Human Plasma Cohn Fraction Research Articles

A Specific Radioimmunoassay for the Growth Hormone (GH)-Dependent Somatomedin-Binding Protein: Its Use for Diagnosis of GH Deficiency

The acid-stable subunit of the GH-dependent large mol wt somatomedin-binding protein (SmBP) was isolated from human plasma Cohn fraction IV by a three-step procedure, and a specific RIA was developed which allowed measurement in unextracted serum. Although in normal human serum most of immunoreactive material was present as the large mol wt complex (150K), considerable amounts of smaller components were found by high performance liquid exclusion chromatography in the 60K, 42K, and 32K range. Normal serum levels were low at birth, rose sharply during the first weeks of life, and showed a moderate peak at puberty. To assess the diagnostic efficacy of SmBP for GH deficiency (GHD), patients previously diagnosed as GH-deficient by conventional criteria (n = 132) were compared to short statured children without GHD (n = 130). Taking the fifth centile as a limit of normality the majority of patients with GHD had subnormal levels, yielding high sensitivity of the test (0.97). In contrast, most of the non-GH-deficient children had SmBP levels within the normal range, resulting in high specificity (0.95) and, consequently, high accuracy (0.96). These results suggest that the large mol wt SmBP is an excellent screening parameter and is highly informative for GHD.

Insulin-Like Growth Factor I (IGF-I)-Binding Protein Complex Is a Better Mitogen than Free IGF-I*

The insulin-like growth factors (IGF)-I and -II are bound to specific carrier proteins in the circulation. For investigation of their physiological role, the acid-stable subunit of the major binding protein (SmBP) was isolated from human plasma Cohn fraction IV. Its effect on the mitogenic activity of IGF-I was studied with baby hamster kidney fibroblasts (BHK-21) and human skin fibroblasts. While free IGF-I had no effect on thymidine incorporation into DNA with BHK-21 cells and only a moderate effect with human fibroblasts under standard conditions, DNA synthesis was significantly enhanced with both cell lines if IGF-I was complexed with SmBP before the experiment. The enhancement was optimal at an approximately equimolar ratio of both peptides. In contrast to experiments in which large concentrations of IGF-I were added at the beginning, repeated addition of small quantities of free IGF-I at hourly intervals clearly stimulated DNA synthesis in BHK-21 cells. Binding studies with radiolabeled SmBP revealed no evidence for direct interaction with either cell line. It is concluded that SmBP acts as a reservoir, releasing continuously low amounts of IGF-I and thereby creating a steady state situation of receptor occupancy, which appears to be a better mitogenic stimulus than temporary large concentrations of IGF-I.

31 SOMATOMEDIN BINDING PROTEIN (SmBP) IN NORMALS AND IN GROWTH HORMONE DEFICIENCY (GHD)

The major SmBP in serum is a 150 kD glycoprotein. Its acid-stable subunit was isolated from human plasma Cohn fraction IV by a 3 steps procedure. With the purified protein a RIA was developed which recognizes the complete 150 kD complex in nativ serum. In normal controls there was a considerable increase from 25.7 nmol/l (median, range 14.7 - 44.7) in newborns to 49.4 nmol/l (28.8 - 84.7) after 8 weeks of age. Thereafter SmBP increased continuously to 110.5 nmol/l (77.9 - 156.9) at puberty and declined to 98.9 nmol/l (73.4 - 132.9) in adults. Correlation with IGF-I and IGF-II (by RIA) was non-linear. The sum of both IGF-I and IGF-II correlated linearly with SmBP (r= 0.89) with a regression line intersecting the abscissae at 19.7 nmol/l (about 148 ng/ml) of total IGF-I and -II. In 70 patients with GHD (max. GH < 10 ng/ml in 2 standard tests) 68 had subnormal levels of SmBP (mean SDS for age: -5.52). SmBP increased significantly on treatment with hGH. Conclusions: 1. The 150 kD SmBP is the major BP form in postnatal life. 2. Total serum Sm levels (IGF-I + IGF-II) are primarily determined by SmBP concentration. 3. SmBP is strongly GH dependent. 4. SmBP is a more specific parameter for the diagnosis of GHD compared to IGF-I or -II particularly below age 5.

Isolation of acid-stable insulin-like growth factor binding protein from human plasma Cohn fraction IV

Isolation of acid-stable insulin-like growth factor binding protein from human plasma Cohn fraction IV

Isolation of insulin-like growth factors I and II from human plasma Cohn fraction IV: An improved method

Isolation of insulin-like growth factors I and II from human plasma Cohn fraction IV: An improved method

Isolation and partial characterization of six somatomedin-like peptides from human plasma Cohn fraction IV.

Six somatomedin-like peptides were purified from human plasma Cohn fraction IV by a six-step procedure which included ethanol precipitation, reversed-phase extraction, gel filtration, chromatofocusing and reversed-phase high pressure liquid chromatography (HPLC). Purification was monitored with a competitive protein binding assay using a crude preparations of somatomedin carrier protein. The peptides isolated were homogeneous by reversed-phase HPLC and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Their apparent isoelectric points determined by chromatofocusing were 9.2 (Sm I), (Sm II), 8.2 (Sm III), 6.7 (Sm IV), 6.3 (Sm V), and 6.15 (Sm VI). SDS-PAGE under reducing conditions revealed that they are composed of a single peptide chain with apparent molecular weights of 6800 for Sm I, II and IV and 6400 for Sm III, V, and VI. They were equally potent in the porcine costal cartilage in vitro bioassay. The basic peptides (Sm I-III) were significantly more active in radioimmunoassays for somatomedin C (SmC) and insulin-like growth factor I C-peptide (IGF-I (30 - 41], while only the slightly acidic peptides were active in a radioimmunoassay for insulin-like growth factor II C-peptide (IGF-II (33-40]. When receptor binding was tested with human placental cell membranes and Sm III as tracer, the basic peptides were significantly more potent than Sm IV-VI. With rat liver cell membranes and Sm V as tracer the slightly acidic peptides were more potent. These findings suggest 1) that human plasma may contain other somatomedin-like peptides besides the major components IGF-I/SmC and IGF-II, and 2) that the basic peptides are structurally related to IGF-I/SmC and the slightly acidic peptides are related to IGF-II.

Structure of somatomedin-binding protein: alkaline pH-induced dissociation of an acid-stable, 60,000 molecular weight complex into smaller components.

The association of somatomedin (Sm) peptides with their specific serum binding proteins (SmPBs) and the preservation of SmBP integrity are both pH dependent. Acid extracts of human plasma Cohn fraction IV-4 chromatographed at pH 5.0 after incubation with [125I]iodoinsulin-like growth factor I yield predominantly a 60,000 molecular weight complex of specifically bound radiolabeled peptide. Alkaline exposure (pH 8.0) of either the initial acid extract of Cohn fraction IV-4 or the isolated 60,000 molecular weight chromatographic peak shifts the recovery of bound [125I]iodoinsulin-like growth factor I on rechromatography to two smaller complexes of approximately 46,000 and 30,000 molecular weight. These results support the existence of two or more forms of human plasma SmBP that may possess a common 30,000 molecular weight component.

Release of somatomedin-like activity by cultured WI-38 human fibroblasts.

Confluent cultures of normal diploid WI-38 human embryonic lung fibroblasts released somatomedin (SM)-like activity into their incubation medium during culture in serum-free medium. This postculture medium (conditioned medium) stimulated cell division in these same cultured WI-38 fibroblasts and 35SO4 uptake by hypophysectomized rat cartilage in vitro. The conditioned medium also contained immunoreactive SM (IRSM) activity which yielded parallel dose-response curves to human serum in a RIA for SM. The IRSM activity measured in conditioned medium was not the artifactual result of effects of possible SM-binding proteins or proteolytic enzymes in conditioned medium. These studies suggest that cultured WI-38 fibroblasts produce and release SM-like activity which has SM-like biological activity and is immunoreactive with a basic SM purified from human plasma Cohn fraction and having similarity with SM-C and insulin-like growth factor-I. Human GH appears to stimulate production and release of IRSM activity by these cells.

Purification of a basic somatomedin, from human plasma Cohn fraction IV-1, with physicochemical and radioimmunoassay similarity to somatomedin-C and insulin-like growth factor.

A basic somatomedin (SM) was purified from human plasma Cohn fraction IV-1 using an initial acid--ethanol--acetone extraction procedure followed by alternating molecular size or charge protein separation techniques. The final recovery of SM bioactivity was approximately 2% of that present in the starting Cohn fraction. The purified SM has an approximate molecular weight of 7500, pI 8.6, 4000 SM bioactivity units per milligram (as measured by a hypophysectomized rat bioassay) and a parallel approximately equipotent radioimmunoassay dose--response curve to SM-C and insulin-like growth factor-1 (IGF-I). Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of this purified SM revealed a single protein band. The preliminary determination of the amino acid sequence of the N terminus suggested that this SM preparation was over 75% pure and the first five N-terminal amino acids were identical with those of IGF-I.

A simple radioimmunoassay for the measurement of testosterone glucosiduronate in unextracted urine

A simple, reliable and rapid radioimmunoassay (RIA) for the determination of testosterone glucosiduronate (TG) in crude urine is described. Two protein-TG complexes were investigated in raising antibodies: a) Bovine serum albumin (BSA)-TG and b) human plasma Cohn's fraction IV-4 (CF)-TG. In rabbits, high liters of antibodies were obtained after the injection of CF-TG. The specificity of the antiserum was sufficiently high (cross reaction with free testosterone 27%, with 5α-dihydrotestosterone-glucosiduronate 20%). TG was estimated in small aliquots of male and female urine after evaporation overnight at 50° C in order to eliminate interfering material. The intraassay coefficient of varation (CV) was found to be 6% and the interassay CV 11%. TG has been determined in 40 samples of urine simultaneously by “direct” RIA and by a “classical” RIA following hydrolysis with β-glucuronidase. The coefficient of correlation was found to be 0.89. The mean excretion of TG in the urine of 26 healthy men amounted to 164 ± 51 μg/24 hours with a range from 97 to 546 μg/24 hours. In a group of 16 women a mean urinary excretion of TG of 24 ± 10 μg/24 hours was determined. The method allows a technician to assay 40 samples per day.