Insulin-like growth factor binding protein-3 protease activity in the urine of children with chronic renal failure.

The insulin-like growth factors, IGF-1 and IGF-II, are polypeptides that potentiate cellular growth. In addition to binding to specific cell surface receptors, the IGFs bind with high affinity to a family of proteins, the insulin-like growth factor binding proteins (IGFBPs). Serum and urine IGFBP patterns are altered in individuals with chronic renal failure (CRF). We recently reported that the urinary IGFBP pattern of CRF patients is unique for increased insulin-like growth factor binding protein-1 (U-IGFBP-1) levels. In this study, we used western ligand blotting (WLB), western immunoblotting (WIB), and radioimmunoassay (RIA) to further evaluate serum and urine IGFBP profiles of children with CRF (n = 14). Five patients with CRF displayed decreased serum IGFBP-3 profiles by WLB. Serum IGFBP-3 WIB profiles were remarkable for 30- and 20-kDa fragments of IGFBP-3 not seen in control serum. Serum IGFBP-3 levels, as determined by RIA, were slightly elevated. Serum levels of IGFBP-2 also were increased, although not at a level reaching statistical significance. WLB of CRF urine revealed a large increase in U-IGFBP-1 and a complete absence of urinary IGFBP-3. Recent studies of serum from pregnant women and seminal plasma have demonstrated a similar absence of intact IGFBP-3, due to the presence of a specific IGFBP-3 protease. To evaluate whether an IGFBP-3 protease accounts for the absence of intact U-IGFBP-3 in children with CRF, urine and serum samples from individuals with CRF and controls were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of urinary somatomedin C by a chemiluminescent method.

We present a method for the measurement of the total Somatomedin C (SmC) content in human early morning urine samples after dialysis, extraction, and concentration. We modified a chemiluminescence immunoassay, previously developed for SmC determination in serum, for analysis of SmC in urine. Appropriate sensitivity was obtained by the preparation of a new chemiluminescent tracer (AEEI-COOH-SmC) and the optimization of a competitive non-equilibrium immunoassay system which had a detection limit of 0.24 fmol SmC per tube.

Quantitation of urinary somatomedin-C and growth hormone in preterm and fullterm infants and normal children.

Urinary GH and somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) excretion were measured in 12-h urine collections obtained from 43 infants (27 stable preterm infants and 16 healthy fullterm infants) and 31 normal children, aged 3-17 yr. Urinary Sm-C/IGF-I was excreted as the free hormone, since no binding of radiolabeled Sm-C/IGF-I to any urine protein with a mol wt similar to those described for plasma Sm-C/IGF-I-binding proteins was found. The preterm infants excreted significantly more urinary GH [13.5 +/- 2.1 (+/- SE) ng/kg.12 h] than either the fullterm infants (5.3 +/- 1.6 ng/kg.12h) or the children (0.27 +/- 0.02 ng/kg.12 h; P less than 0.01). The mean urinary Sm-C/IGF-I excretion in the preterm infants (98.9 +/- 7.5 mU/kg.12 h) was comparable to that in fullterm infants (87.6 +/- 9.7 mU/kg.12 h); both groups excreted significantly more urinary Sm-C/IGF-I than children (28.4 +/- 2.1 mU/kg.12 h; P less than 0.01). The group differences were similar when the results were expressed in terms of creatinine excretion. Urinary GH excretion correlated positively with urinary Sm-C/IGF-I excretion (r = 0.68). The higher output of these peptides in rapidly growing infants and their positive correlation in urine provide additional support for the Sm hypothesis.

Immunoreactive somatomedin C/insulin-like growth factor I in urine from normal subjects, pituitary dwarfs, and acromegalics.

Using antibodies to somatomedin C/insulin-like growth factor I (SmC) produced in rabbits using the recombinant hormone, we have developed a radioimmunoassay for SmC. Gel-chromatography of urine revealed that the vast majority of immunoreactive SmC was eluted coincident with 125I-SmC and a small portion eluted with fractions having a mol. wt. range of 30,000-40,000. The SmC concentration in urine was determined by radioimmunoassay after ammonium sulfate extraction. Values did not ordinarily exceed 1 ng/ml. When the values from normal subjects were expressed as ng/mg creatinine, high levels were observed in the neonatal period. These values fell rapidly in infancy, declined more gradually in childhood, were slightly elevated at early puberty, and were lowest in adulthood. Urine SmC concentrations in 15 pituitary dwarfs were lower than the averages obtained from agematched control subjects, and six of them showed abnormally low values. Three patients with active acromegaly had high SmC values in urine. In conclusion, 1) SmC, mainly of monomeric form, was immunologically detected in urine. 2) Radioimmunoassay for urine SmC revealed that values varied considerably with age in normal subjects and were partially dependent on the human growth hormone status. However, the full meaning of the findings remains to be elucidated.

