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ChE 143/144

ChE 143/144 Undergraduate Research


Project
Development of a Pre-Diagnostic Test for Preeclampsia
E.J.T Battung, H.D.A. Talaro*
Biomedical and Separations Laboratory, Department of Chemical Engineering, University of
the Philippines-Diliman, Quezon City, Philippines

REPORT INFO

ABSTRACT

Report History:
Received 16 April 2014

Preeclampsia is a pregnancy-specific multisystem disorder which is


among the leading causes of maternal and infant mortality
worldwide, especially in developing countries. Its key diagnostics are
hypertension and proteinuria. This study seeks to help develop a
test for preeclampsia by documenting the color changes produced
by a bromophenol blue assay across urine samples containing
varying concentrations of albumin and check for the consistency of
the colors and determine if the color change for the set threshold
values are apparent. Two different sample-to-indicator ratios were
used in the methodology, one for mild proteinuria, 0.4g/L, and the
other for severe proteinuria, 3.0g/L. It was found out that the latter
gave promising results, with the BPB assay consistently forming a
vivid blue complex with a solution containing 3.0g/L albumin. The
test, however, also yielded false positives. Th results of the
experiment may be used in the design of a home-based,
inexpensive pre-diagnostic test for preeclampsia.

Keywords:
preeclampsia,
bromophenol blue,
albuminuria
*Corresponding author.
Tel.: +63
Email address

I.

INTRODUCTION
Preeclampsia
is
a
pregnancy-specific
multisystem disorder which affects 3-8 % of
pregnancies. It is among the leading causes
of maternal and perinatal morbidity and
mortality, especially because it can progress
to
widespread
endothelial
dysfunction
affecting mainly the liver, the brain, and the
kidney. It has no cure, except for delivery of
placenta (Karumanchi & Cerdeira, 2012).
Preeclampsia
has many
risk
factors,
including family history of preeclampsiaeclampsia, obesity, multifetal gestation,
pregestational
diabetes
mellitus,
hypertension and renal disease, among
others (Wagner, 2004). It also manifests
itself through many symptoms such as
edema, thrombocytopenia (platelet count
less than 100,000/mm3), persistent severe
central nervous system symptoms (altered

mental status, headaches, blurred vision or


blindness), but its key diagnostics are
gestational hypertension plus proteinuria
(300 mg or more per 24-hour period). 24hour urine collection is the usual procedure
for testing for proteinuria. However, the
procedure may be considered tedious and
time-consuming. Proteinuria may then be
defined as a concentration of at least 30
mg/dL in at least two random urine samples
collected at least 6 hours apart. At present,
there is no single screening test that is
considered reliable and cost-effective for
predicting preeclampsia (Wagner, 2004).
Preeclampsia may be misdiagnosed as
gestational hypertension when tests for
proteinuria are not undergone by the
patients. This may occur especially in
developing
countries,
where
pregnant
women do not go to the doctor for regular

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)

checkups. With the current state of health


care system in the Philippines, mothers
are often discouraged to go to their
doctors, and instead, rely on midwives,
who may be ill-equipped for testing for
cases of preeclampsia and delivering the
babies of patients with preeclampsia.
Presently, preeclampsia is the third
leading cause of maternal mortality in the
Philippines according to the Philippine
Department of Health (Center for Health
Development, 2010). Preeclampsia may
even escalate to eclampsia, which
accounts for 27% of maternal mortality in
the Philippines or more than 50 deaths for
every 100 000 live births (Morse & Mosher,
2013).
There have been numerous studies about
the role of proteinuria in the diagnosis of
preeclampsia. Among the most common
proteins studied is albumin. Because of its
small size, it is one of the first proteins
able to pass through the kidneys into the
urine, which a healthy kidney normally
doesnt allow (The Cleveland Clinic
Foundation,
2013).
Proteinuria
in
preeclampsia predominantly involves highmolecular weight proteins such as albumin
(Schiff, Friedman, Kao, & Sibai, 1996)
Several studies have investigated the
potential value of measuring urine albumin
concentration as a screening method for
subsequent
development
of
preeclampsia. A study by Salako, et al gave a
detection rate of 89% and a false positive
rate of 32 % for a single estimation of
microalbuminuria (2003). The findings of
separate studies by Bar, et al and Poon, et
al also suggested that the proteinuric
phase in preeclampsia is preceded by a
microalbuminuric phase (1996, 2008).
However, the latter also reported a high
false positive rate or low specificity.
Moreover, it has been concluded that the

