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Chapter – 1 The Body’s water

1. Body water and its determination


2. Nature and composition of ECF and ICF
3. Electrolyte balance
4. pH balance

1. BODY WATER AND ITS DERMINATION

1.1 INTRODUCTION
1.2 CALCULATION OF BODY WATER
1.3 DETERMINATION OF BODY WATER

1.1 INTRODUCTION

Body water is all of the water content of the human body. A significant fraction of the human body
is water. Lean muscle tissue contains about 75% water. Blood contains 95% water, body fat contains
14% water and bone has 22% water. The human body is about 60% water in adult males and 55% in
adult females.

In diseased states where body water is affected, the compartment or compartments that have changed
can give clues to the nature of the problem. Body water is regulated by hormones, including anti-
diuretic hormone (ADH), aldosterone and atrial natriuretic peptide. There are many methods that can
be used to determine body water. One way to get a simple estimate is by calculation.

1.2 CALCULATION OF BODY WATER

In individuals of normal weight, water is abundant in most parts of the body, except in adipose tissue
(fat). These calculations are for adults of average build, and are inappropriate for obese or overly
muscular people. These proportions are very simplified and use round numbers for quick calculation.

The largest component of the body is water. Water makes up between 45 and 75% of body weight,
with the variability due primarily to differences in body fat. While most tissues including muscle,
skin, and visceral organs are over 70% water, adipose tissue contains less than 10% water. The
percentage of body weight that is water therefore varies inversely with body fat. In the average lean
adult male around 60% of the body weight is water. The remaining body weight consists of 16-18%
fat with 22-24% protein, carbohydrate and other solids. In the female the percentage of body weight
that is water is lower due to a relatively greater amount of subcutaneous fat.

Body water is broken down into the following compartments:


Intracellular fluid (2/3 of Body Water)
Extracellular fluid (1/3 of Body Water)
o Plasma (1/5 of Extracellular fluid)
o Interstitial fluid (4/5 of Extracellular fluid)
o Transcellular fluid (normally ignored in calculations)
 Contained inside organs, such as the gastrointestinal, cerebrospinal, and ocular
fluids.

The simplest calculation is the 60-40-20 rule.


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Total Body Water = 60% of Body Weight
Intracellular fluid = 40% of Body Weight
Extracellular fluid = 20% of Body Weight

This is consistent with the above relations between total body water and the compartmental fluids.

1.3 DETERMINATION OF BODY WATER

A) Dilution and equilibration

Total body water can be determined using Flowing afterglow mass spectrometry FA-MS
measurement of deuterium abundance in breath samples from individuals. A known dose of
deuterated water (Heavy water, D2O) is ingested and allowed to equilibrate within the body water.
The FA-MS instrument then measures the deuterium-to-hydrogen (D:H) ratio in the exhaled breath
water vapour. The total body water is then accurately measured from the increase in breath
deuterium content in relation to the volume of D2O ingested.

Different substances can be used to measure different fluid compartments:


total body water: tritiated water or deuterium
extracellular fluid: inulin
blood plasma: Evans blue

Fig 1. Schematic diagram of the FA-MS method

The principle of the FA-MS technique is that it exploits ion chemistry coupled with fast flow tube
techniques and quantitative mass spectrometry. However, FA-MS is specifically used for the on-
line determination of the deuterium content of water vapour in exhaled breath and the vapour
above aqueous liquids. Thus, for example, the deuterium content of single breath exhalations can
be measured following the ingestion of a small known amount of deuterium oxide when, following
the principle of isotope dilution, the total body water of the subject can be obtained, which is
proving to be particularly useful in nephrology.

B) Bioelectrical impedance analysis

Another method of determining total body water percentage (TBW%) is via Bioelectrical
Impedance Analysis (BIA). In the traditional BIA method, a person lies on a cot and spot
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electrodes are placed on the hands and bare feet. Electrolyte gel is applied first, and then a current
of 50 kHz is introduced. BIA has emerged as a promising technique because of its simplicity, low
cost, high reproducibility and noninvasiveness. BIA prediction equations can be either generalized
or population-specific, allowing this method to be potentially very accurate. Selecting the
appropriate equation is important to determining the quality of the results.

For clinical purposes, scientists are developing a multi-frequency BIA method that may further
improve the method's ability to predict a person's hydration level. New segmental BIA equipment
that uses more electrodes may lead to more precise measurements of specific parts of the body.

***

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2. NATURE AND COMPOSITION OF ECF AND ICF

2.1 INTRODUCTION
2.2 EXTRA CELLULAR FLUID
2.3 INTRA CELLULAR FLUID
2.1 INTRODUCTION

Water accounts for 60% of body mass in males, i.e. over 40 L in a 70 kg adult. Total body water is
divided between intracellular (two-thirds of total body fluid) and extracellular compartments (one-
third of total body fluid), with plasma forming a major sub-compartment (one-quarter of ECF)
within the extracellular space.

Water can diffuse freely between these fluid spaces and this exchanges ins controlled by pressure
gradients across the cell membrane (which separates ECF and ECF spaces) and the capillary wall
(which separates plasma from interstitial fluid). In each case the distribution of fluid depends on the
net effect of hydrostatic and osmotic forces. To solutes controlling osmosis at the two sites,

2.1 EXTRA CELLULAR FLUID-nature and composition

Extracellular fluid a general term for all the body fluids outside the cells, comprising about one
third of the body fluids. Extracellular fluid circulates in the spaces between the cells and brings to
the cells the nutrients and other substances needed for their functioning. The extracellular fluid also
includes the transcellular fluid; making up only about 2.5 percent of the ECF.

In humans, the normal glucose concentration of extracellular fluid that is regulated by homeostasis is
approximately 5 mM. The pH of extracellular fluid is tightly regulated by buffers around 7.4. The
volume of ECF is typically 15 L (of which 12 L is interstitial fluid and 3 L as plasma)

In some animals, including mammals, the extracellular fluid can be divided into two major
subcompartments, interstitial fluid and intravascular fluid (blood plasma).

 Interstitial fluid is the extracellular fluid bathing most tissues, constituting the environment of
body cells, excluding the fluid within the lymph and blood vessels. It is low in protein, is
formed by filtration through the capillaries, and drains away as lymph.
 Intravascular fluid a term sometimes used to refer to that part of the extracellular fluid that is
within the blood vessels, in other words, the plasma.

Ionic composition of ECF


The extracellular concentrations of a number of important ions are commonly determined suing
plasma from venous blood samples. National values are summarized in Table.1. Although the
total charge carried by all the cations and anions must be equal, a significant fraction of the anion
loaded is not accounted for in routine measurements. It can be seen that the sum of the measures
cations exceeds the sum of the measured anions, i.e.

[Na+] + [K+] + [Ca+] = 142 mmol L-1


[Cl-] [HCO3-] = 130 mmol L-1 (103 + 27)

The difference between these totals is called the anion gap; (17 mmol L-1 in this case). This
represents the contribution made by anions which are not normally measured in the clinical
laboratory, e.g. sulphate, phosphate, proteins and other organic anions of this sort, e.g. the anions

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released by dissociation of keto-acids in diabetes mellitus, will be assosicated with an increase in
the anion gap.

2.2 INTRA CELLULAR FLUID-nature and composition

The cytosol or intracellular fluid (or cytoplasmic matrix) is the liquid found inside cells. In
eukaryotes this liquid is separated by cell membranes from the contents of the organelles suspended
in the cytosol, such as the mitochondrial matrix inside the mitochondrion. The entire contents of a
eukaryotic cell, minus the contents of the cell nucleus, are referred to as the cytoplasm.

The ICF is a complex mixture of substances dissolved in water. Although water forms the large
majority of the ICF, its structure and properties within cells is not well understood. The
concentrations of ions such as sodium and potassium are different in the ICF than in the extracellular
fluid; these differences in ion levels are important in processes such as osmoregulation and cell
signaling. The ICF also contains large amounts of macromolecules, which can alter how molecules
behave, through macromolecular crowding.

Normal human ICF pH ranges between 7.3 - 7.5, depending on the cell type involved, whereas the
pH of the extracellular fluid is 7.4. The viscosity of cytoplasm is roughly the same as pure water,
although diffusion of small molecules through this liquid is about four-fold slower than in pure
water, due mostly to collisions with the large numbers of macromolecules in the cytosol.

Ionic composition of ICF


Most of the ICF is water, which makes up about 70% of a typical cell by volume. The
concentrations of the other ions in ICF are quite different from those in extracellular fluid and the
ICF also contains much higher amounts of charged macromolecules such as proteins and nucleic
acids than the outside of the cell.

The major anions and cations are different from those found outside the cell.

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Intra cellular [Na+] is low (about 10 mmol L-1) and [K+] is high (about 160 mmol L-1).
Intracellular [Cl-] is very low (2 mmol L-1) while proteins, phosphate and sulphate account for
the majority of intracellular cations.

In contrast to extracellular fluid, ICF has a high concentration of potassium ions and a low
concentration of sodium ions.[20] This difference in ion concentrations is critical for
osmoregulation, since if the ion levels were the same inside a cell as outside, water would enter
constantly by osmosis - since the levels of macromolecules inside cells are higher than their levels
outside. Instead, sodium ions are expelled and potassium ions taken up by the Na⁺/K⁺-ATPase,
potassium ions then flow down their concentration gradient through potassium-selection ion
channels, this loss of positive charge creates a negative membrane potential. To balance this
potential difference, negative chloride ions also exit the cell, through selective chloride channels.
The loss of sodium and chloride ions compensates for the osmotic effect of the higher
concentration of organic molecules inside the cell.

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3. ELECTROLYTE BALANCE

3.1 INTRODUCTION
3.2 ELECTROLYTE BALANCE
3.1 INTRODUCTION

Electrolytes are minerals in your body that have an electric charge. They are in your blood, urine and
body fluids. Maintaining the right balance of electrolytes helps your body's blood chemistry, muscle
action and other processes. Sodium, calcium, potassium, chlorine, phosphate and magnesium are all
electrolytes. You get them from the foods you eat and the fluids you drink.

Levels of electrolytes in your body can become too low or too high. That can happen when the
amount of water in your body changes. Causes include some medicines, vomiting, diarrhea,
sweating or kidney problems. Problems most often occur with levels of sodium, potassium or
calcium.

To function normally, the body must keep fluid levels from varying too much in the areas of the
body that contain fluid (called compartments). The three main compartments are
Fluid within cells
Fluid in the space around cells
Blood

Electrolytes, particularly sodium, help the body maintain normal fluid levels in these compartments
(called fluid balance), because how much fluid a compartment contains depends on the
concentration of electrolytes in it. If the electrolyte concentration is high, fluid moves into that
compartment. If the electrolyte concentration is low, fluid moves out of that compartment. To adjust
fluid levels, the body can actively move electrolytes in or out of cells. Thus, having electrolytes in
the right concentrations (called electrolyte balance) is important in maintaining fluid balance among
the compartments.

The kidneys help maintain electrolyte concentrations by filtering electrolytes from blood, returning
some electrolytes, and excreting any excess into the urine. Thus, the kidney help maintain a balance
between daily consumption and excretion.

If the balance of electrolytes is disturbed, disorders can develop. An electrolyte imbalance can result
from the following:

Becoming dehydrated
Taking certain drugs
Having certain heart, kidney, or liver disorders
Being given intravenous fluids or feedings in inappropriate amounts

3.2 BALANCE OF ELECTROLYTES

Individual cells work in conjunction with the kidneys


and lungs to regulate and maintain normal water and
electrolyte balance within the body. The kidneys
have the largest responsibility for maintaining blood
chemistry, and in concert with the lungs are
responsible for regulating the acid-base balance
within the blood. These processes involve both
electrolytes and water.
Fig.2. Electrolyte balance in cells by
Na pump mechanism
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3.2.1 Electrolyte balance In Cells (Fig. 2)
Sodium (Na+) is the major ion, or electrolyte, outside of the cell. Potassium (K+) is the major ion
inside the cell. Since cell membranes are permeable to Na+, it diffuses into the cell where its
concentration is lower. To maintain the low intracellular Na+ concentration, the cell quickly
pumps Na+ back into the extracellular fluid. Sodium pumps located in the cell membrane pump
Na+ out of and K+ into the cell, maintaining normal osmotic gradients.

3.2.2 Electrolyte balance In Kidneys (Fig 3)


The kidneys filter the blood to remove harmful metabolic acids and wastes and reabsorb those
substances the body needs. They help control plasma ion concentration and maintain pH by
removing strong ions such as Na+ and Cl- from plasma into urine. By removing more Na+ than
Cl-, for example, the kidneys lower the plasma Strong Ion Difference [SID]. SID is simply the
difference between the concentrations of positive and negative strong ions (Fig 3). As Na+ is
removed, the number of positive ions decreases causing a relative increase in the concentration of
negative ions. To offset the increase of negative charges created by Na+ removal, the
concentration of hydrogen ions, [H+], increases. As [H+] increases, pH decreases, making plasma
more acidic. On the other hand, removing more Cl- than Na+ raises plasma [SID], which lowers
[H+] and raises pH.

Fig 3. Electrolyte balance in kidney tubules

The kidneys also control blood volume by regulating the amount of water in extracellular fluid. (Fig
3). Two hormones, aldosterone and antidiuretic hormone (ADH) help the kidneys control the fluid
volume of blood. When water is lost from the body, blood volume decreases. This leads to increased
production of aldosterone and ADH. Elevated aldosterone increases Na+ pump activity in the kidney
tubules. As a result, more Na+ is removed from the distal tubules and concentrated within the kidney
rather than being excreted. This high concentration of Na+ in kidney tissue creates an osmotic
gradient that pulls water out of the tubules. Elevated ADH works in conjunction with aldosterone by
increasing the permeability of the tubules to water, allowing water to follow Na+ out of the tubules,
thereby reducing urinary water loss.

3.2.3 Electrolyte balance In Lungs


The lungs play an important role in regulating plasma CO2 and pH. Since CO2 is a gas, the term
partial pressure, PCO2, is used to describe its concentration in liquids. Changes in respiration rate
relate to changes in the partial pressure of CO2. For example, CO2 is produced during exercise and
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is removed from muscle tissue by the blood, increasing PCO2. To quickly rid the blood of excess
CO2, respiration rate increases. In this process, CO2 from the muscle combines with water to form
carbonic acid in the blood, which then dissociates to H+ and bicarbonate ions (HCO3-).
Bicarbonate is the primary storage and transportation form of CO2 in plasma. In the lungs, this
process is reversed with CO2 and water being exhaled.

Fig 4. Electrolyte balance by Co2 transport in blood

As the rate of CO2 production increases, PCO2, [HCO3-] and respiration rate increase. If CO2
production outstrips the lungs’ ability to convert HCO3- to water and CO2, plasma [HCO3-] will
continue to rise. Plasma [H+] will also rise, offsetting these, causing plasma pH to decrease.

Acidosis = ↑PCO2 , ↑HCO3- , ↓pH


Alkalosis = ↓PCO2 , ↓HCO3- , ↑pH

***

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4. pH BALANCE (ACID-BASE BALANCE)

4.1 INTRODUCTION
4.2 pH BALANCE BY METABOLIC REGULATION
4.3 pH BALANCE BY RESPIRATORY REGULATION

4.1 INTRODUCTION

By regulating blood chemistry, the kidneys and lungs maintain an electrically neutral environment.
The processes involved in maintaining electrical neutrality are referred to as acid-base balance. The
power of the lungs to excrete large quantities of carbon dioxide enables them to compensate rapidly,
while the smaller capacity of the kidneys corresponds to a relatively slower rate of compensation
through metabolic means. Both must work in concert to maintain an acid-base/pH balance.

4.2 pH BALANCE BY METABOLIC REGULATION

Strong ions are fully dissociated from each other in aqueous solutions such as plasma and form
charged water complexes. For example, Na+ and Cl- do not associate with each other to form NaCl
in plasma. Instead, Na+ associates with the O- component of water while Cl- associates with the H+
component.

The orientation of water molecules to a strong ion counterbalances and isolates the ion’s charge, and
exposes either the positive or negative portion of the water molecules. Charged water complexes of
Na+ have an overall positive charge that attracts OH-, while negative charges associated with Cl-
water complexes attract H+.

By selective removal of Na+ or Cl-, the kidneys adjust the relative proportion of H+ to OH- in
plasma. As Na+ is removed, the amount of OH- required to offset positive charged water complexes
decreases. Consequently, the amount of OH- in the solution decreases. As OH- decreases, the
relative amount of H+ increases, bringing about a reduction in pH. On the other hand, removal of
negative charged water complexes reduces the amount of H+ needed in the solution to offset
negative charges. As the relative amount of OH- increases, the solution becomes more basic and pH
rises. These processes are summarized below.

Fig 5. Electroneutrality maintained by kidney

4.3 pH BALANCE BY RESPIRATORY REGULATION

Since bicarbonate (HCO3-) is the plasma transport form of CO2, its regulation falls under the
jurisdiction of the lungs, not the kidneys. As previously discussed, the lungs quickly adjust the
partial pressure of carbon dioxide in plasma (PCO2) by either increasing or decreasing respiration
rate. As a result, plasma bicarbonate either decreases or increases.

Combined Effect of Respiratory & Metabolic Regulation :


Although the lungs and the kidneys have their own regulatory processes, they work together to
maintain the electrical neutrality of plasma. As an example, consider a baby calf that is undergoing
the common summertime problem of heat stress. In an attempt to get rid of extra heat, the calf’s
respiration rate increases. Although rapid breathing rids the body of some excess heat, it also
causes a loss of CO2, which lowers plasma PCO2. This lowers [HCO3-] and reduces the
associated [H+] required to offset the negative charges associated with bicarbonate. As plasma
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[H+] decreases, the relative [OH-] increases, causing plasma to become more alkaline. Plasma pH
rises. This situation is generally referred to as respiratory alkalosis.

Figure 6 summarizes the changes that occur in the calf as a result of increased respiration rate due
to heat stress.

Fig 6. Effect of increased respiration rate


To compensate, the kidneys remove Na+. As Na+ is removed, the [OH-] required to offset the
positive charge of sodium is reduced. This reduction in plasma [OH-] increases the relative [H+],
bringing plasma pH down to normal. Figure 7 summarizes the kidney response to the calf’s
respiratory alkalosis caused by heat stress.

Fig 7. Respiratory alkalosis

Through their combined actions the lungs and kidneys may have averted a couple of potentially
life-threatening situations. Nevertheless, the calf has lost body water during the heat stress and the
removal of Na+ has lowered the plasma [SID] below normal in order to maintain electrical
neutrality. Oral electrolyte therapy is an obvious remedy for both the water and electrolyte loss. If
administered early enough, the electrolyte treatment could avert or at least lessen the heat stress
and resulting physiological changes in the calf.

pH in Body fluids : The pH of intracellular fluid is about 7.0 and about 7.4 for extracellular fluid. At
normal body temperature, the pH of a solution is 6.8. Therefore, body fluids are actually maintained at
a slightly alkaline pH. The pH range of physiological solutions is small, with a pH change of 1.0 being
fatal. Ion movements in body fluids cause changes in pH, making it a dependant variable. As
demonstrated above, significant changes can occur in plasma chemistry that result in virtually no
change in pH. Furthermore, equal changes in [H+] and [OH-] in physiological solutions do not bring
about equal changes in pH. To summarize, a change in pH indicates a problem. It does not, however,
indicate what is causing the problem or what needs to be corrected. pH is not a very sensitive measure
for evaluating the acid-base status or changes in status of body fluids.

***

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Chapter – 2 BLOOD

5. Formation, composition and functions of blood


6. Mechanism of blood coagulation
7. Actions and uses of anticoagulants
8. Thrombolytic therapy and Anti-coagulant therapy

5. FORMATION, COMPOSITION AND FUNCTIONS OF BLOOD

5.1 INTRODUCTION
5.2 FORMATION OF BLOOD
5.3 COMPOSITION AND FUNCTIONS OF BLOOD

5.1 INTRODUCTION

Blood is a specialized bodily fluid that delivers necessary substances to the body's cells—such as
nutrients and oxygen—and transports waste products away from those same cells. In vertebrates it is
composed of blood cells suspended in a liquid called blood plasma. Plasma, which comprises 55%
of blood fluid, is mostly water (90% by volume), and contains dissolved proteins, glucose, mineral
ions, hormones, carbon dioxide (plasma being the main medium for excretory product
transportation), platelets and blood cells themselves. The blood cells present in blood are mainly red
blood cells (also called RBCs or erythrocytes) and white blood cells, including leukocytes and
platelets (also called thrombocytes).

Haemopoietic cells (those which produce blood) first appear in the yolk sac of the 2-week embryo.
By 8 weeks, blood making has become established in the liver of the embryo, and by 12-16 weeks
the liver has become the major site of blood cell formation. It remains an active haemopoietic site
until a few weeks before birth. The spleen is also active during this period, particularly in the
production of lymphoid cells, and the foetal thymus is a transient site for some lymphocytes. The
highly cellular bone marrow becomes an active blood making site from about 20 weeks gestation
and gradually increases its activity until it becomes the major site of production about 10 weeks
later.

At birth, active blood making red marrow occupies the entire capacity of the bones and continues to
do so for the first 2-3 years after birth. The red marrow is then very gradually replaced by inactive,
fatty, yellow, lymphoid marrow. The latter begins to develop in the shafts of the long bones and
continues until, by 20-22 years, red marrow is present only in the upper ends of the femur and
humerus and in the flat bones of the sternum, ribs, cranium, pelvis and vertebrae. However, because
of the growth in body and bone size that has occurred during this period, the total amount of active
red marrow (approximately 1000-1500 g) is nearly identical in the child and the adult.

Adult red marrow has a large reserve capacity for cell production. In childhood and adulthood, it is
possible for blood making sites outside marrow, such as the liver, to become active if there is
excessive demand as, for example, in severe haemolytic anaemia or following haemorrhage. In old
age, red marrow sites are slowly replaced with yellow, inactive marrow. Red marrow forms all types
of blood cell and is also active in the destruction of red blood cells. Red marrow is, therefore, one of
the largest and most active organs of the human body, approaching the size of the liver in overall
mass although as mentioned it is distributed in various parts of the body.

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About two-thirds of its mass functions in white cell production (leucopoiesis), and one-third in red
cell production (erythropoiesis). However as we have already seen there are approximately 700
times as many red cells as white cells in peripheral blood. This apparent anomaly reflects the shorter
life span and hence greater turnover of the white blood cells in comparison with the red blood cells.

5.2 FORMATION OF BLOOD

Hemopoiesis (hematoiesis) is the process that produces the formed elements of the blood.
Hemopoiesis takes place in the red bone marrow found in the epiphyses of long bones (for example,
the humerus and femur), flat bones (ribs and cranial bones), vertebrae, and the pelvis. Within the red
bone marrow, hemopoietic stem cells (hemocytoblasts) divide to produce various ―blast‖ cells. Each
of these cells mature and becomes a particular formed element.

It is now generally accepted that all blood cells are made from a relatively few 'uncommitted' cells
which are capable of mitosis and of differentiation into 'committed' precursors of each of the main
types of blood cell.

Fig. 5.2 Hematopoiesis:


The formation of the different types of blood cells from a common source the stem cell
5.2.1 Erythropoiesis (Formation of red blood cells)

The production of red blood cells is referred to as erythropoiesis. Mature red blood cells develop
from haemocytoblasts. This development takes about 7 days and involves three to four mitotic cell
divisions, so that each stem cell gives rise to 8 or 16 cells.

The various cell types in erythrocyte development are characterised by

the gradual appearance of haemoglobin and disappearance of ribonucleic acid (RNA) in the
cell
the progressive degeneration of the cell's nucleus which is eventually extruded from the cell
the gradual loss of cytoplasmic organelles, for example mitochondria
a gradual reduction in cell size

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The young red cell is called a retlculocyte because of a network of ribonucleic acid (reticulum)
present in its cytoplasm. As the red cell matures the reticulum disappears. Between 2 and 6% of a
new-born baby's circulating red cells are reticulocytes, but this reduces to less than 2% in the
healthy adult. However, the reticulocyte count increases considerably in conditions in which rapid
erythropoiesis occurs, for example following haemorrhage or acute haemolysis of red cells. A
reticulocyte normally takes about 4 days to mature into an erythrocyte.

In health, erythropoiesis is regulated so that the number of circulating erythrocytes is maintained


within a narrow range. Normally, a little less than l% of the body's total red blood cells are
produced per day and these replace an equivalent number that have reached the end of their life
span. However that still represents a huge 200,000,000,000 cells.

Erythropoiesis is stimulated by hypoxia (lack of oxygen). However, oxygen lack does not act
directly on the haemopoietic tissues but instead stimulates the production of a hormone,
erythropoietin. This hormone then stimulates haemopoietic tissues to produce red cells.

Erythropoietin is a glycoprotein. It is inactivated by the liver and excreted in the urine. It is now
established that erythropoietin is formed within the kidney by the action of a renal erythropoietic
factor erythrogenin on plasma protein, erythropoietinogen. Erythrogenin is present in the
juxtaglomerular cells of the kidneys and is released into the blood in response to hypoxia in the
renal arterial blood supply.

In anaemia there is a reduction in blood haemoglobin concentration due to a decrease in the


number of circulating erythrocytes and/or in the amount of haemoglobin they contain. Anaemia
occurs when the erythropoietic tissues cannot supply enough normal erythrocytes to the
circulation. In anaemias due to abnormal red cell production, increased destruction and when
demand exceeds capacity, plasma erythropoietin levels are increased. However, anaemia can also
be caused by defective production of erythropoietin as, for example, in renal disease.

5.2.2 Monocyte formation

Monocytes are produced in the bone marrow,


developing from nucleated precursors, the
monoblast and promonocyte. Mature cells have
a life in blood of approximately 3-8 hours and,
like granulocytes, there is a circulating and
marginating pool.

Fig. 5.2.2 Formation of monocytes from


phagocytic cells

Monocytes are actively phagocytic (engulf


other cells) and, on migration into the tissues,
they mature into larger cells called
macrophages (Derives from the Ancient Greek:
macro = big, phage = eat), which can survive
in the tissues for long periods. These cells form the mononuclear phagocytic cells of the
mononuclear phagocytic system (reticuloendothelial system) in bone marrow, liver, spleen and
lymph nodes.

Tissue macrophages (sometimes called histiocytes) respond more slowly than neutrophils to
chemotactic stimuli. They engulf and destroy bacteria, protozoa, dead cells and foreign matter.
They also function as modulators of the immune response by processing antigen structure and

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facilitating the concentration of antigen at the lymphocyte's surface. This function is essential in
order that full antigenic stimulation of both T and B lymphocytes can take place.

5.2.3 Granulocyte formation (Granulopoiesis/Leucopoiesis)

Granulocytes are the collective name given to three types of white blood cell. Namely these are
neutrophils, basophils and eosinophils.

In terms of their formation (granulopoiesis) they all derive from the same type of committed stem
cells called myeloblasts. After birth and into adulthood granulopoiesis occurs in the red marrow.
The process of producing granulocytes is characterised by the progressive condensation and
lobulation of the nucleus, loss of RNA and other cytoplasmic organelles, for example
mitochondria, and the development of cytoplasmic granules in the cells involved.

The development of a polymorphonuclear leukocyte make take a fortnight, but this time can be
considerably reduced when there is increased demand, as, for example, in bacterial infection. The
red marrow also contains a large reserve pool of mature granulocytes so that for every circulating
cell there may be 50-100 cells in the marrow.

Mature cells pass actively through the endothelial lining of the marrow sinusoid into the
circulation. In the circulation, about half the granulocytes adhere closely to the internal surface of
the blood vessels. These are called marginating cells and are not normally included in the white
cell count. The other half circulate in the blood and exchange with the marginating population.

Within 7 hours, half the granulocytes will have left the circulation in response to specific
requirements for these cells in the tissues. Once a granulocyte has left the blood it does not return.
It may survive in the tissues for 4 or 5 days, or less, depending on the conditions it meets. The
turnover of granulocytes is, therefore, very high. Dead cells are eliminated from the body in faeces
and respiratory secretions and are also destroyed by tissue macrophages (monocytes).

5.2.4 Lymphocyte formation

Lymphocytes are round cells containing large round nuclei. The cytoplasm stains pale blue and
appears non-granular under light microscopy. However, some cytoplasmic granules and organelles
are present.

Morphologically, lymphocytes can be divided into two groups: the more numerous small
lymphocytes, with a diameter of 7-10 mm; and large lymphocytes, which have a diameter of 10-14
mm. Lymphocytes are produced in bone marrow from primitive precursors, the lymphoblasts and
prolymphocytes. Immature cells migrate to the thymus and other lymphoid tissues, including that
found in bone marrow, and undergo further division, processing and maturation.

5.2.5 Platelete formation (Thrombopoeisis)

Platelets are produced in bone marrow by a process known as thrombopoiesis. They are formed in
the cytoplasm of a very large cell, the megakaryocyte. The cytoplasm of the megakaryocyte
fragments at the edge of the cell. This is called platelet budding. Megakaryocytes mature in about
10 days, from a large stem cell, the megakaryoblast.
It is likely that there are thrombopoietic feedback mechanisms as the platelet count remains fairly
constant in health, and platelet production is reduced following an infusion of platelets and
increased following removal of platelets. However, these feedback mechanisms have not been
discovered yet.

15 | P G D C B , P a p e r - I Dr. M. Estari
At any one time, about two-thirds of the body's platelets are circulating in the blood and one-third
are pooled in the spleen. There is constant exchange between the two populations. The life span of
platelets is between 8 and 12 days. They are destroyed by macrophages, mainly in the spleen and
also in the liver.

5.3 COMPOSITION AND FUNCTIONS OF BLOOD

The average human being has 5 liters of blood in his body. 55% of the blood is made up of plasma
constituting the fluid part of the blood. The cells and platelets that are present in our blood make up
the other 45%.

5.3.1 Blood plasma

Plasma is a pale yellowish fluid with a total volume of 2-3 liters in a normal adult. Its contents are
:
Water 90.0%
Protein 8.0%
Inorganic Ions 0.9%
Organic Substances 1.1%

Plasma Proteins: Plasma proteins are made up of 3 main types.


Serum Albumin
Serum Globulin
Fibrinogen
Prothrombin

Most of the proteins are produced by the liver apart from the Serum Globulin which is
produced by the body's immune system. Serum Albumin composed of 60% of the total plasma
protein. It is the smallest of the 4 plasma proteins and can pass through capillary walls. Hence
this will usually lead to a small leakage into the intestinal fluid. Nonetheless, this has been
exploited by medical science to be an effective medium of testing for abnormalities in the body
and damaged organs or diseases. Serum Globulins make up 36% of the total plasma protein.
This can be further split into 3 fragments, the alpha, beta and gamma. Globulins aid in the
inflammatory response of the body. Fibrinogen and prothrombin are important in the clotting
process of blood.

Protein Functions Include:


Transportation of insoluble substances around the body by allowing them to bind to protein
molecules.
Protein reserve for the body
Blood clotting
Responses in accordance to disease (inflammatory response)
Protection from infection the gamma globulins function
Striking balance for the pH of the blood

Inorganic Ions : Inorganic ions play a very important role in the blood.

CONCENTRATION
ION SYMBOL
(mmol/l)
Sodium Na+ 135-146

16 | P G D C B , P a p e r - I Dr. M. Estari
Potassium K+ 3.5-5.2
Calcium Ca++ 2.1-2.7
Chloride Cl- 98-108
Hydrogen Carbonate HCO3- 23-31
Phosphate PO4-- 0.7-1.4

Without sodium, the body will lack extracellular fluid and might affect the blood pressure,
leading to insufficient circulation of oxygen causing drowsiness, nausea etc. Lack of potassium
will result in muscle abnormalities and weakness, leading to vomiting and diarrhoea. Hence all
the inorganic ions facilitate in a very important role as do other cells within the blood.

Organic Substances: Blood plasma carry organic substances such as nutrients. They may include
digested food substances like glucose, sucrose and amino acids. Other nutrients in transit in the
plasma include glycerol, triglycerides, cholesterol and vitamins. Waste products of the body are
also transmitted in the blood plasma. They include urea and cellular waste that will be excreted out
of the body. Hormones, such as cortisol and thyroxine are also transported around the body in
plasma attached to plasma proteins. Medicine and drugs also circulate within the plasma.

5.3.2 Red Blood Cells

Red blood cells are the most common cells found in blood. There are about 5 million red blood
cells in each cubic millimeter of blood or approximately 250 million red blood cells in every drop
of blood. This number varies with individuals in accordance to heredity, gender and state of health.
These cells are produced by the bone marrow and have a lifespan of 3-4 months. When they die,
they are destroyed by macrophages in the liver and spleen. This process releases iron to be stored
in the liver and bile pigments to be excreted.

Structure of A Red Blood Cell : Red blood cells have a bi-concave shape with a flattened center. It
has a diameter less than 0.01 millimeters and do not have a nucleus. Red blood cells contain a
protein chemical known as haemoglobin, which gives it the red color. Haemoglobin contains iron,
which can easily transport gases such as oxygen and carbon dioxide. Red blood cells are highly
elastic, rendering it able to squeeze through capillary walls bigger than itself.

Functions of Red Blood Cells: Red blood cells are important in the process of respiration. Gases
involved in respiration are carried around the body through these cells. Oxygen readily combines
with haemoglobin to form oxy-haemoglobin in the lungs where there is high concentration of
oxygen. However, oxy-haemoglobin is an unstable compound and will break down to release
oxygen when there is low concentration of oxygen in the surroundings. Hence there will be an
even distribution of oxygen to all parts of the body. Red blood cells also carry part of the carbon
dioxide waste from the cells through most is transmitted through plasma as soluble carbonates.
Fig. 5.3.2 Red blood cells
and White blood cells

17 | P G D C B , P a p e r - I Dr. M. Estari
5.3.3 White Blood Cells (Leucocytes)

White blood cells are responsible for the defense system in the body. There are approximately
6,000 white blood cells per millimeter of blood or a million white blood cells in every drop of
human blood. White blood cells fight infections and protect our body from foreign particles, which
includes harmful germs and bacteria. White blood cells, the red blood cells are formed from the
stem cell of the bone marrow. It has a life-span of a couple of days. When they die, they are
destroyed by surrounding white blood cells and replaced with new ones.

Structure of White Blood Cells : White blood cells are colorless without haemoglobin. It contains
a nucleus and has an irregular shape. Though there are fewer white blood cells than red blood
cells, they are much bigger in size. They can change their shape easily and this allows them to
squeeze through walls of the blood vessels into the inter-cellular spaces.

Types of White Blood Cells: Unlike the Red blood cells or platelets, there are 5 different types of
white blood cells, each serving a different purpose in our body’s immune system.
Neutrophils
Eosinophils
Basophils
Monocytes
Lymphocytes

Neutrophils : Neutrophils make up 55%-70% of the total white blood count in the bloodstream.
They have a segmented nuclei and it is said to be C shaped. Neutrophils can be most commonly
found near sites of infection or injury where they will stick to the walls of the blood vessels and
engulf any foreign particles that try to enter the bloodstream. They can also be found in the pus
of wounds.

Eosinophils: Eosinophils make up 2%-5% of the total blood count and mainly attacks parasites
and any antigen complexes. These cells are also responsible for allergic response within the
blood.

Basophils: Basophils make up less than 1% of the total white blood count. They secrete anti-
coagulant and antibodies, which mediate hypersensitivity reactions within the blood. They are
known to have phagocytory features though they are more often related to immediate immune
reaction against external germs and diseases.
Monocytes: Monocytes, though having only 5%-8% in the total white blood count, are the
largest of the 5 types of white blood cells. They act as tissue macrophages and remove foreign
particles and prevent the invasion of germs which cannot be effectively dealt with by the
neutrophils. They have been known to have phagocytic function

Lymphocytes: Lymphocytes produce anti-bodies against toxins secreted by bacteria and


infecting germs. These antibodies will be excreted into the plasma to kill bacteria in the blood as
well as act as anti-toxins. These anti-bodies will cause the foreign particles to cluster together,
rendering them easily engulfed by the phagocytes. However, the nature of lymphocytes is highly
specific and they can only recognize certain antigens

5.3.4 Blood platelets

Normal platelet count in a healthy person is between 150,000 and 400,000 per mm³ of blood (150–
400 x 109/L). Blood Platelets are granular non-nucleated fragments of cytoplasm in the form of

18 | P G D C B , P a p e r - I Dr. M. Estari
oval discs. A platelet consists of two parts, a clear outer ground susbstance occupying the greater
part of the platelet and a central part that contains granules.

Functions of Blood Platelets: They secrete a hormone called serotonin which constricts torn blood
vessels. They also have a major role in accumulating at sites of injury sticking together to plug
gaps in broken blood vessels. They are rich in a certain activator that activates some proteins found
in plasma. These proteins are thrown out in the form of fibers as a network. This network traps the
escaping RBCs and forms a clot that will seal the cut blood vessels and so bleeding is stopped.

***

19 | P G D C B , P a p e r - I Dr. M. Estari
6. MECHANISM OF BLOOD COAGULATION

6.1 INTRODUCTION
6.2 MECHANISM OF BLOOD COAGULATION

6.1 INTRODUCTION

Coagulation is a complex process by which blood forms solid clots. It is an important part of
hemostasis (the cessation of blood loss from a damaged vessel) whereby a damaged blood vessel
wall is covered by a platelet- and fibrin-containing clot to stop bleeding and begin repair of the
damaged vessel. Disorders of coagulation can lead to an increased risk of bleeding and/or clotting
and embolism.

Coagulation is highly conserved throughout biology; in all mammals, coagulation involves both a
cellular (platelet) and a protein (coagulation factor) component. The system in humans has been the
most extensively researched and therefore the best understood.

Coagulation is initiated almost instantly after an injury to the blood vessel damages the endothelium
(lining of the vessel). Platelets immediately form a hemostatic plug at the site of injury; this is called
primary hemostasis. Secondary hemostasis occurs simultaneously—proteins in the blood plasma,
called coagulation factors, respond in a complex cascade to form fibrin strands which strengthen the
platelet plug.

6.2 MECHANISM OF BLOOD COAGULATION

The mechanism of blood coagulation completed in three steps:

Production of Thromboplastin by extrinsic and intrinsic pathways


Formation of Thrombin
Formation of Fibrin clot

6.2.1 Production of Thromboplastin


Thromboplastin is produced by extrinsic and intrinsic pathway.

Extrinsic pathway (Tissue factor pathway): The extrinsic pathway is initiated at the site of
injury in response to the release of tissue factor (factor III). Tissue factor is a cofactor in the
factor VIIa-catalyzed activation of factor X. Factor VIIa, a gla residue containing serine
protease, cleaves factor X to factor Xa in a manner identical to that of factor IXa of the intrinsic
pathway. The activation of factor VII occurs through the action of thrombin or factor Xa.

The ability of factor Xa to activate factor VII creates a link between the intrinsic and extrinsic
pathways. An additional link between the two pathways exists through the ability of tissue factor
and factor VIIa to activate factor IX. The formation of complex between factor VIIa and tissue
factor is believed to be a principal step in the overall clotting cascade. Evidence for this stems
from the fact that persons with hereditary deficiencies in the components of the contact phase of
the intrinsic pathway do not exhibit clotting problems. A major mechanism for the inhibition of
the extrinsic pathway occurs at the tissue factor--factor VIIa--Ca2+--Xa complex.

Intrinsic pathway: The intrinsic pathway requires the clotting factors VIII, IX, X, XI, and XII.
Also required are the proteins prekallikrein (PK) and high-molecular-weight kininogen (HK or
HMWK), as well as calcium ions and phospholipids secreted from platelets. Each of these
pathway constituents leads to the conversion of factor X (inactive) to factor Xa (―a‖ signifies
active).
20 | P G D C B , P a p e r - I Dr. M. Estari
Initiation of the intrinsic pathway occurs when prekallikrein, high-molecular-weight kininogen,
factor XI and factor XII are exposed to a negatively charged surface. The assemblage of contact
phase components results in conversion of prekallikrein to kallikrein, which in turn activates
factor XII to factor XIIa. Factor XIIa can then hydrolyze more prekallikrein to kallikrein,
establishing a reciprocal activation cascade. Factor XIIa also activates factor XI to factor Xia.

In the presence of Ca2+, factor XIa activates factor IX to factor IXa. Factor IX is a proenzyme
that contains vitamin K-dependent g-carboxyglutamate (gla) residues, whose serine protease
activity is activated following Ca2+ binding to these gla residues. Several of the serine proteases
of the cascade (II, VII, IX, and X) are gla-containing proenzymes. Active factor IXa cleaves
factor X at an internal arg-ile bond leading to its activation to factor Xa.

The activation of factor Xa requires assemblage of the tenase complex (Ca2+ and factors VIIIa,
IXa and X) on the surface of activated platelets. One of the responses of platelets to activation is
the presentation of phosphatidylserine (PS) and phosphatidylinositol (PI) on their surfaces. The
exposure of these phospholipids allows the tenase complex to form.

The role of factor VIII in this process is to act as a receptor, in the form of factor VIIIa, for
factors IXa and X. Factor VIIIa is termed a cofactor in the clotting cascade. At the presence of
calcium ions VIIIa factor produced Thromboplastin.

6.2.2 Production of Trombin


The common point in both pathways is the activation of factor X to factor Xa. Factor Xa activates
prothrombin (factor II) to thrombin (factor IIa). Thrombin, in turn, converts fibrinogen to fibrin.
The activation of thrombin occurs on the surface of activated platelets and requires formation of a
prothrombinase complex. This complex is composed of the platelet phospholipids,
phosphatidylinositol and phosphatidylserine, Ca2+, factors Va and Xa, and prothrombin.

Factor V is a cofactor in the formation of the prothrombinase complex, similar to the role of factor
VIII in tenase complex formation. Like factor VIII activation, factor V is activated to factor Va by
means of minute amounts and is inactivated by increased levels of thrombin. Factor Va binds to

21 | P G D C B , P a p e r - I Dr. M. Estari
specific receptors on the surfaces of activated platelets and forms a complex with prothrombin and
factor Xa.

Prothrombin is a 72,000-Dalton, single-chain protein containing ten gla residues in its N-terminal
region. Within the prothrombinase complex, prothrombin is cleaved at 2 sites by factor Xa. This
cleavage generates a 2-chain active thrombin molecule containing an A and a B chain which are
held together by a single disulfide bond.

6.2.3 Formation of fibrin clot

Fibrinogen (factor I) consists of 3 pairs of polypeptides ([A-a][B-b][g])2. The 6 chains are


covalently linked near their N-terminals through disulfide bonds. The A and B portions of the A-a
and B-b chains comprise the fibrinopeptides, A and B, respectively. The fibrinopeptide regions of
fibrinogen contain several glutamate and aspatate residues imparting a high negative charge to this
region and aid in the solubility of fibrinogen in plasma. Active thrombin is a serine protease that
hydrolyses fibrinogen at four arg-gly bonds between the fibrinopeptide and the a and b portions of
the protein.

The initial event in mechanism of fibrin polymerization is the thrombin activation at only one of
the Aa-chains in fibrinogen. The resulting highly reactive intermediate is the true fibrin monomer
and it rapidly, and irreversibly, self-associates to form the stable fibrin dimer (s20.w=12S). Fibrin
dimer possesses the N-terminal pattern alanine/glycine/tyrosine (1:1:2) per 340000 molecular
weight. The fibrin dimer is a soluble inert molecule, but additional thrombin activation of its
remaining intact Aa-chains leads to new associations into larger inert soluble fibrin polymers.

Factor Trivial Name(s) Pathway Characteristic

Functions with HMWK and


Prekallikrein (PK) Fletcher factor Intrinsic
factor XII

Co-factor in kallikrein and


High molecular
contact activation cofactor; Fitzgerald, factor XII activation,
weight kininogen Intrinsic
Flaujeac Williams factor necessary in factor XIIa
(HMWK)
activation of XI, precursor

22 | P G D C B , P a p e r - I Dr. M. Estari
for bradykinin (a potent
vasodilator and inducer of
smooth muscle contraction

I Fibrinogen Both -

Contains N-term. gla


II Prothrombin Both
segment

III Tissue Factor Extrinsic -

IV Calcium Both -

Proaccelerin, labile factor, accelerator (Ac-)


V Both Protein cofactor
globulin

VI This is Va, redundant to


Accelerin Both
(same as Va) Factor V

Proconvertin, serum prothrombin conversion Endopeptidase with gla


VII Extrinsic
accelerator (SPCA), cothromboplastin residues

Antihemophiliac factor A, antihemophilic


VIII Intrinsic Protein cofactor
globulin (AHG)

Christmas Factor,
Endopeptidase with gla
IX antihemophilic factor B,plasma Intrinsic
residues
thromboplastin component (PTC)

Endopeptidase with gla


X Stuart-Prower Factor Both
residues

XI Plasma thromboplastin antecedent (PTA) Intrinsic Endopeptidase

XII Hageman Factor Intrinsic Endopeptidase

Protransglutaminase,
XIII Both Transpeptidase
fibrin stabilizing factor (FSF), fibrinoligase

Table: Different coagulation factors and its characterisitics

***

23 | P G D C B , P a p e r - I Dr. M. Estari
7. ACTION AND USES OF ANTICOAGULANTS

An anticoagulant is a substance that prevents coagulation; that is, it stops blood from clotting. A
group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic
disorders. Some chemical compounds are used in medical equipment, such as test tubes, blood
transfusion bags, and renal dialysis equipment.

Anticoagulants are given to people to stop thrombosis (blood clotting inappropriately in the blood
vessels). This is useful in primary and secondary prevention of deep vein thrombosis, pulmonary
embolism, myocardial infarctions and strokes in those who are predisposed.

7.1 VITAMIN-K ANTAGONISTS

The oral anticoagulants are a class of pharmaceuticals that act by antagonizing the effects of vitamin
K. Examples include warfarin. It is important to note that they take at least 48 to 72 hours for the
anticoagulant effect to develop fully. In cases when any immediate effect is required, heparin must
be given concomitantly. Generally, these anticoagulants are used to treat patients with deep-vein
thrombosis (DVT), pulmonary embolism (PE), atrial fibrillation (AF), and mechanical prosthetic
heart valves.

7.1.1 Adverse effects


Patients aged 80 years or more may be especially susceptible to bleeding complications with a rate
of 13 bleeds per 100 person-years. These oral anticoagulants are used widely as poisons for
mammalian pests, especially rodents.

7.1.2 Available agents


Warfarin (Coumadin) This is the main agent used in the U.S. and UK
Acenocoumarol and phenprocoumon This is used more commonly outside the U.S. and the UK
Brodifacoum Rat poison, not used medically
Phenindione

7.2 HEPARIN AND DERIVATIVE SUBSTANCES

Heparin is a biological substance, usually made from pig intestines. It works by activating
antithrombin III, which blocks thrombin from clotting blood. Heparin can be used in vivo (by
injection), and also in vitro to prevent blood or plasma clotting in or on medical devices. Vacutainer
brand test tubes containing heparin are usually colored green.

7.2.1 Low molecular weight heparin

Low molecular weight heparin is a more highly processed product that is useful as it does not
require monitoring of the APTT coagulation parameter (it has more predictable plasma levels) and
has fewer side effects.

