-binding protein-3 (IGFBP-3).
E2 infusion study
Each boy was studied twice, 1 week apart. Boys spent the first day of each study acclimatizing to the unit. On the following day, an iv access was established in one forearm for administration of saline or E2, and a second heparinized iv cannula was placed in the opposite forearm to obtain blood samples. During the first study, saline was infused at 10 cc/h beginning at 2200 h and continuing through 0800 h. Blood was obtained at 15-min intervals from 1200–0800 h for serum GH determinations. E2 and T concentrations were determined every 60 min. The boys were readmitted 1 week later for an identical protocol, except that saline was replaced with an infusion of E2. The E2 (Sigma Chemical Co., St. Louis, MO) was dissolved in ethanol, diluted into saline to a final concentration of 4.6 nmol/m2 body surface area·10 cc saline and infused at a rate of 10 cc/h to approximate the adult blood production rate (26). For blood samples obtained during saline or E2 infusion, an equal aliquot of each serum sample from 2200–0800 h was also pooled for determination of immunoreactive and bioactive T, E2, LH, and IGF-I concentrations.
Hormone assays
For all patients in the E2 infusion study, except patient 7, the serum immunoreactive GH concentration was determined using a double antibody RIA (27). Standards and antibodies were obtained from the National Hormone and Pituitary Program (Baltimore, MD) and the NIDDK. The sensitivity of the assay was 0.5 µg/L, and the intra- and interassay coefficients of variation (CVs) were less than 4% and 10%, respectively. For patient 7 in the E2 infusion study, the serum immunoreactive GH concentration was determined by IFMA using a Delfia kit purchased from Wallac (Gaithersburg, MD). The immunoreactive GH concentrations in the samples assayed every 15 min overnight were used only to study the GH pulse characteristics and were not used to make comparisons with GH concentrations detected by bioassay. A comparison of the RIA and the IFMA was presented previously (28), and the assays were found to be comparable in terms of peaks detected and GH profile characteristics. For immuno- and bioassay comparisons, an equal aliquot of each sample was pooled for a single determination of serum GH concentration by IFMA and bioassay for all patients in the cross-sectional or E2 infusion studies.
Serum GH bioactivity was determined based on the ability of GH to suppress glucose metabolism in 3T3-F442A adipocytes as described previously (18). The standard was 22,000-dalton recombinant DNA-derived human GH and was a gift from Lilly Research Laboratories (Indianapolis, IN). The assay sensitivity was 3 µg/L, and the intra- and interassay CVs were 9% and 17%, respectively.
Serum E2 and T concentrations were determined by RIA using a kit obtained from Diagnostic Products Corp. (Los Angeles, CA). The assay sensitivity for E2 was 18 pmol/L, and the intra- and interassay CVs were 8% and 15%, respectively. The assay sensitivity for T was 0.35 nmol/L, and intra- and interassay CVs were 8% and 15%, respectively.
In the E2 infusion study, serum IGF-I concentrations were determined using a double antibody RIA after first extracting the serum samples with acetic acid and ethanol as described previously (29). The polyclonal antibody to IGF-I was obtained from the National Hormone and Pituitary Program, and the standard was purchased from Mallinkrodt Specialty Chemicals (St. Louis, MO). The intraassay CV was 8.8%, and all samples were measured in a single assay. The assay sensitivity was 1.0 ng/mL. Serum IGF-I and IGFBP-3 measurements in the cross-sectional study were performed using kits obtained from Endocrine Sciences (Calabasas Hills, CA). The assay sensitivity for IGF-I was 15 ng/mL, and the intra- and interassay CVs were 9% and 13%, respectively. The assay sensitivity for IGFBP-3 was 0.37 mg/L, and intra- and interassay CVs were 9% and 12%, respectively.
Serum LH and FSH concentrations were determined by IFMA using Delfia kits. The assay sensitivity for LH was 0.05 IU/L, and the intra- and interassay CVs were 5% and 6%, respectively. The assay sensitivity for FSH was 0.05 IU/L, and the intra- and interassay CVs were 3.0% and 4.5%, respectively.
Statistical analyses
All data were transformed logarithmically before analysis. Comparisons between each group in the cross-sectional study were made using factorial ANOVA. The data are represented as the mean \ SE. For the E2 infusion study, comparisons between treatments were made by Student’s paired t test for single comparisons or repeated measures ANOVA for multiple comparisons.
