(Semester-3, CEMA)

ANALYTICAL CLINICAL BIOCHEMISTRY (SEC-A2)

Biochemistry of Diseases

Dr. Jishnunil Chakraborty

 BLOOD
Composition and functions of blood:
Blood is a body fluid in human that delivers necessary substances such as nutrients and
oxygen to the cells and transports metabolic waste products away from those same cells.
In terms of anatomy and histology, blood is considered to be a specialized from of
connective tissue, given its origin in the bones and the presence of potential
%molecular fibers in the form of fibrinogen. Blood accounts for 7% of the human
body weight, with an average density of 1060 kg/m3, very close to the density of pure
water (1000 kg/m3). The average adult has a blood volume of roughly 4.5-5.5 liters
which contain plasma and several kinds of cells. These blood cells which are also
called corpuscles or ‘formed elements’ consist of erythrocytes (RBCs), leukocytes
(WBCs) and thrombocytes (platelets). By volume, RBCs constitute about 45% of
whole blood, the plasma about 54.3% and white cells about 0.7%. Human blood is red
in color because it contains a red pigment called hemoglobin in its red cells and the
blood has a pH of 7.35-7.45- slightly alkaline. The pH of blood in arteries is more
than in veins.

Dr. Jishnunil Chakraborty 2

Functions of:
1. Plasma:
Plasma is a straw colored fluid matrix in which blood cells are suspended. It is
slightly alkaline (pH  7.4) and slightly viscous with specific gravity around 1.026. It is
made up of approximately 90% water as well as electrolytes such as sodium,
potassium and proteins. It also contains some organic substances in dissolved from
like glucose, amino acids, fats, urea, hormones, enzymes etc. Plasma carries these
dissolved substances from one part to another part in the body. The specific
composition and function of its components are as follows:
a) Proteins:
These are the most abundant components in plasma by weight and play a part in a
variety of roles including clotting, defense and transport. The protein in plasma
includes antibodies to assist in the body’s defense system against diseases and
infections. There are three major categories of plasma proteins, and each individual
type of proteins has its own specific properties and functions:
Albumins:
These are the smallest and most abundant plasma proteins which regulate colloidal
Dr. Jishnunil Chakraborty 3
osmotic pressure of blood. Albumins also play an important role in the
transportation of drugs, hormones and fatty acids.
Globulins:
The globulins include high density lipoproteins (HDL) and low density lipoproteins
(LDL). Both HDL and LDL play important roles in the regulation of cholesterol and
have a large impact on cardiovascular disease.
Fibrinogen:
This is a soluble precursor of a sticky protein called fibrin, which forms the
framework of blood clot. Fibrin plays a key role in the coagulation of blood.
b) Inorganic salts:
These are about 1-2% and occur as electrolytes such as chlorides, bicarbonates,
phosphates of sodium, potassium and calcium. The most abundant of these are Na+
ions, which account for more of osmolarity and any other solute. Plasma osmolality
measures electrolyte-water balance of the body. Osmolality and osmolarity terms are
technically different but are same functionally for normal use.

