Gihan Gawish.Dr
Plasma chemical composition
It consists of 90 % water and 10%
10%solutes.
solutes.
1. Proteins ≈ 7 %
2. Inorganic salts ≈ 0.9%
3. The reminder consisting
of
diverse
organic
compounds other than
proteins
Gihan Gawish.Dr
1. Organic (Nonprotein)
Constituents of Human Blood
Plasma
Amino acids
Urea
Carbohydrates
Bilirubin
Creatinine
Organic acids
Polysaccharides
Gihan Gawish.Dr
Creatin
Lipids
2. Inorganic Constituents of
Human Blood Plasma
Anions





Bicarbonate
Chloride
Phosphate
Sulfate
Iodine
Gihan Gawish.Dr
Cations






Calcium
Magnesium
Potassium
Sodium
Iron
Copper
3. Composition of plasma
proteins
Fibrinogen Transthyrein
(Clotting)
α1 Globulins
Albumin
α2 Globulins
β Globulins
Serum proteins
Gihan Gawish.Dr
Globulins
γ Globulins
Gihan Gawish.Dr

Serum is blood plasma without fibrinogen
and other clotting factors.
The serum proteins can be separated by:
1. Salting out.
2. Tiselius electrophoresis.
3. Zone electrophoresis.
4. Immunoelectrophoresis.
Gihan Gawish.Dr
1. Salting out
 Salting out is a method of separating proteins
based on the principle that proteins are less
soluble at high salt concentrations.
 The salt concentration needed for the protein to
precipitate out of the solution differs from
protein to protein.
Gihan Gawish.Dr
 Precipitation, separation, or coagulation of a
protein from its solution by saturation or partial
saturation with a neutral salt such as sodium
chloride or ammonium sulfate.
 The separation of protein fractions in the serum
or plasma by precipitation in increasing
concentrations of neutral salts.
Gihan Gawish.Dr
Electrophoresis
 Proteins are large molecules composed of covalently
linked amino acids.
 Depending on electron distributions resulting from
covalent or ionic bonding of structural subgroups,
proteins have different electrical charges at a given pH.
 serum proteins have been fractionated on the basis of
their electrical charge to separate the serum protein
components into five classifications by size and
electrical charge.
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Tiselius electrophoresis
 Moving
boundary
electrophoresis is
electrophoresis in a free
solution.
 The principle is the motion
of
charged
particles
through a stationary liquid
under the influence of an
electric field.
Gihan Gawish.Dr
59% 8% 5%12%
16%
 The apparatus includes a U-shaped cell filled
with buffer solution and electrodes immersed at
its ends.
 The sample applied could be any mixture of
charged components like a serum. On applying
voltage, the compounds will migrate to the anode
or cathode depending on their charges.
Gihan Gawish.Dr
Zone electrophoresis.

The Serum Protein Electrophoresis procedure is
intended for the separation and quantitation of serum
proteins using cellulose acetate electrophoresis.

A drop of serum is applied in a band to a thin sheet of
supporting material, like paper, that has been soaked in
a slightly-alkaline salt solution .

At pH 8.6, which is commonly used, all the proteins
are negatively charged, but some more strongly than
others.
Gihan Gawish.Dr
 A direct current can flow through the paper
because of the conductivity of the buffer with
which it is moistened .
 The serum proteins move toward the positive
electrode .
 The stronger the negative charge on a protein,
the faster it migrates .
Gihan Gawish.Dr
 After a time (typically 20 min),
the current is turned off and the
proteins stained to make them
visible
 The separated proteins appear
as distinct bands .
 The most prominent of these
and the one that moves closest
to the positive electrode is
serum albumin .
Gihan Gawish.Dr
Immunoelectrophoresis
 Serum immunoelectrophoresis is a
test
that
measures
immunoglobulins in the blood
using agar gel.
 Immunoglobulins are proteins
that function as antibodies.
 There are various types of
immunoglobulins. Some can be
abnormal.
 If you do have these abnormal
proteins, this test can also help
identify their specific type.
Gihan Gawish.Dr
 γ Globulins (IgA)
Multiple Myloma
 Albumine + α1,2 Globulins
 Albumin + γ Globulins
 β Globulins + γ Globulins
Nephrosis
Cirrhosis of liver
Chronic rheumatoid arthritis
 α1 Globulins + Albumin+ γ Globulins Hodgkin’s
Gihan Gawish.Dr
Gihan Gawish.Dr
Transthyretin
 The amount in normal plasma is10-40 mg/dl
 Function: Binding and transport of thyroxin
and retinol binding protein
Gihan Gawish.Dr
Gihan Gawish.Dr
Albumin
 Serum albumin is the most abundant blood
plasma protein.
 The amount in normal plasma is 35004500mg/dl
Gihan Gawish.Dr
Structure and properties of albumin
 Human serum albumin is a single peptide chain
of 585 amino acids, held in three homologous
domains by 17 disulfide bonds.
 The S-S bonds provide stability while the
intervening peptide strands allow for flexibility.
Gihan Gawish.Dr
 It is characterized by its solubility in water
 Albumin is one of the few plasma proteins that is
not a glycoprotein.
 It has the lowest molecular weight of almost of
plasma proteins
Gihan Gawish.Dr
Synthesized of albumin
 Albumin is synthesized in the liver as
prealbumin which has an N-terminal peptide that
is removed before the nascent protein is released
from the rough endoplasmic reticulum.
 The product, proalbumin, is in turn cleaved in
the Golgi vesicles to produce the secreted
albumin.
Gihan Gawish.Dr
Function of Albumin
1.Role of albumin in osmotic pressure

