CLINICAL CHEMISTRY

MTAP 1
surface. In normal lipid metabolism, chylomicrons
LIPIDS & LIPOPROTEINS enter the circulation and are metabolized to remnant
particles for uptake and further modification by the
LIPID STRUCTURE liver.

3. Very-low-density-lipoprotein carries endogenous

1. Fatty acids exist as short, medium and long chains of triglycerides synthesized in the liver. VLDL
molecules that are major constituents of triglycerides molecules are composed of 55% triglycerides, 19%
and phospholipids. Minimal amounts of fatty acids are cholesterol, 18% phospholipid, 8% apolipoprotein and
bound to albumin and circulate free (unesterified) in have apolipoproteins B-100 mainly and C-I, C-II, C-
plasma. III and E on their surface. In normal lipid metabolism,
2. Triglyceride VLDLs are secreted into the blood by the liver for
• Triglyceride is formed from one glycerol molecule metabolism in peripheral tissues.
with three fatty acid molecules attached via ester
bonds. 4. Intermediate-density lipoprotein (IDL) is a
• Triglycerides comprise 95% of all fats stored in transitional form as it is formed form VLDL and
adipose tissue. then further modified in the liver to LDL. IDLs
• Triglycerides are transported through the body carry endogenous triglycerides and cholesterol
by chylomicrons and VLDL (very-lo w- esters. IDL molecules are composed of 23%
density-lipoproteins) triglycerides, 38% cholesterol, 19% phospholipid,
• Metabolism involves releasing the fatty acids the 19% apolipoprotein and have apolipoproteins B-100
cells for energy, then recycling the glycerol into mainly and some E on their surface.
triglyceride.
• Lipase, lipoprotein lipase, epinephrine and 5. Low-density lipoprotein is the body’s major
cortisol break down triglycerides. cholesterol carrier and transports a large amount
3. Cholesterol of endogenous cholesterol. Known as “bad
• Unsaturated steroid alcohol, exists in the cholesterol”, LDL is easily taken up by cells, so
esterified form, where a fatty acid forms an ester elevated levels are associated with increased risk
bond at carbon-3, and the free (unesterified) form for atherosclerosis. LDLs are composed of 50%
• Precursor for synthesis of bile acids, steroid cholesterol, 22% phospholipids, 6% triglycerides, 22%
hormones, and vitamin D proteins and have apoproteins B-100 on their
• Low-density-lipoprotein (LDL) is the primary surface. In normal lipid metabolism, this lipoprotein
carrier of cholesterol brings cholesterol to peripheral cells for membrane
4. Phospholipid synthesis and formation of renal and reproductive
• Composed of one glycerol molecule and two fatty hormones.
acid molecules attached via ester bonds
• Found on the surface of lipid layers, they are
major constituents of cell membranes and 6. High-density lipoprotein (HDL) is also known as
outer shells of lipoprotein molecules “good cholesterol”. HDL is synthesized in the
intestine and liver cells. HDL molecules are recycled
CLASSIFICATION OF LIPOPROTEINS chylomicron and VLDL molecules. HDL is composed
of 50% protein, 28% phospholipids, 19% cholesterol
1. Lipoproteins are molecules that combine water and 3% triglycerides. HDL has apoproteins A-I
insoluble dietary lipids and water-soluble proteins mainly and A-II on its surface. In normal lipid
(apolipoproteins) so that lipids can be transported metabolism, HDL removes excess cholesterol from
throughout the body. Micelles are spherical and have peripheral tissues and transports it to other catabolic
an inner core of neutral fat. sites. This function has an antiatherogenic effect.
2. Chylomicrons are the largest lipoproteins and
have the lowest density. They are found in the 7. Lp(a) is composed primarily of cholesterol esters,
intestines and transport triglycerides after a meal, phospholipids and apolipoprotein (a) and B-100.
giving serum a “turbid/milky” appearance. Elevated levels of Lp(a) are associated with
Because of their low density, chylomicrons will float to increased risk for coronary heart disease,
the top and form a creamy layer when plasma is myocardial infarction and cerebrovascular
stored overnight. Chylomicrons are composed of 86% disease.
triglycerides, 5% cholesterol, 7% phospholipid and
2% apolipoprotein.

CLINICAL SIGNIFICANCE
Chylomicrons have apoproteins B-48 mainly and
lesser amounts of A-I, C-I, C-II and C-III on their

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1. Abnormal lipid metabolism can be due to genetic -Serum appearance: Turbid
defects or it can be acquired. Abnormal lipid -Total Cholesterol: Normal to slightly elevated
metabolism is associated with risk of coronary -Triglyceride: Moderately to severely elevated
heart disease and other disorders. -Apo C-II increased/ decreased; Apo B-100
2. The National Cholesterol Education Program increased
established the Adult Treatment Panel III f) Type V hyperlipoproteinemia: Increased VLDL +
Classification (ATP III) which sets cutoff values for chylomicrons
cholesterol and triglyceride levels based on a 9- to 12- -Serum appearance: Turbid with creamy layer
hour fasting: -Total Cholesterol: Slightly to moderately elevated
-Triglyceride: Severely elevated
TOTAL CHOLESTEROL REFERENCE RANGE: -Apo C-II increased/decreased; Apo B-48 and Apo
Desirable : <200 mg/dL B-100 increased
Borderline High : 200-239 mg/dL
High : >240 mg/dL 4. The most common familial form is called familial
combined hyperlipidemia (FCHL). FCHL is
HDL CHOLESTEROL REFERENCE RANGE: characterized by increased plasma levels of total and LDL
Protective against Heart Disease : >60 mg/dL cholesterol (type IIa) or triglyceride (type IV), or a
The Higher the Better : 40-59 mg/dL combination of both (type IIb). Also, Apo B-100 is
Major Risk Factor for Heart Disease : <40 mg/Dl increased. The level of HDL cholesterol may be
decreased.
LDL CHOLESTEROL REFERENCE RANGE:
Optimal : <100 mg/dL 5. Hyperapobetalipoproteinemia is associated with
Near Optimal : 100-129 mg/dL VLDL and Apo B-100 overproduction in the liver. It is
Borderline High : 130-159 mg/dL characterized by normal or moderate elevation of LDL
High : 160-189 mg/dL cholesterol with an elevated Apo B-100. Total cholesterol
Very High : >190 mg/dL and triglyceride are generally elevated but may be normal.
HDL cholesterol and Apo A-I levels are decreased.
TRIGLYCERIDE REFERENCE RANGE:
Normal : <150 mg/dL 6. Familial hypertriglyceridemia is characterized by a
Borderline High : 150-199 mg/dL moderate elevation of triglyceride with excess
High : 200-499 mg/dL production of VLDL. Both triglyceride and cholesterol are
Very High : >500 mg/dL present in higher concentrations than normal in VLDL,
Conversion factors: LDL cholesterol and Apo B-100 are within their reference
Total Cholesterol ranges. HDL cholesterol is decreased.
Triglycerides
7. Type V hyperlipoproteinemia is characterized by
3. Hyperlipoproteinemias have been classified using the increased VLDL and chylomicrons.
Friedrickson-Levy classification system, which is not
commonly used today. However, some of the abnormal 8. Familial hypercholesterolemia is characterized by
lipid types are still referenced up to present which increased LDL cholesterol. The plasma triglyceride
includes: level maybe normal or slightly increased and plasma
HDL cholesterol level is slightly decreased
a) Type I Hyperlipoproteinemia: Elevated
chylomicrons 9. Secondary lipoproteinemia. Many conditions cause
-Serum appearance: Creamy layer of chylomicrons lipoproteins to be abnormally metabolized. Some of
over clear serum those conditions include diabetes mellitus,
-Total cholesterol: Normal to moderately elevated hypothyroidism, obesity, pregnancy, nephrotic
-Triglyceride: Extremely elevated syndrome, pancreatitis, alcoholism, and myxedema.
-Apo B-48 increased, Apo A-IV increased
b) Type IIa hyperlipoproteinemia: Increased LDL
-Serum appearance: Clear 10. Hypolipoproteinemias
-Total Cholesterol: Generally elevated a) Abetalipoproteinemia: Total cholesterol level is
-Triglyceride: Normal very low; triglyceride level nearly undetectable; LDL & Apo
-Apo B-100 increased B-100 absent
c) Type IIb hyperlipoproteinemia: Increased LDL and b) Hypobetalipoproteinemia: Unable to synthesize
VLDL Apo B-100 and Apo B-48, low total cholesterol level and
-Serum appearance: Clear or slightly turbid normal to low triglyceride level
-Total Cholesterol: Elevated c) Hypoalphalipoproteinemia: Severely elevated
-Triglyceride: Elevated triglyceride level and low HDL level
-Apo B-100 increased d) Tangier disease: HDL absent; Apo A-I, A-II, LDL,
d) Type III hyperlipoproteinemia: Increased IDL total cholesterol very low; triglyceride level normal to
-Serum appearance: Creamy layer sometimes slightly increased
present over a turbid layer
-Total Cholesterol: Elevated
-Triglyceride: Elevated
-Apo E-II increased, Apo E-III and Apo E-IV
decreased
e) Type IV hyperlipoproteinemia: Increased VLDL CHOLESTEROL TEST METHODOLOGY