Urinary IGF-I measurement and its clinical application.

Immunoreactive and receptor-reactive IGF-I was found to be present in human urine; 30% of the IGF-I immunoreactivity in urine was in its free form and the remainder was a high molecular weight form (approximately 40,000 MW). Urinary IGF-I was quantified by radioimmunoassay after extraction by Sep-Pak C18 cartridge, a method that measures only the free form of IGF-I. Daily (24-hour) urinary IGF-I excretion was measured in 3 hypopituitary children and 16 short normal children. The IGF-I level in the 24-hour urine samples correlated with the plasma IGF-I level and the mean 24-hour plasma GH concentration. The mean 24-hour plasma GH concentration, however, correlated better with the GH level in the 24-hour urine samples and the plasma IGF-I level than with the urinary IGF-I value. The mean IGF-I levels in single urine samples from normal subjects lay between those from patients with acromegaly (which were high) and those from patients with hypopituitarism (which were low). There were overlaps, however, in individual values between the normal and hypopituitary patients. These data indicate that urinary IGF-I values are altered by the GH secretion state, though the clinical application of urinary IGF-I measurement may be limited.

Quantitation of urinary somatomedin-C in children with normal and abnormal growth.

The renal excretion of radioimmunoassayable somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was measured in 12-h overnight urine samples obtained from 88 subjects, aged 3-19 yr. The participants included 34 healthy children (group 1), 29 children with idiopathic growth failure and normal GH stimulation tests (group 2), and 25 GH-deficient subjects (group 3). The mean (+/- SEM) urinary Sm-C/IGF-I excretion in group 1 (28.4 +/- 2.1 mU/kg) was significantly greater than that in group 2 (8.1 +/- 1.6 mU/kg) or group 3 (8.6 +/- 1.3 mU/kg). Twenty-two of the 29 subjects in group 2 had urinary Sm-C/IGF-I values less than 8 mU/kg. After the administration of biosynthetic GH to 12 GH-deficient subjects, urinary Sm-C/IGF-I excretion rose from 10.3 +/- 2.3 to 21.4 +/- 4.2 mU/kg within 12 h (P less than 0.05), indicating that renal excretion of Sm-C/IGF-I is GH dependent. One woman with acromegaly had markedly elevated urinary Sm-C/IGF-I excretion (420 mU/kg). The authenticity of urinary Sm-C/IGF-I was confirmed by high pressure liquid chromatography (HPLC). Assay of serial dilutions of urinary Sm-C/IGF-I demonstrated a direct proportionality between concentration and dilution. Although it is not possible to identify whether urinary Sm-C/IGF-I reflects local or generalized synthesis of the peptide, we hypothesize that quantitation of Sm-C/IGF-I in timed urine collections will yield additional information about GH production and action in children with normal and abnormal growth.

Demonstration of insulin-like growth factor I in human urine.

Immunoreactive and receptor-reactive insulin-like growth factor I (IGF-I) was demonstrated in human urine. Thirty percent of the IGF-I immunoreactivity in urine was free, and the remainder was a high mol wt form (approximately 43K). Urinary IGF-I was quantitated by RIA after extraction with octadecylsilyl silica cartridges (Sep-Pak C18 cartridge), a method that measures only free IGF-I. The mean urinary immunoreactive IGF-I levels in normal adults (n = 8) and patients with acromegaly (n = 10) or hypopituitarism (n = 9) were 72 +/- 7 (+/- SEM), 225 +/- 34, and 19 +/- 4 pg/mg creatinine, respectively; these mean values were significantly different from one another. The results indicate that IGF-I is present in human urine and that the quantity in urine is altered in patients with GH excess and deficiency.

Immunoreactive somatomedin B (RIA-B) in the circulation of healthy adults and patients with endocrine disorders.

The serum and cerebrospinal fluid (CSF) levels of immunoreactive somatomedin B (RIA-B) were examined throughout life in healthy adult humans. A significant decline in serum RIA-B was observed in subjects over 60 years of age. No significant diurnal, daily, or monthly serum variation was observed in healthy subjects aged 20-60 years. However, women taking oral contraceptives had elevated RIA-B values. No significant decline in CSF RIA-B was observed in subjects over 60 years of age. A significantly lower level of RIA-B in CSF was observed in subjects sampled at 20.00 h compared to subjects sampled in the morning. A significant decline in serum RIA-B was observed in patients with hypopituitarism and diabetes mellitus and a significant elevation of serum RIA-B levels was observed in patients with hyperthyroidism. CSF RIA-B was significantly elevated in patients with Cushing's syndrome.

PIP: The polypeptide called somatomedin B was radioimmunoassayed in serum or cerebrospinal fluid from volunteers throughout the lifespan, the daily and menstrual cycles, in oral contraceptive users, and in several patients with endocrinological disorders. This peptide, of 5000 molecular weight, is said to have insulin-like growth promoting activity, but its actual origin and function are unknown. In serum, there were no significant variations between the sexes, over the ages 20-60, in the diurnal or menstrual cycle, or by height, weight or body surface. There were significant declines after the age of 60 and 70 years in serum. The serum level in 8 women aged 20-40 taking oral contraceptives was significantly higher than that of 46 drug free women (p.001). In cerebrospinal fluid, there was a diurnal variation, a nadir at 2000 hours, but there was no decline in older individuals. Somatomedin B was significantly lower in patients with hypopituitarism and diabetes mellitus, and higher in those with hyperthyroidism and Cushing's syndrome. This is the first demonstration of somatomedin B in cerebrospinal fluid.

Somatomedin inhibitor in uremia.

In uremia, poor growth occurs despite normal to increased levels of insulin and GH. Since serum somatomedin levels measured by RIA and radioreceptor assay are normal in patients with renal failure, while serum somatomedin activity measured by bioassay is low but increased by dialysis, we asked if somatomedin activity could be decreased due to the presence of a low mol wt inhibitor(s). Serum was obtained from eight normal adults and eight uremic patients before hemodialysis treatment and was fractionated by gel filtration. Somatomedins and high mol wt inhibitors were separated on Sephadex G-50, pH 2.4, and high and low mol wt inhibitors were separated on Sephadex G-25, pH 7. Somatomedins were measured by stimulation of SO4 uptake by hypophysectomized rat costal cartilage in vitro, and inhibitor levels were determined by the blunting of stimulation produced by somatomedins in normal serum. Total biologically active somatomedin levels were comparable in uremic and normal sera. High mol wt somatomedin inhibitors (as found in malnutrition and diabetes) also were detected at similar levels in uremic and normal sera. In contrast, serum from uremic patients had increased levels of a low mol wt somatomedin inhibitor(s) [151 +/- 23% (mean +/- SEM) of serum stimulation inhibited vs. 47 +/- 9%; P less than 0.001]. Peak inhibitory activity was found at approximately 940 mol wt (range, 800-1100); an inhibitor of similar size was found in normal urine. Uremic serum fractions blunted cartilage sulfate uptake that was stimulated by whole serum, somatomedins (dissociated from serum carrier proteins), and insulin and lowered uridine and thymidine uptake that was stimulated by whole serum (all P less than 0.005). Lineweaver-Burk analysis indicated that somatomedin-inhibitor interactions on cartilage were noncompetitive, consistent with observations that direct exposure of cartilage to inhibitor decreased SO4 uptake to 30 +/- 3% below buffer levels (P less than 0.001). Despite these marked effects on cartilage, no alterations in basal or insulin-stimulated glucose oxidation occurred after addition of inhibitory serum fractions to adipose tissue incubations. Exposure of the inhibitor to proteolytic enzymes led to a significant decrease in inhibitory activity, indicating that the inhibitor may be a peptide. These studies suggest that decreased circulating somatomedin activity and impaired growth in uremia may reflect the accumulation of a circulating peptide inhibitor that would normally be cleared by the kidneys. Measurements of this factor may provide an index of growth potential in uremic children and help guide therapy of renal failure in both children and adults.

Serum and urine somatomedin B in pediatrics.

Somatomedin B was determined by the radioimmunological method in the serum and urine of 104 normal subjects divided into seven age groups. The serum and urine of each patient were divided into two fractions of which one was heated at 60 degrees C for 30 min. SmB serum levels were significantly higher in heated than in unheated samples in all age groups. Moreover, SmB levels in unheated samples increased slowly, but significantly, up to the age of four years, then decreased until nine years of age, when they started increasing again. In heated serum, however, a rapid and significant increase occurred from the age of six months; later SmB levels showed slight, not significant variations. In urine no significant difference was shown in SmB levels either between unheated and heated samples or between age groups. The difference in the behavior of SmB in serum and urine may be explained by assuming the presence of carrier proteins in the blood.

Immunoreactive somatomedin B in urine.

Urinary excretion by 8 normal adult subjects of immunoreactive somatomedin B was 27.6 +/- 4.4 mug between 1000 h and 1400 h compared to a mean plasma concentration at 1200 h of 5.9 +/- 0.9 mug/ml. Free somatomedin B in urine averaged 85.9%, although in the plasma of the same subjects all but less than 5% was bound to serum proteins.