Characterization of a Low Molecular Mass Form of Insulin-Like Growth Factor Binding Protein-3 (17.7 Kilodaltons) in Urine and Serum from Healthy Children and Growth Hormone (GH)-Deficient Patients: Relationship with GH Therapy

Anna Spagnoli, Sharron E. Gargosky, Gian Luigi Spadoni, Margaret Macgillivray, Youngman Oh, Brunetto Boscherini, Ronald (Ron) Rosenfeld

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Abstract

The insulin-like growth factor binding proteins (IGFBPs) are the carriers for insulin-like growth factor (IGF0-I and IGF-II. IGFBP-3 is GH-dependent and circulates associated with IGFs and an acid-labile subunit to form a 150-kilodalton (kDa) complex. In human serum, two immunoreactive molecular weight forms of IGFBP-3 have been identified. In human urine, radioimmunoassayable levels of IGFBP-3 have been detected. The objectives of this study were to characterize the molecular weight forms of IGFBP-3 in urine and serum of healthy children and adults and in children with GH deficiency (GHD), to quantify the urinary molecular weight forms of IGFBP-3, and to evaluate the relationship of these forms with GH therapy. Urine and serum were obtained from 12 prepubertal children with GHD, before and after 6 months of GH therapy, from 30 prepubertal healthy children, and from 8 healthy adults. Western immunoblotting (WIB) with IGFBP-3 antiserum (αIG-FBP-3g1) showed that in urine the most representative IGFBP-3 was a 17.7-kDa form. The 17.7-kDa IGFBP-3 was high in urine of healthy children compared with healthy adults and was low in children with GHD but increased after GH therapy. Urinary IGFBP-3 immunoreactive profile was determined by neutral-size exclusion chromatography, followed by IGFBP-3 RIA analysis of the fractions. Urine showed a major peak of IGFBP-3 immunoreactivity around 17 kDa. The 17-kDa urinary IGFBP-3 chromatographic peak averaged 8461 ± 367 ng/12 h·m2 of body surface in healthy children, 3415 ± 739 in adults (P <0.001), 2294 ± 354 in children with GHD before GH therapy (P <0.001), and 7940 ± 1874 in children with GHD after GH therapy. Urinary IGFBP-3 was also measured by RIA in unfractionated urine; healthy children showed levels significantly higher (14575 ± 460 ng/12 h·m2) than adults (7823 ± 1083, P <0.001) and higher than children with GHD before GH therapy (4710 ± 703, P <0.001). Again, however, immunoreactive IGFBP-3 increased after GH treatment (12294 ± 3394). In the serum of the healthy children we characterized by specific IGFBP-3 WIB analysis, a 17.7-kDa immunoreactive form of IGFBP-3 that was absent in the serum of healthy adults and low in patients with GHD, increased during GH therapy. Serum samples were subjected to neutral-size exclusion chromatography and the fractions were analyzed by WIB. The distribution of the 41- to 39-kDa and 29-kDa IGFBP-3 forms between the 150- and 44-kDa IGFBP-complexes were similar in sera from adults and children. In sera of children, the 17.7-kDa IGFBP-3 form showed a peak of immunoreactivity in the 110-kDa chromatographic region. The 17.7-kDa IGFBP-3 was assessed to be glycosylated, able to bind IGFs, and capable of forming a ternary complex. We demonstrated in both urine and serum a 17.7-kDa IGFBP-3 form that is age and GH dependent. Although a greater number of children with GHD and healthy children need to be evaluated before urinary IGFBP-3 is indicated as screening test for the GHD, we suggest that the measurement of IGFBP-3 in an overnight urinary collection may be useful for the diagnosis of GHD or for monitoring GH treatment.

Original languageEnglish (US)
Pages (from-to)3668-3676
Number of pages9
JournalJournal of Clinical Endocrinology and Metabolism
Volume80
Issue number12
Publication statusPublished - Dec 1995

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ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

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