Lab practical report on cholesterol determination

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER: Mr NJOGU STEPHENE/DR OKUN
HANDING DATE: 5 /11/2015
SUBJECT: PRACTICAL REPPORT ON TEST FOR DETERMINATION OF
CHOLESTEROL.
10/30/2015
LANDOELVIS OTIENO
P29S/16344/2015
©

2. Introduction:
Cholesterol is a member of a large group of substances called steroid, which include vitamin D.
Cholesterol is an essential component of cell membrane, brain and nerve cells, and bile, which
helps the body absorb fats and fat soluble vitamins. The body uses cholesterol to make vit.D
and various hormones, such as estrogen, testosterone, and cortisol. The body can produce all
the cholesterol that it is needs, but it also obtains cholesterol from food. The recommended
daily dietary intake is 300mg.
Cholesterol is found in all cells of the body . The adrenal gland contain 6% cholesterol (by
weight). It is a precursor of the adrenal and sex hormones . The brain and spinal cord contain
2% cholesterol :in these tissues cholesterol forms part of the lipid "insulation "which separate
individual nerve fibers . The precise function of cholesterol in all other cells is unknown , but it
is may be related to the structure and permeability of the cell membrance . Cholesterol may be
synthesized from two - carbon units (acetyl - CoA) in many body tissues , particularly liver ,
intestine , and skin . Most cholesterol is excreted from the body via the bile , liver cells oxidize
the molecule by adding hydroxyl and carboxyl groups (the ring structure remain intact )to from
cholic acid. These are excreted in the bile where they are instrumental in the absorption of fasts
, including cholesterol it self, from the diet . Cholesterol serves as a precursor of other steroids ,
bile acids , vitamin D3 biosynthesis . Measurement of total cholesterol includes both the
esterified and free forms of the steroid . In plasma or serum, two - thirds of the total
cholesterol exists in the esterified form , with the rest in the free forms . The measurement of
serum cholesterol is one of the most frequently performed tests in the clinical chemistry .
It is important to do a quantitative analysis of the cholesterol levels in the blood for evaluation
of the coronary arterial occlusion, artherosclerosis, liver function, intestinal absorption and
adrenal disease. Total serum cholesterol is the measurement routinely taken. Doctors
sometimes order a complete lipid profile to better evaluate the risks for atherosclerosis
(coronary artery disease, or CAD).
Method:
CHOD – POD Enzymatic method, End Point. The cholesterol present in the sample originates a
colored complex, the intensity of the color formed is proportional to the cholesterol
concentration in the sample.

3. Principle Theory:
Cholesterol esterases (CHE) hydrolyze the cholesterol esters into free cholesterol. Cholesterol
oxidase (CHOD) oxidizes the cholesterol into cholest-4-en-3-one and hydrogen peroxide.
Hydrogen peroxide reacts with a mix. of 4- aminoantipyrene and phenol in the presence of
peroxidase enzyme (POD) & converts the reactants into a red quinone dye.
-Cholesterol Ester + H2O—cholesterol esterase—→ Cholesterol + Fatty Acids
-Cholesterol +O2—cholesterol oxidase—→ Cholest-4-en-3-one + H2O2
-H2 O2 + 4 aminoantipyrene + Phenol →peroxidase—→ Quinoneimine dye + 4H2O
Absorbance of quinoneimine is directly proportional to the cholesterol concentration, when
measured at 505 nm.
Preparation:
Working reagent; dissolve the contents of one vial R 2 Enzymes in one bottle of R1 Buffer. Cap
and mix gently to dissolve contents. This reagent is stable for 4months at 2 – 8°c or 40days at
15 – 25°c.avoid direct sunlight.
Samples; serum or plasma.
Procedure:
1.Assay conditions,
Wavelength 505nm(500 – 550)
Cuvette 1cmlight path
Temperature 37°c/15-25°c
2.Adjust the instrument to zero with distilled water.

4. 3.pipette into a cuvette:
sample Standard Blank
1.0 1.0 1.0 WR (mL)
-- 10 -- Standard(µL)
10 -- -- Sample(µL)
4.mix and incubate for 5min at 37°c or 10min at room temperature.
5.Read the absorbance of samples and standard, against the blank. The color is stable for
60minutes.
Calculation:
Cholesterol concentration in the sample can be calculated using the following formula:
A(sample)/A(standard) x 200(standard conc:)=mg/dlcholesterol in sample.
Example: If the absorbance of sample is 0.200 and the absorbance of standard is 0.18. The
calculation shall be: 0.200 / 0.180 x 200 = 222.22 mg/dl
Reference value Reference range varies from population to population; therefore, each
laboratory establishes its own normal range.
Normal – less than 200mg/dl
Borderline- 200-239mg/dl
High – 240mg/dl and above.

5. Observations and results:
Conc: of standard 200mg/dl or 5.1mmol/l
Optical density(OD) of standard 0.485
Conc: of sample ?
OD of sample 0.262
OD of blank 0.137
Calculating concentration of sample:
Conc: of sample= 200mg x 0.262/0.485 =108.041mg/dl. Or (108.041mg/dl x
0.259) = 2.798mmol/L.
-Therefore the serum sample cholesterol level is within the normal range
Discusions and clinical importanse:
Hypercholesterolemia : Increases of total serum cholesterol levels are found most
characteristically in the primary hyper lipoproteinaemas type I-V, and in the nephrotic
syndrome, myxoedema , obstructive jaundice and diabetes mellitus , but less consistently and
markedly in some other conditions . In the nephrotic syndrome when oedema is present values
up to (600 - 700 mg/100 ml) are common and may occasionally reach and exceed (1000 mg/100
ml) . Values up to between(400 - 500 mg/100 ml) are found in diabetes mellitus while in
jaundice increases occur most commonly when there is obstruction in the large bile ducts. The
increase roughly parallels the rise in serum bilirubin . Very high values are found in primary
biliary cirrhosis rising to (1000-2000 mg/100 ml) and somewhat smaller rises are associated

6. with drug - induced cholestasis . Values up to (500-700 mg/100 ml) are seen in myxoedema and
the determination may give , useful help in diagnosis . Smaller increases may be found in
hypopituitarism , usually in the range (250- 350 mg/100 ml) . Values between (300-400
mg/100ml) are rather frequent findings in coronary thrombosis and angina pectoris.
Atherosclerosis which refers to the deposition of fatty substances , largely but not entirely
cholesterol , in the walls of the arteries . Statistical studies have shown that the tendency to
develop atherosclerosis and coronary heart diseases is directly related to serum cholesterol
concentration which was increased in all heart diseases.
In pregnancy there is an increase in total cholesterol which may reach to 20-25 percent above
the normal at the 30th week.
Hypocholesterolemia :- Cholesterol is the plasma tend to fall during starvation and as the result
of prolonged debilitating illness. Hyperthyroidism (excess thyroid gland activity) also reduce
serum cholesterol, so that below (100 mg/l00ml) may be found in the severest cases, but this
change is not dose of diagnostic significance . Very low values occurs in abetalipoproteinemia
and to lesser extent in familial hypoabetalipoproteinemia in which the cholesterol level in
extremely low and can be regarded by neurological abnormalities and abnormalities in the
absorption of fats from the intestines. Free cholesterol normally forms about 30% of the total
(ranging from 20 - 40%). While changes in this in diabetes, the nephrotic syndrome and
myxoedema, the percentage of free cholesterol rise in liver disease The tendency when liver
cell damage is present , as in infective hepatitis, is that during the development of jaundice, the
total cholesterol either remains within normal limits or falls a little while the proportion of ester

7. falls appreciably and may reach very low levels . As the condition improves , the total
cholesterol are somewhat raised for a time after the jaundice has disappeared. In severe acute
hepatic necrosis , total cholesterol is usually low and may fall below (100 mg/100 ml) with
concomitant marked reduction in the ester proportion . In extra hepatic obstruction jaundice
although total cholesterol is increased, the ratio of free to ester change little . In primary biliary
cirrhosis the increase , however , is almost entirely in the free cholesterol which may as high as
90% .
Limitations
1.The reagent and sample volumes can be altered proportionately so that the sample:reagent,
ratio remains same.
2. Hemolytic and lipemic samples may result in false elevated results. To avoid false results
sample blank may be used as mentioned above: Add 10 l of serum sample to 1000 l of DI water
and read absorbance at 505nm. Subtract the absorbance obtained as above, from the
absorbance of test. this corrected absorbance is used for calculation. Reagents are sensitive to
light and temperature. Reagents may develop a slight pink color on ageing. This does not
interfere in the results. Quality Control The patient results obtained for each batch can be
validated by using normal and abnormal control sera with assayed values for cholesterol.

8. References:
1. Tietz NW. Clinical guide to laboratory tets, 2nd ed. Saunders Co., 1991.
2. Allain CC, Poon LS, Chan CSG, Richmond W and Fu PC. Enzymatic determination of total
serum cholesterol.Clin Chem 1978; 20: 470 – 475.
3.Daniel Steinberg (2007). The Cholesterol Wars: The Cholesterol Skeptics vs the
Preponderance of Evidence. Boston: Academic Press. ISBN 0- 12 373979-9
4- Varley . H. ; Gowenlock , A.M. and Bell, M. (1980) In "Practic Clinical Biochemistry " 5th ed.
Vol. I, WilliamHeinemann Medic Books LTD . , London .
5- Tietz , W.N. (1982) In "Fund, of Clinical Chemmistry " 2nd . ed| W.B. Saunders Co.,
Philadelphia .
6- Kaplan , L.A. and pesce , A.J. (1984) In " Clinical Chemistry " 1st , C.V. Mosby Co., St. Louis
7.Kenyatta university-laboratory-practical-manual- for-MBCHB-Year 1-semester1.