Determination Of Protein In Urine Research Articles

2‐D t‐ITP/CZE determination of clinical urinary proteins using a microfluidic‐chip capillary electrophoresis device

To replace the time-consuming sample pretreatment procedure, a microfluidic chip-CE device incorporating on-chip sample desalting/preconcentration with transient isotachophoresis (ITP)/capillary zone electrophoresis (CZE) was fabricated to perform sequential on-chip sample pretreatment and CE determination of four urinary proteins in clinical samples. On-chip sample desalting, clean-up and analyte preconcentration enable removing interfering sample matrix prior to transferring analytes to separation capillary for transient ITP/CZE determination. Four important urinary proteins transferrin, β2-microglobulin, human serum albumin (HSA) and immunoglobulin G (IgG), investigated were shown to achieve quantitation limits sufficiently high to meet medical requirements, sensitivity enhancement up to 40-fold and detection limits down to 0.3, 0.05, 0.6, 0.5 mg/L, respectively. Compared to the stacking effect, the use of a large sample size was found to be the major factor for sensitivity enhancement. The method reliability is established by close to 100% recoveries and statistical agreement of results from the method developed with currently used clinical radio-immunoassay method for all four proteins investigated. Moreover, an assay time of less than 10 min is needed in the method developed as compared to 7 h for the radio-immunoassay method.

A Flow Injection Chemiluminescence Method for the Determination of Protein Using Copper(II)-Alizarin Red S Complex as an Efficient Chemiluminescent Probe

A sensitive flow injection chemiluminescence (CL) method is proposed for the determination of bovine serum albumin (BSA) using Copper(II)-Alizarin Red S (ARS) complex as an efficient chemiluminescent probe. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II)-ARS in the luminol-H2O2 CL system. Under the selected conditions, the CL intensity is linear with the concentration of BSA in the range of 5.0 × 10−11 to 1.0 × 10−9 mol · L−1. The detection limit was 2.0 × 10−11 mol · L−1. The method is successfully applied to the determination of protein in urine.

Inductively coupled plasma mass spectrometry for determination of total urinary protein with CdTe quantum dots label

In the present work, a novel methodology for sensitive quantification of human total urinary protein by using CdTe quantum dots label and inductively coupled plasma mass spectrometry (ICP-MS) was developed. It combined the intrinsically high sensitivity of elemental mass spectrometric analysis with the remarkable quantity of metallic ions from the nanoparticles label. Through the direct conjugation of CdTe quantum dots and proteins, the ICP-MS signal intensity of cadmium is proportional to the concentration of proteins. The optimization of bio-conjugation parameters was investigated and discussed in detail. The proposed method featured a low detection limit (0.008 μg mL−1 for HSA) and wide linear range response over 4 orders of magnitude. Moreover, there are very little interferences from common inorganic ions and other coexisting compounds. The developed method was successfully applied for detecting total protein in 50 human urine samples and the results were in good agreement with those reported by the hospital, which indicated a great potential for routine clinical application.

Flow Injection Determination of Urinary Protein Using Fluorescence Quenching of Tetraiodofluorescein

Flow Injection Determination of Urinary Protein Using Fluorescence Quenching of Tetraiodofluorescein

Influence of kidney function on urinary excretion of albumin and retinol-binding protein in dogs with naturally occurring renal disease

To evaluate excretion of urinary albumin (UAlb) and urinary retinol-binding protein (URBP) in dogs with naturally occurring renal disease. 64 client-owned dogs. Dogs were assigned to groups according to plasma creatinine concentration, urinary protein-to-urinary creatinine ratio (UP:UC), and exogenous plasma creatinine clearance (P-Cl(Cr)) rates: group A (n = 8), nonazotemic (plasma creatinine < 125 μmol/L) and nonproteinuric (UP:UC < 0.2) with P-Cl(Cr) rate > 90 mL/min/m²; group B (26), nonazotemic and nonproteinuric with P-Cl(Cr) rate 50 to 89 mL/min/m²; group C (7), nonazotemic but proteinuric with P-Cl(Cr) rate 53 to 98 mL/min/m²; group D (8), azotemic and borderline proteinuric with P-Cl(Cr) rate 22 to 45 mL/min/m²); and group E (15), azotemic and proteinuric (P-Cl(Cr) not evaluated). The UAlb and URBP concentrations were measured via ELISA; UAlb-to-urinary creatinine (UAlb:UC) and URBP-to-urinary creatinine (URBP:UC) ratios were determined. UAlb:UC and URBP:UC did not differ between groups A and B. Increased UAlb: UCs and URBP:UCs were paralleled by increased UP:UCs in groups C, D, and E relative to values from groups A and B, independent of azotemia. There were significant positive correlations of UP:UC with UAlb:UC and of UAlb:UC with URBP:UC (r = 0.82 and 0.46, respectively). However, UP:UC, UAlb:UC, and URBP:UC were not significantly correlated with P-ClCr rate. UAlb and URBP concentrations were paralleled by urinary protein concentrations and may be useful in assessing renal management of plasma proteins. Determination of urinary protein, UAlb, or URBP concentration was not sufficiently sensitive to detect reduced P-Cl(Cr) in nonazotemic dogs.

