Creatinine, substances such as glucose, pyruvate, acetoacetate, bilirubin, foetalhaemoglobin

Creatinine, is a waste outcome of phosphocreatine metabolism which is excreted through urine. Since it depends on determinants like
muscle 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). Various
methods often employed for the estimation of creatinine are: Jaffe’s method, Enzymatic
method, High performance liquid chromatography, Gas-chromatography with mass
spectrometry and Isotope-dilution mass spectrometry (IDMS) (2,3). Although the IDMS
method is considered to be the gold standard for creatinine estimation, however
because of its cost and cumbersome nature, it cannot be routinely used in
Clinical Biochemistry labs (4). Due to its simplicity and low cost of the
reagents involved in the  assay, the
Jaffe’s method, with or without modification, even today remains the most
widely used method for creatinine estimation in various clinical laboratories
world-wide (2,5,6). However, this being a non-enzymatic estimation, is subject
to interference by various small molecular weight substances such as glucose,
pyruvate, acetoacetate, bilirubin, foetalhaemoglobin (HbF) and drugs like
cefoxitin etc. The presence of glucose, bilirubin and HbF in test samples are
known to cause negative interference while acetoacetate, ascorbic acid or
cefoxitin (a first generation cephalosporin) have been shown to cause 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 at its low and high
concentrations causes negative and positive interference 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 resulting in negative
interference at its lower concentrations (10, 11). Since, during in any
chemical reaction, substrates and chromogen react on mole to mole basis, there
is 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 with
that of creatinine-picrate complex of 520 nm, hence, at higher concentrations
of serum bilirubin, where the concentration of either NaOH and/or picrate
becomes a limiting factor, the presence of unreached / free bilirubin will
result in positive interference by it in creatinine estimation by the Jaffe’s
method (12).

It is a conventional and widely accepted concept that that bilirubin
can easily be isomerized on light exposure. The therapeutic setting, known as
phototherapy, converts bilirubin into its hydrophilic isomers that can be
excreted by the body. The normal bilirubin (4Z,15Z-bilirubin) absorbs light to form two isomers
of bilirubin: configurational isomer (4Z,15 E -bilirubin)
and structural isomer (Z-lumirubin). Both these isomers of bilirubin (configurational
and structural) have notable contrast in chemical and light absorption
properties than bilirubin. They are comparatively more hydrophilic than normal
bilirubin and can be easily excreted into bile without undergoing any
conjugations like glucuronidation in the liver. Also, the phenomena of light
absorption by bilirubin also leads to origination of an excited-state bilirubin molecules that
react with oxygen to produce photooxidation products which are colorless. The rate of formation of bilirubin photoproducts is highly
dependent on the intensity and wavelengths of the light used (13). The most
efficient wavelength for the isomerization of bilirubin is approximately 450
nm, whether applied to the fluid samples for testing or the treatment of
jaundice. Wavelengths that fall within the range of 400 nm-500 nm, and more
specifically 445 nm-475 nm are known to effect isomerization (14). The blue
lights are chosen for light emission wavelengths of approximately 450 to 530
nm, 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

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Based upon the above information available in literature, the
question that naturally arises is if by converting bilirubin to products which
do not have the absorption maxima in the range used for the estimation of
creatinine by the Jaffe’s method, can interference caused by bilirubin in
creatinine estimation by Jaffe’s method be eliminated? The above speculate formed
the basis of the following objective of the present study: