Alternative Receptacle for Bilirubin Transport

Frances Colline T. Jaranilla, Agila L. Alivia, Jane Pauline F. Coralde, John Gambit B. Garcia,
Maria Cia Cirelle L. Panol, Nathaniel E. Solis, & Cesar D. Turiano Jr.
University of Santo Tomas, Faculty of Pharmacy, Department of Medical Technology

ABSTRACT
Background: Bilirubin, a photosensitive yellow breakdown product of normal heme catabolism caused by
the bodys clearance of aged red blood cells, encounters various problems when determining its amount.
Bilirubin undergoes both isomerization and oxidation in serum when exposed to visible light. This results
to as much as 30% to 50% decrease per hour in bilirubin values.
Objective: The objective of this study is to compare the different receptacle glasses on how the two
manufactured receptacles efficiently prevent light from interfering with the bilirubin levels.
Method: The researchers pooled various serum samples. The serum was then distributed into the four
components namely positive control, negative control, polarized glass and one-way mirror then was
exposed to a constant intensity of light at various time intervals. Bilirubin levels were measured using the
Jendrassik-Grof method.
Results: No statistical significant change (p-value = 0.05) was found within the one-way mirror and
polarized glass receptacles. The most significant effect of light on the alternative receptacles was on the
direct bilirubin level at (p=0.059) for the polarized glass and at (p=0.246) for the one-way mirror.
Conclusion: The researchers concluded that the higher p-value of the one-way mirror, as compared to the
lowest p-value of the polarized glass, indicates that the one-way mirror acts as a more protective
alternative receptacle against photolysis.
Keywords: Polarized glass, One-way mirror, Alternative, Receptacle, Light, Photolysis, Bilirubiin

1 INTRODUCTION
Bilirubin is a bile pigment that is the
major heme waste product from the destruction
of red blood cells. It is produced primarily in the
liver but is also produced in small amounts by the
bone marrow and spleen. This photosensitive
bile pigment may be in two forms Direct
(conjugated or B2) and Indirect (unconjugated or
B1) bilirubin. Unconjugated bilirubin is insoluble
in water, and once released, it will bind to plasma
albumin with high affinity. The albumin-bound
bilirubin is transported to the hepatocytes,
where it is mono- (15%) or di-esterified (~85%)
with glucuronic acid. The resulted conjugated
bilirubin is water-soluble and secreted through
the biliary system.

Bilirubin and its components (direct and

indirect) are measured by determining the Total
Bilirubin and Direct Bilirubin. Indirect bilirubin is
obtained by subtracting the direct bilirubin from
the total bilirubin. The most commonly used test
in determining Bilirubin is the Jendrassik-Grof
method, which involves the use of diazotized
sulfanilic acid. It has been recommended as the
procedure of choice for total Bilirubin estimation
by the U.S. National Committee for Clinical
Laboratory Standards. This Candidate Reference
Method for total Bilirubin was further developed
and validated by the Committee on Standards of
the American Association for Clinical Chemistry
and is now being used worldwide. Other
methods for bilirubin determination include the

Malloy-Evelyn, an enzymatic assay method. One

of the factors that affect the accuracy of the test
is the stability of bilirubin in blood samples.
Increased serum bilirubin levels may
indicate liver diseases such as cirrhosis, hepatitis,
and biliary stones. In addition, it may signify
hemolytic anemias and other diseases. When
bilirubin concentration increases, the yelloworange pigment accumulates in the skin and
sclera, thus causing jaundice. The determination
of bilirubin concentrations has been used to
differentiate the types of jaundice Pre-hepatic,
Hepatic, and Post-hepatic. Pre-hepatic jaundice
is determined when there is an increase in
indirect bilirubin. Hepatic jaundice occurs when
there is an abnormality in the take up,
conjugation, and/or secretion of bilirubin. Lastly,
Post-hepatic jaundice is determined when the
direct bilirubin increased significantly.
1.1 Background of the study
Various problems may be encountered
when determining the amount of bilirubin in the
serum of patients, with the most common one
concerning exposure to light. Bilirubin
undergoes both isomerization and oxidation in
serum exposed to visible lights. This results to
decreased bilirubin values (Rehak, 2008). Upon
exposure to light, bilirubin concentrations may
decrease by 30% to 50% per hour (Bishop, et al.,
2010).
Several studies have been published
about the effects of the different factors of
serum bilirubin level, such as distance, time,
temperature, and intensities of light. According
to Bansil, et. al. (2015), there was a significant
effect on the serum bilirubin levels of icteric
samples after an hour, two hours, and three
hours of light exposure, respectively.
Meanwhile, Zhu, Sofronescu, and Loebs (2012)
determined the effects of temperature and
artificial light to bilirubin. Their study showed
that bilirubin remains stable without light
exposure for at least 24 hours at 3 C (fridge
temperature) and 22 C (room temperature),
respectively. When there is a delay of up to