presence of microalbuminuria in some


otherwise symptom-free patient is a
confirmation that changes in renal function
occur to mothers who will eventually
develop preeclampsia (Fatema, Khatun,
Akter, & Ali, 2011). A study by Fatema, et
al reported good values for the sensitivity
and negative predictive value but the
specificity was rated at around 50%. It
further closed with a statement that early
pregnancy levels of microalbuminuria can
be used as predictors of preeclampsia.
The gold standard for assessment of
microalbuminuria
is
24-hr
sample
collection. This method, however, is
inconvenient for the woman, costly and
may also be inaccurate due to incomplete
collection. Thus, random urine specimens
are often collected and tested, as this
method is more acceptable to pregnant
women. A positive result still needs
confirmation from other tests due to the
low positive predictive value of this
method (Kieler, Zettergren, Svensson,
Dickman, & Larsson, 2003).
Several studies have used random spot
urine samples in their methodologies, due
not
only
to
its
convenience
in
experimentation but also because it is the
more convenient method of testing for
pregnant women (Sheela, Beena, & Askar,
2011).
There are many ways to detect albumin in
a urine sample, such as colorimetry,
radioimmunoassay,
immuno-enzymatic
assay and dipstick tests. Most of these
technologies, however, are costly and not
home-based. (Sharma, Bala, Tulsani,
Sehgal, & Kumar, 2002)
Meanwhile,
bromophenol
blue
or
3',3",5',5"tetrabromophenolsulfonphthalein (BPB) is
used as an acid-base indicator, a color

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)

marker, and a dye. It has been used as a


staining reagent in the determination of
urinary proteins, including albumin. BPB
has long been known to interact with
proteins, forming colored complexes.
Protein binding results in a shift from a
yellow to a green or blue color, depending
on the specific protein used and the dye to
protein ratio (Waldmann-Meyer & Schilling,
1956). It has been reported that the color
change upon mixing is due to the
transformation of dye species of free
acidic form into bound basic form, as well
as to the bathochromic and hyperchromic
effects of conjugation (Yong-Ju Wei, 1996).
II.OBJECTIVES
The study seeks to help develop a test for
preeclampsia by documenting the color
changes produced by a bromophenol blue
assay across urine samples containing
varying concentrations of albumin. It also
aims to examine the validity of the sample
to indicator ratios suggested by Hwang, et
al. It specifically aims to check for the
consistency of the colors and determine if
the color change for the set threshold
values are apparent.
III. METHODOLOGY
A bromophenol blue dye-indicator solution
was first prepared using the method of
Schosinsky et al which is shown below.
1 Preparation of glycine buffer (pH
3.0)
Dissolve 17.26g of glycine in about
800mL of distilled water in a 1000-mL
volumetric flask. Add 100mg of sodium
azide, adjust to pH3.0 with HCl, and
dilute to the mark. Store the solution in
a tightly closed bottle. (This reagent can
be used for six months if stored at room
temperature.)

2 Preparation of Bromophenol blue


(BPB) stock solution
Dissolve 125.7mg of BPB in 3mL of
0.1mol/L NaOH and dilute to 150mL with
distilled water. Store the solution in a
tightly closed bottle. (This reagent is
stable for at least a week at 4C.)
3 Preparation of bromophenol blue
working solution ( pH 3.0 )
Mix 150mL of BPB stock solution with
about 800mL of glycine buffer solution
(pH 3.0) in a 1000-mL volumetric flask,
add 4mL of Brij-23 surfactant solution
and fill to the mark with glycine buffer.
4. Preparation of stock albumin
standard (60g/L):
Dissolve 6g (or 24mL of BUMINATE 25%)
of human serum albumin and 50mg of
sodium azide in 100mL of isotonic NaCl
solution in a small beaker, with gentle
stirring. This solution should be stored in
a tightly closed bottle at 2-4C.
Several sets of working human albumin
standard are prepared at the following
concentrations: 0.1 g/L, 0.4 g/L, 1 g/L, and
3 g/L.
To prepare these standard
solutions:
1 Select one urine specimen.
2 Add 9mL of the urine to 1mL of the
stock human albumin standard
(60g/L) to dilute the stock solution
to an albumin standard of 6g/L
(Solution X).
3 Add 1mL of urine to 1mL of Solution
X to get 3g/L concentration
(Solution A).
4 Add 2mL of urine to 1mL of Solution
A to get 1g/L concentration
(Solution B).
5 Add 1.5mL of urine to 1mL of
Solution
B
to
get
0.4g/L
concentration (Solution C).

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)

Add 3mL of urine to 1mL of Solution


C to get 0.1g/L concentration
(Solution D).
Test 1
0.5 mL of Solutions A, B, C and D were
separately added to their respective
cuvettes. 1.5mL of BPB working solution
was measured into each of the cuvettes. A
control solution, Solution E contains no
albumin. This is made by mixing 1.5 mL
BPB working solution and 0.5 mL of urine
sample into a cuvette. The color changes
were
visually
compared
and
photographed.
Test 2
Ten drops of Solutions A, B, C, and D were
added to separate cuvettes. Fifteen drops
of BPB working solution was measured into
each of the cuvettes. A control solution,
Solution E contains no albumin. This is
made by mixing 15 drops of the BPB
working solution and 10 drops of urine
sample into a cuvette.
The color changes were visually compared
and photographed. The photographs were
taken under artificial light indoors to limit
errors due to changes in the environment.
The results were analyzed by taking the
RGB and HSL values of the photos using
Microsoft Office.
The ratios used in the two tests were
based on a preliminary study by Huang, et
al.
IV. RESULTS AND DISCUSSION

The RGB (Red Green Blue) and HSL


(Hue Saturation Luminance) values of
every sample were averaged across
the three trials, thereby giving a single set
of RGB value and a single set of HSL value
for every concentration in a sample per
test.
RGB values are based on additive mixing.
RGB stands for the three primary colors of
light, red, green and blue. A higher R value
means the color is closer to red; a higher B
value means the color is closer to blue.
Each value is an integer from 0 to 155.
The color black is formed when all three
values are 0, while white is formed when
all are 255. When the B value equals 255
and the other two values are 0, a deep,
vivid shade of blue is formed. When the
values are reversed (R= 255, G=255,
B=0), a bright, vivid yellow is produced.

Figure 1. Left: R=0, G=0, B=255; Right: R=


255, G=255, B=0

The RGB values for every sample were


plotted in a triangular graph. The graph is
shown below. The legend for the data
points are shown in the upper left corner
of the figure, with the letters A, B, C, D,
and E corresponding to the solutions
referred to in the methodology, where
solution E is the control and solution A has
the highest albumin concentration ( 3.0 g/
L). The arrows above the axis labels show
the direction of increasing concentration.

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In figure 3, the cuvettes are arranged in
increasing albumin concentration, such
that the leftmost cuvette corresponds to
the control, solution E, while the rightmost
cuvette, to solution A.

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)

Figure 2. RGB values for Test 1 including


outliers

Figure 2 shows that there is a general


linear trend for the RGB values in Test 1.
Majority of the data points for solution E lie
on the upper right side of the scatter plot
while the data points for solution A lie on
the lower left side of the scatter plot. Data
points for solutions B, C and D lie on the
middle following the linear trend. It may
be
observed
that
with
lower
concentrations, higher R and G values and
lower B values are obtained and viceversa.
This
implies
that
lower
concentrations of albumin give colors
close to yellow when mixed with the BPB
assay while higher concentrations yield
blue solutions when mixed with BPB. This
trend may be visually observed as
evidenced by the following figure.

A similar photograph is obtained for


majority of the samples. By visual
inspection, it was determined that 50 out
of 70 samples adhered to the pattern
formed by these colors. However, it may
be observed in Figure 2 that some data
points corresponding to solutions D and E
may be seen lying on the same area of the
graph occupied by data points for
solutions A and B. These points account
for the remaining 20 samples, whose
control solution turned blue upon addition
of BPB. Of these 20 outliers, 16 control
solutions turned blue upon addition of
BPB. This is indicative of the higher
sensitivity of Test 2 for low concentrations
of albumin. It is important to mention that
some of these samples came from donors
with known urinary system disorders such
as UTI. This accounts for the high albumin
concentration in their urine sample, as
albuminuria is a symptom of renal
diseases, aside from preeclampsia. A
photo representative of these errant
samples is shown below.

Figure 4.An Outlier sample for Test 1 taken


from Sample 24 (L to R: Solution E, D, C, B, A)

Figure 3. Sample Picture for Test 1 taken from


Sample 8
(L to R: Solution E, D, C, B, A)

A new triangular graph was generated


using only the values for the 50 samples

6
for different H values was superimposed in
the graph to help visualize the color of the
solutions based on the graph.

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)

which showed the expected color changes.


The graph is shown below.

Figure 6. H values for Test 1 including outliers


Figure 5. RGB values for Test 1 without
outliers

Figure 5 shows a trend similar to that in


Figure 2 but in this graph, the different
areas on the plot occupied by different
concentrations are even more obvious.
The square points, representing solution E
is clearly lying on the upper right end. The
lower left end is occupied solely by cross
markers, representing solution A.
Meanwhile, another graph is generated
using the H values of the different
solutions in every sample. In the HSL color
representation scheme, the H value is an
integer from 0 to 239 representing a single
hue in the color spectrum. The S and L
values are both integers from 0 to 240
with the former telling something about
the saturation or vividness of the color and
the latter telling how light or dark the color
is. Low values for H show a grey image. An
S value of 0 represents the color black
(minimum luminance) while a value of 240
represents the color white (maximum
luminance).
The H values against concentration were
plotted in a graph, as seen in Figure 6. The
spectrum showing the corresponding hues

Figure 6 shows that most of the data


points are scattered across the spectrum,
except for those of solution A. This, at
least, indicates that the color change
obtained when BPB is added to a urine
sample containing 3 g/L albumin is
consistent. Its H value is the area of 131 to
176, with the densest area occurring at
153 to 164. This color lies along the blue
area on the spectrum, which is consistent
with the values from the triangular graph
and visual observations.
In order to make a more conclusive graph,
a new graph was generated by removing
the data points corresponding to the 20
outlier samples. This graph is shown as
Figure 7a.

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)


3.5
3
2.5
2
1.5
1
Figure 7a. H values for test 1 without outliers

With the outliers removed, the points


noticeably
become
more
cohesive,
especially the points representing solution
E. Hence, the trend becomes more visible
in Figure 7a. H values increase with
increasing concentration, as seen in the
graph.

Solution E is cohesively located along the


orange to yellow area, with H values from
39 to 50. Solution D is also cohesive, with
many points overlapping with solution E.
This may be explained by the fact that a
random urine sample normally contains 0
to 8 mg/dL (0.08 g/L) which is close to 0.1
g/L. (Gerber & Brendler, 2011)The plot
points for solution C are more scattered
with the densest area lying on the yellowgreen area with H values from 50 to 80.
The points for solution D is quite scattered
and even the dense region has a wide
range.

0.5
0
20

40

60

80 100 120 140 160 180 200

Figure 7b. Trendlines of H values of Samples


for Test 1 without outliers

Figure 7b presents the same data as Figure


7a but with the trend lines for every
sample superimposed over the graph. It
confirms that the general trend for all
samples is an increasing one. As the
albumin concentration increases, the H
value increases within the range of about
35 to 180.

Albumin C oncentration in g/L

H ave

Linear (H ave)

3.5
3
2.5
2C
1.5
1
0.5
0

R = 0.98
D

40 120200
0 80 160240
H value

Figure 7c. Trendline for H value of Test 1


without outliers

Meanwhile, Figure 7c above shows a single


trend line for the average H value outliers
superimposed over Figure 7a. The H

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The positive results of the experiment may
be used in the design of a home-based
test for proteinuria, similar to a dipstick. It
is recommended that the experiment in
this study be replicated using urine
samples from pregnant women. In this
way, the sensitivity, specificity, positive
predictive value and negative predictive
value of the test may also be quantified.
Moreover, it is also suggested that a
prototype for the test using acid-free
paper be designed and field-tested. A
sample legend for such a test is shown
below, based from the data gathered in
this study.

E.T. Battung, H.A. Talaro/ University of the Philippines Diliman (2014)

values for Test 1 were averaged after


excluding the outliers. The trend is an
increasing one and it shows good linearity
with a high R-squared value. Solution E
has an average value of 40.41, D of 46.03,
C of 61.95, B of 92.47, and A of 150.05.
As test 1 is designed to detect severe
proteinuria, with an assigned threshold
concentration of 3 g/L, the points we are
most concerned with are the points for
solution A and E. In both Figures 6 and 7a,
it is apparent that the color of solution A
after the addition of BPB is consistently in
the blue area. Figure 6 shows a sharp
difference between the H values of
solution A and the control, solution E. The
former are represented by the markers
lying primarily on the 140 to 180 range
while the latter are represented by
markers lying on the range of 39 to 50.
Their average values are also far from
each other. The colors corresponding to
these values contrast greatly so it may be
said that the color change is easy to
observe visually.

V. CONCLUSIONS AND
RECOMMENDATIONS
It has been found out that a 1.5-mL BPB
assay dropped in 0.5-mL urine sample is a
good indicator of significant amount of
protein levels in the urine. A dark, vivid
blue color may be observed when the
albumin level is about 3g/L, which is the
threshold value used for severe proteinuria
and preeclampsia, as well. The color
change at this level is very apparent. This
color is consistent across all samples,
though there are some errant samples,
where even the control solutions turned
blue, which may be indicative of some
renal disease from the sample donor.

Such a test would make it more


convenient for pregnant women in remote
areas to check if they are at risk for
preeclampsia. As pre-diagnostic tests go, it
will still require an individual who tests
positive to seek confirmation from a
medical expert. This study is good step in
the development of such a test.

ACKNOWLEDGMENT

VII. REFERENCES

1. Bar, J., Hod, M., Erman, A.,


Friedman, S., Gelerenter, I., Kaplan,
B., et al. (1996). Microalbuminuria
as an early predictor of
hypertensive complications in
pregnant women at high risk.

9
http://www.pop.org/content/why-dofilipino-women-die-childbirth

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American Journal of Kidney


Diseases , 220-225.
2. Center for Health Development.
(2010). Regional Profile. Quezon
City: Department of Health.
3. Fatema, K., Khatun, M., Akter, S., &
Ali, L. (2011). Role of Urinary
Albumin in the Prediction of
Preeclampsia. Faridpur Medical
College Journal , 14-18.
4. Gerber, G., & Brendler, C. (2011).
Evaluation of the urologic patient:
history, physical examination, and
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Campbell-Walsh Urology (p. chap 3).
Philadelphia, PA: Elsevier.
5. Hwang, K., Lagundi, J., & ZE
Mendoza, R. C. (2012). Feasibility of
a Bromophenol Blue Assay as a
Low-Cost Portabl Method To Assess
th Risk of Preeclampsia. Quezon
City.
6. Karumanchi, S. A., & Cerdeira, A. S.
(2012). Angiogenic Factors in
Preeclampsia and Related Disorders.
Cold Spring Harbor Perspectives in
Medicine , 65-85.
7. Kieler, H., Zettergren, T., Svensson,
H., Dickman, P. W., & Larsson, A.
(2003). Assessing urinary albumin
excretion in pre-eclamptic
women:Which Sample to Use?
BJOG: an International Journal of
Obstetrics and Gynaecology , 12-17.
8. Morse, A., & Mosher, S. (2013). Why
Do Filipino Women Die in
Childbirth? Retrieved March 31,
2014, from Population Research
Institute:

9. Poon, L., Kametas, N., Bonino, S.,


Vercellotti, E., & Nicolaides, K.
(2008). Urine albumin concentration
and albumin-to creatinine ratio at
11+0 to 13+6 weeks in the
prediction of pre-eclampsia. BJOG:
An International Journal of
Obstetrics and Gynaecology , 866873.
10.Salako, B., Odukogbe, A., Olayemi,
O., Adedapo, K., Aimakhu, C., Alu, F.,
et al. (2003). Serum Albumin,
Creatinine, Uric Acid and
Hypertensive Disorders of
Pregnancy. East African Medical
Journal , 424-428.
11.Salako, B., Olayemi, O., A.-T. O.,
Adedapo, K., Aimakhu, C., Alu, F., et
al. (2003). Microalbuminuria in
pregnancy as a predictor of
preeclampsia. West Afr J Med , 295300.
12.Schiff, E., Friedman, S. A., Kao, L., &
Sibai, B. M. (1996). The importance
of urinary protein excretion during
conservative management of severe
preeclampsia. American Journal of
Obstetrics and Gynecology , 13131316.
13.Schosinsky, K., Vargas, M., Esquivel,
A., & Chavarria, M. (1987). Simple
Spectrophotometric Determination
of Urinary Albumin by Dye-Binding
with Use of Bromphenol Blue. Clin
Chem , 223-226.
14.Sharma, S., Bala, M., Tulsani, N.,
Sehgal, N., & Kumar, A. (2002).
Albumin Test Strip for Quick
Detection of Albuminuria in

10
17.Wagner, L. K. (2004). Diagnosis and
Management of Preeclampsia.
American Family Physician , 2317 2324.

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Humans. Indian Journal of Chemical


Technology , 496-498.
15.Sheela, C., Beena, S., & Askar, M.
(2011). Calcium-Creatinine Ratio
and Microalbuminuria in Prediction
of Preeclampsia. The Journal of
Obstetrics and Gyncology of India ,
72-76.
16.The Cleveland Clinic Foundation.
(2013). Urine Protein
(Microalbuminuria/Proteinuria) Test.
Retrieved October 22, 2013, from
Cleveland Clinic:
http://my.clevelandclinic.org/service
s/urine_protein_test/hic_urine_protei
n_microalbuminuriaproteinuria_test.aspx

18.Waldmann-Meyer, H., & Schilling, K.


(1956). The interaction of
bromophenol blue with serum
albumin and gamma-globulin in acid
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19.Yong-Ju Wei, K.-a. L.-Y. (1996). The
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Acidic Solution. Talanta , 1-10.

APPENDIX
A. Photos taken for Test 1
Labels below the photos are in the following format: T<test number>_s<sample
number>_t<trial number>_af

B. Test 1 Pictures taken with Flash


The following photos were taken under the same conditions as the photos in Appendix A but
with flash. It was decided that the photos in Appendix A (without flash) be analyzed because
in some of the following photos, the cuvette reflected the flash, resulting in white dots in the
photos, which would have made the photo more difficult to analyze numerically.

C. Summary of Numerical Data


Given below is a table summarizing the data used in making the graphs. The values in the
table are averages of the three trials.