Creatinine, complex observed at 520 nm, thus
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 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 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). 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 react on mole to mole basis, there is 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). It is well known that bilirubin is sensitive to photo-isomerisation which converts it into water soluble isomers that can be excreted by the body. The absorption of light by normal bilirubin (4Z,15Z-bilirubin) results in the creation of 2 isomeric forms of bilirubin: structural isomer (Z-lumirubin) and configurational isomer (4Z,15 E -bilirubin). Both the configurational and structural isomers of bilirubin are less lipophilic than normal bilirubin and can be excreted into bile without undergoing glucuronidation in the liver. The absorptions of light by bilirubin also results in the generation of excited-state bilirubin molecules that react with oxygen to produce colorless oxidation products, or photooxidation products. These effects are used for treatment of neonatal jaundice. Causes of hyperbilirubinemia in newborn infants. Adapted from Maisel MJ. Neonatal Jaundice. Pediatrics in Review. 2006; 27: p. 445.. If this effect of blue light exposure can be used to treat the samples and eliminate the interference caused by bilirubin in the estimation of creatinine by Jaffe’s method.
With 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, on creatinine levels estimated by Jaffe’s method respectively