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Clinical Chemistry 46: 392-398, 2000;
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(Clinical Chemistry. 2000;46:392-398.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

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

Thomas Marshalla and Katherine M. Williams

a Author for correspondence. Fax 44-191-515-3747; e-mail tom.marshall{at}sunderland.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The total protein concentration in urine can be determined by biuret assay (1)(2)(3), precipitation with trichloroacetic acid-Ponceau S (TCA-PS)1 (4), turbidimetry [using TCA (5), sulfosalicylic acid (6), or benzethonium chloride (BEC) (7)], or protein dye-binding assays utilizing Coomassie Brilliant Blue (CBB) (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) or Pyrogallol Red-molybdate (PRM) (20)(21)(22). Urinary protein determinations are notoriously unreliable (23)(24)(25)(26)(27), and comparative evaluation of a range of assay methods (28)(29)(30)(31) has failed to establish a convenient method of choice. However, protein dye-binding assays are simple, rapid, and readily automated (10)(12)(13)(15)(16)(18)(19)(20)(22) and are becoming increasingly popular for the clinical determination of total protein in urine. The present study indicates a lack of agreement in the urine protein concentrations measured by the Sigma CBB and PRM test kits as recommended by the manufacturer. The use of an alternative protein calibrator prepared from Sigma urinary protein lyophilizate (UPL) overcomes this problem and simultaneously improves the comparability of the protein concentration values obtained with the dye-binding assays relative to the TCA and BEC methods.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
CBB protein dye reagent (cat. no. 610-A), Microprotein-PRTM dye reagent (cat. no. 611-A), human albumin (HA) protein calibrators (cat. nos. 610-30 and 610-50), the albumin/globulin (A/G) protein calibrator (cat. no. 540-10), urine control (UC) Levels 1 and 2 (cat. nos. U9506 and U9631), and UPL (cat. no. U8126) were purchased from Sigma Diagnostics. Bovine serum albumin (BSA) calibrator was purchased from Pierce & Warriner.

samples
Urine.
Specimens (n = 60), covering a wide range of protein concentrations, were collected without preservatives, centrifuged (2500g for 10 min), and stored at -70 °C. Immediately before protein assay, the samples were thawed at room temperature.

Controls.
Sigma urine controls Levels 1 and 2 (UC 1 and UC 2) were reconstituted in 10 mL of ultrapure water (MilliQ Plus Water Purification System; Millipore) and stored at 4 °C (for up to 5 days) as recommended by the manufacturer.

UPL.
Sigma UPL (5 mg protein) was reconstituted in 2.5 mL of 0.15 mol/L sodium chloride containing 1 g/L sodium azide, and the mixture was stirred gently for 2 h at room temperature. After centrifugation (10 000g for 5 min), the protein content of the supernate (UPL stock solution, 1.9 g/L) was determined by Lowry assay (32), and the UPL was diluted (in saline/sodium azide) to give 300 mg/L and 500 mg/L UPL protein calibrators, which were stored at -20 °C in 100-µL aliquots. Immediately before protein assay, the UPL protein calibrator was thawed at room temperature and gently vortex-mixed for 1 min.

protein assays
CBB protein dye-binding assay.
The assay procedure was performed manually as described by the manufacturer (33), except that the volume of the assay mixture was scaled down from 2.55 mL to 1.02 mL. Briefly, 20 µL (rather than 50 µL) of sample (urine, diluted urine, or UC) or protein calibrator (HA, BSA, A/G, or UPL at 300 mg/L) was gently mixed with 1.0 mL (rather than 2.5 mL) of Protein Assay Solution (Protein Dye Reagent diluted with four volumes of ultrapure water in a plastic container), and after 2–30 min, the absorbance of the assay mixture was measured (A595 nm) in a plastic cuvette by a Jenway 6100 spectrophotometer (Dunmow) zeroed with a reagent blank. Protein concentrations were calculated as recommended by the manufacturer from the ratio of the absorbance value of the sample and 300 mg/L calibrator. In subsequent studies, UC 1 was assayed at 20, 50, 100, 150, and 200 µL, and UC 2 was assayed at 20, 40, 50, 60, 80, and 100 µL; protein concentrations were derived by extrapolation of the absorbance values from a calibration curve.

