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 breakdown becomes a significant interferant for creatinine estimationin patients suffering from jaundice especially the pediatric patients.
Studieshave shown that bilirubin at its low and high concentrations causes negativeand positive interference respectively, in the estimation of creatinine byJaffe’s method. In Jaffe’s method, bilirubin gets converted to biliverdin underalkaline conditions. Biliverdin thus formed has ?max at 630 nm whichsignificantly decreases the absorbance of the creatinine–picrate complexobserved at 520 nm, thus resulting in negative interference at its lowerconcentrations (10, 11). Since, during in any chemical reaction, substrates andchromogen react on mole to mole basis, there is always a specific upper limitfor the substrate where it obeys Beer’s Law.
As the absorption maxima (?max) ofbilirubin (510 nm) almost coincides with that of creatinine-picrate complex of520 nm, hence, at higher concentrations of serum bilirubin, where theconcentration of either NaOH and/or picrate becomes a limiting factor, thepresence of unreached / free bilirubin will result in positive interference by itin creatinine estimation by the Jaffe’s method (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). Light wavelengths within the spectrum of 400 nm-500 nm, and morespecifically 445 nm-475 nm are known to effect conversion. The wavelength of nearly450 nm is considered best for the transition of bilirubin, whether appliedto the fluid samples for testing or the treatment of jaundice. (14). The blue lights are chosen for light emission wavelengths ofapproximately 450 to 530 nm, which is the optimal range of light absorption forbilirubin.
In comparison, the isomer lumirubin absorbs maximum light around 315nm (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: