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Original Studies |
Department of Pediatrics, Okayama University Medical School (N.K., S.K., S.T.W., C.T., Y.Y., Y.S.), Okayama 700-8558; the Department of Pediatrics, Okayama Red Cross Hospital (N.K.), Okayama 700-8607; Cosmic Corp., Inc. (T.M.), Tokyo 112-0002; and Diagnostic Development, SRL, Inc. (M.O.), Tokyo 192-0032, Japan
Address all correspondence and requests for reprints to: Susumu Kanzaki, M.D., Ph.D., Department of Pediatrics, Okayama University Medical School, 2–5-1, Shikata-cho, Okayama 700-8558, Japan. E-mail: smkanzak{at}cc.okayama-u.ac.jp
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Abstract |
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We obtained the following results. 1) In the normal children, the serum free IGF-I levels were low in infancy (<1 yr of age; males, 0.71 ± 0.26 µg/L, mean ± SD; females, 1.05 ± 0.49 µg/L), increased during puberty (males, 5.84 ± 2.18 µg/L; females, 5.80 ± 1.49 µg/L), and declined thereafter. 2) Free IGF-I in the serum occupied about 0.95–2.02% of the total IGF-I values, with the highest ratio occurring in infancy (males, 1.77 ± 0.60%; females, 2.02 ± 0.87%). 3) The SD scores of serum free IGF-I in the 21 GHD children ranged from -3.30 to 0.30, and the 5 complete GHD children had free IGF-I values more than -2 SD below those of age-matched normal subjects. 4) There was a significant correlation between the SD scores of free IGF-I and those of total IGF-I (r = 0.715; P < 0.0005) in the GHD children. 5) In the 16 partial GHD children receiving GH treatment, the serum free IGF-I levels were elevated to 209% of pretreatment levels after 1 month of GH treatment and remained high during GH therapy. The GH-induced increase in the serum free IGF-I levels was significantly higher than those of the total IGF-I and IGF binding protein-3 levels. 6) The percent increase in the serum free IGF-I level after 1 month of GH treatment showed a significant positive correlation with that of the GH-induced improvement in the percent increase in the height velocity during 1 yr of GH therapy (r = 0.526; P < 0.05).
These results show that free IGF-I in the serum has an essential role in bone formation because the higher free IGF-I levels were observed when the growth rate accelerated. The measurement of serum free IGF-I may become a useful tool for both diagnosing GH deficiency and predicting growth responses to long term GH therapy.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A small, but significant, proportion of IGF-I in biological fluids exists in an unbound or readily dissociated form (9, 13, 14, 15, 16, 17, 18, 19, 20). Although only a small fraction, this free or unbound form of IGF-I (free IGF-I), like that of sex and adrenal steroids and thyroid hormones, has been suggested to have more potent biological action than the bound form of IGF-I (8, 14, 18, 21). A specific immunoradiometric assay (IRMA) for serum free IGF-I was recently developed (15, 17, 22). The development of this convenient assay for free IGF-I will help to further elucidate the complex GH-IGF-IGFBP systems.
Many studies have confirmed the GH dependence of serum total IGF-I and IGFBP-3 levels. Therefore, the indicators of total IGF-I and IGFBP-3 in serum have been widely accepted in the diagnosis of GH deficiency (GHD) (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). At present, however, very little is known about the clinical and physiological value of the determination of free IGF-I. In the present study, we examined age-related changes in serum free IGF-I in normal children using a sensitive free IGF-I IRMA to investigate the relationship between free IGF-I levels and the annual height gain of children. In addition, to evaluate the effect of GH on serum free IGF-I levels and the diagnostic value of free IGF-I determinations, we measured the serum free IGF-I levels in prepubertal GH-deficient children before and during the first year of GH treatment.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We studied 354 healthy Japanese infants, children, adolescents, and adults (193 males and 161 females, aged 0–40 yr) whose height, weight, and annual height gain were normal for age. No subject was receiving any medication at the time of blood sampling. In this study, the developmental stage was defined as follows: infant, less than 1 yr of age (male, n = 26; female, n = 22); child, 1–10 yr in males (n = 94) and 1–8 yr in females (n = 54); puberty, 11–15 yr in males (n = 48) and 9–13 yr in females (n = 48); and adult, 18–40 yr (male, n = 16; female, n = 20).
Patients with GHD
Twenty-one GH-deficient Japanese children (11 boys and 10 girls,
aged 4–14 yr) were studied. The diagnosis of GHD was based on the
short stature (<2.0 SD below the mean height of age- and
sex-matched children), low annual height gain (<5.0 cm/yr), and
failure to show serum GH levels above 10 µg/L after at least 2
provocation tests (2) in each of the children. Five children (1 boy and
4 girls, aged 7–14 yr) were diagnosed as having complete GHD (peak GH
values of < 5 µg/L after any provocation test) secondary to a
neoplasm of the hypothalamic-pituitary region (craniopharyngioma in 3
and germinoma in 2 subjects). These 5 patients had multiple pituitary
hormone deficiency and were receiving appropriate replacement therapy
with L-T4, cortisol, and/or desmopressin
acetate. However, GH treatment in these children was not
approved during this study period, because GH administration might
enhance the recurrence of their brain neoplasms. The other 16 children,
aged 4–10 yr, were considered to have idiopathic partial GHD (peak GH
values of 
5 µg/L but <10 µg/L after provocation tests). GH
supplementation was initiated for these patients at the Pediatric
Department of Okayama University Medical School (Okayama, Japan). The
GH treatment for these patients was authorized by the Foundation for
Growth Science in Japan.
Experimental protocol
Informed consent was obtained from the subjects, including the children over 10 yr of age who were old enough to understand the study, or their parents. Blood samples were obtained from the nonfasted normal subjects and from the GH-deficient patients between 1000–1100 h, i.e. about 12 h after their GH injection. The 16 idiopathic partial GH-deficient patients were treated with recombinant human GH [0.5 U (0.17 mg)/kg/week] given sc at night (2100–2200 h) 6–7 days/week. The recombinant human GH (rhGH) was purchased from Sumitomo Pharmaceuticals Co. (Osaka, Japan) and Novo Nordisk A/S Pharma (Tokyo, Japan). All 16 of these patients remained prepubertal during the entire year of the study. The annual growth rate was calculated from the height increase observed after 1 yr ear of GH treatment. Blood samples were collected from these GH-deficient patients before and 1, 2, 3, 6, and 12 months after the initiation of GH therapy. All blood samples were promptly centrifuged, and serum was separated and stored at -70 C until assayed.
This protocol was approved by the investigational review board of Okayama University Medical School.
Measurement of serum free IGF-I levels
Serum free IGF-I levels were measured using a solid-phase,
two-site IRMA kit (Diagnostic System Laboratories, Inc., Webster, TX).
The assay method was previously reported in detail (22). This assay was
carried out as follows. One hundred microliters of the serum sample
were added to a tube containing a dense coating of high affinity
anti-IGF-I antibody, which binds free IGF-I. The serum sample was
incubated for 2 h at 2–8 C, washed, and incubated with an
125I-labeled anti-IGF-I antibody directed to a second
epitope of IGF-I for 2 h at room temperature. After washing and
decanting, the radioactivity of the tube was counted in a 
-counter.
The assay standard was rhIGF-I (0.15–20.0 µg/L). The minimal
detection limit was 0.03 µg/L or 3 pg/tube. The intraassay
coefficients of variation (CVs) were 10.3% (at 0.29 µg/L), 5.1% (at
6.26 µg/L), and 3.3% (at 14.2 µg/L). The interassay CVs were 7.7%
(at 0.26 µg/L), 3.6% (at 5.52 µg/L), and 10.7% (at 13.87 µg/L).
Cross-reactivity was undetectable at 0.2 µg/tube for IGF-II, insulin,
proinsulin, and GH. The addition of pure IGFBP-1 or IGFBP-3 caused a
dose-dependent decrease in measurable free IGF-I (22). Free IGF-I
values less than 0.15 µg/L were considered to be below the detection
limit in this study, because the lowest IGF-I standard of this assay
was 0.15 µg/L.
Measurements of serum total IGF-I and IGFBP-3 levels
The serum total IGF-I concentrations were determined by an IRMA
kit (somatomedin-C II Chiron, Chiron, Inc., Tokyo, Japan). In brief,
IGF-I was extracted with acid-ethanol (35) and mixed with a
radiolabeled indicator antibody. Antibody-coated beads were added to
the mixture and incubated for 2 h at room temperature. After
washing, the radioactivity of the beads was counted with a -counter.
The minimal detection limit was 0.3 µg/L, and the intra- and
interassay CVs were 2.0–6.4% and 3.6–6.4%, respectively. The
cross-reactivity of IGF-II in the IGF-I IRMA was less than 0.1%.
Serum IGFBP-3 levels were analyzed with a RIA kit (IGFBP-3 Cosmic,
Cosmic Corp., Inc., Tokyo, Japan). The assay characteristics have been
reported previously (27). Briefly, unprocessed serum (diluted 1:51) was
added to polyclonal rabbit antiserum and 125I-labeled
IGFBP-3 tracer obtained from a 30.5-kDa stable IGFBP-3 fragment
isolated from Cohn fraction IV (27). The reaction mixture was
incubated, and the bound tracer was separated from unbound tracer by
precipitation using goat antirabbit serum. After centrifugation, the
supernatants were decanted, and the radioactivity was determined in a
-counter. The minimal detection limit was 0.2 µg/L, and the intra-
and interassay CVs were 3.8–7.4% and 5.7–8.4%, respectively.
Statistical analysis
The results for the healthy subjects are given as the mean ± SD, and those of the GH-deficient patients treated with rhGH are given as the mean ± SEM. Free IGF-I values below the detection limit were calculated as 0.15 µg/L for the statistical analysis. The Mann-Whitney U test was used to compare groups. Wilcoxon’s nonparametric test was used to compare the effects of GH treatments. The relationships between the different variables were calculated by linear regression analysis. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The age-related changes in serum free IGF-I in normal subjects are
shown in Fig. 1A
and Table 1. In the males, the serum free IGF-I
levels were low in infancy (first year of life; 0.71 ± 0.26
µg/L, mean ± SD) and childhood, then rose and
peaked at 13 yr of age (5.84 ± 2.18 µg/L), and declined
thereafter. In the females, the serum free IGF-I levels were also low
in infancy (1.05 ± 0.49 µg/L) and childhood, began to increase
at the age of 9 yr, peaked at 11 yr (5.80 ± 1.49 µg/L), and
then declined. Therefore, free IGF-I in serum attained its highest
value during puberty in both males and females. The peak level of serum
free IGF-I in the girls appeared about 2 yr earlier than that in the
boys.
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and Table 1).
The males achieved peak values at the age of 13 yr, whereas the females
showed their highest values between the ages of 11–13 yr. The total
IGF-I level then decreased to the adult levels. The age-related changes
in serum total IGF-I in this study closely resembled those in serum
free IGF-I.
Serum IGFBP-3 also showed low values in infancy (males, 1.37 ±
0.28 mg/L; females, 1.55 ± 0.41 mg/L), and thereafter an almost
linear increase in IGFBP-3 was seen from early childhood to puberty
(males, 2.98 ± 0.43 mg/L; females, 3.31 ± 0.37 mg/L; Fig. 1C and Table 1). The serum IGFBP-3 levels in puberty were twice those
in infancy. Therefore, the increase in serum IGFBP-3 was not as
significant compared with those in free IGF-I and total IGF-I.
Relationships among free IGF-I, total IGF-I, and IGFBP-3 in normal children
Free IGF-I in serum made up about 0.95–2.02% of the total serum
IGF-I values (Fig. 2). As shown in Fig. 2, the ratio of free IGF-I to total IGF-I was significantly higher in
infancy (<1 yr of age). As for the relationship between total IGF-I
and IGFBP-3, the total IGF-I to IGFBP-3 ratio was not constant but,
rather, was increased in puberty, suggesting that the increase in IGF-I
was relatively larger than that in IGFBP-3 in puberty (Fig. 3).
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The SD scores of serum free IGF-I levels in the 21
prepubertal GH-deficient patients before GH therapy ranged from -3.30
to 0.30 (mean, -1.59; Fig. 4). All five
complete GH-deficient patients had serum free IGF-I values more than
-2 SD below those of age-matched normal subjects. Among
them, 3 had free IGF-I values less than the assay sensitivity limit
(<0.15 µg/L). As shown in Fig. 4A, a significant correlation was
observed between the SD scores of free IGF-I and those of
total IGF-I for all 21 GH-deficient patients (r = 0.715;
P < 0.0005). The correlation between the
SD scores of free IGF-I and those of IGFBP-3 was not as
significant (r = 0.490; P < 0.05; Fig. 4B). There
were no apparent correlations between the serum free IGF-I levels and
height velocity in the GH-deficient patients before GH therapy (data
not shown).
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With the beginning of GH therapy, the patients’ serum free IGF-I
levels increased significantly (Fig. 5A).
After 1 month of treatment, their serum free IGF-I levels were elevated
to 209% of the pretreatment levels and remained high during the GH
therapy. The maximal free IGF-I level (252% of pretreatment levels)
was seen with 6 months of GH therapy, and the serum free IGF-I levels
remained high during GH therapy.
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). The total IGF-I levels were
increased to only 139% of pretreatment levels after 1 month of GH
treatment and attained their highest values (149% of pretreatment
level) after 6 months of GH treatment. The serum IGFBP-3 levels were
elevated to 114% of pretreatment levels after 1 month of treatment and
remained elevated during the 1 yr of GH therapy (Fig. 5C). Relationships between serum free IGF-I, total IGF-I, and IGFBP-3 levels and height velocity in GH-deficient patients
As shown in Fig. 6A, the percent
increase in the serum free IGF-I level after 1 month of GH treatment
correlated with the GH-induced improvement in height velocity (the
ratio of height velocity during 1 yr of GH therapy to that before GH
therapy; r = 0.526; P < 0.05). However, the
increases in total IGF-I and IGFBP-3 after 1 month of GH therapy did
not show any significant correlation with the improvement in height
velocity (Fig. 5, B and C).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In this study, we demonstrated that free IGF-I in serum attained its highest values in puberty; the peak values of the girls and boys were observed at 11 and 13 yr of age, respectively. The girls experienced peak values approximately 2 yr earlier than the boys. These age-related changes in serum free IGF-I during puberty closely resemble the height velocity curves for Japanese children (36). Consistent with our results, Juul et al. and Hasegawa et al. observed an elevation in serum free IGF-I during puberty (16, 19). A recent report also revealed that boys with precocious puberty have increased free IGF-I concentrations compared to prepubertal boys (17). These findings suggest that sex steroids increase the serum free IGF-I levels directly or by increasing the GH secretion.
The present data also showed that the highest values of total IGF-I and IGFBP-3 occurred during puberty, as previously reported (21, 26, 27, 37, 38). However, there was a considerable difference between total IGF-I and IGFBP-3 in the magnitude of increase; the ratio of total IGF-I to IGFBP-3 increased in puberty, suggesting more marked increases in serum total IGF-I compared to those in IGFBP-3. Juul et al. also found a marked increase in the molar ratio between total IGF-I and IGFBP-3 in puberty (21). Our finding of a pubertal elevation of serum free IGF-I supports the hypothesis that the increased total IGF-I/IGFBP-3 ratio in puberty reflects an increase in free, biologically active IGF-I.
An acceleration of height gain takes place in infancy as well as in puberty (2). In agreement with these accelerations, we found that several serum bone formation markers, i.e. the bone-specific alkaline phosphatase isoenzyme and the carboxyl-terminal propeptide of type I procollagen, showed high values in both infancy and puberty (39, 40). The significant increase in serum free IGF-I observed during puberty certainly contributes to the accelerated bone growth in puberty. However, in contrast to the pubertal period, neither free IGF-I nor total IGF-I showed any significant increase during infancy in the present study. It was reported that children with genetic defects of the GH gene (type IA GHD) or the GH receptor gene (Laron syndrome) present with severe short stature beginning in early infancy (41, 42). A postnatal growth delay was also observed in IGF-I knockout [igf-1(-/-)] mice (4). These facts indicate that the GH/IGF-I axis plays an important role in growth during infancy. Hasegawa et al. (16, 18) reported that the free to total IGF-I ratio was relatively increased in early infancy. Based on their data, they speculated that the increased ratio of free to total IGF-I represents an increased conversion of plasma IGF-I to the free form, and that the resultant increase in IGF-I bioavailability contributes to the rapid growth in early infancy. In our present study, the free to total IGF-I ratio attained the highest values during infancy, as Hasegawa et al. reported (16, 18). This increased free to total IGF-I ratio might contribute to the acceleration of height gain observed in infancy. Of course, further study is necessary to clarify the significance of the free to total IGF-I ratio to growth.
Several reports have confirmed the GH dependence of serum total IGF-I
and IGFBP-3 levels (2, 7, 43). Therefore, the measurements of serum
total IGF-I and IGFBP-3 have been widely accepted as indicators in the
diagnosis of GHD (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). As for the diagnostic usefulness of free
IGF-I, Juul et al. (19) recently reported that the free
IGF-I determination offered no major advantage in the evaluation of
adult GHD compared to total IGF-I or IGFBP-3 measurements. However,
Hasegawa et al. (16) reported that the clinical utility of
plasma free IGF-I measurements is similar to that of the measurements
of total IGF-I in the evaluation of childhood GHD. In our study, the
SD scores of serum free IGF-I in the GH-deficient patients
before GH therapy ranged from -3.30 to 0.30 (mean = -1.59).
There was a considerable overlap between the serum free IGF-I values of
the prepubertal GH-deficient patients and those of the healthy
subjects. However, the complete GH-deficient patients showed free IGF-I
values more than -2 SD below those of age-matched normal
subjects. In agreement with this result, Frystyk et al. (14)
demonstrated that serum free IGF-I levels in GH-deficient patients
were significantly lower than those in healthy volunteers despite some
overlap between the two groups. It may be difficult to conclude which
is more useful in the diagnosis of GHD between free IGF-I and total
IGF-I, because of the small number of patients examined. However, as
shown in Fig. 4A, the SD scores of free IGF-I correlated
significantly with those of total IGF-I in the GH-deficient children.
In addition, the serum levels of both free and total IGF-I in the five
complete GH-deficient patients were quite low (-2 SD or
more). These results suggest that the diagnostic usefulness of free
IGF-I for GHD is almost the same as that of total IGF-I.
With the beginning of GH treatment, our GH-deficient patients showed a rapid and progressive increase in serum free IGF-I. There is only one prior report, to our knowledge, of the effect of GH treatment on serum free IGF-I levels. Skjærbæk et al. reported for the first time that GH administration increased the serum free IGF-I levels in adult GH-deficient patients (20). Although a GH dependency of serum free IGF-I was suspected based on the finding of low serum free IGF-I levels in patients with GHD, our present results provided additional evidence for the GH regulation of serum free IGF-I. As also previously found (44, 45, 46), GH treatment increased the serum total IGF-I and IGFBP-3 levels in our patients. However, the increase in free IGF-I was significantly higher than those in total IGF-I and IGFBP-3. This finding may indicate that the serum free IGF-I level is more sensitive to the GH secretory status than are total IGF-I and IGFBP-3.
Of the various IGF system components studied, the increase in free IGF-I after 1 month of GH treatment was significantly correlated with the increase in height velocity during the first year of GH therapy. This result indicates that the increase in free IGF-I plays an important role in the increase in height during GH therapy. This result also suggests that a GH-induced increase in serum free IGF-I may be used to predict the growth response to 1 yr of GH treatment. In the management of patients receiving GH therapy, the early determination of the long term growth response is important. The measurement of IGF-I has previously been advocated as a tool to assess the magnitude of a growth response to GH therapy (44, 47). However, several researchers reported that serum IGF-I levels have not correlated well with growth velocity (48, 49), because IGF-I is synthesized not only in bone but in many other tissues as well (50). Although the numbers of GH-deficient patients investigated have been small, free IGF-I in the serum is currently a better candidate for predicting the efficacy of GH therapy than are total IGF-I and IGFBP-3.
In conclusion, our results show that serum free IGF-I increases when the height velocity is elevated, such as during puberty and GH treatment. The measurement of serum free IGF-I may become a useful tool both for diagnosing GHD and predicting growth responses to long term GH therapy.
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Acknowledgments |
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Footnotes |
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Received May 12, 1998.
Revised September 15, 1998.
Accepted October 5, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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