Chapter Two

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2-1 Introduction
Biological processes are mainly complex chemical reactions. Chemical properties results from the arrangement and motion of electron and nuclei in molecules determined by the interaction of the magnetic and electrics of these particles making up the molecules. The principles of chemistry and biology are therefore understood in term of electrodynamics and quantum mechanics. Consequently, electric and magnetic fields would seem to be the natural experiment devices to be used to obtain information regarding the basic phenomena themselves [38]. Living cell is electrical in nature, cell possesses a positive electric charge at its nucleus and a negative electrical charge on its outer membrane [1].As cell perform their normal bodily function, this electromagnetic charge wears down. The body attempt to revitalize electromagnetic energy from the brain through nervous system to recharge the cells and strengthen the polarized field. Magnetic field with flux densities much higher than one tesla can influence biochemical reactions in isolated enzyme system and consequently various cellular processes, like proliferation or differentiation. Cellular reactions, which are connected with phase transitions of membrane lipids, are apparently influenced most.This is caused by anisotropy of lipids [38].

2-2 The Atomic Origin of Magnetism

An atom whose shells are not completely filled has a permanent magnetic moment, which arises from the combination of the orbital and spin motions of its electrons. The effect of external magnetic field on such a moment from classical theory and from quantum theory can be explained as shown below:

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From classical theory the potential energy V of a magnetic dipole in a magnetic field is given by: V= B where is the magnetic dipole moment, B magnetic field strength. From quantum theory we can express the magnetic dipole moment of the atom in terms of the total orbital angular momentum L as: = g (e /2m) L (2-2) (2-1)

where e is the electron charge, m mass of the electron, g is a constant known as the Land factor or the spectroscopic splitting factor. The value of the constant depends on the relative orientations of the orbital and spin angular momentum, and is given by: [39] g ={1+j (j+1)+ s (s+1) (1)}/2j(j+1) (2-3)

where j is the total quantum number, is orbital quantum number, s is spin quantum number. The coefficient (e /2m) relating l to total orbital angular momentum L is called gyromagnetic ratio (), where L is given by: L =i Li where the sum is over the incomplete shell. For spin rotation the magnetic moment associated with the spin, this moment s may be related to the spin angular momentum S the relation is s = (e /m) S The total spin angular momentum is S = i Si 13 (2-4)

Chapter Two

Theory

where the sum is over the incomplete shell. The total angular momentum of the atom is J given by J= L+S = J= g (e/2m) J (2-5) (2-6)

where (e/2m) is called Bohr magneton B. The angular momentum L and S interact with each other via the spin-orbital interaction. When a magnetic field is applied to the atom a Zeeman splitting results. The energy levels of the system in a magnetic field are: E =mj g B B (2-7) where mj is the azimuthal quantum number and has a values J , J-1 ,.., -J Fig.(2-1)[40].
+m j B=0 E -m
j

Figure (2-1) Zeeman Effect for precession case [40]

2-3 Magnetic Resonance

The three principle subatomic particles, the proton, neutron and electron, each have spin angular momentum. The proton and electron are charged and, as

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we should expect, possess magnetic dipole moments, while the neutron is uncharged particle. When a charged particle is placed in an magnetic field,

it experiences a torque tending to align the magnetic moment vector along the field direction. The interaction of the particle is, however, complicated by its spin properties, and instead of simply aligning itself with the field it experiences precession Fig(2-2)[41,42].The magnetic moment vector rotates about the external field direction tracing out the surface of the cone. The frequency of the precessional rotation is proportional to the external field strength and is known as Larmor frequency.To explain the resonance phenomena, according to quantum mechanics, subatomic particles must be described by wave functions, the properties for which are quantized. Spin angular momentum is quantized, the angular momentum vector of a particle cannot have any arbitrary magnitude but is constrained to a single value, denoted by J. Magnetic moment is also quantized, since the magnetic moment of a particle of angular momentum J is =J (2-8)

15

Chapter Two B0 B0

Theory

(a)

(b)

Figure: (2-2) Precession of the magnetic moment vector about the magnetic field B0 .In case of a particle with spin quantum number s=1/2 , trace out of the structure of a con with half angle . a) Low energy configuration (spin up state). b) high energy configuration (spin down state) [42]

