Analysis Of Urinary Proteins Research Articles

A strategy of plasma protein quantitation by selective reaction monitoring of an intact protein.

Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was approximately 15 min. An LLOQ of 99.2 ng/mL from a sample volume of 50 microL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlation coefficient (r(2)) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Interassay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research.

Cux-1 transgenic mice develop glomerulosclerosis and interstitial fibrosis

Cux-1 is a murine homeobox gene that is highly expressed in the nephrogenic zone of the developing kidney. Transgenic mice ectopically expressing Cux-1 develop renal hyperplasia associated with down-regulation of the cyclin kinase inhibitor p27. Because the reduction of p27 has been associated with mesangial cell proliferation and glomerular disease, we evaluated glomerular changes in Cux-1 transgenic mice. Adult kidneys from Cux-1 transgenic mice were analyzed morphologically for changes in glomerular cell number and for changes in mesangial and interstitial extracellular matrix deposition. Mesangial matrix expansion was identified by light microscopy. Glomerular cell number was performed following immunohistochemistry. Type IV collagen deposition was analyzed by immunofluoresence and Western blotting. Renal function was evaluated by serum protein, blood urea nitrogen (BUN), creatinine, and electrolyte analysis, and by urine protein and creatinine analysis. In adult transgenic glomeruli, Cux-1 was ectopically expressed in mesangial cells, and this was associated with an increase in mesangial cell number, resulting from an increase in proliferation. There was a marked increase in mesangial matrix area in transgenic mice compared to non-transgenic littermates, related to an increase in type IV collagen. Podocyte foot process effacement was observed in transgenic mice, and this was related to an increase in urinary albumin. Interstitial fibrosis was also observed in transgenic kidneys. These observations indicate that increased expression of Cux-1 in mesangial cells results in cell proliferation and mesangial expansion. In addition, these changes are potentially related to disruption of podocyte architecture leading to loss of filtration. These results suggest that expression of Cux-1 is sufficient to induce the early events of mesangioproliferative glomerulonephritis.

Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis.

Urinary proteins from 14 patients with tubulointerstitial nephritis were analyzed by cellulose acetate membrane electrophoresis. Urinary total protein concentrations were measured, and urinary 15 proteins (prealbumin, albumin, alpha(1)-microglobulin, alpha(1)-antitrypsin, alpha(2)-macroglobulin, haptoglobin, retinol binding protein, transferrin, beta(2)-microglobulin, IgA, IgG, kappa- and lambda-light chains, cystatin C, and lysozyme) were identified by the use of a rapid and highly sensitive colloidal silver staining reagent suited for use with cellulose acetate membranes, as reported previously by Matsuda et al. (J Clin Lab Anal 15:171-174, 2001; Clin Chem47:763-766, 2001) and Hiratsuka et al. (J Clin Lab Anal 10:403-406, 1996). We also analyzed urinary total protein concentration and urinary protein fractions according to the presence of acute or nonacute interstitial nephritis. In addition, the relationship between urinary protein fraction and complications of interstitial nephritis was analyzed. The goal of this work was to find a useful index for the diagnosis of tubulointerstitial nephritis.

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation

Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation

Stress tolerance test and SDS-PAGE for the analysis of urinary proteins in children and youths.

Excretion of urinary proteins (UP) is an important marker for the evaluation of patients with progressive renal disease. In order to analyze quantitative and qualitative variability of UP in relation to physical activity, we used standardized stress tolerance test and SDS-PAGE. Five urine samples were obtained from each patient at rest, during ordinary daily activity and after physical stress. Determination of total proteins was performed using Meulman's classic method with sulfosalicylic acid. UP were separated by ultrathin horizontal gradient SDS-PAGE according to Görg. There were 142 patients; 40 with poststreptococcal glomerulonephritis (PSGN), 11 with diabetes mellitus, 16 with chronic pyelonephritis and 75 who attended for investigation of asymptomatic proteinuria. Functional proteinuria was established in 42 subjects, who displayed maximal UP excretion during stress and the presence of apolipoprotein AI on SDS-PAGE. Children with PSGN showed no significant increase of UP during stress. Some children with diabetes mellitus (27%) and chronic pyelonephritis (47%) displayed microproteinuria or overt proteinuria after stress. Quantitative and qualitative changes in total UP excretion can be detected by stress tolerance test and SDS-PAGE. It remains to be seen whether stress tolerance test can identify children and youths who are at higher risk for disease progression.

