UNIVERSITY OF SANTO TOMAS - LEGAZPI

College of Health Sciences

St. Martin de Porres Bldg., Rawis Campus, Legazpi City 4500 Philippines
Trunklines: 736-0335 / 736-0358 / 736-0368 / 736-0479 local 262 • healthsciences@ust-legazpi.edu.ph

LIPID and LIPOPROTEIN DETERMINATION

1. Define cholesterol and give the components of total cholesterol.
Cholesterol is an unsaturated steroid alcohol that contains four rings: A, B, C, and D. Cholesterol is an
amphipathic lipid found on the surface of lipid layers due to the presence of one hydrophilic hydroxyl group in
the A ring (OH), while the rest of the lipid is hydrophobic. Cholesterol is different from other lipids in that it is not
readily catabolized by most cells. Thus, it is not a source of fuel. However, it can be converted in the liver to
primary bile acids, such as cholic acid and chenodeoxycholic acid, which promote fat absorption in the intestine.

The components of Total Cholesterol % by weight are

Chylomicrons: 7%,
Very Low-Density Lipoproteins (VLDL): 16-22%,
Low-Density Lipoproteins (LDL): 62%, and
High-Density Lipoproteins (HDL): 19%

2. Describe briefly all methods of cholesterol determinations.

Serum or plasma specimens collected in blood collection tubes from patients who have fasted for at least 12
hours are usually preferred for total cholesterol testing. If analysis is delayed, the serum/plasma specimen can be
refrigerated at 4°C for several days. Hemolyzed blood must be avoided because it may falsely increase
cholesterol levels.

Standard Lipid Panel: Utilizes chemistry analyzers

Sequence most common for measuring cholesterol: Enzymatic

The enzyme cholesteryl ester hydrolase cleaves the fatty acid residue from cholesteryl esters, which comprise
about two thirds of circulating cholesterol, converting them to unesterified or free cholesterol. The free
cholesterol is then reacted by the second enzyme, cholesterol oxidase, producing hydrogen peroxide which is a
substrate for a common enzymatic color reaciton using horseradish peroxidase to couple two colorless chemicals
into a colored compound. The intensity of the resulting color, proportional to concentration of cholesterol, can
be measured by a spectrophotometer.
 Chemical methods of Cholesterol Determination: The presence of double bonds and hydroxyl group in the
sterol’s structure makes it possible for cholesterol to carry out a cololrimetric assay

1. Libermann Burchardt Reaction

End product: Cholestadienyl Monosulfonic Acid
End Color: Green

2. Salkowski Reaction
End Product: Cholestadienyl Disulfonic Acid
End Color: Red

The Reference Method for Cholesterol Measurement is Gas Chromatography-Mass Spectrometry (GC-MS)
(Bishop et al., 2023). It shows good agreement with the gold standard method applied at the U.S. National
Institute of Standards and Technology, using isotope dilution mass spectrometry (IDMS).

The Reference Method for Cholesterol Determination according to CDC is the Abell, Levy Brodie Method which
uses hexane extraction after hydrolysis with alcoholic KOH followed by reaction with Libermann Burchardt color
reagent.

3. Describe the different types of familial hyperlipoproteinemia.

Hyperlipoproteinemia type 1 (HLP-1) or Primary hyperchylomicronemia syndrome is a genetic disorder
characterized by markedly increased triglyceride and chylomicron levels in blood which causes a high risk of
pancreatitis and other complications. The syndrome is caused by mutations in the gene that encodes the enzyme
lipoprotein lipase (LPL), or, less commonly, by mutations in genes encoding other proteins required for LPL
function. The disorder is usually diagnosed in childhood.

Type 2a Hyperlipoproteinemia or Familial Hypercholesterolemia (FH): It is one of the forms of

Hypercholesterolemia characterized with genetic abnormalities that predispose affected individuals to elevated
cholesterol levels. Patients with this condition can have their first heart attack in their teenage years.
Homozygotes of this disease are rare (about 1:1 million), but heterozygotes are seen more frequently (1:310).
This is because its an autosomal codominant disorder. A defect in just one or two copies of the LDL receptor can
adversely affect lipid levels. Heterozygotes tend to have total cholesterol concentrations in the range of 200 to
550 mg/dL and if not treated, become symptomatic for heart disease in before age 55. In fact, approximately 5%
of patients youngre than 50 years old with coronary artery disease are FH heterzygotes. Other symptoms
associated with FH include tendinous and tuberous xanthomas, which are cholesterol deposits in tendons and
under the skin, respectively.

Familial dysbetalipoproteinemia (Hyperlipidemia type III): This disease results from an accumulation of
cholesterol-rich VLDL and chylomicron remnants as a result of defective catabolism of those particles due to a
defect in the apolipoprotein E gene (apoE). Individuals with hyperlipidemia type III will frequently have total
cholesterol values of 200 to 300 mg/dL and triglycerides of 300 to 600 mg/dL, and palmar xanthomas and
tuberoeruptive xanthomas. Additionally, this disorder is associated with an increased risk of PVD and coronary
disease. Patients with this disease have an increased risk of attaining peripheral vascular disease and coronary
disease.

Hyperlipidemia type III can be distinguished form other forms of hyperlipidemias by isolating the VLDL fraction
with ultracentrifugation. A ratio derived from the cholesterol concentration in VLDL to total serum triglycerides
will be greater than 0.30 in the presence of hyperlipidemia type III. If the VLDL fraction is analyzed by agarose
 electrophoresis, the particles will migrate in a broad beta region than in the normal pre-beta region. Definitive
diagnosis requires a determination of Apo E isoforms by isoelectric focusing or DNA typing.

Type 4 Hyperlipoproteinemia or Familial Hypertriglyceridemia: Familial hypertriglyceridemia (type IV familial

dyslipidemia) is a disorder characterized by the overproduction of very low-density lipoproteins (VLDL) from the
liver. As a result, the patient will have an excessive number of triglycerides and VLDL on the lipid profile that can
cause acute pancreatitis. The common mutation involved in the development of familial hypertriglyceridemia is a
heterozygous inactivating mutation of the LPL gene. This disease is inherited in a dominant manner.

Type 5 hyperlipoproteinemia or Familial Combined Hyperlipidemia (FCH): It is a hereditary metabolic disorder

characterized by elevated levels of total cholesterol, triglycerides, LDL cholesterol, and decreased levels of HDL
cholesterol. The genetic element of FCH has not been fully understood to date. The pattern of inheritance of FCH
was initially reported to be autosomal dominant, then later research proposed that FCH be a multigenic mode
and complex inheritance. Individuals from an affected family may only have elevated cholesterol, whereas others
monly have elevated triglycerides, and others, elevations of both. Laboratory evaluations of patients with FCH
may present with elevated levels of serum triglycerides, total cholesterol, mixed triglycerides, increased VLDLs,
increased LDLs, or reduced levels of HDLs.

4. Why should blood specimen for lipid studies be drawn in fasting state of at least 12 – 14 hours?
Blood specimens for lipid studies must be drawn in fasting state because of four reasons:
 Post prandial triglycerides remain elevated for several hours;
 Most reference values for serum lipids are established on fasting blood specimens;
 NCEP and European guidelines recommend doing lipid profile in fasting blood specimens; and
 The Friedewald equation, used for calculation of LDL cholesterol, uses fasting triglycerides values. If non-
fasting triglycerides value is used in this equation, the LDL cholesterol will be undersetimated.

5. Give the principle of all the methods for triglyceride estimation.

Hantzsch Method (Chemical)

First, potassium hydroxide in ethanol + heat hydrolyzes triglycerides to form glycerol and free fatty acids. Then,
glycerol is oxidated using periodate anion forming formaldehyde and formic acid. The following reaction of
formaldehyde with chromotropic acid forms a chromogen which can be measured at 570 nm.
which is measured at 412 nm.

Gas Chromatographic Isotope Dilution Mass Spectrometry (GC-IDMS)

All glycerides (triglycerides, diglycerides, and monoglycerides) are chemically reduced to glycerol which is
subjected to gas chromatography. Then, to enhance the accuracy of the analysis, a “spike”, a known amount of
an isotopically enriched standard, is added to the sample. The ratios of the spike and the sample are then
determined through Mass spectrometry, hence the name gas chromatographic isotope dilution mass
spectrometry.
TRIGLYCERIDES GPO METHOD (Enzymatic):

Serum triglycerides are hydrolyzed to glycerol and free fatty acids by lipase. In the presence of ATP and glycerol
kinase (GK), the glycerol is converted to glycerol-1-phosphate. The glycerol-1-phosphate is then oxidized by
glycerol phosphate oxidase (GPO) to yield hydrogen peroxide. The condensation of hydrogen peroxide with 4-
chlorophenol and 4-aminophenazone (4-AA) in the presence of peroxidase (POD) produces a red colored
quinonimine dye which absorbs at, or near 500nm. The intensity of the colored complex
formed is directly proportional to the triglycerides concentration of the sample.

In addition, the following equations estimate LDL cholesterol using triglycerides:

Friedewald’s equation: Usually done on a fasting sample; commonly used in the estimation of LDL cholesterol.
The Friedewald’s equation assumes a fixed ratio of triglycerides to very low-density lipoprotein cholesterol
(VLDL-C).
Martin/Hopkins equation: Also used to estimate the LDL cholesterol levels. Compared to Friedewald’s equation,
Martin/Hopkins’ equation is a more flexible equation with adjustable values. The fixed factor of 5 used to
estimate VLDL-C is instead replaced by an adjustable factor based on a patient’s non-HDL-C and TG values.