BIOCHEMISTRY OF BLOOD

BIOCHEMISTRY OF BLOOD. RESPIRATORY FUNCTION OF ERYTHROCYTES. PATHOLOGICAL FORMS OF HEMOGLOBIN. ACID-BASE STATE OF BLOOD.

Biochemical components of blood in normal and pathological conditions. Blood proteins. Non-protein nitrogenous containing and non- nitrogenous containing components of blood.

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

  neutrophils

  eosinophils

  basophils

o          Two kinds of leukocytes without granules in their cytoplasm

  lymphocytes

  monocytes

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.

The formation of blood cells (cell types and acronyms are defined below)

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.

Examples:

• 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).

 

Biological chemistry of blood cells

   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.

White Blood Cells (leukocytes)

 

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.

 

Neutrophils

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*Í -OXYDASE catalyzes formation of super oxide anion     

2. An enzyme NADH- OXYDASE is responsible for formation of hydrogen peroxide

3. Ìyeloperoxydase 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.

 

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

• serotonin

• 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.

Lymphocytes

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.

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

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.

                                                    Red Blood Cells (erythrocytes)

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 18,000 feet may have as many as 8.3 x 106 RBCs per µl.)

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.

 

Oxygen Transport

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.

 

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 Transport

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:

• It probably enters (and leaves) the cell by diffusing through transmembrane channels in the plasma membrane. (One of the proteins that forms the channel is the D antigen that is the most important factor in the Rh system of blood groups.)

• Once inside, about one-half of the CO2 is directly bound to hemoglobin (at a site different from the one that binds oxygen).

• 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).

Only about 5% of the CO2 generated in the tissues dissolves directly in the plasma. (A good thing, too: if all the CO2 we make were carried this way, the pH of the blood would drop from its normal 7.4 to an instantly-fatal 4.5!)

When the red cells reach the lungs, these reactions are reversed and CO2 is released to the air of the alveoli.

Anemia

Anemia is a shortage of

• RBCs and/or

• the amount of hemoglobin in them.

Anemia has many causes. One of the most common is an inadequate intake of iron in the diet.

Blood Groups

Red blood cells have surface antigens that differ between people and that create the so-called blood groups such as the ABO system and the Rh system.

 

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)

 

Hemoglobin is the protein that carries oxygen from the lungs to the tissues and carries carbon dioxide from the tissues back to the lungs. In order to function most efficiently, hemoglobin needs to bind to oxygen tightly in the oxygen-rich atmosphere of the lungs and be able to release oxygen rapidly in the relatively oxygen-poor environment of the tissues. It does this in a most elegant and intricately coordinated way. The story of hemoglobin is the prototype example of the relationship between structure and function of a protein molecule.

 

  Hemoglobin Structure

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.)

 

Each a or b globin chain folds into 8 a helical segments (A-H) which, in turn, fold to form globular tertiary structures that look roughly like sub-microscopic kidney beans. The folded helices form a pocket that holds the working part of each chain, the heme.

A heme group is a flat ring molecule containing carbon, nitrogen and hydrogen atoms, with a single Fe2+ ion at the center. Without the iron, the ring is called a porphyrin. In a heme molecule, the iron is held within the flat plane by four nitrogen ligands from the porphyrin ring. The iron ion makes a fifth bond to a histidine side chain from one of the helices that form the heme pocket. This fifth coordination bond is to histidine 87 in the human a chain and histidine 92 in the human b chain. Both histidine residues are part of the F helix in each globin chain.

 

The Bohr Effect

The ability of hemoglobin to release oxygen, is affected by pH, CO2 and by the differences in the oxygen-rich environment of the lungs and the oxygen-poor environment of the tissues. The pH in the tissues is considerably lower (more acidic) than in the lungs. Protons are generated from the reaction between carbon dioxide and water to form bicarbonate:

CO2 + H20 -----------------> HCO3- + H+

This increased acidity serves a twofold purpose. First, protons lower the affinity of hemoglobin for oxygen, allowing easier release into the tissues. As all four oxygens are released, hemoglobin binds to two protons. This helps to maintain equilibrium towards the right side of the equation. This is known as the Bohr effect, and is vital in the removal of carbon dioxide as waste because CO2 is insoluble in the bloodstream. The bicarbonate ion is much more soluble, and can thereby be transported back to the lungs after being bound to hemoglobin. If hemoglobin couldn’t absorb the excess protons, the equilibrium would shift to the left, and carbon dioxide couldn’t be removed.

In the lungs, this effect works in the reverse direction. In the presence of the high oxygen concentration in the lungs, the proton affinity decreases. As protons are shed, the reaction is driven to the left, and CO2 forms as an insoluble gas to be expelled from the lungs. The proton poor hemoglobin now has a greater affinity for oxygen, and the cycle continues. 

 

Lymphocytes

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

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.

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 .

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.

Plasma

Plasma is the straw-colored liquid in which the blood cells are suspended.

 

Plasma transports materials needed by cells and materials that must be removed from cells:

• various ions (Na+, Ca2+, HCO3−, etc.

• glucose and traces of other sugars

• amino acids

• other organic acids

• cholesterol and other lipids

• hormones

• urea and other wastes

Most of these materials are in transit from a place where they are added to the blood (a "source")

• exchange organs like the intestine

• depots of materials like the liver

to places ("sinks") where they will be removed from the blood.

• every cell

• exchange organs like the kidney, and skin.

Serum Proteins

Proteins make up 6–8% of the blood. They are about equally divided between serum albumin and a great variety of serum globulins.

After blood is withdrawn from a vein and allowed to clot, the clot slowly shrinks. As it does so, a clear fluid called serum is squeezed out. Thus:

Serum is blood plasma without fibrinogen and other clotting factors.

The serum proteins can be separated by electrophoresis.

• A drop of serum is applied in a band to a thin sheet of supporting material, like paper, that has been soaked in a slightly-alkaline salt solution.

• At pH 8.6, which is commonly used, all the proteins are negatively charged, but some more strongly than others.