Diffusion and Osmosis: Laboratory Applications PDF

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PA PAGE 1 G E 1 https://edu.umch.de www.umfst.ro Diffusion and osmosis. 2024 Lecturer Dr. Florina Gliga Assist. Dr. Horațiu Sabău Assist. Dr. Andreea Tinca  PAGE 2 Introduction THE BODY AS AN ORGANIZED “SOLUTION” The total body water: 1. Extracellular fluid (ECF) enclosed within the integument = 1/3 of the total body water from the ECF: the cells take up O2 and nutrients into it ECF: the cells discharge metabolic waste products. In animals with a closed vascular system, the ECF is divided into two components: the interstitial fluid: is outside the vascular system, bathing the cells the circulating blood plasma: the plasma + the cellular elements of the blood-> the total blood volume. The special fluids considered together as transcellular fluids are another small category. 2. Intracellular fluid (ICF) = 2/3 of the total body water.  PAGE 3 Introduction THE BODY AS AN ORGANIZED “SOLUTION” Relationship Between the Volumes of the Various Body Fluid Compartments. The actual values shown are for an individual weighing 70 kg. Levy MN, Koeppen BM, Stanton BA. Berne & Levy's Principles of Physiology. 4th ed. St. Louis: Mosby; 2006  PAGE 4 Introduction THE BODY AS AN ORGANIZED “SOLUTION” In the average young adult male: 18% of the body weight is protein and related substances 7% is mineral 15% is fat 60% is water: ICF 40% and ECF 20% (25% in the vascular system and 75% outside the blood vassels- interstitial fluid) The total blood volume is about 8% of body weight. Flow between these compartments is tightly regulated!  PAGE 5 Introduction THE BODY AS AN ORGANIZED “SOLUTION” The water balance. Crash Course Anatomy and Physiology, Hall, Samuel; Stephens, Jonny. © 2019. The cell membrane PAGE 6 Cell membrane major components: 1. Lipids 2. Proteins. Physiology, Sixth Edition, Costanzo, Linda S., PhD, 2018 by Elsevier The cell membrane PAGE 7 1. The lipid component consists of: phospholipids cholesterol glycolipids The phospholipid molecules that make up the lipid bilayers are amphiphilic= they consist of: a polar, hydrophilic (water-loving) head a non-polar, hydrophobic (water-hating) tail Wheater's Functional Histology, Young, Barbara, BSc Med Sci (Hons), PhD, MB BChir, MRCP, FRCPA; O'Dowd, Geraldine, BSc (Hons), MBChB (Hons), FRCPath; Woodford, Phillip, MB BS, FRCPA. © 2014. The cell membrane PAGE 8 Role of the cell membrane components The lipid component of cell membranes is responsible for: 1.The high permeability to lipid-soluble substances such as carbon dioxide, oxygen, fatty acids, and steroid hormones. 2.The low permeability of cell membranes to water-soluble substances such as ions, glucose, and amino acids. The cell membrane PAGE 9 2. The protein component of the membrane consists of: Water channels Ion channels Transporters Enzymes Hormone receptors Cell-surface antigens Wheater's Functional Histology, Young, Barbara, BSc Med Sci (Hons), PhD, MB BChir, MRCP, FRCPA; O'Dowd, Geraldine, BSc (Hons), MBChB (Hons), FRCPath; Woodford, Phillip, MB BS, FRCPA. © 2014. Transport across cell membranes PAGE 10 Permeability properties of a typical lipid bilayer The lipid bilayer is a diffusion barrier. To penetrate a lipid bilayer, a dissolved substance has to pass through the array of hydrophilic head groups, then across the hydrophobic core, and out between the head groups on the opposite side. 1. Soluble substances such as inorganic ions, sugars, amino acids, and proteins cannot penetrate the bilayer because they do not dissolve in lipid. 2. Triglycerides and other water-insoluble lipids cannot pass because they form fat droplets that are repelled by the hydrophilic head groups. Only small molecules that are at least somewhat soluble in both lipid and water can pass freely. Principles of Medical Biochemistry Meisenberg, Gerhard, PhD; Simmons, William H., PhD. © 2017. Transport across cell membranes PAGE 11 Permeability properties of a typical lipid bilayer 3.Oxygen, carbon dioxide, and other gases diffuse freely across membranes. 4.Most nutrients, metabolic intermediates, and coenzymes are water soluble and cannot cross the lipid bilayer. Their transport requires specialized membrane proteins that act as solute carriers. 5.Inorganic ions cannot cross either; therefore, the electrical conductivity of lipid bilayers is very low. Membranes contain ion channels, formed by membrane proteins, which regulate ion permeabilities and thereby membrane potential and excitability. 