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Aminoglycoside interference in the pyrogallol red-molybdate protein assay is increased by the addition of sodium dodecyl sulfate to the dye reagent.

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 [gamma]-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 [micro]L of sample (protein calibrator, aminoglycoside, or urine control with or without aminoglycoside), adjusted to 20 [micro]L 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).

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 [greater than or equal to] 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.

DOI: 10.1373/clinchem.2003.025254

References

(1.) Fujita Y, Mori I, Kitano S. Color reaction between Pyrogallol red-molybdenum(VI) complex and protein. Bunseki Kagaku 1983;32:E379-86.

(2.) Watanabe N, Kamel S, Ohkubo A, Yamanaka M, Ohsawa S, Makino K, et al. Urinary protein as measured with a pyrogallol-red-molybdate complex manually and in a Hitachi 726 automated analyzer. Clin Chem 1986;32:1551-4.

(3.) Orsonneau JL, Douet P, Massoubre C, Lustenberger P, Bernard S. An improved pyrogallol red-molybdate method for determining total urinary protein. Clin Chem 1989;35:2233-5.

(4.) Koerbin G, Taylor L, Dutton J, Marshall K, Low P, Potter JM. Aminoglycoside interference with the Dade Behring Pyrogallol red-molybdate method for the measurement of total urine protein [Letter]. Clin Chem 2001;47:2183-4.

(5.) Marshall T, Williams KM. Total protein determination in urine: aminoglycoside interference [Letter]. Clin Chem 2003;49:202-3.

(6.) Macart M, Gerbaut L. An improvement of the Coomassie Blue dye binding method allowing an equal sensitivity to various proteins: application to cerebrospinal fluid. Clin Chim Acta 1982;122: 93-101.

(7.) Parfitt K, ed. Martindale: the complete drug reference, 32nd ed. Taunton: Pharmaceutical Press, 1999:112-270.

(8.) Dollery C, ed. Therapeutic drugs, Vol. 1. London: Churchill Livingstone, 1991:A1-1131.

Thomas Marshall *

Katherine M. Williams

Analytical Biochemistry Group

Sunderland Pharmacy School

The University of Sunderland

Sunderland SR1 3RG, UK

* Author for correspondence. Fax 44191-515-3747; e-mail tom.marshall@sunderland.ac.uk.
Table 1. Effect of SDS on aminoglycoside interference in the PRM
assay as indicated by absorbance at 600 nm. (a)

 SDS, mg/L

 0 25 50 100
Aminoglycoside

 Neomycin (2 g/L) 0.305 0.350 0.540 0.640
 Gentamicin (5 g/L) 0.318 0.367 0.580 0.837
 Tobramycin (10 g/L) 0.292 0.342 0.575 0.890
 Paromomycin (10 g/L) 0.140 0.172 0.275 0.523
 Kanamycin (10 g/L) 0.060 0.062 0.068 0.295
 Geneticin (10 g/L) 0.033 0.045 0.051 0.286
 Streptomycin (10 g/L) 0.030 0.031 0.032 0.030
 Amikacin (10 g/L) 0.020 0.023 0.021 0.023
 Dihydrostreptomycin (10 g/L) 0.020 0.024 0.023 0.024

Protein calibrator

 BSA (2 g/L) 0.712 0.450 0.290 0.152
 BGG (2 g/L) 0.393 0.350 0.240 0.063

(a) The values correspond to the mean absorbance ([A.sub.600 nm])
obtained in the assay for 20 [micro]L of sample (at the concentration
indicated) with 1 mL of dye reagent (n = 4; CV <8%).
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Title Annotation:Letters
Author:Marshall, Thomas; Williams, Katherine M.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Dec 1, 2003
Words:1145
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