eight hours in the measurement of bilirubin left

unprotected from light at room temperature,
the result is not affected and not clinically
significant. Another study by Buan et al. (2014),
entitled The effects of various distances of light
source to serum samples on bilirubin levels at
different time intervals, a significant decrease
was observed in total and direct bilirubin after
three (3) and 24 hours, and in total bilirubin at
different distances or pedestals. It was also
observed previously that total and indirect
bilirubin levels of covered samples were higher
compared to that of the uncovered ones.
In this study, the researchers will
conduct a comparative analysis of the alternative
bilirubin transport mediums using the gathered
and proven facts from previous researches. The
researchers will determine if polarized glass and
one-way mirror can be an effective alternative
transport medium for bilirubin.

1.2. Objectives of the Study

1.2.1. General Objective
The general objective of this study is to compare
the different transparent tubes on how the two
created tubes efficiently prevent light from
interfering with the bilirubin levels.
1.2.2 Specific Objective/s
To create a glass using polarized glass and a oneway mirror that could act as an alternative
transport receptacle for bilirubin.
To determine the bilirubin level prior to using the
alternative transport receptacles
To determine the pre- and post-bilirubin levels of
every sample used.
To determine the difference of bilirubin levels
among the samples based on the duration of
light exposure.

1.3. Significance of the Study

The tube to be created in this study will
have several contributions to the medical field.
First, it may be used as a universal balance
measurement for icteric assessment and
contamination or hemolysis detection.
Second, this experiment will be
beneficial to the field of medical technology, as
it can help improve laboratory practices by
introducing a new transport container for
bilirubin. For medical technologists, these tubes
made from polarized mirror and a one-way
mirror can help them in handling serum samples
without covering them with carbon paper or
placing them in amber bottles.
Third, physicians will benefit from this
experiment because they will be able to identify
and give appropriate medication to their
patients using the results of the tests, which are
more reliable and accurate.
Future researchers and manufacturers
may also use this study as a stepping stone for
further innovations, inventions, and discoveries
regarding other alternative transport media for
handling serum samples for bilirubin
determination. This study may provide them
with new information that may be used to
strengthen their own research about bilirubin as
well.

1.4. Scope and Limitations

The study focused only on the
measurement of the exposure of serum bilirubin
Total Bilirubin, Direct Bilirubin and Indirect
Bilirubin in various duration of exposure to
light. The researchers used a fluorescent bulb as
the light source. Pooled serum was used as a
specimen. The distance of the light source to the
specimens was set to 1.5 meters. The serum
bilirubin levels were measured prior to exposure
and after 30, 60, 120, 180 minutes, respectively.
Automated Spectrophotometer was used to
measure the bilirubin.

1.5. Definition of Terms

Adult A person aged 21 and above
Analyte Substance or chemical constituent that
is of interest in an analytical procedure
Anneal - to heat and then slowly cool (metal,
glass, etc.) in order to make it stronger
Bilirubin Yellow breakdown product of normal
heme catabolism caused by the bodys clearance
of aged red blood cells which contain
haemoglobin
Diazotize - to cause (an aryl amine) to react with
nitrous acid to produce a diazonium salt
Direct bilirubin Water soluble variant of
bilirubin that passes out to the liver
Electromagnetic radiation Form of radiant
energy released by certain electromagnetic
processes, and synchronized oscillations of
electric and magnetic fields that propagate at
the speed of light
Fluorescence The emission of light by a
substance that has absorbed light or other
electromagnetic radiation
High performance liquid chromatography A
technique in analytical chemistry used to
separate the components in a mixture
Indirect bilirubin Insoluble variant of bilirubin.
It passes through the bloodstream to the liver,
where unconjugated bilirubin is converted to
conjugated bilirubin.
Isomerization the process by which one
molecule is transformed into another molecule
which has exactly the same atoms, but the atoms
have a different arrangement
Fluorescent light Low pressure mercury-vapor
gas-discharge lap that uses fluorescence to
produce visible light
Light An electromagnetic radiation within a
certain portion of the electromagnetic spectrum
Luminescence Emission of light by a substance
not resulting from heat; a form of cold body
radiation
One-way mirror a mirror that is partially
reflective and partially transparent that allows
viewing from the darkened side but not viceversa
Plasma Yellow-colored liquid component of
blood in which blood cells are suspended.

Photoisomerization the light-initiated process

of change from one isomeric form of a
compound, radical, or ion to another
Photosensitive Amount to which an object
reacts upon receiving photons, especially visible
light
Phototherapy Exposure to daylight or specific
wavelengths of light using polychromatic or nonpolychromatic sources of light.
Photolysis Breakdown of molecules into
smaller units through light absorption
Polarization Property of waves that can
oscillate with more than one orientation
Polyvinyl alcohol A colorless, water-soluble
synthetic resin employed principally in the
treating of textiles and paper.
Resin Any natural or synthetic organic
compound consisting of a non-crystalline or
viscous liquid substance
Serum Blood component that is neither a blood
cell nor a clotting factor; blood plasma no
including the fibrinogens
TAC films Also known as cellulose triacetate
films. They are manufactured from cellulose and
are sources of acetate esters, typically acetic
anhydride.
Total bilirubin Sum of the direct and indirect
bilirubin

2 REVIEW OF RELATED LITERATURE

2.1. Formation and Structure of Bilirubin and Its
Functions
When red blood cells are destroyed,
hemes are released. The yellow breakdown
product of normal heme catabolism is bilirubin.
It is formed by the breakdown of heme present
in hemoglobin, myoglobin, cytochromes,
catalase, peroxidase, and tryptophan pyrrolase.
It can be derived from two main sources. The
80% of bilirubin produced in the body comes
from the heme secreted from senescent red
blood cells. The remaining originates from
various heme-containing proteins found in other
tissues, notably the liver and the muscles

(Barrett, 2006). It binds to albumin and is

transported in the bloodstream to the liver as
unconjugated bilirubin, which is insoluble in
water. The liver converts unconjugated bilirubin
into a water soluble substance in the form of
conjugated bilirubin, which can then be excreted
via urine or feces (Strasinger, 2008). It is
accountable for the yellow
color of bruises and the yellow discoloration in
jaundice. It is also responsible for the brown
color of feces (via its conversion to stercobilin),
and for the straw-yellow color of urine through
its breakdown product, urobilin. Bilirubin
consists of an open chain of four pyrrole-like
rings (tetrapyrrole). In heme, by contrast, these
four rings are connected into a larger ring called
a porphyrin ring as shown in Figure 1 and the
molecular formula of bilirubin is C33H36N4O6.
Bilirubin is created by the activity of biliverdin
reductase on biliverdin, a green tetrapyrrolic bile
pigment which is also a product of heme
catabolism.

Fig.1. The atomic structure of bilirubin.

2.2. Methods for Bilirubin Determination
According to Choosongsang, et al (2009),
numerous methods for bilirubin assay have been
described, including; direct spectrophotometry,
colorimetric method using diazotization reaction
by Malloy-Evelyn, an enzymatic assay,
Jendrassik-Grof and High Performance Liquid
Chromatography (HPLC). But the Jendrassik-Grof
method or the Malloy-Evelyn procedure is the
most frequently used methods to measure
bilirubin because they both have acceptable
precision and are adapted to many automated
instruments (Bishop, et al., 2010).

Although both methods are considered

acceptable for measuring bilirubin, JendrassikGrof method is considered to be more complex
and has a number of advantages over the
Malloy-Evelyn procedure, and to name a few;
Jendrassik-Grof is not affected by pH changes, it
is insensitive to 50-fold variation in protein
concentration; it maintains optical sensitivity
even when bilirubin levels are low; it has
minimal turbidity and a relatively constant
serum blank; and it is not affected by
hemoglobin concentrations that reaches 750
mg/dL (Bishop, et al., 2010).