The magnetic moments of the proton and electron are p = 1.41 10-26 JT-1 and for electron e = -9.28 10-24JT-1. When a particle is placed in a magnetic field, the Larmor precessional frequency 0 is related to the external field strength, B0, by the expression: 0 = B0 /2 (2-9)

since is a fixed property for each particle, the Larmor frequencies of other field strength can easily be determined. Angular momentum and magnetic moment are quantized not only in magnitude, in an external magnetic field their vectors are also quantized in direction. The orientation of each vector is constrained in such way that the component of the vector along the field direction may only take certain values. The component of the angular momentum vector J along the field direction may only take one of the following values s , (s-1) ,.,- (s-1) , - s , where is h/2 Planks 16

Chapter Two

Theory

constant and s is called the spin quantum number, s is related to the magnitude of the angular momentum vector, by the expression: J = [s(s+1)] (2-10)

for the proton, neutron and electron, s= 1/2 while other particles including atomic nuclei, all have half integer or integer spin quantum numbers. There are (2s+1) possible orientations of a particle with spin quantum number s, so each of the fundamental subatomic particles has only two possible spin states, known as spin-up and spin-down states. In each state the particle has a different energy, the spin-up representing the lower energy state. The difference in energy is h0 and is therefore proportional to B0, the two energy levels merging into one in the absence of an external field. The magnetic moment undergoing transition from the spin-up state to the spin-down state, with energy

being absorbed from electromagnetic field. Since absorption of energy can only occur at the Larmor frequency, the phenomenon is known as magnetic resonance [42].

2-4 Hyperfine Interaction

The hyperfine interaction is the magnetic interaction between the magnetic moment of nucleus and the magnetic moment of an electron. The interaction is caused by the magnetic field produced by the motion of the electron about the nucleus. There is an electron current about the nucleus if the electron is in a state with orbital angular momentum about the nucleus. Even if the electron is in a state of zero orbital angular momentum, there is an electron spin current about the nucleus, this current give rise to contact hyperfine interaction [41]. 17

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2-5 Magnetization and Magnetic Field

Magnetism is the study of phenomena involving magnetic field and effect on materials exposed to a magnetic field. Magnetization M is a moment per unit volume (Am-1 ) or per unit mass(Am2 Kg-1). Subatomic charges such as proton and electrons can be thought as tracing out tiny circuits and behaving as tiny magnetic moments. They respond to external magnetic fields and give rise to a magnetization that is proportional to them. The relationship between M and the external field H is defined as M=H be a complicated function of orientation, temperature, state of stress and applied field.The relation between the induction B and the field H is given by B = 0 (H+M) (2-12) (2-11) the parameter is known as the magnetic susceptibility of the material and can

where 0 = 1.256 10-6 Vs A-1m-1 is the magnetic permeability of vacuum][43]. Fields from permanent magnets and from electromagnets have the same effect on the surrounding objects, both fields from both sources are simply called the electromagnetic field. Magnetic field from permanent magnet arises from two atomic sources, the spin and the orbital motions of electron. Therefore, the magnetic characteristic of material may change as a function of alloying with other element [42]. All materials were found to have a permeability which is the ability of a material to modify a magnetic field that is applied to it. Permeability can to be define as the amount of magnetic flux density produced in a material divided by the strength of the magnetic field applied to it.

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= B/H

(2-13)

This led to the magnetic behavior into diamagnetic, paramagnetic, ferromagnetic and anti-ferromagnetic.

2-6 Static Magnetic Field

A magnetic field with field strength H (Am-1) causes magnetic induction, or magnetic flux density B in a body in Tesla (Vsm-2) B = 0 H where 0 = 1 in air. The elevation of the magnetic permeability for non ferromagnetic material from 1 is very small. It is more convenient to use the (2-14)

relative permeability r.Therefore the magnetic susceptibility = r-1 is introduced. Magnetic properties of various materials are classified according to their properties as Diamagnetic r<1 Paramagnetic r>1 Ferromagnetic r>>1 <0 >0 >>0

Cells and tissues in general have diamagnetic properties [38]. Highly organized structures, like the cell membrane show magnetic anisotropy. Its magnetic susceptibility depends on the direction on which it is measured. Anisotropic magnetic structure, like cell membrane or chromosomes, can be oriented in strong magnetic fields [42].

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2-7 The Mechanism of Interaction Between Magnetic Field and Biological System
In general the following interaction or effects are possible: 1. Orientation of particle in static magnetic fields. 2. Translation of particle in inhomogeneous static magnetic field. 3. Action of Lorentz force on moving charged particles in magnetic field (e.g. ions, molecules, cell in moving blood). 4. Induction of electric current in the electrolyte milieu of the body in case of movements in a magnetic field or in gradient of a magnetic field [38]. In inhomogeneous magnetic fields diamagnetic bodies move in the direction of decreasing field strength, whereas para and especially ferromagnetic particles are driven in the opposites direction. Charges moving in a magnetic field are mechanically influenced by so called Lorentz force. This force also acts on ions and other charged particles under the influence of strong magnetic fields in the flowing blood. The inverse of this effect is the induction of an electric current if a conductor crosses lines of a magnetic field. This can occur in limbs moving in the magnetic field of a Nuclear Magnetic Resonance Technique (NMR).The result is a painful nerve excitation. Magnetic fields with flux densities much higher than one tesla can influence biochemical reactions in isolated enzyme system and consequently various cellular processes, like proliferation and differentiation. Cellular reaction, which is composed with phase transition of membrane lipids are apparently, influenced most. This is caused by the anisotropy of lipids [43].

2-8 How Magnetic Field Affects a Living System

Some of the way magnetic field affects living organisms are:[44] 1. Increased blood flow with resultant increased oxygen carrying capacity.

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Theory

2. Changes in migration of calcium ions which can help moving calcium a way from painful, arthritic. 3. The PH balance (acid/ alkaline) of various body fluids (often out of balance in conject in with illness of abnormal condition) can apparently be altered by magnetic field. 4. Hormone production from the endocrine gland can be either increased or decreased by magnetic stimulation. 5. Altering of enzyme activity and other biochemical processes. To better understand the implications of providing the body with an adequate magnet environment, it is important to understand the basic movement of certain body fluids and their role in health and disease. In some what simplified explanation, as the heart pump approximately 80 times per minute, blood in the arteries forces nutrient liquid through the pores in the capillaries into the cell area to nourish the cells, this liquid is called plasma while it is in the blood stream and is renamed Lymph once it leaves the blood stream. Any thing that slows down the blood movement, even for a fraction of a second, can result in oxygen depletion, leading to sever damage to organs [43,44].

2.9 The Effect of Water on Cells

Cells separate from one another; in intercellular space water is the media of exchanging information. Water that can be found in the water cover of protein has orderly structure in each instance. On inner and outer cell membrane due to ion difference, an electric duality starts out. The electrostatic effect of this duality has its effect on the cells as well as on the intercellular entities [45]. Fluid fluctuation affects blood volume and cellular function. When the amount of fluid within various compartments is relatively constant, there is an exchange of solutes and water between compartments to maintain unique compositions. 21

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Health and aging depend on the quality, content and structure of the water in our body [46]. Since our body is 70% water, total body water percentage decreases with age, resulting in inadequate cellular hydration. Hydration is the process whereby a substance interacts or combines with water [47]. Most critical is the decrease of the ratio of intracellular hydration. The normal ratio is 60% intracellular, 40% extracellular. All body functions take place in water, interacting with cells. Because water is an excellent solvent, assimilation of vital nutrients and proteins take place through absorption. Movement of water in the body between cells (extracellulr fluid) is caused by osmosis, this is created by magnetic forces, which keep the movement in balance. As water flows, changes in pressure create movement across the cell membranes. Any change in pressure will allow proteins, minerals and other nutrient being carried by the blood to escape into spaces between vessels and deprive the cells of their vital needs to sustain life. When water in the blood is contaminated with chemical,it enters the cells and changes their structure which in turn could lead to changes in DNA. This is the start of disease process [46].

2.10 Effect of Magnetization on the Bodys Water

We can observe a magnetic influence in different organisms both in cell and molecular level. Water structure is dynamic and not static, its molecules are not rigidly clamped together [47].Water in our body is randomly ordered and cluster with neighboring water molecules to form large molecular cluster [46]. Water has been known to be affected by magnetism. When water is exposed to a magnetic field, technically, it increases the speed of sedimentation of suspended particles in water (and other liquids) and enhances conductivity and the process of ionization or dissociation of atoms and molecules into electrically charged particles. Also the influence of magnetic field on water 22

Chapter Two

Theory

more hydroxyl (OH ) ions are created to form calcium bicarbonate and other alkaline molecules, so the PH of water will be altered. Magnet also affects the bonding angle between the hydrogen and the oxygen atom in the water molecule, it will cause the hydrogen-oxygen bond angle within the water molecule to be reduced from 104 to 103 degrees. This is in turn causes water molecule to cluster together in groups of 6-7 rather than 10-12.The smaller cluster leads to better absorption of water across cell membrane into the cell nucleus by osmosis [44,48]. This means the diffusion of water across a membrane, from a region of higher water concentration to one of lower water concentration. However, the concentration of a solution is not expressed in tern of water, but in terms of solute (i.e. sugars, amino acid and ions). Thus water diffuses from less concentrated solution, which has fewer solute molecules and more water molecules, into the more concentrated solution with more solute molecules and fewer water molecules [49]. All processes within living organism are regulated by very weak magnetic gravitational forces; its connection with other cells becomes loose. Cell multiplication requires the alteration of the water structure of the cell. If

the orderly structure of cells is not restored by the end of cell multiplications a pathological change or even cancerous disease can start out [45]. Since the amorphous structure of the water cluster is responsible for the formulation of cancer, every alteration in the water cluster of the cells, that is, cell isolation, is a source of dangers. The magnetic field affects the ions making their way through it and brings about the Lorentz forces, which can be calculated by the way following equation: F = K q v Bsin (2-15)

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where F is , Lorentz force, K coefficient of proportionality, v ion velocity, B magnetic field strengthened and angle of magnetic field direction with flow [50]. Quantum electrodynamics calls for the existence of long range electromagnetic fields that can be transmitted by large coherent domains existing in water[51,52]. These long range electromagnetic fields may transmit the electromagnetic signals (message) coming from molecules, thus generating specific alteration between molecules with matching spectra, excluding nonresonating. The electromagnetic signal resulting from the aggregation of the two co-resonating molecules would obviously exhibit a different frequency that co-resonate with the next molecules or cluster of molecules which intervene is the next step of the biochemical reaction, and so forth [51].

2.11 Effect of Environment On Natural Molecular Structure of Water

The basis of molecular biology in the cell, it's DNA and how critical water is to its order.The molecular parts which compose the cells of our bodies are tied together by a matrix of water molecules. Like the atoms that compose them, they are tied together by ionic bonds, consisting of positive and negative charges on their outer surface. The basis of molecular biology in the cell, it's DNA and how critical water is to its order. If DNA is dehydrated, like other bonding component of the cell, it loses its orderly, repeating structures and takes more random shapes and forms. In other words, DNA looses its normal functions and properties. DNA operating in harmony with water, which determines shapes that are adopted as they are produced and move within the cell. It appears that 24

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molecules themselves are the code and water the medium where the code is expressed. As long as environment and temperature are maintained within the cell, and there is adequate magnetization to keep cell biology functioning normally, then cellular reproduction, along with normal DNA coding, will continue. If, the content and effectiveness of water inside and outside the cell contain destructive toxins, the degradation and deterioration of cells will increase quickly, resulting in rapid aging and disease [48]. Any change in the physical or chemical conditions required for the optimum function of living organisms introduces a stress in the organism must either resist or adapt to. Water stress that results from changes in the intermolecular nature of water and / or solute hydration induced by changes in solute concentration. Cells have to maintain itself in osmotic equilibrium visavis its external environment. Dr. Hempling used the term Osmotic death in connection with the absolute requirement for osmoregulation.A physiological stress can be defined as external factor acting on a cell or an organism in such a way that it interferes with its normal functioning. The responses of living organisms to water stress involve a wide range of physical and chemical principles, including osmotic regulation, specific hydration phenomena, promotion or inhibition of nucleation and redistribution of water within organism [53].

2.12 Cell Membrane

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Cell membrane or plasma membrane, encloses the cytoplasm and forms the boundary between material inside the cell and material outside the cell, Figs (2-3,2-4)[49].Substances outside the cell are called extracellular substances, and substances inside the cell are called intracellular substances. The cell membrane is a selective barrier that determines what moves into and out of the cell, the major molecules that make up the cell membrane are phospholipids and proteins. In addition the membrane contains other molecules, such as, carbohydrates, water and ions [42], which can be illustrated as shown below

Figure (2-3) Generalized cell showing the major organelles No single cell contains all organelle types. In addition, some kinds of cell contain many organelles of one type, and another kind of cell contains very few [49].

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Figure (2-4): The cell membrane The cell membrane is composed of a double layer of phospholipids molecules with protein. The nonpolar end of each phospholipids molecule is directed toward the center of the membrane, and the polar end of each phospholipids molecule is directed toward the water environment either outside or inside the cell. Cholesterol molecules are interspersed among the phospholipids molecules. Groups of proteins can form membrane channels, carrier molecules, receptor molecules, enzymes, or structural supports [49].

a) Water: It has been selected by nature as the biological solvent because of its physical properties, which are different from the solvents commonly used by chemist. Water structure contains one oxygen atom bonds with two hydrogen atoms Fig.(2-5)[54]. Each OH bond is formed by a sharing electrons and it is a covalent bond. The electropositive oxygen nucleus attracts the electron more strongly than the hydrogen atom which results in a slight negative charge () 27

Chapter Two
+

Theory

in the region of oxygen atom and positive charge ( ) in the region of hydrogen atom. Although the water has no net charge, but it has two region of a partial

positive and one region of partial negative which results in the molecule being an electric dipole .i.e. polar. This fact is largely responsible for the electrostatic attraction between the partial negative charge on the oxygen atom of one water and a partial positive charge on the hydrogen atom of its neighbor. This for electrostatic attraction is called hydrogen bond, which is much weaker than covalent bonds. This hydrogen bond is not unique to water and has an important role is determining the structure of macromolecules [55]. H + O-----H + H A Hydrogen bond O2H H H O+ H Hydroxyl ion + OH

Hydronium ion

Figure (2-5): The structure and ionization of water [54] Water is a diamagnetic, the magnetic susceptibility for water is 9.05 10 6 [38]. b) Protein: The protein are organized in the membrane according to the hydrophobic (i.e. apolar) and hydrophilic (polar) regions. Many proteins proceed to the external surface of the membrane as glycoprotein. They are located with their C-terminal end in the cytoplasm. Their N-terminal end, which is modified by various carbohydrates, extends over along distance into the outer environment of the cell. Protein posses four levels of organization: primary, secondary, tertiary and quaternary. The primary structure of a protein 28

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is its amino acid sequence. All these amino acids have common structure Fig. (2-6)[55]. H R C COOH

NH2 Figure (2-6): Amino acid structure

where R is a side chain groups that differ to give the 20 known amino acids. The secondary structure is the local three dimensional conformation of polypeptide chain. The tertiary structure of molecular species comprises several polypeptide chain held together by chemical bonds to form a single molecule. Quaternary structure results from the association, without chemical bonding, of Individual protein molecules into a aggregates of two or more molecules [42]. c) Carbohydrate: The name carbohydrate suggests carbon and water, most carbohydrates have the empirical formula Cx(H2O)Y. Carbohydrate constitute up to 10% of the total dry weight of mammalian cell membranes, functioning as receptors, antigens and other recognition sites, also being involved in intracellular adhesion. They are composed of chains of six membrane ring structures (saccharides). They may be classified as monoses or monosaccharides (simple sugars), bioses or disaccharides (more complex sugars) . Carbohydrate is also responsible for the most of the negative charge on the cell surface, since a charged saccharide, silica acid, form the terminal residue of many polysaccharide chains [42, 55].

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d) Lipids: Lipids are a varied group of substances, which are water insoluble organic substance. The most important constituents of biological membranes are phospholipids Fig. (2-4).This is a group of compounds which consists of two fatty acids and phosphate group, cell attached to a polyhydric alcohol such as glycerol [42].The lipid in biological membranes shows a high lateral mobility because they can easily exchange their position. The mobility of protein, in contrast, is strongly limited by their fixation on the cytoskeleton. In the mechanisms of lymphocyte activation the surface glycoproteins function as stimulating receptors which have a transmembrane orientation. These integral proteins are linked to components of the cytoskeleton thus providing a structure which enables the direct transfer of an extracellular signal

to component in side the cell. Also the carbohydrate moieties of membrane glycoproteins are an absolute requirement for stimulation [56]. The receptors on the lymphocyte cell surface are necessary for mediating signals by which many cells respond to external stimuli. Interaction of these receptors with biochemical messenger initiates chemical and physical changes in membranes. The biochemical changes in membranes after stimulation of receptor are:[57] 1-Changes in the mobility of receptor on cell surface probably due to increased membrane fluidity [58]. 2-Increased Ca+2 influx [59]. 3-Rapid rise and fall of cyclic nucleotides [60]. 4-Increased turnover of phospholipids [61]. 30

