Investigation of acid-base state of blood and respiratory function of
erythrocytes. Pathological
forms of hemoglobin.
Investigation of blood plasma proteins of
inflammation acute phase, own and indicator enzymes. Non-protein
nitrogenous containing and nitrogen not containing organic components of blood.
residual nitrogen
Blood is a liquid tissue. Suspended in the watery plasma are seven types of cells and cell fragments.
·
red blood cells (RBCs)
or erythrocytes
·
platelets or thrombocytes
·
five kinds of white blood cells
(WBCs) or leukocytes
o
Three kinds of granulocytes
o
Two kinds of leukocytes without
granules in their cytoplasm
If
one takes a sample of blood, treats it with an agent to prevent clotting, and
spins it in a centrifuge, ·
the red cells settle to the bottom ·
the white cells settle on top of
them forming the "buffy coat". The
fraction occupied by the red cells is called the hematocrit. Normally
it is approximately 45%. Values much lower than this are a sign of anemia. |
|
Biological functions of the blood
The blood is the most specialized fluid tissue which
circulates in vascular system and together with lymph and intercellular space compounds
an internal environment of an organism.
The blood executes such functions:
1. Transport of gases –
oxygen from lungs is carried to tissues and carbon dioxide from tissues to
lungs.
2. Transport of nutrients to all cells of
organism (glucose, amino acids, fatty acids, vitamins, ketone bodies, trace
substances and others). Substances such as urea, uric acid, bilirubin and
creatinine are taken away from the different organs for ultimate excretion.
3. Regulatory or hormonal function – hormones
are secreted in to blood and they are transported by blood to their target
cells.
4. Thermoregulation
function - an exchange of heat between tissues and blood.
5. Osmotic function-
sustains osmotic pressure in vessels.
6. Protective function- by
the phagocytic action of leucocytes and by the actions of antibodies, the blood
provides the most important defense mechanism.
7. Detoxification function
- neutralization of toxic substances which is connected with their
decomposition by the help of blood enzymes.
Blood
performs two major functions:
·
transport through the body of
o
oxygen and carbon dioxide
o
food molecules (glucose, lipids,
amino acids)
o
ions (e.g., Na+, Ca2+,
HCO3−)
o
wastes (e.g., urea)
o
hormones
o
heat
·
defense of the body against
infections and other foreign materials. All the WBCs participate in these
defenses.
All the
various types of blood cells
·
are produced in the bone marrow
(some 1011 of them each day in an adult human!).
·
arise from a single type of cell
called a hematopoietic stem cell — an "adult" multipotent stem cell.
These stem cells
·
are very rare (only about one in
10,000 bone marrow cells);
·
are attached (probably by adherens junctions)
to osteoblasts
lining the inner surface of bone cavities;
·
express a cell-surface protein
designated CD34;
·
produce, by mitosis, two kinds of
progeny:
o
more stem cells (A mouse that has had
all its blood stem cells killed by a lethal dose of radiation can be saved by
the injection of a single living stem cell!).
o
cells that begin to differentiate
along the paths leading to the various kinds of blood cells.
Which
path is taken is regulated by
·
the need for more of that type of
blood cell which is, in turn, controlled by appropriate cytokines
and/or hormones.
·
Interleukin-7 (IL-7)
is the major cytokine in stimulating bone marrow stem cells to start down the
path leading to the various lymphocytes
(mostly B cells
and T cells).
·
Erythropoietin (EPO),
produced by the kidneys, enhances the production of red blood cells
(RBCs).
·
Thrombopoietin (TPO),
assisted by Interleukin-11 (IL-11), stimulates the production of megakaryocytes.
Their fragmentation produces platelets.
·
Granulocyte-macrophage
colony-stimulating factor (GM-CSF), as its name
suggests, sends cells down the path leading to both those cell types. In due
course, one path or the other is taken.
o
Under the influence of granulocyte
colony-stimulating factor (G-CSF), they differentiate into neutrophils.
o
Further stimulated by interleukin-5 (IL-5)
they develop into eosinophils.
o
Interleukin-3 (IL-3)
participates in the differentiation of most of the white blood cells but plays a
particularly prominent role in the formation of basophils
(responsible for some allergies).
o
Stimulated by macrophage
colony-stimulating factor (M-CSF) the granulocyte/macrophage
progenitor cells differentiate into monocytes, macrophages,
and dendritic cells
(DCs).
Two types of blood cells can be distinguished
- white and red blood cells. White blood cells are called leucocytes. Their
quantity in adult is 4-9 x 109/L.
Red blood cells
are called erythrocytes. Their quantity
in peripheral blood is 4,5-5 x 1012/L.
Besides that, there are also thrombocytes or platelets in blood.
Leucocytes
(white blood cells) protect an organism from
microorganisms, viruses and foreign substances, that provides the immune status
of an organism.
·
are much less numerous than red (the
ratio between the two is around 1:700),
·
have nuclei,
·
participate in protecting the body
from infection,
·
consist of lymphocytes and monocytes
with relatively clear cytoplasm, and three types of granulocytes, whose
cytoplasm is filled with granules.
Leucocytes are divided into
two groups: Granulocytes and agranulocytes. Granulocytes consist of
neutrophils, eosinophils and basophils. Agranulocytes consist of monocytes and
lymphocytes.
http://www.youtube.com/watch?v=8ytkFqAMoa8
http://www.youtube.com/watch?v=ce0Xndms1bc
Neutrophils comprise of
60-70 % from all leucocytes. Their main function is to protect organisms from
microorganisms and viruses. Neutrophils have segmented nucleus, endoplasmic
reticulum (underdeveloped) which does not contain ribosomes, insufficient
amount of mitochondria, well-developed Golgi apparatus and hundreds of
different vesicles which contain peroxidases and hydrolases. Optimum condition
for their activity is acidic pH. There are also small vesicles which contain
alkaline phosphatases, lysozymes, lactopherins and proteins of cationic origin.
Glucose is the main source
of energy for neutrophils. It is directly utilized or converted into glycogen.
90 % of energy is formed in glycolysis, a small amount of glucose is converted
in pentosophosphate pathway. Activation of proteolysis during phagocytosis as
well as reduction of phosphatidic acid and phosphoglycerols are also observed.
The englobement is accompanied by intensifying of a glycolysis and
pentosophosphate pathway. But especially intensity of absorption of oxygen for
neutrophils - so-called flashout of respiration grows. Absorbed oxygen is spent
for formation of its fissile forms that is carried out with participation
enzymes:
1. NADP*Н -OXYDASE catalyzes formation of super oxide
anion
2. An enzyme
NADH- OXYDASE is responsible for formation
of hydrogen peroxide
3. Мyeloperoxydase catalyzes
formation of hypochloric acid from chloride and hydrogen peroxide
Neutrophils
are motile phagocyte cells that play a key role in acute inflammation. When
bacteria enter tissues, a number of phenomena occur that are collectively known
as acute inflammatory response. When neutrophils and other phagocyte cells
engulf bacteria, they exhibit a rapid increase in oxygen consumption known as
the respiratory burst. This phenomenon reflects the rapid utilization of oxygen
(following a lag of 15-60 seconds) and production from it of large amounts of
reactive derivates, such as O2-, H2O2,
OH. and OCl-
(hypochlorite ion). Some of these products are potent microbicidal
agents. The electron transport chain system responsible for the respiratory
burst contains several components, including a flavoprotein NADPH:O2-oxidoreductase
(often called NADPH-oxidase) and a b-type cytochrome.
The most
abundant of the WBCs. This photomicrograph shows a single neutrophil surrounded
by red blood cells.
Neutrophils
squeeze through the capillary walls and into infected tissue where they kill
the invaders (e.g., bacteria) and then engulf the remnants by phagocytosis.
This
is a never-ending task, even in healthy people: Our throat, nasal passages, and
colon harbor vast numbers of bacteria. Most of these are commensals,
and do us no harm. But that is because neutrophils keep them in check.
However,
·
heavy doses of radiation
·
chemotherapy
·
and many other forms of stress
can
reduce the numbers of neutrophils so that formerly harmless bacteria begin to proliferate.
The resulting opportunistic infection can be life-threatening.
http://www.youtube.com/watch?v=EpC6G_DGqkI&feature=related
Some
important enzymes and proteins of neutrophilis.
Myeloperoxidase
(MPO). Catalyzed following reaction:
H2O2
+ X-(halide) + H+®
HOX + H2O (where X- = Cl-, Br-, I-
or SCN-; HOX=hypochlorous acid)
HOCl,
the active ingredient of household liquid bleach, is a powerful oxidant and is
highly microbicidial. When applied to normal tissues, its potential for causing
damage is diminished because it reacts with primary or secondary amines present
in neutrophils and tissues to produce various nitrogen-chlorine (N-Cl)
derivates; these chloramines are also oxidants, although less powerful than
HOCl, and act as microbicidial agents (eg, in sterilizing wounds) without
causing tissue damage. Responsible for the green color of pus.
