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 body’s 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 yellow-orange 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 one-way 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 body’s 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 vice-versa 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 non-polychromatic 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, Jendrassik-Grof 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 half-silvered 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 one-way 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 specimen’s 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.

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

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 4.3.1 Mean percentage change of total bilirubin after one to three hours at different intensities

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. 4.3.2 Direct Bilirubin Percentage Change

Figure 4.3.2 Mean percentage change of direct bilirubin after one to three hours at different intensities

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. 4.3.3. Indirect Bilirubin Percentage Change

Figure 4.3.4 Mean percentage change of indirect bilirubin after one to three hours at different intensities

Group Baseline 30

mins

60

mins

120

mins

180

mins

F4,4

stat

p-

valu

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

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

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

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

Group Baseli

ne

30

mins

60

mins

120

mins

180

mins

F

sta

t

p-

valu

e

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

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

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

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 4.3.5 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

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 didn’t achieve the 0.05 p-value and wasn’t 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.

Group Baseline

30 mins

60 mins

120 mins

180 mins

F stat

p-value

Positive Control

2.4 ± 0.6

2.8 ± 0.0

2.8 ± 0.0

2.3 ± 0.3

2.8 ± 0.4

0.548

0.713

One Way Mirror

2.4 ± 0.1

2.5 ± 0.1

2.5 ± 0.6

2.2 ± 0.2

1.9 ± 0.1

0.924

0.530

Polarized Glass

2.8 ± 0.3

2.9 ± 0.4

2.6 ± 0.6

2.7 ± 0.3

2.5 ± 0.1

0.196

0.928

Negative Control

3.1 ± 0.1

1.8 ± 0.1

3.1 ± 0.8

2.0 ± 0.0

1.9 ± 0.3

3.068

0.152

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 pre-experimental 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 one-way 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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