PRM protein dye-binding assay.
The assay procedure was performed manually as described by the manufacturer (34). Briefly, 20 µL of sample (urine, diluted urine, or UC) or protein calibrator (HA, BSA, A/G, or UPL at 500 mg/L) was gently mixed with 1 mL of Microprotein-PR Reagent, and after 3–15 min, the absorbance of the assay mixture was measured (A600 nm) against water, followed by subtraction of the absorbance of a reagent blank. Protein concentrations were calculated as recommended by the manufacturer from the ratio of the absorbance values of the sample and the 500 mg/L calibrator. In subsequent studies, UC 1 was assayed at 20, 50, 100, 150, and 200 µL, and UC 2 was assayed at 20, 40, 50, 60, 80, and 100 µL; protein concentrations were derived by extrapolation of the absorbance values from a calibration curve.

Lowry protein assay (32).
Reagent C (1 mL) was mixed with 0.1 mL of HA calibrator or 10 µL of UPL stock solution (adjusted to 100 µL with 0.15 mol/L sodium chloride containing 1 g/L sodium azide) followed, after 15 min, by 0.1 mL of reagent E. The absorbance of the assay mixture was measured (A540 nm) against a reagent blank after 30 min, and protein concentrations were derived by extrapolation of the absorbance values from a calibration curve.

TCA protein assay (35).
TCA (0.25 mL of a 125 g/L solution) was mixed with 1 mL of sample (UC) or protein calibrator (A/G or UPL at 300 mg/L), and after 10 min, the absorbance of the assay mixture was measured (A620 nm) against a sample blank (1 mL of sample plus 0.25 mL of 0.15 mol/L sodium chloride containing 1 g/L sodium azide). Protein concentrations were calculated from the ratio of the absorbance values of the sample and the 300 mg/L calibrator. In subsequent studies, UCs 1 and 2 were each assayed at 0.2, 0.4, 0.6, 0.8, and 1 mL, and protein concentrations were derived by extrapolation of the absorbance values from a calibration curve.

BEC protein assay (7).
Eight hundred microliters of 0.5 mol/L sodium hydroxide containing 33 mmol/L EDTA was mixed with 20 µL of sample (UC) or protein calibrator (HA or UPL), followed immediately by the addition of 0.2 mL of 2 g/L BEC. The absorbance of the assay mixture was measured (A660 nm) against a reagent blank after 50 min. Protein concentrations were derived by extrapolation of the absorbance values from a calibration curve. In subsequent studies, UC 1 was assayed at 25, 50, 75, and 100 µL, and UC 2 was assayed at 10, 20, 30, 40, and 50 µL.

TCA-PS protein assay (4).
TCA-PS concentrated reagent (50 µL) was mixed with 0.5 mL of sample (UC 1 or UC 2 diluted 1:5 with saline/azide) or protein calibrator (A/G or UPL at 100 mg/L), and the mixture was centrifuged at 2500g for 5 min before removal of the supernate and resolubilization of the pellet in 1 mL of 0.8 g/L sodium hydroxide. The absorbance of the assay mixture was measured (A560 nm) against a reagent blank. Protein concentrations were derived by extrapolation of the absorbance values from a calibration curve. In subsequent studies, UC 1 was assayed at 100, 200, 300, 400, and 500 µL, and UC 2 was assayed at 50, 100, 150, and 200 µL.

Biuret protein assay (1).
Ice-cold ethanolic phosphotungstic acid (5 mL) was added to duplicate 5-mL aliquots of ice-cold sample (0.8 mL of UPL calibrator diluted with 4.2 mL of saline/azide) or calibrator (A/G) and allowed to stand for 15 min in ice before centrifugation at 1000g for 10 min. The supernates were decanted, and the protein pellets were washed with 2.5 mL of ice-cold ethanol. After centrifugation (1000g for 10 min), the supernates were discarded, and the pellets of duplicate aliquots were dissolved in either 1 mL of biuret reagent or reagent without copper sulfate. After 15 min, the absorbances