NADPH-oxidase.
2O2
+ NADPH ®
2O2- + NADP + H+
Key
component of the respiratory burst. Deficiency may be observed in chronic
granulomatous disease.
Lysozyme.
Hydrolyzes
link between N-acetylmuramic acid and N-acetyl-D-glucosamine found in certain
bacterial cell walls. Abundant in macrophages.
Defensins.
Basic
antibiotic peptides of 29-33 amino acids. Apparently kill bacteria by causing
membrane damage.
Lactoferrin.
Iron-binding
protein. May inhibit growth of certain bacteria by binding iron and may be
involved in regulation of proliferation of myeloid cells.
Neutrophils
contain a number of proteinases (elastase, collagenase, gelatinase, cathepsin
G, plasminogen activator) that can hydrolyze elastin, various types of
collagens, and other proteins present in the extracellular matrix. Such
enzymatic action, if allowed to proceed unopposed, can result in serious damage
to tissues. Most of these proteinases are lysosomal enzymes and exist mainly as
inactive precursors in normal neutrophils. Small amounts of these enzymes are
released into normal tissues, with the amounts increasing markedly during
inflammation. The activities of elastase and other proteinases are normally
kept in check by a number of antiproteinases (a1-Antiproteinase,
a2-Macroglobulin,
Secretory leukoproteinase inhibitor, a1-Antichymotrypsin,
Plasminogen activator inhibitor-1, Tissue inhibitor of metalloproteinase)
present in plasma and the extracellular fluid.
Basophiles
Basophiles make up 1-5% of all blood leukocytes. They
are actively formed in the bone marrow
during allergy. Basophiles take part in
the allergic reactions, in the blood coagulation and intravascular
lipolysis. They have the protein synthesis mechanism, which works due to the
biological oxidation energy . They synthesize the mediators of allergic
reactions – histamine and serotonin, which during allergy cause local
inflammation. Heparin, which is formed in the basophiles, prevents the blood
coagulation and activates intravascular lipoprotein lipase, which splits triacylglycerin.
The
number of basophils also increases during infection. Basophils leave the blood
and accumulate at the site of infection or other inflammation. There they
discharge the contents of their granules, releasing a variety of mediators such
as:
·
histamine
·
prostaglandins
and leukotrienes
which
increase the blood flow to the area and in other ways add to the inflammatory
process. The mediators released by basophils also play an important part in
some allergic responses such as
·
hay fever and
·
an anaphylactic response
to insect stings.
Eosinophiles
They make up 3-6% of all leukocytes. Eosinophiles as
well as neutrophiles defend the cells from microorganisms, they contain
myeloperoxidase, lysosomal hydrolases. About the relations of eosinophiles with
testifies the growth of their amount during the sensitization of organism, i.e.
during bronchial asthma, helminthiasis. They are able to pile and splits
histamine, “to dissolve” thrombus with the participation of plasminogen and
bradykinin-kininase.
Monocytes
They are formed in the bone marrow. They make up 4-8% of all leukocytes.
According to the function they are called macrophages. Tissue macrophages
derive from blood monocytes. Depending on their position they are called: in
the liver – reticuloendotheliocytes, in the lungs - alveolar macrophages, in
the intermediate substance of connective tissue – histocytes etc. Monocytes are
characterized by a wide set of lysosomal
enzymes with the optimum activity in the acidic condition. The major
functions of monocytes and macrophages are endocytosis and phagocytosis.
The amount – 20-25%, are formed in the lymphoid tissue
or thymus, play important role in the formation of humoral and cellular
immunity. Lymphocytes have powerful system of synthesis of antibody proteins,
energy is majorily pertained due to glycolysis, rarely – by aerobic way.
http://www.youtube.com/watch?v=cD_uAGPBfQQ&feature=related
There
are several kinds of lymphocytes (although they all look alike under the
microscope), each with different functions to perform . The most common types
of lymphocytes are
·
B lymphocytes
("B cells"). These are responsible for making antibodies.