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 a) Apo A-I is the major protein found in HDL. It
1. Elevated cholesterol concentrations have been activates lecithin-cholesterol acyltransferase
linked to atherosclerosis, coronary artery disease, (LCAT) and removes free cholesterol from
and increased risk for myocardial infarction. extrahepatic tissues. Thus, it is considered
2. Decreased cholesterol levels are present in antiatherogenic.
various forms of liver disease, most notably b) Apo B-100 is the major protein found in LDL. It
alcoholic cirrhosis. is associated with increased risk of coronary
3. Enzymatic methodology: artery disease.
c) Lp(a) is an independent risk factor associated
with impaired plasminogen activation and thus
decreased fibrinolysis. A high level suggest
increased risk for coronary heart disease and
stroke.

2. Test methodology
*Apo-A, Apo-B and Lp(a) are measured by
HDL TEST METHODOLOGY immunochemical methods such as immunochemical
1. HDL decreases the atherosclerotic process. methods such as immunoturbidimetric and
Increased HDL cholesterol is associated with immunonephelometric
decreased risk of coronary artery disease, and 3. Reference ranges:
decreased HDL cholesterol is associated with a) Apo-A : 120-160 mg/dL
increased risk of coronary artery disease b) Apo-B : <120 mg/dL
2. Test methodology: c) Lp(a) : <30 mg/dL
a) Precipitate LDL and VLDL cholesterol with
dextran-sulfate magnesium chloride or heparin
sulfate-manganese chloride, then assay the PROTEINS
supernatant for cholesterol using an enzymatic
technique. Cholesterol present is HDL. PROTEIN FUNCTIONS
1. Tissue repair
b) Homogenous assay uses an antibody to Apo B- 2. Coagulation
100 to bind LDL and VLDL. An enzymatic cholesterol 3. Transport carrier
analysis can then be performed with only HDL 4. Maintenance of osmotic pressure
cholesterol able to react. 5. Inflammation and anaphylaxis
6. Immunity
LDL TEST METHODOLOGY 7. Biocatalyts
1. LDL is directly associated with atherosclerosis
and coronary heart disease. PLASMA PROTEINS
2. Test methodology: LDL cholesterol may be Prealbumin
calculated or measured directly • Fraction that migrates in a position faster than
*Friedewald Formula (indirect, not valid for albumin toward the anode.
triglycerides over 400 mg/dL) • It is also called thyroxine-binding prealbumin
LDL cholesterol = total cholesterol – (HDL (TBPA) or transthyretin (TTR).
cholesterol + TAG/5) • Transport thyroxine and retinol (Vitamin A)
*Homogenous assay uses detergents to block • It is a good indicator for nutritional status of the body.
HDL and VLDL from reacting with the dye to form a • It crosses more easily into the CSF than other serum
colored chromogen product. An enzymatic cholesterol proteins.
analysis is performed with only LDL cholesterol able
to react. Albumin
• It is the most abundant protein in the plasma.
TRIGLYCERIDE TEST METHODOLOGY • It is the general transport protein or carrier.
1. Elevated triglyceride levels may be seen in • Measurements of albumin concentrations are
Friedrickson Type I, Iib, IV and V important in interpreting calcium and magnesium
hyperlipoproteinemias, pancreatitis, alcoholism, levels because these ions are bound to albumin.
obesity, hypothyroidism, nephrotic • Dye binding methods for albumin measurements
syndrome, and storage diseases (Gaucher, employ
Niemann-Pick) i. Bromcresol green (BCG)
2. Enzymatic methodology: ii. Hydroxyazobenzene Benzoic Acid (HABA)

DECREASED ALBUMIN INCREASED ALBUMIN

APO A-1, APO-B & LP(A) • Nephrotic • Dehydration
Syndrome • Prolonged
1. Clinical Significance

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 • Renal Diseases tourniquet • Prevents lost of ion through the kidney
• Inflammation application
• Liver Disease Beta 2-microglobulin
• Malnutrition • Indicator of renal tubular damage
• Malabsorption • Appears in urine when reabsorption is incomplete
and maldigestion
• Burns Immunoglobulins
• Synthesized by the plasma cells
• IgG>IgA>IgM>IgD>IgE
• IgG synthesized by the mother crosses then placenta
Globulins
• Consists of alpha1, alpha2, beta and gamma Fibrinogen
functions • It is the most abundant of the coagulation factors.
• GLOBULIN= TOTAL PROTEIN-ALBUMIN • 04104144It forms fibrin clot.
• It is elevated with other acute phase reactants, in
Alpha 1-Antitrypsin pregnancy and the use of contraceptive medications.
• It is the major component of the alpha1-globulins
(90%). C-Reactive Protein
• Major inhibitor of Protease activity- prevents • The earliest indicator of inflammation.
muscle destruction • CRP levels are sometimes used as rapid test for
• It is one of the serum glycoproteins that rise in presumptive diagnosis of bacterial infection (high)
response to acute inflammation. versus viral infection (low CRP)
• Cardiac marker: determine risk for CAD.
Alpha-1-fetoprotein (AFP)
• Tumor marker (hepatic and gonodal cancer) Myoglobin
• AFP is increased in amniotic fluid and serum in neural • A small heme protein found in skeletal and cardiac
tube defects (spina bifida). muscles
• Decreased level is associated with Down’s Syndrome. • Toxic to renal tubules
• First to increase after the onset of AMI
Alpha1-Acid Glycoprotein
• It is also known as orosomucoid. Troponins
• It is elevated during pregnancy. • Most important marker for cardiac injury
• It binds to progesterone and could be important in its • TnC,TnI,TnT= present in both cardiac and skeletal
transport or metabolism. muscles

Hemopexin PROTEIN DETECTION & QUANTIFICATION

• It binds heme released by degradation of hemoglobin.
• It is most profoundly decreased in intravascular Kjeldahl Technique
hemolysis. • Standard reference method
• Quantifies protein by its nitrogen content
Alpha 1- antichymotrypsin • Total serum protein is obtained by multiplying the
• Binds and inactivates PSA value of total protein nitrogen TPN) by 6.25.
• May also form part of the amyloid plaques found in
Alzheimer’s disease

Haptoglobin
• It is another major protein migrating in the alpha2
region.
• Binds free hemoglobin
• Decreased in IH and hemoglobinuria

Ceruloplasmin
• It is a copper-binding protein
• Indicator of Wilson’s disease

Alpa 2-macroglobulin
• Largest major nonimmunoglobulin protein in pasma.
• Its concentration rises 10-fold or more in nephrotic
syndrome.

Serum Protein Electrophoresis

Transferrin • Principle: Migration of charged particles in an
• Siderophilin electrical field
• It is the major B-globulin. • Separates protein according to their electrical charge
• It transports ferric ions • At pH 8.6, proteins are negatively charged

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 • Migration of proteins: CATHODE→ANODE
• Fastest to Slowest: Albumin> Alpha1- globulin>
Alpha2- globulin> Beta Globulin> Gamma
Globulin

ELECTROPHORETIC PATTERNS

Gamma Spike
• Monoclonal gammopathy (Multiple myeloma and
Waldenstrom macroglobulinemia)

Alpa1- globulin flat curve

• Emphysema and juvenile cirrhosis

Alpa2- macroglobulin spike

• Nephrotic syndrome
Serum Protein Electrophoresis
Spikes of Alpha1, Alpha2, and Beta globulin bands
• Regions are stained using:
• Inflammation
• Bromphenol blue
• Ponceau S
Beta- gamma region bridging
• Amido black 10B
• Hepatic cirrhosis (increased IgA)
• Lissamine green
• Coomassie brilliant blue
Small spikes in the beta region
• The bands are scanned by DENSITOMETER after
• IDA
staining to quantitate the amount of proteins present
on each band