Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins.

BACKGROUND: Analysis of urinary proteins using cellulose acetate membrane electrophoresis (CAME) is a useful and challenging method for the recognition of damaged sites in the kidney. However, protein content of each CAME fraction is still not completely understood. METHODS: In this study, an effective method of protein extraction from each band fractionated by CAME was established, which enabled us to examine the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. RESULTS: Proteins were extracted from the gel and analyzed by mass spectrometry. In all, 31 proteins were identified, including 20 urinary proteins that were newly identified in the CAME-based analysis. CONCLUSION: This methodology was useful for identifying the proteins responsible for creating unique bands on CAME in a urine sample of a patient with drug-induced interstitial nephritis. These findings provide in-depth characterization of urinary protein contents in each CAME fraction.

Direct analysis of airborne mite allergen (Der f1) in the residential atmosphere by chemifluorescent immunoassay using bioaerosol sampler.

Dermatophagoides farinae allergen (Der f1) is one of the most important indoor allergens associated with allergic diseases in humans. Mite allergen Der f1 is usually associated with particles of high molecular weight; thus, Der f1 is generally present in settled dust. However, a small quantity of Der f1 can be aerosolized and become an airborne component. Until now, a reliable method of detecting airborne Der f1 has not been developed. The aim of this study was to develop a fiber-optic chemifluorescent immunoassay for the detection of airborne Der f1. In this method, the Der f1 concentration measured on the basis of the intensity of fluorescence amplified by an enzymatic reaction between the labeled enzyme by a detection antibody and a fluorescent substrate. The measured Der f1 concentration was in the range from 0.49 to 250 ng/ml and a similar range was found by enzyme-linked immunosorbent assay (ELISA). This method was proved to be highly sensitive to Der f1 compared with other airborne allergens. For the implementation of airborne allergen measurement in a residential environment, a bioaerosol sampler was constructed. The airborne allergen generated by a nebulizer was conveyed to a newly sampler we developed for collecting airborne Der f1. The sampler was composed of polymethyl methacrylate (PMMA) cells for gas/liquid phases and some porous membranes which were sandwiched in between the two phases. Der f1 in air was collected by the sampler and measured using the fiber-optic immunoassay system. The concentration of Der f1 in aerosolized standards was in the range from 0.125 to 2.0 mg/m(3) and the collection rate of the device was approximately 0.2%.

[Molecular heterogeneity of alpha 1-antitrypsin in the urine of the urolithiasis].

Randall's plaque theory is regarded as the most plausible mechanism of urinary stone formation; however, we speculated that urine proteins are necessarily involved in the process of stone formation. We focused on alpha 1-antitrypsin (alpha1-AT), a protein verified to be present in urinary calculi, and which is considered as a protein of inflammation, comparing its presence in healthy subjects and patients with urolithiasis. Quantitative analysis of alpha1-AT was performed with ELISA, whereas qualitative analysis was performed with SDS PAGE, two-dimensional electrophoresis, and western blotting. The results revealed a molecular heterogeneity in alpha1-AT, which can be classified into four patterns, a concentration-independent difference in alpha1-AT molecules found in the urine of patients and healthy subjects. A wider distribution of protein isoelectric points was found in urolithiasis (3.0-8.0) than in healthy subjects (4.0-5.0). We suggest that this new finding with molecular heterogeneity was due to the urolithiasis.

Fluorescence immunoassay using an optical fiber for determination of Dermatophagoides farinae (Der f1).

Immunoassay methods are generally used for measuring of allergenic substances. However, they need special facilities, skilled handling, and time-consuming procedure. In this work, a fiber-optic immunoassay system which could measure allergen by fluorescent intensities of immune complexes formed by allergens and fluorescently labeled antibodies was established. Immune complexes absorbed on the optical fiber probe surface, and excitation light was injected into the probe, then evanescent field is created in the proximity of the probe. The fluorophores were excited by the evanescent light, and fluorescence was detected by a photo diode. The target allergen detected by our system was Der f1 derived from Dermatophagoides farinae that is one of the house dust mite and major source of inhaled allergens. The fluorophore used labeling on detecting antibody was cyanine 5. The system enabled to detect and quantitatively determine of Der f1. The measurement range was from 0.24 to 250 ng/ml, and the result competes with ELISA. The measurement time was 16 min/sample. The immunoassay system was applied to measurement of Der f1 from actual dust samples. Calculated values of Der f1 showed good correlations between the fiber-optic fluoroimmunoassay and ELISA.

Redox state of urinary albumin in patients with IgA nephropathy.

We examined the relationship between oxidative damage and molecular instability of urinary albumin in the urine of patients with IgA nephropathy (IgAN). As measure of oxidation, we detected the free thiol group at Cys34 in albumin (albumin-Cys34) using maleimide-PEG2-biotin reagent; because decreased levels of albumin-Cys34 are correlated with increased oxidation. The urine albumin-Cys34 level in all 30 patients with IgAN was decreased to varying extents. No correlations were found between urinary albumin/creatinine ratio and decreased urinary albumin-Cys34 level. Furthermore, decreases in urinary albumin-Cys34 were not accompanied by changes in serum albumin-Cys34. In diagonal-two-dimensional SDS PAGE analysis which reveals reduction-induced degradation of H2O2-treated human serum albumin, a similar pattern of albumin degradation was also detected in urine samples from patients with IgAN, indicating that structural alterations resulting from oxidative stress may be involved. Our findings suggest that redox state, in addition to urinary albumin concentration, may be an important indicator of post-translational modification and a potentially useful predictor of the kidney disease.

Development of a new measurement method for serum calcium with chlorophosphonazo-III.

BACKGROUND: Although serum calcium has been measured using the o-cresolphthalein complexone (oCPC) method in the clinical laboratory, this method still has some problems regarding linearity and reagent stability. We developed a new measurement procedure using chlorophosphonazo-III (CPZ-III: 2,7-bis (4-chloro-2-phosphonophenylazo) -1,8- dihydroxy-3, 6-naphthalenedisulphonic acid, disodium salt) as a chelator with an acid medium for serum calcium measurement. The present method showed better linearity and reagent stability compared with the oCPC method. METHODS: Characteristics were studied in optimized conditions measuring wavelength by absorption spectra analysis, and interference of protein and metals with Mg(2+), Fe(2+), Cu(2+) and Zn(2+). The method was applied to an automated analyser (7170; Hitachi High Technologies Corp). The measurement performance was evaluated for accuracy, precision, recovery rate, linearity and reagent stability with a comparison study against atomic absorption spectrophotometry (AAS). RESULTS: The within-run and between-run variations (coefficient of variation [CV]) were 0.92-1.01% and 0.75-1.43%, respectively. The linearity was 0-7.0 mmol/L. The comparison study obtained y = 1.002x (AAS) - 0.10, Sy/x = 0.18 mmol/L, n = 50. Reagent stability was at least 20 d at 4 degrees C without daily calibration. CONCLUSION: The new calcium measurement method in serum was demonstrated to have reliable and acceptable performances as a routine test in clinical laboratory.

[Urinary albumin fragmentation and immunoreactivity].

Urinary albumin (ALB) has been measured as a marker for the early detection of diabetic nephropathy. In 2004, Comper et al. developed a gel-filtration high-performance liquid chromatography (HPLC) procedure for the determination of urinary ALB. They demonstrated the presence in its albumin fraction of non immunoreactive ALB with the total molecular weight of a monomeric ALB that was non-reactive with the existing anti-ALB antibody, and reported that the level of urinary non-immunoreactive ALB was higher in diabetic patients than in normal subjects. In this study, we isolated urinary ALB from diabetic patients using an anti-ALB antibody-coupled affinity column to test its immunoreactivity. In some diabetic patients, the results of HPLC and turbidimetric immunoassay for urinary ALB were discrepant. Western blot analysis showed that ALB samples from such patients were contaminated with proteins other than ALB, and contained ALB, whose molecular weight became lower using a reductive procedure. In addition, the reactivity of ALB with anti-ALB antibody differed depending on whether it was in a reduced or non-reduced state. These results indicate that ALB in such patients is susceptible to structural changes due to disease-induced urinary factors and, thus, their urine contains ALB with an altered reactivity to antibody.

Molecular heterogeneity of urinary albumin in glomerulonephritis: comparison of cardiovascular disease with albuminuria.

BACKGROUND: Despite the unstable structure of urinary albumin in kidney diseases, urinary albumin fragments have been identified by denaturing methods such as two-dimensional electrophoresis. This study examined the relationship between the structural heterogeneity of urinary albumin and protease effects. METHODS: Urine samples from patients with glomerulonephritis (GN), cardiovascular diseases (CVD), and healthy subjects were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Western blot, diagonal 2-dimensional non-reducing/reducing (d2D) SDS PAGE, and albumin zymography. RESULTS: The major band was monomer albumin in CVD and healthy subjects; however, 13 urinary albumin bands ranging from 55 to 172 kDa were identified by non-reducing SDS PAGE in GN. The results from d2D SDS PAGE showed urinary albumin polymerization between disulfide bridges, interactions with other proteins, and reduction induced degradation in GN patients. The results from albumin zymography showed that low-molecular mass forms of albumin did not necessarily correspond to high protease activity. Furthermore, concentrated healthy urine showed similar protease digestion as in GN without low-molecular mass of albumin. CONCLUSIONS: The molecular alterations observed cannot be explained only by urinary proteases. The specific alteration of urinary albumin molecules in GN can be attributed to different mechanisms to CVD.

