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1 Analytical Biochemistry Group Institute of Pharmacy, Chemistry & Biomedical Science School of Health, Natural & Social Sciences The University of Sunderland Sunderland SR1 3RG, United Kingdom
aAuthor for correspondence. Fax 44-191-515-3747; e-mail tom.marshall{at}sunderland.ac.uk.
To the Editor:
The aminoglycosides gentamicin and tobramycin interfere in the Dade Behring pyrogallol red-molybdate (PRM) assay used for determination of urinary protein (1). This is an important finding because aminoglycosides are nephrotoxic and accurate determination of urinary protein is necessary to detect renal damage in patients receiving aminoglycosides. We have investigated interference in the PRM assay (2)(3) from other aminoglycosides (dihydrostreptomycin, geneticin, kanamycin, neomycin, paromomycin, and streptomycin) and extended the study to the Coomassie Brilliant Blue (CBB) and the benzethonium chloride (BEC) protein assays, which are also used routinely for urinary protein determination (4)(5)(6).
Dihydrostreptomycin (cat. no. D7253), geneticin (cat. no. G5013), gentamicin (cat. no. G3632), kanamycin (cat. no. K4000), neomycin (cat. no. N6386), paromomycin (cat. no. P9297), streptomycin (cat. no. S6501), and tobramycin (cat. no. T1783) were purchased from Sigma-Aldrich Co. Ltd., and solubilized in 0.1 mol/L phosphate buffer (pH 7) at concentrations of 10, 5, 1, 0.5, and 0.1 g/L. Bovine serum albumin (BSA; cat. no. A7906; Sigma) was solubilized at 10 g/L, calibrated with the Sigma biuret assay, and diluted to 2 g/L for calibration of the PRM assay and 1 g/L for the CBB and BEC assays. Sigma urine control (cat. no. U9631) was reconstituted in water or aqueous aminoglycoside (final concentration, 0.2 g/L).
We mixed 20 µL of aminoglycoside, 20 µL of urine control (± aminoglycoside), or 5–20 µL of protein calibrator (adjusted to 20 µL with phosphate buffer) with 1 mL of either Sigma Microprotein-PR Reagent (PRM assay) or Sigma Protein Assay Solution (CBB assay). The absorbance at 600 nm (PRM assay) or 595 nm (CBB assay) was measured with a Jenway 6100 spectrophotometer zeroed against a reagent blank. For the BEC assay, we mixed the samples (as above) with 0.8 mL of 0.5 mol/L sodium hydroxide containing 33 mmol/L EDTA. We then added 0.2 mL of 2 g/L BEC and measured the turbidity (A660 nm) after 50 min.
Aminoglycoside interference in the PRM assay (calculated as absorbance/µg) was determined using the concentration of a sample that gave an absorbance value within the linear range of the assay and was expressed relative to the absorbance/µg of BSA (corresponding to the gradient of the calibration curve):
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Neomycin, gentamicin, tobramycin, and paromomycin gave higher responses (442%, 304%, 259%, and 135%, respectively) than BSA (100%), whereas geneticin, kanamycin, streptomycin, and dihydrostreptomycin gave lower responses (26%, 21%, 7%, and 3%, respectively; n = 5; CV <5%). The aminoglycosides gave negligible interference in the CBB assay (<0.2% relative to BSA) and failed to generate turbidity in the BEC assay.
Aminoglycoside interference in urine was demonstrated with urine control containing the aminoglycosides at 0.2 g/L. Although the data are based on model experiments, they reflect aminoglycoside concentrations that could reasonably be present in urine from patients (1). The PRM control value (0.36 ± 0.01 g/L protein) increased 244% with neomycin (1.24 ± 0.01 g/L), 142% with gentamicin (0.87 ± 0.02 g/L), 111% with tobramycin (0.76 ± 0.02 g/L), 53% with paromomycin (0.55 ± 0.02 g/L), 11% with geneticin (0.40 ± 0.01 g/L), 8% with kanamycin (0.39 ± 0.01 g/L), 3% with streptomycin (0.37 ± 0.01 g/L), and 3% with dihydrostreptomycin (0.37 ± 0.02 g/L; n = 5; CV <5.5%). The interference was significant (P <0.01) with neomycin, gentamicin, tobramycin, paromomycin, geneticin, and kanamycin but not significant (P >0.1) with streptomycin or dihydrostreptomycin. In contrast, neither the CBB control value (0.21 ± 0.01 g/L protein) nor the BEC control value (0.28 ± 0.01 g/L) were affected by aminoglycosides at 0.2 g/L.
Thus, in contrast to the Roche and Cobas Fara PRM assays, which are resistant to aminoglycoside interference, the Sigma PRM assay resembles the Dade Behring PRM assay in its susceptibility to interference (1). This may reflect the formulation of the reagents or the volume ratio of sample to reagent. The clinical importance of this interference is evident for tobramycin [at urinary concentrations >0.2 g/L (1)] but more difficult to assess for the other aminoglycosides, whose urinary concentrations do not appear to have been reported.
In conclusion, we have confirmed interference in the PRM assay by gentamicin, neomycin, and tobramycin (1)(2)(3); reported interference from additional aminoglycosides; and demonstrated a susceptibility of the Sigma PRM assay to interference from aminoglycosides in urine at 0.2 g/L. In contrast to the PRM assay, the CBB and BEC assays are resistant to aminoglycoside interference.
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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T. Marshall and K. M. Williams Aminoglycoside Interference in the Pyrogallol Red-Molybdate Protein Assay Is Increased by the Addition of Sodium Dodecyl Sulfate to the Dye Reagent Clin. Chem., December 1, 2003; 49(12): 2111 - 2112. [Full Text] [PDF]
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