A major function of albumin is its role in
osmotic regulation.

It gives a much greater osmotic effect at
the Ph 7.4 of blood
Gihan Gawish.Dr
 Albumin is responsible for about 75- 80 % of
the osmotic effect of plasma because:
1.It constitutes slightly> half the plasma proteins
by weight
1.It has the lower molecular weight of the major
plasma proteins.
Gihan Gawish.Dr
Mechanism of osmotic pressure
 At pH 7.4 albumin
charges/molecule
has
20
negative
 These
markedly
influence
the
positive
concentration of plasma
the osmotic
pressure
movement of water between
plasma and extra vascular fluid.
 Changes in plasma protein concentration and
impaired water balance
Edema
Gihan Gawish.Dr
2. Transport function of albumin
 About 40% of albumin is contained in the
circulation
 The reminder being in the extra vascular space of
tissue ( skin, muscle & intestine)
 Albumin transports
aldosterone.
of
fatty
acids,
bilirubin&
 Many drugs like sulfonamides, penicillin G and
aspirin also bind tightly to albumin.
Gihan Gawish.Dr
Gihan Gawish.Dr
α Globulins
α1 globulins
• α1 Acid glycoprotein
• Retinol binding
• α1 Fetoglbuline
Gihan Gawish.Dr
α2 globulins
• Ceruloplasmin
• Haptoglobin
Type1-1
Type 2-1
Type 2-2
α1 globulins
α1 Acid glycoprotein
 Unknown function.
 It has extraordinarily high carbohydrate
content(42%).
 There are intrachain disulfide bond.
Gihan Gawish.Dr
α1 globulins
α1 Fetoglobulin
 A glycoprotein, is the major protein in the human fetal plasma and
amniotic fluid.( the same function of albumin in adult)
 It is very low amounts in adults.
 After birth its conc. decrease with the increase in albumin.
 The sequences of fetoglobulin and albumin are homologous.
 Fetoglobulin levels are elevated in patients with hepatoma and
during pregnancy.
Gihan Gawish.Dr
α1 globulins
Retinol binding protein
 It serves in retinol transport.
 Its presence in such a complex could prevent
renal excretion of the small retinol binding
protein in the urine.
Gihan Gawish.Dr
α2 globulins
Ceruloplasmin
 It is MW about 151000
 It is an enzyme synthesized in the liver containing 8 atoms
of copper in its structure.
 Ceruloplasmin carries 90% of the copper in our plasma.
The other 10% is carried by albumin.
 Ceruloplasmin exhibits a copper-dependent oxidase
activity, which is associated with possible oxidation of Fe2+
(ferrous iron) into Fe3+ (ferric iron), therefore assisting in
its transport in the plasma in association with transferrin,
which can only carry iron in the ferric state.
Gihan Gawish.Dr
 Wilson’s disease:
1. It is rare inherited disease
2. Plasma ceruloplasmin is markedly reduced
2+
and Cu levels increase in brain and liver
with resultant neurological changes and
liver damage.
Gihan Gawish.Dr
α2 globulins
Haptoglobulin(Hp)
 Hp constitute about ¼ of the α2 globulins
 It is a glycoprotein, produced by the liver
 They form specific, stable 1:1 molecular complexes with
hemoglobin, Such complexes form in vivo as the result of
intravascular hemolysis of erythrocytes
 Because of their high molecular , the complexes can’t be
excreted by the kidney; this prevent excretion of iron in the
urine and at the same time protects the kidney from
damage by hemoglobin .
Gihan Gawish.Dr
 The haptoglobin- hemoglobin complexes are
degraded by reticuloendothleial cells, and iron is
reutilizes for heme synthesis after degradation of
globins and excretion of degraded heme as bile
pigment.
 Patients with a
have haptoglobin
variety
of
hemolytic
anemias
 There three genetic types of haptoglobulin designated
Hp 1-1(MW about 100000), Hp 2-1 (MW about 9000),
and Hp 2-2 (MW about 42600), differs in the structure
according to disulfide bonds (They are distinguished
electrophoretically).
Gihan Gawish.Dr
β Globulins
1- Hemopexin
 It binds heme and prevents its urinary excretion,
thereby retaining heme iron for further use.
 Neither hemoglobin, cytochrome c, nor bilirubin binds
to hemopexin.
 The hemopexin isolated from normal individuals does
not have a full complement of heme, but when isolated
from patients with hemolytic anemia it is almost fully
saturated.
Gihan Gawish.Dr
 2- Transferrin
1. It is the major component of β Globulins.
2. It makes up 3% of total plasma proteins
3+
3. The main function to transport Fe ion to tissue where
it is required like bone marrow
2+
2+
4. Transferrin may also transport Cu & Zn
5. Also, regulates the concentration of free iron in
plasma
6. The concentration of transferrin increases during
pregnancy and iron deficiency
3+
7. Free iron is toxic, but binding of Fe to transferrin
reduces iron toxicity and transports iron to tissues
where it is required
Gihan Gawish.Dr
 3- C reactive protein
 It is normally present at a concentration of < 1mg/dl
in adults but increases markedly after acute
infections.
 The function of these protein is unknown, but it has
been suggested that it promotes phagocytosis.
Gihan Gawish.Dr
 4- β2 microglobulin
 It is present in very small amount in plasma
because of it is low molecular weight .
 It is excreted in the urine where it is found
normally in a concentration of about 0.1mg/l
 It is the smaller subunit of the HLA
histocompatipility antigen complex, which regulates
rejection of transplanted tissue.
Gihan Gawish.Dr
γ Globulins
 This fraction of
immunoglobulins .
plasma
contains
the
 They are often found during inflammatory
illness e.g. rheumatoid arthritis and multiple
myloma.
Gihan Gawish.Dr
Lipoprotein
 Classes of conjugated proteins consisting of a
protein combined with a lipid.
 The normal functioning of higher organisms
requires movement of insoluble lipids, such as
cholesterol,
steroid
hormones,
bile,
and
triglycerides, between tissues.
 To accomplish this movement, lipids are
incorporated into macromolecular complexes
called lipoproteins.
Gihan Gawish.Dr
Function of lipoprotein
 Lipoproteins serve as transport vehicles for fatty
acids and cholesterol in the blood and lymph.
A combination of a lipid and protein
Gihan Gawish.Dr
Classification of lipoprotein
 They are classified according to their density as highdensity lipoproteins, low-density lipoproteins, and verylow-density lipoproteins.
