TOTAL SERUM CHOLESTEROL

2015-03257
Department of Biology, College of Science, University of the Philippines Baguio
November 24, 2017

ABSTRACT

The experiment requires the student to have a grasp of the mechanisms

and principles of the lipid extraction and determination and the ability to
apply the methods used in the experiment to other biological samples.
Blood samples [2-5 mL] were collected using syringes, cooled and
centrifuged thrice for five minutes at 5000 rpm for the first centrifugation
and 10 minutes at 10000 rpm for the last second and third. The
supernatant was then diluted to 8.2 mL with glacial acetic acid. The
Liebermann-Burchard method was used to quantify the total serum
cholesterol and required making five standard solutions [analytical grade
cholesterol dissolved in glacial acetic acid] with concentrations of: 19.17,
27, 36.67, 42.33, and 56.67 measured in mg/dL. Half a milliliter of the
sample and standard solutions were spectrophotometrically analyzed at
640 nm but 2.5 mL of color development mixture (CDM), prepared by
mixing a defined ratio of glacial acetic acid, acid anhydride, sulfuric acid,
and sodium sulfate, was added to the sample and standard solutions
before spectrophotometric analysis. A calibration curve was derived from
the absorbance readings of the standard solutions and the following
equation of the line was derived:

= 0.0002 + 0.0004 Eq. 7.1

The absorbance reading of the blood sample was 2.593 and the
concentration, determined by using a modification of equation 7.1, is 12,
963 mg/dL, multiplying two dilution factors [5.125 for the first and 6 for
the second] the true value is found to be 398, 612 mg/dL identifying the
donor of the sample to be hypercholesteremic based on 200mg/dL for a
normal healthy person. Other blood samples from other groups also
were hypercholesteremic. The results however were determined to be
erroneous due to the possible presence of steroid hormones and failure
to follow the proper procedure during the addition of the CDM to the
standard solutions.
I. INTRODUCTION
Total serum cholesterol is a measure of how much cholesterol is in your blood in milligrams per
deciliter (mg/dL) and it is used in the medical field as in indication of the likelihood of some diseases such
as heat diseases (Sheehan, n.d.), and is also an important component in the cell membranes of living
organisms as it contributes to the mosaic lipid bilayer (Stoker, 2013). Total serum cholesterol can be
defined as the total of the high density lipoprotein, low density lipoprotein, and triglyceride concentration
of the blood (Sheehan, n.d.) but it can also be referred to as the total of the concentration of the amount
of cholesterol and cholesterol esters the structures of both are illustrated in figure 9.1. The normal total
serum cholesterol for normal human is said to be 200mg/dL while anything above or lower than this value
means that a person can be hypercholesteremic or hypocholesteremic, respectively (Sheehan, n.d.).
H 3C CH3 H3C
H 3C CH3
CH3 CH3
CH3
CH3 H3C
O

HO H 3C O

Cholesterol Cholesterol Ester

Figure 9.1 The structures of cholesterol and a cholesterol ester

The method used for the determination of free cholesterol for this experiment was the Liebarman-
Burchard method which makes use of a spectrophotometer and is reliant on a color development mixture
(CDM) (Burke, Diamondstone, Velapoldi, & Menis, 1974).
The student should have a grasp of the underlying mechanisms and principles of lipid extraction
and quantification and should be able to apply he methods used in the experiment to quantify cholesterol
in other biological samples by knowing how to make the necessary and applicable adjustments based on
the understanding of the student of the principles.
II. EXPERIMENTAL PROCEDURE
A medical practitioner was commissioned to extract six 2-5 mL blood samples (one person from
each group) that were collected into dry, clean test tubes and then cooled for 5 minutes in an ice bath.
The blood sample was then divided into several Eppendorf tubes which were then centrifuged for 5
minutes with 5000 rounds per minute (rpm). The supernatant was then collected and transferred into
another set of Eppendorf tubes and centrifuged for 10 minutes for 10000 rpm and then repeated again.
The supernatant was then collected into a labelled test tube and saved for later for spectrophotometric
analysis.
Five standard solutions were prepared to make the standard curve. The five standard solutions
by weighing 11.5 mg, 16.2 mg, 22 mg, 25.1 mg and 31.6 mg of analytical grade cholesterol and
dissolving them with 10 mL glacial acetic acid in 5 test tubes marked 1-5 respectively with 1 for the 11.5
mg sample and 5 for the 31.6 mg sample.
 For the spectrophotometric analysis, 20 mL of color development mixture (CDM) was made by
mixing 12 mL of acetic anhydride (Ac 2O), 6 mL of glacial acetic acid (glacial HoAc), 2 mL of sulfuric acid
(H2SO4), and 0.12 g of anhydrous sodium sulfate (Na2SO4) in a dry beaker placed on an ice bath inside a
fume hood.
Six test tubes were calibrated and marked by wrapping the outside with a masking tape on the
height of the test tube that corresponds to three milliliters. The test tubes were then labeled blank, 1, 2, 3,
4, and 5. Test tubes 1-5 were filled with 0.5 mL of the standards 1-5 respectively while the blank was filled
with 0.5 mL glacial HoAc. All test tubes were then filled with 2.5 mL of the CDM. The resulting solutions
were then spectrophotometrically analyzed using a cuvette and a UV-Vis spectrophotometer at 640 nm
wavelength to construct a calibration curve through linear regression with the help of Microsoft Word
Excel.
The 1.6 mL sample collected was diluted to 8.2 mL of which 0.5 mL was placed in a test tube was
added with 2.5 mL of CDM and then read in a UV-Vis spectrophotometer. The absorbance reading was
then used to determine the total serum cholesterol of the sample using the derived equation from linear
regression.
III. RESULTS AND DISCUSSION
The blood sample collected was cooled to slow down the clotting of the blood (Polderman, 2012)
and facilitate the transfer of the blood samples to the Eppendorf tubes. The several rounds of
centrifugation separated the cholesterol and cholesterol esters from the heavier entities in the blood such
as platelets, red blood cells, white blood cells, and proteins among others. The separation of the latter
entities was crucial since they may cause unintended reactions with the CDM and thus cause
interferences.
The Liebermann-Burchard (L-B) method was used to determine the total serum cholesterol of the
blood sample collected. The L-B method made use a CDM composed of Ac2O, H2SO4, glacial HoAc, and
Na2SO4. The ratios of the components are strictly followed as a variation in the ratios in the components
can significantly alter the sensitivity of the test (Burke, Diamondstone, Velapoldi, & Menis, 1974). The L-B
method is dependent upon the formation of a pentaenylic cation whose reaction is illustrated by figure
9.2.
H 3C CH3 CH3
H 3C CH3
H 3C
H3C H 3C
CH3 CH3 H3C
CH3
CH3 HoAc CH3
Ac2O
CH3 +
H2SO4 SO3
HO +

Figure 9.2 Formaton of Cored Product of Liebermann-Burchard method

The first step involves the protonation of the hydroxyl group of the cholesterol or the OR group
of a cholesterol ester upon reaction of the cholesterol wth glacial HoAc and H 2SO4 which results to the
formation of a 3,5-diene. The product from the preceding undergoes a series of oxidation by Ac 2O and
SO3 released from H2SO4 and produces a cholestopolyene with initially a carbonium ion but due to the
low acidity of the L-B reaction the carbonium ion is converted to a carbocation (Burke, Diamondstone,
Velapoldi, & Menis, 1974). The formation of the carbocation is favored by the increased conjugation of the
cholestopolyene and the stabilizing effect of the terminal cyclopentinyl ring (Burke, Diamondstone,
Velapoldi, & Menis, 1974). The final product of the L-B reaction (refer to figure 9.2) gives the color of the
solution to be read due to the extended conjugation of the cholestopolyene. The same product mentioned
also has the highest absorbance at wavelengths near 620 nm (Burke, Diamondstone, Velapoldi, & Menis,
1974).
In the procedure it was instructed that the glassware should be dry since water has an effect on
the results of the determination. The main trend in all the reasons why water must not be present in the
materials is it is a component of unwanted reactions that will occur at the moment that water is present in
the materials. First, water can affect the ratio of glacial HoAc and H2SO4 in the CDM. The presence of
water will increase the amount of glacial HoAc in the CDM more than what is ideal since water will lead to
the breakdown of the Ac2O which give glacial HoAc as a product. A deviation in the ratio of glacial HoAc
and H2SO4 will result to a change in the maximum absorbance of the analyte in which too much and too
less glacial HoAc will result to having peak absorbances at lower wavelengths (Burke, Diamondstone,
Velapoldi, & Menis, 1974) thus increasing error. Water may also cause esterification of cholesterol with
unwanted species such as the glacial HoAc present in the CDM. This will increase the detected
cholesterol esters in the solution thus increasing the number of milligrams detected for each 0.03 deciliter
sample. However the hydrolysis of cholesterol ester may also occur as a result of the presence of water
which will have the opposite effect to the preceding statement since cholesterols are lighter than
cholesterol esters.
Glacial HoAc was used as solvent for the standard solutions and as diluent for the serum
extracted from the blood samples as not to counteract the reagents needed for the L-B method and yield
optimum results.
The standard curve was constructed using the absorbances of the standard solutions with
increasing concentration to establish and derive a linear equation that expresses the absorbance as a
function of the concentration of a solution and thus provide a way the calculate the concentration of a
solution based on the absorbance of the said solution. Thus table 9.1 displays the data collected from
creating the standard curve and below it (figure 9.3) shows the standard curve.
Table 9.1 Concentration and Absorbance of the Standard Solutions
Cholesterol
solution concentration (mg/dl) Absorbance
(mg)
blank 0 0 0
Standard 1 11.5 19.17 0.0055
Standard 2 16.2 27 0.0063
Standard 3 22 36.67 0.0071
Standard 4 25.1 41.83 0.0105
Standard 5 31.6 52.67 0.0117
 0.015
abs vs conc

absorbance
0.01
Linear (abs vs
0.005 conc)

0 y = 0.0002x + 0.0004
0 20 40 60 R = 0.9596
Concentration (mg/dL)

Figure 9.3 The linear relationship between concentration and absorbance

As we can see in figure 9.3 the derived line is quite reliable as indicated by the R 2 of 0.9572,
which means that the data points from which the line was regressed from approximate the line. The
absorbance reading of the blood serum sample was 2.593. Since the absorbance reading far above the
standard curve the equation of the derived equation of the line (refer to figure 9.3) was used to
extrapolate the concentration of total cholesterol in the blood serum. Applying two dilution factors [one for
the dilution of the blood serum from 1.6 mL to 8.2 mL and another for dilution of a 0.5 mL, taken from the
previous solution, diluted with 2.5 mL of CDM] the total serum cholesterol of the blood serum was
computed to be 398, 612 mg/dL.
Assuming that the other blood samples underwent the process and volumes involved in dilution,
the following table (Table 9.2) shows the total cholesterol of the other blood samples from other groups.
Table 9.2 Total Serum Cholesterol of other groups
Concentration Concentration
group # Absorbance in Analyte in Serum
(mg/dL) (mg/dL)
2 1.1373 5, 684.5 174, 798
3 2.3948 11, 972 368, 139
5 1.4631 7, 313.5 224, 890
As can be seen in table 9.2 and the first result from the preceding statements (398, 612 mg/dL)
all the donors of the blood samples were hypercholesteremic at the time of the testing.
However, the obtained results are abnormally high for all the groups. This is an indication that
there may have been an error in the performed test. One possible source of error is the presence of other
sterols that may be present in the blood such as hormones that may have reacted with the CDM. Sterols
may be in the form of hormones such as testosterone, progesterone, and estradiol among others
(Encyclopaedia Britannica, 2017) which have similar structural properties as cholesterol. The preceding
error may have raised the concentration of detected as an effect being an interference with similar
properties as cholesterol. Another possible source of error resulted when the CDM was dropped a
distance away from the standard solutions. This possibly caused the CDM to stick to the walls of the test
tube or some droplets may have splashed to the side of the test tube. Since there is less CDM to react
with the standard solutions, lesser absorbance readings that do not reflect the actual concentration of the
standard solutions were recorded.
IV. CONCLUSION
It was introduced that total serum cholesterol was used in the medical field as an indicator of
certain aspects of the state of a persons health. Total serum cholesterol was defined as the total
concentration as the total concentration of cholesterol and cholesterol esters in the blood. The total serum
cholesterol for a normal healthy person was indicated as less than or equal to 200 mg/dL. The
Liebermann-Burchard method, a spectrophotometric method, was used in determining the total serum
cholesterol in the experiment.
The blood was extracted by centrifugation to separate the serum from the rest of the blood. The
test involved the making of five cholesterol standards dissolved in glacial HoAc and CDM which is made
up of glacial HoAc, Ac2O, H2SO4 and NaSO4. A calibration curve was made from the data given by the
standard solutions and used to make a linear equation with the absorbance as a function of
concentration. The equation derived was then used to determine that the total serum cholesterol of the
blood sample is 398, 612 mg/dL.
It was that the absorbance readings in the Liebermann-Burchard method is dependent on the
reaction of the CDM with cholesterol and cholesterol esters which eventually leads to highly conjugated
cholestopolyene which exhibits maximum absorbance at wavelengths near 620 nm. Water was
determined to affect the test greatly in three ways: upsetting the glacial HoAc: H 2SO4 ratio which affects
the maximum absorbance, provides possibility for esterification of free cholesterol, and hydrolysis of
cholesterol esters both of the latter upsets the actual weight of total cholesterol per 0.3 dL since both
have different molecular weights.
The obtained total serum cholesterol from the blood sample and the blood samples of the other
groups were all hypercholesteremic compared with a normal healthy person. However the abnormally
high values for total serum cholesterol was attributed to errors performed during the experiment such as
the possibility of the presence of steroid hormones in the blood which may increase the concentration and
the failure to follow the correct procedure correctly pertaining to the addition of CDM to the standard
solutions which may have resulted to the lower absorbance readings of the standard solutions.
Having been able to explain the principles of the methods used in the experiment, it is concluded
that the student has met the objectives of the experiment.
V. LITERATURE CITED

Burke, R., Diamondstone, B., Velapoldi, R., & Menis, O. (1974). Mechanisms for the Lieberman-Burchard
and Zak Color Reactions for Cholesterol. Clinical Chemistry, 20(7), 794-801.
Encyclopaedia Britannica. (2017). Steroid Hormones. Retrieved November 23, 2017, from
https://www.britannica.com/science/steroid-hormone
Polderman, K. (2012). Hypothermia and Coagulation. Retrieved November 23, 2017, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389480/
Sheehan, J. (n.d.). Normal Serum Cholesterol. Retrieved November 23, 2017, from
http://healthyeating.sfgate.com/normal-serum-cholesterol-5334.html
Stoker, S. (2013). General, Organic, and Biological Chemistry. 6th ed., Belmont: Brooks/Cole.
VI. APPENDIX
Sample calculations:
Total serum cholesterol for blood sample of group 1:
= 0.0002 + 0.0004
0.0004
=
0.0002
Using y=absorbance of sample=2.593
2.5930.0004
=
0.0002

= 12, 963 /

Using a dilution factor for the dilution of the sample from 1.6 mL to 8.2 mL and a second dilution from 0.5
mL of the previous dilution to 3 mL:
8.2 3
= 12, 963
1.6 0.5

= 398, 612 /

Thus the total serum cholesterol of the blood sample is 398, 612 mg/dL.