2.2.1. Jendrassik-Grof Method

The Jendrassik - Grof procedure uses a
combination of caffeine-benzoate as a
solubilizer. With sodium acetate as an
accelerator at pH 13.4 to couple bilirubin with
diazo reagent to form alkaline azobilirubin. This
method uses bilirubin pigments in serum or
plasma that will react with a diazo reagent
(sulfanilic acid in hydrochloric acid and sodium
nitrite) that will produce a purple product of
azobilirubin (Bishop, et al., 2010). The
Jendrassik-Grof method, involving the use of
diazotized sulfanilic acid is currently the used
method and has been recommended as the
procedure of choice for total bilirubin estimation
by the U.S. National Committee for Clinical
Laboratory Standards. This Candidate Reference
Method for total bilirubin was further developed
and validated by the Committee on Standards of
the American Association for Clinical Chemistry
and is now being used worldwide (Nagaraja, et
al., 2010).
Bilirubin + sodium acetate + caffeine-sodium
benzoate + diazotized sulfanilic acid -> purple
azobilirubin + alkaline tartrate -> green-blue
azobilirubin (600 nm) (Ciulla, et al.,2010).
The reaction without the accelerator will yield
conjugated bilirubin only. After a short period of
time, the reaction of the aliquots with the diazo
reagent is terminated by the addition of ascorbic
acid. The ascorbic acid destroys the excess diazo

reagent. The solution is then alkalinized using an

alkaline tartrate solution, which shifts the
absorbance spectrum of the azobilirubin to a
more intense blue color that is less subject to
interfering substances in the sample. The final
blue product is measured at 600 nm with the
intensity of color produced directly proportional
to
bilirubin
concentration.
Indirect
(unconjugated) bilirubin may be calculated by
subtracting
the
conjugated
bilirubin
concentration from the total bilirubin
concentration (Bishop, et al., 2010).

2.2.2. Malloy-Evelyn Procedure

Bilirubin pigments in serum are reacted
with a diazo reagent. The diazotized sulfanilic
acid reacts at the central methylene carbon of
bilirubin to split the molecule into two molecules
of azobilirubin. This method is typically
performed at pH 1.2 where the azobilirubin
produced is red-purple in color with a maximal
absorption of 560 nm. Methanol is most
commonly used accelerator to solubilize
unconjugated bilirubin (Bishop, et al., 2010).
2.3. Factors Affecting Serum Bilirubin Levels
Several studies were published to
determine the factors that affect the serum
bilirubin levels, such as the stability of bilirubin in
blood samples, intensities of light, temperature,
and time. According to McDonough, bilirubin is a
photosensitive substance that when it
undergoes both photoisomerization and photo
oxidation the latter is much slower than the
former. To prevent the reactions, laboratories
protect the bilirubin specimen from light
exposure. However, Tanner et al. found out that
bilirubin was stable when stored at 15, 25 or
35OC for up to 24 hours prior to
centrifugation. Also, the study that Boyanton et
al. have made, it was shown that both total and
direct bilirubin were stable when both plasma
and serum were maintained in contact with
blood cells at room temperature for up to 56
hours. Nevertheless, in the present study it was
conducted that both plasma and serum must be

separated from contact with cells within 2 hours

from time of collection.
Another study showed by Rehak et al.
has stated that bilirubin is a substance absorbing
light within the visible spectrum, and it is well
recognized to undergo both isomerization and
oxidation in serum exposed to visible light,
resulting in decreased measured bilirubin values.
Although protection of specimens from light has
been recognized as important for accurate
bilirubin analysis and that most studies have
examined stability of hyperbilirubinemic
specimens, and data on the stability of
normobilirubinemic specimens is limited. They
sought to determine the rates of bilirubin
photolysis, as measured by the diazo reaction,
which is the most commonly used clinical
methods, to establish the requirements for the
handling of normobilirubinemic specimens.
Photolysis of specimens may have different
effects on other methods of bilirubin analysis
which do not form diazo dyes. Detection of
changes in photoisomers requires more
sophisticated assays, such as chromatographic
analysis, that are not typically used in clinical
laboratories (Cecco et al., 2008). Moreover, Dr.
McDonagh rightly points out that the
wavelength as well as intensity of light is an
important factor in photoisomerization of
bilirubin, although most available data is for the
in vivo clearance of bilirubin in patients exposed
to phototherapy rather than in vitro stability of
serum specimens exposed to light. Laboratories
can influence the stability of bilirubin by the type
of lighting used in the laboratory. Ideally, all
specimens for bilirubin analysis should be
protected from light completely. However,
except for specimens collected specifically for
bilirubin analysis, such precautions are not easy
to implement in routine laboratory practice.
Routinely used collection tubes do not provide
light protection, and it is not practical to shield
all specimens from light (Cecco et al., 2008).