 Health care workers are interested in the concentration
of the different types of lipoproteins in the blood
because it has implications for health:
– A high concentration of low-density lipoproteins appears to
present a health risk and is associated with a high incidence of
heart disease.
Gihan Gawish.Dr
Classification by density
 General categories of lipoproteins, listed in order
from larger and less dense (more fat than protein)
to smaller and denser (more protein, less fat):
1. Chylomicrons - carry triacylglycerol (fat) from the
intestines to the liver, skeletal muscle, and to
adipose tissue.
2. Very low density lipoproteins (VLDL) - carry (newly
synthesized) triacylglycerol from the liver to
adipose tissue.
Gihan Gawish.Dr
3. Intermediate density lipoproteins (IDL) - are
intermediate between VLDL and LDL. They are
not usually detectable in the blood.
4. Low density lipoproteins (LDL) - carry cholesterol
from the liver to cells of the body. Sometimes
referred to as the "bad cholesterol" lipoprotein.
5. High density lipoproteins (HDL) - collects
cholesterol from the body's tissues, and brings it
back to the liver. Sometimes referred to as the
"good cholesterol" lipoprotein.
Gihan Gawish.Dr
Classification according to
alpha and beta
 It is also possible to classify lipoproteins as
"alpha" and "beta", akin to the classification
of proteins in serum protein electrophoresis .
Moving to cathode (lipoprotein have more
proteins)
Gihan Gawish.Dr
Serum lipids (plasma lipoproteins)
 Because of their relationship to cardiovascular disease, the
analysis of serum lipids has become an important health
measure.
LIPID
Typical values mg/dl
Desirable mg/dl
Cholesterol (total)
210–170
<200
LDL cholesterol
140–60
< 100
HDL cholesterol
85–35
>40
Triglycerides
160–40
<160
Gihan Gawish.Dr
Total cholesterol is the sum of
 HDL cholesterol
 LDL cholesterol and
 20% of the triglyceride value
 Note that
 high LDL values are bad, but
 high HDL values are good.
Gihan Gawish.Dr
Gihan Gawish.Dr
Plasma Enzymes
 Enzymes are biological catalysts responsible
for supporting almost all of the chemical
reactions in the body.
 Enzymes are found in all tissues and fluids
of the body.
 Intracellular enzymes catalyze the reactions
of metabolic pathways.
Gihan Gawish.Dr
 Plasma membrane enzymes regulate
catalysis within cells in response to
extracellular signals, and enzymes of the
circulatory system are responsible for
regulating the clotting of blood.
 Most plasma enzymes don’t have metabolic
roles in plasma, except for the enzymes
concerned in blood coagulation.
Gihan Gawish.Dr
1- Lipoprotein lipase
 It
is
an enzyme that hydrolyzes lipids in
lipoproteins ,like those found in chylomicrons and
very low-density lipoproteins (VLDL), into three
free fatty acids and one glycerol molecule.
 Lipoprotein
lipase
is
specifically
endothelial cells lining the capillaries
Gihan Gawish.Dr
found
in
Lipoprotein lipase deficiency
 Severe mutations that cause LPL deficiency result
in type I hyperlipoproteinemia, while less extreme
mutations in LPL are linked to many disorders of
lipoprotein metabolism.
 High-fat diets have been shown to cause tissuespecific over expression of LPL: This has been
implicated in tissue-specific insulin resistance and
consequent development of type 2 diabetes
mellitus
Gihan Gawish.Dr
2.Cholinesterase
 It is an enzyme that catalyzes the hydrolysis
of the neurotransmitter acetylcholine into
choline and acetic acid,
 A reaction necessary to allow a cholinergic
neuron to return to its resting state after
activation.
Gihan Gawish.Dr
There are two types of
cholinesterase
 1- Acetyl cholinesterase, also known as RBC
cholinesterase, found primarily in the blood and
neural synapses
 2- Pseudocholinesterase, also known as plasma
cholinesterase, found primarily in the liver.
The difference between the two types of
cholinesterase has to do with their respective
preferences for substrates: RBC cholinesterase
hydrolyses acetylcholine more quickly
Gihan Gawish.Dr
3. Acid phosphatase
 Acid phosphatase is a phosphatase, a type
of enzyme, used to free attached phosphate
groups from other molecules during
digestion.
 Different forms of acid phosphatase are
found in different organs, and their serum
levels are used as a diagnostic for disease
in the corresponding organs.
Gihan Gawish.Dr
 For example, elevated prostatic acid
phosphatase levels may indicate the
presence of prostate cancer.
 The optimal pH for the enzyme activity is
below 7
Gihan Gawish.Dr
4. Alkaline phosphatase
 (ALP) is a hydrolase enzyme responsible for
removing phosphate groups from many types of
molecules, including nucleotides, proteins, and
alkaloids.
 The process of removing the phosphate group is
called dephosphorylation.
 As the name suggests, alkaline phosphatases are
most effective in an alkaline environment.
Gihan Gawish.Dr
Physiology of alkaline phosphatase
 In humans, alkaline phosphatase is present
in all tissues throughout the entire body, but
is particularly concentrated in liver, bile duct,
kidney, bone, and the placenta.
 The optimal pH for the enzyme activity is10
Gihan Gawish.Dr
Diagnostic use of alkaline phosphatase
 Concentrations blood serum levels of ALP
are typically 39-117 Units per liter in adults
 Levels are significantly higher in children
and pregnant women.
 Lowered levels of ALP are less common
than elevated levels.
Gihan Gawish.Dr
 The following conditions
abnormal levels of ALP:
can
cause
 Elevated levels (hyperphosphatasemia)
 Lowered levels (hypophosphatasemia)
Gihan Gawish.Dr
Isoenzymes
 Isoenzymes )are enzymes that differ in
amino acid sequence but catalyze the same
chemical reaction).
 In many cases, they are coded for by
homologous genes that have diverged over
time.
Gihan Gawish.Dr
5. Lactate dehydrogenase
 LDH is found in many cells, but especially in
muscle cells.
 It catalyses interconversions of pyruvic acid and
lactic acid.
 The level of LDH is commonly used in exercise
physiology as a measure of the capacity of
glycolysis

Gihan Gawish.Dr
There are different forms of LDH
 One form called heart-specific LDH (H-LDH)
preferentially catalyses lactate oxidation to
pyruvate and predominates in slow-twitch
muscle fibers.
 Another form, muscle-specific LDH (MLDH), preferentially catalyses the reduction
of pyruvate to lactate and predominates in
fast-twitch muscle fibers.
Gihan Gawish.Dr
 LDH-1 (4H) - in the heart
 LDH-2 (3H1M) - in the reticuloendothelial
system
 LDH-3 (2H2M) - in the lungs
 LDH-4 (1H3M) - in the kidneys
 LDH-5 (4M) - in the liver and striated muscle
Gihan Gawish.Dr
6. Transaminase or
an aminotransferase
 It is an enzyme that catalyzes a type of
reaction between an amino acid and an αketo acid .
Gihan Gawish.Dr
I. Aspartate transaminase
Aspartate transaminase
called serum glutamic
transaminase (SGOT)
Gihan Gawish.Dr
(AST) also
oxaloacetic
Function of AST(SGOT)
 It facilitates the conversion of
Aspartate + α –ketoglutarate
oxaloacetate
Gihan Gawish.Dr
AST
glutamate+
 Isozymes
 Two isoenzymes are present in humans. They
have high similarity.
 GOT1 ,the cytosolic isoenzyme derives mainly
from red blood cells and heart
 GOT2 ,the mitochondrial
isoenzyme
predominantly present in liver .
Gihan Gawish.Dr
is
Clinical significance of AST