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5-Membrane protein phosphorylation [62]. All these events have been postulated to play some roles in the initiation of lymphocyte activation, namely, proliferation and/ or expression of function. Membranes are diamagnetic, when an external magnetic field applied, the orbital motion of the electrons in the constituent molecules is modified in such way as to induce local Magnetic fields opposing the external field .These induced fields are very small, and do not produce any appreciable effect on a gross scale. An exception occurs in membranes containing densely packed helical protein, in which quite strong fields are induced when the axes of the helices lie a long the field direction. Despite the lack of gross reaction by the majority of membranes, magnetic field affects the subatomic level. Subatomic particles have an associated spin, and since spinning charge generates a magnetic field, charged Particles possess a magnetic dipole moment.Although this subatomic magnetism is weak, but it give rise to resonance phenomena [42].

2-13 Relationship of Magnetism to Cell Stability

The stability of a particular molecule is the electromagnetic bond of energies between atoms. The electrons circling around atoms resist attempt to disturb their usual orbits, so they can create their own magnetic field. As a result, the atoms behave like little magnetic needles, all which point in a direction opposite to the field which is being applied. Magnetism takes place 31

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when electrons orbit a round their atoms. Electrons have a property called spin, which makes them acts like miniature magnets with two opposite poles. If neighboring electrons are made to spin so their poles are aligned in the same direction, it becomes magnetic. This is how bacteria and cancer strives. This is believed to be the activity creating free radicals, which is unstable, strong magnetic activity result of cell with electron imbalance aggressively seeking another electron, thereby creating imbalance in another cell. Electrons of a cell are established in pairs, magnetism affects the electron stability, when electron is taken by oxygen which becomes unpaired, and free radicals form and destroy cells [44].

2-14 Cell Activity, Energy and Cell Metabolism

The conversion of energy is essential to all life forms. All living things assimilat energy and use it to grow. This process is called metabolism [48]. The interaction between organelles must be considered to fully understand cell function, which will illustrate the cell activity. Adenosine triphosphates (ATP) are the major energy source for most chemical reaction within the cell. The ATP is produced in the cell by mitochondria. The transport of many food molecules into the cell by the plasma membrane requires ATP and plasma membrane protein [11].The production of plasma membrane protein requires amino acids to be transported into the cell

by the plasma membrane. Instructions provided by the nucleus cause the amino acids to be combined at ribosome to form proteins [49]. 32

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2-15 Movement Through the Plasma Membrane

The plasma membrane (Cell membrane) is selectively permeable, allowing some substance, but not others to pass into or out of the cell. Intracellular material has a different composition from extracellular material, and the survival of cells depends on maintaining the difference. Rupture of the membrane, alteration of its permeability characteristic or inhibition of transport processes can disrupt the normal intracellular concentration of molecules and lead to cell death. Molecules can pass through the plasma membrane by: 1-Passing through the phospholipids layers. 2-Moving through membrane channels. 3-Being transported by carrier molecules Fig.(2-7). 4-Being transported with vesicles. Lipid soluble molecules such as oxygen pass through the plasma membrane by dissolving in the double layer of phospholipids. The phospholipids layers acts as a barrier to most substances that are not lipid soluble; but non lipid soluble molecules such as water, carbon dioxide can diffuse between the phospholipids molecules of the plasma membrane [49]. Large number of different transport processes in a single cell appears closely meshed together. By PH and volume regulation, and by control of internal Ca++ concentrations, transport processes may be control complex cellular functions, such as protein expression, cell proliferation etc.

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Figure (2-7): Mediated transport mechanism A) A molecule binds to a protein carrier molecule on one side of the cell membrane. B) The carrier molecule changes shape and releases the molecule on the other side of the cell membrane [49].

2-16 Immunity and Immune System

Immunity is the ability to resist damage from foreign substances, such as microorganism and harmful chemicals, such as toxin released by microorganism [49]. Immune system has involved to protect us from pathogens, as soon as an organism is invaded by foreign agents, usually viruses or bacteria, the immune system is activated. Those immune system cells that carry the specific immune receptors for interaction with the foreign agent are stimulated to proliferate.

2-17 Leukocytes

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Leukocytes and cell derived from leukocyte are the most important cellular component of immune system. Leukocytes protect the body against microorganisms and remove dead cells. There are two types of Leukocytes:

a) Granulocytes that contain cytoplasmic granules, and these are of three types: neutrophils are small phagocytic cells, eosinophils reduce inflammation and basophile promote inflammation.
b)

Agranulocytes: have a very small granules, and there are two types: Lymphocytes are involved in antibody production and other immune system responses, monocyte and tissue macrophages that ingest microorganism and cellular debris [49].

