Creatinine, is a waste outcome of phosphocreatine metabolism which is excreted through urine. Since it depends on determinants likemuscle mass, sex, diet, exercise and age, creatinine is produced at a fairly constant rate by the body,hence, its measurement is commonly employed 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. 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 a conventional and widely accepted concept that that bilirubincan easily be isomerized on light exposure. The therapeutic setting, known asphototherapy, converts bilirubin into its hydrophilic isomers that can beexcreted by the body. The normal bilirubin (4Z,15Z-bilirubin) absorbs light to form two isomersof bilirubin: configurational isomer (4Z,15 E -bilirubin)and structural isomer (Z-lumirubin). Both these isomers of bilirubin (configurationaland structural) have notable contrast in chemical and light absorptionproperties than bilirubin. They are comparatively more hydrophilic than normalbilirubin and can be easily excreted into bile without undergoing anyconjugations like glucuronidation in the liver. Also, the phenomena of lightabsorption by bilirubin also leads to origination of an excited-state bilirubin molecules thatreact with oxygen to produce photooxidation products which are colorless.
The rate of formation of bilirubin photoproducts is highlydependent on the intensity and wavelengths of the light used (13). The mostefficient wavelength for the isomerization of bilirubin is approximately 450nm, whether applied to the fluid samples for testing or the treatment ofjaundice. Wavelengths that fall within the range of 400 nm-500 nm, and morespecifically 445 nm-475 nm are known to effect isomerization (14). The bluelights are chosen for light emission wavelengths of approximately 450 to 530nm, which is the optimal range of light absorption for bilirubin. In contrast,the optimal range of light absorption 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: