DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404

DATE:
17 JANUARY 2007
TITLE:
• MEASUREMENT OF LIPID
AIMS & OBJECTIVES:
• To understand the principles involved in cholesterol measurement
• To measure the cholesterol level in the samples given.

MEASUREMENT OF CHOLESTEROL
• CHOD-PAP method

○ Test principle
Cholesterol esterase
Cholesterol ester + HbO ---------------------► cholesterol + RCOOH (fatty acid)

Cholesterol oxidase
Cholesterol + 02 _______________► Cholesteroi-3-one + H202

Peroxidase
2H2O2 + 4-aminophenazone + phenol -----------► 4 - (p-benzoquinone - monoimino) -
phenazone + 4H20

SAMPLE:
• Serum, heparinized plasma or EDTA plasma
PROCEDURES:
• Wavelength: 500nm
• Cuvette: 1 cm light path

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 • Incubation temperature: 20-25°C or 37°C
• Measure against reagent blank
• One reagent blank is sufficient for each assay series.

Pipette into test tubes

Blank Sample/control/standard
Distilled water 0.01 ml -
Sample material - 0.01 ml
Cholesterol Reagent 1.00 ml 1.00 ml

• Mix, and incubate blank and sample for 20 min at 20-25°C

• Or 10 min at 37°C.
• Read absorbance of sample against reagent blank within 1 hour.
RESULTS:
TUBES ABSORBANCE READING
NORMAL CONTROL 0.150
STANDARD 0.232
SAMPLE 1 0.201
SAMPLE 2 0.242

CALCULATIONS:

Cholesterol (mg/dl) = A sample / A Std. X Conc. Std. (mg/dl)

Cholesterol (mmol/l) = Cholesterol (mg/dl) X 0.02586

• Concentration Standard (mg/dL) : 200 mg/dL

• Standard Absorbance : 0.232

TUBES CALCULATION
NORMAL CONTROL  Cholesterol (mg/dL)
○ A sample / A Std. X Conc. Std.

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 (mg/dl)
○ 0.150 /0.232 X 200
○ 129.31 mg/dL
 Cholesterol (mmol/l)
○ Cholesterol (mg/dl) X 0.02586
○ 129.31 X 0.02586
○ 3.34 mmol/L
 Cholesterol (mg/dL)
○ A sample / A Std. X Conc. Std.
(mg/dl)
○ 0.201 /0.232 X 200
SAMPLE 1 ○ 173.28 mg/dL
 Cholesterol (mmol/l)
○ Cholesterol (mg/dl) X 0.02586
○ 173.28 X 0.02586
○ 3.34 mmol/L
 Cholesterol (mg/dL)
○ A sample / A Std. X Conc. Std.
(mg/dl)
○ 0.242 /0.232 X 200
SAMPLE 2 ○ 208.62 mg/dL
 Cholesterol (mmol/l)
○ Cholesterol (mg/dl) X 0.02586
○ 208.62 X 0.02586
○ 5.39 mmol/L

CONTROL RANGE:
• Cholesterol: 164.35 +/- 14.76 (2SD) mg/dL.

DISCUSSION:

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 On this experiment, method CHOD-PAP is for the quantitative
determination of total cholesterol in serum and plasma. The CHOL method is
based on the principle first described by Stadtman. Cholesterol esterase (CE)
catalyzes the hydrolysis of cholesterol esters to produce free cholesterol which,
along with preexisting free cholesterol, is oxidized in a reaction catalyzed by
cholesterol oxidase (CO) to form cholest-4-ene-3-one and hydrogen peroxide.
In the presence of horseradish peroxidase (HPO), the hydrogen peroxide
thus formed is used to oxidize N,N-diethylaniline-HCI/4-aminoantipyrine (DEA-
HCI/AAP) to produce a chromophore that absorbs at 500 nm. The absorbance is
due to oxidized DEA-HCI/AAP and directly proportional to the total cholesterol
concentration.
TUBES INTERPRETATION
• The result shown low than
NORMAL CONTROL
cholesterol range.
• The result shown high than
SAMPLE 1& SAMPLE 2
cholesterol range.

Below are the list interferences or error during the experiment.