7.2.2 Synthetic pentasaccharide inhibitors of factor Xa


Fondaparinux is a synthetic sugar composed of the five sugars (pentasaccharide) in heparin that
bind to antithrombin. It is a smaller molecule than low molecular weight heparin.
Ex: Idraparinux

7.2.3 Major pharmaceutical Heparin


In March 2008 major recalls of Heparin were announced by pharmaceuticals due to a suspected
and unknown contamination of the raw Heparin stock imported from China. The U.S. Food and
Drug Administration was quoted as stating that at least 19 deaths were believed linked to a raw
24 | P G D C B , P a p e r - I Dr. M. Estari
Heparin ingredient imported from the People's Republic of China, and that they had also received
785 reports of serious injuries associated with the drug’s use. According to the New York Times:
'Problems with heparin reported to the agency include difficulty breathing, nausea, vomiting,
excessive sweating and rapidly falling blood pressure that in some cases led to life-threatening
shock'.

7.3 DIRECT THROMBIN INHIBITORS

Another type of anticoagulant is the direct thrombin inhibitor.[5] Current members of this class
include argatroban, lepirudin, bivalirudin, and dabigatran. An oral direct thrombin inhibitor,
ximelagatran (Exanta) was denied approval by the Food and Drug Administration (FDA) in
September 2004 and was pulled from the market entirely in February 2006 after reports of severe
liver damage and heart attacks.

***

25 | P G D C B , P a p e r - I Dr. M. Estari
8. THROMBOLYTIC AND ANTICOAGULANT THERAPY

8.1 THROMBOLYTIC THERAPY


8.2 ANTICOAGULANT THERAPY

8.1 THROMBOLYTIC THERAPY

8.1.1 Definition
Thrombolytic therapy is the use of drugs that dissolve blood clots.

8.1.2 Purpose
When a blood clot forms in a blood vessel, it may cut off or severely reduce blood flow to parts of
the body that are served by that blood vessel. This can cause serious damage to those parts of the
body. If the clot forms in an artery that supplies blood to the heart, for example, it can cause a
heart attack. A clot that cuts off blood to the brain can cause a stroke. Thrombolytic therapy is used
to dissolve blood clots that could cause serious, and possibly life-threatening, damage if they are
not removed. Research suggests that when used to treat stroke, thrombolytic therapy can prevent or
reverse paralysis and other problems that otherwise might result.Thrombolytic therapy also is used
to dissolve blood clots that form in tubes put into people's bodies for medical treatments, such as
dialysis or chemotherapy.

8.1.3 Description
Thrombolytic therapy uses drugs called thrombolytic agents, such as alteplase (Activase),
anistreplase (Eminase), streptokinase (Streptase, Kabikinase), urokinase (Abbokinase), and tissue
plasminogen activator (TPA) to dissolve clots. These drugs are given as injections, only under a
physician's supervision.

8.1.4 Recommended dosage


The physician supervising thrombolytic therapy decides on the proper dose for each patient. He or
she will take into account the type of drug, the purpose for which it is being used, and in some
cases, the patient's weight.

8.1.5 Precautions
For thrombolytic therapy to be effective in treating stroke or heart attack, prompt medical attention
is very important. The drugs must be given within a few hours of the beginning of a stroke or heart
attack. However, this treatment is not right for every patient who has a heart attack or a stroke.

Thrombolytic therapy may cause bleeding. Usually this is not serious, but severe bleeding does
occur in some people. This is especially likely in older people. To lower the risk of serious
bleeding, people who are given this drug should move around as little as possible and should not
try to get up on their own unless told to do so by a health care professional. Following all the
instructions of the health care providers in charge is very important.

Thrombolytic therapy may be more likely to cause serious bleeding in people who have certain
medical conditions or have recently had certain medical procedures. Before being given a
thrombolytic agent, anyone with any of these problems or conditions should tell the physician in
charge about it:

blood disease or current or past bleeding problems in any part of the body
heart or blood vessel disease
stroke (recent or in the past)
high blood pressure

26 | P G D C B , P a p e r - I Dr. M. Estari
After being treated with thrombolytic therapy, women who are breastfeeding should check with
their physicians before starting to breastfeed again.

8.1.6 Side effects


Anyone who has fever or who notices bleeding or oozing from their gums, from cuts, or from the
site where the thrombolytic agent was injected should immediately tell their health care provider.
People who are given thrombolytic therapy should also be alert to the signs of bleeding inside the
body and should check with a physician immediately if any of the following symptoms occur:

blood in the urine, blood or black, tarry stools, constipation, coughing up blood, vomiting
blood or material that looks like coffee grounds, nosebleeds, unexpected or unusually heavy
vaginal bleeding, dizziness, sudden, severe, or constant headaches and Pain or swelling in the
abdomen or stomach.

Fig. 8.1 Thrombolytic therapy

8.2 ANTICOAGULANT THERAPY

8.2.1 Definition
Anticoagulant therapy is the use of Anticoagulant drugs to prevent clot formation or to prevent a
clot that has formed from enlarging. They inhibit clot formation by blocking the action of clotting
factors or platelets. Anticoagulant drugs fall into three categories: inhibitors of clotting factor
synthesis, inhibitors of thrombin and antiplatelet drugs.

8.2.2 Purpose
Anticoagulant drugs reduce the ability of the blood to form clots. Although blood clotting is
essential to prevent serious bleeding in the case of skin cuts, clots inside the blood vessels block
the flow of blood to major organs and cause heart attacks and strokes. Although these drugs are
sometimes called blood thinners, they do not actually thin the blood.

27 | P G D C B , P a p e r - I Dr. M. Estari
Anticoagulant drugs are used for a number of conditions. The four most common conditions for
which anticoagulant therapy is prescribed are atrial fibrillation, deep vein thrombosis, pulmonary
embolism, and mechanical heart valves.

8.2.3 Description
Anticoagulant drugs, also called anticlotting drugs or blood thinners, are available only with a
physician's prescription. They come in tablet and injectable forms. They fall into three groups:

Inhibitors of clotting factor synthesis. These anticoagulants inhibit the production of certain
clotting factors in the liver. One example is warfarin (brand name: coumadin).
Inhibitors of thrombin. Thrombin inhibitors interfere with blood clotting by blocking the
activity of thrombin. They include heparin, lepirudin (Refludan).
Antiplatelet drugs. Antiplatelet drugs interact with platelets, which is a type of blood cell, to
block platelets from aggregating into harmful clots. They include: aspirin, ticlopidine (Ticlid),
clopidogrel (Plavix), tirofiban (Aggrastat), and eptifibatide (Integrilin).

8.2.4 Recommended dosage


The recommended dosage depends on the type of anticoagulant drug and the medical condition for
which it is prescribed. The prescribing physician or the pharmacist who filled the prescription can
provide information concerning the correct dosage. Usually, the physician will adjust the dose
after checking the patient's clotting time.

Anticoagulant drugs must be taken exactly as directed by the physician. Larger or more frequent
doses should not be taken, and the drug should also not be taken for longer than prescribed. Taking
too much of this medication can cause severe bleeding. Anticoagulants should also be taken on
schedule. A record of each dose should be kept as it is taken.

8.2.5 Precautions
Persons who take anticoagulants should see a physician regularly while taking these drugs,
particularly at the beginning of therapy. The physician will order periodic blood tests to check the
blood's clotting ability. The results of these tests will help the physician determine the proper
amount of medication to be taken each day.

Time is required for normal clotting ability to return after anticoagulant treatment. During this
period, patients must observe the same precautions they observed while taking the drug. The
length of time needed for the blood to return to normal depends on the type of anticoagulant drug
that was taken. The prescribing physician will advise as to how long the precautions should be
observed. Alcohol can change the way anticoagulant drugs affect the body. Anyone who takes this
medicine should not have more than one to two drinks at any time and should not drink alcohol
every day.

8.2.6 Side effects


The most common minor side effects are bloating or gas. These problems usually go away as the
body adjusts to the drug and do not require medical treatment. More serious side effects may
occur, especially if excessive anticoagulant is taken. If any of the following side effects occur, a
physician should be notified immediately:

bleeding gums, sores or white spots in the mouth or throat, unusual bruises or purplish areas on
the skin, cloudy or dark urine, painful or difficult urination or sudden decrease in amount of
urine, black, tarry, or bloody stools, yellow eyes or skin and severe or continuing headache.

***
28 | P G D C B , P a p e r - I Dr. M. Estari
Chapter – 3 CEREBROSPINAL FLUID (CSF)

9. Composition and functions of CSF


10. Composition and functions of Lymph

9. COMPOSITION AND FUNCTIONS OF CSF

9.1 INTRODUCTION
9.2 COMPOSITION OF CSF
9.3 FUNCTIONS OF CSF
9.1 INTRODUCTION

Cerebrospinal fluid (CSF), Liquor cerebrospinalis, is a clear bodily fluid that occupies the
subarachnoid space and the ventricular system around and inside the brain. Essentially, the brain
"floats" in it.

More specifically the CSF occupies the space between the arachnoid mater (the middle layer of the
brain cover, meninges) and the pia mater (the layer of the meninges closest to the brain). Moreover it
constitutes the content of all intra-cerebral (inside the brain, cerebrum) ventricles, cisterns and sulci
(singular sulcus), as well as the central canal of the spinal cord.

It is produced in the brain by modified ependymal cells in the choroid plexus. It circulates from the
choroid plexus through the interventricular foramina (foramen of Monro) into the third ventricle,
and then through the mesencephalic duct (cerebral aqueduct) into the fourth ventricle, where it exits
through two lateral apertures (foramina of Luschka) and one median aperture (foramen of
Magendie). It then flows through the cerebromedullary cistern down the spinal cord and over the
cerebral hemispheres.

It is an approximately isotonic solution and acts as a "cushion" or buffer for the cortex, providing
also a basic mechanical and immunological protection to the brain inside the skull.

9.2 COMPOSITION OF CSF

The cerebrospinal fluid is produced at a rate of 500 ml/day. Since


the brain can only contain from 135-150 ml, large amounts are
drained primarily into the blood through arachnoid granulations in
the superior sagittal sinus. This continuous flow into the venous
system dilutes the concentration of larger, lipoinsoluble molecules
penetrating the brain and CSF.

The CSF contains approximately 0.3% plasma proteins, or 15 to 40


mg/dL, depending on sampling site.[3] CSF pressure ranges from 60
- 100 mmH2O or 4.4 - 7.3 mmHg, with most variations due to
coughing or internal compression of jugular veins in the neck.
Fig. 9.1 Cerebrospinal fluid
9.3 FUNCTIONS OF CSF

Protection
The CSF protects the brain from damage by "buffering" the brain. In other words, the CSF acts to
cushion a blow to the head and lessen the impact.
29 | P G D C B , P a p e r - I Dr. M. Estari
Buoyancy
Because the brain is immersed in fluid, the net weight of the brain is reduced from about 1,400 gm
to about 50 gm. Therefore, pressure at the base of the brain is reduced.

Excretion of waste products


The one-way flow from the CSF to the blood takes potentially harmful metabolites, drugs and
other substances away from the brain.

Endocrine medium for the brain


The CSF serves to transport hormones to other areas of the brain. Hormones released into the CSF
can be carried to remote sites of the brain where they may act.

***

30 | P G D C B , P a p e r - I Dr. M. Estari
10. COMPOSITION AND FUNCTIONS OF LYMPH

10.1 INTRODUCTION
10.2 COMPOSITION OF LYMPH
10.3 FUNCTIONS OF LYMPH
10.1 INTRODUCTION

Lymph is the fluid that is formed as the interstitial fluid enters the lymph vessels by filtration. The
lymph then travels to at least one lymph node before emptying ultimately into the right or the left
subclavian vein, where it mixes back with blood.

The lymph is a transparent, usually slightly yellow, often opalescent liquid found within the
lymphatic vessels, and collected from tissues in most parts of the body and returned to the blood via
the lymphatic system.

Lymph is the name given to interstitial fluid which enters the lymphatic vessels. Lymphatic
capillaries are present in nearly all tissues. Significant exceptions are the central nervous system and
bone. Small interstitial channels are present in the brain and the fluid flows into the CSF and then
passes back into the circulation via the arachnoid villi.

The lymph capillaries are blind-ending and possess flap valves between adjacent lymphatic
endothelial cells. These functional valves permit entry of ISF but prevent its return to the
interstitium. The pressure inside the lymph capillary is about 1 mmHg at rest and the flap valves are
closed. The lymph capillaries interconnect and join together to form lymph venules, and then large
lymph veins which drain via lymph nodes into the thoracic duct (on the left) and the right lymphatic
duct. By these two final pathways, lymph returns into the circulation.

10.2 COMPOSITION OF LYMPH

Lymph has a composition comparable to that of plasma, but it is different in various parts of the
body depending upon the tissue drained. It is about 95% water; the remainder consists of plasma
proteins and other chemical substances contained in the blood plasma, but in a slightly smaller
percentage than in plasma.

In particular, the lymph that leaves a lymph node is richer in lymphocytes (about 8,000 per cubic
millimeter), proteins, and fats. Likewise, the lymph formed in the digestive system called chyle is
rich in triglycerides (fat), and looks white.

10.3 FUNCTIONS OF LYMPH

Lymph plays an important part in the immune system and in absorbing fats from the small intestine.
The three functions of the lymphatic system are:

Return of protein and fluid from the ISF to the circulation to maintain a low interstitial fluid
protein concentration and maintain the oncotic pressure gradient across the capillary
membrane. Oedema will occur if ISF oncotic pressure is not kept low.
Role in absorption and transport of fat from the small intestine.
Immunological role -lymph glands & circulation of immune cells such as lymphocytes and
dendritic cells, removal of bacteria.

Lymph from most parts of the body usually has a low protein concentration. Liver lymph is different
because:

31 | P G D C B , P a p e r - I Dr. M. Estari
It normally has a high protein concentration (due to low reflection coefficient)
It contributes more than half of all the thoracic duct lymph

Consequently, the average lymph protein concentration in thoracic duct lymph is much higher than
expected based on protein concentration in lymph from other body tissues.

The thoracic duct carries about 80% of the total lymph flow. This total flow at rest is about 120
mls/hr. If interstitial hydrostatic pressure rises (ie becomes less negative) due to excess fluid
filtration & accumulation, the total lymph flow can increase quite markedly.

Chyle is lymph from the intestines which has a milky-while appearance due to the presence of large
numbers of chylomicrons. Chylomicrons are 100nm diameter complexes of mostly triglycerides
(containing the long chain fatty acids) enclosed in a hydrophobic protein coat. Chylomicrons enter
the lymphatic lacteals in the villi, travel in the lymph and then enter the circulation via the thoracic
duct.

***

32 | P G D C B , P a p e r - I Dr. M. Estari
Chapter – 4 DIGESTIVE SECRETIONS

11. Formation, composition and functions of


digestive secretions

11. FORMATION, COMPOSITION AND FUNCTIONS OF DIGESTIVE SECRETIONS

11.1 INTRODUCTION
11.2 DIGESTIVE SECRETIONS

11.1 INTRODUCTION

Digestive secretions are enzymes in the alimentary tract that break down food so that the organism
can absorb it. The main sites of action are the oral cavity, the stomach, the duodenum and the
jejunum. They are secreted by different glands: the salivary glands, the glands in the stomach, the
pancreas, Liver and the glands in the small intestines.

In the oral cavity, salivary glands secrete saliva, which digests starch into small segments of multiple
sugars and into individual soluble sugars. The secretions that from the stomach are called gastric
juice, which digest the proteins.

The liver secretes bile juice, which emulsifies the fat substances. The pancreas at the region of
duodenum secreted pancreatic juice, which digest the proteins, lipids and carbohydrates. The small
intestine (Duedenum/jejunum) secretes succus entericus (Interstinal juice), which digest all type of
food materials.

11.2 DIGESTIVE SECRETIONS

The following are the digestive secretions which secreted from the different digestive glands like
salivary glands, gastric glands of stomach, pancreas, liver and the glands in the small intestines.
Saliva
Gastric juice
Bile juice
Pancreatic juice
Succus entericus (Intestinal juice)

11.2.1 Saliva:

a) Formation of saliva
The production of saliva is stimulated both by the sympathetic nervous system and the
parasympathetic.
The saliva stimulated by sympathetic innervation is thicker, and saliva stimulated
parasympathetically is more watery.
The formation of saliva is a multi-step process. Initially formed of an aqueous solution (water
based solution) of electrolytes, proteins (mostly enzymes), and mucus, saliva undergoes several
chemical changes before it is release from the glandular collecting ducts into the oral cavity. The
sodium content is reduced and potassium levels increase along with the addition of bicarbonate
ions that make the saliva alkaline.
The average person produces approximately 700mL of saliva per day, which is much less than
was once thought.
33 | P G D C B , P a p e r - I Dr. M. Estari
b) Composition of saliva
Produced in salivary glands, saliva is 98% water, but it contains many important substances,
including electrolytes, mucus, antibacterial compounds and various enzymes.
It is a fluid containing:

Water
Electrolytes:
o 2-21 mmol/L sodium (lower than blood plasma)
o 10-36 mmol/L potassium (higher than plasma)
o 1.2-2.8 mmol/L calcium
o 0.08-0.5 mmol/L magnesium
o 5-40 mmol/L chloride (lower than plasma)
o 25 mmol/L bicarbonate (higher than plasma)
o 1.4-39 mmol/L phosphate
Mucus. Mucus in saliva mainly consists of mucopolysaccharides and glycoproteins;
Antibacterial compounds (thiocyanate, hydrogen peroxide, and secretory immunoglobulin A)
Various enzymes. There are three major enzymes found in saliva.
o α-amylase . Amylase starts the digestion of starch and lipase fat before the food is even
swallowed. It has a pH optima of 7.4.
o lysozyme. Lysozyme acts to lyse bacteria.
o lingual lipase. Lingual lipase has a pH optimum ~4.0 so it is not activated till entering
an acidic environment.
o Minor enzymes include salivary acid phosphatases A+B, N-acetylmuramyl-L-alanine
amidase, NAD(P)H dehydrogenase-quinone, salivary lactoperoxidase, superoxide
dismutase, glutathione transferase, class 3 aldehyde dehydrogenase, glucose-6-
phosphate isomerase, and tissue kallikrein.
Cells: Possibly as much as 8 million human and 500 million bacterial cells per mL. The
presence of bacterial products (small organic acids, amines, and thiols) causes saliva to
sometimes exhibit foul odor.
Opiorphin, a newly researched pain-killing substance found in human saliva.

c) Functions of saliva
Digestion: The digestive functions of saliva include moistening food, and helping to create a food
bolus, so it can be swallowed easily. Saliva contains the enzyme amylase that breaks some
starches down into maltose and dextrin. Thus, digestion of food occurs within the mouth, even
before food reaches the stomach. Salivary glands also secrete enzymes (salivary lipase) to start fat
digestion.[4]

Disinfectants: A common belief is that saliva contained in the mouth has natural disinfectants,
which leads people to believe it is beneficial to "lick their wounds". However, researchers find
human saliva contains such antibacterial agents as secretory IgA, lactoferrin, and lactoperoxidase.
It has not been shown that human licking of wounds disinfects them, but licking is likely to help
clean the wound by removing larger contaminants such as dirt and may help to directly remove
infective bodies by brushing them away. Therefore, licking would be a way of wiping off
pathogens, useful if clean water is not available to the animal or person.

Cleaning: Saliva is an effective cleaning agent used in art conservation. Cotton swabs coated
with saliva are rolled across a paintings surface to delicately remove thin layers of dirt that may
accumulate.

34 | P G D C B , P a p e r - I Dr. M. Estari
11.2.2 Gastric secretion

a) Formation of Gastric secretion


Mucus, ions and water make up the bulk of the gastric juice secreted by the proximal and distal
regions. The composition of the gastric secretions is not constant but varies with the flow rate.
The basal secretion produced by the surface epithelial cells is plasma-like in composition built its
modified extensively as gastric secretion increases through secretion of acid by the oxyntic cells
of the proximal region.

HCl is secreted by oxyntic cells. As rest these cells contain numerous tubulovesicles that are
rich in H+, K+ -ATPase and the adjacent to canaliculi which open into the lumen of the gastric
glands. When the cells are stimulated to secrete acid the vesicles fuse with the canaliculi, and at
the same time there is an increase in canalicular membrance permeability to K+ and Cl-. The
movement of K+ into the lumen activates the H+, K+, -ATPase so that intracellular H+ is
exchanged with K+ and there is therefore a net secretion of HCl. The water accompanying the
HCl is driven out of the cell by the efflux of ions.

The composition of the acidic fluid secreted by the parietal cell Is therefore largely isosmotic
HCl with a little KCl. For each mole of H+ secreted, an equivalent amount of base (OH-) is
producec. In the oxynitic cell carbonic anhydrase catalyses the reaction that combines OH- with
CO2 to give HCO3- , The HCO3- then passes out of the cell into the blood via a Cl-/HCO3-
exchanger. Tlhis provides the sourceof Cl- that accompanies the secretion of H+.

Pepsinogens are synthesized in the chief cells, stored in granules and secreted by exocytosis is
inactive precursors. This prevents digestion of the chief cells. Intrinsic factor is synthesized and
secreted by the oxyntic cells and argentiferin cells.

c) Composition and functions of Gastric secretion


Mucus: The most abundant epithelial cells are mucous cells, which cover the entire lumenal
surface and extend down into the glands as "mucous neck cells". These cells secrete a
bicarbonate-rich mucus that coats and lubricates the gastric surface, and serves an important role
in protecting the epithelium from acid and other chemical insults.
Acid: Hydrochloric acid is secreted from parietal cells into the lumen where it establishes an
extremely acidic environment. This acid is important for activation of pepsinogen and
inactivation of ingested microorganisms such as bacteria.
Proteases: Pepsinogen, an inactive zymogen, is secreted into gastric juice from both mucous
cells and chief cells. Once secreted, pepsinogen is activated by stomach acid into the active
protease pepsin, which is largely responsible for the stomach's ability to initiate digestion of
proteins. In young animals, chief cells also secrete chymosin (rennin), a protease that coagulates
milk protein allowing it to be retained more than briefly in the stomach.
Hormones: The principal hormone secreted from the gastric epithelium is gastrin, a peptide that
is important in control of acid secretion and gastric motility.

11.2.3 Bile juice

a) Formation of Bile juice

Bile is produced by hepatocytes in the liver, draining through the many bile ducts that penetrate
the liver. During this process, the epithelial cells add a watery solution that is rich in bicarbonates
that dilutes and increases alkalinity of the solution. Bile then flows into the common hepatic duct,
which joins with the cystic duct from the gallbladder to form the common bile duct.

35 | P G D C B , P a p e r - I Dr. M. Estari
The common bile duct in turn joins with the pancreatic duct to empty into the duodenum. If the
sphincter of Oddi is closed, bile is prevented from draining into the intestine and instead flows into
the gall bladder, where it is stored and concentrated to up to five times its original potency between
meals. This concentration occurs through the absorption of water and small electrolytes, while
retaining all the original organic molecules. Cholesterol is also released with the bile, dissolved in
the acids and fats found in the concentrated solution. When food is released by the stomach into
the duodenum in the form of chyme, the gallbladder releases the concentrated bile to complete
digestion.

The human liver can produce close to one litre of bile per day (depending on body size). 95% of
the salts secreted in bile are reabsorbed in the terminal ileum and re-used. Blood from the ileum
flows directly to the hepatic portal vein and returns to the liver where the hepatocytes resorb the
salts and return them to the bile ducts to be re-used, sometimes two to three times with each meal.

b) Composition of Bile juice


Bile has various components, some of which are produced by hepatocytes (liver cells). Its
constituents include:

Cholesterol
Phospholipids (mainly Lecithin)
Bile pigments (bilirubin diglucoronoide)
Bile salts (sodium glycocholate & sodium taurocholate)
Bicarbonate ions and other ions
Metabolized red blood cells

The bile acids cholate and chenodeoxycholate are typically conjugated with taurine or glycine and
are produced by the liver from cholesterol. Ordinarily the concentration of bile salts in bile is
0.8%, however the gall bladder removes water from the bile, concentrating it between meals. It
concentrates it up to 5 times (increasing concentration to 4%), before contracting the walls and
releasing it into the duodenum once chyme has entered the small intestine.

c) Functions of Bile juice


Role of Bile Acids in Fat Digestion and Absorption: Bile acids are derivatives of cholesterol
synthesized in the hepatocyte. Cholesterol, ingested as part of the diet or derived from hepatic
synthesis is converted into the bile acids cholic and chenodeoxycholic acids, which are then
conjugated to an amino acid (glycine or taurine) to yield the conjugated form that is actively
secreted into cannaliculi.

Their amphipathic nature enables bile acids to carry out two important functions:

Emulsification of lipid aggregates: Bile acids have detergent action on particles of dietary fat
which causes fat globules to break down or be emulsified into minute, microscopic droplets.
Emulsification is not digestion per se, but is of importance because it greatly increases the
surface area of fat, making it available for digestion by lipases, which cannot access the inside
of lipid droplets.

Solubilization and transport of lipids in an aqueous environment: Bile acids are lipid carriers
and are able to solubilize many lipids by forming micelles - aggregates of lipids such as fatty
acids, cholesterol and monoglycerides - that remain suspended in water. Bile acids are also
critical for transport and absorption of the fat-soluble vitamins.

Role of Bile Acids in Cholesterol Homeostasis: Hepatic synthesis of bile acids accounts for the
majority of cholesterol breakdown in the body. In humans, roughly 500 mg of cholesterol are
36 | P G D C B , P a p e r - I Dr. M. Estari
converted to bile acids and eliminated in bile every day. This route for elimination of excess
cholesterol is probably important in all animals, but particularly in situations of massive
cholesterol ingestion.

Besides its digestive function, bile serves as the route of excretion for the hemoglobin breakdown
product (bilirubin) created by breakdown of erythrocytes, which are conjugated by glucuronidation
in the liver ; it also neutralises any excess stomach acid before it enters the ileum, the final section
of the small intestine. Bile salts are also bacteriocidal to the invading microbes that enter with
food.

11.2.4 Pancreatic secretions

a) Formation of Pancreatic secretions


Pancreatic secretion is composed of two secretory products critical to proper digestion: digestive
enzymes and bicarbonate.

Bicarbonate ions are secreted by centro-acinar cells in higher concentrations than present in
plasma, while Na+ and K+ concentrations are identical to that in the plasma. Since chloride
concentration in the centro-acinar secretion is lower than in the plasma, it is exchanged for
bicarbonate ions to maintain iso-osmolarity of the composite secretion. With increase in secretory
rate, the available time for this exchange during the passage in the ducts diminishes considerably
and hence the chloride concentration in the final secretion falls. This bicarbonate-chloride shift in
responsible for the reciprocal relationship between the concentration of these two ions in the
secretion.

The enzymes are synthesized and stored as zymogen granules in the acinar cells. Under adequate
stimuli, these zymogen granules are discharged into the central cavity by a process called
emiocytosis (reverse pinocytosis).

b) Composition and functions of Pancreatic secretion


Pancreatic juice is a juice produced by the pancreas. It contains water (97.6%) and organic
constituents like a variety of enzymes, including trypsinogen, chymotrypsinogen, elastase,
carboxypeptidase, pancreatic lipase, and amylase (1.8%).

Pancreatic juice is composed of two secretory products critical to proper digestion: digestive
enzymes and bicarbonate. The enzymes are synthesized and secreted from the exocrine acinar
cells, whereas bicarbonate is secreted from the epithelial cells lining small pancreatic ducts.

Digestive Enzymes:
1. Proteases: Digestion of proteins is initiated by pepsin in the stomach, but the bulk of protein
digestion is due to the pancreatic proteases. Several proteases are synthesized in the pancreas and
secreted into the lumen of the small intestine. The two major pancreatic proteases are trypsin and
chymotrypsin, which are synthesized and packaged into secretory vesicles as an the inactive
proenzymes trypsinogen and chymotrypsinogen.

As you might anticipate, proteases are rather dangerous enzymes to have in cells, and packaging
of an inactive precursor is a way for the cells to safely handle these enzymes. The secretory
vesicles also contain a trypsin inhibitor which serves as an additional safeguard should some of
the trypsinogen be activated to trypsin; following exocytosis this inhibitor is diluted out and
becomes ineffective - the pin is out of the grenade.

37 | P G D C B , P a p e r - I Dr. M. Estari
Once trypsinogen and chymotrypsinogen are released into the lumen of the small intestine, they
must be converted into their active forms in order to digest proteins. Trypsinogen is activated by
the enzyme enterokinase, which is embedded in the intestinal mucosa.

Once trypsin is formed it activates chymotrypsinogen, as well as additional molecules of


trypsinogen. The net result is a rather explosive appearance of active protease once the
pancreatic secretions reach the small intestine.

Trypsin and chymotrypsin digest proteins into peptides and peptides into smaller peptides, but
they cannot digest proteins and peptides to single amino acids. Some of the other proteases from
the pancreas, for instance carboxypeptidase, have that ability, but the final digestion of peptides
into amino acids is largely the effect of peptidases on the surface of small intestinal epithelial
cells. More on this later.

2. Pancreatic Lipase: A major component of dietary fat is triglyceride, or neutral lipid. A


triglyceride molecule cannot be directly absorbed across the intestinal mucosa. Rather, it must first
be digested into a 2-monoglyceride and two free fatty acids. The enzyme that performs this
hydrolysis is pancreatic lipase, which is delivered into the lumen of the gut as a constituent of
pancreatic juice.

Sufficient quantities of bile salts must also be present in the lumen of the intestine in order for
lipase to efficiently digest dietary triglyceride and for the resulting fatty acids and monoglyceride
to be absorbed. This means that normal digestion and absorption of dietary fat is critically
dependent on secretions from both the pancreas and liver.

Pancreatic lipase has recently been in the limelight as a target for management of obesity. The
drug orlistat (Xenical) is a pancreatic lipase inhibitor that interferes with digestion of triglyceride
and thereby reduces absorption of dietary fat. Clinical trials support the contention that inhibiting
lipase can lead to significant reductions in body weight in some patients.

3.Amylase: The major dietary carbohydrate for many species is starch, a storage form of glucose
in plants. Amylase (technically alpha-amylase) is the enzyme that hydrolyses starch to maltose (a
glucose-glucose disaccharide), as well as the trisaccharide maltotriose and small branchpoints
fragments called limit dextrins. The major source of amylase in all species is pancreatic
secretions, although amylase is also present in saliva of some animals, including humans.
i.

38 | P G D C B , P a p e r - I Dr. M. Estari
Other Pancreatic Enzymes: In addition to the proteases, lipase and amylase, the pancreas
produces a host of other digestive enzymes, including ribonuclease, deoxyribonuclease,
gelatinase and elastase.

Bicarbonates and Water


Epithelial cells in pancreatic ducts are the source of the bicarbonate and water secreted by the
pancreas. Bicarbonate is a base and critical to neutralizing the acid coming into the small
intestine from the stomach. The mechanism underlying bicarbonate secretion is essentially the
same as for acid secretion parietal cells and is dependent on the enzyme carbonic anhydrase. In
pancreatic duct cells, the bicarbonate is secreted into the lumen of the duct and hence into
pancreatic juice

11.2.5 Intestinal secretions (Succus entericus)

a) Formation/secretion of intestinal secretions


Enzymes, Mucus, water and electrolytes are the main constituents of intestinal secretion. The
intestinal enzymes are secreted from small intestinal mucosa, these enzymes are: sucrase,
maltase, lactase, lipases, amylases and erypsins. The secretion of enzymes in the small intestine
does not occur in the usual manner. Instead, the digestive enzymes are formed in the epithelial
cells lining the intestinal wall, and much of the digestive process occurs either inside these cells
or on their surfaces.

The another substance of intestinal secretion is mucus, which is secreted by Brunner’s glands,
which are located within the duodenum and goblet cells located along the length of the intestinal
epithelium and in the intestinal crypts, called the crypts of lieberkuhn.

The final constituents of intestinal secretions are water and electrolytes. These are secreted by all
the epithelial cells of the intestine. The watery secretion proves a solvent into which the products
of digestion are dissolved.

b) Composition and functions of intestinal secretions


The small intestinal juice is a colorless, straw colored fluid. It is cloudy in appearance due to the
shed epithelial cells and flecks of mucus. Due to rapid exchange of fluid in both directions of the
intestinal mucosa, it has been difficult to determine the actual amount secreted. It is estimated
that daily secretions may range between 1000 to 3000 ml. Succus entericus is composed of water,
inorganic salts (1%) and organic material (0.6%).

The organic matter consists mainly of enzymes, cellular debris and mucus. A number of the
enzymes are located intracellularly (within the shed epithelial cells). Peptidases are the most
important group of proteolytic enzymes in succus entericus. They bring about the final breakdown
of polypeptides to amino acids. Di- and tripeptidases break up di- and tripeptides respectively,
while aminopeptidases act on peptide linkages of terminal amino acids possessing free amino
groups.

Enteropeptidase or enterokinase activates trypsinogen. Its release in Succus entericus is triggered


by the presence of pancreatic juice in the duodenum.

Lipase is present in the intestinal juice along with a large groups of amylolytic enzymes mainly
the disaccharides like lactase, sucrose, maltase and isomaltose. Most of the latter group of
enzymes is located intracellularly.

***

39 | P G D C B , P a p e r - I Dr. M. Estari
Chapter – 5 URINE

12. Formation of urine


13. Composition of urine

12. FORMATION OF URINE

12.1 INTRODUCTION
12.2 FILTRATION
12.3 REABSORPTION
12.4 SECRETION
12.1 INTRODUCTION

Urine is composed of water, certain electrolytes, and various waste products that are filtered out of
the blood system. Remember, as the blood flows through the body, wastes resulting from the
metabolism of foodstuffs in the body cells are deposited into the bloodstream, and this waste must
be disposed of in some way. A major part of this "cleaning" of the blood takes place in the kidneys
and, in particular, in the nephrons, where the blood is filtered to produce the urine. Both kidneys in
the body carry out this essential blood cleansing function.

Normally, about 20% of the total blood pumped by the heart each minute will enter the kidneys to
undergo filtration. This is called the filtration fraction. The rest of the blood (about 80%) does not
go through the filtering portion of the kidney, but flows through the rest of the body to service the
various nutritional, respiratory, and other needs that are always present.

For the production of urine, the kidneys do not simply pick waste products out of the bloodstream
and send them along for final disposal. The kidneys' 2 million or more nephrons (about a million in
each kidney) form urine by three precisely regulated processes:

1. Glomerular Filtration also called "Ultra-filtration",


2. Tubular Reabsorption also called "Selective Re-Absorption" and
3. Tubular Secretion.

12.2 GLOMERULAR FILTRATION

Blood enters the kidney via the renal artery. This seperates many times (Renal Artery ->
Segmental Arteries -> Interlobar Arteries -> Arcuate Arteries -> Interlobular Arteries -> Afferent
Arterioles), eventually forming many afferent arterioles, each of which delivers blood to an
individual kidney nephron.

The diameter of the afferent (incoming) arteriole is greater than the diameter of the efferent
arteriole (by which blood leaves the glomerulus). The pressure of the blood inside the glomerulus
is increased due to the difference in diameter of the incoming and out-going arterioles. This
increased blood pressure helps to force the following components of the blood out of the
glomerular capillaries:

Most of the water;


Most/all of the salts;

40 | P G D C B , P a p e r - I Dr. M. Estari
Most/all of the glucose;
Most/all of the urea.

The above are filtered in preference to other components of blood based on particle size. (Water
and solutes of relative molecular mass less than 68,000 form the filtrate.) Blood cells and plasma
proteins are not filtered through the glomerular capillaries because they are relatively larger in
physical size.

In a healthy person at rest almost 25% of cardiac output passes the two kidneys (1200 ml each
min). The blood reaches the first part of the nephron through the afferent arteriole to the
glomerular capillaries.

The pressures governing the glomerular ultrafiltration rate (GFR) are called the Starling forces The
filtration pressures are as follows:

In the glomerular capillaries the hydrostatic pressure (Pgc) = +60 mmHg


Hydrosatatic pressure within the tubule (Pt) = -20 mmHg
Hydrostatic pressure gradient across the capillary wall (delta P) = Pgc - Pt
Delta P = 60 – 20 = +40 mmHg
Colloid osmotic pressure of the blood (Pco) = -30 mmHg
Net filtration pressure (Pnet) = Pgc – Pco – Pt
(Pnet) = +10 mmHg

The net ultrafiltration pressure (Pnet) is +10 mmHg through the glomerular capillaries, and
provides the force for ultrafiltration of a fat- and protein- free fluid across the glomerular barrier
into Bowmans space and flow through the renal tubules.

12.3 TUBULAR REABSORPTION

Only about 1% of the glomerular fitrate actually leaves the body because the rest (the other 99%)
is reabsorbed into the blood while it passes through the renal tubules and ducts. This is called
tubular reabsorption and occurs via three mechanisms. They are:

41 | P G D C B , P a p e r - I Dr. M. Estari
Osmosis
Diffusion, and
Active Transport.

Reabsorption varies according to the body's needs, enabling the body to retain most of its nutrients.
The processes of tubular reabsorption occur in the following order.

a) In the PCT
Most (80%) of the volume of the fitrate solution is reabsobed in the proximal convoluted
tubule (PCT). This includes some water and most/all of the glucose (except in the case of
diabetics). Most of the energy consumed by the kidneys is used in the reabsorption of sodium
ions (Na+), which are solutes - that is, they are dissolved in the water component of the fitrate
solution.

Symporters simultaneously facilitate passage through the PCT membrane of both Na+ and
another substances/solutes. Other such substances that are reabsorbed with Na+ in this way
include glucose (an important type of sugar), amino acids, lactic acid, and bicarbonate ions
(HCO3-). These then move on through cells via diffusion and/or other transport processes.

A short way to summarize the above is to say that solutes are selectively moved from the
glomular filtrate to the plasma by active transport. (However, almost all glucose and amino
acids, and high but variable amounts of ions, are reabsorbed again later).

Following the movement of solutes (including Na+ ), water is then also reabsorbed by osmosis.
About 80% of the filtrate volume is reabsorbed in this way. As this part of the reabsorption
process is not controlled by the proximal tubule itself, it is sometimes called obligatory water
reabsorption.

b) In the Loop of Henle


The remaining water (together with the dissolved salts and urea) passes from the PCT into the
descending limb of Henle. It then passes along the Loop of Henle, and up the ascending limb
of Henle.

The different permeability properties of the two limbs of the Loop of Henle, together with their
counterflow arrangement, allows a countercurrent multiplication to generate a high solute
concentration in the tissue fluid of the medulla (that is, outside of the tubules). The highest
solute concentrations are generated deep in the medulla. This is explained as follows:

i) Descending Limb of Loop of Henle


The epithelium lining of the descending limb of Henle is relatively permeable to water -
but much much less permeable to the salts Na+ and Cl-, and to urea. Therefore water
gradually moves from the descending limb and into the interstitium (surrounding the
tubules) as fluid flows through this part of the system of renal tubules.

ii) Thin Ascending Limb of Loop of Henle


The thin ascending limb of Henle differs from the descending limb in that it is
impermeable to water (so the water that is inside the tubule at this stage generally remains
inside it), but is highly permeable to Na+ and Cl-, and somewhat permeable to urea.

Therefore while the tubular fluid flows back towards the renal cortex, Na+ and Cl- (which
are more concentrated in the tubular fluid than in the interstital fluid) diffuse from the
tubules into the interstitium.

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Some urea also enters the tubules at this stage - but the loss of NaCl from the tubular fluid
greatly exceeds the gain in urea.

iii) Thick Ascending Limb of Loop of Henle

The thick ascending limb of Henle (and its continuation into the first part of the DCT),
reabsorbs NaCl from the tubular fluid via a different transport process from that of the thin
ascending limb of Henle.

The overall effect of the processes outlined above is that the concentation of the fluid inside the
renal tubules that form the Loop of Henle is highest at the deepest part of the renal medulla, and is
less concentrated in the renal cortex. This is what is meant by the "concentration gradient" of the
Loop of Henle.

The term "counter-current" is also used in descriptions of the Loop of Henle - and refers to the
tubular fluid flowing in opposite directions along the descending and ascending limbs (as indicated
by the thin red arrows in the diagram above.

c) In the DCT
The water, urea, and salts contained within the ascending limb of Henle eventually pass into
the distal convoluted tubule (DCT).

The DCT reacts to the amount of anti-diuretic hormone (ADH) in the blood:

The more ADH is present in the blood, the more water is re-absorbed into it. This happens
because the presence of ADH in the blood causes the cells in the last section of the DCT
(and associated tubules and collecting ducts) to become more permeable to water, therefore
they allow more water to pass from the tubular fluid back into the blood. This results in
more concentrated urine.

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12.4 TUBULAR SECRETION

The third process by which the kidneys clean blood (regulating its composition and volume) is
called tubular secretion and involves substances being added to the tubular fluid. This removes
excessive quantities of certain dissolved substances from the body, and also maintains the blood at
a normal healthy pH (which is typically in the range pH 7.35 to pH 7.45).

Tubular secretion of H+ is important in maintaining control of the pH of the blood.


When the pH of the blood starts to drop, more hydrogen ions are secreted.
If the blood should become too alkaline, secretion of H+ is reduced.
In maintaining the pH of the blood within its normal limits of 7.3–7.4, the kidney can produce
a urine with a pH as low as 4.5 or as high as 8.5.

The substances that are secreted into the tubular fluid (for removal from the body) include:
Potassium ions (K+),
Hydrogen ions (H+),
Ammonium ions (NH4+),
creatinine,
urea,
some hormones, and
some drugs (e.g. penicillin).

Tubular secretion occurs from the epithelial cells that line the renal tubules and collecting ducts. It
is the tubular secretion of H+ and NH4+ from the blood into the tubular fluid (i.e. urine - which is
then excreted from the body via the ureter, bladder, and urethra) that helps to keep blood pH at its
normal level. The movement of these ions also helps to conserve sodium bicarbonate (NaHCO3).

The typical pH of urine is about 6. Urine formed via the three processes outlined above trickles
into the kidney pelvis. At this final stage it is only approx. 1% of the originally filtered volume but
includes high concentrations of urea and creatinine, and variable concentrations of ions.

***
44 | P G D C B , P a p e r - I Dr. M. Estari
13. COMPOSITION OF URINE

13.1 INTRODUCTION
13.2 FILTRATION
13.3 REABSORPTION
13.4 SECRETION
13.1 INTRODUCTION

Urine is a transparent solution that is clear to amber in color, and usually is light yellow. It is the
byproduct or waste fluid secreted by the kidneys, transported by the ureters to the urinary bladder
where it is stored until it is voided through the urethra. Urine is made up of a watery solution of
metabolic wastes (such as urea), dissolved salts and organic materials. Fluid and materials being
filtered by the kidneys, destined to become urine, comes from the blood or interstitial fluid.

The composition of urine is adjusted in the process of reabsorption when essential molecules needed
by the body, such as glucose, are reabsorbed back into the blood stream via carrier molecules. The
remaining fluid contains high concentrations of urea and other excess or potentially toxic substances
that will be released from the body via urination. Urine flows through these structures: the kidney,
ureter, bladder, and finally the urethra. Urine is produced by a process of filtration, reabsorption, and
tubular secretion.

Urine contains large amounts of urea, an excellent source of nitrogen for plants. As such it is a
useful accelerator for compost. Urea is 10,000 times less toxic than ammonia and is a byproduct of
deamination (2 NH3 molecules) and cellular respiration's. (1 CO2 molecule) products combining
together. Other components include various inorganic salts such as sodium chloride (the discharge of
sodium through urine is known as "natriuresis".)

13.2 CHEMICAL COMPOSITION OF URINE

The composition of normal urine is:

1) Volume: 600- 2500 ml/24 hrs. Average: 1,200 ml.


2) Specific gravity: 1.003 - 1.030
3) Reaction: Acidic (pH: 4.7 - 7.5) Average pH: 6.0
4) Total solids: 30 - 70 g/liter.

Urine is aprox. 95% water. The other components of normal urine are the solutes that are dissolved
in the water component of the urine. These solutes can be divided into two categories according to
their chemical structure (e.g. size and electrical charge).

Organic molecules are electrically neutral and can be relatively large (compared with the 'simpler'
ions - below). These include:

Urea - Urea is an organic (i.e. carbon-based) compound whose chemical formula is: CON2H4
or (NH2)2CO. It is also known as carbamide. Urea is derived from ammonia and produced by
the deamination of amino acids. The amount of urea in urine is related to quantity of dietary
protein.
Creatinine - Creatinine is a normal (healthy) constituent of blood. It is produced mainly as a
result of the breakdown of creatine phosphate in muscle tissue. It is usually produced by the
body at a fairly constant rate (which depends on the muscle mass of the body).
Uric acid - Uric acid is an organic (i.e. carbon-based) compound whose chemical formula is:
C5H4N4O3.

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Due to its insolubility, uric acid has a tendency to crystallize, and is a common part of kidney
stones.
Other substances/molecules - Example of other substances that may be found in small
amounts in normal urine include carbohydrates, enzymes, fatty acids, hormones, pigments, and
mucins (a group of large, heavily glycosylated proteins found in the body).

Ions are atoms or groups of atoms that have either, lost some outer electrons, hence have a positive
electric charge, or have gained some outer electrons (to the atom or group of atoms), and hence have
a negative electric charge. Even in the cases of ions formed by groups of atoms (they are ions due to
the few lost or gained electrons), the groups are formed from only a small number of particles and
therefore tend to be relatively small. These include:

Individual elements:

Sodium (Na+) : Amount in urine varies with diet and the amount of aldosterone (a steroid
hormone) in the body.
Potassium (K+) : Amount in urine varies with diet and the amount of aldosterone (a steroid
hormone) in the body.
Chloride (Cl-) : Amount in urine varies with dietart intake (chloride is a part of common salt,
NaCl).
Magnesium (Mg2+) : Amount in urine varies with diet and the amount of parathyroid hormone
in the body. (Parathyroid hormone increases the reabsorption of magnesium by the body,
which therefore decreases the quantity of magnesium in urine.)
Calcium (Ca2+) : Amount in urine varies with diet and the amount of parathyroid hormone in
the body. (Parathyroid hormone increases the reabsorption of calcium by the body, which
therefore decreases the quantity of calcium in urine.)
Small groups formed from a few different elements:

Ammonium (NH4+) : The amount of ammonia produced by the kidneys may vary according to
the pH of the blood and tissues in the body.
Sulphates (SO42-) : Sulphates are derived from amino acids. The quantity of sulphates excreted
in urine varies according to the quantity and type of protein in the person's diet.
Phosphates (H2PO4-, HPO42-, PO43-) : Amount in urine varies with the amount of parathyroid
hormone in the body - parathyroid hormone increases the quantity of phosphates in urine.

Following inorganic constituents (listed in table) are excreted per 24 hours.


Note: These values generally vary with diet.

Constituent Quantity excreted/24 hrs.

1) Sodium 3-4g

2) Potassium 1.5 – 2 g
3) Chlorides 9 - 16g
4) Calcium 0.1 - 0.3 g
5) Inorganic phosphorus 1 - 1.5 g
6) Sulfur 0.7 - 3.5 g
7) Magnesium 0.05 - 0.2 g
8) Ammonia 0.3 - 1.0 g
9) Iodine 50 - 250 g
10) Arsenic less than 50 µg

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11) Lead less than 50 µg
12) Urea 25 - 30 g
13) Creatinine 1 - 1.8 g
14) Uric acid 0.3 - 1.0 g
15) Creatine 60 – 150 mg
16) Hippuric acid 0.1 - l.0 g
17) Purine bases 7 – 10 mg
18) Ketone bodies 3 - 15 mg
19) Oxalic acid 15 - 20 mg
20) Indican 4 - 2 mg
21) Allantoin 20 - 30 mg
22) Coproporphyrins 60 - 280 µg
23) Phenols 0.2 - 0.5 g
24) Vitamins, hormones, and enzymes Detected in small quantities

***

47 | P G D C B , P a p e r - I Dr. M. Estari