[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.

Urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining.

We previously reported a rapid and highly sensitive colloidal silver staining solution suitable for the cellulose acetate membrane. This method was useful for detecting even very small amounts of urinary protein. In the present study, we examined urinary protein fractions in healthy subjects, using cellulose acetate membrane electrophoresis (CAE) with a highly sensitive colloidal silver staining, in an attempt to determine the clinical relevance of urinary protein fractions. Sixty unconcentrated spot urine specimens were analyzed by CAE and calculated by densitometry. All of the samples were separated into five fractions by CAE. The mean +/- 1 SD of the percentage of five fractions was 28.37 +/- 8.51 in albumin, 4.30 +/- 4.19 in alpha1-globulin, 14.41 +/- 6.14 in alpha2-globulin, 19.45 +/- 7.10 in beta-globulin, and 33.46 +/- 8.24 in gamma-globulin. The albumin/globulin (A/G) ratio was 0.41 +/- 0.17. These six items and the concentrations of total protein, albumin, and beta-N-acetyl-D-glucosaminidase (NAG) did not significantly differ between males and females. NAG is the marker of tubulointerstitial nephropathy. The results suggest that there are no gender-dependent differences in the urinary protein fractions of healthy subjects.

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.

Analysis of an expanded width of albumin fraction by cellulose acetate membrane electrophoresis in IgA nephropathy urine before treatment.

Cellulose acetate membrane electrophoresis with colloidal silver stain re-vealed that the width of the albumin fraction in IgA nephropathy (IgAN) urine before treatment was significantly expanded. This phenomenon was not shown in IgAN urine after treatment or in non-IgAN urine. There was a reverse correlation between the width of the albumin fraction and the albumin con-centration in IgAN urine. By immuno-fixation, Tamm-Horsfall protein (THP) was located in the same position as the albumin band in IgAN urine before treatment; however, in the urine of a healthy subject it was located in the same position as alpha(1)-globulin. By ELISA, the THP-albumin complex concentration in IgAN urine before treatment was significantly higher than in the other two diseases. The width of the albumin fraction and the sodium ion concentra-tion of the urine were significantly correlated. The THP/albumin ratio in IgAN urine before treatment was significantly higher than in the other two groups. This suggests that the characteristic expanded width of albumin found by immunofixation indicates a THP-albumin complex, and that the sodium concentration of urine is involved in the formation of this complex.