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 Osmolality (l) = number of osmoles (osm) of solute per kg of solvent (Osmol/kg)
Osmolarity (r) = number of osmoles of solute per liter (L) of solvent (Osmol/L)
The larger number indicates a greater concentration of solutes in the plasma.
c) Dissolved nutrients:
These include glucose, lipids, vitamins, cholesterol and amino acids.
Beside these, plasma also contains dissolved gases, nitrogenous waste and enzymes.
2. Formed elements:
Human blood contains several kinds of cells called corpuscles or formed elements.
These consist of RBCs, WBCs and platelets. The formed element are so named because
they are enclosed in a plasma membrane and have a definite structure and shape.
All formed elements are cells except for the platelets, which are tiny fragments of
bone marrow cells.
a) Erythrocytes (RBCs):
An erythrocyte is a disc-shaped cell having no nucleus and containing a pigment
called hemoglobin- made up of Fe(II) containing pigment known as heme and a
protein called globin. If all human hemoglobin were free in plasma rather than
Dr. Jishnunil Chakraborty 5
being contained in RBCs, the fluid would be too viscous for the cardiovascular
system to function effectively. This combines with molecular oxygen to form
oxyhemoglobin. The hemoglobin of RBC facilitates oxygen transport by reversibly
binding to this respiratory gas and greatly increasing its solubility in blood. RBCs
are produced in spleen and in the bone marrow, having a life span of 120 days.
There are approximately 4.5-5 million RBCs per L of blood.
The plasma membrane of a mature RBC has glycoproteins and glycolipids
that determine a person’s blood type. RBCs die in spleen, where these become
trapped in narrow channels, broken up and destroyed. Hemolysis refers to the
rupture of RBCs where hemoglobin is released leaving empty plasma membranes
which are easily digested by cells known as macrophages in the liver and spleen.
b) Leukocytes (WBCs):
These are round or irregular, semi-transparent cells containing a nucleus and
visible under a microscope. These are little larger than RBCs. They are known as
soldiers of the body’s defense mechanism because they fight against infections and
thus they provide immunity to the body. They typically have a life span of a few days

Dr. Jishnunil Chakraborty 6

and there are only 5-10 thousand WBCs per L of blood.
Leukocytes can be divided into granulocytes and agranulocytes. Granulocytes
consist of neutrophils, eosinophils and basophils. Agranulocytes consist of
lymphocytes and monocytes. Each variety of WBC performs specific functions, such
as neutrophils (65-70% of total WBC) attack the invading bacteria and engulf them.
Lymphocytes (25% of WBC) produce antibodies which protects the body against
antigen, and thus provides immunity against infection. They also function in
destroying cancer cells. Basophils secret histamine which increases tissue blood flow
via dilating the blood vessels, and also secret heparin which is an anticoagulant that
promotes mobility of other WBCs by preventing clotting. Eosinophils and monocytes
also assist in defense mechanism of the body by becoming active against specific
antigens.
c) Thrombocytes (platelets):
These are tiny, circular or oval, colorless cells formed in the bone marrow. These lack
nuclei and help in the coagulation (clotting) of blood in a cut or wound, due to which
bleeding stops. The main function of platelets is to secret vasoconstrictors which
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constrict blood vessels causing vascular spasms, form temporary platelet plugs and
secret clotting factors to promote blood clotting. All the blood cells made in the bone
marrow from the cells are called stem cells.
Evidently blood has three main functions: transportation, maintaining
homeostasis and protecting by providing immunity and defense.

Blood coagulation:
Blood is a necessary component of human body and loss of this fluid may be life-
threatening. Hemostasis (commonly known as blood coagulation, heme means blood
and stasis means to hault) is the physiological process that stops bleeding at the site
of an injury while maintaining normal blood flow elsewhere in the circulation. Blood
loss is stopped by formation of a hemostatic plug. The endothelium in blood vessels
maintains an anticoagulant surface that serves to keep blood in its fluid state, but if
the blood vessel is damaged, the components of the subendothelial matrix are
exposed to blood. Several of these components activate the two main processes of
hemostasis to initiate the formation of a blood clot, composed primarily of platelets
and fibrin. This process is strictly regulated so that it is activated within seconds of
Dr. Jishnunil Chakraborty 8
an injury but must remain localized at the site of injury.
There are two main components of hemostasis. Primary hemostasis refers to
platelet aggregation and platelet plug formation. Secondary hemostasis refers to
the deposition of insoluble fibrin that forms a mesh which is incorporated into and
around the platelet plug. This mesh serves to strengthen and stabilize the blood
clot. These two processes occur simultaneously and are mechanically intertwined.
in essence, blood clotting is achieved by conversion of a soluble plasma
protein fibrinogen to the insoluble mesh-like cross-linked network of fibrins and
this process is mediated by a serine protease called thrombin. Thrombin is present
in circulating blood as an inactive proenzyme prothrombin which needs to be
activated by an enzymatic cascade involving a host of coagulation-promoting
proteins called clotting factors.

Collection of blood samples:

Blood is the body fluid that is used most frequently for analytical purposes. Three
general procedures for obtaining blood are venipuncture, arterial puncture and skin

Dr. Jishnunil Chakraborty 9

puncture. The technique used to obtain the blood specimen is critical in order to
maintain its integrity. Arterial and venous blood differ in important respects. Arterial
blood is essentially uniform in composition throughout the body, while the
composition of venous blood varies and it dependent on the metabolic activity of
perfused organ or tissue. Site of collection can affect the venous composition. Blood
collected by skin puncture (incorrectly called capillary blood) is an admix of
arterioles, venous and capillaries. Skin puncture blood also contains interstitial and
intracellular fluids. Usually for common biochemical tests, blood is collected through
venipuncture.

Techniques for venipuncture:

Usually a suitable vein of left arm is selected for puncture. Veins of antecubital fossa,
in particular, the median cubital and cephalic veins are preferred. If one arm has an
intravenous line, the other arm is used to draw a blood specimen. At first the
venipuncture site is cleaned with 70% isopropanol alcohol solution or 1%iodine-
saturate d swab-stick. Then a tourniquet is applied several inches above the
puncture site only for one minute. Then the needle of the syringe at approximately
Dr. Jishnunil Chakraborty 10
15 angle to the arm is inserted into the vein. The tourniquet is released when
blood begins to flow. Capillary blood is collected from the tip of the thumb or finger
or from ear lobe by using a sterile needle.

Preservation of blood samples:

Alteration in the concentration of a constituent in a stored specimen can result from
various processes such as: adsorption on the glass container, evaporation if the
constituent is volatile, water shift due to the addition of anticoagulants; metabolic
activities of the erythrocytes and leukocytes (accelerated by hemolysis) inducing O2
consumption and CO2 production, hydrolysis, glycolysis and finally degradation,
microbial (fungal/bacterial) growth.
Changes in concentration of volatile substances such as O2 and CO2 are
prevented or at least hindered by collection and storage of sample under anaerobic
conditions. The problem of microbial growth appears when the sample is to be
stored for longer than one day either at room or refrigerator temperature. This can
be solved by four alternative courses of action:
Dr. Jishnunil Chakraborty 11
1. collection and storage under sterile condition;
2. freezing of the sample;
3. extreme alteration of pH;
4. addition of an antibacterial agent.
Often after collection of blood it is preserved by using chemical preservatives
for prevention of chemical changes such as glycolysis and for prevention of
microbial growth.
In 1923, Sander introduced the combination of 10 mg NaF + 1 mg thymol/mL
of blood for preservation. The presence of thymol essentially controlled microbial
growth so that non-sterile specimens are stable for all determinations (except non-
protein nitrogen) for at least two weeks. Chlorobenzene and bromobenzene have
also been coupled with F and have been claimed to be superior to thymol.
Antibiotics can be used to prevent bacterial growth. Potassium oxalate is often used
as anticoagulant. Here oxalate ion forms the insoluble calcium oxalate which
precipitates from the medium, thereby stopping the coagulation cascade. It is used
together with NaF which inhibits glycolysis in collected blood. Among others, EDTA
and citrate also chelate Ca2+ ion and prevent clotting.
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 To carry out precise estimation, dilution of blood sample is often necessary.
For this purpose, normal saline is used but not distilled water. If distilled water is
used to dilute blood, then all the RBCs will explode. The blood will become hemolyzed.
This is because of osmosis. The RBC’s membrane is semipermeable and its interior
has a lot of substances dissolved. So if we put a cell into distilled water then by
osmosis water will penetrate into the cell, increasing its volume until it bursts.
Normal saline is isotonic with blood plasma to avoid this to happen.

