Total Protein Determination in Urine: Elimination of a Differential Response between the Coomassie Blue and Pyrogallol Red Protein Dye-binding Assays

Abstract

AbstractBackground: The total protein content of urine is a good index of renal function, but its determination is unreliable. Protein dye-binding assays are simple, but they characteristically lack a uniform response to different proteins.Methods: We investigated a differential response of the Sigma Microprotein Coomassie Brilliant Blue (CBB) and Pyrogallol Red-molybdate (PRM) protein dye-binding assays to urine, using human albumin, albumin/globulin, or urinary protein as calibrator.Results: The urine protein values (n = 60) obtained with the CBB assay were 110–13 500 mg/L (mean, 2390 mg/L) compared with 160–18 300 mg/L (mean, 3470 mg/L) obtained with the PRM assay (CBB:PRM protein concentration ratio, 0.46–0.88, mean, 0.69 ± 0.10). The differential response was highly reproducible as indicated by Sigma urine control Level 1 (within-day CBB:PRM ratio, 0.68 ± 0.02; between-day CBB:PRM ratio, 0.67 ± 0.04) and Sigma urine control Level 2 (within-day CBB:PRM ratio, 0.60 ± 0.01; between-day CBB:PRM ratio, 0.59 ± 0.02). The use of urinary protein as a calibrator (rather than human albumin) greatly improved the agreement between the assays when applied to urine (yCBB = 0.972xPRM − 16 vs yCBB = 0.685xPRM + 17). In studies using urine controls, this calibrator also improved agreement between the CBB, PRM, trichloroacetic acid (TCA), and benzethonium chloride protein methods and, to a lesser extent, agreement with the TCA-Ponceau S method.Conclusion: The use of a urinary protein calibrator improves the agreement between different methods used to determine total protein in urine.