Guilherme Póvoa

The somatomedin-binding protein isolated from a human hepatoma cell line is identical to the human amniotic fluid somatomedin-binding protein.

Serum-free medium conditioned by the human hepatoma cell line HEP G2 was shown to contain a somatomedin-binding protein with a relative molecular mass of about 35,000. This binding protein was purified to homogeneity by the use of immunoaffinity chromatography and subsequent size exclusion chromatography. Antibodies for the immunoaffinity step were raised in rabbits against a previously isolated human amniotic fluid somatomedin-binding protein. The total composition and N-terminal amino acid sequence showed the protein to be identical to the binding protein from human amniotic fluid. Both have the N-terminal structure Ala-Pro-Trp-Gln-. The HEP G2 cell line offers a useful model to study the regulation of the synthesis and secretion of human somatomedin-binding proteins.

Affinity chromatography with amniotic fluid somatomedin binding protein in the purification of insulin−like growth factor I

A simplified procedure has been developed for the isolation of insulin-like growth factor I from human plasma by use of affinity chromatography with the somatomedin binding protein. After acidification of human plasma and separation of insulin-like growth factor I and endogenous binding protein by cation exchange chromatography on SP-Sephadex the material was passed through a column packed with pure human amniotic fluid binding protein covalently coupled to Sepharose. The bound insulin-like growth factors I and II were eluted by 1M acetic acid and separated on a Mono S cation exchange column by use of a salt gradient. The 30 micrograms insulin-like growth factor I and 18 micrograms insulin-like growth factor II recovered from 1 liter plasma gave an overall recovery of 30% for insulin-like growth factor I but only 2.5% for insulin-like growth factor II.

Levels of Somatomedin-Binding Protein in Amniotic Fluid from Pregnancies with Small and Large for Gestational Age Infants

Levels of immunoreactive somatomedin-binding protein (SMBP) were determined in amniotic fluid from early gestation (week 14-20) in pregnancies resulting in newborns who were small (SGA) or large (LGA) for their gestational age. The mean level of SMBP (77 Mg/ml) in the SGA group was slightly elevated (p > .05) compared to the mean levels for the LGA and the control groups (48 Mg/ml and SI Mg/ml respectively). No correlations were found with parameters such as length or weight at birth, sex, levels of α-feto-protein and hCG in amniotic fluid, gestational age at sampling and the age of the mother.

SERUM LEVELS OF SOMATOMEDINS AND SOMATOMEDIN BINDING PROTEIN IN PREGNANT WOMEN AND THEIR INFANTS

Serum levels of IGF-I, IGF-II and the low molecular form of somatomedin binding protein (SMBP) were determined in pregnant women and their infants. Longitudinal studies during pregnancy were performed in healthy women, women with GH deficiency (n=3) and women with diabetes or gestational diabetes (n=44). IGF-I, IGF-II and SMBP were determined by radioimmunoassays using polyclonal antibodies.The serum levels of IGF-I and SMBP, but not IGF-II, increased during pregnancy in both healthy women and women with diabetes. In GH deficient women the levels rose to normal pregnant levels. The birth weight percentile of the infants increased with the maternal IGF-I levels and decreased with the maternal SMBP levels. The ratio between maternal IGF-I and SMBP levels improved the correlation to the birth weight percentile. No correlation was found between the birth weight and the isolated values of IGF-I, IGF-II, SMBP and C-peptide in cord blood. The IGF-II levels in cord blood from infants of diabetic women were 50% higher than those of nondiabetic women.The correlation between maternal levels of the birth weight of the infants indicates that maternal IGF-I levels may play a role in the transfer of nutrients to the fetus. The elevated IGF-II levels in cord blood of infants of diabetic women suggest that IGF-II could participate in the glucose homeostasis of the fetus.

51 REGULATION OF SOMATOMEDIN-BINDNING PROTEIN

The low molecular form of somatomedin-binding protein (SMBP) in serum crossreacts in a radioimmuno-assay developed for the SMBP isolated from human amniotic fluid. The SMBP in medium conditioned by hepatoma cells, HEP G2, was isolated by use of immunoaffinity chromatography and shown to have a N-terminal amino acid sequence identical with the amniotic SMBP. The HEP G2 cells were used as a model for the regulation of SMBP and the result was compared with those in humans.The levels of immunoreactive SMBP are tenfold higher in newborns than in adults, in which they are inverse related to GH. Increased levels are found during pregnancy and in patients with uremia. Estrogens increase the levels whereas GH causes a suppression.