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Table of Contents
Year : 2018  |  Volume : 7  |  Issue : 2  |  Page : 97-98

Evaluation of an automated method for the measurement of salivary creatinine

1 Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
2 Department of Nephrology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

Date of Web Publication26-Mar-2019

Correspondence Address:
P. V L. N Srinivasa Rao
Senior Professor and Head, Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JCSR.JCSR_40_18

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How to cite this article:
Kiranmayi V S, Srinivasa Rao PL, Kumar V S. Evaluation of an automated method for the measurement of salivary creatinine. J Clin Sci Res 2018;7:97-8

How to cite this URL:
Kiranmayi V S, Srinivasa Rao PL, Kumar V S. Evaluation of an automated method for the measurement of salivary creatinine. J Clin Sci Res [serial online] 2018 [cited 2020 Feb 21];7:97-8. Available from: http://www.jcsr.co.in/text.asp?2018/7/2/97/254982

Frequent measurement of serum creatinine is used for the monitoring of renal function in patients with chronic kidney disease (CKD) which requires repeated collection of blood samples which is associated with discomfort for patients. Moreover, patients with CKD undergoing dialysis are at an increased risk of hepatitis B and C,[1] thus posing potential health risk for healthcare professionals. This has resulted in search of the diagnostic role of other body fluids. Saliva, which includes secretions from major and minor salivary glands, consists of various components of serum including enzymes, proteins, hormones, antibodies and cytokines.[2] Since recent times, saliva is being considered as an alternate biological fluid of choice as it was found to reflect the changes occurring in serum.[3] The ease and non-invasiveness of collection as well as the minimal handling required for its transportation and storage have resulted in considering saliva as a potential alternate diagnostic fluid for various clinical conditions including renal disease.[4] Although studies have demonstrated association between serum and salivary creatinine levels, the concentration of creatinine in saliva was found to be very low when compared to that in serum.[5]

Measurement of serum creatinine is based on modified Jaffe's method which includes the formation of an orange–red-coloured creatinine picrate (Janovsky) complex as a result of the reaction between creatinine in serum and picric acid in alkaline medium, provided by sodium hydroxide. The reaction sequence of modified Jaffe's reaction is as follows:[6]

Picrate + hydroxide →Picrate-OH

Picrate-OH + creatinine →Picrate-creatinine (orange–red colour).

The kinetics of the reaction follows first-order reaction.[6] Salivary creatinine level was found to be 10%–15% of that in blood.[7] The concentrations of picric acid and sodium hydroxide which were used to measure serum creatinine levels were unable to detect creatinine levels in saliva. Deep insight into the kinetics of Jaffe's reaction at various concentrations of picric acid and sodium hydroxide resulted in the observation that the specificity of the method can be improved by increasing picric acid concentration and by increasing the alkalinity of reaction, i.e. increasing the concentration of sodium hydroxide.[6] These modifications could help in detecting lower ranges of creatinine concentration. The application of this scientific knowledge to detect lower concentrations of creatinine observed in saliva was the focus of the present study. The present study intended to evaluate a modified Jaffe's rate reaction method by automation for the measurement of creatinine concentrations in saliva.

Salivary creatinine was measured using modified Jaffe's rate reaction method[7] on Beckman Synchron CX5 autoanalyzer (Beckman Synchron, Illinois, USA). The assay was performed using a picric acid concentration of 12 μmol/L and sodium hydroxide at 2.5 mol/L. When picric acid reacts with creatinine under alkaline environment of NaOH, it forms an orange–red-coloured complex. The intensity of orange–red colour was measured at 520 nm wavelength. Sample volume utilised for the present study was 25 μL. Calculation of creatinine concentration was done by endpoint rate reaction method using linear mathematical model. The method was calibrated using two-point calibration using calibrator solutions at concentrations of 6.54 μmol/L (0.07 mg/dL) and 415 μmol/L (4.69 mg/dL). The performance characteristics of the assay were checked for linearity, intraassay precision, interassay precision and recovery. Intraassay precision was performed using twenty replicates for two different concentrations at medical decision levels. Interassay precision was performed for 7 days at two different medical decision levels concentrations run in triplicates. Recovery of the study was performed using an aqueous standard creatinine solution with a concentration of 176.8 μmol/L (2.00 mg/dL) of creatinine added to saliva. The data were recorded and analysis was done using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).

