Transport rate of RBCs

Question 1

Preparation of Controls

Answer 1

• Positive control: Max lysis, 3ml distilled water + 100μl rat blood = BLANK
• Negative control: No lysis, 3ml physiological saline + 100μl rat blood
  (both to be placed in cuvettes)

Question 2

Experiment setup

Answer 2

1) Turn on photometer and set to 620nm, measuring absorption
2) Shake the rat blood samples to re-suspend the sedimented RBCs & make mixtures
3) Pipette prepared mixtures into cuvette
3) Use positive control of blood + water to Blank the photometer

Question 3

Preparation of other solutions

Answer 3

1) 3ml of chosen solution (urea, glucose, …)
2) 100μl rat blood
3) Mix in a test tube
4) Pour into cuvette and start timer immediately
5) Measure absorption every 10 seconds for 3 mins

Question 4

Acetazolamide

Answer 4

Inhibition of Carbonic anhydrase enzyme which converts
Co2 + H2O <–> H2Co3 <–> HCO3 + H

Question 5

Urea 300mM expected result

Answer 5

• Rapid lysis
• Due to Urea transporter (UT-B) being very fast
• Large gradient, urea enters cell, water follows = Lysis

Question 6

Glucose 300mM expected result

Answer 6

• Much slower lysis
• High gradient, but low permeability, slow facilitated diffusion (GLUT-1)

Question 7

NH4Cl 150mM expected result

Answer 7

• Slow or no lysis
• NH3 permeable but turns into NH4+ inside cell with H+
• H+ limited inside due to pH regulation so stops eventually
• Slowly gets trapped, lysis

Question 8

NH4Cl 150mM + NaHCO3 after 1 min expected result

Answer 8

• Faster lysis
• NaHCO3 supplies bicarbonate, carbonic anhydrase can convert into Co2, passes freely into RBC, and turns into H+ inside cell
• Replenishes H+ store in cell so more and more NH4+ forms, faster accumulation
• More NH3 diffuses in so more NH4+ forms inside the cell, faster lysis

Question 9

Difference in Results for Acetazolamide treated blood for the NH4Cl & NaCl

Answer 9

There is no conversion of substrates into H+ inside the cell, no replenished H+ either, so lysis does not occur