Optimization and Validation of a Capillary MEKC Method for Determination of Proteins in Urine

Proteinuria, i.e. increased excretion of proteins in urine, is a common sign indicating renal or urinary tract diseases. In this study, a fast and simple procedure for urine sample preparation and capillary micellar electrokinetic chromatographic analysis is presented, without any sample pretreatment prior to the analysis. The developed MEKC method was employed for simultaneous determination of albumin (ALB), haemoglobin (HGB), and myoglobin (MYO) in human urine samples obtained from patients with diagnosed proteinuria. Optimum conditions for detection and separation of ALB, HGB, and MYO are 50 mmol L−1 borate buffer containing 20 mmol L−1 SDS (pH 9.3), injection 40 mbar × 20 s, voltage 25 kV, temperature 30 °C, and detection wavelength 200 nm. The method was shown to be specific, accurate, linear (correlation coefficients r2 > 0.99), and precise (RSD below 3.75 and 7.23% for migration time and peak area, respectively). Multi-variable-at-a-time (MVAT) approach for robustness testing shows no significant variations in accuracy, specificity, and precision as RSD values were lower than 5 and 10% for migration time and peak area, respectively. The presented method is applicable for routine analyses of urine samples as a screening method for patients with excess ALB, HGB, and MYO.

The Role of Calcium Correction during Normal Pregnancy at Third Trimester in Mosul.

To assess calcium status in healthy pregnant women during limited sun exposure time in winter, and to demonstrate the possible effect of serum albumin alterations on serum total calcium level and the role of albumin adjusted calcium concentration. Subjects enrolled in the study included 160 apparently healthy women divided equally into four groups (I - IV), group I was considered as the control group, composed of non-pregnant women. Groups II-IV were composed of pregnant women in the first, second and third trimesters respectively. Semiquantitative urine protein determination and measurement of serum total calcium, ionized calcium, albumin, phosphorous and creatinine with calculation of corrected calcium were performed in all groups. The results were statistically evaluated by standard statistical methods. There was no significant difference in serum ionized calcium, corrected calcium and phosphorous during pregnancy. However, there was a significant reduction of serum total calcium, albumin and creatinine in pregnant women at second and third trimesters. In each group, a significant positive correlation was observed between total calcium with corrected and ionized calcium. In healthy pregnant women even during limited sun exposure time in winter, there was no need for calcium supplementation in spite of the continuous and progressive reduction of serum measured total calcium during the second and third trimesters due to dilutional hypoalbuminemia. During pregnancy, measured calcium is parallel to both corrected and ionized calcium and since there was no significant difference between measured and corrected calcium, therefore, measured calcium is a useful test in assessing calcium status and suggests the need to establish a reference range for pregnant women.

Successive determination of urinary protein and glucose using spectrophotometric sequential injection method

A new sequential injection (SI) system with spectrophotometric detections has been developed for successive determination of protein and glucose. The protein assay is based on ion-association of protein with tetrabromophenolphthalein ethyl ester (TBPE) in the presence of Triton X-100 at pH 3.2. The blue product is monitored for absorbance at 607 nm. For glucose, hydrogen peroxide, generated by the oxidation of glucose in the presence of glucose oxidase immobilized on glass beads packed in a minicolumn, is monitored using iron-catalyzed oxidation reaction of p-anisidine to form a red colored product (520 nm). The SI procedure takes advantage in performing the protein assay during the incubation period for glucose oxidation. Linear ranges were up to 10 mg dL −1 human serum albumin (HSA) with a limit of detection (LOD) (3 σ) of 0.3 mg dL −1, and up to 12.5 mg dL −1 glucose with LOD of 0.08 mg dL −1. R.S.D.s ( n = 11) were 2.7% and 2.5% (for 1 mg dL −1 and 5 mg dL −1 HSA) and 1.4% (9 mg dL −1 glucose). Sample throughput for the whole assay of both protein and glucose is 6 h −1. The automated system has been demonstrated for the successive assay of protein and glucose in urine samples taken from diabetic disease patients, with good agreement with the other methods. This developed SI system is an alternative automation for screening for diabetic diagnosis.

Influence of urine pH on accurate urinary protein determination in Sprague‐Dawley rats

Rat urinary protein concentration is commonly measured during safety assessment studies to evaluate potential drug-induced nephrotoxicity. It has been reported that impregnated reagent test strips (dipsticks) can yield false-positive urinary protein results for alkaline urine samples. The objective of this study was to determine if urinary dipsticks accurately assess protein concentrations, especially in alkaline rat urine. Ten male Sprague-Dawley rats were treated with 2% sodium bicarbonate and 2% ammonium chloride to alkalinize and acidify the urine, respectively. Urine pH was measured in treated and control rats using a pH meter and urinary dipsticks with the Clinitek 500. Quantitative urinary protein results were compared to urinary dipstick protein evaluations obtained with the Clinitek 500 and sulfosalicylic acid precipitation test methods. The urinary dipstick pH measurement had a very high correlation (r = .98) with the pH meter technique. Samples with alkaline pH (>or=7.5) analyzed for protein by dipstick analysis were in complete agreement 34.7% of the time with the quantitative technique, which was very similar to the 39.3% agreement for samples with neutral and acidic pH (<or=7.0). Samples with alkaline pH analyzed by the sulfosalicylic acid method were in agreement 0% of the time with the quantitative technique, compared to 7.1% of the time with samples with neutral or acidic pH. The urine dipstick method does not lead to gross overestimation of urinary protein concentration in alkaline urine and provides a rapid and reasonably accurate way of determining the presence or absence of urinary protein in rats.

Diagnostic performance of combined specific urinary proteins and urinary flow cytometry in urinary tract pathology

Urinalysis comprises three techniques: urinary flow cytometry, test strip analysis and determination of specific urinary proteins. We investigated the diagnostic possibilities of combining these methods for a cohort of patients with a variety of well-documented urological and nephrological pathology. Urinary samples from 407 in- and out-patients with nephrological or urological pathology were retrospectively included in our study. Test strip analysis (URISYS 2400), urinary flow cytometry (UF-100) and urinary protein analysis [albumin, total protein, alpha1-microglobulin (A1M), alpha2-macroglobulin (A2M)] were performed. In discriminating upper and lower urinary tract infections, A1M and A1M/log(white blood cells) can be used, whereas pathological casts only give poor discrimination. The ratio A2M/log(red blood cells; RBC) allows differentiation between cystitis and pyelonephritis, while glomerular diseases can be recognised by the log(RBCxurinary total protein). Combining A2M and urinary albumin allows the determination of acute prostatitis. Using combined parameters provided by various urinalysis techniques, ratios can be proposed with diagnostic value for classifying renal and urological conditions. Possible integration in computer-based knowledge systems may offer valuable information for clinicians dealing with these pathologies.

A New Spectrophotometric Method for Determination of Urinary Protein Using Erythrosin B

Abstract A new sensitive spectrophotometric method for human serum albumin (HSA), the main protein in urine, has been developed using Erythrosin B. The proposed method is not based on conventional spectral shift but on an enhanced absorbance of the protein–bound dye and suppression of reagent blank in the presence of Triton X-100. The detection limit for HSA is 0.06 mg L−1 (S/N = 3), which is superior to conventional methods by a factor of 50–100. This is the first ever spectrophotometric method applicable for urinary protein even in healthy subjects.

Comparison of various methods for the determination of total protein in urine

Comparison of various methods for the determination of total protein in urine

Spectrophotometric Determination of Urinary Protein with o-Sulfophenylfluorone-Metal Complex

A simple and sensitive spectrophotometric method for the determination of human serum albumin (HSA) was established based on the ternary complex-formation reaction of HSA with o-sulfophenylfluorone (SPF) as a xanthene dye and metal ion (niobium(V) and bismuth(III)) in the presence of a dispersion agent. This new method enabled the determination of HSA in the range of 1 - 15 microg/ml HSA by measuring the difference of the absorbance at 530 nm between HSA-SPF-metal ion and SPF-metal ion solutions. In the determination of HSA, this method is about 2-times more sensitive than the Pyrogallol Red-molybdenum(VI) method (PR method), which accounts for more than 80% of the quantification methods for urinary protein assays in Japan. There was no significant difference between the results obtained by the present method and the PR method for human urine samples. The binding process between the SPF-metal complex and HSA was studied by determining the binding parameters and the thermodynamic parameters.

Problems with the estimation of urine protein by automated assays

Objectives: Most clinical laboratories replaced their manual precipitation techniques for the determination of urinary protein with automated dye binding assays or benzethonium chloride-turbidimetric assays. Few studies have validated these assays for the measurement of urinary proteins in the normal range. Design and methods: This study compares four automated assays for the measurement of urinary protein to a manual Ponceau S/TCA precipitation assay. We evaluated the linearity, the precision, the analytical sensitivity, the accuracy and the recovery of different proteins for each assay. Results: All assays showed good linearity with the theoretical concentration of albumin present in the sample. The coefficient of variation was below 10% at a concentration of 0.142 g/L. However, the manual Ponceau S/TCA assay demonstrated superior analytical sensitivity. Accuracy determinations showed a variable positive bias and poor correlations at concentrations below 0.1 g/L when compared to the Ponceau S/TCA assay. Small molecular weight peptides particularly affected the pyrogallol red assays but other urinary components also interfered with the automated assays. Conclusions: Most automated assays show high imprecision and poor accuracy for the measurement of urinary protein in the normal range. The Ponceau S/TCA offers a precise and accurate manual alternative to these automated assays.

Colorimetric and Visual Methods for Urinary Protein Determination with Tetrabromophenol Blue

Colorimetric and Visual Methods for Urinary Protein Determination with Tetrabromophenol Blue

Elimination of the interference from aminoglycoside antibiotics in the pyrogallol red-molybdate protein dye-binding assay.

The Pyrogallol Red-molybdate (PRM) protein dye-binding assay is used for urinary protein determination (1)(2)(3). The assay is sensitive to interference from aminoglycoside antibiotics, and the extent of interference varies with different PRM reagents (1)(2)(4)(5)(6)(7). Thus, the Dade Behring PRM and the Sigma PRM assays are sensitive to aminoglycoside interference, whereas the Cobas Fara and Roche Integra 700 PRM assays are not (4)(5). Furthermore, the PRM reagents used by Fujita et al. (1) and Orsonneau et al. (3) are more sensitive to interference than the reagent used by Watanabe et al. (2). We have previously shown that the addition of sodium dodecyl sulfate (SDS) to the PRM reagent increases the interference, whereas the addition of sodium oxalate reduces the interference (6)(7). In the present …

A study on the relationship between homocysteine and diabetic nephropathy in rats

Hyperhomocysteinemia is known to be associated with many of the occlusive vascular diseases including ischemic heart disease. Elevated plasma total homocysteine (t-Hcy) is also remarkably common among patients with moderate to severe renal failure. The purpose of this study was to investigate the role of homocysteine (Hcy) in the pathogenesis of diabetic nephropathy in the rat. Additionally, any effect of aminoguanidine (AG), an inhibitor of advanced glycation end product (AGE) formation, on the onset of nephropathic symptoms and on the concentrations of Hcy was searched for. Diabetes was induced in male Wistar albino rats (6 months old) by a single injection of 50 mg kg −1streptozotocin (STZ) into the penile vein. Animals with blood glucose levels higher than 350 mg dl −172 h after STZ injection were included in the study. Age-matched rats receiving a single dose of citrate buffer served as controls. One half of the control and diabetic groups received AG via drinking water (1 g l −1). The experimental period lasted for ten weeks. Animals were killed by cardiac venipuncture after 24 hour urine samples were collected. Serum t-Hcy was quantified using HPLC, and urinary GAGs using the spectrophotometric 1,9-dimethyl methylene blue dye method. Serum glucose, protein, creatinine and total sulfydryl (t-SH) measurements, and urinary protein determinations were carried out spectrophotometrically. In diabetic rats, serum t-Hcy levels were significantly decreased (P< 0.001), and were negatively correlated with the urinary protein concentration (r= −0.67, P< 0.05). Urinary GAG levels were also increased in diabetic rats (P< 0.001). AG neither affected the t-Hcy levels, nor ameliorated the nephropathic symptoms. These results indicate that diabetic nephropathy is not linked to homocysteinemia in the rat.

Serum uric acid and standardized urinary protein: Reliable bioindicators of lead nephropathy in Nigerian lead workers

The question as to whether lead causes renal damage still remains largely controversial. Eighty-five male lead workers and 51 control subjects who had never been occupationally exposed to lead were studied. They were also classified according to duration of exposure. The mean age of the lead workers was similar to that of control subjects. The mean duration of occupational exposure to lead was 16.7 ± 2.13 years. Blood lead level was significantly higher in Pb workers than in controls (P 0. 05). Serum uric acid level was significantly raised in lead workers than in controls (P significantly correlated with blood lead level (r = 0.24, P < 0.0026). Standardized total urinary protein was also significantly raised in lead workers compared with control (P < 0. 001). Serum potassium level was equally significantly higher in lead workers than in controls (P < 0.01). In contrast serum total calcium level was significantly decreased in lead workers than in controls (P < 0.01), while serum phosphate level did not differ significantly. Serum uric acid level and standardized urinary protein determination may prove a readily available, reliable marker of lead nephropathy in Nigerians.

Application of an improved biuret method to the determination of total protein in urine and cerebrospinal fluid without concentration step by use of Hitachi 7170 auto-analyzer.

A biuret automated colorimetric assay for total protein in urine and cerebrospinal fluids was established. The procedures were as follows. Acidify all urine sample before analysis. Add precipitant Na(2)WO(4) to urine samples. After 10 min, centrifuge, decant the supernatant fluid, drain the inverted tubes on absorbent tissue, dissolve the precipitation with 0.1 mol/L NaOH, and finally adapt the reconstituted urine to the Hitachi 7170 analyzer. A cell-free cerebrospinal fluid sample produced by centrifugation can be inserted in an auto-analyzer for protein measurement directly. The program: mix 35 microl sample (CSF or reconstituted urine) and standard with 0.2 mol/L NaOH; incurable at 37 degrees C for 5 min, and real A1. Add concentrated biuret reagent, and 10 min later measure absorbance A2 at 546 nm vs. reagent blank. Secondary wavelength was 700 nm. The test results were calculated against a one-point standard. This biuret colorimetric method was relatively simple, fast, and accurate for the determination of protein in urine and cerebrospinal fluid, with a wide linearity extending from 0.125 g/L up to 6 g/L, had a good correlation with Benzethonium chloride turbidimetry technique, and was a practical routine method.

Recommended method of the Japanese Association of Medical Technologists for the determination of protein in urine by HPLC

A recommended method for the determination of total urinary proteins as a designated comparative method (DCM) is described. The method is based on gel permeation high performance liquid chromatography (HPLC). A total of 20 μl of urine diluted 5-fold with saline solution were applied to a column, eluted with 0.13 mol/l phosphate buffer (pH 7.0) at a flow rate of 1.0 ml/min at 35°C, and the absorbance is monitored at 220 nm. The fraction eluted in the void volume was quantified for urinary protein, using bovine serum albumin (NIST SRM 927) as the standard. The linear range of the assay was 10–5000 mg/l, and the precision was 1.5% in two samples with concentrations of 1037 and 4338, respectively. The analytical recovery was 96–104%. Low molecular weight substances such as glucose, ascorbic acid and bilirubin did not interfere the assay. The assay was satisfactorily applied to a variety of proteins regardless of their nature or molecular weights (12.4 kDa) including β2-microglobulin and Bence Jones Proteins. Surveys in five clinical laboratories, employing different types of HPLC apparatus with the same column type, showed satisfactory measurement precision (CV 2.1%) for three urine samples with different protein concentrations.