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Technical Briefs |
1 Analytical Biochemistry Group, Sunderland Pharmacy School, The University of Sunderland, Sunderland SR1 3RG, UK;
aauthor for correspondence: fax 44-191-515-3405, e-mail tom.marshall{at}sunderland.ac.uk
The Pyrogallol Red-molybdate (PRM) protein dye-binding assay is used for urinary protein determination (1)(2)(3). The assay is sensitive to interference from aminoglycoside antibiotics, and the extent of interference varies with different PRM reagents (1)(2)(4)(5)(6)(7). Thus, the Dade Behring PRM and the Sigma PRM assays are sensitive to aminoglycoside interference, whereas the Cobas Fara and Roche Integra 700 PRM assays are not (4)(5). Furthermore, the PRM reagents used by Fujita et al. (1) and Orsonneau et al. (3) are more sensitive to interference than the reagent used by Watanabe et al. (2). We have previously shown that the addition of sodium dodecyl sulfate (SDS) to the PRM reagent increases the interference, whereas the addition of sodium oxalate reduces the interference (6)(7). In the present study, we indicate that the extent of interference also depends on the concentration of dye in the reagent and demonstrate that a decrease in the dye concentration combined with an increase in the sodium oxalate concentration eliminates the interference.
The aminoglycosides (Sigma-Aldrich) were dissolved in water at a concentration of 10 g/L. The neomycin and gentamicin were further diluted to 1 g/L. Pierce Prediluted Protein Assay Standard (1 g/L bovine serum albumin) was purchased from Perbio Science UK Ltd. Urine control (cat. no. AU2353) was purchased from Randox Laboratories Ltd. and reconstituted in deionized water or aqueous aminoglycoside (final concentration, 0.2 or 1.0 g/L).
The Watanabe dye reagent was freshly prepared by dissolving 5.9 g of succinic acid and 0.5 g of sodium benzoate in 900 mL of water and mixing with 40 mL of dye solution [60 mg of Pyrogallol Red (cat. no. P8759; Sigma-Aldrich Co. Ltd.) in 100 mL methanol]. We then added 4 mL of 2.4 g/L disodium molybdate and 4 mL of 35 g/L sodium oxalate, adjusted the mixture to pH 2.5 with 0.5 mol/L hydrochloric acid, and brought the final volume to 1 L with deionized water (2). We modified the Watanabe reagent (0.06 mmol/L dye and 1.0 mmol/L sodium oxalate without SDS) by varying the concentration of the dye (0.03 or 0.12 mmol/L), sodium oxalate (0 or 2 mmol/L), and SDS (25 or 100 mg/L) in all combinations. For protein assays, we mixed 20 µL of sample, calibrator, or water (blank) with 1 mL of PRM reagent and measured the absorbance at 600 nm after 30 min against the blank.
The effects of the aminoglycosides on the absorbance readings in the PRM assay (in the absence of protein) with reagents at three different dye concentrations are shown in Table 1
. Doubling the concentration of the dye from 0.06 to 0.12 mmol/L increased the absorbance attributable to aminoglycoside by up to 273% (geneticin), whereas halving the concentration (0.03 mmol/L) decreased the absorbance by up to 87% (tobramycin). Doubling the concentration of the dye in combination with addition of SDS and omission of sodium oxalate increased the aminoglycoside interference by up to 2500% (amikacin). Halving the concentration of the dye and doubling the sodium oxalate concentration (with omission of SDS) eliminated the interference (Table 1
). We confirmed the results, using urine control supplemented with aminoglycosides at the maximum concentrations expected in patients’ urines: 0.2 g/L amicakin, gentamicin, neomycin, or tobramycin; 1.0 g/L kanamycin or streptomycin (4)(8)(9). The measured protein concentration in the urine control was falsely increased by neomycin (67% higher), gentamicin (33%), tobramycin (26%), amikacin (9%), streptomycin (7%), and kanamycin (6%) when we used the Watanabe reagent but was unaffected by the aminoglycosides (respective values <1%) when we used the modified reagent (n = 4; CV <6%).
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In conclusion, the extent of aminoglycoside interference in the PRM assay is dictated by the composition of the dye reagent. SDS increases the interference (6), whereas sodium oxalate decreases the interference (7), and these effects are compounded by changes in the concentration of the dye. Thus, increased dye combined with the omission of sodium oxalate (plus SDS) gives very high interference, whereas decreased dye combined with increased sodium oxalate (minus SDS) eliminates detectable interference.
References
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