An Improved Prediction Model for Ovarian Cancer Using Urinary Biomarkers and a Novel Validation Strategy.

This study was designed to analyze urinary proteins associated with ovarian cancer (OC) and investigate the potential urinary biomarker panel to predict malignancy in women with pelvic masses. We analyzed 23 biomarkers in urine samples obtained from 295 patients with pelvic masses scheduled for surgery. The concentration of urinary biomarkers was quantitatively assessed by the xMAP bead-based multiplexed immunoassay. To identify the performance of each biomarker in predicting cancer over benign tumors, we used a repeated leave-group-out cross-validation strategy. The prediction models using multimarkers were evaluated to develop a urinary ovarian cancer panel. After the exclusion of 12 borderline tumors, the urinary concentration of 17 biomarkers exhibited significant differences between 158 OCs and 125 benign tumors. Human epididymis protein 4 (HE4), vascular cell adhesion molecule (VCAM), and transthyretin (TTR) were the top three biomarkers representing a higher concentration in OC. HE4 demonstrated the highest performance in all samples withOC(mean area under the receiver operating characteristic curve (AUC) 0.822, 95% CI: 0.772-0.869), whereas TTR showed the highest efficacy in early-stage OC (AUC 0.789, 95% CI: 0.714-0.856). Overall, HE4 was the most informative biomarker, followed by creatinine, carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), and TTR using the least absolute shrinkage and selection operator (LASSO) regression models. A multimarker panel consisting of HE4, creatinine, CEA, and TTR presented the best performance with 93.7% sensitivity (SN) at 70.6% specificity (SP) to predict OC over the benign tumor. This panel performed well regardless of disease status and demonstrated an improved performance by including menopausal status. In conclusion, the urinary biomarker panel with HE4, creatinine, CEA, and TTR provided promising efficacy in predicting OC over benign tumors in women with pelvic masses. It was also a non-invasive and easily available diagnostic tool.

An extensive targeted proteomic analysis of disease-related protein biomarkers in urine from healthy donors.

The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1 ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development.

Urinary proteome analysis in congenital bilateral hydronephrosis.

OBJECTIVE: A proteomics strategy was applied to map protein changes in urine after relief of congenital bilateral hydronephrosis to identify proteins correlated with the pathophysiological processes in congenital obstructive nephropathy as potential urinary biomarkers. MATERIAL AND METHODS: Urine samples from 10 infants with bilateral abnormal drainage from the kidneys were collected at the time of relief from obstruction, and after 2 and 4 weeks. Proteomics techniques were used on samples from three patients for identification of protein changes between the three time-points, and enzyme-linked immunosorbent assay (ELISA) was used on samples from all 10 patients for validation of five selected proteins. RESULTS: Mass spectrometry quantified 315 protein hits, out of which 33 proteins showed significantly changed urinary excretion between the time-points. Validation by ELISA showed high urinary excretion of fibrinogen, plasminogen, transthyretin and transferrin at the time of relief from obstruction, followed by a significant reduction. In contrast, Tamm-Horsfall protein exhibited the reverse pattern. CONCLUSION: Using a mass spectrometry-based proteomics approach, this study identified 33 proteins related to congenital bilateral hydronephrosis, and pinpointed a panel of five proteins consistently linked to this congenital kidney disorder as potential urinary biomarkers.

Urinary vitamin D-binding protein is elevated in patients with endometriosis.

BACKGROUND: Recently, proteomic technologies have demonstrated that several proteins are differently expressed in various body fluids of patients with endometriosis compared with those without this condition. The aim of this study was to investigate proteins secreted in urine of patients with endometriosis using proteomic techniques in order to identify potential markers for the clinical diagnosis of endometriosis. METHODS: Urine samples were collected from women undergoing laparoscopy for different indications including pelvic masses, pelvic pain, suspicious endometriosis, infertility and diagnostic evaluation. Proteomic techniques and mass spectrometry were used to identify proteins secreted in the urine of the patients with and without endometriosis and quantification of identified protein was performed using western blot and specific commercial sandwich enzyme-linked immunosorbent assays (ELISA). RESULTS: Twenty-two protein spots were differentially expressed in the urine of patients with and without endometriosis, one of which was identified as urinary vitamin D-binding protein (VDBP). ELISA quantification of urinary VDBP corrected for creatinine expression (VDBP-Cr) revealed that urinary VDBP-Cr was significantly greater in patients with endometriosis than in those without (111.96 ± 74.59 versus 69.90 ± 43.76 ng/mg Cr, P = 0.001). VDBP-Cr had limited value as a diagnostic marker for endometriosis (Sensitivity 58%, Specificity 76%). When combined with serum CA-125 levels (the product of serum CA-125 and urinary VDBP-Cr), it did not significantly increase the diagnostic power of serum CA-125 alone. CONCLUSIONS: Urinary VDBP levels are elevated in patients with endometriosis. They have limited value as a potential diagnostic biomarker for endometriosis but suggest it would be worthwhile to investigate other urinary proteins for this purpose.

Proteomic identification of human urinary biomarkers in diabetes mellitus type 2.

BACKGROUND: During the proteomic era, one of the most rapidly growing areas in biomedical research is biomarker discovery, particularly using proteomic technologies. The urinary proteome is known to be a valuable field of study and has become one of the most attractive subdisciplines in clinical proteomics for human diseases. We have described the levels of protein biomarkers specific to diabetes mellitus type 2 in the Pakistani population using proteomic technology. METHODS: One hundred type 2 diabetes patients with 50 age- and sex-matched normal healthy controls were recruited from Sheikh Zayed Hospital, Lahore, Pakistan. Urinary proteins were analyzed by two-dimensional liquid chromatography, using chromatofocusing in the first dimension and reverse-phase chromatography in the second, followed by mass spectrometric analysis. Levels of the proteins, which were found to vary in the diabetes type 2 patients compared to the controls, were then determined by enzyme-linked immunosorbent assay in all the samples. RESULTS: Levels of transthyretin, α-1-microglobulin/bikunin precursor, and haptoglobin precursor decreased by 30.8%, 55.2%, and 81.45%, whereas levels of albumin, zinc α2 glycoprotein, retinol binding protein 4, and E-cadherin increased by 486.5%, 29.23%, 100%, and 693%, respectively, in the diabetes patients compared to the controls. CONCLUSIONS: Variation in the levels of these identified protein biomarkers have been reported in other pathological states. Assessment of the levels of these biomarkers will be helpful not only in early diagnosis but also in prognosis of diabetes mellitus type 2.

Microalbuminuria is a major determinant of elevated plasma retinol-binding protein 4 in type 2 diabetic patients.

Plasma retinol-binding protein 4 (RBP4) may be a new adipokine linked to obesity-induced insulin resistance and type 2 diabetes. The impact of diabetic nephropathy on plasma RBP4 levels, however, is not known. We tested the hypothesis that microalbuminuria is associated with elevated plasma concentrations of RBP4 in type 2 diabetic subjects. Retinol, its binding protein and transthyretin (TTR) were measured in the plasma and urine of 62 type 2 diabetic subjects, 26 of whom had microalbuminuria. The results were compared to 35 healthy control subjects. Despite no differences in plasma retinol, concentrations of the RBP4 were significantly elevated in plasma of diabetic patients and significantly higher in those with microalbuminuria. The higher plasma levels of the binding protein in subjects with microalbuminuria were accompanied by both significantly elevated plasma TTR and increased urinary levels of RBP4. There were no correlations of plasma-binding protein levels and parameters of insulin resistance. Our study suggests that plasma RBP4 levels in type 2 diabetic patients are affected by incipient nephropathy. Therefore, further studies evaluating RBP4 as a regulator of systemic insulin resistance and type 2 diabetes will need to take renal function into consideration.

[Protein markers in evaluation of nephroprotective effects of antihypertensive drugs in patients with arterial hypertension]. / Belkovye markery otsenki nefroprotektivnogo deistviia gipotenzivnykh sredstv u bol'nykh s arterial'noi gipertoniei.

Fifty patients with stable slight and moderate uncomplicated essential hypertension, treated by ramipril, atenolol, or isradipine, were examined. Total protein and urinary excretion of individual proteins were studied before and after treatment. Urinary concentrations of apolipoproteins A1 and B1, alpha 1-acid glycoprotein, alpha 1-antitrypsin, prealbumin, albumin, beta 2-microglobulin, transferrin, haptoglobin, IgG and IgA, and C3 and C4 complement components were measured. Index of proteinuria selectiveness was calculated for each portion of urine. All three drugs exerted a nephroprotective effect, atenolol being the most active of them. Apolipoproteins, IgG, and complement components were the most valuable for diagnosis. Their excretion correlated with the severity of arterial hypertension and efficiency of treatment. Use of protein markers helps reliably assess the renal function and monitor the treatment efficiency.

Duplicate prealbumin bands in urine. Association with hepatorenal failure.

By electrophoresis, prealbumin is seen as a discrete band anodic to the albumin fraction. Cases of duplicate bands in the prealbumin region have been described previously in serum, cerebrospinal fluid, and urine, and have had an association with varied clinical entities. The authors describe a novel association of severe hepatic and renal failure with the presence of prominent duplicate prealbumin bands in urine.

Clinical associations of an increased transthyretin band in routine serum and urine protein electrophoresis.

The 9697 electrophoretograms performed over an 8 year period were reviewed to identify the frequency and clinical associations of the finding of a prominent transthyretin band in serum or urine, the concentration of which was equal to or greater than a 64 mg/dL protein calibrator. All samples were electrophoresed at a constant 90 V using agarose gels with a barbital buffer pH 8.6 and Ponceau S staining. Reference calibrators were used as standards to identify increased transthyretin bands and the patients' clinical records were subsequently reviewed. High values were found in 46 patients' sera and a further nine patients' urines representing 0.57% of the total workload. Renal impairment was present in 58% of cases including those with chronic renal failure, the nephrotic syndrome and paraproteinaemia. The high levels were not persistent in three myeloma cases where there was a recovery in renal function following chemotherapy. In some nephrotics, a high urine transthyretin may be secondary to a general hepatic albumin and transport protein synthesis response to severe proteinuria. Why the serum transthyretin was elevated in many other cases remains unclear.

A radioimmunoassay for human urinary prealbumin.

We describe a validated radioimmunoassay for prealbumin in urine. Using timed overnight urine samples, the normal reference range was less than 10-148 micrograms/L; or less than 1.8-9.6 micrograms/mmol creatinine, excretion in women being significantly greater than in men (P < 0.05); or less than 7.3-114 ng/min with no significant difference in excretion rate between the sexes. Urines exhibited loss of immunoreactivity after storage at -20 degrees C and thawing. No such loss occurred after storage for 4 weeks at 4 degrees C or room temperature. The urinary excretion of prealbumin was highly correlated with that of albumin (r = 0.85), and clearance relative to creatinine was 2 x 10(-6), the same order as that of albumin.

Automated immunoturbidimetric methods for the determination of retinol binding protein, prealbumin and transferrin in urine.

We describe the development of rapid and simple automated methods for the estimation of retinol binding protein (RBP), prealbumin (PA) and transferrin (TRF) in urine. These methods measure the turbidity formed as a result of reaction between the protein and a specific antibody in a phosphate buffer containing 70 g/L polyethyleneglycol as accelerator for the antigen antibody reaction. The method for retinol binding protein (RBP) correlated well when compared with a method based on enzyme-linked immunosorbent assay (ELISA) (r = 0.978). The recoveries were 93-104%, 84-95% and 84-100% for RBP, PA and TRF respectively. The between-batch coefficients of variation were 2.6-10.6, 2.8-12.6 and 4.2-8.7% respectively. The analytical range of the method was 0.0625 to 40 mg/L for RBP, 0.28 to 45 mg/L for PA and 0.14 to 70 mg/L for TRF. The methods can be performed manually using a simple spectrophotometer or can be easily automated.

