Saint Louis University School of Medicine Baguio City

Physiology (Laboratory) MOLECULAR MOTION AND CELL TRANSPORT MECHANISM

Submitted to: Dr. Lizalyn B. Revilla Dr. Bunag

Submitted by: Group 10 Verceles, Timotheo Joy

S.Y. 2012-2013

1.1.1 Diffusion Results: In the first beaker: a mixture of methylene blue crystals and warm water we observed that the methylene blue crystals diffused and spread throughout the container and maintained a diffused form longer. In the second beaker: a mixture of methylene blue crystals and cold water we observed that the methylene blue crystals became stationary and maintained a suspended form and did not spread out unlike the one in the first beaker The Theory Behind:

Questions: 1. What is meant by diffusion? - Diffusion is one of several transport phenomena that occur in nature. A distinguishing feature of diffusion is that it results in mixing or mass transport without requiring bulk motion. Thus, diffusion should not be confused with convection or advection, which are other transport mechanisms that use bulk motion to move particles from one place to another. InLatin word "diffundere" means "to spread out". - Diffusion refers to the process by which molecules intermingle as a result of their kinetic energy of random motion. - Diffusion is simply the transfer of substance from higher level of concentration to a lower level of concentration 2. The Factors that affect the rate of diffusion - Size o Small molecules can slip by the polar heads of the phospholipids and through the membrane to the other side. Oxygen gas, carbon dioxide and water can move in this manner. Very large molecules like proteins cannot diffuse across the membrane at all. - Shape o Glucose is able to get into cells much faster than other sugars. This is accomplished by facilitated diffusion. A carrier protein specific for glucose (not other sugars) combines with it on the outer surface, closes around it, and then opens to the inside of the cell where the glucose is released. The carrier then returns to its original shape and is ready to transport another glucose molecule. These carriers can move up to 100 glucose molecules per second across the cell membrane. - Concentration o The greater the concentration gradient between the outside and inside of the membrane the greater the rate of diffusion. If the concentration of oxygen outside the cell increases then it will diffuse more quickly into the cell. The opposite is also true. If a muscle cell for example is working hard and using up large quantities of oxygen in cellular respiration producing ATP, then the low levels inside the cell will increase the concentration gradient compared to outside and the rate of diffusion of oxygen into the cell will increase. The same conditions in a muscle cell would create high concentrations of carbon dioxide inside the cell and increase the rate of diffusion from inside to outside. - Charge (+/-)

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Ions or molecules with a charge cannot pass through the lipid bilayer by diffusion. Other mechanisms involving protein carriers and ATP energy are required. The sodium/potassium ion pump is an example of this type of transport. Lipid Solubility o Lipid soluble molecules can move through the lipid bilayer. Generally these molecules are other lipids. Steroid hormones like testosterone and estrogen are examples of such molecules. This easy access to cells explains the powerful and wide ranging effects of such hormones. Temperature o In general, increases in temperature cause all molecules to move faster. Diffusion is a passive movement of molecules so quicker molecule movement translates into quicker diffusion. Rate of diffusion increases as: * Diffusion distance decreases * Concentration gradient increases * Surface area increases

3. What is the principal driving force of diffusion? - In chemical systems other than ideal solutions or mixtures, the driving force for diffusion of each species is the gradient of chemical potential (concentration Gradient) of this species. - Concentration gradient - the gradual difference in the concentration of solutes in a solution between two regions. In biology, agradient results from an unequal distribution of ions across the cell membrane. When this happens,solutes move along a concentration gradient. This kind of movement is called diffusion. 1.1.2 Osmosis Results: In slide A (blood and distilled water) The red blood cells shrink and seemingly became small as observed under the microscope. In slide B (blood and 0.5% NaCl solution) we observed that the red blood cells maintained their form and no change was seen In slide C (blood and 0.9% NaCl solution) The cells seemingly became large, and some are likely to explode. In slide D (blood and 1.5% NaCl solution) The cells, like in slide C, became large and are likely to explode.

1.1.3 Adsorption and Absorption Results: We prepared 2 beakers, In beaker A containing brown sugar and methylene blue crystals, and in beaker B - containing active charcoal and methylene blue crystals. As we filtered beaker A, we observed that the color of the solution/mixture did not change, as compared to beaker B, were in the filtrate became clear/transparent as we filtered it.

Questions: 1. Define adsorption and differentiate from absorption: - Adsorption is the adhesion of atoms, ions, or molecules from a gas, liquid, or dissolved solid to a surface. This process creates a film of the adsorbate on the surface

of the adsorbent. This process differs from absorption, in which a fluid (the absorbate) permeates or is dissolved by a liquid or solid (the absorbent). Note that adsorption is a surface-based process while absorption involves the whole volume of the material. The term sorption encompasses both processes, while desorption is the reverse of adsorption. It is a surface phenomenon. - Adsorption, the binding of molecules or particles to a surface, must be distinguished from absorption, the filling of pores in a solid. The binding to the surface is usually weak and reversible. Just about anything including the fluid that dissolves or suspends the material of interest is bound, but compounds with color and those that have taste or odor tend to bind strongly. Compounds that contain chromogenic groups (atomic arrangements that vibrate at frequencies in the visible spectrum) very often are strongly adsorbed on activated carbon. Decolorization can be wonderfully efficient by adsorption and with negligible loss of other materials. - Adsorption is different from absorption in the sense that it focuses not on the volume but on the surface. If fluids and gases settle on the surface of another material (liquids or solids) rather than be diffused into the said material, then this forms a solution. The said process is an adsorption. To use another explanation, adsorption is the process when an outside contaminant (atoms) gets attracted to the outside surface of a piece of material while absorption involves the uptake of the contaminant into the literal structure of the material. Similarly, absorption occurs when the outside contaminant has merged or has become part of the other material. A similar analogy occurs when you drink water. In drinking such, you are therefore absorbing fluid making it a part of you. Adsorption occurs when you accidentally spill water on your shirt. The water did not actually become part of you but just fell on you. It just bonded physically with a certain surface (your shirt). Overall, although both absorption and adsorption are sorption processes they still differ in the following areas: 1. Absorption happens when atoms pass through or enter a bulky material like sponges. 2. Adsorption happens when the atoms settle or accumulate on the surface of a material rather than literally entering or diffusing into that same material. 2. Give one significant therapeutic use of activated charcoal - Activated charcoal is used in water filters, medicines that selectively remove toxins, and chemical purification processes. Activated charcoal is carbon that has been treated withoxygen. The treatment results in a highly porous charcoal. These tiny holes give the charcoal a surface area of 300-2,000 m2/g, allowing liquids or gases to pass through the charcoal and interact with the exposed carbon. The carbon adsorbs a wide range of impurities and contaminants, including chlorine, odors, and pigments. Other substances, like sodium, fluoride, and nitrates, are not as attracted to the carbon and are not filtered out. Because adsorption works by chemically binding the impurities to the carbon, the active sites in the charcoal eventually become filled. Activated charcoal filters become less effective with use and have to be recharged or replaced. Several factors influence the effectiveness of activated charcoal. The pore size and distribution varies depending on the source of the carbon and the manufacturing process. Large organic molecules are absorbed better than smaller ones. Adsorption tends to increase as pH and temperature decrease. Contaminants are also removed more effectively if they are in contact with the activated charcoal for a longer time, so flow rate through the charcoal affects filtration. - Activated carbon, also called activated charcoal, activated coal or carbo activatus, is a form of carbon that has been processed to make it

extremely porous and thus to have a very large surface area available for adsorption or chemical reactions. The word activated in the name is sometimes replaced with active. Due to its high degree of microporosity, just 1 gram of activated carbon has a surface area in excess of 500 m2, as determined typically by nitrogen gas adsorption. Sufficient activation for useful applications may come solely from the high surface area, though further chemical treatment often enhances the adsorbing properties of the material. Activated carbon is usually derived from charcoal.

Case 1: Permeability and Simple Diffusion Question: 1. What equation describes the diffusion coefficient for a solute? What is the relationship between molecular radius and diffusion coefficient? - The Stokes-Einstein equation is the equation first derived by Einstein in his Ph.D thesis for the diffusion coefficient of a "Stokes" particle undergoing Brownian Motion in a quiescent fluid at uniform temperature. The result was formerly published in Einstein's (1905) classic paper on the theory of Brownian motion (it was also simultaneously derived by Sutherland (1905) using an identical argument). Einstein's result for the diffusion coefficient D of a spherical particle of radius a in a fluid of dynamic viscosity h at absolute temperature T is:

where is the gas constant and NA is Avogadro's Number. The formula is historically important since it was used to make the first absolute measurement of NA so confirming molecular theory. Although the formula can be derived alternatively using the Langevin equation of motion for a Brownian particle [Chandrasekhar (1943)] the derivation of Einstein is a powerful and ingenious one, correct even when the Langevin equation is only approximate. Einstein assumed that van't Hoff's law for the osmotic pressure exerted by solute molecules in a solvent fluid at equilibrium was equally applicable to the pressure p associated with a suspension of Brownian particles at equilibrium in the same fluid, i.e., where nM is the number of gram moles of fluid per unit volume and f the 'molar fraction' defined here as the ratio of the number of particles to the number of fluid molecules. Einstein then argued that a suspension of Brownian particles at equilibrium under their own weight could be viewed in two ways both of which were equivalent: a balance between the net weight of the particles and the gradient of the particle pressure in the direction of gravity; or a balance between the diffusion flux and the settling flux due to gravity. Using the Stokes drag formula for the settling velocity (see Stokes Law) and the formula for p above gives the formula for D given above. A similar argument allows one to deduce a form for the particle pressure in a turbulent gas knowing the form for the particle turbulent diffusion coefficient (see Particle Transport in Turbulent Fluids). - The relation between molecular radius and diffusion is inversely proportional, because, when molecular radius increase, diffusion coefficient decreases 2. What equation relates permeability to diffusion coefficient? What is the relationship between molecular radius and permeability?

- Fick's laws of diffusion describe diffusion and can be used to solve for the diffusion coefficient, D - Diffusion equation for moisture migration. This is analogous to Fourier's equation of heat conduction. - R=D X A Dp / d R=the rate of diffusion D=diffusion coefficient, which is a characteristic of the medium and varies exponentially with temperature A=the surface area and dC/dx Is the concentration gradient over the diffusion distance Fick's first law suggests that the rate of diffusion in a given direction across and exchange surface: 1. is directly proportional to the concentration gradient- the steeper the concentration gradient, the faster the rate of diffusion 2. is directly proportional to the surface area- the greater the surface area of a membrane through which diffusion is taking place, the faster the rate of diffusion this is one of the factors which limits cellsize. 3. is inversely proportional to the distance- the rate of diffusion decreases rapidly with distance. diffusion is thus effective only over short distances. this limits cell size. Fick's second law predicts how diffusion causes the concentration to change with time. It is a partial differential equation which, within the character limitations of Wikianswers, the second law is: /t = (D) where

is being used as the symbol for partial differential is the concentration in dimensions of [(amount of substance) length3] t is time is the "dot product" is the del or gradient operator D is is the diffusion coefficient in dimensions of [length2 time1]

Note that when is at steady state, this equation reduces to Fick's first law. - The relation between molecular radius and permeability is inversely proportional, because, when molecular radius increase, permeability decreases 3. What is the relationship between oil-water partition coefficient and permeability? - There is a linear relationship between oil-water partition coefficient and permeability, as proven by the Overton rule.

Case 2: Osmotic pressure Questions: 1. What is osmolarity and how is it calculated? - Osmolarity is the measure of solute concentration, defined as the number of osmoles (Osm) of solute per litre (L) of solution (osmol/L or Osm/L). The osmolarity of a solution is usually expressed as Osm/L (pronounced "osmolar"), in the same way that the molarity of a solution is expressed as "M" (pronounced "molar"). Whereas molarity measures the number of moles of solute per unit volume of solution, osmolarity measures the number ofosmoles of solute particles per unit volume of solution.

- The osmolarity of a solution can be calculated from the following expression: Where

is the osmotic coefficient, which accounts for the degree of non-ideality of the solution. In the simplest case it is the degree of dissociation of the solute. Then, is between 0 and 1 where 1 indicates 100% dissociation. However, can also be larger than 1 (e.g. for sucrose). For salts, electrostatic effects cause to be smaller than 1 even if 100% dissociation occurs (see Debye-Hckel equation); n is the number of particles (e.g. ions) into which a molecule dissociates. For example: glucose has n of 1, while NaCl has n of 2; C is the molar concentration of the solute; the index i represents the identity of a particular solute.

2. What is osmosis? What is the driving force for osmosis? - Osmosis is the net movement of solvent molecules through a partially permeable membrane into a region of higher solute concentration, in order to equalize the solute concentrations on the two sides. It may also be used to describe a physical process in which any solvent moves, without input of energy across a semipermeable membrane (permeable to the solvent, but not the solute) separating two solutions of different concentrations. Although osmosis does not require input of energy, it does use kinetic energy and can be made to do work. - Osmosis is the net movement of water across a selectively permeable membrane driven by a difference in solute concentrations on the two sides of the membrane. A selectively permiable membrane is one that allows unrestricted passage of water, but not solute molecules or ions. - The driving force for this separation is an osmotic pressuregradient, such that a "draw" solution of high concentration (relative to that of the feed solution), is used to induce a net flow of water through the membrane into the draw solution, thus effectively separating the feed water from its solutes. In contrast, the reverse osmosis process uses hydraulic pressure as the driving force for separation, which serves to counteract the osmotic pressure gradient that would otherwise favor water flux from the permeate to the feed. 3. What is osmotic pressure, and how is it calculated? What is effective osmotic pressure, and how is it calculated? - The osmotic pressure of a dilute solution is found to obey a relationship of the same form as theideal gas law:

In chemistry texts, it is usually expressed in terms of the molarity of the solution and given the symbol . -

Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane. It is also defined as the minimum pressure needed to nullify osmosis.
Effective osmotic pressure - that part of the total osmotic pressure of a solution that governs the tendency of its solvent to pass across a boundary, usually a semipermeable membrane; it

is commonly represented by the product of the total osmotic pressure of the solution and the ratio (corrected for activities) of the number of dissolved particles that do not permeate the bounding membrane to the total number of particles in the solution; equivalent in meaning to tonicity; commonly expressed in equivalent units of osmolality rather than pressure itself.

The part of the total osmotic pressure of a solution that governs the tendency of its solvent to pass across a boundary, usually a semipermeable membrane. the effective osmotic pressure due to the presence of the solute is given by the intercept atJn=0. Thus, the slope of the line represents both the osmotic conductancePos=Jn(os)/ and the hydraulic conductance Pf=dJn/dp. Linearity is therefore a demonstration that Pos=Pf

Case 4: Hypokalemic Paralysis 1. Propose a mechanism whereby a decrease in serum K concentration could lead to skeletal muscle weakness. - During attacks of muscle weakness, potassium moves from the blood into muscle cells. This makes the cell unable to contract properly. When the level of potassium in the blood falls these ion channels fail to regulate the flow of ions properly. The ratio of sodium and potassium inside and outside the cell become unbalanced. The muscle responds less when asked to move, which is felt as weakness. If the imbalance becomes pronounced the muscle quits responding at all, i.e. is paralysed. This person with HypoKPP is very sensitive to drops in serum potassium. Some patients with HypoKPP may become paralyzed while their potassium levels remain within "normal" limits. - Action potentials from the central nervous system cause end-plate potentials at the NMJ which causes sodium ions to enter and depolarise the muscle cells. This depolarisation propagates to the T-tubules where it triggers the entry of calcium ions via Cav1.1 as well as from the sarcoplasmic reticulum through the associated ryanodine receptor RyR1. This causes contraction (tensing) of the muscle. Depolarisation of the motor end plate causes potassium ions to leave the muscle cells, repolarising the muscle and closing the calcium channels. Calcium is pumped away from the contractile apparatus and the muscle relaxes. Mutations altering the usual structure and function of these channels therefore disrupts regulation of muscle contraction, leading to episodes of severe muscle weakness or paralysis. Mutations have been identified inarginine residues making up the voltage sensor of Nav1.4. This voltage sensor comprises the S4 alpha helix of each of the four transmembrane domains (I-IV) of the protein, and contains basic residues that only allow entry of the positive sodium ions at appropriate membrane voltages by blocking or opening the channel pore. In Cav1.1, mutations have also been found in domains II and IV. These mutations are loss-of-function, such that the channels cannot open normally. In patients with mutations in SCN4A or CACNA1S, therefore, the channel has a reduced excitability and signals from the central nervous system are unable to depolarise the muscle. As a result, the muscle cannot contract efficiently (paralysis). The condition is hypokalemic because a low extracellular potassium ion concentration will cause the muscle to repolarise to the resting potential more quickly, so even if calcium conductance does occur it cannot be sustained. It becomes more difficult to reach the calcium threshold at which the muscle can contract, and even if this is reached then the muscle is more likely to relax. Because of this, the severity would be reduced if potassium ion concentrations are kept high.

2. Why did Mannys weakness occur after exercise? Why did eating carbohydrates exacerbate (worsen) the weakness? - Other common triggers include unusual activity or exercise - usually the day before the attack. The mechanism involves the increase need for carbohydrate and excitation of muscles. Were in the pancreas responds to this rapid rise in blood sugar by producing a lot of insulin. Insulin drives potassium from the blood into the muscle cell, which triggers weakness. - Food triggers include sweet or starchy foods like candy, cakes, pie and other desserts, soft drinks which are sweetened with sugar, fruit juices, bread, cereal products, rice, potatoes, and pasta. Foods like these are digested very quickly and raise blood sugar rapidly. The pancreas responds to this rapid rise in blood sugar by producing a lot of insulin. Insulin drives potassium from the blood into the muscle cell, which triggers weakness. 3. How would K supplementation be expected t improve Mannys condition? - Potassium that is given during an attack may stop the attack. It is preferred that potassium be given by mouth, but if weakness is severe, potassium may need to be given through a vein (IV). These supplements are given to restore and maintain the normal level of potassium in the blood and cells thus maintaining equilibrium and relieving the patient of the signs. 4. Another inherited disorder, called primary hyperkalemic periodic paralysis, involves an initial period of spontaneous muscle contractions (spasms), followed by prolonged muscle weakness. Using your knowledge of the ionic basis for the skeletal muscle action potential, propose a mechanism whereby an increase in serum K concentration could lead to spontaneous contraction followed by prolonged weakness. - Action potentials from the central nervous system cause end-plate potentials at the NMJ which causes sodium ions to enter via Nav1.4 and depolarise the muscle cells. This depolarisation triggers the entry of calcium from the sarcoplasmic reticulum to cause contraction (tensing) of the muscle. To prevent the muscle from being perpetually contracted, the channel contains a fast inactivation gate which plugs the sodium pore very quickly after it opens. This prevents further entry of sodium. In time, potassium ions will leave the muscle cells, repolarising the cells and causing the pumping of calcium away from the contractile apparatus to relax the muscle. - Mutations altering the usual structure and function of this sodium channel therefore disrupt regulation of muscle contraction, leading to episodes of severe muscle weakness or paralysis. Mutations have been identified in residues between transmembrane domains III and IV which make up the fast inactivation gate of Nav1.4. Mutations have also been found on the cytoplasmic loops between the S4 and S5 helices of domains II, III and IV, which are the binding sites of the inactivation gate. - In patients with mutations in SCN4A, therefore, the channel is unable to inactivate, sodium conductance is sustained and the muscle remains permanently tense. Since the motor end plate is depolarised, further signals to contract have no effect (paralysis). The condition is hyperkalemic because a high extracellular potassium ion concentration will make it even more unfavourable for potassium to leave the cell in order to repolarise it to theresting potential, and this further prolongs the sodium conductance and keeps the muscle contracted. Hence, the severity would be reduced if extracellular (serum) potassium ion concentrations are kept low.