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.
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.
We designed a semiquantitative analysis of urinary low protein levels using silver dot blot assay. In this method, 3 microl of urine are blotted to one dot onto a cellulose acetate membrane, which is stained by a colloidal silver staining reagent, and the optical density of the silver stained urinary protein is measured at 500 nm using a densitometer. There was a good linearity between 2.5 mg/L and 100 mg/L and a gentle linearity between 100 mg/L and 200 mg/L, and the minimum sensitivity was 2.5 mg/L. This method is suitable for semiquantitative analysis of urinary protein levels less than 300 mg/L, which can not be determined precisely by dipstick.
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