2-18Lymphocyte Transformation
Lymphocyte phenotype can be described by the nuclear to cytoplasmic ratio (N:C). The nucleus has a spherical shape occupying almost the entire cell and condensed chromatin and intersely basophilic cytoplasm [63]. Nowell,1960 was the first scientist used the term lymphocyte transformation, and described the morphologic changes that result when small, rest lymphocytes were transformed into lymphoblast on exposure to mitogen phytoheamagglutinin (PHA),or lymphocyte stimulated by either nonspecific mitogen or antigens[64]. Activation can be triggered by a varity of stimuli. Any multivalent ligands that cross-link immunoglobulin molecules can induce rest lymphocyte cell to enter the cell cycle that begin with resting phase (G0), and the first growth phase (G1), DNA synthesis ( protein and nucleic synthesis phase ) (S-phase) and finally the second growth phase (G2)[64]. The interaction of multivalent ligand with cell surface immunoglobulin triggers a series of metabolic events in lymphocyte cell. These include activation of membrane enzyme, that are responsible for the activity of the cell such as 35

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cyclic-Guanidin monophosphate (CGMp) and whose percentage increased during the first time or minute of activation which lead to morphological changes as a result of the transformation of rest, small cell (lymphocyte) to

blast cell (lymphoblast).An increase in the size and number of lymphocyte cells, increase in number of vacuoles, the nuclei become more visible in side the cell due to accumulation of the nucleus protein. On the other hand, the biochemical changes events include increased synthesis of phospholipids, increase cell membrane permeability to cations such as the increase the penetration of K+ to inside the cells, and resultant elevation of intracellular CAMP (cyclic adenosine monophosphate ), at this point the cells inter the (G1) phase and also there is a continues penetration of both glucose and Ca+2 ions that is essential for synthesis of other enzymes in the nucleus this phase is characterized by the protein synthesis, RNA and lymphokines this phase occur during 12-24 hours. After that the cell enter synthesis of DNA phase (S-phase) and duplication of chromatin, this phase takes 48 hours and finally the activated cells inter (G2-phase) after 72 hours of activation with specific antigen or mitogen [64]. Binding of mitogens to lymphocyte cell surface receptors triggers a sequence of metabolic events which must take place in the plasma membrane. Cell mediated immune response is dependent on interaction between growth factors and other hormone like substance and receptor proteins on cell membranes. Substances, secreted by some immune cells, include cytokines, which are protein hormones that enhance their own function and the function of many other immune cells; they also stimulate inflammatory responses. The mitogen produces their effect on DNA synthesis in 72 hours, whereas mitogen stimulated large number of lymphocytes, antigen stimulated far fewer cells which are specifically sensitized to the antigen. As in the case of mitogen induced activation, the degree of lymphocyte activation is also a function of the cellular regulatory influences that are present in the culture. Time and dose response kinetics are of crucial importance in generating reliable data. In 36

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contrast to mitogen induced lymphocyte activation, antigen stimulated ion results in lower total DNA synthesis. Furthermore the time of maximal response does not occur until the culture has been allowed to continue for 5-7 days [64].The growth of cells can be controlled by the interaction of growth factors with their receptors on the plasma membrane. Such interaction may for example cause somatic cells to leave G0, traverse to G1 , and enter S-phase, whereupon they are normally committed to at least on round of cell cycle[64]. The lymphocyte transformation percentage can be measured either by: a) Determination of the percentage of the formation of lymphocyte cells [65] b)Using method for the determination of the cellular up take of thymidine-H3 enzymatic activity (fluoremetric assay) [66].

2-19 Phagocytosis
Phagocytosis is the ingestion and destruction of a particles by cells called phagocytes, Fig.(2-8)[49].It is an important defense mechanism that occur when a foreign antigens or microorganisms or foreign substances or dead cells from the individual body inter the body. Phagocyte cells play an important role in the immune response by secretion of specific material called (monokines) which in addition to activation of both T-and B lymphocyte cells, do many biological functions [67].The most important phagocytes are neutrophils and macrophages. 2-19-1 Monocyte-Macrophage Cell Monocytes are larger than granulocytes and most lymphocyte, macrophage cells are monocytes that leave the blood, inter tissues. They have round or kidney shaped nuclei with fine, lightly stained granules.

