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Original Studies |
Research Center for Endocrinology and Metabolism, Department of Internal Medicine (C.L.B., B.C., L.M.S.C.), and International Pediatric Growth Research Center, Department of Pediatrics (C.J., M.C.S.B., S.R., K.A.W.), University of Göteborg, Göteborg, Sweden
Address all correspondence and requests for reprints to: Dr. Cesar L. Boguszewski, Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital, Bruna Stråket 16, S-413 45 Göteborg, Sweden. E-mail: cesar.boguszewski{at}ss.gu.se
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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GH is involved in the regulation of many aspects of growth, and short stature is a well established consequence of GH deficiency. However, the mechanisms of growth failure in a majority of short children who are not GH deficient (GHD) are much less clear and may involve abnormalities in different parts of the GH system. The fact that several different isoforms and fragments of GH are present in the circulation and that the activation of the GHR requires a ligand in which both site 1 and site 2 are intact suggests that an altered proportion of 22-kDa and non-22-kDa GH isoforms in the circulation may be one of such abnormalities that explains growth failure in some children with short stature.
The aim of this study was to evaluate the proportion of circulating non-22-kDa GH isoforms at the time of spontaneous GH peaks in samples from three groups of prepubertal short children and from a group of healthy prepubertal children of normal stature using the 22-kDa GH exclusion assay (GHEA), an immunomagnetic-extraction method for measurements of non-22-kDa GH isoforms in human blood (16). We have also studied the relationships among non-22-kDa GH isoforms, auxology, and spontaneous GH secretion.
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Subjects and Methods |
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A total of 66 short prepubertal children were studied. The group
consisted of 17 girls with Turner’s syndrome (TS), aged 3–13 yr (6.9
yr); 25 children (19 boys and 6 girls) born small for gestational age
(SGA) without postnatal catch-up growth, aged 3–13 yr (7 yr); and 24
children (15 boys and 9 girls) born appropriate for gestational age
(AGA) with idiopathic short stature (ISS), aged 4–15 yr (9.4 yr).
Twenty-three normally growing prepubertal children born AGA (13 boys
and 10 girls), aged 4–13 yr (10.2 yr), were used as a reference group.
Table 1
shows the clinical
characteristics of the study groups. Height and weight were converted
into a SD score using the childhood component (17) of the
Swedish growth reference values for healthy children (18). The weight
for height SD score was determined as previously reported
(19), and body mass index was calculated as body weight
(kilograms)/height (meters)2. The diagnosis of TS was
confirmed by karyotype analysis of peripheral lymphocytes: 13 girls
were 45,X, and the other 4 girls were 45,X/46,XX, 45,X/47,XXX,
45,X/46,XX/47,XXX and 45,X/46,X,i. SGA was defined as a birth weight
and/or birth length below -2 SD score compared with the
Swedish reference values of newborn infants (20, 21). One child born
SGA had signs of Silver-Russell syndrome (small and triangular facies,
body asymmetry, and clinodactyly of the fifth finger). The diagnosis of
ISS (height below -2 SD score for Swedish reference
values) (17, 18) was established in non-GHD children [GH response to
an arginine-insulin tolerance test >20 mU/L (
7.7 µg/L)] in whom
no cause of growth failure was identified.
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The children stayed at the hospital for at least 24 h, during which time they received an ordinary diet and were allowed normal activity and sleep. Blood samples were collected with a heparinized catheter (Carmeda, Stockholm, Sweden) and a constant withdrawal pump (Swemed, Göteborg, Sweden), as previously described (22). The heparinized tubes were changed at intervals of 20 or 30 min, and all samples were centrifuged and stored at -20 C. The 24-h GH profiles were analyzed with the Pulsar program (23), as previously reported (22, 24). None of the children had received any treatment for their short stature at the time of the 24-h GH profiles. GH levels were measured using a polyclonal immunoradiometric assay (IRMA; Pharmacia & Upjohn, Uppsala, Sweden). The GH standards provided in the IRMA were calibrated against International Reference Preparation (IRP) 80/505 from the WHO, in which 1 mg = 2.6 IU GH (25). The samples were analyzed either with IRP 66/217 or IRP 80/505. A conversion factor of 1.55 was used to transform the values from IRP 66/217 to those for IRP 80/505 (26). The study was approved by the ethical committee of the Medical Faculty, University of Göteborg. Informed consent was obtained from the children and/or their parents.
