References used in Clinical Biochemistry labs (4). Due

References ID- 2018-00028

Effect
of photoisomerisation on the serum concentration of bilirubin and its ability to
interfere in creatinine estimation by the Jaffe’s method.   

Introduction

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 generated at a fairly constant rate by the body, hence, its measurement
is usually 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 have been shown to
cause positive interference in creatinine estimation by the Jaffe’s method. (7,8,9).
Bilirubin, a product of heme breakdown 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. Intensity
and wavelengths of the light used are the major factors on which the rate of
formation of bilirubin photoproducts depends (13). Light wavelengths within the
spectrum of 400 nm-500 nm, and more specifically 445 nm-475 nm are known to
effect conversion. The wavelength of nearly 450 nm is considered best for the
transition of bilirubin, whether applied to the fluid samples for testing or
the treatment of jaundice. (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 comparison, the isomer lumirubin
absorbs maximum light around 315 nm (15).

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:

Objectives

1.    
To compare serum
creatinine levels (by Jaffe’s method) before and after exposure to blue light,
at different levels of serum bilirubin.

2.    
To study the significance
of photoisomerisation of bilirubin on its ability to interfere in creatinine estimation by Jaffe’s method

Methodology

After obtaining approval from the Institutional Ethics committee,
the study will be conducted in an MCI recognized teaching hospital over a
period of 3 months.

Study Design: Interventional (experimental)

Type of the study:  Prospective

Study Population: The study would be conducted on serum samples, received from
patients visiting outpatient and inpatient departments, in the Clinical Biochemistry
section of the hospital for the estimation of serum creatinine and bilirubin
(total and direct).

Sample Size: Minimum of 100 serum samples would be analyzed during the study.

Inclusion
criteria:
The serum samples received in the laboratory for serum creatinine and bilirubin
(total and direct) estimation having serum creatinine levels within analytical measurement range
(0.2 -25 mg/dl) and bilirubin different concentrations shall be included in the
study

Samples from patients having normal
plasma glucose levels and ketone bodies negative in urine samples.

Exclusion
criteria:
The serum samples with serum creatinine greater than the analytical measurement range

Samples from diabetic patients i.e. high plasma glucose and ketone
bodies positive in urine

Methods

All the tests will be done on an auto-analyzer at an NABL
accredited reference laboratory attached to the hospital. Quality controls for
the tests will be run and maintained as per the SOPs.

·      
Creatinine Estimation:

The creatinine
concentration will be estimated by the rate modified Jaffe’s reaction. Under
alkaline conditions, picrate reacts with the creatinine in the sera to form a
creatinine-picrate complex having significantly increased absorbance at 520nm,
this increase in absorbance at 520nm is directly proportional to the serum creatinine
concentration (16).

Alkaline Medium

Reaction Scheme:

Creatinine + Picric acid                                                                      Creatinine
picrate

(Red
colored         complex)

·        
Total Bilirubin Estimation: The total bilirubin estimation in the samples will be done
by the timed endpoint diazo method. Bilirubin reacts with diazo in the presence
of caffeine, benzoate and acetate as accelerators yielding azobilirubin having
absorption maxima at

Caffeine, Benzoate, Acetate

520nm (17).

       Total Bilirubin +
Diazo + H+                                                              
Azobilirubin (blue color)  

 

·      
Direct Bilirubin Estimation: The direct bilirubin estimation in the samples will be done
by the timed endpoint diazo method. Bilirubin reacts with diazo yielding azobilirubin
having absorption maxima at 560 nm).

Direct Bilirubin +
Diazo + H+                                                          Azobilirubin
(blue color)                                                                                   

 

Study
Protocol:

·      
Serum samples that
are the leftover aliquots of blood, collected from patients visiting outpatient
and inpatient departments and received in the laboratory for serum creatinine
and bilirubin (total and direct), fulfilling the inclusion and exclusion criteria
will be used for the study.

·      
The serum samples
thus selected will be coded to maintain anonymity of the patients and to
eliminate any associated bias.

·       The sample vacutainers will then be exposed to the blue light (light intensity of 410-490 nm wavelength
range) for a period of 1 to 60
minutes per milliliter of fluid (14).

·      
After exposure to
blue light for the requisite time the sample vacutainers will be again analysed
for serum creatinine and bilirubin (total and direct) levels.

Data Management & Statistical
Analysis: For statistical analysis
SPSS version 22.0 will be employed.

Study Tools: Data
collected from patients’ test reports will be collected on SPSS spread sheet in
the form of creatinine levels at different concentrations of bilirubin before
and after exposure to blue light.

Implications

As
bilirubin is known to show negative interference at its low concentration and
positive interference at its higher concentration, in the creatinine estimation
by Jaffe’s method, the elimination of this interference with the exposure of
the serum samples to blue light can be of great help in preventing the
influence of bilirubin in the estimation of creatinine thus providing more
accurate test results.

The present study
assumes clinical significance in the management of Liver /kidney ailments.

 

References

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2.    
Vaishya, R., Arora, S., Singh, B. et al.Modification of Jaffe’s
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4.    
Srivastava, Tarak, et al. Impact of Standardization of Creatinine
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Bowers LD, Wong ET. Kinetic serum creatinine assays. II. A
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8.    
Pardue HL, Bacon BL, GroegerNevius M, Skoug JW. Kinetic study of
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9.    
Spencer K. Analytical reviews in clinical biochemistry: the
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10.  Sandra Boot, Nicholas LaRoche and
Edward F Legg Elimination of bilirubin interference in creatinine assays by
routine techniques: comparisons with a high performance liquid chromatography
method Ann ClinBiochem 1994; 31: 262-266

11.  Abraham Sam Rajan PM. Elimination of
bilirubin interference by photolysis in the analysis of creatinine, glucose and
alkaline phosphatase. International Journal of Clinical Biochemistry and
Research. 2017;4(1):1-5

12.  Kakkar
M, Kakkar R. A comparative
study on the estimation of serum Creatinine levels by Jaffe’s and Enzymatic
methods at different levels of serum Bilirubin. International Journal of Clinical
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13.  Maisel
MJ, McDonagh AD. Phototherapy for Neonatal Jaundice. N Engl J Med. 2008; 358: 920-928

14.  Mane V, Wong E. Device and method for bilirubin photoisomerization to reduce laboratory
test interference.US; US 9541482 B2, 10th Jan 2017

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