References nm, thus causing negative interference at its lower

References ID- 2018-00028Title- Effect of photolytic degradation of Bilirubin on the creatinine estimation by Jaffe’s method Introduction- Creatinine, a breakdown product of creatine phosphate is a waste product excreted through urine.

Its produced at a fairly constant rate by the body depending on muscle mass, age, sex, diet and exercise and hence measurement of creatinine is commonly used to assess the GFR (1) Estimation of creatinine level can be done using various methods like alkaline picrate (Jaffe’s) method, high performance liquid chromatography, gas-chromatography with mass spectrometry (2) and enzymatic method using creatinine deaminase (3). Isotope-dilution mass spectrometry (IDMS) method being the gold standard, for creatinine assay, is both expensive and cumbersome for routine use in Clinical Biochemistry labs (4) The kinetic alkaline picrate (Jaffe’s) method, with or without modification, even today remains the most widely used method for creatinine estimation in both blood and serum in various clinical laboratories world-wide mainly due of the easy availability and the low cost of the reagents required for the test besides the simplicity of performing the test making it suitable for all kinds of lab settings . (2,5,6). This being a non-enzymatic estimation, is subject to interference by various small molecular weight substances such as glucose, pyruvate, acetoacetate, bilirubin, foetal haemoglobin (HbF) and drugs like cefoxitin etc. The presence of glucose, bilirubin and HbF in test samples causes negative interference and acetoacetate, ascorbic acid or cefoxitin (a first generation cephalosporin) result in positive interference in creatinine estimation by the Jaffe’s method. (7,8,9).

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Bilirubin, a product of heme catabolism becomes a significant interferant for creatinine estimation in patients suffering from jaundice especially the pediatric patients. Studies have shown that bilirubin can cause negative and positive interference at its low and high concentration respectively, in the estimation of creatinine by Jaffe’s method. In Jaffe’s method, bilirubin gets converted to biliverdin under alkaline conditions. Biliverdin thus formed has ?max at 630 nm which significantly decreases the absorbance of the creatinine–picrate complex observed at 520 nm, thus causing negative interference at its lower concentration (10,11). Since, substrates and chromogen in the two methods react on mole to mole basis, each method has a specific upper limit for the substrate where it obeys Beer’s Law. As the ?max of bilirubin absorbance (510 nm) almost coincides with that of creatinine-picrate complex i.e.

520 nm, hence, at higher concentrations of serum bilirubin, the concentration of either NaOH and/or picrate may become a limiting factor resulting into the positive interference by unreacted bilirubin in the Jaffe’s method (12)The  total bilirubin and direct bilirubin levels decrease in the values were because of the substantial changes in the bile pigment composition of the sample because of photoisomerization reactions, which is an unimolecular processes that occurs many orders of magnitude faster. (5) Nadja N. Rehak et al have shown in their the measured values of total bilirubin in normobilirubinemic specimens decreased by an average of 59% and that for moderately hyperbilirubinemic specimens decreased by 41%, the direct bilirubin values decreased by 38% for normobilirubinemic and decreased by 31% for moderately hyperbilirubinemic specimens, when these samples were exposed to fluorescent lighting at ambient temperature approximately 6 feet below a light fixture for 9 hours.

(6) The wavelength as well as the intensity of light is an important factor in photoisomerisation of bilirubin, although most of the available data is for the in vivo clearance of bilirubin in patients exposed to phototherapy rather than in vitro stability of serum specimens exposed to light. (7) Photoisomerisation of bilirubin is enhanced by exposure to shorter wavelengths of light, near bilirubin absorbance maximum of about 455nm, at the blue end of the visible spectrum. Tan reported that the decline of bilirubin is most rapid with special blue lights and duration of exposure is also less, when compared to fluorescent day light lampsWith this background the question naturally arises that if the bilirubin can be converted to a product which does not have the absorption maxima in the range used for estimation of creatinine by Jaffe’s method, can interference caused by bilirubin in creatinine estimation by Jaffe’s method be eliminated? The present study aims to study the effect of photolytic conversion of serum bilirubin to its isoforms by exposing the serum samples to special blue light of 440nm for six hours on serum bilirubin (Total and Direct) and creatinine levels estimated by timed endpoint diazo method and Jaffe’s method respectively.  Objectives- 1.      To conduct a comparative study on the estimation of serum creatinine levels by Jaffe’s method at different levels of serum bilirubin before and after exposure to blue light for 6 hours. 2.

