Experiment 1: Estimation of Total Serum Cholesterol by Zak and Henlys Method

2014, 201428982, 201435733

Group 3, Biochemistry 35.1, MEG
Instructor: Ms. Ciara Christianne Lim
Date submitted: January 30, 2017 Monday

I. Abstract
Cholesterol, a steroid alcohol, regulates the fluidity of plasma membrane so right amount of
cholesterol in the body must be maintained because too much or too low amount of it may
cause different types of diseases. This study aims to quantify cholesterol level of serum
samples and compare it with clinically established cholesterol level value. To quantify the
amount of cholesterol in serum sample, a standard calibration curve was obtained by
preparing different concentrations of cholesterol and measuring its absorbance known as
Zak and Henlys method. From the equation obtained, it was found that the serum 1 and
serum 2 contains 0.921 and 0.821 mg/dL cholesterol, respectively.

II. Keywords cholesterol

III. Introduction
Cholesterol is a steroid alcohol found in
abundance in animals. It has a branched
aliphatic side-chain and OH group
attached to the C17 and C3 of the steroid
nucleus, making it an amphiphatic
molecule (Voet & Voet, 2011). Its structure
is shown on figure 1.
artheroscelerosis and coronary heart
disease (Carmena, MD, Duriez, PhD, &
Fruchart, PhD, 2004). Low levels of
cholesterol in the blood have been
connected to an increased risk for liver
and pancreatic cancer, hepatic cirrhosis,
suicide and alcohol dependency syndrome
(Neaton, PhD, Blackburn, MD, Jacobs, PhD,
& Lee, MD, 1992). It is therefore important
to be able to quantify the total serum
cholesterol in order for one to assess
whether or not their blood cholesterol is
within safe levels.

The objectives of the experiment are to

Figure 1. The structure of cholesterol (Voet & Voet,
2011)
Cholesterol functions in the regulation of
the fluidity of animal plasma membranes.
It is also the precursor to steroid
hormones, which are important regulators
of several physiological functions, such as
the development of secondary sex
characteristics (Voet & Voet, 2011).
Cholesterol, however, is widely known for
the health hazards attributed to it. High
levels of cholesterol in the blood, a
condition called hypercholesterolemia,
increases the risk for the development of
cardiovascular diseases such as
Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
Page 1 of 6
prepare test serum samples from blood, to
generate a standard calibration curve for
cholesterol, to quantify the cholesterol
level of the test serum samples and to
assess and compare the cholesterol level
with clinically established values.
IV. Experimental
A Blood Serum Preparation
Ten milliliters of whole blood was
incubated at room temperature for 15-
30 minutes. Coagulated masses/ Blood
clots were then removed via
centrifugation at 1500 xg for 10
minutes. *The supernatant (serum)
was transferred into a falcon tube and
 any turbid samples were further
centrifuged and had their supernatant
collected. Aliquots of 0.5 mL were
placed in microcentrifuge tubes and
stored at -80C.
B Generation of the Cholesterol Standard
Calibration Curve
//The cholesterol stock was prepared
by dissolving enough cholesterol in
chloroform to reach 1mg/mL
concentration. The ferric chloride
solution was prepared by dissolving
enough of Kilianis reagent with glacial
acetic acid to form a 1% v/v solution.//
**this can be replaced by:
mixtures of the 1mg/ml cholesterol
stock and 1% v/v ferric chloride
solutions, with volumes shown on
***Im not sure which would be better
though
Mixtures of the cholesterol stock and
ferric chloride solution, with volumes
shown on Table 1, were allowed to
stand at room temperature for 15
minutes.
Table 1 The volumes of the cholesterol stock
and ferric chloride solution mixed for the
generation of the standard curve
Std Cholesterol Ferric
No. Stock (mL) Chloride (mL)
1 0.00 5.00
2 0.05 4.95
3 0.10 4.90
4 0.20 4.80
5 0.30 4.70
6 0.40 4.60
Solutions which had precipitation were
centrifuged at 2000 xg for 15 minutes,
and had their supernatant collected.
Three milliliters of concentrated
sulfuric acid were added to each of the
solutions. The solutions were incubated
at room temperature for 30 minutes.
The absorbances of the solutions were
measured at 560 nm, and the standard
calibration curve was constructed from
this data.
Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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C Total Serum Cholesterol Measurement
A 0.05 mL aliquot of test serum sample
was mixed with 4.95 mL ferric chloride
reagent to form a 1% v/v serum
solution. This was done in duplicate.
The solutions were allowed to stand for
15 minutes, with occasional mixing.
(Charles, pasabi na lang ditto na inulit
natin yung ginawa. Hehe. Di ko
maformulate ng maayos kung ano
sasabihin e hahaha. Salamat)
Solutions which had precipitation were
centrifuged at 2000 xg for 15 minutes,
and had their supernatant collected.
Three milliliters of concentrated
sulfuric acid were added to each of the
solutions. The solutions were incubated
at room temperature for 30 minutes.
The absorbances of the solutions were
measured at 560 nm.
*** copy-paste lang to from the
previous part. Tell me if kailangan kong
ireword for this part
The cholesterol level (in mg/dL) of the
test sample was determined from the
standard calibration curve *previously
constructed.
V. Results and Discussion
Humans intake cholesterol-rich food in
their day to day lives. In fact, cholesterol
is needed for the structural components in
biological membranes and as a precursor
in the production of several steroids and
hormones as well as uses in hormone
signalling. However, too much or too little
cholesterol in blood may lead to several
complications (Campbell & Farrell, 2010;
Cox & Nelson, 2013).
Blood was obtained an hour prior to the
experiment. This was done to ensure that
the blood has sufficient time to coagulate.
The blood was allowed time to coagulate
as the specimen needed for the
experiment was the blood serum. Blood
serum is essentially blood plasma after it
undergoes adequate coagulation. Blood
serum is preferred over plasma as
Cholesterol Concentration ( mgdL )= Volume
Total Volume
(m
Stock Cholesterol Sol' n

Mixture ( mL

interference from proteins and ions are (Eq. 1)

minimized, both of which may affect the
results of biochemical analysis. The blood
serum sample was aliquoted to Sample computation for cholesterol
microcentrifuge tubes in 0.5mL volumes in concentration for Standard # 2,
order to prevent contamination and stored
in a refrigerator prior to use. The protein 0.05mL 1 mg 100 mL mg
fractional values of serum varies greatly =0.625
8 mL 1 mL 1 dL dL
as to what temperature it is stored.
Storage of serum at a temperature of 5 O Paa=lagay ng introduction dito about doon sa
10OC would ensure a negligible change in table. Salamat hehe.
protein fraction over a period of seven
Di ko gets Meh - Beah
days (Guder, et al., 2002; Kawai, 1973).
Guder, et al. (2002) reported that High Concentration vs. Absorbance Calibration Curve
Density Lipoproteins and Low Density
Lipoproteins would be stable for 7 days 0.990
within the temperature range of 4 O 8OC 0.790
and lesser at 20O 25OC. f(x) = 0.18x - 0.13
0.590
R = 0.89
Absorbance (560 nm)
After sample preparation, cholesterol 0.390
standard calibration curve was plotted.
0.190
The table below shows the absorbances
and their corresponding cholesterol -0.010
concentrations of the cholesterol standard 0.000 5.000
calibration curve as well as that of the [Cholesterol] in mg/dL
serum samples.

Table 1. Absorbances and their Corresponding Figure 1. Cholesterol Standard Calibration Curve.
Concentrations of the Cholesterol Standard
Calibration Curve and Serum Samples.
The obtained line equation (2) was
Average y=0.184 x 0.132
[Cholestero (Eq. 2)
Std. # Absorbance
l] (mg/dL)
(A)
1 0.000 0.000
2 0.625 -0.003 with an R value of 0.8871. Using
3 1.250 0.051 Equation 2, the total serum concentration
4 2.500 0.120 can be calculated.
5 3.750 0.539
6 5.000 0.917 Sample computation of total serum
Serum 1 0.921 0.038 cholesterol of Serum sample 1 using Eq. 2,
Serum 2 0.821 0.019
A=0.184[Cholesterol]0.132
Cholesterol concentration (in mg/dL) of
the standard calibration curve can be A +0.132
determined using Equation 1,
[ Cholesterol ] =
0.184

[ Cholesterol ] dilution factor=Total Serum Cholesterol

Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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 mg 8.00 mL
0.921
dL 0.05 mL

mg
147.4
dL

Table 2. Total Serum Cholesterol of the Serum

Samples.
Total Serum
Sampl [Cholestero
Cholesterol
e# l] (mg/dL)
(mg/dL)
1 0.921 147.4
2 0.821 131.3

Total serum cholesterol (TSC) was
estimated using the Zak and Henlys
method. Zak and Henlys method involves
a colorimetric reaction between
cholesterol and the ferric ions. Cholesterol
reacts in the presence of sulphuric acid to
form 3, 5 cholestadiene (Figure 3) due to
a dehydration reaction. In the presence of
ferric (Fe3+) ion, 3, 5-cholestadiene
undergoes oxidation and is then
sulphonated to form the red/purple-
colored cholestapolyene sulphonic acid
(reaction pathway shown in Figure 4).
Absence of ferric ions will result to a
solution that is green-colored, which is the
principle behind of the Liebermann
Burchard reaction. The intensity of the
color is dependent upon the amount of
cholesterol present in the serum
(Prasannan, Rajan, & Ramakrishnan, 2004;
Shivaraja Shankara, 2008).
Figure 3. Products of Cholesterol that underwent
the LiebermannBurchard Reaction (Xiong, Wilson,
& Pang, 2007).
Errors in this method of analysis may
arise due to several factors. First is due to
the interference of several compounds.
Several examples are bromide, bilirubin,
tryptophan, and nitrate. It was studied
that bromide would give a positive error as
it enhances the color reaction with ferric
chloride resulting to a darker solution than
what is true. On the other hand, bilirubin is
oxidised by a ferric chloride reaction to
form biliverdin that shows a minimum in
spectral characteristics as well as lowered
absorptivity resulting to a negative error.
Tryptophan may interfere with the analysis
given that glyoxal is present due to the
resulting Hopkins-Cole reaction but
purification of acetic acid can be used to
prevent this. Interference of nitrate may
also be presumed however no further
information about how or why this
happens was available (Zak, 1965).

In blood, 30% of cholesterol exists in its

Figure 2. Structure of 3, 5-cholestadiene.
(Retrieved
http://www.pherobase.com/pherobase/gif/3,5-
cholestadiene.GIF.)
free form while the rest exists in its
esterified form. Regulation and monitoring
of cholesterol concentration in the body
especially of that in the blood is important
in order to prevent several diseases.
from
Normal serum cholesterol levels for young
adults are usually in the range of 150-220
mg/dL though it is preferable to have a

Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
Page 4 of 6
concentration below 200 mg/dL (Shivaraja
Shankara, 2008).
Too little serum cholesterol
(Hypocholesterolemia) may result in
anemia, hyperthyroidism, hemolytic
jaundice, etc. Hypocholesterolemia is
often a symptom of a serious underlying
problem rather than a disease itself.
Currently, there is still no definable
cholesterol level below which clinically
significant hypocholesterolemia is
diagnosed. Most agree on diagnosing
hypocholesterolemia to those levels below
150 mg/dL. Others, to levels below 100
mg/dL. Total serum cholesterol of the
sample was found to be below 150 mg/dL
however this may not signify that the
sample donor is hypocholesterolemic as
standard TSC is also affected by gender
and race. Nevertheless, awareness of the
condition may ensure a healthier lifestyle
and prevention of any serious diseases
(Elmehdawi, 2008; Shivaraja Shankara,
2008).
On the other hand, too much serum
cholesterol or Hypercholesterolemia may
lead to uncontrollable Diabetes mellitus,
atherosclerosis and coronary heart disease
(Shivaraja Shankara, 2008).
Table 3. Total Serum Cholesterol Standards
(Dominiczak, Rifai, & Warnick, 2000).
Treatment of hypercholesterolemia
usually include the intake of drugs which
have statins. Statins are inhibitors of 3-
hydroxy-3-methylglutarylcoenzyme A method should be an effective way on
(HMG-CoA) reductase, an enzyme that
converts HMG-CoA into mevalonic acid
which is a cholesterol precursor.
Additionally, they also change the
conformation of the enzymes active site
rendering it unusable to the normal
Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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substrate. This process is further
enhanced due to the statins having an
affinity with the enzyme in the nanomolar
range versus that of the normal
substrates affinity which in the millimolar
range. Furthermore, it also increases the
activity of hepatic LDL receptors which
helps lead to the reduction of circulating
LDL and its precursors (Sima & Stancu,
2001).
VI. Conclusions and
Recommendations
In this experiment, Zak and Henlys
method was used to create a standard
calibration curve to quantify the amount of
cholesterol in serum sample. It is
important to monitor the amount of
cholesterol in blood because too low or too
much amount of it causes several types of
diseases to arise like
hypercholesterolemia.
It is recommended to perform other
tests in measuring cholesterol level in
serum like enzymatic colorimetric method
that uses cholesteryl ester hydrolase
(Simpson, 2012). Another possible
technique that can be used is the alcohol-
acetone technique (Zak, 1965).
The ferric-chloride solution is
unstable at room temperature thus the
color-formation capacity of the reagent is
being lessen. This can be prevented by
adding Rosenthal, or using ferric
ammonium chloride dissolved in 80%
acetic acid. Bromide and tryptophan
causes interference by enhancing the
color of the complex that will yield to a
higher reading in the spectrometer. To
eliminate these interferences, serum must
be treated with ethanol and alkali and will
be extracted with chloroform (Zak, 1965).
Theoretically, Zak and Henleys
measuring cholesterol in total serum
samples but due to some errors that arise,
cholesterol level was not accurately
measured.
Tanong lang, kailangan ba ilagay ko
equation, seru cholsetreol, at
icompare sa standard?
VII. References
Campbell, M. K., & Farrell, S. O. (2010).
Biochemistry (Seventh ed.). CA:
Cengage Learning: Brooks/Cole.
Cox, M. M., & Nelson, D. L. (2013).
Lehningers Principles in
Biochemistry (Sixth ed.). NY: W. H.
Freeman and Company.
Dominiczak, M. H., Rifai, N., & Warnick, G.
R. (2000). Handbook of Lipoprotein
Testing (Second ed.). DC,
Washington: Amer. Assoc. for
Clinical Chemistry.
Elmehdawi, R. R. (2008). Hypolipidemia: A
Word of Caution. Libyan Journal of
Medicine, 3(2), 84-90.
Guder, W., Ehret, W., Heil, W., Darmstadt,
Y. S., Tpfer, G., Wisser, H., & Zawta,
B. (2002). Use of anticoagulants in
diagnostic laboratory investigations
& stability of blood, plasma and
serum samples. WHO/DIL/LAB/99.1
Rev.2.
Kawai, T. (1973). Clinical Aspects of The
Plasma Proteins (First ed.). New
York, USA: Springer Science &
Business Media.
Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
Page 6 of 6
Prasannan, K. G., Rajan, R., &
Ramakrishnan, S. (2004). Textbook
of Medical Biochemistry (Third ed.).
Hyderabad, India: Orient Blackswan.
Shivaraja Shankara, Y. M. (2008).
Laboratory Manual for Practical
Biochemistry (First ed.). New Delhi,
India: Jaypee Brothers Medical
Publishers.
Sima, A., & Stancu, C. (2001). Statins:
mechanism of action and effects.
Journal of Cellular and Molecular
Medicine, 5(4), 378387.
Simpson, W. (2012). Cholesterol (blood,
plasma, serum). Retrieved from
Association for Clinical Biochemistry
2012: http://www.acb.org.uk/Nat
%20Lab%20Med
%20Hbk/Cholesterol.pdf
Xiong, Q., Wilson, W. K., & Pang, J. (2007).
The LiebermannBurchard Reaction:
Sulfonation, Desaturation, and
Rearrangment of Cholesterol in
Acid. Lipids, 42(1), 8796.
Zak, B. (1965). Total and Free Cholesterol.
Standard Methods of Clinical
Chemistry, 5, 7989.