Pulse detection. Pulses of GH were determined using the DETECT method of Oerter et al. (30). The false positive peak detection level was set at less than 1% using the predicted variance model. All values less than assay sensitivity were assigned a value of assay sensitivity. Missing values were not replaced. Peak amplitudes were derived by calculating the difference between the peak and the prepeak nadir. All accepted peaks had an amplitude at least twice the assay sensitivity.
Results
Cross-sectional study
Mean overnight serum LH, FSH, E2, and T
concentrations.
In addition to physical signs, biochemical markers
of pubertal maturation in the subjects were assessed by determining
serum LH, FSH, T, and E2 concentrations in the
overnight pools. Serum LH, FSH, E2, and T concentrations
were the lowest in young prepubertal and prepubertal groups and
increased as pubertal maturation advanced, peaking in the mid- to late
pubertal group (Table 3).
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).
The mean overnight serum IGF-I concentration
increased from 106 \ 27 µg/L in group 1 to 169.9 \ 15
µg/L in group 2 to 195.7 \ 34.7 µg/L in group 3, and to a
peak concentration of 325.4 \ 54.7 µg/L in group 4 boys
(P <0.05 vs. group 1). Boys in the mid- to late
puberty (group 4) also had the greatest concentration of mean overnight
serum IGFBP-3 (P < 0.05 vs. group 1).
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). The
mean overnight serum bioactive GH concentration was 48 \ 13
µg/L in group 1, decreased from 39 \ 8 µg/L in group 2 to a
nadir of 31 \ 3 µg/L in group 3, and then increased 2-fold
in group 4 (64 \ 15 µg/L; P < 0.05
vs. group 3; Fig. 1). The ratios of bioactive GH to
immunoreactive GH were 23 \ 4, 23 \ 2, 13 \ 3, and
14 \ 3 in groups 1, 2, 3, and 4, respectively (P
< 0.05, group 2 vs. groups 3 and 4). E2 infusion study
To determine whether the changes we observed in serum immunoreactive and bioactive GH concentrations throughout male pubertal maturation in the cross-sectional study were related to increased circulating E2 concentrations, we compared the effects of a 20-h infusion of saline vs. E2 on serum GH and IGF-I concentrations in eight boys with stage II and III puberty.
Mean overnight serum LH, E2, and T
concentrations.
The effectiveness of the E2
infusion was assessed by measurement of E2, T, and LH
concentrations during saline and E2 infusions. The mean
overnight serum E2 concentration increased significantly
from 22 \ 4 pmol/L during saline infusion to 81 \ 26 pmol/L
during E2 infusion (P = 0.032), whereas the
mean serum LH and T concentrations decreased about 2-fold during the
E2 infusion compared to those during the saline infusion
(Table 4).
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Individual and mean data representing the effect of E2
infusion on immunoreactive and bioactive GH and IGF-I concentrations
are shown in Fig. 2. The mean overnight serum bioactive
GH concentration decreased from 48 \ 10 to 16 \ 3.0 µg/mL
during saline and E2 infusions, respectively
(P = 0.0487). Similarly, the mean serum IGF-I
concentration decreased from 116 \ 17 µg/L during saline
infusion to 93 \ 15 µg/L during E2 infusion
(P = 0.021).
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Increased linear growth during puberty occurs coincidentally with an increase in the mean serum GH concentration and GH pulse amplitude (1, 2, 3, 4, 5, 6, 7). It has been thought that sex steroids and, in particular, E2 increase GH secretion (31), yet a direct relationship among E2, serum GH, and pubertal growth has not been established even in thorough longitudinal studies in boys (32). We hypothesized that the isoform profile of secreted GH might change in the face of rising sex steroid concentrations at puberty, such that E2 might increase the production of a highly bioactive species of GH with limited immunoactivity. This hypothesis was examined in a cross-sectional study of serum GH bioactivity in boys across pubertal transition. Serum GH bioactivity in these boys increases abruptly between early and mid- to late puberty, but the ratio of bioactive to immunoreactive GH concentrations suggests that pubertal GH secretion is not characterized by a secretion of GH isoforms with high biological potency. It is of interest that serum bioactive GH concentrations are relatively greater in prepubertal boys with bone ages of 7 yr or less and decrease to a nadir in early puberty. This tendency of serum GH bioactivity to decline with age parallels the reported decline in growth velocity with advancing age in prepubertal boys, which reaches a nadir at 10–11 yr when pubertal signs first begin to appear (33). At this same time, the serum IGF-I concentration, another biological marker of GH activity, increases. Although serum IGF-I concentrations are not governed solely by GH, if the decline in GH bioactivity preceding the pubertal growth spurt is borne out in additional studies, then the current views regarding the interrelationship among GH, IGF-I, and growth will require reexamination.