      To study the usefulness of photolytic conversion of bilirubin for the elimination of bilirubin interference in the estimation of creatinine by Jaffe’s methodMethodology- The study will be conducted in Department of Biochemistry in MCI recognized Hospital located in the of Uttarakhand, over a period of 2 months. The study group would comprise of samples received in the Clinical Biochemistry section of the hospital from patients visiting outpatient and inpatient departments, fulfilling the inclusion and exclusion criterion after obtaining approval from the institutional Ethics committee. Study Design: Selection of Subject:      Inclusion Criteria      Exclusion Criteria                                                                                        Type of the study:  Prospective interventional    Study Protocol: All serum samples were the leftover aliquots of blood draw collected from patients in serum tubes that were submitted to the laboratory for routine chemistry analysis was collected. The specimens were used as anonymous samples irrespective of factors like age, sex, medication, pathological problems etc. The samples were analysed for the estimation of total bilirubin, direct bilirubin, creatinineAfter the estimation, the samples were transferred into glass tubes and were subjected to photolysis under the blue light at 440nm for 6hrs duration for photolysis.Methods·       Creatinine Estimation: Jaffe’s method will be used for creatinine estimation in all the serum samples.

o   Jaffe’s Method: The creatinine concentration will be estimated by the rate modified Jaffe’s reaction () using Beckman Coulter, Synchron DXC System. Principle:  Under alkaline conditions, picrate reacts with the creatinine in the sera to form a creatinine-picrate complex having significantly increased absorbance at 520nm, this increase in absorbance at 520nm is directly proportional to the serum creatinine concentration. The SYNCHRON DXC system automatically maintains the ratio of 1:11 between the sample and the reagent.

Reaction Scheme: Creatinine + Picric acid                                                         Creatinine picrate (Red colored         complex)·       Total Bilirubin Estimation: The total bilirubin estimation in the samples is done by the timed endpoint diazo method () using Beckman Coulter, Synchron DXC SystemPrinciple:  Study Tools: Structured Study formats and subject proformas will be developed, and used to generate data.Data Management & Statistical AnalysisInterpretation and statistical analysis will be done by using SPSS 22 version.    Implication HbA1c is widely used as an important marker of glycemic control, and it is of great importance to exclude factors which could spuriously elevate its levels. The present study would help clinicians assess the glycemic control of their patients better in light of their iron deficient status which in turn would help them decide the course of treatment of their patient.Describes all the procedures that will be used to achieve the objectives and justify the study design including any techniques and procedures to be used. This may include: type of study and study design, study population, sample size and selection criteria, Proposed intervention (if applicable), Data collection procedures & instruments used, quality control, confidentiality, plan of analysis/ statistical tools, ethical considerations with all required forms.  Implications- Describes what is expected to be achieved or gained from the proposed research.

This could be given in terms of knowledge gained by the student or in terms of scientific advancement.  References- Provide appropriate references from recently published journals/ literature supporting the proposed research. Sumitra Chauhan, Neeta Malukar, Modification of Jaffe’s Kinetic Method for Serum Creatinine Estimation to Decrease Bilirubin Interference .Int J Res Med Sci 2017 ; 6(1); 35-37 Vaishya, R., Arora, S., Singh, B. et al.

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Pediatric Research, vol. 65, no. 1, 2009, pp. 113–116. Toora BD, and Rajagopal G. Measurement of Creatinine by Jaffe’s Reaction–Determination of Concentration of Sodium Hydroxide Required for Maximum Color Development in Standard, Urine and Protein Free Filtrate of Serum.

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A critical evaluation and review. Clin Chem. 1980;26:555–61.PubMed Pardue HL, Bacon BL, Groeger Nevius M, Skoug JW. Kinetic study of the Jaffe reaction for quantifying creatinine in serum:1. Alkalinity controlled with NaOH.

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Sandra Boot, Nicholas LaRoche and Edward F Legg Elimination of bilirubin interference in creatinine assays by routine techniques: comparisons with a high performance liquid chromatography method Ann Clin Biochem 1994; 31: 262-266 Abraham Sam Rajan PM. Elimination of bilirubin interference by photolysis in the analysis of creatinine, glucose and alkaline phosphatase. International Journal of Clinical Biochemistry and Research. 2017;4(1):1-5 Kakkar M, Kakkar R. A comparative study on the estimation of serum Creatinine levels by Jaffe’s and Enzymatic methods at different levels of serum Bilirubin.

International Journal of Clinical Biochemistry and Research. July-September 2017;4(3):305-308