Permeability properties II:

Mammalian Erythrocytes

Diamos, Pineda, Rañola, Saballo and Tancio

Schematic Diagram

Prescribed NaCl Concentrations

0.15M

0.13M

0.12M

0.11M

0.10M

0.08M

0.06M

0.04M

Methodology

Acquire blood sample from a

volunteer

Prepare prescribed concentrations of

NaCl solutions

Turn on and warm up the

spectrophotometer

Suspend 0.10mL blood in 5mL 0.15M

NaCl

Suspend 0.10mL blood in 5mL distilled H2O.

Set wavelength to 620nm

Cover and invert to mix settled blood

with solution

Measure absorbance and transmittance

Repeat steps 6-8 for the other

concentrations

Results

Table 1. Salt concentration vs. absorbance and transmittance

Concentration of NaCl (M) Absorbance Transmittance (%)

0.04 -0.076 115.8

0.06 1.383 4.1

0.08 0.823 15.0

0.10 1.397 9.0

0.11 1.412 3.9

0.12 1.395 4.0

0.13 1.460 3.5

0.15 1.509 3.1

Figure 1. Salt concentration (M) as a function of absorbance (nm)

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

NaCl Concentration (M)

Ab

sorb

an

ce (

nm

)

Figure 2. Salt concentration as a function of transmittance

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160

20

40

60

80

100

120

140

NaCl Concentration (M)

Tra

nsm

itta

nce (

%)

Study Question #2: Explain the appearance of the curve on the graph showing absorbance (or transmittance) values versus NaCl concentration).

Why oh why?

RBC Ghost

Low NaCl concentration=hemolysis=less intact cells=less absorbance=higher transmittance

Study Questions1. Do all RBCs in any

one solution hemolyze at the same time? Explain.

No, they don’t, because at any one solution, the cells and the ions would not interact simultaneously, thus, hemolysis will not occur at the same time for all erythrocytes.

3. What is the importance of the absorbance values in relation to the permeability in the erythrocyte?

More permeable=greater movement of water and solutes into and out of RBC=more prone to lysing and crenation=less absorbance values

Less absorbance value, more permeable!

Study Questions

4. Predict what would happen to the absorbance values obtained if you used saline solutions at a concentration greater than 0.15M?

- Normal physiological saline concentration- O.15 M or 9% NaCl - Hypertonic - All erythrocytes present in the solution would shrink (crenation), releasing all contents from the cells less intact cells less absorbance values

Study Questions

Figure 3. Theoretical graph of absorbance at increasing NaCl concentrations

0.04 0.06 0.08 0.1 0.11 0.12 0.13 0.15

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

NaCl Concentration (M)

Ab

sorb

an

ce (

nm

)

Figure 4. Theoretical graph of absorbance at increasing NaCl concentrations

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.220

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

NaCl Concentration (M)

Ab

sorb

an

ce (

nm

)

Table 2. Salt concentration vs. absorbance and transmittance (Grp 1 -

female)

Concentration of NaCl (M) Absorbance Transmittance (%)

0.04 0.019 95.6

0.06 0.286 5.2

0.08 0.634 23.2

0.10 1.265 5.5

0.11 1.353 4.5

0.12 1.426 3.8

0.13 1.394 4.1

0.15 1.447 3.6

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

NaCl Concentration (M)

Ab

sorb

an

ce (

nm

)

Figure 5. Salt concentration (M) as a function of absorbance (nm)

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160

20

40

60

80

100

120

NaCl Concentration (M)

Tra

nsm

itta

nce (

%)

Figure 6. Salt concentration as a function of transmittance

Table 3. Salt concentration vs. absorbance and transmittance (Grp 3 –

female with fever)

Concentration of NaCl (M) Absorbance Transmittance (%)

0.04 0.044 90.4

0.06 1.430 3.7

0.08 1.224 6.0

0.10 1.459 3.5

0.11 1.435 3.7

0.12 1.682 2.1

0.13 1.491 3.2

0.15 1.537 2.9

Figure 7. Salt concentration (M) as a function of absorbance (nm)

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

NaCl Concentration (M)

Ab

so

rba

nce

(n

m)

Figure 8. Salt concentration as a function of transmittance

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.160

10

20

30

40

50

60

70

80

90

100

NaCl Concentration (M)

Tra

nsm

itta

nce

(%

)

5. What is the fragility test? How is this test clinically applied?

This is a test to determine if the RBCs are more likely to break down. It is done by subjecting the RBCs in decreasing strengths of hypotonic saline solutions. This is clinically applied in evaluating hemolytic anemia, especially hereditary spherocytosis, and in evaluating hemolytic states.

Study Questions

Blood is drawn from a vein In the laboratory, Red blood cells are tested with a solution that makes them swell, in order to determine how fragile they are.

Normal RBCs – initial hemolysis at 0.45 – 0.39% salt concentrations

- complete hemolysis at 0.33 – 0.30% salt concentration

Increased fragility -> hereditary spherocytosis Reduced fragility -> thalassaemia, sickle cell

anemia, obstructive jaundice

Typical Graphs for RBC Osmotic Fragility

An increase in osmotic fragility is caused by decreasing the surface area of the cell (such as in a spherocyte).

References

Franklin Institute. 2004. Red Blood Cells: Riding on the Road. The Franklin Institute: The Human Heart. Retrieved November 24, 2012 from http://www.fi.edu/learn/heart/blood/red.html.

Liang, B. 2010. Red Blood Cells. Wisc-Online. Retrieved November 24, 2012 from http://www.wisc-online.com/objects/ViewObject.aspx?ID=ap14604. Fall. 2009. Red Blood Cell Membrane Permeability. Home.Zhaw.Ch. Retrieved November 24, 2012 from https://home.zhaw.ch/~fusa/PSS_VLE/PSS_CASE_04/ACTIVITIES/INVESTIGATIONS/EXPERIMENTS/Exp_11/Files/Blood_Cells_Permeability.pdf

Von Blum, R. 2003. Experimental Studies of Permeability in Red Blood Cells. Ableweb.org. Retrieved November 24, 2012 from http://www.ableweb.org/volumes/vol-2/5-von_blum.pdf.