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.

[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.

[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.

[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.

[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.

[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.

Changes in salivary components by drug administration in patients with heart diseases.

In this study, patients with heart diseases were classified into 2 groups: Warfarin user and Warfarin non-user, and six salivary components were determined to assess intraoral pathologic conditions. Groups of healthy subjects and patients with periodontal disease without receiving any medication were set as control groups, and they were compared with those of the 2 groups with heart diseases. In patients with heart diseases in both the groups, albumin (ALB) level was found to be significantly higher compared to that in the control groups, and it was significantly higher in the patient group receiving Warfarin user and Warfarin non-user compared to that in the patient group with periodontal disease. C-reactive protein (CRP) levels were found to be higher in both the groups with heart diseases than those in the healthy group. Correlations between various salivary components and the clinical parameters were examined, showing significant correlations between ALB and gingival index (GI) and clinical attachment level (CAL), and between alanine aminotransferase (ALT) and GI, probing depth (PlI), bleeding on probing (BOP) and CAL. Significant correlations were also found between creatine kinase (CK) and PlI, GI and BOP. Thus, it was suggested that ALB and CRP might serve as the markers of intraoral pathologic conditions, and CK and ALT might serve as those alternative to GI.

[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.

[Analysis of proteins in urinary tract stones and urine of urolithic patients].

In order to investigate the mechanism of urinary tract stone formation, we analyzed protein components in urine and the stone. Urinary proteins of healthy subjects and urolithic patients as well as protein components urinary tract stone of the urolithic patients were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic patterns of urinary proteins of the patients differed from those of healthy subjects after separating protein patterns into those larger than 66kDa or smaller than 30kDa. Protein constituents of urinary tract stone were mainly separated into 18 bands ranging from 26.8 to 143 kDa. Major bands among these 18 bands differed among stones from different patients. On western blotting, the developed intensities of Tamm-Horsfall protein (THP) were fainter than those of healthy subjects. Whereas intensities of albumin (ALB) were stronger than those of healthy subjects. Moreover, blotting patterns of THP of the patients on non-reducing SDS-PAGE were obviously broad. Thus, we suggest that analysis of fractionated urinary proteins or protein components of urinary tract stone may provide a tool for monitoring the prognosis or relapse in the patients.

[Education and research toward the development of clinical laboratory techniques].

We have entered an era to discuss the modalities of the education of the students who are qualified as medical technologists. Continual acquisition of new knowledge and information, and the development of new diagnostic laboratory methods and new laboratory techniques coping with acquired knowledge and information are the research themes that are very adequate for the postgraduate students to whom the places for education and research are provided to aim top-ranking leading medical technologists. If they could progress toward the application for patent and commercialization of the research results, they would be encouraged to advance their researches further.

Reinvestigation of clinical value of Rivalta reaction of puncture fluid.

Rivalta reaction is still used as a puncture fluid test for differentiation of exudate and transudate. However, the test method of Rivalta reaction has not been standardized, or positive precipitates for the reaction have not been investigated. Thus, we clarified the measurement method, and investigated Rivalta reaction-positive proteins. Rivalta reaction-positive punctuates converted to negative when pH increased to 4.6 or higher, showing the necessity of pH adjustment of acetic acid solution to 3.6-4.2 in Rivalta reaction. Using pH 4.0 acetic acid solution, 8 types of proteins were identified in Rivalta reaction-positive turbid precipitates: C-reactive protein (CRP), alpha1-antitrypsin (alpha1-AT), alpha1-acid glycoprotein (alpha1-AG), haptoglobin (Hp), transferrin (Tf), ceruloplasmin (Cp), fibrinogen (Fg), and hemopexin (Hpx). Since these are acute reactive proteins, or proteins increased in malignant tumors and infections, positivity for Rivalta reaction at the specified pH may suggest pathological inflammation.

[Differences in whole salivary total protein concentration and protein fractions among the groups of dentulous subjects, edentulous subjects and periodontitis patients].

PURPOSE: To apply salivary proteins to the diagnosis of periodontal disease in partial denture supporting teeth, total protein concentrations and protein fractions in whole saliva were compared among dentulous subjects, edentulous subjects and periodontitis patients. MATERIALS AND METHODS: Eighty-five subjects/patients in total were studied, who consisted of 52 dentulous subjects with diagnosed normal periodontal tissue, 18 edentulous subjects using complete dentures and 15 patients with diagnosed periodontal disease. Total protein concentration in whole saliva was measured using dot blotting-silver staining, and protein fractions were analyzed using cellulose acetate membrane electrophoresis and silver staining. Based on the results obtained and some oral cavity examination items, the correlation between the periodontal pocket depth and the number of residual teeth was studied. RESULTS: Total protein concentration was highest in the group of periodontal disease patients and significantly different from that in the group of dentulous subjects (p<0.01). The percentage area and concentrations of albumin fraction were largest in periodontal patients, followed by dentulous subjects and edentulous subjects in this order. A significant difference (p<0.01) was observed in albumin concentration between periodontal patients and edentulous or dentulous subjects. Concentrations of immunoglobulin A and Gamma-globulin were significantly higher in periodontal patients than in dentulous subjects (p<0.01). In addition, among oral cavity examination items, the total periodontal pocket depth, the number of residual teeth and the number of teeth with periodontal pocket depth of over 4 mm were significantly correlated with the percentage area of albumin fraction or albumin concentration. CONCLUSIONS: Albumin concentration in whole saliva was suggested to be most effective for diagnosing periodontal disease in supporting teeth.

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 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.