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Original Studies |
Endocrine Sciences Research Group, Department of Medicine, University of Manchester (M.S.G., P.E.C.), Manchester, United Kingdom M13 9PT; the Department of Endocrinology, Federal University of Sergipe (M.H.A.-G., E.S.d.A.B, M.R.S.A., C.A.M., A.H.O.S., F.A.P.), 49060-100 Aracaju, Brazil; Faculty of Medicine of Ribeirao Preto, University of Sao Paulo (C.E.M.), 14049-900 Sao Paulo, Brazil; the Department of Endocrinology and Chemical Endocrinology, St. Bartholomew’s Hospital (F.M.-M., C.C.-H.), EC1A 7BE London, United Kingdom; the Department of Endocrinology, John Hopkins University (R.S., M.A.L.), Baltimore, Maryland 21287; and the Department of Endocrinology, Christie Hospital National Health Service Trust (S.M.S.), Withington, M20 4BX Manchester, United Kingdom
Address all correspondence and requests for reprints to: Dr. Peter E. Clayton, Department of Medicine, Endocrine Sciences Research Group, University of Manchester, Stopford Building, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: pclayton{at}man.ac.uk
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Abstract |
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All components of the IGF axis, measured and theoretical, showed complete separation between GHD and control subjects, except IGFBP-1 and IGFBP-2 concentrations, which did not differ. The most profound effects of GHD were on total IGF-I, IGF-I in the ternary complex, and ALS. The proportion of IGF-I associated with IGFBP-3 remained constant throughout life, but was significantly lower in GHD due to an increase in IGF-I/IGFBP-2 complexes. IGF-I in the ternary complex was determined principally by concentrations of ALS in GHD and IGFBP-3 in controls, implying that ALS has greater GH dependency. In the controls, IGF-II was associated primarily with IGFBP-3 and to a lesser extent with IGFBP-2, whereas in GHD the reverse was found. There was also a dramatic decline in the proportion of free ALS in GHD adults that was not evident in controls. As diagnostic tests, IGF-I in the ternary complex and total IGF-I provided the greatest separation between GHD and controls in childhood. Similarly, in older adults the best separation was achieved with IGF-I in the ternary complex, with free ALS being optimal in younger adults.
Severe GHD not only reduces the amounts of IGFs, IGFBP-3, and ALS, but also modifies the distribution of the IGFs bound to each IGFBP. Diagnostic tests used in the investigation of GHD should be tailored to the age of the individual. In particular, measurement of IGF-I in the ternary complex may prove useful in the diagnosis of GHD in children and older adults, whereas free ALS may be more relevant to younger adults.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Variation in the severity and etiology of GHD and in the age and pubertal status of individuals are further confounding factors in assessing the performance of tests for GHD. In routine clinical pediatric practice GHD generally presents as a heterogeneous disorder with the etiology of the majority of patients fulfilling the criteria for this diagnosis being classed as idiopathic, with or without abnormalities of the hypothalamic-pituitary axis on magnetic resonance imaging scan. Congenital GHD arising from gene defects is rare and usually presents in consanguineous unions. However, we have recently identified a large kindred in northeast Brazil with severe familial isolated GHD (17). All affected individuals have a novel homozygous donor splice mutation (G to A at position +1) in intron 1 of the GHRH receptor gene, which disrupts the highly conserved consensus GT of the 5'-donor splice site, generating a truncated GHRH receptor. Mutations in the GHRH receptor as the cause of GHD in humans have been identified previously in small kindreds (18, 19, 20). However, the Itabaianinha kindred differs in that there is a total of 71 affected individuals between 5–82 yr of age. Thus, this large group provides a unique opportunity to evaluate the impact of the absence of GH throughout life on the performance of serum markers of the GH-IGF axis in a genetically homogenous population.
We have therefore measured serum concentrations of components of the GH-IGF axis in affected individuals and in matched controls, both children and adults. To assess the usefulness of novel markers of the GH-IGF axis we have also calculated theoretical concentrations of IGFs bound in the binary and ternary complexes and concentrations of free ALS.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study was approved by the institutional review board at the Federal University of Sergipe (Sergipe, Brazil), and all subjects gave informed consent. All subjects were members of an extended kindred resident in the county of Itabaianinha, in the state of Sergipe, northeast Brazil. The diagnosis of IGHD in 12 children [4 males and 8 females; aged 5–20 yr; body mass index (BMI), 17.0 kg/m2 (range, 14.0–22.8); 6 pubertal stage (PS) 1 and 6 PS 2–5] was based on early growth failure, proportionate short stature and evidence of delayed bone age. Height SD scores (HtSDS) were calculated from United Kingdom standards (21). The median HtSDS in affected children was -6.0 (range, -8.1 to -3.2). The diagnosis of GHD in 15 adults [6 males and 8 females; aged 29–82 yr; BMI, 22.3 kg/m2 (range, 16.5–28.9); HtSDS, -7.9 (-9.5 to -6.1)] was based on a history of early growth failure and proportionate short stature. Affected subjects were compared with 55 controls recruited from the same area and included 39 children and young people [13 males and 26 females; aged 5–18 yr; BMI, 16.2 kg/m2 (range, 13.2–22.3); 17 PS 1 and 22 PS 2–5; HtSDS, -1.0 (range, -3.1 to 1.3)] and 16 adults [10 males and 6 females; aged 33–80 yr; BMI, 21.6 kg/m2 (range, 16.4–25.4); HtSDS, -2.4 (-3.5 to -0.2)]. In the analysis, adult subjects were stratified into 2 age bands: younger adults (aged <50 yr; GHD, n = 9; median age, 40 yr; control, n = 8; median age, 40 yr) and older adults (aged >50 yr; GHD, n = 6; median age, 63 yr; control, n = 8; median age, 63 yr). The homozygous splice site mutation at the boundary exon 1/intron 1 of the GHRH receptor gene responsible for GHD in this population was detected in DNA samples from 6 children and 10 adults by denaturing gradient gel electrophoresis (17).
The 12 affected children had previously been admitted to the Clinical Research Unit at the Federal University of Sergipe for GH stimulation tests. The children underwent both an insulin tolerance test (0.1 IU/kg insulin, iv, as a single bolus injection) and a clonidine test (0.15 mg/m2, orally). The median response to the ITT was 0.01 ng/mL (range, 0.0–1.0 ng/mL), and that to clonidine stimulation was 0.2 ng/mL (range, 0.02–2.2 ng/mL). Estimation of bone age was also performed in these children using the method of Greulich and Pyle. The median bone age delay was 3.5 yr (range, 2.0–6.0 yr).
Assays
Serum IGF-I was measured by immunoradiometric assay (IRMA) after acid-alcohol extraction (Diagnostic Systems Laboratories, Inc., Webster, TX). The intraassay coefficients of variation (CVs) for mean IGF-I concentrations of 9.4, 55.4, and 263.6 ng/mL were 3.4%, 3.0%, and 1.5%, respectively. The interassay CVs for mean IGF-I concentrations of 10.4, 53.8, and 255.9 ng/mL were 8.2%, 1.5%, and 3.7%, respectively. The sensitivity of this assay was 0.8 ng/mL.
Serum IGF-II was measured using an in-house IRMA as previously described (22). The sensitivity of the assay was 30 ng/mL. The intra- and interassay CVs for serum concentrations of 200 and 4500 ng/mL were less than 10%.
Serum IGFBP-1 was measured using an in-house RIA as previously described (23). The sensitivity of the assay was 5 ng/mL. The intra- and interassay CVs were 6.8% and 8%, respectively.
Serum IGFBP-2 was measured by RIA (Diagnostic Systems Laboratories, Inc.). The sensitivity of the assay was 0.5 ng/mL. The intraassay CVs for serum concentrations of 13.0, 32.2, and 94.4 ng/mL were 8.5%, 6.2%, and 4.7%, respectively. The interassay CVs for serum concentrations of 2.7, 13.2, and 69.7 ng/mL were 7.4%, 4.5%, and 7.2%, respectively.
Serum IGFBP-3 was measured using an IRMA (Diagnostic Systems Laboratories, Inc.). The sensitivity of this assay was 0.5 mg/L. The intraassay CVs for mean serum concentrations of 0.7, 2.8, and 8.3 mg/L were 3.9%, 3.2%, and 1.8%, respectively. The interassay CVs for mean IGFBP-3 concentrations of 0.8, 2.2, and 8.0 mg/L were 0.6%, 0.5%, and 1.9%, respectively.
Total serum ALS was measured using an enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories, Inc.). The sensitivity of the assay was 0.07 mg/L. The intraassay CVs for mean serum concentrations of 1.7, 7.7, and 29.2 mg/L were 6.1%, 7.5%, and 3.8%, respectively. The interassay CVs for mean ALS concentrations of 2.2, 7.9, and 30.1 mg/L were 8.6%, 2.8%, and 8.9%, respectively.
Results were expressed as nanomoles per L using a molecular mass of 7.5 kDa for IGF-I and IGF-II, 28 kDa for IGFBP-1, 35 kDa for IGFBP-2, 42 kDa for IGFBP-3, and 85 kDa for ALS.
Estimation of binary and ternary complex concentrations
Theoretical concentrations of IGF-I and IGF-II associated with the IGFBPs and ALS were calculated by the method of Fant et al. (24), using previously published estimates of affinity constants for IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, and ALS (24, 25, 26) as follows: Ka(IGF-I/IGFBP-1) = 6.6 x 109 mol/L-1 Ka(IGF-II/IGFBP-1) = 3.2 x 109 mol/L-1; Ka(IGF-I/IGFBP-2) = 5 x 109 mol/L-1 Ka(IGF-II/IGFBP-2) = 4.1 x 1010 mol/L-1; Ka(IGF-I/IGFBP-3) = 5.6 x 109 mol/L-1 Ka(IGF-II/IGFBP-3) = 1.8 x 1010 mol/L-1; and Ka(IGF-I/IGFBP-3/ALS) = 1.55 x 1010 mol/L-1, Ka(IGF-II/IGFBP-3/ALS) = 1.55 x 1010 mol/L-1.
The following variables were derived and used in subsequent analysis: IGF-I/IGFBP-1, -2 or -3, IGF-I in a binary complex with IGFBP-1, IGFBP-2, or IGFBP-3; residual IGF-I, IGF-I not associated with IGFBP-1, IGFBP-2, or IGFBP-3; IGF-II/IGFBP-1, -2, or -3 = IGF-II in a binary complex with IGFBP-1, IGFBP-2, or IGFBP-3; residual IGF-II, IGF-II not associated with IGFBP-1, IGFBP-2, or IGFBP-3; TC[IGF-I], ternary complex containing IGF-I (IGF-I/IGFBP-3/ALS); TC[IGF-II], ternary complex containing IGF-II (IGF-II/IGFBP-3/ALS); and free ALS, ALS not associated with the ternary complex.
Statistical analysis
Nonparametric statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, Inc., Chicago, IL). Groups were compared using the Kruskal-Wallis and Mann-Whitney U tests, and relationships were assessed using Spearman’s rank correlation coefficient. Statistical significance was indicated by P < 0.05, except for multiple pairwise comparisons where P < 0.01 was used.
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Results |
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Concentrations of IGF-I, IGF-II, IGFBP-3, and ALS were
significantly lower in GHD compared with control subjects at all ages
(all P < 0.001; Fig. 1
and Table 1). In contrast, concentrations
of IGFBP-1 and IGFBP-2 were not different between GHD and control
subjects within any age group (Table 1).
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). In the childhood
controls, IGF-I, IGFBP-3, and ALS concentrations were positively
correlated with age, whereas IGFBP-1 was inversely correlated with age
(Table 2). In the adults, only IGFBP-3
was inversely correlated with age. HtSDS was positively correlated with
IGF-I, IGF-II, IGFBP-3, and ALS and was inversely correlated with
IGFBP-2 concentrations in control children (Table 2). In the
control adults, HtSDS only correlated inversely with IGFBP-1 and
IGFBP-2 (Table 2).
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).
However, IGFBP-3 concentrations exhibited significant changes
throughout life. ALS concentrations were identical in prepubertal and
pubertal GHD children, but were significantly lower in the younger
adults (Table 1). There was no significant difference in serum ALS
concentrations between younger and older adults. In contrast to the
controls, none of the peptides was correlated with age in the children,
whereas in the GHD adults, IGFBP-1 and IGFBP-2 increased with age, and
ALS concentrations decreased (Table 2). In addition, there was no
relationship between IGF-I, IGF-II, IGFBP-1, IGFBP-2, or IGFBP-3
concentrations and HtSDS. In the children, only ALS concentrations
exhibited a positive association with HtSDS (Table 2). In the adults,
there was an inverse relationship between both IGFBP-1 and IGFBP-2 and
HtSDS. Molar ratios of IGF-I, IGF-II, and IGFBP-3
In children, the ratio of IGF-I to IGFBP-3 was significantly lower
in the GHD subjects than the controls. In contrast, the ratio of IGF-II
to IGFBP-3 was higher in GHD children compared with controls, and
consequently, the ratio of IGF-I plus IGF-II (total IGF) to IGFBP-3 was
identical in the two groups (Fig. 2A).
Similarly, in adults the IGF-I to IGFBP-3 ratio was reduced, and the
ratio of IGF-II to IGFBP-3 was increased in the GHD subjects. However,
the increase in the ratio of IGF-II to IGFBP-3 was such that the ratio
of total IGF to IGFBP-3 was significantly higher in GHD adults compared
with control adults (Fig. 2B).
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To examine the influence of the IGFBP profile on
partitioning of IGFs, we examined the theoretical concentrations of
IGF/IGFBP binary complexes. In control subjects, the concentration of
IGF-I/IGFBP-3 complexes increased into puberty and declined thereafter
(Fig. 3A) and paralleled the independent
changes in IGF-I and IGFBP-3 (Table 1). However, when expressed as a
percentage of total IGF-I concentrations, the amount of IGF-I bound in
this complex was relatively constant throughout life, varying between
73.7–88.7% (Fig. 3A). The amount of IGF-I associated with IGFBP-1 and
IGFBP-2 accounted for only a small fraction of the total IGF-I
concentration. Residual IGF-I increased to 23.2% in pubertal children
and declined through adulthood to 0.8% in older adults.
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). IGF-I bound to IGFBP-3 accounted for
between 50.8–69% of the total IGF-I concentration, and this
proportion was significantly lower than that observed in controls
(P < 0.01). The proportion of total IGF-I
concentrations bound to IGFBP-2 was significantly elevated in GHD
subjects compared with controls (P < 0.001), as was
the percentage of IGF-I associated with IGFBP-1 in GHD adults
(P < 0.001). Residual IGF-I remained unchanged through
life in GHD subjects and was significantly lower in GHD children
compared with control children (P < 0.001). Partitioning of IGF-II among IGFBP-1, IGFBP-2, and IGFBP-3
In control subjects the major fraction of total IGF-II
concentrations (68–79%) were in the form of IGF-II/IGFBP-3 binary
complexes, with 14.6–30% of IGF-II associated with IGFBP-2 (Fig. 4A), and only 0.2–1.4% associated with
IGFBP-1. In common with IGF-I, residual IGF-II concentrations
increased with puberty and declined through adulthood (Fig. 4A). This
distribution of IGF-II between the binding proteins was reversed in GHD
subjects, with a significantly higher proportion of IGF-II associated
with IGFBP-2 and a reduction in the proportion of
IGF-II/IGFBP-3 complexes compared with controls (Fig. 4B).
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Using theoretical concentrations, between 54.9–71.8% of the
total ALS was not associated with the ternary complex in controls (Fig. 5A). In GHD subjects, the percentage of
free ALS was comparable with that in controls in childhood; however,
there was a marked reduction in the percentage of free ALS in GHD
adults compared with controls (Fig. 5B). The proportion of TC[IGF-I]
ranged between 7.9–18.4% of the total ALS in controls and was
significantly reduced in GHD to between 1.7–5.8% (P
< 0.001). The proportion of total ALS associated with binary complexes
containing IGF-II was not different between GHD children and control
children, but was markedly elevated in GHD adults compared with control
adults, such that TC[IGF-II] accounted for 88.2% and 74.1% of the
total ALS concentration in younger and older subjects, respectively
(P < 0.001).
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To compare the relative impact of GHD on the levels of the
measured peptides and the theoretical concentrations of the ternary
complex, the minimum value in the control group was divided by the
maximum value in the GHD group, as an index of the degree of separation
between the two groups (Table 3). The
maximum IGF-I concentration in the prepubertal GHD children was 8 times
lower than the minimum value in controls, whereas TC[IGF-I]
concentrations were 8.4 times lower than controls. Similarly, in
pubertal children, TC[IGF-I] and IGF-I concentrations were 12.3 and
12 times lower, respectively, in GHD subjects compared with controls.
In the younger adults, free ALS concentrations were most severely
affected by GHD, with concentrations approximately 36 times lower in
the GHD group. In the older adults, the greatest separation between the
two groups was provided by TC[IGF-I] concentrations, with levels in
controls approximately 46 times higher than those in GHD adults (Table 3).
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Discussion |
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In the control group, IGF-I, IGFBP-3, and ALS showed significant age- and puberty-related changes consistent with those described previously for healthy children and adolescents (15, 28). IGF-II and IGFBP-2 concentrations did not exhibit any significant developmental changes, whereas IGFBP-1 concentrations decreased significantly with age in children as expected (29). In normal healthy adults, IGF-I, IGFBP-3, and ALS concentrations are inversely correlated with age (15, 30), but in our cohort of controls only IGFBP-3 decreased significantly with age. The lack of any significant decrease in IGF-I and ALS concentrations with age is probably due to the relatively small sample size.
Serum concentrations of IGF-I, IGF-II, IGFBP-3, and ALS were all severely reduced in the GHD children and adults compared with levels in their matched controls, with the levels of IGF-I, IGF-II, and IGFBP-3 in the GHD subjects comparable to those reported for the patients from Sindh (19). In the GHD subjects there was no change in IGF-I and IGF-II, whereas IGFBP-3 and ALS concentrations were significantly different across the age groups. ALS concentrations were not different in childhood, but there was a marked decrease after puberty, which continued in the older adults. Interestingly, HtSDS did not correlate with IGFBP-3 in the GHD children, as has been reported for GHRD (27, 31), but there was a significant correlation with ALS concentrations. An additional contrasting feature of the Ecuadorian GHRD individuals was that IGF-I and IGFBP-3 concentrations were significantly higher in adults than in children, with lower GH secretion in the adults (27). This was thought to represent an independent action of sex steroids on IGF-I and IGFBP-3 during puberty, which reduced GH secretion by negative feedback (27). In contrast, there was no significant pubertal rise in IGF-I, IGF-II, IGFBP-3, or ALS concentrations in our GHD subjects. This observation is difficult to explain in the context of the GHRD individuals, as pubertal development in our cohort was normal, although slightly delayed.
After IGFBP-3, IGFBP-2 accounts for the major portion of IGF-binding activity in the circulation, and IGFBP-2 concentrations have been reported to be regulated by GH, albeit indirectly (32). In contrast to other GH-dependent peptides, concentrations of IGFBP-2 are elevated in GHD (13). Furthermore, Smith et al. (13) found that the ratio of IGF-I to IGFBP-2 was useful in the diagnostic evaluation of children with GHD, whereas in adults over the age of 60 yr, IGFBP-2 is not an effective test (33). In our study IGFBP-2 concentrations were not different between GHD and control subjects at any age. IGFBP-2 concentrations in our control subjects did not exhibit any developmental changes, in agreement with the report by Juul et al. (29), whereas IGFBP-2 levels declined with age in the normal range reported by Smith et al. (13). Thus, the lack of any difference between our GHD and control subjects may be due to differences in the IGFBP-2 assays used or in the control subjects. Although IGFBP-2 concentrations were not elevated in GHD subjects compared with controls, the molar concentration of IGFBP-2 was significantly higher than the molar concentration of IGFBP-3 in GHD subjects, whereas in controls the reverse was true. IGFBP-2 and IGFBP-3 concentrations were similar in the GHD subjects from Sindh (19), indicating that IGFBP-2 accounts for a significant proportion of the total IGF-binding activity in GHD subjects.
In the circulation, IGFBP-3 and either IGF-I or IGF-II combine with the ALS to form the 150-kDa ternary complex, which is thought to act as a reservoir for IGFs, preventing their rapid clearance (14). It has previously been noted that the molar concentration of IGFBP-3 is approximately equal to the sum of IGF-I plus IGF-II (8). It was suggested that IGFBP-3 synthesis is regulated according to IGF availability or, alternatively, that it is the concentration of circulating IGFs that determines the level of IGFBP-3. To compare the relative proportions of IGF-I, IGF-II, and IGFBP-3, we calculated their molar ratios in GHD and control subjects, both children and adults. In GHD children and adults, there was a profound reduction in the ratio of IGF-I to IGFBP-3 compared with that in controls, indicating that IGF-I was more severely affected than IGFBP-3 by the absence of GH. Conversely, the ratio of IGF-II was significantly higher in GHD subjects. As a consequence, the combined IGF-I plus IGF-II to IGFBP-3 ratio was identical in control and GHD children, whereas in adults the ratio in GHD was almost double that in controls. These findings therefore suggest a differential effect of GHD on the level of these peptides, as IGF-I and IGFBP-3 are not proportionately reduced. This may be a result of IGF-I being more GH dependent or because IGFBP-3 concentrations also reflect the amount of IGF-II, which is not as severely reduced as that of IGF-I. Despite the reduction in concentration, there does appear to be an increase in the amount of IGF-II relative to IGFBP-3 in GHD that continues into adult life. The reason for this is unclear, but it may represent an up-regulation of IGF-II to compensate for the diminished IGF-I concentrations. It is noteworthy that the IGF-I-deficient child with an IGF-I gene deletion described by Woods et al. (34) had IGF-II concentrations that, when expressed as a molar quantity, were 2-fold greater than the concentration of IGFBP-3.
To examine the impact of alterations in the IGFBP profile on IGF partitioning we calculated theoretical concentrations of free and bound IGFs using the method of Fant et al. (24). The advantage of this approach over simple ratios is that it provides an integrated measure of the GH-IGF axis, taking into account not only relative concentrations but also differences in binding affinities. The theoretical proportion of IGF-I associated with IGFBP-3 remained constant throughout life at about 85% in controls and was significantly lower in GHD at about 65%. This reduction was due principally to an increase in the proportion of IGF-I associated with IGFBP-2 and IGFBP-1 in GHD. In controls, IGF-II was associated mainly with IGFBP-3, followed by IGFBP-2, whereas in GHD, more than 50% of IGF-II was associated with IGFBP-2, and the proportion of IGF-II/IGFBP-3 complexes was significantly reduced. These theoretical data indicate that despite an increase in the amount of IGFBP-2 relative to IGFBP-3 in GHD, the impact on IGF-I binding is diminished by preferential binding of IGF-II to IGFBP-2.
We also used partitioning analysis to examine the theoretical distribution of IGFs in the ternary complex. Although there is some evidence to suggest that binary complexes of IGFBP-3 bound to ALS exist in the circulation, which have increased binding affinity for IGFs over IGFBP-3 alone (35), we assumed the sequential binding model of Baxter and Martin (14), according to which only binary complexes of IGF and IGFBP-3 can associate with ALS to form the ternary complex. The theoretical percentage of free ALS in controls ranged from approximately 50–70%, values consistent with estimates from neutral gel filtration studies (15) and direct measurement by specific enzyme-linked immunosorbent assays (16). Free ALS concentrations in GHD children were similar to levels in controls, because the total molar concentrations of IGFBP-1, -2 and -3 in GHD were approximately twice those of the total IGF concentrations. Consequently, using the partitioning model for IGF associating with the IGFBPs, only 50% of total ALS in GHD children would form ternary complexes with IGF-IGFBP-3, leaving 50% as free ALS. In contrast, theoretical concentrations of free ALS were substantially lower in GHD adults compared with controls. In young adults this was a result of total IGF concentrations being similar to total IGFBP levels. This would mean that the majority of IGFBP-3 would form a binary complex with IGF, and as IGFBP-3 and ALS concentrations were similar, there would be very little free ALS. However, in older adults, low free ALS levels resulted from total ALS concentrations being lower than IGFBP-3 concentrations.
Recent data suggest that IGFBP-3 is not unique in its ability to associate with the ALS, as IGFBP-5 has been shown to circulate in a ternary complex with IGF and ALS (36). In addition, IGFBP-5 exhibits similar developmental changes as IGFBP-3, with increased levels during puberty and a decline through adulthood, although circulating concentrations of IGFBP-5 are about 7 times lower than those of IGFBP-3 (37). The lower levels of IGFBP-5 and the molar excess of ALS would suggest that IGFBP-5 ternary complexes would not impact on the amount of IGFBP-3 ternary complexes derived from our theoretical calculations in control subjects. In GHD, the reduced level of IGFBP-3 may lead to an increased proportion of IGFBP-5 ternary complexes. However, there is some evidence that IGFBP-5 may be GH dependent (38), and thus, concentrations in GHD subjects might be expected to be reduced in parallel with IGFBP-3.
The identification of a single biochemical test that is able to completely differentiate between abnormal and normal GH secretion has been the goal of many investigations over recent years. This has not been achievable, and thus, the diagnosis of GHD remains a contentious issue (1, 2). In this study we have evaluated the performance of markers of the GH-IGF axis in severe GHD, a diagnosis that is relatively easy to make. However, the potential success of any test rests upon how well it performs in subjects with milder degrees of GH deficiency, i.e. GH peak between 5–10 ng/mL by conventional RIA. It is in this situation that many tests used in the diagnosis of GHD perform badly, due to the overlap between GHD and non-GHD individuals. Thus, for a new test to be useful in the diagnosis of GHD it must provide the greatest separation between severe GHD and non-GHD subjects. To address this issue we quantified the degree of separation as the minimum value in controls divided by the maximum value in GHD. In children, theoretical concentrations of IGF-I in the ternary complex provided the greatest separation, although the degree of separation was only marginally better than that with total IGF-I. Serum concentrations of IGFBP-3 were only between 2.5–5.1 times lower in prepubertal children and younger adults with GHD, respectively, compared with controls. Moreover, IGFBP-3 did not rank in the three parameters generating the greatest separation in any group, confirming recent studies that have found IGFBP-3 to be a poor discriminator of GHD (11, 12). This appears to be attributable to the fact that IGFBP-3 concentrations are also a reflection of IGF-II levels. The relatively poor performance of ALS in children suggests that further evaluation of this test in a more heterogeneous population may not be of use. Indeed, a recent comprehensive assessment of the diagnostic potential of ALS measurements in young adults with childhood-onset GHD found the sensitivity and specificity of ALS to be similar to those of IGF-I and IGFBP-3 (39). However, in adults free ALS was approximately 36 and 20 times lower in younger and older GHD adults, respectively, than in their controls, suggesting that free ALS may be useful in the diagnosis of GHD in adults. In the older adults the greatest separation was achieved with TC[IGF-I], with about 50 times greater TC[IGF-I] in controls than in GHD patients. Immunoassays that specifically measure free ALS (16) and the ternary complex (40) have been described, and our findings using theoretical concentrations suggest that such assays may be useful in the diagnosis of GHD.
Historically, the diagnosis of GHD has been made using GH stimulation tests and, more recently, also by measurement of IGF-I and IGFBP-3. In this study we have evaluated tests of the GH-IGF axis in a large, genetically homogenous population of all ages, with severe GHD arising from a novel mutation in the GHRH receptor. In addition, using affinity constants and measured concentrations, we have evaluated theoretical concentrations of the ternary complex as potential novel markers of the GH axis. Our findings suggest that measurement of IGF-I in the ternary complex in children and free ALS in adults may be useful adjuncts to conventional diagnostic tests of GHD.
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Footnotes |
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2 These authors contributed equally to this work.
Received April 15, 1999.
Revised July 13, 1999.
Accepted July 22, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) subunit of the high molecular
weight insulin-like growth factor-binding protein complex. J Clin
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R. Coutant, F. B. de Casson, S. Rouleau, O. Douay, E. Mathieu, F. Gatelais, N. Bouhours-Nouet, C. Voinot, M. Audran, and J. M. Limal Divergent Effect of Endogenous and Exogenous Sex Steroids on the Insulin-Like Growth Factor I Response to Growth Hormone in Short Normal Adolescents J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6185 - 6192. [Abstract] [Full Text] [PDF]
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M. Cisternino, M. Draghi, S. Lauriola, D. Scarcella, S. Bernasconi, L. Cavallo, F. De Luca, A. Lomeo, and L. Tato The Acid-Labile Subunit of Human Ternary Insulin-Like Growth Factor-Binding Protein Complex in Girls with Central Precocious Puberty before and during Gonadotropin-Releasing Hormone Analog Therapy J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4629 - 4633. [Abstract] [Full Text] [PDF]
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J. A. S. Barreto-Filho, M. R. S. Alcantara, R. Salvatori, M. A. Barreto, A. C. S. Sousa, V. Bastos, A. H. Souza, R. M. C. Pereira, P. E. Clayton, M. S. Gill, et al. Familial Isolated Growth Hormone Deficiency Is Associated with Increased Systolic Blood Pressure, Central Obesity, and Dyslipidemia J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2018 - 2023. [Abstract] [Full Text] [PDF]
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R. G. Gondo, M. H. Aguiar-Oliveira, C. Y. Hayashida, S. P. A. Toledo, N. Abelin, M. A. Levine, C. Y. Bowers, A. H. O. Souza, R. M. C. Pereira, N. L. Santos, et al. Growth Hormone-Releasing Peptide-2 Stimulates GH Secretion in GH-Deficient Patients with Mutated GH-Releasing Hormone Receptor J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3279 - 3283. [Abstract] [Full Text] [PDF]
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