Effects of Solute Permeability on Hemolysis in the Red Blood Cells of the Domestic Sheep, Ovis aries. Lab section: 1B- Biology 3151

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1 Effects of Solute Permeability on Hemolysis in the Red Blood Cells of the Domestic Sheep, Ovis aries Lab section: 1B- Biology 3151 Date: 11/30/2012 o

2 Results: Q, y = -0.05x R 2 = (.) C o ~ ---5\ ~ - -~ ~~ ~ j o -100 o [NaCl] (mosm) 600! Figure 1: The mean cell response (measured in % transmittance, T) in relation to the extracellular concentrations of Sodium Chloride solution (in milli-osmolar) in the erythrocytes of Domestic sheep (Ovis ariesi. The error bars represent ±1 standard deviation. (R2 = , p- value <0.05),. // l,../ ~~O~; ge~G-41!.~ ~o _ i--,._--_._-----_._------_...._ &O~ ~ ~ ~~ ±o~ " " -. o! -100 o [Urea] (mosm) / V Figure 2: Mean Cell response (measured in % transmittance, T) in relation to extracellular concentrations of Urea (measured in milli-osmolar) in erythrocytes of domestic sheep (Ovis aries). The error bars represent ± standard deviation. (p-value > 0.05) 1

3 _.... i ~ ['-l '-" ['-l -Vi 25 "0..:: o Q,) e.... =10 ~Q,) :;s 5 o i ~-.-.~ Cold Room Warm ---- Temperature Treatments -.1 Figure 3: The average time to hemolysis (in seconds, s) of Domestic Sheep (Ovis aries) in relation to various temperatures of Ethylene Glycol (cold, room, and warm). The error bars represent ± 1 standard deviation. (p-value <0.05) Discussion There were great variances in solute permeability to the red blood cell membranes of Domestic Sheep (Ovis aries). The Different concentrations of sodium chloride and urea and the different temperatures of ethylene glycol caused the red blood cell to respond in a volume change due to the movement of water into or out of the cell. n order to measure the effects of the solute permeability to the membrane, the turbidity of cell suspensions were measured using a spectrophotometef/rlvlanalis, 1996)..As the cells shrink, they reflect greater amounts of light. " fi That light which :~ ~.eete'lf;;;:dsorbed by the cells does not pass through to the light sensor in the spectrophotometer; therefore, the percent transmittance (%T) decreases as the turbidity of the solution increases. (Manalis, 1996) Consequently the percent of transmittance increases as the 2

4 cell volume increases due to the movement of water into the cell. f the water from the extracellular solution continues to move into the cell it will eventually cause the cell to burst in an event called hemolysis. Hemolysis of the cell will greatly increase the percent of transmittance. When the concentrated sodium chloride solution is hypertonic to the red blood cell, water moves by osmosis to the area of higher solute concentration (Sherwood et. al, 2005) from the cell, through the aquaporins, to the outside of the cell and causes the cell to crenate. Crenation of the erythrocytes is the cause of turbidity in the solution and as a result the percent transmittance decreases. When the red blood cells were exposed to higher concentrations of sodium chloride solution (measured in milli-osmolar) hemolysis occurred faster. Hemolysis occurs slower as the concentrations of sodium chloride solutions are decreased.this effect is due to the impermeability of sodium chloride and the high permeability of water to the cell membrane. The membrane permeability of sodium is mper second, and the membrane permeability for water is 30 11mper second. (nouye, 2012). When the cells were exposed to a hyposmotic solution of sodium chloride, the water moved down its concentration gradient into the cell. When the cell is in a hypotonic solution the cell will swell and eventually hemolyze. (nouye, 2012) This causes the transmittance percentage to be higher because there is no longer a cell membrane present to block the light sensor in the spectrophotometer. n the second experiment we exposed the red blood cells to various concentrations of urea. Urea is highly permeable across most plasma membranes due to the presence of numerous urea transporters (integral proteins in the plasma membrane). (nouye 2012) Based on our data, 3

5 which is expressed by Figure 2, we see that the transmittance percentage remains at around 99% regardless of the concentration of Urea. As said by Goodman (2002), red blood cells lyse very rapidly (too fast to measure the time) when placed in an isosmotic solution of urea. Both compounds enter the cell rapidly causing the cell to swell and hemolyze instantly. n the third experiment we were able to conclude that there is a relationship between temperature and the time for the cells to reach hemolysis, see figure 3. We must first acknowledge that there are no ethylene glycol membrane transporters and that ethylene glycol is moderately lipid soluble. (nouye 2012) We must also keep in mind that membrane fluidity can change according to temperature. According to Gershfeld and Muramaya (1988) studies indicate that a transformation of the membrane bilayer occurs when the normal ambient temperature of the cell is exceeded. n the warm ethylene glycol treatment the cell membrane increases in fluidity and allowes for the ethylene glycol to move more easily into the cell which causes the time to hemolysis to be much faster than in room temperature. When the red blood cells were placed in the cold treatment, the time to hemolysis was slowed down significantly due to the structure of the bilayer which changes from a liquid to a more rigid, gel like (semi crystalline) state. (nouye 2012) Our results are in agreement with a similar experiment done by Gershfeld and Muramaya (1988) which concluded that temperature is a dependent factor in hemolysis 4

6 Literature Cited: Gershfeld N. L., & Murayama, M (1988) Thermal nstability of Red Blood Cell Membrane Bilayers Temperature Dependence of Hemolysis. Journal of Membrane Biology, 101(1), Goodman, B.E Transport of small molecules across cell membranes: urea transporters. Advan. Physiol. Educ. 26: water channels and nouye, C.Y Water and solute movement through red blood cell membranes. Biology 3151 Laboratory Manual. Manalis, R.S Water and solute movement through red blood cell membranes. ABLE 17: Sherwood, L., H. Klandorf, and P.B. Yancey Animal Physiology Organisms, 2ndedition. Thomson Brooks/Cole, California. From Genes to 5

7 BOL Fall 2012 SCORNG RUBRC - PHASE RBC OSMOSS PAPER Additional Points Earned TTLE PAGE (up to 2 pts. for Phase + Phase ) Original, appropriately descriptive title of the study including both common and scientific names of the study species with the title page formatted according to the standards set for Phase. ncludes the suggested revisions. [up to 2 pts.] RESULTS (up to 18 pts. for Phase + Phase ) Text of the Results Section Thorough verbal, factual description of the results provided per the standards set / for Phase. ncludes the appropriate revisions. [up to 8 pts.] e; Figures Graphic depiction of results provided per the standards set for Phase. ncludes -(- LF the appropriate revisions. [up to 10 pts.] uk! DSCUSSON (up to 12 pts. for Phase + Phase ) Discussion including accurate physiological explanation of results and integration of relevantbackground references provided per the standards set for Phase. ncludes the appropriate revisions. [up to 12 pts.] LTERATURE CTED (up to 3 pts. for Phase + Phase ) All references cited in the Discussion section listed in proper format. Does not include any references not cited in the Discussion section. [up to 3 pts.] -1- jt~ flt-; -s Writing Proficiency (up to 5 pts. for Phase + Phase ) Overall report demonstratesproper grammar, punctuation, spelling, conciseness, and clarity and incorporates the appropriate revisions. [up to 5 pts.] Level of mprovement/demonstration of Effort [5 pts.] +- J +3~ - ' Electronic Upload to Blackboard/Turnitin by 11:59 PM on Fri., 11/30/12 [2 pts.] :.// Past Deadline (if report not received by due date, -1 pt./day) - / TOTAL ADDTONAL PONTS FOR PHASE 'Ll SCORE FROM PHASE -: TOTAL SCORE (45 pts. possible for Phase + Phase ) (0 ',. /--r' Comments:

Lab 4: Osmosis and Diffusion

Page 4.1 Lab 4: Osmosis and Diffusion Cells need to obtain water and other particles from the fluids that surround them. Water and other particles also move out of cells. Osmosis (for water) and diffusion