Development of a new method for measuring total urinary protein using acid violet 6B pigment

Abstract

We have developed a new method for measuring total urinary protein using acid violet 6B (AV6B) pigment. Nine purified components of human urinary proteins and urine samples collected randomly from 123 diabetic outpatients were used. There were 62, 36, and 25 cases of prenephropathy, early nephropathy, and overt nephropathy, respectively. All samples were measured by Coomassie brilliant blue G 250 (CBB), pyrogallol red-molybdate (PRM), and AV6B methods using an optical photometer. In healthy subjects, the major components of urinary proteins, such as gamma-globulins, IgG, IgA Tamm-Horsfall protein, and transferrin, the reactivity values of the AV6B and PRM methods were similar. The CBB method was the least sensitive of the three methods. In the urine samples from diabetic patients, the urinary protein values measured by the AV6B method were higher than those measured by the CBB method in the prenephropathy stage. The values obtained by the AV6B method (y) correlated well with those from the CBB method (x) (y=1.243x+3.61, r=0.904). When the values from the AV6B method (y) were compared to those from the PRM method (x), correlation was low (y=1.406x-29.15, r=0.786). In conclusion, the AV6B method was more useful than the CBB and PRM methods for low levels of urinary protein.

Measurement of proteinuria in dogs: analytic and diagnostic differences using 2 laboratory methods

“…In contrast to an earlier report 11 , our results demonstrate that there was overestimation of UP using CBB compared with PRM. In that study, the authors attributed the underestimation to adhesion of dye-protein complex to test tubes.…”

“…Preliminary data were presented as a poster abstract at the 16th annual ESVCP/ECVCP Congress, Milan, Italy, October 1-3, 2014. complexes readily adhere to the surface of test tubes, leading to falsely low measurements. 11 In contrast, PRM does not absorb onto the wall of cuvettes but characteristically lacks a uniform response to different proteins compared with CBB. 12,13 Specifically, PRM underestimates albumins and globulins due to the characteristics of the reagents used.…”

“…Nevertheless, all dye‐binding methods suffer from analytic limitations in that CBB–protein complexes readily adhere to the surface of test tubes, leading to falsely low measurements . In contrast, PRM does not absorb onto the wall of cuvettes but characteristically lacks a uniform response to different proteins compared with CBB .…”

Hypoglobulinemia in a dog with disseminated plasma cell neoplasia: Case report and review of the diagnostic criteria

“…Some urinary proteins are soluble in SSA but react with Pyrogallol red, including alpha 1-acidoglycoprotein (variable) and Tamm-Horsfall mucoprotein. 34 Precipitation of Ig light chains in the renal tubules, renal amyloidosis, glomerulonephropathy, or renal infiltration with neoplastic cells are also possibilities. 35 Unfortunately, a postmortem examination was declined; however, histologic evaluation and special staining for the presence of amyloid would be warranted in future suspect cases.…”