22-kDa GHEA
The GHEA, which has been previously described (16), was used to determine the serum levels of non-22-kDa GH isoforms. The assay is based on the extraction of monomeric and dimeric 22-kDa GH from a 100-µL aliquot of serum using an anti-22-kDa GH monoclonal antibody (MCB; Genentech, South San Francisco, CA) (27) and magnetic beads coated with rat anti-mouse IgG (Dynal, Oslo, Norway). After extraction, non-22-kDa GH levels were measured using a polyclonal IRMA (Pharmacia & Upjohn), in which 1 mg = 2.6 IU GH (25). In the GHEA, total GH levels were determined in another 100-µL aliquot of serum incubated with assay buffer (without addition of MCB), and the non-22-kDa GH levels were expressed as a percentage of total GH concentration in the samples. The detection limit was 0.02 µg/L, and the intraassay CV was below 1.5% for 22-kDa GH extraction (16). In this study, 2 serum pools with total GH levels ranging between 9–13.2 µg/L and between 2.5–3 µg/L were used to determine the interassay CVs, which were 2.2% and 2.8%, respectively. Non-22-kDa GH levels were measured in a single sample in 44 children or in pools of 2 or 3 samples in 37 and 8 children, respectively, collected at the time of a GH peak in the 24-h GH profile.
Statistical analysis
The descriptive statistical results are presented as the median
and range, unless otherwise stated. Comparisons between groups were
performed using the nonparametric Mann-Whitney U test, and correlations
were sought by calculating the nonparametric Spearman rank correlation
coefficient (Spearman’s 
, denoted r in the correlations).
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Results |
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The median proportion of non-22-kDa GH isoforms was 8.1% (range,
3.2–13.9%) in the normal prepubertal children, 9.9% (4.1–39.5%) in
the girls with TS, 9.8% (2.4–18.4%) in the children born SGA, and
8.9% (3.1–24.9%) in children with ISS (Fig. 1). As a group, children born SGA and
girls with TS had an increased proportion of circulating non-22-kDa GH
isoforms compared with normal children (P = 0.05 and
0.01, respectively). Considering all short children together (n =
66), a negative correlation was observed between non-22-kDa GH isoforms
and height SD score (r = -0.3; P =
0.02).
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Of the girls with TS, 14 (82.3%) had a proportion of non-22-kDa
GH isoforms above the 50th percentile of the reference group. In 5
girls, the values were more than 2 SD above the mean for
the normal children (equivalent to the maximum value; Fig. 2), and all of them were 45,X. In girls
with karyotype 45,X/46,XX, 45,X/47,XXX, 45,X/46,XX/47,XXX, and
45,X/46,X,i, the proportions were 10.3%, 12.4%, 8.7%, and 4.1%,
respectively. No significant correlations were seen in TS among
non-22-kDa GH, auxological data, and spontaneous GH secretion.
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Of the children born SGA, 17 (68%) had a proportion of non-22-kDa
GH isoforms above the 50th percentile of the reference group. Five
children born SGA had values more than 2 SD above the mean
for the normal children (Fig. 2
). In the SGA group, the proportion of
non-22-kDa GH isoforms was negatively correlated to height
SD score (r = -0.42; P = 0.04; Fig. 3). Moreover, the amount of non-22-kDa GH
isoforms was directly correlated with different estimates of
spontaneous GH secretion: mean 24-h GH concentration (r = 0.41;
P = 0.04), area under the curve over baseline (r =
0.41; P = 0.04), and GH peak area (r = 0.61;
P = 0.003). As shown in Fig. 3, the GH secretion
pattern in SGA children younger than 7 yr (n = 13) was different
from that in the older SGA group (n = 12), characterized by a
lower mean GH secretion rate, an increased number of GH peaks with
lower amplitudes, and higher baseline GH levels (P <
0.01 for all variables). The proportion of non-22-kDa GH isoforms was
similar in these two subgroups of SGA children (Table 2). The inverse correlation between
non-22-kDa GH isoforms and height SD score observed in the
whole SGA group was more pronounced in the younger children (r =
-0.68; P = 0.01), whereas it was not seen in the older
ones (r = -0.21; P = 0.5). Such age-related
differences regarding GH secretory pattern and correlation between
non-22-kDa GH and height SD score observed in children born
SGA were not seen in the other groups.
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The proportion of non-22-kDa GH isoforms in children with ISS was
not significantly different from that in normal children. However, four
children with ISS had altered proportions of non-22-kDa GH isoforms,
more than 2 SD above the mean for the reference group (Fig. 2). The proportion of non-22-kDa GH isoforms was not correlated with
auxology in this group, whereas it was directly correlated to the
number of GH peaks in the 24-h profile (r = 0.49;
P = 0.02).