·
T lymphocytes
("T cells"). There are several subsets of these:
o
inflammatory T cells
that recruit macrophages and neutrophils to the site of infection or other
tissue damage
o
cytotoxic T lymphocytes
(CTLs) that kill virus-infected and, perhaps, tumor cells
o
helper T cells
that enhance the production of antibodies by B cells
Although bone
marrow is the ultimate source of lymphocytes, the lymphocytes that will become
T cells migrate from the bone marrow to the thymus where they mature. Both B cells and T cells
also take up residence in lymph nodes, the spleen and other tissues where they
·
encounter antigens;
·
continue to divide by mitosis;
·
mature into fully functional cells.
Monocytes
leave the blood and become macrophages and dendritic cells.
This
scanning electron micrograph (courtesy of Drs. Jan M. Orenstein and Emma
Shelton) shows a single macrophage surrounded by several lymphocytes.
Macrophages
are large, phagocytic cells that engulf
·
foreign material (antigens) that
enter the body
·
dead and dying cells of the body.
Thrombocytes
(blood platelets)
Platelets
are cell fragments produced from megakaryocytes.
Blood
normally contains 150,000–350,000 per microliter (µl) or cubic millimeter (mm3).
This number is normally maintained by a homeostatic (negative-feedback)
mechanism .
The amount – less than 1%, they play the main role in
the process of hemostasis. They are formed as a result of disintegration of
megakaryocytes in the bone marrow. Their
–life-time is 7-9 days. In spite of the fact that thrombocytes have no nucleus,
they are able to perform practically all functions of the cell, besides DNA
synthesis.
If
this value should drop much below 50,000/µl, there is a danger of uncontrolled
bleeding because of the essential role that platelets have in blood clotting.
Some
causes:
·
certain drugs and herbal remedies;
·
autoimmunity.
When
blood vessels are cut or damaged, the loss of blood from the system must be
stopped before shock
and possible death occur. This is accomplished by solidification of the blood,
a process called coagulation or clotting.
A blood
clot consists of
·
a plug of platelets enmeshed
in a
·
network of insoluble fibrin
molecules.
The
most numerous type in the blood.
·
Women average about 4.8 million of
these cells per cubic millimeter (mm3; which is the same as a
microliter [µl]) of blood.
·
Men average about 5.4 x 106
per µl.
·
These values can vary over quite a
range depending on such factors as health and altitude. (Peruvians living at
RBC
precursors mature in the bone marrow closely attached to a macrophage.
·
They manufacture hemoglobin until it
accounts for some 90% of the dry weight of the cell.
·
The nucleus is squeezed out of the cell
and is ingested by the macrophage.
·
No-longer-needed proteins are
expelled from the cell in vesicles called exosomes.
Human blood contains 25 trillion of erythrocytes.
Their main function – transportation of O2 and CO2 – they
perform due to the fact that they contain 34% of hemoglobin, and per dry cells
mass – 95%. The total amount of
hemoglobin in the blood equals 130-160 g/l. In the process of erythropoesis the
preceding cells decrease their size. Their nuclei at the end of the process are
ruined and pushed out of the cells. 90% of glucose in the erythrocytes is
decomposed in the process of glycolysis and 10% - by pentose-phosphate way.
There are noted congenital defects of enzymes of these metabolic ways of
erythrocytes. During this are usually observed hemolytic anemia and other
structural and functional erythrocytes’ affections.
This
scanning electron micrograph (courtesy of Dr. Marion J. Barnhart) shows the
characteristic biconcave shape of red blood cells.
Thus RBCs are
terminally differentiated; that is, they can never divide. They live about 120
days and then are ingested by phagocytic cells in the liver and spleen. Most of
the iron in their hemoglobin is reclaimed for reuse. The remainder of the heme
portion of the molecule is degraded into bile pigments
and excreted by the liver. Some 3 million RBCs die and are scavenged by the
liver each second.
Red
blood cells are responsible for the transport of oxygen and carbon
dioxide.
In
adult humans the hemoglobin (Hb) molecule
·
consists of four polypeptides:
o
two alpha (α)
chains of 141 amino acids and
o
two beta (β)
chains of 146 amino acids
·
Each of these
is attached the prosthetic group heme.
·
There is one atom of iron at the center
of each heme.
·
One molecule of oxygen can bind to
each heme.
http://www.youtube.com/watch?v=WXOBJEXxNEo&feature=related
The
reaction is reversible.
·
Under the conditions of
lower temperature, higher pH, and increased oxygen pressure in the capillaries
of the lungs, the reaction proceeds to the right. The purple-red deoxygenated
hemoglobin of the venous blood becomes the bright-red oxyhemoglobin of
the arterial blood.
·
Under the conditions of
higher temperature, lower pH, and lower oxygen pressure in the tissues, the
reverse reaction is promoted and oxyhemoglobin gives up its oxygen.
Carbon dioxide (CO2) combines with water forming carbonic acid, which dissociates into a hydrogen ion (H+) and a bicarbonate ions
CO2
+ H2O ↔ H2CO3 ↔ H+ +
HCO3−
95%
of the CO2 generated in the tissues is carried in the red blood
cells:
·
The
rest is converted — following the equation above — by the enzyme carbonic
anhydrase into
o
bicarbonate
ions that diffuse back out into the plasma and
o
hydrogen
ions (H+) that bind to the protein portion of the hemoglobin (thus
having no effect on pH).
·
the
amount of hemoglobin in them.
Anemia
has many causes. One of the most common is an inadequate intake of iron
in the diet.
An
Essay on Hemoglobin Structure and Function:
Figure 1 is a model of human
deoxyhemoglobin. It was created in RasMol version 2.6 by Roger Sayle
using the pdb coordinates from the pdb file 4hhb. The 3D coordinates were
determed from x-ray crystallography by Fermi, G., Perutz, M. F., Shaanan, B.,
Fourme, R.: The crystal structure of human deoxyhaemoglobin at 1.74 A
resolution. J Mol Biol 175 pp. 159 (1984)
A
hemoglobin molecule consists of four polypeptide chains: two alpha chains, each
with 141 amino acids and two beta chains, each with 146 amino acids. The
protein portion of each of these chains is called "globin". The a and b globin chains are very similar in
structure. In this case, a and b refer to the two types of globin. Students
often confuse this with the concept of a helix and b sheet secondary
structures. But, in fact, both the a and b globin chains contain primarily a
helix secondary structure with no b sheets.
Figure 2 is a close up view of one
of the heme groups of the human a chain from dexoyhemoglobin. In this
view, the iron is coordinated by a histidine side chain from amino acid 87
(shown in green.)
http://www.youtube.com/watch?v=eor6EK_JP40
CO2
+ H20 -----------------> HCO3- + H+
The
three logical possibilities are:
3)
Low-spin Fe4+ binds to O22-. Both are
diamagnetic.
CO2
+ H2O → H2CO3 → HCO3-
+ H+
·
Pregnant
women: 11 to 12 g/dl
Because
of the large buffering capacity, the actual change in [H+] is so
small it can be ignored in any quantitative assessment, and instead, the magnitude
of a disorder has to be estimated indirectly from the decrease in the total
concentration of the anions involved in the buffering. The buffer anions,
represented as A-, decrease because they combine stoichiometrically
with H+ to produce HA.
A decrease in A- by 1
mmol/l represents a 1,000,000 nano-mol/l amount of H+ that is hidden from view and this is
several orders of magnitude higher than the visible few nanomoles/l change in
[H+] that is visible.) - As noted above in the comments about the
Swan & Pitts experiment, 13,999,994 out of 14,000,000 nano-moles/l of H+ were hidden on buffers and just to
count the 36 that were on view would give a false impression of the magnitude
of the disorder.
Henderson-Hasselbalch
Equation
pH = pK’a + log10 (
[HCO3] / 0.03 x pCO2)
The
pK'a is derived from the Ka value of the following reaction:
CO2 + H2O <=> H2CO3 <=> H+ + HCO3-
(where
CO2 refers to
dissolved CO2)
Ka
= [H+] . [HCO3-] / [CO2] . [H20]
K’a
= Ka x [H2O] = [H+] . [HCO3-] / [CO2]
K'a
= 800 nmol/l (value for plasma at 37C)
[CO2]
= 0.03 x pCO2 (by
Henry’s Law) [where 0.03 is the solubility coefficient]
into
the equation yields the Henderson Equation:
[H+]
= (800 x 0.03) x pCO2 /
[HCO3-] = 24 x pCO2 /
[HCO3-] nmol/l
Taking
the logs (to base 10) of both sides yields the Henderson-Hasselbalch equation:
pH
= log10(800) - log (0.03 pCO2 /
[HCO3-] )
pH
= 6.1 + log ( [HCO3] / 0.03 pCO2 )
The
other buffer systems in the blood are the protein and phosphate buffer systems.
Phosphoric
acid is triprotic weak acid and has a pKa value for each of the three
dissociations: