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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 1 156-158
Copyright © 1997 by The Endocrine Society

Pediatric Endocrinology

High Ratios of Free to Total Insulin-Like Growth Factor-I in Early Infancy

Yukihiro Hasegawa, Tomonobu Hasegawa, Katsura Fujii, Hideko Konii, Makoto Anzo, Taiji Aso, Shinobu Koto, Makoto Takada and Yutaka Tsuchiya

Division of Endocrinology and Metabolism, Tokyo Metropolitan Kiyose Children’s Hospital (Y.H., K.F., H.K., M.A., T.A., S.K.) Tokyo, Japan 204; Yuka Medias, Research Center (M.T.); and Keio University School of Medicine (T.H., Y.T.)

Address all correspondence and requests for reprints to: Yukihiro Hasegawa, Division of Endocrinology and Metabolism, Tokyo Metropolitan Kiyose Children’s Hospital, 1–3-1 Umezono Kiyose, Tokyo, Japan 204.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor-I (IGF-I) is a major effector of somatic growth and metabolism. In normal, nonpregnant plasma, most of the IGF-I is complexed to specific IGF-binding proteins (IGFBPs), particularly IGFBP-3; only a minor fraction of plasma IGF-I exists in a free form. Recently, we have reported that free IGF-I levels, as measured using a new immunoradiometric assay, are relatively high in maternal plasma during pregnancy because of increased IGFBP-3 proteolytic activity. These high free IGF-I levels are physiologically important for the growth of maternal tissues such as uterus and placenta, which are related to the fetal growth. Growth during early infancy may be a continuation of fetal growth. In the present study, we have analyzed free and total plasma IGF-I and IGFBP-3 proteolytic activity in early infancy. Although the levels of free and total IGF-I were not significantly different in early infancy as compared with prepubertal periods, the ratio of free to total IGF-I (mean = 2.04%) was relatively increased and was similar to the ratio in pregnancy plasma (1.86%). However, unlike in maternal plasma, the high ratios were not totally caused by increased IGFBP-3 proteolytic activity. Our results suggest that there may be an increased conversion of plasma IGF-I to a free form in early infancy. The resultant increase in IGF-I bioavailability could contribute to the rapid somatic growth in early infancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor-I (IGF-I) mediates most of the physiological actions of GH and is a major effector of somatic growth. Most of the plasma IGF-I is complexed to specific IGF-binding proteins (IGFBPs). Only a small fraction of plasma IGF-I is present in a free form (1, 2).

We have recently reported a new, direct immunoradiometric assay for plasma free IGF-I (3, 4, 5). This assay appears to have advantages over other methods because the capture antibody does not cross-react with bound forms of IGF-I. In addition, the assay is specifically designed to avoid disturbances of the endogenous equilibrium between IGF-I and the IGFBPs.

Using this assay, we have found that free IGF-I levels are higher in plasma from pregnant women as compared with nonpregnant, age-matched controls (5). Total IGF-I levels were not different between the groups, leading to relatively high ratios of free to total IGF-I in pregnancy plasma. The increased free IGF-I levels were related to increased IGFBP-3 proteolytic activity in pregnancy serum (6, 7, 8) and could play a role in the growth of maternal tissues such as the uterus and placenta, which are related to the growth of the fetus.

In postnatal life, the most rapid rates of somatic growth occur during early infancy. In many respects, growth during early infancy appears to be a continuation of fetal growth patterns. Therefore, we examined plasma free and total IGF-I levels and serum IGFBP-3 proteolytic activity in early infancy to clarify the possible role of IGF-I during this rapid growth period.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sample collection

The involvement of human subjects in this study was approved by the institutional Review Board of Tokyo Metropolitan Kiyose Children’s Hospital. Written informed consent was obtained from the subjects and/or their parents or legal guardians before entry into the study. The subjects included 52 normal infants (male = 21), postnatal age 32–180 days, approximately equally distributed by month of age. The comparison population included normal, healthy, prepubertal children (n = 28, male = 15, 4–7 yr old); normal, healthy, nonpregnant adults (n = 61, male = 23, 19–45 yr old); and pregnant women (n = 44, 22–35 yr old, 10–38 weeks gestation). EDTA plasma and serum were collected by peripheral venipuncture and stored at -70 C until assay.

Assays

Free IGF-I was measured by immunoradiometric assay (3, 4, 5). In brief, 0.2 mL EDTA plasma sample or standard (recombinant human IGF-I: Toyobo Co., Ltd., Osaka, Japan) was added to assay tubes, each containing a polystyrene bead precoated with anti-IGF-I monoclonal antibody. After a 5-min incubation at 37 C, the beads were washed three times with distilled H2O, and then 125I-anti-IGF-I monoclonal antibody directed to second epitope was added for an additional 3-h incubation at room temperature. The beads were then washed and counted in an automatic {gamma}-counter. We have previously demonstrated that the capture antibody is specific for IGF-I, and that the epitope consists of amino acids 24–31 of the mature peptide. As studied by immunoblot and by cross-linking experiments, the capture antibody does not recognize IGF-I/IGFBP complexes (tested with IGFBP-1, 2, 3, 4, and partially proteolyzed IGFBP-3). The capture reaction does not disturb the equilibrium between IGF-I and IGFBPs within the first 5-min incubation period (3). The functional range of the assay is 0.03–10.00 ng/mL, with interassay and intraassay coefficients of variation of <10%.

Total IGF-I was measured using the same reagents as in the free IGF-I assay with the addition of an extraction step (using 12.5% vol/vol 0.1 N HCl in absolute ethanol) and slight technical modifications (3). The functional assay range is 6.3–2100.0 ng/mL, with interassay and intraassay coefficients of variation of <10%.

IGFBP-3 proteolytic activity was analyzed by Western immunoblot (9). Briefly, 0.002 mL of each serum sample was analyzed directly or preincubated for 2–4 h at 37 C with 0.002 mL of the control (normal adult) pooled serum. The samples were then separated by SDS-PAGE under nonreducing conditions. The separated proteins were transferred to nitrocellulose and blocked with 3% BSA, followed by incubation with goat anti-hIGFBP-3 antiserum (1:1000: Diagnostic System Laboratories, Webster, TX) for 30–60 min at 37 C or overnight at 4 C and developed using a horseradish peroxidase-linked second-antibody system with chemiluminescent substrate (Amersham, Tokyo, Japan). For samples analyzed directly, the ratio of intact to proteolyzed IGFBP-3 was used as a marker of IGFBP-3 proteolytic activity. For samples incubated with the control pooled serum, the degree of decrease in the intact IGFBP-3 band was analyzed by densitometer (Advantec, Tokyo, Japan). The control pooled serum served as a source for intact IGFBP-3. For normal nonpregnant test serum, <10% of intact IGFBP-3 was proteolyzed, whereas >50% of the IGFBP-3 was proteolyzed after incubation with pregnant serum (9). The values of IGFBP-3 proteoltic activity were the means of three separate experiments.

Statistical analysis

Descriptive data are shown as the mean and SD. Statistical comparisons were performed using the Mann-Whitney U-test. P values of <0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Free and total IGF-I levels are shown in Table 1Go. Although mean free and total levels were lower in early infancy as compared with the other age groups, the differences in free IGF-I levels between early infants and prepubertal children or adults were not statistically significant. However, the ratios of free to total IGF-I were significantly higher in early infancy and pregnancy as compared with the prepubertal and nonpregnant adult groups (P < 0.001). There was no difference between the free to total IGF-I ratios for the early infancy and pregnancy groups.


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  		Table 1. Free IGF-I and total IGF-I levels

 
Figure 1Go shows representative data for IGFBP-3 proteolysis on Western immunoblot of test samples with or without incubation of control pooled serum (Fig. 1Go, Incubation with C). The three infancy samples are shown in this figure because they had the highest IGFBP-3 proteolytic activity among 18 infants studied. Control pooled serum alone (Fig. 1Go, C; lanes 1 and 2) show the intact ~43 to 45 kilodalton (kDa) IGFBP-3 doublet, a less intense band at ~29 kDa, and relatively faint lower molecular weight bands. The intact IGFBP-3 bands are virtually absent in pregnancy serum (Fig. 1Go, P; lane 3). The intact doublet band is present in the early infancy serum (Fig. 1Go, Infancy; lanes 4–6); the lower molecular weight bands are more intense in these samples as compared with controls (Fig. 1Go, C; lanes 1 and 2), but the ratios of the major lower molecular weight band (~29 kDa) to intact IGFBP-3 bands were not as high as in the pregnancy sample lane. The intact IGFBP-3 bands of control pooled serum almost completely proteolyzed after incubation with pregnant pooled serum (Fig. 1Go, P; lane 7), whereas those were not proteolyzed after incubation with control pooled serum itself (Fig. 1Go, C; lane 11), and those were only partially proteolyzed after incubation with infancy serum (Fig. 1Go, Infancy; lanes 8–10).



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  		Figure 1. Representative Western immunoblot of serum samples with or without preincubation with control pooled serum (Incubation with C). IGFBP-3 immunoreactivity was examined in serum samples separated by SDS-PAGE. Lanes 1 and 2 show control pooled serum (C) alone; lane 11 shows control pooled serum (C) after preincubation period. Early infancy serum alone (Infancy; lanes 4–6) or after preincubation with control pooled serum (Infancy; lanes 8–10) and pregnancy pooled serum (P) alone (lane 3) or after preincubation with control plasma (lane 7) are also shown. See text for additional details.

 
Using densitometer, IGFBP-3 proteolytic activity was examined in a total 18 of the early infancy samples. Six of 18 did not have any significant IGFBP-3 proteolytic activity (significant; >10% decrease in the intact IGFBP-3 band (9)). Twelve of these samples had measurable IGFBP-3 proteolytic protease activity (means of three separate experiments; 18–64% decrease in the intact IGFBP-3 band), but this activity was less than the level observed in pregnancy serum. Only three infants (as shown in Fig. 1Go) showed IGFBP-3 proteolytic activity more than 50%. All of the pregnancy serum samples examined (n = 22) had significant IGFBP-3 proteolytic activity (69–100% decrease in the intact IGFBP-3 band, data not shown); this result is consistent with our previous reports (5, 9). None of the prepubertal childhood (n = 12) or nonpregnant adult (n = 15) serum samples studied had significant IGFBP-3 proteolytic activity (data not shown).

We have previously reported that free to total IGF-I ratios are significantly correlated (n = 41, r = 0.86, P = 0.001) with IGFBP-3 proteolytic activity in serum from various subjects not including infants (10). However, this correlation was not observed for the early infancy samples in the current study.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The free fraction of IGF-I is postulated to account for the biological activity of IGF-I in the circulation (11). We have previously found that both free IGF-I and free to total IGF-I ratios are higher in plasma from pregnant as compared with nonpregnant women (5 and our unpublished data). This increase was related to higher levels of IGFBP-3 proteolytic activity in the pregnancy serum. The increased amount of plasma free IGF-I in pregnancy could increase the tissue bioavailability of IGF-I (11) and could play an important physiological role in the growth of maternal tissues, which are related to fetal growth.

The rapid postnatal growth that occurs in infancy can be divided into two phases; an initial GH-independent phase, as evidenced by the relatively normal growth of infants with GH-1 gene deletion or Pit-1 gene abnormalities during this period, and a second phase that is relatively GH dependent. The initial phase of rapid growth suggests a continuation of fetal growth patterns, whereas the second phase of infant growth provides a gradual deceleration into the lower postinfancy childhood growth rates.

Our results indicate that the rapid growth during early infancy, which is relatively GH independent, cannot be explained by increased levels of total or free IGF-I. However, the increased ratio of free to total IGF-I could theoretically represent a cross-sectional view of a system in which there is an increased rate of free IGF-I release, thereby providing a mechanism for increased delivery of IGF-I to the growing tissues.

The reason for the increased flux of free IGF-I remains unknown. Our results also indicate that this increased flux of free IGF-I cannot be explained solely by increased IGFBP-3 proteolytic activity. An alternate explanation is the possible presence of an acid-protease in infant serum that cleaves IGF-I to form des-(1–3) IGF-I, a form of IGF-I with full affinity for the receptor(s) and low affinity for IGFBPs (12, 13). In rat serum, the acid-protease was reported to be present (14). Our free IGF-I assay detects both intact and des-(1–3) IGF-I; therefore, the contribution of des-(1–3) IGF-I to our free IGF-I results cannot be assessed. Additional studies will be needed to further define the reason for increased ratio of free to total IGF-I in early infants.


   Acknowledgments
 
The authors gratefully acknowledge the assistance of Dr. Phillip D.K. Lee (U.S A.) in the preparation of this manuscript.

Received March 11, 1996.

Revised July 13, 1996.

Accepted July 19, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jones JI, Clemmons DR. 1995 Insulin-like growth factors, and their binding proteins: biological actions. Endocr Rev. 16:3–34.[Abstract/Free Full Text]
  2. Cohen P, Fielder PJ, Hasegawa Y, Frisch H, Giudice LC, Rosenfeld RG. 1991 Clinical aspects of insulin-like growth factor binding proteins. Acta Endocrinol (Copenh). 124:74–85.
  3. Takada M, Nakanome H, Koshida M, Hirose S, Hasegawa T, Hasegawa Y. 1994 Measurements of free IGF-I using immunoradiometric assay. J Immunoassay. 15:263–276.[Medline]
  4. Hasegawa Y, Takada M, Hasegawa T, Tsuchiya Y. 1996 Clinical utility of newly developed free insulin-like growth factor-I measurements in the diagnosis of growth hormone deficiency. Eur J Endocrinol. 134:184–189.[Abstract/Free Full Text]
  5. Hasegawa T, Hasegawa Y, Takada M, Tsuchiya Y. 1995 The free form of insulin-like growth factor-I increases in circulation during normal human pregnancy. J Clin Endocrinol Metab. 80:3284–3286.[Abstract]
  6. Fielder PJ, Thordarson G, Talamates F, Rosenfeld RG. 1990 Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice: effects of hypophysectomy and an IGFBP-specific serum protease activity. Endocrinology. 127:2270–2280.[Abstract/Free Full Text]
  7. Guidice LC, Farrell EM, Pham H, Lamson G, Rosenfeld RG. 1990 Insulin-like growth factor binding proteins in maternal serum throughout gestation, and on the puerperium: effects of a pregnancy-associated serum protease activity. J Clin Endocrinol Metab. 71:806–816.[Abstract/Free Full Text]
  8. Hosselopp P, Segovia B, Lassarre C, Roghani M, Bredon M, Binoux M. 1990 Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy. J Clin Endocrinol Metab. 71:797–805.[Abstract/Free Full Text]
  9. Hasegawa Y, Hasegawa T, Konii N, et al. 1995 Proteolytic activity of IGFBP-3 in various clinical conditions during childhood using western immunoblot technique. Endocrine J. 42:569–576.[CrossRef]
  10. Hasegawa Y, Hasegawa T, Konii H, Tsuchiya Y. 1995 Correlation between IGFBP-3 proteolytic activity and the ratio of free IGF-I to total IGF-I levels in circulation. Clin Endocrinol (Oxf). 43:943–945 (in Japanese).
  11. Guler HP, Zapf J, Schmid C, Froesch ER. 1989 Insulin-like growth factors I and II in healthy man. Estimates of half lives and production rates. Acta Endocrinol (Copenh). 121:753–758.[Abstract/Free Full Text]
  12. Bagley CJ, May BL, Szabo L, et al. 1987 A key functional role for the insulin-like growth factor I N-terminal pentapeptide. Biochem J. 259:665–671.
  13. Ballard FJ, Francis GL, Ross M, Bagley CJ, May BL, Wallace JC. 1987 Natural and synthetic forms of insulin-like growth factor-I (IGF-I) and the potent derivative, destripeptide IGF-I: biological activities and receptor binding. Biochem Biophys Res Commun. 149:398–404.[CrossRef][Medline]
  14. Yamamoto H, Murphy LJ. 1994 Generation of des-(1–3) Insulin-like growth factor-I in serum by an acid protease. Endocrinology. 135:2432–2439.[Abstract]



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