Electrophoretic analysis of urinary proteins in diabetic adolescents.

Pathological changes in the urine sodium dodecyl sulphate gel electrophoresis (SDS PAGE) patterns often precede the occurrence of any sign of renal involvement in diabetes. However, data concerning the most frequent SDS PAGE pattern of the urine in early stages of type I diabetes mellitus are controversial. In the present study an SDS PAGE technique has been used that provides an adequate sensitivity for the detection of the abnormal pattern. Urinary proteins have been analyzed by SDS PAGE in twenty two diabetic adolescents and twenty four age matched controls. Albumin concentration, and N acetyl-beta-D-glucosaminidase (NAG) activity were also measured in the same samples. There was no significant difference in urine albumin concentration and NAG activity between diabetic children and controls. However twelve patients showed an electrophoretic pattern characteristic for glomerulopathy, two had a pattern indicating tubular dysfunction and another two patients had a mixed pattern. Among the twenty four controls only three showed abnormal electrophoretic patterns. The results support the view that early stages of diabetic nephropathy may involve both glomerular and tubular dysfunction. However the exact clinical and prognostic significance of the information provided by SDS PAGE analysis remains to be elucidated.

Analysis of urinary proteins by SDS-agarose gel electrophoresis in multiple myeloma patients

Analysis of urinary proteins by SDS-agarose gel electrophoresis in multiple myeloma patients

Analysis of untreated human urinary proteins utilizing capillary zone electrophoresis

Analysis of untreated human urinary proteins utilizing capillary zone electrophoresis

Clinical Applications of Capillary Electrophoresis. Stephen M. Palfrey, ed., Totowa, NJ: Humana Press, 1999, 212 pp., $79.50. ISBN 0-89603-639-1.

For clinical laboratories, electrokinetic capillary assays represent an emerging technology whose popularity and importance are increasing steadily. This book brings together for the first time a collection of detailed capillary electrophoresis (CE) protocols for the clinical laboratory. Included are assays for analysis of serum, urinary, and cerebrospinal fluid proteins; hemoglobin variants, DNA fragments associated with certain diseases; serum lamotrigine; drugs of abuse in urine; urinary oxalate and citrate; and plasma nitrite and nitrate. The book tries to give a “representative rather than a comprehensive look” at those assays. Chapters are written in …

URINE PROTEIN ANALYSIS BY GEL ELECTROPHORESIS AND LASER DENSITOMETRY AFTER CHEMOTHERAPY IN PEDIATRIC CANCER PATIENTS

A common side effect of chemotherapy is reversible or nonreversible nephrotoxicity. SDS polyacrylamide gel electrophoresis combined with laser densitometry was evaluated as a suitable method to analyze pathologic urine proteins. A total of 52 pediatric patients were followed during and 63 patients were followed after therapy. During therapy renal damage was recorded in 43% of the leukemia patients, in 56% of nephroblastoma patients, and 75% of patients with other tumors. Three or more months after therapy pathologic patterns were seen in 25% of acute lymphoblastic leukemia patients, in 35% of patients with nephroblastoma, and in 62% of other patients. Patients with persistent complete tubular proteinuria and mixed glomerular/tubular proteinuria were found to have a high risk for irreversible renal damage and should be controlled periodically. This method permits a rapid and reliable analysis of urine proteins and is suitable for follow-up tests of renal function during and after chemotherapy.

Strategy for urinary protein analysis

SUMMARY: Recent studies have highlighted the intrinsic toxicity of filtered proteins and their contribution to deterioration of renal function. In‐depth analysis of proteinuria may assess the degree of glomerular and/or tubular involvement and help in the prognostication of an individual patient. One should consider not only the quantity but also the quality of the urinary protein. Apart from quantitating the total urinary protein, assessment of the quality of the protein may be achieved by performing sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) for an overview assessment follow by automated nephelometry or simple radial immunodiffusion measurement of specific proteins for calculating the selectivity index and detecting tubular dysfunction.

Clinical analysis of human urinary proteins using high resolution electrophoretic methods.

The application of isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE) and capillary electrophoresis (CE) for high resolution electrophoretic analysis of human urinary proteins is reviewed. In each case, the information is tabulated chronologically with details of sample preparation, electrophoretic system, detection method and clinical application. The text includes an historical perspective of the use of each method for urinalysis and a detailed review of the application of the methods to the investigation of renal disease, renal transplantation, Bence Jones proteinuria (BJP), diabetes mellitus, cadmium toxicity, nephrolithiasis and cancers of the urogenital tract.

Sodium dodecyl sulfate-agarose gel electrophoresis of urinary proteins: application to multiple myeloma

We evaluated a new sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) for urinary protein analysis in patients with multiple myeloma (MM; n = 47; ages, 62 +/- 2 years, mean +/- SE). Abnormal proteinuria (mean = 1872 +/- 360 mg/24 h) was present in 95% of the samples; 75% of the patients had some sign of renal dysfunction (glomerular and/or tubular) according to their SDS-AGE pattern. A band suggesting Bence Jones proteinuria (BJP) was detected in 40 vs 33 specimens by routine AGE. Immunofixation identified BJP in 38 patients; the calculated sensitivity of SDS-AGE for BJP was 97%. Excellent correlation (P <0.0001) was obtained with routine AGE (r = 0.994) and immunonephelometry (r = 0.963) for light chain quantification. SDS-AGE allows easy evaluation of renal dysfunction and shows high sensitivity for BJP detection. In a specialized laboratory, it is useful for following the progress of MM patients through the semiquantification of BJP.

Renal Function in Adult Beta-Thalassemia/Hb E Disease

β-Thalassemia hemoglobin E (β-thal/Hb E) is the commonest form of hemoglobinopathy in Thailand. Shortened red cell life span, rapid iron turnover and tissue deposition of excess iron are major factors responsible for functional and physiological abnormalities found in various forms of thalassemia. Increased deposition of iron had been found in renal parenchyma of thalassemic patients, but no systematic study of the effect of the deposits on renal functions has been available. The purpose of this study is to describe the functional abnormalities of the kidney in patients with β-thal/Hb E and provide evidence that increased oxidative stress might be one of the factors responsible for the damage. Urine and serum samples from 95 patients with β-thal/Hb E were studied comparing with 27 age-matched healthy controls. No difference in the creatinine clearance was observed. β-thal/Hb E patients excreted significantly more urinary protein (0.8 ± 0.5 vs. 0.3±0.1 g/day, p < 0.001). Aminoaciduria was found in 16% of the patients. Analysis of urinary protein by SDS-PAGE electrophoresis and silver staining revealed abnormal pattern of protein with increased small molecular weight (<45 kD) bands. Morning urine analysis showed significant lower urine osmolality (578.3 ± 164.6 vs. 762.4 ± 169.9 mosm/kg, p < 0.001) in patients. Patients excreted more NAG (N-acetyl beta-D-glucosaminidase, 26.3 ± 41.3 vs. 8.4 ± 3.9 U/g Cr, p < 0.0001) and β₂-microglobulin, 124.3 ± 167 vs. 71 ± 65.5 µg/g Cr, p = 0.001. Plasma and urine MDA (malonyldialdehyde) levels were both raised (p < 0.0001). Nine patients were selected for renal acidification study. All were found to be normal, but showed poor response to DDAVP challenge (urine osmolality 533 ± 71). This is the first report of renal tubular defects found associated with β-thal/Hb E disease. The mechanism leading to the damage is not known but it might be related to increased oxidative stress secondary to tissue deposition of iron, as indicated by the raised levels of serum and urine MDA. It is not known whether these functional defects would have any long-term effects on the patients. Further studies are warranted and means of prevention of these defects should urgently be sought.

Urinproteinanalyse mit der Sodium‐Dodecyl‐Sulfat‐Polyacrylamid‐Gradientengel Elektrophorese (SDS‐PAGE) bei gesunden und nierenkranken Katzen

In this investigation, the value of urine protein analysis by means of molecular-weight related sodium dodecyl-polyacryl gradient gel electrophoresis (SDS-PAGE) was examined with regard to its applicability and diagnostic significance in nephropathy in the cat. A total of 87 cats was included in the study, 30 of them that were clinically healthy served as the control group. The urine protein pattern of this group had, besides the band representing the market albumin, and additional broad band within the size of the marker transferrin. In some cases, weak bands were present within the range of the Tamm-Horsfall-protein and immunoglobulin G. Micromolecular protein bands were not demonstrable. The remaining 57 animals had a histologically proven nephropathy. Thirty-eight cats had elevated urea and/or creatinine values in the plasma (group 1), and 19 animals had values within the reference range (group 2). The urine protein pattern as evidenced by SDS-urine electrophoresis was altered in all cats with histologically proven nephropathy, and it is thus concluded that with this technique a nephropathy can be diagnosed very early and prior to changes of plasma urea and creatinine (group 2). Moreover, in most of the cases, the nephrological changes can be classified as glomerular or tubulo-interstitial (group 1 and group 2). However, it is not possible to draw exact conclusions concerning the underlying morphological changes, nor can the severity of the disease be correctly assessed.

