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Clinical Biochemistry 46 (2013) 177–179

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Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem

Streptomycin interference in Jaffe reaction — Possible false positive creatinine

estimation in excessive dose exposure
Kirtimaan Syal a, Anand Srinivasan b, Dibyajyoti Banerjee a,⁎
a
Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India
b
Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: To study the potential of commonly used aminoglycoside antibiotics to form non-creatinine
Received 16 March 2012 chromogen with alkaline picrate reagent.
Received in revised form 5 August 2012 Design and methods: We studied the non-creatinine chromogen formation of various concentrations of strep-
Accepted 21 October 2012 tomycin, amikacin, kanamycin, netilmicin, gentamicin and tobramycin added to known creatinine concentrations
Available online 31 October 2012
by the Jaffe reaction based creatinine estimation.
Results: Only streptomycin above therapeutic concentrations of 10 mg/mL interfered in the Jaffe reaction and
Keywords:
Creatinine
acted as non-creatinine chromogen.
Streptomycin Conclusions: Therapeutic doses of the aminoglycosides do not form non-creatinine chromogens.
Aminoglycosides © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Kidney function tests
False positive reactions

1. Introduction 2. Material and methods

Aminoglycosides have long been one of the commonest causes of 2.1. Materials
drug-induced nephrotoxicity and its therapeutic administration re-
quires accurate assessment of renal function and pharmacokinetic Creatinine (Ananlytical Reagent (AR); assay: ≥99.8%), picric acid (AR;
monitoring [1]. Glomerular filtration rate (GFR) is the gold- assay: ≥99.8%) and sodium hydroxide (AR; assay: ≥98%) were pur-
standard for the measurement of renal function and serum chased from HiMedia Laboratories Pvt. Ltd, India. The aminoglycoside an-
creatinine remains the most commonly used biomarker of GFR [2]. tibiotics were purchased as injectable preparation from the local drug
The Jaffe reaction that depends on color complex formation of creat- shop. Streptomycin sulfate, amikacin and netilmicin were manufactured
inine with alkaline picrate solution forms one of the most by Cipla Limited, India. Gentamicin sulfate, tobramycin and kanamycin
commonly-used methods for the measurement of renal function. were manufactured by Biochem Pharmaceuticals Industries Ltd., India,
Many biomolecules and drugs like cephalosporin antibiotics form Solitaire Pharmacia Pvt. Ltd. and Macleods Pharmaceuticals Pvt. Ltd.,
non-creatinine chromogen with alkaline picrate reagent and pro- India respectively. The glassware was cleaned with detergent solution
duce false positive results for creatinine [3,4]. There is a paucity of thoroughly followed by chromic acid and rinsed in a flow of distilled
studies analyzing the formation of non-creatinine chromogens by water overnight before each experiment. The absorbance of colored com-
aminoglycosides. To address this knowledge gap, we performed an plex formed in Jaffe reaction was measured by spectrophotometry using
interference study of the more commonly used aminoglycoside UV–visible double beam spectrophotometer manufactured by Beckman
antibiotics gentamicin, amikacin, streptomycin, tobramycin, kana- Coulter International (517507-DU-640UV–Vis Scanning Spectrophotom-
mycin and netilmicin with Jaffe reagent to evaluate their potential eter). Double distilled water was used for the preparation of reagents
to produce false creatinine measurements. and other purposes throughout the study.

2.2. Preparation of reagents

Picric acid reagent containing 0.04 M picric acid was prepared by

⁎ Corresponding author at: Department of Experimental Medicine and Biotechnology,
Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India.
mixing 9.1 g of picric acid to 1 L of double distilled water. Alkali
Fax: +91 172 2744401. reagent containing 0.75 N NaOH was prepared by mixing 30 g of
E-mail address: dibyajyoti5200@yahoo.co.in (D. Banerjee). NaOH in 1 L of double distilled water. The alkali reagent was stored

0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.clinbiochem.2012.10.031
 Author's personal copy

178 K. Syal et al. / Clinical Biochemistry 46 (2013) 177–179

Fig. 1. a. Bland–Altman plot showing the agreement between the two creatinine concentrations before and after spiking it with 5 mg/mL of streptomycin. X-axis represents the average of
the readings before and after spiking the creatinine sample and Y-axis represents the difference of the readings before and after spiking the creatinine sample. LLA — lower limit of agree-
ment and ULA — Upper limit of agreement. Middle line represents the mean of the differences. b. Bland–Altman plot showing the agreement between the two creatinine concentrations
before and after spiking it with 10 mg/mL of streptomycin. X-axis represents the average of the readings before and after spiking the creatinine sample and Y-axis represents the differ-
ence of the readings before and after spiking the creatinine sample. LLA — lower limit of agreement and ULA — upper limit of agreement. Middle line represents the mean of the differ-
ences. c. Figure showing increasing absorbance at 515 nm (mean±SD, n=10) with increasing streptomycin concentration beyond 10 mg/mL while performing Jaffe reaction in absence
of creatinine. Streptomycin concentration is plotted in the X-axis and absorbance is plotted in the Y-axis. d. Figure showing increasing absorbance (mean±SD, n=10) at 515 nm of in-
creasing concentration of streptomycin (beyond 10 mg/mL) in presence of 6 μg/mL and 24 μg/mL of creatinine while performing Jaffe reaction. With other concentrations of creatinine
this pattern of positive interference of streptomycin are also observed. Streptomycin concentration is plotted in the X-axis and absorbance is plotted in the Y-axis.

in a plastic container. Stock solution of creatinine containing 1 mg of solution with aminoglycoside antibiotics if not mentioned otherwise.
creatinine per mL was prepared by dissolving 1 g of pure dry creati- The agreement of the data before and after spiking the creatinine solution
nine in 1 L of 0.1 N HCl. Standard creatinine (S1) solution containing with aminoglycosides was analyzed using Bland–Altman method [6]. The
0.02 mg/mL (i.e. 177 μmol/L) creatinine was prepared by diluting bias (difference in the creatinine concentration before and after spiking
2 mL of stock creatinine solution to 100 mL with double distilled the creatinine sample with the aminoglycosides) created by the
water .Standard creatinine (S2) solution containing 0.04 mg/mL aminoglycosides was correlated with their concentration with Spearman
(i.e.354 μmol/L) creatinine was prepared by diluting 4 mL of stock correlation method. The distribution of the dataset was analyzed with
creatinine solution to 100 mL with double distilled water. Likewise Kolmogorov–Smirnov (KS) test, since it was believed to generate mean-
up to S50 of standard creatinine solution was prepared which ingful results with small sample size [7] and based upon its results appro-
contained 1 mg/mL (i.e.8800 μmol/L) of creatinine. priate tests of significance were chosen.

2.3. Biological sample 3. Results

Left over serum and urine sample were collected randomly from the The standard curve of creatinine by alkaline picrate method based on
clinical laboratory and used in the study. Urine sample was diluted to Jaffe reaction was observed to be linear up to 50 μg/mL (440 μmol/L) of
1:50 with double distilled water and that was used as the test solution. creatinine at 515 nm. There was no statistically significant difference in
the absorbance between the groups of aliquots containing standard cre-
2.4. Methods atinine alone and aliquots containing different concentrations of the
aminoglycosides tested other than streptomycin along with standard
1 mL of picric acid reagent, 1 mL of alkali reagent and 100 μL of test creatinine. Fig. 1a and b shows a good agreement of the dataset before
(diluted urine or serum)/standard solution were mixed immediately and after spiking the creatinine sample with the streptomycin and simi-
followed by recording of absorbance at 515 nm in a quartz cuvette at lar agreement was observed with all the other aminoglycosides. In the
20 s and 80 s and creatinine concentration was calculated from the ab- absence of creatinine the above aminoglycosides were not observed to
sorbance difference as per published protocol [5]. The above experi- show elevated absorbance at 515 nm when incubated with the alkaline
ment was repeated in the presence of the given aminoglycosides in picrate reagent. In the presence of streptomycin up to 5 mg/mL, no sta-
various concentrations (5, 10, 20, 30, 40…200 μg/mL) and (5, 10, 20, tistically significant interference was observed in the above reaction.
30, 40…..200 mg/mL) both in presence and absence of creatinine. From 10 mg/mL onward positive interference of streptomycin was
noted in Jaffe reaction while taking the absorbance at 515 nm
2.5. Statistical analysis (Table 1a and 1b, Fig. 1a–d). The bias created by streptomycin was
strongly correlated to the concentration of streptomycin whereas other
All the values were represented as mean and standard deviation of the aminoglycosides did not show any such correlation and the bias at
difference of values measured before and after spiking the creatinine 10 mg/mL streptomycin was significantly more than at 5 mg/mL
 Author's personal copy

K. Syal et al. / Clinical Biochemistry 46 (2013) 177–179 179

Table 1a
Mean (standard deviation) of difference of the creatinine concentration (μmol/L) before and after spiking the samples with aminoglycosides. The agreement of the data before and
after spiking with aminoglycosides was analyzed with Bland–Altman plot. There was a significant difference in the bias created by streptomycin between 5 and 10 mg/mL concen-
trations while measuring the creatinine concentration whereas such a difference in the bias was not observed with the other aminoglycosides.

Creatinine Streptomycin (mg/mL) Amikacin (mg/mL) Gentamicin (mg/mL) Kanamycin (mg/mL) Netilmicin (mg/mL) Tobramycin (mg/mL)
concentration
5 10 5 10 5 10 5 10 5 10 5 10
(μmol/L)

176.8 0.5* (1.57) 43.55* (1.15) 0.55 (2.26) 2.4 (2.17) 0.95 (1.28) 0.8 (2.74) 1.6 (2.06) 0.7 (3.13) 2.2 (2.73) 1.5 (1.27) 0.75 (1.92) 1.6 (2.5)
353.6 6.95# (2.46) 53.35# (1.9) 4.85 (2.43) 2.60 (3.41) 1 (1.59) 0.5 (2.27) 3.8 (1.61) 3.3 (2.41) 3.35 (2.15) 3.6 (2.84) 3.7 (3.82) 3.1 (3.37)

*,# p b 0.001 (Mann–Whitney test after assessing the distribution of the dataset with KS test). In other cases the p value is not less than 0.05.

Table 1b 5. Conclusion
Correlation between the bias and the aminoglycoside concentration in 353.6 μmol/L cre-
atinine. With increasing concentration of streptomycin the bias in the creatinine measure-
ment created by the streptomycin correlated well with the concentration of streptomycin
Streptomycin in toxic concentration above 10 mg/mL can act as
whereas such a significant correlation was not observed with the other aminoglycosides. non-creatinine chromogen while performing Jaffe reaction based creat-
inine estimation. But gentamicin, amikacin, kanamycin, netilmycin and
Aminoglycoside Correlation coefficient p
tobramycin do not interfere in creatinine estimation by Jaffe method
Streptomycin 0.93 b0.001 due to formation of non-creatinine chromogen as a result of interaction
Amikacin 0.23 0.09
of the aminoglycosides with the alkaline picrate reagent. Hence in acci-
Gentamicin 0.04 0.88
Kanamycin 0.19 0.12 dental overdose of streptomycin appropriate method for creatinine es-
Netilmicin 0.18 0.47 timation in clinical biochemistry laboratory should be worked out
Tobramycin 0.09 0.68 although this level in biological samples is not possible in the usual
doses given to the patients.

Acknowledgements
concentration which was not observed in the case of other
DB acknowledges PGIMER, Chandigarh for financial assistance
aminoglycosides (Table 1a and 1b). When we added the aminoglycoside
under New Institute Research Scheme (No. 71/3-Edu/10/765 dated
antibiotics in the biological samples the measured creatinine concentra-
16.6.2011).
tions before and after such addition corroborated with the above findings
(data not shown).
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