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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) reagent of Fujita et al. (1) has been modified by Watanabe et al.(2) for urinary protein determination. The assay has been widely adopted for this purpose, but interference from aminoglycoside antibiotics can produce falsely increased urinary protein values in acute-care patients, and the extent of interference varies with different PRM reagents (3)(4). Thus, the Dade Behring and Sigma reagents are susceptible to interference, whereas the Cobas Fara and Roche Integra 700 reagents are resistant (3)(4). The present study compares the PRM reagents of Fujita et al. (1) and Watanabe et al. (2) and demonstrates that the aminoglycoside interference varies with the concentration of sodium oxalate in the dye reagent.
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) were purchased from Sigma-Aldrich. Aqueous aminoglycoside solutions (10 g/L) were prepared gravimetrically and diluted to 1 g/L before assay. Pierce Prediluted Protein Assay Standard (1 g/L bovine serum albumin 23208) was purchased from Perbio Science UK Ltd. Urine control (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 Fujita dye reagent was freshly prepared by mixing 300 mL of 0.2 mol/L HCl–0.2 mol/L sodium acetate buffer (pH 2.5) with 100 mL of 20 g/L gum arabic, 5 mL of 2.4 g/L disodium molybdate, 67 mL of dye solution [60 mg of Pyrogallol Red (cat. no. P8759; Sigma Aldrich) in 100 mL of methanol], and 33 mL of methanol and adjusting the final volume to 1 L with deionized water (1). The reagent was modified to include 0.1, 0.5, 1.0, 2.0, or 4.0 mmol/L sodium oxalate by adding 0.4, 2.0, 4.0, 8.0, or 16.0 mL of 35 g/L sodium oxalate before adjusting the volume. 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 in 100 mL of methanol). Four milliliters of 2.4 g/L disodium molybdate and 4 mL of 35 g/L sodium oxalate (final concentration, 1 mmol/L) were added; the mixture was adjusted to pH 2.5 with 0.5 mol/L hydrochloric acid and brought to a volume of 1 L with deionized water (2). The reagent was modified by omitting the sodium oxalate or varying its concentration between 0.1 and 4.0 mmol/L. For protein assay, 20 µL of aminoglycoside, urine control (with or without aminoglycoside), or protein calibrator was mixed with 1 mL of PRM reagent. After 30 min, the absorbance (600 nm) was measured with a Jenway 6100 spectrophotometer zeroed with a water/reagent blank.
The PRM reagent of Fujita et al. (1) was more sensitive to aminoglycoside interference than the reagent of Watanabe et al. (2) (Table 1
). Modification of the Fujita reagent by adding sodium oxalate (0.1–4.0 mmol/L) progressively reduced the interference (Table 1
). Thus, modified reagent containing 1.0 mmol/L sodium oxalate had a susceptibility to interference comparable to the Watanabe reagent, whereas reagent containing 2.0 or 4.0 mmol/L sodium oxalate was less susceptible to interference than the Watanabe reagent (Table 1
). In contrast, modification of the Watanabe reagent by reducing the sodium oxalate concentration below the recommended 1.0 mmol/L (2) progressively increased the aminoglycoside interference (Table 1
). Thus, modified reagent prepared without sodium oxalate gave high interference comparable to the Fujita reagent (Table 1
). Conversely, modification of the Watanabe reagent by increasing the sodium oxalate concentration to 2.0 or 4.0 mmol/L progressively reduced the interference (Table 1
).
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The effect of sodium oxalate on aminoglycoside interference was confirmed with a urine control supplemented with prescribed aminoglycosides at concentrations consistent with the maximum concentrations expected in patients’ urine based on pharmacokinetic data, i.e., 0.2 g/L (amikacin, gentamicin, neomycin, and tobramycin) or 1.0 g/L (kanamycin and streptomycin) (3)(5)(6). The Fujita reagent gave negative absorbance values with the urine control. Modification of the Fujita reagent by adding 0.5 mmol/L sodium oxalate produced positive absorbance values with strong aminoglycoside interference. Thus, in the presence of neomycin, gentamicin, tobramycin, amikacin, streptomycin, or kanamycin, the urine control protein value was falsely increased by 197%, 130%, 85%, 18%, 36%, and 70%, respectively (n = 4; CV <10%). The addition of 1.0 mmol/L sodium oxalate to the Fujita reagent reduced the interference (respective values, 81%, 59%, 37%, 9%, 26%, and 35%). The interference was further reduced with 2.0 mmol/L sodium oxalate (respective values, 48%, 41%, 31%, 9%, 25%, and 30%). In comparison with the Fujita reagent, the Watanabe reagent gave low aminoglycoside interference with the urine control. Thus, in the presence of neomycin, gentamicin, tobramycin, amikacin, streptomycin, or kanamycin, the protein value was falsely increased by only 59%, 35%, 20%, 5%, 4%, and 4%, respectively (n = 4; CV <10%). Modification of the Watanabe reagent by decreasing the sodium oxalate concentration from the recommended 1.0 mmol/L (2) to 0.5 mmol/L increased the interference (respective values, 130%, 79%, 44%, 9%, 11%, and 15%). The interference was further increased with 0.1 mmol/L sodium oxalate (respective values, 346%, 246%, 154%, 11%, 20%, and 45%). Conversely, modification of the Watanabe reagent by increasing the sodium oxalate concentration from 1.0 mmol/L (2) to 2.0 mmol/L decreased the interference (respective values, 27%, 22%, 13%, 3%, 1%, and 1%).
In conclusion, the reagent of Fujita et al. (1) is more prone to aminoglycoside interference than the reagent of Watanabe et al. (2), but the relative response depends on the concentration of sodium oxalate in the dye reagents. Although Watanabe et al. (2) recommended the addition of sodium oxalate to the PRM reagent to "eliminate negative errors caused by chelating substances in urine", its beneficial effect on aminoglycoside interference has not been reported. The effect of sodium oxalate contrasts with the addition of sodium dodecyl sulfate to the PRM reagent (7), which increases aminoglycoside interference (8). Because commercial PRM reagents are based on different formulations (1)(2)(7) and vary in their susceptibility to aminoglycoside interference (3)(4), it would be helpful if suppliers specified the exact composition of their reagents to avoid unnecessary problems with acute-care patients.
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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