Urinary transcript quantitation of CK20 and IGF2 for the non-invasive bladder cancer detection.

PURPOSE: Cytokeratin 20 (CK20) and insulin-like growth factor 2 (IGF2) were previously proposed to be elevated in clinical samples from patients with bladder cancer (BCa). A two cohort design validation study was used to assess the relevance for BCa detection by transcript quantitation of both markers in urine samples. Their diagnostic value was assessed in comparison with voided urine cytology (VUC). METHODS: RNA isolation was carried out using cellular sediments of urine samples from 196/103 histologically positive BCa patients, as well as 97/50 control subjects for the test (TC) and validation cohort (VC), respectively. Urinary transcript levels of CK20 and IGF2 were determined by qPCR. RESULTS: Relative transcript levels were significantly elevated 3.4/11-fold for CK20 and 188/64-fold for IGF2 (p < 0.001) in urine sediments of BCa patients compared to controls in the TC and VC, respectively. In a combined analysis, the resulting sensitivity (SN) (SNTC: 77.9; SNVC: 90.3%) and specificity (SP) (SPTC: 88.0; SPVC: 84.0%) were similar to that of VUC. The sensitivity of VUC in combination with CK20 and IGF2 was considerably increased (SNTC: 94.6; SNVC: 93.2%) while specificity was reduced (SPTC: 72.0; SPVC: 82.0%) compared to VUC alone in the test and validation cohort. CONCLUSIONS: Transcript levels of IGF2 and CK20 enabled the detection of BCa with a diagnostic performance similar to VUC. Combined analysis of voided urine cytology together with altered transcript levels of CK20 and IGF2 enhanced sensitivity, but did not improve overall test performance.

Insulin-like growth factors and kidney disease.

Insulin-like growth factors (IGF-1 and IGF-2) are necessary for normal growth and development. They are related structurally to proinsulin and promote cell proliferation, differentiation, and survival, as well as insulin-like metabolic effects, in most cell types and tissues. In particular, IGFs are important for normal pre- and postnatal kidney development. IGF-1 mediates many growth hormone actions, and both growth hormone excess and deficiency are associated with perturbed kidney function. IGFs affect renal hemodynamics both directly and indirectly by interacting with the renin-angiotensin system. In addition to the IGF ligands, the IGF system includes receptors for IGF-1, IGF-2/mannose-6-phosphate, and insulin, and a family of 6 high-affinity IGF-binding proteins that modulate IGF action. Disordered regulation of the IGF system has been implicated in a number of kidney diseases. IGF activity is enhanced in early diabetic nephropathy and polycystic kidneys, whereas IGF resistance is found in chronic kidney failure. IGFs have a potential role in enhancing stem cell repair of kidney injury. Most IGF actions are mediated by the tyrosine kinase IGF-1 receptor, and inhibitors recently have been developed. Further studies are needed to determine the optimal role of IGF-based therapies in kidney disease.

Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS.

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.

Effect of physical fitness and endurance exercise on indirect biomarkers of growth hormone and insulin misuse: Immunoassay-based measurement in urine samples.

Indirect biomarkers of recombinant human growth hormone (rhGH), insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBP-2 and IGFBP-3) and insulin (C-peptide) were measured together with urinary parameters of renal damage (beta(2)-microglobulin and proteinuria) by immunoassays, in house validated for the purpose, in 61 subjects (36 elite athletes, 18 recreational athletes and 7 sedentary individuals) with different levels of physical fitness and endurance exercise. Validation parameters were good for the evaluated assays, excluding a high inter-assay imprecision and inaccuracy of 24 and 26% obtained for GH assay. The range of concentrations found in urine samples under investigation was generally covered by the calibration curves of the studied immunoassays. However, for the samples below or above the calibration curve, opportune dilution or concentration were performed. Particularly, C-peptide samples had to be diluted 1:5 and beta(2)-microglobulin ones assayed using a triple sample volume, to fall within the calibration range. Urinary C-peptide was the only biomarker statistically higher in samples of elite athletes when compared to recreational athletes and sedentary individuals. Among elite athletes, tae-kwon-do athletes showed the highest IGF-II basal values while weightlifting athletes showed the lower IGF-I and IGFBP-3 basal values. The trend observed in weightlifters' basal samples was confirmed in their training samples: IGF-I, IGF-II, IGFBP-3 and beta(2)-microglobulin were lower in with respect to those from synchronised swimming. Over the training season, within athlete variability was observed for IGFBP-3 for weightlifting athletes. In the studied subjects, no direct associations were found between biomarkers of GH or insulin misuse and urinary parameters of renal damage, eventually due to high-workload endurance training. The variations observed in different biomarkers should be taken in consideration in the hypothesis of setting reference concentration ranges for doping detection.

Urinary insulin-like growth factor 2 identifies the presence of urothelial carcinoma of the bladder.

OBJECTIVE: To examine urinary insulin-like growth factor 2 (IGF-2) levels in patient urine samples and determine the potential of IGF-2 as a marker for the presence of urothelial carcinoma of the bladder (UCB). PATIENTS AND METHODS: The current gold standard for diagnosis of UCB is cystoscopy and cytological analysis. The identification of an accurate urine marker for UCB with the potential to replace unnecessary cystoscopy would benefit patients with UCB and others investigated after detecting haematuria. In the present study, we analysed 65 urine samples, and optimized an enzyme-linked immunosorbent assay-based approach to measure urinary levels of IGF-2. RESULTS: Based on a threshold of 5.4 ng/mL, patients with UCB have significantly elevated levels of urinary IGF-2 (P = 0.009) and this difference remained significant after adjustment for age and sex (P = 0.04). Sensitivity and specificity values of 80% and 52%, respectively, were determined for urinary IGF-2 alone and when combined with nuclear matrix protein 22 (NMP22; an approved biomarker for detection of UCB). There was a positive correlation between urinary IGF-2 levels and NMP22 levels in patient urine samples and the combined assay improved the detection of UCB (sensitivity 85% and specificity 52%). CONCLUSION: Substantiated evidence has identified IGF-2 as a valuable marker for UCB. In addition, the novel observations of the present study have shown that aberrant levels of IGF-2 occurring in the presence of UCB, can now be determined through a simple and inexpensive urine assay.

Clearance of IGFs and insulin from wounds: effect of IGF-binding protein interactions.

We have examined the role binding proteins have in regulating the clearance of exogenous growth factors from wounds. Hunt-Schilling chambers were subcutaneously implanted in rats, and the clearance of insulin-like growth factor (IGF) I from the chamber wound fluid was compared with IGF-II, LR3-IGF-I, which binds poorly to IGF-binding proteins (IGFBP), or insulin. Elimination rate constants of the slow phase of the decay curves did not differ between IGF-I and IGF-II. However, LR3-IGF-I and insulin were cleared more rapidly from wound fluid than IGF-I so that the half-lives for IGF-I, IGF-II, LR3-IGF-I, and insulin were 872, 861, 563, and 324 min, respectively. In wound fluid, minimal degradation of the IGFs occurred, whereas insulin was degraded considerably. The increased clearance of LR3-IGF-I and insulin equated with a reduced association with wound fluid IGFBPs, and increased amounts of radioactivity of these peptides were detected in the circulation and urine. These results show that this model of wound repair may be of use in examining the kinetics of growth factors and other bioactive molecules in extravascular spaces and support the hypothesis that IGFBPs can be significant regulators of IGF bioavailability in vivo.

Quantification of urinary insulin-like growth factors (IGFs) and IGF binding protein 3 in healthy volunteers before and after stimulation with recombinant human growth hormone.

We examined excretion of urinary insulin-like growth factors I and II (IGF-I and IGF-II) and their major binding protein IGFBP-3 in comparison to their respective serum concentration in nine healthy female volunteers (median age 25 years, range 22-27) under baseline conditions and after stimulation with recombinant human growth hormone (rhGH), 4.5 IU twice daily subcutaneously for a period of 3 days. The IGFs were measured in unconcentrated urine by use of recently developed, highly sensitive radioimmunoassays. The IGFBP-3 was measured by a specific radioimmunoassay. The mean (+/- SD) urinary concentrations of IGF-I (0.08 +/- 0.07 micrograms/l), IGF-II (1.02 +/- 0.47 micrograms/l) and IGFBP-3 (19.1 +/- 6.9 micrograms/l) were two to three orders of magnitude lower than in serum. The ratio of IGF-II over IGF-I concentration in urine (13:1) was five times higher than in serum (2.5:1), and the ratio of IGFBP-3 over the sum of IGF-I and IGF-II in urine (17:1) was four times higher than in serum (4:1). Urinary excretion was 63.3 +/- 46.6 ng.m-2.24h-1 for IGF-I, 1002 +/- 598 ng.m-2.24h-1 for IGF-II and 18039 +/- 4983 ng.m-2.24h-1 for IGFBP-3. Using fast protein liquid exclusion chromatography, only immunoreactive IGFBP-3 components of less than 60 kD were detected in urine, with a major peak at 20 kD. Urinary IGFBP-3 excretion correlated with serum IGFBP-3 (r = 0.61, p < 0.01) and the glomerular filtration rate (r = 0.56, p < 0.05) measured by steady-state inulin infusion clearances.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary insulin-like growth factor-II excretion in healthy infants and children with normal and abnormal growth.

The output of urinary IGF-II was measured by RIA in 12-h overnight urine samples obtained from 22 preterm and 15 full-term infants, 40 normal children, 18 children with growth hormone (GH) deficiency, and 25 patients with idiopathic short stature. GH deficiency was defined as a peak to GH provocative tests < or = 9.9 micrograms/L during two provocative tests. The authenticity of urinary IGF-II was confirmed by size exclusion chromatography. Statistical analysis was performed by one-way analysis of variance using the Student Neuman-Keuls test to detect intergroup differences at the level of p < 0.05. The preterm and full-term infants excreted significantly higher amounts of urinary IGF-II (18.4 +/- 1.7 and 5.7 +/- 1.0 pmol/kg, respectively) compared with normal children (2.4 +/- 0.25 pmol/kg; p < 0.001). The output of urinary IGF-II in preterm infants was greater than that observed in full-term infants (F = 84.7, p < 0.001). The control children excreted significantly more IGF-II (2.4 +/- 0.2 pmol/kg) than children with GH deficiency (0.9 +/- 0.1 pmol/kg) or idiopathic short stature (1.0 +/- 0.1 pmol/kg; F = 13.5; p < 0.001). Analysis of urinary IGF-II excretion based on creatinine output yielded similar results. Data on urinary IGF-I and GH previously published were correlated and compared with the excretion pattern of urinary IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary insulin-like growth factors (IGF) and IGF-binding proteins in normal subjects, growth hormone deficiency, and renal disease.

Recent studies in our laboratories have shown that urine from healthy adults contains immunoreactive and intact insulin-like growth factor-binding protein-3 (IGFBP-3). The aim of this study was to assess urinary IGF-I, IGF-II, and IGFBP-3 in a cross-sectional study of healthy subjects, as well as characterize urinary IGFBPs (uIGFBps) in patients with GH deficiency (GHD) and renal disease, such as, Alport syndrome, immunoglobulin A nephropathy, focal segmental glomerulosclerosis, and systemic lupus erythematosus. Urinary concentrations of IGF-I and IGF-II in pooled spot morning urines of healthy subjects, measured by RIA, were low and relatively unaltered throughout age, when expressed as either nanograms per milliliter or nanograms per milligram creatinine. To determine the complement of IGFBPs in urine of healthy subjects, spot morning urine samples were subjected to Western ligand blot and immunoblot analysis. IGFBP-3 was detected at 40-50 kDa, possibly due to variable glycosylation of uIGFBP-3. In addition, a 32-kDa IGFBP-2 and smaller unclassified IGFBPs were detected. Unlike uIGFs, urinary concentrations of IGFBP-3 (uIGFBP-3; nanograms per milligram creatinine) were age-, but not sex-related. Levels of uIGFBP-3 ranged from 40-60 ng/mL in children between 4 and 10 yr of age. After 11 yr, immunoreactive uIGFBP-3 progressively declined, attaining a plateau after 26 yr of age to approximately 18 ng/mg creatinine. uIGFBP-3 did not correlate with uIGF levels. Regulation of IGFBP-3 in the urine of normal subjects and of renal disorders was examined by RIA, Western ligand blot (WLB), and protease assay. Intact uIGFBP-3 was consistently found in normal urine and little urinary protease was identified. In GHD patients, IGFBP-3 by WLB was low or undetectable, whereas RIA levels of uIGFBP-3 were normal or high, consistent with the presence of IGFBP-3 proteolytic activity. In Alport syndrome, both RIA measures and WLB analysis were high, as was the IGFBP-3 proteolytic activity. Patients with immunoglobulin A nephropathy, focal segmental glomerulosclerosis and systemic lupus erythematosus measured low-normal levels of IGFBP-3 by WLB and RIA, and displayed little protease activity. This study provides normative data concerning radioimmunoassayable levels of IGFBP-3 in urine. The presence of normal-elevated levels of uIGFBP-3 by RIA in GHD indicates that uIGFBP-3 levels are not under GH control and are unlikely to represent filtered serum IGFBP-3.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of insulin-like growth factor-II in physiological fluids and tissues. I. An improved extraction procedure and radioimmunoassay for human and rat fluids.