Allergy to rats: quantitative immunoelectrophoretic studies of rat dust as a source of inhalant allergen.

The antigenic composition of an extract of rat dust, as a source of aeroallergens for rat-sensitive individuals, has been investigated and compared to the antigenic composition of rat saliva and urine. Of four main antigenic peaks identified by crossed immunoelectrophoresis, one antigenic peak (Ag 4) was demonstrated to be antigenically closely related to and with similar molecular weight (approximately 22 kd) and isoelectric point values as urinary prealbumin, already recognized as a major rat allergen. Ag 4 was present in all dusts studied and was also identified as a minor component of saliva. However, no component with the same electrophoretic mobility or physicochemical characteristics of the alpha 2-euglobulin of male rat urine that shares partial identity with the prealbumin was detected, even in dust collected from a male rat room. A second high molecular weight (greater than 200 kd) component, Ag 1, present in most of the dust extracts, could not be detected in either urine or saliva. Crossed immunoelectrophoresis and skin prick tests confirmed the allergenicity of both these antigens. Analysis of an air filter sample taken within a male rat room revealed significant amounts of the "prealbumin" component, and a monospecific antiserum to this component was used to quantitate levels in dusts collected from various locations. These findings suggest that a major inhalant allergen present in rat dust is closely related to urinary prealbumin but that this and other allergenic components may not be derived predominantly from rat urine or saliva but possibly from secretions originating from the skin of the animals and present in the fur.

Allergy to mice. I. Identification of two major mouse allergens (Ag 1 and Ag 3) and investigation of their possible origin.

An extract of dust from the outlet filters of a mouse isolator was used as a basis for determining the source of inhalant allergens for subjects sensitive to this species. The antigenic components, identified by crossed immunoelectrophoresis (XIE), were compared to those found in extracts of other mouse-derived source materials, i.e. urine, fur, dander and saliva. Of the eight dust components, one (Ag 1) was identified as antigenically identical to the major urinary pre-albumin whilst the others were detected in fur, and to a lesser extent dander and saliva. None of the dust antigens was detected as a component of food or bedding. Crossed radio-immunoelectrophoresis (XRIE), performed using sera from a group of fifteen mouse-allergic subjects (positive by RAST to mouse extracts), identified seven of the dust antigens as IgE-binding components. Antigens 1 and 3 were reactive with all the sera tested and have, therefore, been termed the 'major' allergens. Varied responses were obtained to the other 'minor' antigens. Ag 1 (urinary pre-albumin) and Ag 3 were detected in all samples of mouse dust studied. RAST and RAST inhibition also indicated the presence of urinary pre-albumin. These findings suggest that the major mouse inhalant allergens may be derived predominantly from urine and secretions originating in the skin and present on the fur.

Biochemical marker in familial amyloidotic polyneuropathy, Portuguese type. Family studies on the transthyretin (prealbumin)-methionine-30 variant.

A transthyretin variant with a methionine for valine substitution at position 30 [TTR(Met30)] is found in Portuguese patients with familial amyloidotic polyneuropathy (FAP). Effective, rapid, small- and semimicro-scale (immunoblotting) procedures were developed to determine whether or not TTR(Met30) is present in the plasma of an individual subject. The immunoblotting procedure employs only 0.10 ml of serum and can serve as a reliable procedure for the screening of large numbers of persons for the presence of TTR(Met30). In family studies of seven FAP kindreds, TTR(Met30) was found in 21 out of 41 asymptomatic FAP offspring, and its presence was not related to either age or sex. Thus, the mutant TTR segregated in accordance with the known autosomal dominant mode of inheritance of FAP. Total plasma TTR levels were not reduced in asymptomatic FAP offspring who were carriers of TTR(Met30), and no difference was observed between carriers and noncarriers of the mutant TTR. The ratios of the variant to normal TTR in plasma were estimated in asymptomatic FAP offspring and were similar to those found in FAP patients. In contrast, TTR(Met30) was relatively enriched in cerebrospinal fluid samples from two FAP patients. The significance of this finding is not known, but might relate to the preferential deposition of amyloid in the nervous system in FAP. A limited study was conducted involving simultaneous analysis of both stored (collected in 1975) and fresh serum from 20 FAP offspring, all of whom had been asymptomatic in 1975. In every subject, the results obtained with the stored and the fresh serum samples were in agreement. Six of these subjects developed clinical FAP since 1975; TTR(Met30) was present in each of these subjects. These several studies strongly suggest that the presence of TTR(Met30) in plasma constitutes a predictive biochemical marker of FAP in the preclinical phase of the disease.