2.4. Phototherapy
Phototherapy (light treatment) is the
most effective process of using light to eliminate
bilirubin in the blood. It undergoes
photochemical reaction through the light
absorption by dermal and subcutaneous
bilirubin which having a light fraction (Maisels,
2008).
Blue light is the most commonly used for a more
rapid response than green light which takes time
to see remarkable results. The special blue and
the tungsten halogen lamps produce rapid rates
of isomerization and are therefore probably the
most effective of lamps currently used clinically
(Ennever, 1984).
Bilirubin can be "conjugated" with a
molecule of glucuronic acid which makes it
soluble in water. This is an example of
glucuronidation. Bilirubin is very similar to the
pigmentphycobilin used by certain algae to
capture light energy, and to the pigment
phytochrome used by plants to sense light. All of
these contain an open chain of four pyrrolic
rings. Like these other pigments, some of the
double-bonds in bilirubin isomers when exposed
to light. This is used in the phototherapy of
jaundiced newborns: the E,Z-isomers of bilirubin
formed upon light exposure are more soluble
than the unilluminated Z,Z-isomer, as the
possibility of intramolecular hydrogen bonding is
removed. This allows the excretion of
unconjugated bilirubin in bile.
The role of cross polarized glass in the
management of the photosensitivity of patients
who have epilepsy was exhibited in this
experiment, the most sensitive light flicker
frequency causing a photic response was
determined. The results showed that the cross
polarized glass were more effective than
conventional glass in terms of avoiding
photosensitive epilepsy (Jain, 2001).

2.5. Definition of Light

Light is visually perceived radiant in the
bile. This visible light is a small part of the
Electromagnetic Spectrum that is a form of
energy, exhibiting wavelike behavior as it travels
through space and ranges in wavelengths from
380 nm to 780 nm. Also, it is what energizes our
visual system and reflected from objects into our
eyes, which enables us to see.
Speed of light, defined as is the fastest
anything has been observed to move. In a
vacuum, the speed is 300 million meters per
second. At that speed, it takes light one ten
thousandth of a second to travel around the
earth. When light enters a material, it slows
down. The amount depends on the material it
enters and its density. For example, light travels
about 30% slower in water than it does in a
vacuum, while in diamonds, which is about the
densest material, it travels at about half the
speed it does in a vacuum. This slowing down of
light plays a role in another property, refraction.
Refraction means that light bends when it passes
from one medium to another. When light enters
a denser medium from one that is less dense, it
bends toward a line normal to the boundary
between the two media. The greater the density
difference between the two media, the more the
light bends. This property is used with respect to
optical devices such as microscopes, corrective
lenses for vision and magnifying lenses.

2.6. Polarized Glass

Generally, light travels in a transverse
direction perpendicular to the line of
propagation of the light waves. It is polarized
vertically and horizontally. Light is considered
polarized when the electric vibrations are
horizontal and the vibrations are vertical. When
light passes through a beam, the first polarizer
divides the light into two components: one is
transmitted or passed through the polarizer
while the other one is blocked. The remaining
light has either vertically or horizontally
polarized. A second polarizer will maintain a
parallel path to the first one, the polarized light

will be transmitted through the rotation of the

second polarizer. The amount of light passed
through decreases proportionally to the amount
of rotation of the second polarizer. Theoretically,
all of the light is absorbed by the second
polarizer if they are at right angles to one
another. This phenomenon is employed to a
substantial advantage in the use of polarized
sunglasses to substantially reduce the annoying
effects of glare, since reflected sunlight (glare)
has its polarization rotated ninety degrees or at
right angles to direct sunlight.