(A540 nm) of the assay tubes containing biuret reagent were measured against the duplicate assay tubes containing reagent without copper sulfate. Protein concentrations were derived by extrapolation of the absorbance values of the samples from a calibration curve.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Sigma CBB Microprotein assay gave consistently lower protein concentration values than the corresponding PRM assay when applied to urine and Sigma urine controls. The values for the urine were 110–13 500 mg/L (mean, 2390 mg/L) with the CBB assay compared with 160–18 300 mg/L (mean, 3470 mg/L) with the PRM assay (CBB:PRM protein concentration ratio, 0.46–0.88; mean, 0.69 ± 0.10). The effect was highly reproducible as demonstrated using Sigma urine controls UC 1 and UC 2 (Table 1 ). Thus, UC 1 gave within-day (between-day) protein values of 86 ± 2 mg/L (88 ± 3 mg/L) by CBB assay and 127 ± 4 mg/L (131 ± 8 mg/L) by PRM assay. The corresponding values for UC 2 were 262 ± 5 mg/L (259 ± 5 mg/L) by CBB assay and 438 ± 13 mg/L (440 ± 15 mg/L) by PRM assay. The respective within-day (between-day) CBB:PRM protein concentration ratios were 0.68 ± 0.02 (0.67 ± 0.04) for UC 1 and 0.60 ± 0.01 (0.59 ± 0.02) for UC 2. These ratios were unaffected by the use of BSA (rather than HA) as a protein calibrator, and A/G only slightly improved the comparability of the assays (Table 1 ). The use of UPL as a protein calibrator increased the CBB protein concentration values of the controls and decreased the corresponding PRM values to greatly improve comparability between the assays, i.e., within-day CBB:PRM ratio, 1.0 ± 0.03 for UC 1 and 0.85 ± 0.02 for UC 2 (Table 1 ). However, the latter was further improved when protein concentrations were derived using a calibration curve, i.e., the calculation of protein concentration from an absorbance ratio relative to a single protein calibrator (see Table 1 ) fails to take into account the nonlinear response of UPL to the assays (Fig. 1 ). Thus, when UC 1 and UC 2 were each assayed over a range of sample volumes (to give varying color yields within the calibration curves) and the protein concentration values were derived by extrapolation of absorbance from a UPL calibration curve, then agreement between the assays was obtained with UC 2 as well as UC 1 (Fig. 2 ). Repeat assays of the urine using a UPL calibration curve gave protein concentration values of 120–15 200 mg/L (mean, 2700 mg/L) with the CBB assay and 140–15 500 mg/L (mean, 2800 mg/L) with the PRM assay. Thus, the CBB:PRM protein concentration ratio was greatly improved (0.69–1.20; mean, 0.96 ± 0.11). The correlation plots for the CBB and PRM assay of the urine are shown in Fig. 3 , and the respective statistical data are tabulated in Table 2 . The agreement between the assays was dramatically improved when UPL (slope, 0.972; intercept, -16 mg/L) was used as protein calibrator rather than HA (slope, 0.685; intercept, 17 mg/L; Fig. 3 ).


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  		Table 1. Protein concentrations for the urine controls (UC 1 and UC 2) as determined by the CBB and PRM protein dye-binding assays calibrated against HA, BSA, A/G, or UPL.



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  		Figure 1. The response of the Sigma CBB and PRM protein dye-binding assays to the urinary protein calibrator.

The calibration curves correspond to increasing amounts of HA (•) or urinary protein ({blacksquare}) assayed with the Sigma CBB (A) or PRM (B) test kits. Urinary protein demonstrated a nonlinear response and gave lower color yields than HA with the CBB assay but higher color yields with the PRM assay.



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  		Figure 2. Protein concentration of the Sigma urine controls as determined by protein dye-binding assays.

Increasing volumes of UC 1 (• and {blacksquare}) and UC 2 ({diamondsuit} and {blacktriangleup}) were assayed with the Sigma CBB (• and {diamondsuit}) or PRM ({blacksquare} and {blacktriangleup}) test kits using HA (A) or urinary protein (B) as a protein calibrator. The differences in the protein concentration values resulting from calibration against HA (A) are eliminated when urinary protein is used as a calibrator (B).



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  		Figure 3. Correlation plots of the CBB and PRM protein concentration values for human urine (n = 60).

The urine was assayed with the Sigma CBB or PRM test kits with HA (A) or urinary protein (B) as calibrator. Linear regression analysis indicated an improved agreement between the assays when urinary protein (yCBB = 0.972xPRM - 16) rather than HA (yCBB = 0.685xPRM + 17) was used as calibrator.


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  		Table 2. Correlation between the CBB and PRM protein dye-binding assays using HA or UPL as calibrator for the assay of human urine (n = 60).

We investigated the comparability between the CBB, PRM, TCA, BEC, and TCA-PS protein assays by determining the protein concentration of the urine controls over a range of sample volumes, using calibration curves (Fig. 4 ). When the recommended calibrator (HA or A/G) was used, the protein concentration values obtained with the CBB, PRM, TCA, BEC, and TCA-PS assays for UC 1 were 81 ± 7, 122 ± 5, 89 ± 4, 115 ± 8, and 79 ± 8 mg/L, respectively; and the UC 2 values were 264 ± 17, 455 ± 15, 369 ± 23, 350 ± 12, and 305 ± 13 mg/L, respectively. With UPL as a calibrator, the values obtained with the CBB, PRM, TCA, BEC and TCA-PS assays for UC 1 were 89 ± 8, 88 ± 4, 84 ± 4, 93 ± 9, and 61 ± 6 mg/L, respectively; and the UC 2 values were 295 ± 16, 303 ± 12, 322 ± 6, 289 ± 12, and 243 ± 11 mg/L, respectively. Thus, UPL improved the CBB:PRM ratio for UC 1 from 0.66 to 1.01, the TCA:PRM ratio from 0.73 to 0.95, and the BEC:PRM ratio from 0.94 to 1.06; UPL improved the CBB:PRM ratio for UC 2 from 0.58 to 0.97, the TCA:PRM ratio from 0.81 to 1.06, and the BEC:PRM ratio from 0.77 to 0.95.



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  		Figure 4. The response of the TCA and BEC protein assays to the urinary protein calibrator.

The calibration curves correspond to increasing amounts of HA ({blacktriangleup}), A/G (•), or urinary protein ({blacksquare}) assayed with the TCA (A) or BEC (B) methods. Urinary protein gave a higher color yield than the recommended calibrators (HA or A/G) and demonstrated a nonlinear response in both assays.

The protein composition of the UPL calibrator was investigated by electrophoresis and densitometry. The UPL consisted predominantly of albumin (57.5%), {gamma}-globulin (6.5%), and transferrin (6.9%), plus a diverse range of minor proteins (29.1%), the latter including protein bands corresponding to {alpha}1-microglobulin, immunoglobulin light chain, and retinol-binding protein (36) (Fig. 5 )



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  		Figure 5. Protein composition of the urinary protein calibrator as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry.

The proteins were separated under nonreducing conditions on ExcelGel® SDS homogeneous 12.5% gels (A) using the Multiphor II electrophoresis system (Amersham Pharmacia) and scanned using an LKB Ultroscan laser densitometer (B). The patterns detected by CBB staining correspond to the low-molecular weight calibration proteins (lane 1) and UPL (lanes 2, 3, and 4, which contain 5, 10, and 15 µg of protein, respectively). Mr, relative molecular weight (x 10-3). Proteins (bands in A and peaks in B) identified by comparison with reference patterns (36) include: 1, immunoglobulin; 2, transferrin; 3, albumin; 4, {alpha}1-microglobulin; 5, immunoglobulin light chain; and 6, retinol-binding protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Methods for the determination of total urinary protein that use precipitation of protein and indirect biuret are accurate, but they also are time-consuming and, therefore, not widely used. Protein dye-binding assays are rapid, simple, and readily automated (10)(12)(13)(15)(16)(18)(19)(20)(22), but they characteristically lack a uniform response to different proteins (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(22)(28). This problem has been addressed by modification of the purity/concentration of the dye and the addition of sodium dodecyl sulfate to the reagent (14)(15)(18)(19)(22). However, the preparation of modified reagents (and the inherent problems of reproducibility and stability) is troublesome, and it is advantageous to use optimized test kits that are commercially available worldwide in a standardized form.

The results of the present study indicate that the Sigma Microprotein CBB assay and the corresponding PRM assay give different protein concentration values when applied to urine and Sigma urine controls. This effect is unrelated to freezing/thawing of the samples and is a potential source of confusion because it is desirable to obtain the same protein concentration values with both tests. Consequently, we have aimed to equalize the response of the assays (without modifying the dye reagents) by use of a urinary protein calibrator and commercial urine controls to minimize the compounding effects of pathological variation. Our results indicate that the disagreement between the protein concentration values obtained with the different assays reflects a difference in the response of the CBB and PRM assays to urinary protein (compared with HA) and that the problem is overcome by the use of UPL as a protein calibrator. Previous studies have indicated similar differences in the urine protein values obtained with other protein assay methods (28)(29)(30)(31). Thus, it is interesting to note that in the present study, the use of a urinary protein calibrator (rather than HA or A/G) greatly improved the comparability of the results obtained with the CBB, PRM, TCA, and BEC protein assays when applied to the urine controls (and slightly improved their comparability with the TCA-PS method). This is an important observation because the urine controls are likewise recommended for assessing the reliability of other methods for total protein determination of urine. However, the potential value of UPL as a protein calibrator for other protein assays can only be fully evaluated with actual urine specimens, and this is beyond the scope of the present study.

In conclusion, our results indicate that the use of a urinary protein calibrator will improve the comparability of the Sigma CBB and PRM protein assay methods when used for the determination of total protein in urine. Ideally, such a calibrator should be commercially available in a stabilized form and precalibrated against a reference method such as the biuret. We have attempted biuret assays of submilligram quantities of UPL calibrator, and this suggests a protein concentration of 545 mg/L (rather than 500 mg/L as determined by Lowry assay). However, the biuret assay is insensitive (requiring milligram quantities of protein for accurate determination), and the response of the assay to UPL is nonlinear. Consequently, the accuracy of the UPL biuret value is questionable. This complicates the use of the biuret assay as a reference method for comparison of the CBB and PRM assays. In addition, this is impractical given the expense of UPL and its availability in only milligram amounts, i.e., each UPL calibration curve would require more than the maximum batch amount (10 mg) of UPL as currently supplied. The use of CBB:PRM protein concentration ratios and correlation plots of the CBB and PRM values eliminates any uncertainty relating to the absolute protein concentration of the UPL calibrator and is adequate, for the purpose of the present study, to demonstrate improved agreement between the assays.


   Footnotes
 
Analytical Biochemistry Group, School of Sciences, The University of Sunderland, Sunderland SR1 3 RG, UK.

1 Nonstandard abbreviations: TCA, trichloroacetic acid; PS, Ponceau S; BEC, benzethonium chloride; CBB, Coomassie Brilliant Blue; PRM, Pyrogallol Red-molybdate; HA, human albumin; A/G, albumin/globulin; UC, urine control; UPL, urinary protein lyophilizate; and BSA, bovine serum albumin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Protein:Creatinine Ratio in Random Urine Samples Is a Reliable Marker of Increased 24-Hour Protein Excretion in Hospitalized Women with Hypertensive Disorders of Pregnancy
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M. Maachi, S. Fellahi, A. Regeniter, M.-E. Diop, J. Capeau, J. Rossert, and J.-P. Bastard
Patterns of Proteinuria: Urinary Sodium Dodecyl Sulfate Electrophoresis Versus Immunonephelometric Protein Marker Measurement Followed by Interpretation with the Knowledge-Based System MDI-LabLink
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F. M. Yilmaz, N. Celebi, and D. Yucel
Automated Turbidimetric Benzalkonium Chloride Method for Measurement of Protein in Urine and Cerebrospinal Fluid
Clin. Chem., August 1, 2004; 50(8): 1450 - 1452.
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R. B. Caldwell and C. T. Lattemann
Simple and Reliable Method To Precipitate Proteins from Bacterial Culture Supernatant
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T. Marshall and K. M. Williams
Total Protein Determination in Urine: Aminoglycoside Interference
Clin. Chem., January 1, 2003; 49(1): 202 - 203.
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