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.

[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.

[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.