The overall complexity of the relationship among serum GH, IGF-I, and E2 is highlighted by the unexpected results obtained when E2 was infused in early to midpubertal boys. We had expected that E2, if it had an effect, would increase the serum GH concentration determined by immuno- and bioassays. Instead, the serum GH concentration determined by immunoassay was constant over the 20-h infusion period, but serum GH bioactivity decreased by 67%, and that of IGF-I decreased by 20%. The reason for the acute decline in serum GH bioactivity is unclear. Serum E2 concentrations achieved by the infusions were well within the normal concentration range of adult men (<37 to 210 pmol/L) (26), but were twice the concentrations of mid- to late pubertal boys in our cross-sectional study. E2 infusion also suppressed serum T concentrations to values similar to those in the early pubertal boys in our cross-sectional study, the same boys who exhibited the lowest serum bioactive GH concentrations. This raises the intriguing possibility that the androgen, rather than the estrogen, concentration in boys governs GH secretion, plasma processing, or GH clearance in such a way to enhance GH bioactivity. Borski et al. showed in ovariectomized rats that chronic dihydrotestosterone treatment increases pituitary GH stores and circulating IGF-I levels and decreases circulating GH levels, whereas E2 treatment has the opposite effect (34). Thus, the serum concentration of E2 relative to that of T might be an important factor for the net effect on the GH axis. We have shown in another study that chronic E2 treatment in pubertal aged girls with gonadal dysgenesis leads to a significant increase in serum GH concentrations determined by immuno- and bioassays (35). These observations suggest that there may be sex differences in the control of GH secretion and in the response of the GH axis to E2 exposure during pubertal maturation and that acute and chronic E2 exposures have different effects on serum GH bioactivity. An initial decline in bioactivity with short term exposure to E2, as seen in early pubertal boys of our cross-sectional study, may be followed by an increase in GH bioactivity with prolonged exposure. Alternatively, there may be other factors in serum besides sex steroids that affect the GH axis during pubertal maturation.
Acute E2 infusion results in a small, but significant, decrease in the serum IGF-I concentration. In a similar study, a 4-day infusion of E2 in pubertal boys either did not change or increased the serum IGF-I concentration (36). These conflicting results regarding the response of the serum IGF-I concentration to E2 infusion may be due to differences in the duration of sex steroid exposure. The acute decrease in the IGF-I concentration may be related to either E2-mediated changes in synthesis, as observed in liver (34, 37), or its increased clearance from the circulation.
In this study we have shown that serum immunoreactive and bioactive GH concentrations increase during late puberty in concert with an increase in T and E2 concentrations, but the ratio of bioactive to immunoreactive GH concentrations suggests that pubertal growth in boys is not associated with a shift in GH secretion to isoforms with high biological potency. The relative importance of E2vs. T on GH secretion during pubertal maturation is still unclear. Acute E2 infusion does not alter the serum immunoreactive GH concentration, but serum IGF-I and bioactive GH concentrations decrease, paralleling the decrease in the T concentration, suggesting that the balance between T and E2 concentrations may be important for GH secretion in males. Alternatively, there may be other factors in the serum besides sex steroids that increase during pubertal maturation and cause the changes seen in GH and IGF-I secretion and the linear growth rate.
Acknowledgments
The authors thank Mrs. Maria Borondy and Mrs. Alice Rolfes-Curl for their expert technical assistance, and Dr. Nancy J. Hopwood for her helpful comments.
Footnotes
1 This work was supported by NIH Grants DK-43513 and HD-16000 and the
Kughn Clinical Research Center (M01-RR00042). Presented in part at the
76th Annual Meeting of The Endocrine Society, Anaheim, CA, 1994, and at
65th Annual Meeting of the Society for Pediatric Research, Washington,
D.C., 1996. 
2 Recipient of a fellowship award from the Genentech Foundation for
Growth and Development and supported in part by Eli Lilly Co. Funding
for Fellowship Education and Research.
3 Current address: Department of Pediatrics, University of
Washington, Seattle, Washington 98105.
4 Current address: National Center for Research Resources, National
Institutes of Health, Bethesda, Maryland 20892.
Received January 30, 1997.
Revised March 21, 1997.
Accepted March 31, 1997.
References
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 S. L. S. Drop, W. J. de Waal, and S. M. P. F. de Muinck Keizer-Schrama Sex Steroid Treatment of Constitutionally Tall Stature Endocr. Rev., October 1, 1998; 19(5): 540 - 558. [Abstract] [Full Text]
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