Crossed radio-immunoisoelectric focusing as a method of identifying isoallergens: identification of isoallergens in rat urine extracts.

A method of 2-dimensional radio-immunoelectrophoresis to detect directly the presence of 'isoallergens' in complex allergenic (skin test) extracts is described. This procedure, in which the components are separated by isoelectric focusing in agarose gel in the first dimension is therefore basically similar to that of crossed radio-immunoelectrophoresis, and hence has been termed crossed radio-immunoisoelectric focusing. The method has been applied to the allergens present in rat urine and has verified the presence of the cross-reacting alpha 2-euglobulin and prealbumin components in (at least) 3 and 2 isoallergenic forms respectively.

Proteinuria following nephroangiography. IX. Chemical and morphological analysis in dogs.

Following unilateral selective nephroangiography with diatrizoate, changes in urinary concentrations of prealbumin, albumin, IgG and alpha 2-macroglobulin were analysed in 8 dogs. All reacted with increased proteinuria. The median increase of urinary albumin concentration was 1,300 times (range 27-3 500). The proteinuria was caused mainly by increased glomerular permeability exhibiting some molecular size selectivity in filtration of the proteins measured. Light and electron microscopy did not reveal any glomerular on tubular abnormalities which could be attributed to effects of the angiography. It is suggested that the increased glomerular permeability to proteins might be an effect of disturbance of the electrical hindrance in the glomerular capillary wall.

Interaction between prealbumin and retinol-binding protein studied by affinity chromatography, gel filtration and two-phase partition.

The interaction between prealbumin and apo or holo retinol-binding proteins has been studied by affinity chromatography, gel filtration and two-phase partition. At physiological ionic strength apo and holo retinol-binding protein form 1:1 molar complexes with prealbumin. Mean dissociation constants for the prealbumin compex with apo retinol-binding protein and holo retinol-binding protein with all-trans retinol, retinoic acid, retinal and retinyl acetate were calculated from the partition data as 0.33 +/- 0.11 x 10(-6) M and 0.075 +/- 0.015 x 10(-6) M respectively (mean +/- S.E.M.). The difference was statistically significant. Quantitative data on the amount of retinol, retinol-binding protein and prealbumin in plasma and urine were in good agreement with the ratio of the dissociation constants for the complexes of apo and holo retinol-binding proteins with prealbumin as determined in the partition experiment. The magnitude of the dissociation constants was compatible with previously published data on the turnover of retinol-binding protein.

[Indirect immunoelectrophoresis in the study of urinary proteins]. / L'immunoelettroforesi indiretta nello studio delle proteine urinarie.

The indirect immunoelectrophoresis applied to the proteinurias is a simple and quick method, of usual execution, that makes use of not concentrated urines and discriminant between physiological and pathological proteinurias. The results obtained from a various casistic, collected in many years are presented. For the correlation which exist between morphological isototype and immunoelectropherogram, sometimes, it is possible to go up to the diagnosis of nephropathy also without applying to the renal biopsy.

Respiratory allergy to urine proteins of rats and mice.

Five atopic laboratory workers with asthma and other manifestations of allergy to rats and mice were sensitive to low-molecular-weight urine proteins--an alpha2-globulin in the rat and a prealbumin in the mouse. Positive skin prick and inhalation test reactions to the urine proteins were obtained; and radioallergosorbent tests (R.A.S.T.) for specific IgE antibody were also positive. These findings show that proper control of the collection and disposal of the animals' urine is necessary to minimise the likelihood of sensitisation.