[Construction of urinary Tamm-Horsfall protein assay by enzyme-linked immunosorbent assay and establishment of reference range of healthy subject].

Tamm-Horsfall protein (THP) is the most abundant protein in the urine. THP is expressed in the renal tubule as a precursor protein having 640 amino acid residues and anchored by GPI anchor in the cell membrane. Thereafter, THP is secreted as a glycoprotein is a molecular weight of about 95-100 kD. Despite several investigations from the perspective of renal failure, the physiological role of this protein is not yet clear. It has been reported that THP is also related to certain conditions, such as kidney stone formation and urinary tract infection. To examine the excretion of THP into urine, we constructed an enzyme linked immunosorbent assay(ELISA) for the measurement of THP in the urine. THP was purified from healthy human urine using Diatomaceous Earth. Then we obtained an antibody to purified THP and constructed a sandwich ELISA assay system to test urine samples. The sensitivity of measurement was 0.78 ng/ml. In this assay, the concentration of THP in spot urine can be linearly measured from 0.78 ng/ml to 50 ng/ml. The CV of assay was 2.4 to 4.1%. The measurement was not disrupted by urinary albumin (approximately 15 mg/ml), hemoglobin (approximately 15 microg/ml), glucose (approximately 30 mg/ml) and ascorbic acid (approximately 10 mg/ml). Pretreatment by centrifugation or filtration of urine affected the concentration of THP because of the agglutinated form of THP. We showed that the urinary excretion rate remained almost constant in our test population, 1.00-1.65 mg/hr (average +/- 1SD 1.30 +/- 0.25) for healthy men and 0.61-1.51 mg/hr (0.90 +/- 0.30) for healthy women and 1.10 +/- 0.34 mg/hr overall.

[Establishment of simultaneous measurement method of 8 salivary components using urinary test paper and clinical evaluation of oral environment].

PURPOSE: Clinical findings were compared with glucose, protein, albumin, bilirubin, creatinine, pH, occult blood, ketone body, nitrite, and white blood cells contained in whole saliva to investigate the components that most markedly reflect the periodontal condition. MATERIAL AND METHOD: The subjects were staff of the Prosthodontics Department, Showa University, and patients who visited for dental treatments (57 subjects in total). At the first time, saliva samples were gargled with 1.5 ml of distilled water for 15 seconds and collected by spitting out into a paper cup. At the second time, saliva samples were collected by the same method. At the third time, saliva samples after chewing paraffin gum for 60 seconds were collected by spitting out into a paper cup. Thus whole saliva collecting that was divided on three times. After sampling, 8 mul of the saliva sample was dripped in reagent sticks for the 10 items of urinary test paper and the reflectance was measured using a specific reflectometer. In the periodontal tissue evaluation, the degree of alveolar bone resorption, probing value, and tooth mobility and the presence or absence of lesions in the root furcation were examined and classified into 4 ranks. The mean values in each periodontal disease rank and correlation between the periodontal disease ranks and the components were statistically analyzed. RESULTS: Bilirubin and ketone body were not measurable. The components density of the 8 items was increased as the periodontal disease rank increased. Regarding the correlation between the periodontal disease ranks and the components, high correlations were noted for protein, albumin, creatinine, pH, and white blood cells. CONCLUSION: The simultaneous measurement method of 8 salivary components using test paper may be very useful for the diagnosis of periodontal disease of abutment teeth.

[The effect of air massage on serum constituents].

This study investigated the effect of air massage of the legs on serum constituents. Five volunteers (aged 48.8+/-12.98 years, n=5) served as subjects. The study consisted of three periods as follows: control period for one month(no-mas), massage period receiving the massage for 15 minutes per day for one month(mas), and another massage period receiving the massage for 15 minutes per day along with 40% oxygen uptake for one month (O2-mas). Venous blood samples were drawn every week during the study periods. Serum constituents were examined using an auto analyzer(Hitachi Ltd., JEOL Ltd.). Albumin concentration significantly increased under the O2-mas condition, whereas free-cholesterol concentration was significantly decreased under the same condition. In addition, significant increases in concentrations of creatinine and calcium were seen in men under the O2-mas condition. Furthermore, there was a significant decrease in the activity of alkaline phosphatase in women under both the mas and O2-mas conditions. These findings suggested that massage has positive effects on nutritional recovery and metabolism of lipids, bone and muscle. Thus, it may be argued that massage could potentially be effective as a form of exercise.

Rapid and sensitive electrophoresis of urinary protein clearly reveals the pathophysiological feature of renal diseases.

AIM: The diagnostic approach for renal diseases with the electrophoretic pattern of urinary protein on cellulose acetate (CA) membrane differentiates the causes of proteinuria. However, this method has not been used routinely because of its difficulty in obtaining a clear image. This study was performed in order to re-evaluate this method with an improved system. METHODS: Using the newly developed system of CA membrane electrophoresis and its visualization, we examined fresh urine from patients (n = 100) who subsequently underwent renal biopsy and compared the results with the histological findings. RESULTS: The improved method of urine electrophoresis with CA membrane provided clear images and was sensitive enough for urine samples to be applied without concentration. The profiles of proteinuria were clearly classified into three patterns: glomerular, tubular or mixed. The profiles exhibited a good agreement with the histological findings of renal biopsy. CONCLUSION: The recognition of damaged portions in kidney through the profiles of proteinuria by this system could be practically effective for understanding the kidney disease at bedside.

[Promotion of the systematization of consistent education for medical technologists].

Although only about 35 years have passed since the birth of medical technology, marked advances have been made in the clinical laboratory science field. However, the educational system for technologists attached importance only to the learning of techniques for a long period because special training schools primarily provided medical technologist education. With the passing of time, the need for advanced knowledge has increased, and a plan to change the education system for medical technologists to 4-year colleges was evaluated. In 1989, the Course of Laboratory Sciences as a 4-year system for medical technologist education was established in the Department of Medicine, Tokyo Medical & Dental University. The Doctoral Course of Graduate School (first term) was established in 1993 and the Doctoral Course of Graduate School(second term) in 1995. In 2001, these courses formed a graduate university as the Division of Biomedical Laboratory Sciences, the Graduate School of Allied Health Sciences. Thus, a consistent educational system for medical technologists was established. By March 2005, about 500 students had graduated from this division. Based on this experience, we produced a 4-stage developmental program and provide an advanced educational system for the promotion of the systematization of consistent medical technologist education.

Presence of immunounreactive albumin in the urine of diabetic patients.

Recent studies have demonstrated that conventional immunochemical assays underestimate urinary albumin concentration because of the presence of immunounreactive albumin. It has been reported that intact urinary albumin in 24-hr diabetic urine samples could be detected as total concentration (immunoreactive+immunounreactive) by an HPLC method based on size exclusion chromatography. The aim of this study was to investigate urinary albumin concentration in diabetic spot urine samples by comparing the HPLC method with several other methods. The albumin concentrations on 80 diabetic spot urine specimens were measured by turbidimetric immunoassay (TIA), high performance liquid chromatography (HPLC), and a dipstick method. In addition, they were also analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native polyacrylamide gel electrophoresis (Native PAGE). The albumin concentrations derived from diabetic spot urine samples measured by the HPLC method were higher than those of the other methods except for five of 80 samples. Furthermore, the albumin concentrations analyzed by Native PAGE were higher than SDS PAGE in 61 (76.2%) of 80 samples. This study suggests the need for evaluating diabetes not only by HPLC, but also by combining it with another method.

Urinary protein analysis in pre- and postoperative cancer patients.

Urinary proteins from six patients with esophageal cancer and two with stomach cancer were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analyses were performed on days-1 to 3, 5, 7, 10, 14, and 21 (or 22) after surgery. The protein patterns were scanned by densitometry and divided into nine fractions. The main proteins in the fractions (Fr.) were identified as follows: immunoglobulin G in Fr. A, Tamm-Horsfall glycoprotein (THP) in Fr. B, transferrin in Fr. C, albumin in Fr. D, alpha(1)-acid glycoprotein in Fr. E, alpha(1)-microglobulin in Fr. F, retinol binding protein in Fr. G, and beta(2)-microglobulin in Fr. I. The protein in Fr. H was not identified. The percentage of each fraction was calculated from the densitometry pattern of each lane. The percentage values were averaged among all the patients, and pre- and postoperative data were compared. The percentage of Frs. E, F, and G increased on days 1-7, and the changes in these three proteins were similar to changes in serum C-reactive protein (CRP). In particular, the percentage of Fr. G peaked within 1 day of operation, which was faster than for CRP. Conversely, other fractions decreased. These results suggest that urinary protein analysis is useful for monitoring the response to surgical stress.