Urine protein analysis by capillary electrophoresis.

Increased concentration of proteins in urine as well as abnormal patterns are seen in many disorders such as various renal disorders and light chain disease. At the wavelength of 214 nm used for detection of the peptide bond, numerous compounds interfere in the analysis of urinary proteins. We show that either adsorptive filtration with a wash step or cold ethanol precipitation are two methods which can eliminate many of the interferences. The wash step is rapid, greatly reduces the interfering substances, and decreases the effect of sample matrix. Both of these methods yield results comparable to the traditional agarose method. Capillary electrophoresis (CE) is faster and more cost-effective than agarose electrophoresis.

Clinical Significance of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of Urinary Proteins in Preeclampsia

Objective: To establish the correlation between clinical and biochemical parameters and the urinary patterns of proteinuria detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) in preeclampsia.Methods: 34 severe preeclamptics (BP ± 110 mm Hg diastolic and s= + + proteinuria) who were < 34 weeks gestational age were included in the study. Blood and urinary samples were obtained and the following tests performed: full blood count, serum urea, electrolytes and urates, 24-h proteinuria and creatinine clearance, and SDS PAGE.Results: The patients were divided into two groups depending on whether they excreted only albumin or both tubular and glomerular proteins as detected by SDS PAGE. There were no significant differences in maternal age, parity, gravidity, and gestational age between the groups. Systolic and diastolic blood pressures were significantly higher in those who excreted both glomerular and tubular proteins than in those who excreted albumin only: 173/116 versus 156/106 mm Hg; P = 0.0167. There were no statistical differences between the groups in relation to hemoglobin, platelets, urea, creatinine, or 24-h urinary protein and creatinine clearance values.Conclusion: The present study shows that SDS PAGE analysis of urinary proteins reveals two distinct patterns of proteinuria. Patients with mixed pattern (tubular and glomerular proteins) have higher blood pressure values than those who excrete albumin only. SDS PAGE appears to be a good noninvasive method of assessing severity of preeclampsia. It may be of value in identifying those patients who require postpartum renal biopsies.

High resolution two-dimensional electrophoretic analysis of urinary proteins of patients with prostatic cancer.

In an attempt to identify marker(s) for prostatic cancer, proteins in urines of normal and prostatectomized males and in men with cancerous prostate were analyzed. Only urines collected with protease inhibitors were examined. Two-dimensional (2-D) gel electrophoresis was used for high resolution separation of proteins and the electrophoretograms were either developed by double stain or the proteins were electrophoretically transferred onto nitrocellulose for immunological identification. The pool of each group exhibited similar relative positions of major protein spots. The study of normal and prostatectomized men identified two proteins denoted as A (36 kDa, pI 6-6.5) and B (23 kDa, pI 6.6), which were undetectable in the latter group. A visual comparison of the patterns of normals and patients with cancerous prostate revealed that both these proteins were undetectable in urines of men with malignant prostatic carcinoma (PCA) and benign prostatic hyperplasia (BPH) and hence may be useful in identifying prostatic carcinoma. Also, while protein C 43.5 kDA, pI 6-6.6) was discerned in normals its abundance, along with those of proteins D (40 kDa, pI 6-6.4) and E (26.5 kDa, pI 6-6.7), appeared to be higher in BPA than in PCA. Protein F (18-28 kDa, pI 4-5.5) was found in patients with BPH but was undetected in normals and men with PCA. Hence, it may become useful in distinguishing BPH from PCA. All the proteins, A to F, appear to be previously unidentified. Their further characterization is warranted.

5492834 Method of sample preparation for urine protein analysis with capillary electrophoresis : Liu Cheng-Min; Wang Hann-Pin Yorba Linda, CA, United States assigned to Beckman Instruments Inc

5492834 Method of sample preparation for urine protein analysis with capillary electrophoresis : Liu Cheng-Min; Wang Hann-Pin Yorba Linda, CA, United States assigned to Beckman Instruments Inc