The measurement of serum insulin-like growth factors (IGFs) in serum is complicated by the presence of high affinity IGF-binding proteins. The accurate measurement of IGFs by radioligand binding assays requires that the interference from binding proteins be eliminated. Acid-gel chromatography, the standard method for removing binding proteins, is laborious and time consuming. Alternative methods for extracting serum IGFs include the use of HCl-ethanol treatment and reverse phase minicolumns. However, these methods are unsuitable for use with serum for some species, such as rat and sheep, due to incomplete removal of binding proteins. We developed a fast protein liquid chromatography size-exclusion chromatographic method for characterizing the presence of IGF-binding proteins in physiological fluids and used this method to systematically investigate different combinations of acids and organic solvents as potential extraction methods for IGFs. We developed and validated an improved extraction procedure that uses formic acid, Tween-20, and acetone. The new extraction method was used in conjunction with purified biosynthetic human IGF-II and a commercially available anti-IGF-II monoclonal antibody in the development of an improved RIA for IGF-II. The new RIA is sensitive (5.0 pg/tube), specific (IGF-I cross-reactivity, less than 1%), and reproducible [interassay precision (coefficient of variation), less than 9.2%). We measured the serum concentrations of IGF-II in adults and found a significant difference between normal subjects and individuals with insulin-dependent diabetes mellitus.

Immunoreactive insulin-like growth factor II in urine.

Insulin-like growth factor II and insulin-like growth factor binding protein-1 were identified and quantified in the urine of 23 healthy subjects between 17 and 76 years of age. IGF-II was measured after separation by gel chromatography at low pH and compared with IGF-I levels in the same samples, whereas IGF binding protein-1 was measured in dialysed urine. Urinary IGF-II was found at much higher concentrations than IGF-I (mean +/- SEM: 717 +/- 69 vs 110 +/- 5 ng/mmol creatinine). The chromatographic profile indicates that pro-IGF-II may also be present. The concentrations of IGF-II appear to be less variable than the other reported parameters. The mean IGF binding protein-1 concentrations in these urine samples was 414 +/- 83 ng/mmol creatinine. IGFs in the urine are in part bound to binding proteins.

Insulinlike growth factors in patients with active nephrotic syndrome.

The serum concentrations of insulinlike growth factors 1 and 2 (IGF-1 and IGF-2) were measured by specific radioimmunoassays in 25 nephrotic patients. The serum concentration of IGF-1 in nephrotic patients (mean +/- SEM, 169 +/- 17 ng/mL) was significantly lower than that observed in 20 control subjects matched for sex and age (338 +/- 36 ng/mL). The IGF-2 serum concentration was significantly lower in nephrotic patients (343 +/- 22 ng/mL) than in control subjects (898 +/- 80 ng/mL). The IGF-1 and IGF-2 150-kd and 45-kd carrier protein complexes were found in the urine of nephrotic patients, whereas there was no binding of radiolabeled IGF-1 or IGF-2 to IGF carrier proteins in the urine of control subjects. The low serum IGF-1 and IGF-2 levels observed in nephrotic patients could be partially due to the increased urinary losses of the IGF carrier proteins.