N.S.

BIO-TEC

CHOLESTEROL (CHOD-PAP)
Enzymatic Colorimetric Determination of Serum Cholesterol

INTENDED USE REAGENTS

NS Biotec cholesterol reagent is intended for the in vitro quantitative
R1 Cholesterol standard 200 mg/dl
determination of total cholesterol in serum and plasma on both
automated and manual systems. Pipes buffer,
R2
pH 6.9 90 mmol/l
CLINICAL SIGNIFICANCE Phenol 26 mmol/l
Cholesterol is a steroid with a secondary hydroxyl group in the C3 position, Cholesterol oxidase 500 U/l
and found in blood, bile, and brain tissue. It serves as a precursor to bile Cholesterol esterase 500 U/l
acids, steroids and vitamin D. It is synthesized in many types of tissue, but Peroxidase 1250 U/l
4-Aminoantipyrine 0.4 mmol/l
particularly in the liver and intestinal wall. Approximately, 75% of cholesterol
is newly synthesized and a 25% originates from dietary intake. Reagent Preparation & Stability
All reagents are ready for use and stable up to the expiry date given
Measurements of serum cholesterol levels are important in the diagnosis 0
on label when stored at 2–8 C.
and classification of hyperlipoproteinemias. Elevated cholesterol levels may
occur with hypothyroidism, nephrotic syndrome, diabetes, and various liver SPECIMEN
diseases. There is a correlation between elevated serum cholesterol levels • Serum, or plasma*.
and the incidence of coronary artery diseases. Normal cholesterol levels are • The only acceptable anticoagulants are heparin and EDTA.
affected by stress, diet, age, gender, hormonal balance, and pregnancy.
Specimen Preparation & Stability
Depressed levels are associated with hyperthyroidism and severe liver No special preparation of the patient is necessary, however it is
1, 2
diseases . recommended that prior to collection, patients should be following
their usual diet and be in their usual state of health. Patients who are
ASSAY PRINCIPLE acutely ill, losing weight, pregnant or have had a myocardial infarction
Cholesterol analysis was first reported by Liebermann in 1885 followed by in the previous 3 months should be rescheduled.
Burchard in 1889. In Liebermann-Burchard reaction, cholesterol forms a
blue-gree dye from polymeric unsaturated carbohydrates in an acetic Blood should be collected by venipuncture, after the patient has been
acid/acetic anhydride/concentrated sulfuric acid medium. The Abell and in a seated position for at least 5 minutes. Tourniquet usage should
Kendall method is specific for cholesterol but is technically complex and be kept to a minimum and the specimen should be allowed to clot for
12
9
requires corrosive reagents. In 1974, Allain et al and Roeschlau et al
10 30 minutes at room temperature .
were able to combine cholesterol esterase and cholesterol oxidase into a The best specimen is unhemolysed serum, and should be analyzed
single enzymatic reagent for the determination of total cholesterol. NS 0
on the day of collection. When stored at 4 C, specimens are stable for
Biotec cholesterol reagent combines the use of these enzymes with the 0 2
3-4 days; specimens are stable at –20 C for several months .
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peroxidase/phenol/4-aminoantipyrine system of Trinder for the
measurement of total cholesterol in human serum. PROCEDURE
The series of reactions involved in the assay system are as follows: • Manual Procedure
Wavelength 500 - 550 nm
1. Cholesterol esters are enzymatically hydrolyzed by cholesterol
esterase (CE) to cholesterol and free fatty acids. Cuvette 1 cm light path
0
2. Free cholesterol, including that originally present, is then oxidized Temperature 20-25 or 37 C
by cholesterol oxidase (CHOD) to cholest-4-en-3-one and H2O2. Zero adjustment against reagent blank
3. In presence of peroxidase (POD), the formed hydrogen peroxide Specimen Serum or plasma
formed effects the oxidative coupling of phenol and 4-
aminoantipyrine (4-AAP) to form a red-colored quinoneimine dye.
CHOD Blank Standard Specimen
Cholesterol esters+H2O Cholesterol + fatty acids
CE
Cholesterol +O2 Cholest-4-ene-3-one+H2O2 R2 1.0 ml 1.0 ml 1.0 ml
POD
2 H2O2+ 4-AAP + Phenol Quinoneimine dye + 4 H2O
Standard …… 10 µl ……
Specimen …… …… 10 µl
The intensity of the color produced is directly proportional to cholesterol
concentration. It is determined by measuring the increase in absorbance at
500 – 550 nm. 0 0
Mix, incubate for 5 minutes at 37 C or 10 minutes at 20-25 C. Measure
the absorbance of specimen (Aspecimen) and standard (Astandard) against
EXPECTED VALUES reagent blank.
The color is stable for 60 minutes.
Risk classification Total Cholesterol
• Automated Procedure
Desirable <200 mg/dl (5.2 mmol/l)
Borderline high 200 – 239 mg/dl User defined parameters for different auto analyzers are available
(5.2 – 6.2 mmol/l) upon request
High >240 mg/dl (6.2 mmol/l) CALCULATION
Calculate the cholesterol concentration by using the following
formulae:
Each laboratory should investigate the transferability of the expected
values to its own patient population and if necessary determine its own Cholesterol Concentration=
reference range. For diagnostic purposes, the cholesterol results
Absorbance of Specimen
should always be assessed in conjunction with the patient’s medical X Standard value
history, clinical examination, and other findings. Absorbance of Standard
 • Unit conversion • Valid results depend on an accurately calibrated instrument, timing,
mg/dl x 0.0259 = mmol/l and temperature control.
LINEARITY • The reagent blank will not exceed an absorbance of 0.06 but don’t use the
When run as recommended, the assay is linear up to 800 mg/dl reagent if it is turbid or if the absorbance is greater than 0.2 at 500 nm.
(20.7 mmol/l).
If result exceeds 800 mg/dl (20.7 mmol/l), specimen should be BIBLIOGRAPHY
diluted with 0.9% NaCl solution and reassayed. Multiply the result by
1. Searcy, RL (1969): Diagnostic Biochemistry. McGraw-Hill, New York, NY.
the dilution factor.
2. Ellefson, RD and Garaway, WT (1976): Fundamentals of clinical
SENSTIVITY chemistry. Ed Tietz NW, p 506.
The sensitivity is defined as the change of analytical response per unit 3. Lieberman, C. (1885): Ber. 18: 1803.
change in analyte concentration at a pathlength of 1 cm.
4. Burchard, H (1890): Chem. Zentr. 61: 25.
When run as recommended the sensitivity of this assay is 3.0 mg/dl
5. Flegg, HM (1973): Ann. Clin. Biochem. 10: 79.
(0.08 mmol/l).
6. Richmond, W (1972): Scand. J. Cal. Invest. 29 (Suppl): 126.
QUALITY CONTROL 7. Hernandez, HH and Chaikoff, IL (1957): J. Biol. Chem. 228: 447.
It is recommended that controls (normal and abnormal) be included in: 8. Hyun, J, et al., (1969): J. Biol. Chem. 244: 1937.
• Each set of assays, or
9. Allain, CC, et al., (1974): Clin. Chem. 20: 470.
• At least once a shift, or
• When a new bottle of reagent is used, or 10. Roeschlau, P, Bernt, E and Gruber, F (1974): Clin. Chem. Clin.
Biochem. 12: 226.
• After preventive maintenance is performed or a clinical component
is replaced. 11. Trinder, P (1969): Ann. Clin. Biochem. 6: 24.
Commercially available control material with established cholesterol 12. Henry, RJ (1974): Clinical Chemistry: Principles and Techniques.
values may be routinely used for quality control. Harper & Row, Hagerstown, MD.
Failure to obtain the proper range of values in the assay of control material 13. Young, DS (1990): Effects of Drugs on Clinical Laboratory Tests. Third
may indicate: Edition. 1990: 3: 6-12.
• Reagent deterioration, 14. Young, DS, et al., (1975): Clin. Chem. 21: 5.
• Instrument malfunction, or
• Procedure errors.
The following corrective actions are recommended in such situations:
• Repeat the same controls.
• If repeated control results are outside the limits, prepare fresh
control serum and repeat the test.
• If results on fresh control material still remain outside the limits,
then repeat the test with fresh reagent.
• If results are still out of control, contact NS Biotec Technical
Services.
INTERFERING SUBSTANCES
• Anticoagulants
The only acceptable anticoagulants are heparin and EDTA.
• Bilirubin:
Bilirubin levels higher than 7.5 mg/dl decrease the apparent total
cholesterol concentration significantly.
• Drugs:
Methyldopa causes artificially low total cholesterol values at the
tested drug level. For a more comprehensive review of drugs
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affecting cholesterol assays refer to the publication by Young .
• Haemoglobin:
No interference from haemoglobin up to a level of 500 mg/dl.
• Lipemia:
No significant interference.
• Others:
Ascorbic acid levels higher than 7.5 mg/dl decrease the apparent
total cholesterol concentration significantly.
Other 3-beta-hydroxysteroids cause positive interference but are
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not normally present in significant quantities in human serum .
WARNING & PRECAUTIONS
• NS Biotec cholesterol reagent is for in vitro diagnostic use only.
Normal precautions exercised in handling laboratory reagents should
be followed.
• Warm up working solution to the corresponding temperature before
use.
• The reagent and sample volumes may be altered proportionally to
accommodate different spectrophotometer requirements.