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Letters to the Editor |
1 Analytical Biochemistry Group, Sunderland Pharmacy School, The University of Sunderland, Sunderland SR1 3RG, UK
aAuthor for correspondence. Fax 44-191-515-3747; e-mail tom.marshall{at}sunderland.ac.uk.
To the Editor:
The pyrogallol red-molybdate (PRM) protein dye-binding assay (1) is commonly used for urinary protein determination (2). The addition of sodium dodecyl sulfate (SDS) to the dye reagent is recommended to improve the uniformity of response to different proteins (3). The assay is prone to interference from aminoglycoside antibiotics, particularly gentamicin, neomycin, tobramycin, paromomycin, geneticin, and kanamycin (1)(2)(4)(5), but the degree of interference varies with different PRM methods. Thus, the DADE Behring PRM and the Sigma PRM assays are sensitive to aminoglycoside interference (4)(5), but the Cobas Fara and Roche Integra 700 PRM assays are not (4). The present study indicates that the addition of SDS to the dye reagent increases the susceptibility of the PRM assay to aminoglycoside interference. Thus, the concentration of SDS in commercial PRM reagents may be a contributory factor in determining the differential responses of the assays to aminoglycosides.
We purchased amikacin (cat. no. A1774), dihydrostreptomycin (cat. no. D7253), geneticin (cat. no. G5013), gentamicin (cat. no. G1914), kanamycin (cat. no. K4000), neomycin (cat. no. N5285), paromomycin (cat. no. P9297), streptomycin (cat. no. S6501), and tobramycin (cat. no. T1783) from Sigma-Aldrich Co. Ltd. Aqueous aminoglycoside solutions (10 g/L) were prepared gravimetrically. Gentamicin was further diluted to 5 g/L and neomycin to 2 g/L. Pierce bovine serum albumin (BSA; cat. no. 23208) and bovine 
-globulin (BGG; cat. no. 23213) Prediluted Protein Assay Standards (0.5–2.0 g/L) were purchased from Perbio Science UK Ltd. Commercial urine control (cat. no. AU2353) was purchased from Randox Laboratories Ltd. and reconstituted in either the absence or presence of aminoglycoside (final concentration, 0.2 g/L). The PRM reagent was prepared as described by Watanabe et al. (2) and modified by the addition of 25 mg/L SDS as recommended by Orsonneau et al. (3). For further investigations, the SDS concentration was increased to 50 or 100 mg/L.
For protein assays, 5–20 µL of sample (protein calibrator, aminoglycoside, or urine control with or without aminoglycoside), adjusted to 20 µL with water, was mixed with 1 mL of dye reagent. After 30 min, the absorbance (600 nm) was measured on a Jenway 6100 spectrophotometer zeroed against a water/reagent blank.
The PRM assay using dye reagent without SDS [as recommended by Watanabe et al. (2)] showed strong interference from neomycin, gentamicin, tobramycin, and paromomycin but relatively little interference from kanamycin, geneticin, streptomycin, amikacin, and dihydrostreptomycin (Table 1
). The addition of SDS to the dye reagent [as recommended by Orsonneau et al. (3)] produced progressively increasing interference such that the color yields from kanamycin and geneticin at 100 mg/L SDS were almost equivalent to those of neomycin, gentamicin, and tobramycin in the absence of SDS (Table 1
). The effect of the SDS was compounded by a decrease in the color yields of the BSA and BGG protein calibrators used to calculate the protein concentration values (Table 1
). SDS produces a similar inhibitory effect on the response of BSA and BGG when used as an additive in the Coomassie Brilliant Blue protein dye-binding assay (6).
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We confirmed the effect of SDS on the PRM assay by use of urine control containing 0.2 g/L amikacin, gentamicin, kanamycin, neomycin, streptomycin, or tobramycin. These aminoglycosides are excreted unmodified in patients’ urine at concentrations 
0.2 g/L (4)(7)(8). When we used PRM reagent without SDS (2), the mean (SD) protein value of the urine control [0.189 (0.006) g/L] increased 149% with neomycin, 65% with gentamicin, 40% with tobramycin, 11% with kanamycin, 8% with amikacin, and 6% with streptomycin (n = 5; CV <5.0%). When we used PRM reagent plus 25 mg/L SDS (3), the respective values increased to 367%, 153%, 94%, 17%, 9%, and 7% (n = 5; CV <3.0%).
In conclusion, the protein concentration values of urine containing aminoglycosides will vary with the PRM assay depending on the concentration of SDS in the dye reagent. Consequently, caution is required when the PRM assay is used to monitor urinary protein in acute care situations. Suppliers of commercial PRM reagents usually note the presence of a "surfactant" without revealing its identity or concentration. A clearer declaration of the composition of such reagents is warranted.
References
The following articles in journals at HighWire Press have cited this article:
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  T. Marshall and K. M. Williams Elimination of the Interference from Aminoglycoside Antibiotics in the Pyrogallol Red-Molybdate Protein Dye-Binding Assay Clin. Chem., September 1, 2004; 50(9): 1674 - 1675. [Full Text] [PDF]
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T. Marshall and K. M. Williams Extent of Aminoglycoside Interference in the Pyrogallol Red-Molybdate Protein Assay Depends on the Concentration of Sodium Oxalate in the Dye Reagent Clin. Chem., May 1, 2004; 50(5): 934 - 935. [Full Text] [PDF]
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