Correlation between non-22-kDa GH isoforms and total GH concentrations
There was no correlation between the proportion of non-22-kDa GH isoforms and the total GH concentration in the samples measured by the GHEA either in the whole group or in each group separately. The median total GH concentration in the samples was 9.8 µg/L (5.2–30.1 µg/L) in the normal prepubertal children, 15.8 µg/L (7–41 µg/L) in the girls with TS, 12.1 µg/L (4.5–28 µg/L) in the children born SGA, and 13.2 µg/L (5–25.4 µg/L) in children with ISS. The median values were higher in all groups of short children (P < 0.05 vs. normal children).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In 1984, Spiliotis et al. (35) proposed that some children with ISS had abnormalities in the pattern of spontaneous GH secretion, but this was not observed in another study (36). More recently, a variety of defects in the GH/insulin-like growth factor I (IGF-I) axis have been suggested as causes of growth impairment in children with ISS. Partial insensitivity to GH due to mutations in the GHR gene was identified in a subgroup of children with ISS (37), and a partial deletion of the IGF-I gene was found in a boy with severe prenatal and postnatal growth failure (38). Moreover, Takahashi et al. (39) described a child with severe growth retardation caused by a single missense mutation in the GH gene. Interestingly, the mutant GH was not only biologically inactive, but also inhibited the action of the wild-type GH, because of its greater affinity to the GHR. This finding opens the possibility that the high proportion of non-22-kDa GH isoforms in some children with ISS in our study may be due to the presence of mutant GH molecules not recognized by the monoclonal antibody used for extraction in the 22-kDa GHEA.
In prepubertal girls with TS, GH responses to pharmacological stimulation tests and assessment of spontaneous GH secretion have produced conflicting results, with some studies showing normal and others subnormal secretion (40, 41, 42). Our girls with TS had an increased proportion of circulating non-22-kDa GH isoforms with a wide spectrum of values ranging from 4–39%, which may partly contribute to these divergent results, because of the use of different monoclonal and polyclonal GH assays with diverse epitope specificity (43). Our results are in agreement with the report by Blethen et al. (44), in which the existence of different non-22-kDa GH isoforms in TS was suggested by comparing serum GH immunoreactivity with a panel of three site-specific monoclonal antibodies. Moreover, we observed that all girls with high amounts of circulating non-22-kDa GH isoforms had karyotype 45,X, whereas the values were in the normal range in the girls with mosaicism. However, due to the small number of observations, additional studies are needed to establish whether distinct chromosome abnormalities can influence the molecular nature of GH in the circulation.
The mechanisms of growth retardation in children born SGA who do not show postnatal catch-up growth are not completely known (45). Recent studies have shown that prepubertal children born SGA secrete less GH (21) and have lower serum IGF-I and IGF-binding protein-3 levels (46) compared with prepubertal healthy children born AGA, indicating a suboptimal secretion of GH. In this study, the proportion of circulating non-22-kDa GH isoforms in prepubertal short children born SGA was higher than that in normal children. Moreover, the values increased according to spontaneous GH secretion, suggesting that the daily secretion of non-22-kDa GH isoforms might be augmented in some SGA children.
In this study, children born SGA younger than 7 yr had lower mean GH secretion rate, higher baseline GH levels, and an increased number of GH peaks with lower amplitudes compared with those in children older than 7 yr. These changes in the GH secretory pattern according to age were previously reported by some of us in a much large group of prepubertal short children born SGA (21). As 24-h GH profiles were not available in very young normal children, it is not known whether this observed difference in GH pattern is an age-related phenomenon or an adaptative process in children born SGA. The presence of increased amounts of non-22-kDa GH isoforms was associated with less growth in children born SGA. This finding was mainly seen in the subgroup of SGA children younger than 7 yr, although the proportion of non-22-kDa GH isoforms at GH peaks did not differ between the younger and the older SGA children. The reasons for the lack of correlation between non-22-kDa GH and height in the older SGA children are not known and should be investigated in further studies.
The role of circulating non-22-kDa GH levels in the growth response to GH treatment in short children has not been evaluated. In TS, the absolute height SD score at the end of treatment showed a better outcome in girls who received high doses of exogenous GH (47). It is possible that in some children with elevated levels of non-22-kDa GH in the circulation, the daily administration of high doses of exogenous GH saturates the GHR and inhibits the secretion of less bioactive isoforms and fragments by the pituitary gland through a feedback mechanism. Thus, we believe that the GH heterogeneity should be considered an additional factor influencing the growth response to GH therapy in different groups of short children.
In conclusion, the wide range of values in the proportion of circulating non-22-kDa GH observed in this study may partly explain the discrepant results reported in the investigations of spontaneous GH secretion in short children. Using the GHEA, we identified some non-GH-deficient short children with abnormally high proportions of circulating non-22-kDa GH isoforms. In the group of children born SGA, the proportion of non-22-kDa GH was directly correlated to estimates of spontaneous GH secretion and negatively correlated with height SD score, mainly in those younger than 7 yr. Our results suggest that the ratio of circulating non-22-kDa GH isoforms may have important implications for normal and abnormal growth by modulating the action of 22-kDa GH in vivo.
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Acknowledgments |
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Footnotes |
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Received January 17, 1997.
Revised May 21, 1997.
Accepted June 7, 1997.
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