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2-19-2 Neutrophils Polymorphonuclear neutrophils(PMNs) are bone marrow derived white blood cells, which play a central role in defense of the host against infection the neutrophil plays the primary role as an effectors or killer cell. Defect in neutrophil function can be classified as quantitative or qualitative. In quantitative the total number of normally functioning neutrophils is reduced below a critical level, allowing infection to ensue. In qualitative Europhilic disorders, the total number of circulating PMN is either normal or sometimes elevated, but the cells fail to exert their normal microbicidal functions [63].

2-20 Mechanism of Phagocytosis

Phagocytosis or phagocytic process can be divided into five stages: 1-Chemotaxis 2-Recognition and adherence 3-Ingestion 4-Degranulation 5-Intercellular killing and digestion

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Figure(2-8): Phagocytosis Cell processes extend from the cell and surround the particle to be taken into the cell by phagocytosis. The cell processes surround the particle and fuse to form a vesicle that contains the particle. The vesicle then is internalized within the cell[49]. After the recognition of foreign particles by macrophage it will adhere to the particles and presenting them. The adherences lead to the activation of the cellular membrane, which in turn leads to the surrounding of these foreign particles and their complete ingestion [68]. After ingestion the particles are subjected to digestion by different mechanism and material, such as peroxides, lysozom, lactoferrin [68]. Phagocytic activity can be measured by the percentage of phagocytosis which is calculated as shown; Phagocytosis %= Number of phagocyte cells/ Total (phagocyte +non phagocyte) 100

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Chapter Two

Theory

2-21 Metabolism during Phagocytosis

The ingestion of particles by macrophages as well as by neutrophils is a accompanied by a respiratory burst observed as a dramatic increas in the consumption of oxygen and activation of a membrane associated with oxidias that is dependent on reduced nicotinamide adenine dinucleotide phosphate (NADPH). This oxidase reduces molecular oxygen to superoxide anion (O2 ), which in turn dismutates to hydrogen peroxide (H2O2).The events of the respiratory burst associated with phagocytosis as shown: a) Activation of the hexose monophosphate shunt and the glutathione reductase-glutathion peroxides cycle b) Activation of membrane associated NADPH oxidase, with increased consumption of oxygen and generation of superoxide anion and hydrogen peroxidase. c) Formation of hydroxyl radicals(OH), singlet oxygen(O21), and other metabolites of reactive oxygen [64]. Superoxide, hydrogen peroxide can interact to give rise to hydroxyl radical and possibly, singlet oxygen. These radical are called reactive species oxygen [69].These reactive species can oxidaze sulfhydryls, pyridine nucleotides, and unsaturated fatty acids. In addition they can directly alter deoxyribose moieties of DNA [64]. Hydrogen peroxide (H2O2) induces changes in membranes potential and that hypertonic media partially protects against swelling of renal epithelial cells. An increase in plasma membrane permeability for either Na+ or K+ contributed to depolarization to a minor extent [69].

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Chapter Two

Theory

2-21 Free Radicals and Theory of aging

Free radical is a molecules containing single, unpaired electrons,e.g.CH3 (Methyl).Free radical are highly reactive, and combine quickly with each other, producing stable molecules with paired electrons[40].Free radical can react with and alter the structure of molecules that are critical for normal cell function [70]. Alteration of these molecules can result in cell dies function, cancer, or other type of cellular damage. The unpaired electron of a free radical is represented by a dot on the atom or group in which it predominately resides. Most free radical are very reactive with oxygen. The damage caused by free radicals may accumulate with age. A radical is an electrically neutral species; the electron transfer from O2 to its end product of reduction (H2O) water is a complicated process and is attributed to the formation of free radicals. These radicals can occur either by photodynamic way or by catalysis of transition metal or enzymes. A special role in cell metabolism is played by so called reactive oxygen species (ROS). They trigger chain reaction capable of damaging the different constituents of the cell [36]. One of the most convenient modes of free radical generation is electro magnetic field. In aqueous system it is splits waters H2O RH OH + eaq + H+ R- +H+ In organic media it breaks chemical bonds, e.g. [64] Oxy radicals are a class of free radicals with the unpaired electron residing predominantly on an oxygen atom, the best known are superoxide radical (O2), hydrogen radical (OH) and various peroxy radicals (ROO).

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