It is raised in acute liver damage.
 It is also present in
1. red blood cells,
2. cardiac muscle,
3. skeletal muscle,
4. Kidney,
5. and brain tissue
It may be elevated due to damage to those sources
as well.
Gihan Gawish.Dr
II.ALT or Alanine transaminase
 ALT is found in serum and in various bodily
tissues,
 but is most commonly associated with the
liver
Gihan Gawish.Dr
Function of ALT
 It catalyzes the transfer of an amino group from
alanine to a-ketoglutarate ,the products of this
reversible transamination reaction
being
pyruvate and glutamate.
Alanine + α –ketoglutarate
Gihan Gawish.Dr
ALT
glutamate +pyruvate
Clinical significance of ALT
 It is commonly measured clinically as a part
of a diagnostic liver function test ,to
determine liver health.
 It is also called serum glutamate pyruvate
transaminase( SGPT
Gihan Gawish.Dr
Elevated levels of ALT
Elevated levels of ALT often suggest the existence
of other medical problems such as
1. alcoholic or viral hepatitis ,
2. congestive heart failure,
3. liver damage ,
4. biliary duct problems,
5. infectious mononucleosis ,
6. or myopathy .
 For this reason ,ALT is commonly used as a way
of screening for liver problems. However,
elevated levels of ALT do not automatically mean
that medical problems exist.
Gihan Gawish.Dr
7. Creatine kinase (CK),
 Creatine kinase (CK), also known as
creatine
phosphokinase
(CPK)
or
phosphocreatine kinase,
 It is an enzyme expressed by various tissue
types.
 It catalyses the conversion of
Adenosine diphosphate (ADP)
triphosphate (ATP)
Gihan Gawish.Dr
CK
Adenosine
 Creatine kinase is an
enzyme in such tissues.
important
 Clinically, creatine kinase is assayed
in blood tests as a marker of
1. (heart attack)
2. (severe muscle breakdown),
3. acute renal failure.
Gihan Gawish.Dr
Types of CK
 In most of the cell, the CK enzyme consists of two
subunits, which can be either B (brain type) or M (muscle
type).
 There are, therefore, three different isoenzymes: CK-MM,
CK-BB and CK-MB.
 The genes for these subunits are located on different
chromosomes: B on 14q32 and M on 19q13.
 In addition to those, there are two mitochondrial creatine
kinases, the ubiquitous and sarcomeric form.
Gihan Gawish.Dr
Laboratory testing of CK
 CK is often
routinely
in
patients.
determined
emergency
 In addition, it is determined
specifically in patients with
chest pain and acute renal
failure is suspected.
 Normal values are usually
between 25 and 200 U/L.
Gihan Gawish.Dr
Crystals of creatine kinase
 Elevation of CK is an indication of damage to
muscle
 Lowered CK can be an indication of
alcoholic liver disease and rheumatoid
arthritis.
 Isoenzyme determination has been used
extensively as an indication for myocardial
damage in heart attacks.
Gihan Gawish.Dr