2.7. One-Way Mirror

A one-way mirror has a reflective coating
applied in a very thin, sparse layer -- so thin that
it's called a half-silvered surface. The name halfsilvered comes from the fact that the reflective
molecules coat the glass so sparsely that only
about half the molecules needed to make the
glass an opaque mirror are applied. The glass is
coated with, or has encased within, a thin and
almost-transparent layer
of
metal
(usually aluminium). The result is a mirrored
surface that reflects some light and is penetrated
by the rest. Light always passes exactly equally in
both directions. However, when one side is
brightly lit and the other kept dark, the darker
side becomes difficult to see from the brightly lit
side because it is masked by the much brighter
reflection of the lit side. It may be possible to
achieve something similar by combining
an optical isolator layer with a traditional oneway mirror, which would prevent light coming
from one direction. At the molecular level, there
are reflective molecules speckled all over the
glass in an even film, but only half of the glass is
covered. The half-silvered surface will reflect
about half the light that strikes its surface, while
letting the other half go straight through. It turns
out that half-silvered mirrors are also essential to
many types of lasers.

3 METHODOLOGY
3.1 Research Design
The researchers based the experiment
from traditional concepts that serum samples to
be tested for bilirubin must be wrapped with
carbon paper prior to testing. Sample collection
was done using convenience sampling. Proper
handling and containment of specimens were
followed albeit with slight alteration. The
distance, and intensity of light used were kept
constant. The researchers wanted to determine
on how effective it would to use one-mirrors and
polarized glass as an alternative transport
medium as compared to the traditional method
of handling samples used for bilirubin testing.
The researchers created a glass receptacle using
different materials namely: one-way mirror and
polarized glass. The tube created using the two
materials were tested based from the previous
studies, comparing its results to the normal red
top tube used in most laboratories. The test for
comparison is based on the duration light
exposure and temperature.

3.2 Manufacturing
The components for the glass were
bought from a local glass shop, four glass pieces
had an individual height of 5 inches and a width
of 3 inches. The four glass pieces were put
together by a local glass maker. The
measurement of 2 by 2 inches was done to
compensate for the length of the tube.
3.2.1 One-Way Mirror
After the glass was manufactured, a thin,
reflective silver coating alongside a 5% black film
was placed on the outer side of the tube in order
to prevent the penetration of light.
Hypothetically, doing this prevented the
photosensitive degradation of bilirubin and
allowed the transparent supervision of bilirubin
for purposes such as balance measuring,
photometric examination, and contamination or
hemolysis assessment.

3.2.2 Polarized Glass

While the glass is in progress a polarizing
film is attached to it covering the entire glass.
The polarizing film is made by a dye that mainly
contains polyvinyl alcohol and is being adsorbed
to its surface while stretching and orienting it.
Polyvinyl alcohol is a colourless, water-soluble
synthetic resin employed principally in the
treating of textiles and paper. This gives the film
polarization characteristics that allow only light
with a certain oscillation direction to pass
through it. Furthermore, in order to secure
mechanical strength of the film, backing
materials such as a TAC film or a protective film
is laminated to it.

3.3 Pooling of Serum Specimens

The samples that were used for the
study came from pooled serum samples
provided through convenience sampling. Each
tube was used for the positive and negative
controls, and the polarized and one-way mirror
tube. The volume of each sample was at least 5.0
mL with a carbon paper-cover and was stored in
a refrigerator with a temperature of 3 degrees to
5 degrees Celsius until the start of the study.

3.4 Transportation of Specimens

The specimens used were transported in
an ice filled container in order to preserve the
specimens chemical integrity. The container
does not allow the light to enter hence
preventing photolysis of the specimens prior to
the actual experimentation.

3.5 Experimentation
The study used a total of eight (8)
samples from one mother tube. Each sample was
divided into four tubes, the samples were used
for the one positive control and one negative
control and concluding the four tubes are the
polarized glass and the one-way mirror tube. The
samples were measured using an ILAB 300 Plus
Chemistry Analyzer for their bilirubin levels prior

to the actual experimentation in order to

establish a baseline.
The positive control sample was placed on a rack
and was wrapped with carbon-paper, the
negative control sample was placed on a
different rack and was completely exposed to
light. The serum samples used for polarized glass
was transferred to the polarized glass tubes, the
same was done to the one-way mirror tubes.
Each sample was exposed to fluorescent light
and the bilirubin levels were measured at 30, 60,
120, and 180 minutes. The fluorescent light was
set at a constant light intensity and was at
approximately 1.5 meters from the test samples,
akin to regular laboratory conditions. Figure 3.1
summarizes the experiment.

measured the intensity of light as beam of light

passed through a sample solution. The auto
analyzer used was calibrated before the
experimentation with its known concentration
calibrator material and it measured the serum
using photometry.