INTERFERENCES & ERROR
BILIRUBIN • Bilirubin (conjugated) of 8.1
mg/dL [139 umol/L] and
bilirubin (unconjugated) of 9.4
mg/dL [161 nmol/L] decrease
the CHOL result by 15 mg/dL
[0.4 mmol/L] at CHOL
concentration of 150 mg/dL [3.9
mmol/L].
• Bilirubin (conjugated) of 12.8
mg/dL [219 umol/L] and
bilirubin (unconjugated) of 14.7
mg/dL [251 nmol/L] decrease
the CHOL result by 25 mg/dL
[0.7 mmol/L] at CHOL
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 concentration of 250 mg/dL [6.5
mmol/L].
• The following substances have a
negligible effect (<10 mg/dL [0.3
mmol/L]) on the CHOL method
at the concentrations.
○ Acetaminophen
○ Ampicillin
○ Diazepam
○ Digoxin
○ EDTA
DRUGS
○ Ethanol
○ Gentamicin
○ Hemoglobin
○ Lipemia
○ Nortriptyline
○ Phenobarbital
○ Phenytoin
○ Salicylate
○ Theophylline
• Interference from the statins at

the concentrations indicated,

when added to a 151 mg/dL [3.9
mmol/L] CHOL serum pool
STATINS
respectively, is less than 10%.
○ Atorvastatin
○ Lovastatin
○ Pravastatin
ANTICOAGULANT • Potassium Oxalate/Sodium
Fluoride can decrease cholesterol
results an average of 12%.
• Li Heparin can depress
cholesterol results by an average
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 of 4 mg/dL [0.1 mmol/L] at a
level of 200 mg/dL [5.2
mmol/L].
• Date due of the reagent.
• Not fresh samples.
REAGENT & SAMPLE
• Incubation time not correct,
either prolong or shorten.
• Pipetting error.
HUMAN ERROR
• Sample tubes not mixing well.

MEASUREMENT OF HDL-CHOLESTEROL

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 • Test principle
Chylomicrons VLDL and LDL are precipitated by adding
phosphotungstic acid and magnesium ions to the sample. Centrifugation
leaves only the HDL in the supernatant; their cholesterol content is
determined enzymatically.
• Sample material
○ Serum
○ Heparinized plasma
○ EDTA plasma
* Serum must be separated from the blood clot as rapidly as possible.
• Sample preparation (Semi micro assay)
○ Precipitation
○ Mix, and let stand for 10 min at room temperature, then centrifuge:
 15 min at 4000 rpm or more
 2 min at 12000 rpm.
○ After centrifugation separate the clear supernatant within 2 hours and
determine the cholesterol content by the CHOD-PAP method.
○ The supernatant may be stored for up to five days at +4 to 25°C.
Pipette into test tubes
Sample/control/standard 0.2 ml
Precipitant 0.2 ml
○ Mix, and let stand for 10 min at room temperature, then centrifuge:
 15 min at 4000 rpm or more
 2 min at 12000 rpm.
○ After centrifugation separate the clear supernatant within 2 hours and
determine the cholesterol content by the CHOD-PAP method.
○ The supernatant may be stored for up to five days at +4 to 25°C.

• Assay procedure
○ Determination of cholesterol using the CHOP-PAP method.
 Wavelength: 500nm.
 Cuvette: 1 cm light path.
 Incubation temperature: 20-25°C or 37°C.
 Measure against reagent blank.
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  One reagent blank is sufficient for each assay series.

Pipette into test tubes

Blank Sample/control/standard
Distilled water 0.01 ml -
Sample material - 0.01 ml
Cholesterol Reagent 1.00 ml 1.00 ml

• Mix, and incubate blank and sample for 10 min at 20-25°C or 5 min at 37°C.
• Read absorbance of sample against reagent blank within 1 hour.
CALCULATIONS:
• Calculation of HDL cholesterol
HDL-C(mg/dl) = A sample / A Std. X Conc. Std. (mg/dl)

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 • Calculation of LDL cholesterol mg/dL
LDL cholesterol= total cholesterol – triglycerides / 5 - HDL cholesterol
• Calculation of LDL cholesterol mmol/L
LDL cholesterol= total cholesterol – triglycerides / 2.2 - HDL cholesterol

• Concentration Standard (mg/dL) : 200 mg/dL

• Standard Absorbance : 0.232

PRECAUTION:
• The values obtained are reliable, provided that;
○ No chylomicrons are present in the sample.
○ The triglyceride concentration does not exceed 400 mg/dl
○ The sample does not show signs of type III hyperlipoproteinemia.
NOTES:
• The values obtained are reliable, provided that;
○ The supernatant obtained on centrifugation must be clear.
○ If the sample has a high triglyceride content (above 1000 mg/dl),
lipoprotein precipitation may be incomplete (cloudy supernatant), or part of
the precipitate may float on the surface.

○ In these cases, dilute the sample 1 + 1 with 0.9% NaCI solution and repeat
the precipitation step.
○ The result of the cholesterol assay must then be multiplied by 2.

RESULTS:
TUBES ABSORBANCE READING
NORMAL CONTROL 0.294
STANDARD 0.787
SAMPLE 1 0.125
SAMPLE 2 0.119

TUBES CALCULATION
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  HDL-CHOLESTEROL (mg/dL)
○ A sample / A Std. X Conc. Std.
(mg/dl)
○ 0.294 /0.787 X 200
NORMAL CONTROL ○ 74.71 mg/dL
 HDL-CHOLESTEROL (mmol/l)
○ HDL-Cholesterol (mg/dl) X 0.02586
○ 74.71 X 0.02586
○ 1.93 mmol/L
 HDL-CHOLESTEROL (mg/dL)
○ A sample / A Std. X Conc. Std.
(mg/dl)
○ 0.125 /0.787 X 200
SAMPLE 1 ○ 31.77 mg/dL
 HDL-CHOLESTEROL (mmol/l)
○ HDL-Cholesterol (mg/dl) X 0.02586
○ 31.778 X 0.02586
○ 0.82 mmol/L
 HDL-CHOLESTEROL (mg/dL)
○ A sample / A Std. X Conc. Std.
(mg/dl)
○ 0.119 /0.787 X 200
○ 30.24 mg/dL
SAMPLE 2
 HDL-CHOLESTEROL (mmol/l)
○ HDL-Cholesterol (mg/dl) X 0.02586
○ 30.24 X 0.02586
○ 0.78 mmol/L

CONTROL RANGE:
• HDL-Cholesterol: 43.12 +/- 7.99 (2SD) mg/dL.

DISCUSSION:

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 On this experiment, method HDL is for the quantitative determination of high
density lipoprotein cholesterol (HDL-C) in serum and plasma. Plasma lipoproteins are
spherical particles containing varying amounts of cholesterol, triglycerides, phospholipids
and proteins.
The phospholipid, free cholesterol and protein constitute the outer surface of the
lipoprotein particle, while the inner core contains mostly esterified cholesterol and
triglyceride. These particles serve to solubilize and transport cholesterol and triglyceride in
the bloodstream.
The relative proportions of protein and lipid determine the density of these
lipoproteins and provide a basis on which to begin their classification. These classes are:
chylomicron, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and
high-density lipoprotein (HDL).
The principle role of HDL in lipid metabolism is the uptake and transport of
cholesterol from peripheral tissues to the liver through a process known as reverse
cholesterol transport (a proposed cardioprotective mechanism).
Low HDL-C levels are associated with an increased risk of coronary heart disease and
coronary artery disease. Hence, the determination of serum HDL-C is a useful tool in
identifying high-risk patients. The reference method for the quantitation of HDL-C
combines ultracentrifugation and chemical precipitation to separate HDL from other
lipoproteins, followed by cholesterol measurement using the Abell-Kendall assay.
This method is too time consuming and labor intensive for use in routine analysis.
Therefore, most laboratories utilize one of several methods for selective precipitation and
removal of LDL and VLDL, followed by the enzymatic measurement of HDL-C in the
supernatant fraction. Since these methods require off-line pretreatment and separation
steps the assay procedures cannot be fully automated. As a result, routine determination of
HDL-C has suffered from long handling times and poor reproducibility.
TUBES INTERPRETATION
• The result shown high than
NORMAL CONTROL
HDL-C cholesterol range.
• The result shown low than
SAMPLE 1& SAMPLE 2
HDL-C cholesterol range.
Below are the list interferences or error during the experiment.
INTERFERENCES & ERROR
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DRUGS • Interference from the following
substances at concentrations
indicated, when added to a 40
mg/dL [1.04 mmol/L] HDL-C
serum pool, is less than 10%.
○ Albumin
○ Amikacin
○ Ampicillin
○ Ascorbic Acid
○ Atorvastatin
○ Caffeine
○ Carbamazepine
○ Chloramphenicol
○ Chlordiazepoxide
○ Chlorpromazine
○ Cimetidine
○ Clofibrate
○ Creatinine
○ Furosemide
○ Gemfibrozil
○ Heparin (sodium)
○ Ibuprofen
○ Immunoglobulin G (IgG)
○ LDL-Cholesterol
○ Lidocaine
○ Lithium Chloride
○ Lovastatin
○ Niacin
○ Nicotine
○ Penicillin G
○ Pentobarbital

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 ○ Phenytoin
○ Protein, Total
○ Rheumatoid Factor
○ Urea
○ Uric Acid

• Interference from the following

substances at concentrations
indicated, when added to a 40
mg/dL [1.04 mmol/L] HDL-C
serum pool, is less than
10%.Serum or plasma should be
SAMPLING
removed from cells within 2
hours of venipuncture
• Serum or plasma should be
removed from cells within 2
hours of venipuncture.
• Must test within 8 hour.
• Sampling, mixing and
processing samples.
HUMAN ERROR
• Cuvettes contain human body
contact.

MEASUREMENT OF TRIGLYCERIDES (GPO-PAP method)

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Enzymatic hydrolysis of triglyceride with subsequent determination of liberated glycerol
by colorimetry
• Test principle

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 Lipoprotein lipase
Triglycerides + 3H20---------------► glycerol + 3 RCOOH

Glycerokinase
Glycerol + ATP----------------► glycerol-3-phosphate + ADP

Glycerol-3-phosphate-oxidase
Glycerol-3-phosphate + O2 dihydroxyacetone phosphate + H2O2

peroxidase
2 H2O2 + 4 aminophenazone + 4-chlorophenol 4-(p-benzoquinone-mono-
imino)-phenazone + 2H2O + 2 HCL

• Sample material
○ Serum, heparinized
○ EDTA plasma.
• Procedure
○ Wavelength: 500nm
○ Cuvette: 1 cm light path
○ Incubation temperature: 20-25°C
○ Measure against reagent blank
○ One reagent blank is sufficient for each assay series.

Pipette into test tubes

Blank Sample/control/standard
Distilled water 0.01 ml -
Sample material - 0.01 ml
Triglycerides Reagent 1.00 ml 1.00 ml

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 • Mix and incubate for 20 min at 20-25°C or 10 min at 37°C.
• Read absorbance of sample against reagent solution within 1 hour.

• Calculation via factor

○ Calculate the concentration (c) of triglycerides as follows:

Triglycerides (mg/dl) = A sample / A Std. X Cone. Std (mg/dl)

Triglycerides (mmol/L) = Triglycerides (mg/dL) X 0.01126

• Calculation via factor

○ Calculate the concentration (c) of triglycerides as follows:
* Please note: To correct for free glycerol subs tract 10 mg/dl (0.11 mmol/T) from
the calculated triglyceride value.
• Clinical interpretation according to the recommendations of the European
Atherosclerosis.
Analytes Reference Range Lipid disorder
Cholesterol <200mg/dl
Triglycerides <200 mg/dl No

Yes, if HDL cholesterol

Cholesterol 200-300 mg/dl
< 35 mg/dl

Cholesterol >300 mg/dl

Triglycerides >200 mg/dl Yes

• Values in mmol/l:
TEST UNITS
Cholesterol: 35 mg/dl = 0.9 mmol/L
200mg/dl=5.2 mmol/L
300mg/dl= 7.8 mmol/L
Triglycerides: 200mg/dl= 2.3 mmol/L

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RESULTS:
TUBES ABSORBANCE READING
NORMAL CONTROL 0.377
STANDARD 0.426
SAMPLE 1 0.227
SAMPLE 2 0.403
• Concentration Standard (mg/dL) : 200 mg/dL
• Standard Absorbance : 0.426
• Control range Triglyceride: 130.99 +/ - 14.97 (2SD) mg/dL

TUBES CALCULATION
 TRIGLYCERIDES (mg/dL)
○ A sample / A Std. X Conc. Std. (mg/dl)
○ 0.377 /0.426 X 200
○ 176.99 mg/dL
NORMAL CONTROL
 TRIGLYCERIDES (mmol/L)
○ Triglycerides (mg/dl) X 0.01126
○ 176.99 X 0.01126
○ 1.99 mmol/L
SAMPLE 1  TRIGLYCERIDES (mg/dL)
○ A sample / A Std. X Conc. Std. (mg/dl)
○ 0.227 /0.426 X 200
○ 106.57 mg/dL
 TRIGLYCERIDES (mmol/L)
○ Triglycerides (mg/dl) X 0.01126
○ 106.57 X 0.01126
○ 1.2 mmol/L

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  TRIGLYCERIDES (mg/dL)
○ A sample / A Std. X Conc. Std. (mg/dl)
○ 0.403 /0.426 X 200
○ 189.20 mg/dL
SAMPLE 2
 TRIGLYCERIDES (mmol/L)
○ Triglycerides (mg/dl) X 0.01126
○ 189.20 X 0.01126
○ 2.13 mmol/L

TUBES CALCULATION VIA FACTOR

 LDL CHOLESTEROL (mg/dL)
○ Total Cholesterol - Triglycerides / 5 X
HDL cholesterol (mg/dl)
○ 129.31 – 176.99 /5 X 74.71
○ 19.20 mg/dL
NORMAL CONTROL
 LDL CHOLESTEROL (mmol/L)
○ Total Cholesterol - Triglycerides / 2.2 X
HDL cholesterol (mmol/L)
○ 3.34 – 1.93/2.2 – 1.99
○ 0.47 mmol/L
 LDL CHOLESTEROL (mg/dL)
○ Total Cholesterol - Triglycerides / 5 X
HDL cholesterol (mg/dl)
○ 173.28 – 31.77 /5 X 106.57
○ 60.36 mg/dL
 LDL CHOLESTEROL (mmol/L)
SAMPLE 1 ○ Total Cholesterol - Triglycerides / 2.2 X
HDL cholesterol (mmol/L)
○ 4.48 – 0.82/2.2 – 1.2
2.91 mmol/L

SAMPLE 2  LDL CHOLESTEROL (mg/dL)

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 ○ Total Cholesterol - Triglycerides / 5 X
HDL cholesterol (mg/dl)
○ 208.62 – 30.24 /5 X 189.20
○ 13.37 mg/dL
 LDL CHOLESTEROL (mmol/L)
○ Total Cholesterol - Triglycerides / 2.2 X
HDL cholesterol (mmol/L)
○ 5.39 – 0.78/2.2 – 2.13
○ 2.91 mmol/L

• The amount of free glycerol, 10 mg/dL (0.11 mmol/L) is subtracted from

the calculated triglyceride value.

SAMPLE TRIGLYCERIDE TRIGLYCERIDE

(mg/dL) (mmol/L)
106.57 – 10 1.2 - 0.11
1
= 96.57 mg/dL =1.09 mmol/L
189.20 – 10 2.13 – 0.11
2
=179.20 mg/dL = 2.02 mmol/L

DISCUSSION:
This experiment is a quantitative determination of triglycerides in serum
and plasma. It is useful in diagnosis and treatment of patients with diabetes
mellitus, nephrosis, liver obstruction, other diseases involving lipid metabolism,
or various endocrine disorders.
Triglycerides are water-insoluble lipids consisting of three fatty acids linked
to one glycerol molecule. Triglycerides are transported in the blood as core
constituents of all lipoproteins, but the greatest concentration of these molecules is
carried in the triglycerides-rich chylomicrons and very low density lipoproteins
(VLDL).

Through the action of lipases and bile acids, triglycerides are hydrolyzed
into glycerol and fatty acids which are absorbed by adipose tissue for storage or
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by other tissues requiring a source of energy. A peak concentration of
chylomicron-associated triglycerides occurs within 3-6 hours after ingestion of a
fat-rich meal; however, the rate of absorption of fats is highly variable, depending
on the individual and dietary composition of the fat. After absorption,
triglycerides are resynthesized in the epithelial cells and combined with
cholesterol and a number of apolipoproteins to form chylomicrons.
TUBES INTERPRETATION
• The result shown high than
NORMAL CONTROL
cholesterol range.
• The result shown low than
SAMPLE 1& SAMPLE 2
cholesterol range.

INTERFERENCES & ERROR

DRUGS • The following substances do
not interfere with the TGL
method when present in serum
and plasma at the
concentrations indicated.
Inaccuracies (biases) due to
these substances are less than
10% at triglycerides
concentrations of 200 mg/dL
[2.26 mmol/L].
○ Acetaminophen
○ Albumin
○ Amikacin
○ Ampicillin
○ Ascorbic Acid
○ Bilirubin
○ Caffeine
○ Carbamazepine
○ Chloramphenicol

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○ Chlordiazepoxide
○ Chlorpromazine
○ Cholesterol
○ Cimetidine
○ Creatinine
○ Dextran 75
○ Diazepam
○ Digoxin
○ Erythromycin
○ Ethanol
○ Ethosuximide
○ Furosemide
○ Gentamicin
○ Sodium Heparin
○ Hemoglobine (monomer)
○ Ibuprofen
○ Lidocaine
○ Lithium (lithium chloride)
○ Nicotine
○ Penicillin G
○ Pentobarbital
○ Phenobarbital
○ Phenytoin
○ Primidone
○ Propoxyphene
○ Protein: Total
○ Rheumatoid factors
○ Salicylic Acid
○ Theophylline
○ Urea
○ Uric Acid

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 • Cause a positive interference.
• Sampling, mixing and
Free glycerol & human error processing samples.
• Cuvettes contain human body
contact.

CONCLUSION:
1. These routines test that one mostly done as diagnostic tests and it is a
quantitative determination of all lipids either in serum or plasma.
2. These experiments are based on enzymatic reaction.

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