The assay showed linearity between 6.54 μmol/L (0.07 mg/dL) and 418.5 μmol/L (4.73 mg/dL). The intraassay variability was observed as coefficient of variation of 8% and 7% at mean creatinine concentrations of 12.5 μmol/L (0.14 mg/dL) and 51.3 μmol/L (0.58 mg/dL), respectively. The interassay variations performed at mean creatinine concentrations of 12.5 μmol/L (0.14 mg/dL) and 51.3 μmol/L (0.58 mg/dL) showed a coefficient of variation of 12% and 5%, respectively. Recovery using aqueous solutions of creatinine was found to be 96.7% and 90.46% at creatinine concentration of 12.5 μmol/L (0.14 mg/dL) and 96.9% and 99.4% at creatinine concentration of 53.4 μmol/L (0.60 mg/dL).

Creatinine, which is the waste product of metabolism, is mainly excreted by kidneys. Studies have shown increased salivary creatinine levels in patients with CKD along with serum creatinine levels.[8] The increase in salivary creatinine levels is a result of an increase in the concentration gradient and diffusion of creatinine from serum into salivary fluid,[8] thus reflecting the changes occurring in the serum creatinine levels in patients with CKD. Several preliminary studies have evaluated the utility of saliva in various clinical conditions. In an attempt to further validate the diagnostic utility of saliva in CKD, the present study evaluated an automated method for the measurement of salivary creatinine in terms of linearity, imprecision and recovery. The method was found to be linear up to a creatinine concentration of 418.5 μmol/L (4.73 mg/dL). Lloyd et al.[7] observed a linearity for salivary creatinine estimation between 6 and 1200 μmol/L. The intra and interassay coefficient of variations in the present study were found to be comparable to those obtained by Llyod et al.,[7] although the concentrations used were different.[7] When aqueous solutions of creatinine at different concentrations were added and recovery study performed, the recovery obtained in the present study was above 90%, which was similar to that by Lloyd et al.[7] To conclude, the characteristics of the salivary creatinine assay of the present study indicate that salivary creatinine estimation can be performed on autoanalyzer with slight modifications in the concentration of the reagents used for Jaffe's method. Thus, saliva with its distinct advantages associated with collection, transport, storage and processing provides an attractive alternate to blood sample for the diagnosis and monitoring of renal disease.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Saha D, Agarwal SK. Hepatitis and HIV infection during haemodialysis. J Indian Med Assoc 2001;99:194-9, 203, 213.  Back to cited text no. 1
Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: Current state and future applications. Clin Chem 2011;57:675-87.  Back to cited text no. 2
Malamud D. Saliva as a diagnostic fluid. Dent Clin North Am 2011;55:159-78.  Back to cited text no. 3
Malathi N, Mythili S, Vasanthi HR. Salivary diagnostics: A brief review. ISRN Dent 2014;2014:158786.  Back to cited text no. 4
Yajamanam N, Vinapamula KS, Sivakumar V, Bitla AR, Rao PV. Utility of saliva as a sample to assess renal function and estimated glomerular filtration rate. Saudi J Kidney Dis Transpl 2016;27:312-9.  Back to cited text no. 5
[PUBMED]  [Full text]  
Spencer K. Analytical reviews in clinical biochemistry: The estimation of creatinine. Ann Clin Biochem 1986;23(Pt 1):1-25.  Back to cited text no. 6
Lloyd JE, Broughton A, Selby C. Salivary creatinine assays as a potential screen for renal disease. Ann Clin Biochem 1996;33(Pt 5):428-31.  Back to cited text no. 7
Venkatapathy R, Govindarajan V, Oza N, Parameswaran S, Pennagaram Dhanasekaran B, Prashad KV, et al. Salivary creatinine estimation as an alternative to serum creatinine in chronic kidney disease patients. Int J Nephrol 2014;2014:742724.  Back to cited text no. 8


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