Creatinine, the estimation of creatinine by Jaffe’s

Creatinine, a creatine phosphate metabolism product, is a waste product excreted through urine. Since, dependingon muscle mass, sex, diet, exercise and age; creatinine is produced at a fairly constant rate by thebody, hence, its measurement is commonly used to assess the GFR (1). Variousmethods often employed for the estimation of creatinine are: Jaffe’s method, Enzymaticmethod, High performance liquid chromatography, Gas-chromatography with massspectrometry and Isotope-dilution mass spectrometry (IDMS) (2,3).

Although the IDMSmethod is considered to be the gold standard for creatinine estimation, howeverbecause of its cost and cumbersome nature, it cannot be routinely used inClinical Biochemistry labs (4). Due to its simplicity and low cost of thereagents involved in the  assay, theJaffe’s method, with or without modification, even today remains the mostwidely used method for creatinine estimation in various clinical laboratoriesworld-wide (2,5,6). However, this being a non-enzymatic estimation, is subjectto interference by various small molecular weight substances such as glucose,pyruvate, acetoacetate, bilirubin, foetalhaemoglobin (HbF) and drugs likecefoxitin etc. The presence of glucose, bilirubin and HbF in test samples areknown to cause negative interference while acetoacetate, ascorbic acid orcefoxitin (a first generation cephalosporin) have been shown to cause positiveinterference in creatinine estimation by the Jaffe’s method. (7,8,9).Bilirubin, a product of heme catabolism becomes a significant interferant forcreatinine estimation in patients suffering from jaundice especially thepediatric patients. Studies have shown that bilirubin at its low and highconcentrations causes negative and positive interference respectively, in theestimation of creatinine by Jaffe’s method. In Jaffe’s method, bilirubin getsconverted to biliverdin under alkaline conditions.

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Biliverdin thus formed has?max at 630 nm which significantly decreases the absorbance of thecreatinine–picrate complex observed at 520 nm, thus resulting in negativeinterference at its lower concentrations (10, 11). Since, during in anychemical reaction, substrates and chromogen react on mole to mole basis, thereis always a specific upper limit for the substrate where it obeys Beer’s Law.As the absorption maxima (?max) of bilirubin (510 nm) almost coincides withthat of creatinine-picrate complex of 520 nm, hence, at higher concentrationsof serum bilirubin, where the concentration of either NaOH and/or picratebecomes a limiting factor, the presence of unreached / free bilirubin willresult in positive interference by it in creatinine estimation by the Jaffe’smethod (12). It is well known that bilirubin is sensitive to lightmediated isomerisation known as phototherapy in therapeutic settings whichconverts it into water soluble isomers that can be excreted by the body. Theabsorption of light by normal bilirubin (4Z,15Z-bilirubin) results in thecreation of 2 isomeric forms of bilirubin: structural isomer (Z-lumirubin) andconfigurational isomer (4Z,15 E -bilirubin).Both these structural and configurational isomers of bilirubin have substantiallydifferent chemical and light absorption properties than bilirubin, are lesslipophilic than normal bilirubin and can be excreted into bile withoutundergoing glucuronidation in the liver.

The absorption of light by bilirubinalso results in the generation of excited-state bilirubin molecules that reactwith oxygen to produce colorless oxidation, or photooxidation products. Therate of formation of bilirubin photoproducts is highly dependent on theintensity and wavelengths of the light used (13). The most efficient wavelengthfor the isomerization of bilirubin is approximately 450 nm, whether applied tothe fluid samples for testing or the treatment of jaundice. Wavelengths thatfall within the range of 400 nm-500 nm, and more specifically 445 nm-475 nm areknown to effect isomerization (14). The blue lights are chosen for lightemission wavelengths of approximately 450 to 530 nm, which is the optimal rangeof light absorption for bilirubin.

In contrast, the optimal range of lightabsorption for the isomer lumirubin is around 315 nm (15).Based upon the above information available in literature, thequestion that naturally arises is if by converting bilirubin to products whichdo not have the absorption maxima in the range used for the estimation ofcreatinine by the Jaffe’s method, can interference caused by bilirubin increatinine estimation by Jaffe’s method be eliminated? The above speculate formedthe basis of the following objective of the present study: