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1 Division of Chemical Pathology, Northside Pathology, Queensland Health Pathology Service, The Prince Charles Hospital, Rode Road, Chermside, Brisbane 4032, Australia
aAuthor for correspondence. Fax 61-7-33508553; e-mail gus_koerbin{at}health.qld.gov.au
To the Editor:
We recently became aware of apparently wide within-person, day-to-day fluctuations in total urine protein concentrations measured by the pyrogallol red–molybdate method on the Dade Behring Dimension RxL in some individual patients. In several such patients with suddenly and unexpectedly high proteinuria, electrophoresis of the urine failed to confirm the presence of protein. A retrospective review of 862 total urine protein requests indicated that most patients prescribed aminoglycosides at the time of urine collection had a total urine protein >150 mg/L. Aminoglycosides are recognized as having significant nephrotoxicity (1). Importantly however, some patients showed sufficient variability in results while still receiving aminoglycosides to raise the question of aminoglycoside interference.
We added six to nine concentrations of gentamicin and tobramycin (both from Pharmacia & Upjohn) individually in the interval 0.8–200 mg/L to a donor urine sample and to solutions of human albumin (100 and 2000 mg/L Albumex 4; Commonwealth Serum Laboratories) in 9 g/L saline. Protein analysis of the urine samples was performed on the Dade Behring Dimension RxL (pyrogallol red–molybdate), Cobas Fara [Orsonneau pyrogallol red–molybdate (2)], Roche Modular System [benzethonium chloride (3)], and Roche Integra 700 (pyrogallol red–molybdate). Human albumin solutions were measured on the Dade Behring Dimension RxL (pyrogallol red–molybdate).
At a protein concentration of 100 mg/L, the Dade Behring pyrogallol red–molybdate method showed an overestimate in concentration of >10% at aminoglycoside concentrations of 6.3–12.5 mg/L. Above these aminoglycoside concentrations there was a progressive increase with each aminoglycoside. The effect was similar for both urine and for the albumin solutions. Changes of 10% did not occur with the 2000 mg/L sample until the aminoglycoside concentration was >80 mg/L. The influence of the aminoglycosides on both urine and the human albumin solution was statistically significant (ANOVA, P <0.0001 for both). On the basis of a linear regression model, the aminoglycoside concentrations at which there was a significant increase in measured protein concentration in a urine containing 110 mg/L protein were 4.8 mg/L [confidence interval (CI), 1.2–9.0 mg/L] for gentamicin and 4.1 mg/L (CI, 3.6–12.7 mg/L) for tobramycin. In a solution containing 110 mg/L human albumin, the concentrations were 5.0 mg/L (CI, 2.0–10.1 mg/L) and 4.3 mg/L (CI, 3.23–5.33 mg/L), respectively. The Roche and Orsonneau total urine protein methods showed no changes in concentration with the addition of aminoglycosides.
To confirm the clinical relevance of the in vitro observations, we collected three urine samples over a 24-h period from each of three cystic fibrosis patients (patients A, B, and C) receiving tobramycin once per day. These samples were analyzed on the Dade Behring Dimension RxL for urinary protein with the Dade Behring pyrogallol red–molybdate method and for tobramycin with the Dade Behring PETINIA method (Fig. 1
).
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High concentrations of urinary tobramycin were seen over a 24-h dosing interval. There was an apparent increase in urinary protein concentration after aminoglycoside dosage. That concentration fell in proportion to the decrease in urinary tobramycin concentration. In the absence of aminoglycoside interference, over equivalent 24-h periods, samples from these patients had urinary protein concentrations in the range of 160–240 mg/L (shaded area in Fig. 1B
).
Aminoglycosides are excreted almost entirely by glomerular filtration. With plasma half-lives of 2–3 h, aminoglycoside concentrations in the urine may be >200 mg/L. Up to 25% of patients who receive aminoglycosides for more than a few days will develop mild impairment of renal concentrating ability attributable to accumulation of the drug in the proximal tubules (1). Accurate urinary protein measurement is needed to identify renal damage in these patients. In reviewing the patient data, we saw evidence of concern and confusion about the fluctuating results for urinary protein.
We conclude that aminoglycosides produce positive interference with the Dade Behring pyrogallol red–molybdate method for urinary protein estimation but not with other tested pyrogallol red–molybdate methods. Patients with essentially normal urinary protein concentrations but on aminoglycoside therapy had urinary aminoglycoside concentrations sufficiently high, even at the end of a 24-h dosing interval, to produce artifactually increased urinary protein results. The mechanism of the interference is unknown.
References
The following articles in journals at HighWire Press have cited this article:
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  B. L. West, M. M. Picken, and D. J. Leehey Albuminuria in acute tubular necrosis Nephrol. Dial. Transplant., October 1, 2006; 21(10): 2953 - 2956. [Full Text] [PDF]
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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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T. Marshall and K. M. Williams Total Protein Determination in Urine: Aminoglycoside Interference Clin. Chem., January 1, 2003; 49(1): 202 - 203. [Full Text] [PDF]
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, patient B; 
, patient C.