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

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.