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

The insulin-like growth factor binding proteins (IGFBPs) are the carri ers for insulin-like growth factor (IGF0-I and IGF-II. IGFBP-3 is GH-d ependent and circulates associated with IGFs and an acid-labile subuni t to form a 150-kilodalton (kDa) complex. In human serum, two immunore active molecular weight forms of IGFBP-3 have been identified. In huma n urine, radioimmunoassayable levels of IGFBP-3 have been detected. Th e objectives of this study were to characterize the molecular weight f orms of IGFBP-3 in urine and serum of healthy children and adults and in children with GH deficiency (GHD), to quantify the urinary molecula r weight forms of IGFBP-3, and to evaluate the relationship of these f orms with GH therapy. Urine and serum were obtained from 12 prepuberta l children with GHD, before and after 6 months of GH therapy, from 30 prepubertal healthy children, and from 8 healthy adults. Western immun oblotting (WIB) with IGFBP-3 antiserum (alpha IG-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 healt hy adults and was low in children with GHD but increased after GH ther apy. Urinary IGFBP-9 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 arou nd 17 kDa. The 17-kDa urinary IGFBP-3 chromatographic peak averaged 84 61 +/- 367 ng/12 h(.)m(2) 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 G H therapy. Urinary IGFBP-3 was also measured by RIA in unfractionated urine; healthy children showed levels significantly higher (14575 +/- 460 ng/12 h(.)m(2)) than adults (7823 +/- 1083, P < 0.001) and higher than children with GHD before GH therapy (4710 +/- 703, P < 0.001). Ag ain, however, immunoreactive IGFBP-3 increased after GH treatment (122 94 +/- 3394). In the serum of the healthy children we characterized by specific IGFBP-3 WIB analysis, a 17.7-kDa immunoreactive form of IGFB P-3 that was absent in the serum of healthy adults and low in patients with GHD, increased during GH therapy. Serum samples were subjected t o neutral-size exclusion chromatography and the fractions were analyze d by WIB. The distribution of the 41- to 39-kDa and 29-kDa IGFBP-3 for ms between the 150- and 44-kDa IGFBP-complexes were similar in sera fr om adults and children. In sera of children, the 17.7-kDa IGFBP-3 form showed a peak of immunoreactivity in the 110-kDa chromatographic regi on. 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 bo th urine and serum a 17.7-kDa IGFBP-3 form that is age and GH dependen t. 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 ov ernight urinary collection may be useful for the diagnosis of GHD or f or monitoring GH treatment,