6. Drugs : some sufficiently hydrophobic for passive diffusion across the lipid bilayer, but highly water-soluble drugs cannot enter cells.Principles of Medical Biochemistry, Meisenberg, Gerhard, PhD; Simmons, William H., PhD. © 2017. Diffusion PAGE 12 KEY WORDS Solvent: large amount of a substance which is the dissolving medium (in the body= water) Solute: small amount of a substance which is the dissolved substance and it dissolves in the solvent Solution: homogenous mixture of a solute in a solvent Concentration: the amount of solute dissolved in a specific amount of solution Concentration gradient: difference in the concentration of a solute on two sides of a permeable membrane. Equilibrium: exact balance between two opposing forces. Diffusion PAGE 13 Diffusion is the process by which a gas or a substance in a solution expands, because of the motion of its particles, to fill all the available volume. The particles of a substance (molecules or atoms) dissolved in a solvent are in continuous random movement. The movement of small molecules and ions is dictated by their electrochemical concentration gradient. They move: Encyclopedia of membrane scence and technology-volume3 Eric M. V. Hoek from high to low concentrations (= the net flux of and MaryTheresa M. Pendergast University of California, Los Angeles, CA 2016 solute particles) to neutralize a charge imbalance between two zones. Diffusion PAGE 14 A semipermeable membrane is a barrier that allows passage of certain compounds, but not others. Netter's Essential Physiology, Mulroney, Susan E., PhD; Myers, Adam K., PhD. Published January 1, 2016. Pages 12-22. © 2016. Diffusion PAGE 15 The rate of diffusion depends on: the diffusive gradient (concentration difference between two points) the electrical charge the solubility of the particle in the solvent (if it is not very soluble the concentration will be low) the size of the solute particle (small particles will diffuse faster than large particles) temperature (diffusion is faster at high temperatures than at low temperatures; body temperature is about 37°C in normal human subjects) the shape of the solute the weight of the solute Diffusion PAGE 16 Occurs in: 1. liquids 2. solids 3. gases. The membrane separating the two substances must be permeable for the solute! If the solutions either side of a membrane comprise only diffusible ions, diffusion occurs until equilibrium is reached and the ion distribution on each side is the same. Diffusion PAGE 17 Types of diffusion Substances can be transported: down an electrochemical gradient (downhill) - diffusion, either simple or facilitated against an electrochemical gradient (uphill) - by active transport, which may be primary or secondary. Passive and Active Transport 2011 Elsevier Guyton & Hall 13th Edition Copyright © 2015 by Elsevier, Inc Diffusion PAGE 18 Types of diffusion Most diffusion in living systems takes place in an environment in which water is the solvent. Diffusion is the key factor in providing exchange of gases, substrates, and waste products between capillaries and tissue cells. Medical Pharmacology and Therapeutics, Waller, Derek G., BSc (HONS), DM, MBBS (HONS), FRCP; Sampson, Anthony P., MA, PhD, FHEA, FBPhS. © 2018. Osmosis PAGE 19 The movement of water (solvent) across cell membranes occurs by the process of osmosis. Movement of water through a semipermeable membrane. Occurs between two compartments. Moves from low solute towards a high solute concentration. Does not require energy. Fluid, Electrolyte and Acid-Base Physiology 5th Edition, A Problem-Based Approach, Kamel Kamel Mitchell Halperin 2016, Elsevier Osmosis PAGE 20 The osmotic pressure= the pressure at which water is drawn from the weak solution into the more concentrated solution. Osmotic pressure is determined solely by the number of solute particles in the solution. It is not dependent on factors such as the size of the solute particles, their mass, or their chemical nature (e.g., valence). the higher the solute concentration, the higher the osmotic pressure Note: a low water concentration implies a high Netter's Essential Physiology, Mulroney, Susan E., PhD; Myers, Adam K., PhD. Published January 1, solute concentration. 2016. Pages 2-11. © 2016. Osmosis PAGE 21 Osmolarity= concentration of osmotically active particles, expressed as osmoles per liter or milliosmoles per liter. Osmolality= similar to osmolarity, except that it is the concentration of osmotically active particles, expressed as osmoles (or milliosmoles) per kilogram of water. The tonicity PAGE 22 The tonicity of a solution is related to its effect on the volume of a cell. Solutions that do not change the volume of a cell are said to be isotonic. A hypotonic solution causes a cell to swell, whereas a hypertonic solution causes a cell to shrink. Although it is related to osmolality, tonicity also takes into consideration the ability of the solute to cross the cell membrane. Consider two solutions: 1. a 300 mmol/L solution of sucrose 2. a 300 mmol/L solution of urea. Both solutions have an osmolality of 300 mOsm/kg H 2 O and therefore are said to be isosmotic (they have the same osmolality). The tonicity PAGE 31 PAGE 23 When red blood cells (which, for the purpose of this illustration, also have an intracellular fluid osmolality of 300 mOsm/kg H 2O) are placed in: the sucrose solution-> maintain their normal volume-> isotonic solution the permeability of the urea solution-> swell and eventually burst-> hypotonic solution plasma membrane to sucrose and urea The red blood cell membrane: contains uniporters for urea-> urea easily crosses the cell membrane (i.e., the membrane is permeable to urea), driven by the concentration gradient (extracellular [urea] > intracellular [urea]). does not contain sucrose transporters -> sucrose cannot enter the cell (the membrane is impermeable to sucrose). The tonicity PAGE 32 PAGE 24 To exert an osmotic pressure across a membrane, a solute must not cross the membrane! Because the red blood cell membrane is impermeable to sucrose, it exerts an osmotic pressure equal and opposite to the osmotic pressure generated by the contents of the red blood cell (in this case, 300 mOsm/kg H 2 O). In contrast, urea is readily able to cross the red blood cell membrane and it cannot exert an osmotic pressure to balance that generated by the intracellular solutes of the red blood cell. Consequently, sucrose is termed an effective osmole and urea is termed an ineffective osmole.  Practical approach- experiment 1 PAGE 25 Aim of the experiment: observing the diffusion process of a substance through a semipermeable membrane a semi-permeable membrane is going to allow the solvent and small molecules of solute to diffuse, while it remains impermeable to large molecules (such as proteins). Practical approach- experiment 1 PAGE 26 Principle of the experiment: iodine solution is going to change to a blue or a black color in the presence of starch. If starch is not present, then the color will remain yellow or orange. Practical approach- experiment 1 PAGE 27 Materials: 1. Starch 2. Iodine or lugol’s solution 3. Water 4. Beaker 5. Plastic bag 6. Pipette 7. Spoon Practical approach- experiment 1 PAGE 28 Procedure: 1. Fill a plastic bag with 100 ml of water 2. Add a teaspoon of starch 3. Tie the bag 4. Fill a beaker with water and then add ten drops of iodine 5. Place the bag in the cup 6. Make sure the starch mixture is submerged in the iodine water mixture Practical approach- experiment 1 PAGE 29 Results: Note the color of the solutions at the start of the experiment Note the color of the solutions after 15 minutes Practical approach-experiment 2 PAGE 30 Aim of the experiment: we propose to investigate the relationship between the solute concentration and the movement of water by osmosis through a semi-permeable membrane.  Practical approach-experiment 2 PAGE 31 Principle: There are several concepts we need to detaliate: 1. Isosmotic substances= two solutions with the same osmolarity. 2. Hyperosmotic substance= a substance with a higher osmolarity compared to another one less concentrated. 3. Hyposmotic substance= a substance with a lower osmolarity than another one which is less concentrated. These terms can only be used to compare solutions. If the two solutions are separated by a semi-permeable membrane, a net water flow will occur towards the hyperosmotic solution. Practical approach-experiment 2 PAGE 32 Materials: 1. Sucrose solutions, concentration: 0.2 m, 0.4 m, 0.6 m, 0.8 m, 1.0 m 2. Measuring cylinder 3. Distilled water 4. Plastic bags 5. Beakers 6. Pipette 7. Scale Practical approach-experiment 2 PAGE 33 Procedure: 1. Pour 25 ml of distilled water in a plastic bag and 25 ml of each solution in a separate bag 2. Tie the bags and leave sufficient space for the expansion 3. Rinse each bag gently with water to remove any sucrose spilled during filling 4. Record the initial mass of each bag 5. Fill six 250 ml beakers 2/3 full with distilled water 6. Immerse each bag in one of the beakers of distilled water 7. Submerge each bag 8. After 30 minutes, remove the bags from the water and measure their mass. Practical approach-experiment 2 PAGE 34 Bag Initial mass Final mass Change between masses Water Change between masses=Final mass-Initial 0.2 mass 0.4 Percent=Mass difference / initial mass 0.6 0.8 1.0 Calculate the percent of each change and put them on a graphic. References PAGE 35 1. Guyton and Hall, Textbook ot Medical Physiology thirteen edition, John E. Hall 2. Linda S. Constanzo, Physyology Sixth Edition 3. Encyclopedia of membrane scence and technology-volume3 Eric M. V. Hoek and MaryTheresa M. Pendergast University of California, Los Angeles, CA 2016 4. Wheater's Functional Histology, Young, Barbara, BSc Med Sci (Hons), PhD, MB BChir, MRCP, FRCPA; O'Dowd, Geraldine, BSc (Hons), MBChB (Hons), FRCPath; Woodford, Phillip, MB BS, FRCPA. © 2014. 5. Netter's Essential Physiology, Mulroney, Susan E., PhD; Myers, Adam K., PhD. © 2016. 6. Fluid, Electrolyte and Acid-Base Physiology 5th Edition, A Problem-Based Approach, Kamel Kamel Mitchell Halperin 2016, Elsevier 7. Medical Pharmacology and Therapeutics, Waller, Derek G., BSc (HONS), DM, MBBS (HONS), FRCP; Sampson, Anthony P., MA, PhD, FHEA, FBPhS. © 2018. 8. Cellular Physiology and Neurophysiology, Blaustein, Mordecai P., MD; Kao, Joseph P.Y., PhD; Matteson, Donald R., PhD. © 2020. 9. Principles of Medical Biochemistry, Meisenberg, Gerhard, PhD; Simmons, William H., PhD. 2017. 10. Crash Course Anatomy and Physiology, Hall, Samuel; Stephens, Jonny. © 2019. 11. Levy MN, Koeppen BM, Stanton BA. Berne & Levy's Principles of Physiology. 4th ed. St. Louis: Mosby; 2006