[Usefulness of protein/creatinine ratio in spot urine using test strips].

The influence of concentration and dilution has been the major issue of urinalysis. This study is to evaluate the usefulness of protein (P)/creatinine (C) ratio (P/C ratio) in spot urine by test strips method. For this purpose, the basic performance of creatinine test strips is evaluated and judging criteria for the urine P/C ratio is defined. Our evaluation revealed that the creatinine test strips were basically not affected by substances which have similar composition to creatinine, such as creatine, but by some medical agents and disinfectant. Its minimum detective sensitivity was 6.0 mg/dl, while that of protein test strips was 1.3 mg/dl. We determined judging criteria for the P/C ratio as follows: Below 80 mg/gCr, Normal; 80 to 500mg/gCr, 1 +; over 500 mg/gCr, 2+. The P/C ratio based on the quantitative and test strips methods provided good consistency of 82.2%. In low-creatinine urine samples (11 to 99mg/dl), 181 of 216 samples (83.8%) were tested negative on single-protein tests; while the P/C ratio provided fewer negative results, 134 of 216(62.0%). Moreover, in high-creatinine urine samples (200 to 600 mg/dl), 70 of 122 samples (57.4%) were tested negative on single protein tests; while the P/C ratio provided negative results in almost all the samples, 120 of 122 (98.4%). These urine samples were mostly collected at a medical examination. The results above showed that the P/C ratio obtained by easy and quick test strip method is equivalent to the quantitative method in performance and useful for spot urine testing.

[Evaluation of novel test strip to measure albumin and creatinine in urine].

The purpose of this study was to evaluate the clinical usefulness of novel test strip that simultaneously measure urinary albumin and creatinine. Testing was performed on 95 random urine samples from diabetics. Each sample was assayed with following methods: test strip by instrument (AX4280) reading and by visual interpretation, quantitative method for albumin, creatinine, and alpha1-microglobulin and cellulose acetate membrane electrophoresis. The results of test strip had good correlation with quantitative results. In the case of instrument reading, the sensitivity, specificity and consistency were 91.2%, 78.9% and 86.3% for albumin and 94.5%, 87.5% and 91.6% for albumin index (albumin/creatinine ratio; A/C ratio), respectively. The percent same level agreement for creatinine was 63.2%. After correction using creatinine value on test strip, 18.4% of samples defined as negative by albumin quantitative value were turned to be positive, 12.3% of samples defined as positive were turned to be negative. The same level of creatinine correction effect as the quantitative method was obtained at the test strip. When alpha1-microglobulin/creatinine (alpha1 m/g x Cr) ratio was compared with albumin index, percent positive of alpha1 m/g x Cr ratio was 35.0% for samples with albumin index less than 30 mg/g x Cr, 59.5% for those with albumin index between 30 and 300 mg/g x Cr. In addition, on cellulose acetate membrane electrophoresis, both retinol binding protein (RBP) and beta2-microglobulin (beta2 m) were detected in 12.5% of patients in normal condition or pre-nephropathy. Among patients at early-nephropathy, the detection rate of RBP and beta2 m were 20.0% and 15.0% respectively. These results indicate that the renal tubule is also damaged at the early stages of nephropathy.

Examination of the molecular diversity of alpha1 antitrypsin in urine: deficit of an alpha1 globulin fraction on cellulose acetate membrane electrophoresis.

In the clinical field of nephrology, a noninvasive approach employing the analysis of electrophoretic patterns in urinary protein has been established. In this study a total of 52 urine samples with IgA nephropathy (IgAN), anti-neutrophil cytoplasmic antigen-associated crescentic glomerulonephritis (GN), and other types of GN were analyzed. Patients with high alpha1 globulin (alpha1G) fractions, which contained alpha1AT in cellulose acetate membrane electrophoresis (CAE), tended to have alpha1 antitrypsin (alpha1AT) of normal molecular weight (57 kDa and 49 kDa), while patients with a deficit alpha1G fraction tended to have alpha1AT of low molecular weight (<49 kDa) (P < 0.01). The alpha1G fraction was significantly higher in patients with IgAN, and there were significantly more patients with normal molecular weight alpha1AT compared to patients with other diseases (P < 0.01). The isoelectric point of alpha1AT with lower-weight molecules was more on the alkali side compared to higher-weight molecules in two-dimensional electrophoresis. Detecting changes in alpha1G fractions in CAE may support the differential diagnosis of IgAN from other types of GN.