4 RESULTS & DISCUSSION

4.1. Statistical Analysis
Means and its standard error (SEM)
were used to summarize the total bilirubin,
direct bilirubin and indirect bilirubin of the four
groups (positive control, one-way mirror,
polarized glass, and negative control) from
baseline to 180 minutes. Repeated measures
analysis of variance was used to determine the
time or length of exposure and the created
material. This statistical test was performed
using SPSS ver. 20.0. P-values less than 0.05
indicate significant differences.

4.3. Results
4.3.1 Total Bilirubin Percentage Change

Figure 3.1 Overview of the methodology

Figure 3.2 Actual Bilirubin Set-up

3.6.1 Bilirubin Measurement
The automated spectrophotometric method was
used to measure the total bilirubin, indirect
bilirubin and direct bilirubin. In this method,
spectrophotometry, it determined how much
the chemical substance absorbed light as it

Figure 4.3.1 Mean percentage change of total

bilirubin after one to three hours at different
intensities

Group

Baseline

30
mins

60
mins

120
mins

180
mins

F4,4
stat

pvalu
e

Group

Baseli
ne

30
mins

60
mins

120
mins

180
mins

F
sta
t

pvalu
e

Positive
Control

3.9 0.5

4.2
0.0

4.1
0.2

3.8
0.4

4.2
0.1

0.35
8

0.82
8

Positive
Control

1.6
0.1

1.4
0.0

1.3
0.2

1.5
0.1

1.5
0.3

0.5
70

0.70
0

One Way
Mirror

3.9 0.2

3.7
0.0

3.9
0.7

3.8
0.3

3.4
0.1

0.38
8

0.80
9

One Way
Mirror

1.5
0.1

1.2
0.1

1.4
0.1

1.6
0.1

1.5
0.1

2.0
95

0.24
6

Polarized
Glass

4.4 0.4

4.2
0.4

3.9
0.7

4.3
0.4

4.0
0.1

0.23
5

0.90
5

Polarized
Glass

1.7
0.2

1.3
0.1

1.3
0.1

1.6
0.1

1.6
0.1

5.7
83

0.05
9

Negative
Control

4.5 0.2

3.3
0.2

4.5
0.8

3.3
0.1

3.1
0.1

3.23
6

0.14
1

Negative
Control

1.4
0.1

1.6
0.3

1.4
0.0

1.3
0.1

1.2
0.2

0.5
57

0.70
7

Table 4.3.1 Mean total bilirubin percentage

change after one to three hours at different
intensities
The mean total bilirubin [F4,4=0.358,
p=0.828], one way mirror [F4,4=0.388, p=0.809],
polarized glass [F4,4=0.235, p=0.905], and
negative control [F4,4=3.236, p=0.141] did not
significantly change from baseline to 180
minutes. Moreover, the mean total bilirubin of
the positive control, one way mirror, polarized
glass and negative control did not differ
[F3,4=2.369, p=0.212]. From the above clinical
findings, the researchers are able to compare the
percentage differences of the separate control
groups and rationalize that photolysis does not
as easily occur within specimens of the positive
control (carbon paper), the one-way mirror, and
the polarized glass as it does within a negative
control when exposed to a fixed intensity of light
after a period of 180 minutes.

Table 4.3.3 Mean direct bilirubin percentage

change after one to three hours at different
intensities

The mean direct bilirubin [F4,4=0.570,

p=0.700], one-way mirror [F4,4=2.095, p=0.246],
polarized mirror [F4,4=5.783, p=0.059], and
negative control [F4,4=5.783, p=0.059] did not
significantly change from baseline to 180
minutes. Moreover, the mean direct bilirubin of
the positive control, one-way mirror, polarized
mirror and negative control did not differ
[F3,4=1.133, p=0.436]. From the above clinical
findings, the researchers are able to compare the
percentage differences of the separate control
groups and rationalize that photolysis does not
as easily occur within specimens of the positive
control (carbon paper), the one-way mirror, and
the polarized glass as it does within a negative
control when exposed to a fixed intensity of light
after a period of 180 minutes.

4.3.2 Direct Bilirubin Percentage Change

4.3.3. Indirect Bilirubin Percentage Change

Figure 4.3.2 Mean percentage change of direct

bilirubin after one to three hours at different
intensities

Figure 4.3.4 Mean percentage change of indirect

bilirubin after one to three hours at different
intensities

Group
Positive
Control
One Way
Mirror
Polarized
Glass
Negative
Control

Baselin
e
2.4

0.6
2.4

0.1
2.8

0.3
3.1

0.1

30
mins
2.8
0.0
2.5
0.1
2.9
0.4
1.8
0.1

60
mins
2.8
0.0
2.5
0.6
2.6
0.6
3.1
0.8

120
mins
2.3
0.3
2.2
0.2
2.7
0.3
2.0
0.0

180
mins
2.8
0.4
1.9
0.1
2.5
0.1
1.9
0.3

F
stat
0.5
48
0.9
24
0.1
96
3.0
68

Table 4.3.5 Mean indirect bilirubin percentage

change after one to three hours at different
intensities

The mean indirect bilirubin [F4,4=0.548,

p=0.713], one-way mirror [F4,4=0.924, p=0.530],
polarized mirror [F4,4=0.196, p=0.928], and
negative control [F4,4=3.068, p=0.152] did not
significantly change from baseline to 180
minutes. Moreover, the mean indirect bilirubin
of the positive control, one-way mirror,
polarized mirror and negative control did not
differ [F3,4=2.581, p=0.191]. From the above
clinical findings, the researchers are able to
compare the percentage differences of the
separate control groups and rationalize that
photolysis does not as easily occur within
specimens of the positive control (carbon paper),
the one-way mirror, and the polarized glass as it
does within a negative control when exposed to
a fixed intensity of light after a period of 180
minutes.
The sample was collected and pooled
five (5) days prior to the experimentation day
which were leftover serum samples from the
East Avenue Medical Center. The pooled sample
was already considered old as it is ideal to have
a pooled icteric sample a maximum of two (2)
days prior to the experimentation day. According
to the study done by Rehak et al. (2008), slow
decline of about 15% happen in the bilirubin
level after 24 hours of collection and drastically
changes after 48 hours of collection. The
bilirubin within the sample has already

pvalu
e
0.71
3
0.53
0
0.92
8
0.15
2

undergone isomerization and oxidation due to

its exposure to light thus resulting to a lower
bilirubin level. The fluctuations in the results of
total bilirubin and direct bilirubin may be caused
by the spectrophotometer that was used for the
experiment or because of the age of the
specimen. According to a study done by Rehak et
al. (2008), substantial changes occur to the bile
pigment composition of the sample because of
isomerization particularly photoisomerization.
The researchers didnt achieve the 0.05
p-value and wasnt considered statistically
significant because according to statistical
principles small sample sizes rarely provide
evidence that there was a significant change.

6 Conclusion
The results of the experimentation
showed that when bilirubin was measured at 700
lux and at different intervals in time, namely 30,
60, 120, and 180 minutes, there was no
statistical significant change within the one-way
mirror and polarized glass receptacles. Among
the direct, indirect, and total bilirubin levels,
there were no significant changes in the mean
percentage from the baseline [F3,4=2.581,
p=0.191]. The most significant effect of light on
the alternative receptacles was on the direct
bilirubin level at (p=0.059) for the polarized glass
and at (p=0.246) for the one-way mirror. From
the experimentation the researchers concluded
that the higher p-value of the one-way mirror, as
compared to the lowest p-value of the polarized
glass, indicates that the one-way mirror acts as a
more protective alternative receptacle against
photolysis. In addition to the invention of a
polarized glass and a one-way mirror glass the
researchers concluded that with further
innovations, in time; this product will soon be
sellable because it is easy to use and it helps with
protecting bilirubin from further photolysis.

7 Recommendation
The results of the experimentation
revealed that there were no statistical significant
changes within the serum bilirubin levels after
three (3) hours of exposure to 700 lux of light.
The researchers recommend the use of freshly
collected icteric serum samples to avoid preexperimental photolysis. The researchers also
recommend the use of plasma for international
standardization and to ascertain comparative
analysis between serum and plasma. Should the
future researchers pursue this study, it is
recommended that the volume of the sample be
increased to elicit a significant statistical value
and use other methods of bilirubin testing aside
from the Jendrassik-Grof method to truly prove
the effectiveness of polarized glass and the oneway mirror glass. In line with this, the
researchers would also recommend for the
further testing of the said receptacles as a better
replacement for the commonly used carbon
paper.

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