Anemia:
Anemia is a blood disorder in which there is a deficiency of actual or available
hemoglobin in blood. There is a reduced oxygen capacity leading to oxygen
deficiency. RBCs play great role in anemia. Actually RBCs carry hemoglobin that
attaches to oxygen in the lungs and carries it to the tissues throughout the body.
Anemia occurs when there is a decreased number of RBCs in blood or when RBCs do
not function properly. It is diagnosed when a blood tests shows a hemoglobin value of
less than 13.5 gm/dL in a male or less than 12.0 gm/dL in a female.
Anemia can be temporary or long term, and can range from mild to severe.
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Anemia is associated with the symptoms of weakness, shortness of breath, dizziness,
fast or irregular heartbeat, pounding or whooshing in ears, headache, cold hands or
feet, pale or yellow skin and chest pain.
The causes of anemia may be classified as impaired RBC production,
increased RBC destruction (hemolytic anemia), blood loss and fluid overload
(hypervolemia). The most common cause of anemia is blood loss, but usually this
does not cause any lasting symptoms unless a relatively impaired RBC production
develops, in turn most commonly by iron deficiency. Anemia can be classified into
the following types:
1. Iron-deficiency anemia is the most common type of anemia caused by the
inadequate amount of iron in the body.
2. Vitamin-deficiency anemia may result from low levels of vitamin B12 or folic acid
(B9) usually due to poor dietary intake.
3. Aplastic anemia is a rare, life-threatening, bone-marrow failure disorder in which
the bone-marrow stops producing enough blood cells (RBCs, WBCs and platelets).
This type of anemia occurs as a result of destruction or deficiency of blood-
forming stem cells in bone marrow, in particular, when the body’s own immune
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system attacks the stem cells. Causes of aplastic anemia include infections, certain
medicines, autoimmune diseases and exposure to toxic chemicals.
4. Hemolytic anemia occurs when RBCs are destroyed faster than bone-marrow can
replace them. Hemolytic anemia may due to mechanical causes such as leaky heart
valves or aneurysms, infections, autoimmune disorders or congenital abnormalities
in the RBCs.
5. Sickle cell anemia is an inherited hemolytic anemia. It is caused by a defective
form of hemoglobin that forces RBCs to assume an abnormal crescent (sickle)
shape. These irregular blood cells die prematurely, resulting in a chronic shortage
of RBCs.
Anemia can also be caused by other diseases. Some diseases can affect the body’s
ability to make RBCs. For example, some patients with kidney disease develop
anemia because the kidneys are not making enough of the hormone erythropoietin
to signal the bone-marrow to make new or more RBCs. Chemotherapy for treating
cancer often impairs the body’s ability to make new RBCs, and anemia often results
from this treatment.

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Regulation, estimation and interpretation of data for:

Blood sugar:
Blood sugar regulation is the process by which the levels of blood sugar, primarily
glucose, are maintained by the body within a narrow range. Blood sugar levels are
regulated by negative feedback in order to keep the body in balance. The tight
regulation is referred to as glucose homeostasis. The levels of glucose in blood are
monitored by many tissues, but the cells in the pancreatic islets are among the most
well-understood and important. Insulin, which lowers blood sugar, and glucagon,
which raises it, are the most well-known of the hormones involved.
Insulin is of prime importance in regulating blood sugar. A rise in blood sugar
level stimulates insulin secretion and it lowers the blood sugar in several ways. It
increases glucose uptake by tissues, promotes glycolysis for utilization of glucose,
reduces glycogenolysis and gluconeogenesis in the liver. When sugar levels fall,
secretion of glucagon is stimulated. It is antagonistic to insulin and increases blood
sugar by enhancing glycogenolysis in the liver. Hormones like adrenaline,
glucocorticoids also regulate blood glucose level in the body. As hyperthyroidism
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leads to hyperglycemia (Grave’s disease), thyroid hormones act against insulin and
tend to raise blood sugar by increasing hepatic glycogenolysis and stimulating
gluconeogenesis.
Growth hormone (GH) is also antagonistic to insulin in its effect on
carbohydrate metabolism. It has diabetogenic effects as it raises blood sugar by
inhibiting the uptake and utilization of glucose by muscles. It reduces insulin
sensitivity.
Alcohol rapidly effects glucose levels in the blood stream which can be a
challenge for those dealing with diabetes mellitus. Alcohol affects many parts of the
body and as a result has many pathways for affecting blood glucose levels. The
carbohydrates in beer can be metabolized and thus increase blood glucose levels.
Alcohol dehydrogenase, the main enzyme in the catabolic reaction of alcohol,
produces NADH which triggers lactic acid formation. This increase in NADH leads to
a decrease in glucose level.
Glucose oxidase-peroxidase method: Glucose oxidase (GOD) catalyzes the oxidation of
glucose by oxygen to gluconic acid and H2O2. This H2O2 is broken down to H2O and O2
by a peroxidase (POD) in presence of an oxygen acceptor 4-aminophenazone which
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itself is converted to a pink quinonimine complex, the amount of which is measured
colorimetrically at 510 nm.
H2O2 + 4-aminophenazone Peroxidase Quinonimine complex + H2O
This method is used in various autoanalyzer. Thus, reagents for such estimation are
available in the form of kit supplied by various companies: GOD-POD reagent buffer, GOD-
POD reagent enzymes, standard D-glucose solution (100 mg of D-glucose in 100 mL of
saturated benzoic acid solution).
The reaction mixture is incubated at 37C for 15 minutes and the absorbances are noted
at 510 nm.
Blood glucose (mg/dL) = Absorbance of test solution
X 100
Absorbance of standard solution
For the majority of healthy individuals, normal blood sugar levels are as follows: between
72-99 mg/dL when fasting and up to 140 mg/dL at two hours after a meal. However,
test results vary with age.
Blood urea:
Urea is the major end product of protein and amino acid catabolism and is generated
in the liver through the urea cycle. Urea represents about 45-50% of the non-protein
Dr. Jishnunil Chakraborty 18
nitrogen of blood and 80-90% of the total urinary nitrogen excretion. The reference
range of urea is 15-40 mg/dL.
States associated with elevated levels of urea in blood are referred to as
hypemrureia or azotemia. Azotemia is a biochemical designation referring to any
significant increase of NPN compounds mainly urea and creatinine, in the plasma.
Very high plasma urea concentration accompanied by renal failure is called
uremia. It is evidently fatal if not treated by dialysis or kidney transplantation. In
renal diseases urea concentrations are elevated when the glomerular filtration rate
is markedly reduced and when the protein intake is higher than 200 gm per day. High
level of urea may also occur in a number of diseases in addition to those in which the
kidneys are primarily involved. In diseases such as hydronephrosis, congenital cystic
kidneys, renal tuberculosis, hypervitaminosis D, increase in urea levels is seen, the
extent of which depends on the extent of kidney tissues damaged.
decrease in serum urea level is rare. This usually indicated severe liver disease
and is seen, for example, in viral hepatitis with extensive necrosis. Decreased levels
are found in liver failure and pregnancy.
Diacetyl monoxime method: This direct method involves the formation of diacetyl
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from diacetyl monoxime and the subsequence condensation of diacetyl with urea of
serum or plasma, in presence of acid (H2SO4), oxidizing agent (FeCl3) and
thiosemicarbazide to form a red colored diazine derivative which can be quantitated by
photometry against the blank and the standard.

The reagents for such estimation are available in the form of kit supplied by various
companies.
Blood urea (mg/dL) = Absorbance of test solution
Absorbance of standard solution X 40
This diacetyl reaction although simple to perform, lacks specificity. Other limitations
include instability of the color, deviation from Beer’s law and the irritating odour of
the reagents. However, thiosemicarbazide intensifies the reaction and stabilizes the
Dr. Jishnunil Chakraborty 20
red product towards light. The major advantage of this direct method is that
ammonia does not interfere.
Creatinine:
Creatinine is the metabolic waste product resulting from the breakdown of creatine.
Creatine is a naturally occurring nitrogenous organic acid that helps to supply
energy to muscle cells. Creatine also acts as a buffer. Chemically creatinine is the
anhydride form of creatine that results from the breakdown of creatine and
phosphocreatine in skeletal muscle. It is produced at a constant rate depending on
muscular mass which is higher in male than female.

Creatinine is eliminated by glomerular filtration through the kidneys and excreted

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