Practical Laboratories Physiology I - 2015

EXPLORATION OF OSMOTIC BALANCE

1. Definition

Isotonia (osmotic balance) represents maintenance of a constant number of particles solved

per volume unit in all body fluids compartments. The process of osmosis is the base of isotonia
maintenance within human body.

Osmosis is the net diffusion of water across a selectively permeable membrane from a region
of high water concentration to one that has lower water concentration.

2. Parameters of osmotic balance

Osmotic pressure represents the amount of mechanical pressure required to prevent osmosis.

Calculation: osmotic pressure of a solution is directly proportional to the concentration of

osmotically active particles in that solution, environmental temperature and inversely
proportional to the volume of solution, according to the following formula:

nR T
P= = C×R×T
V

where: P = osmotic pressure;

n = number of solved particles;
R = general constant of gases (0.082);
T = absolute temperature in Kelvin degrees (273 + t°C);
V = solution volume;
C = osmotic concentration of solution.

Normal value of osmotic pressure is 7.6 atm or 5667 mmHg.

Osmotic concentration (osmolarity) represents the number of particles solved in volume unit.
C = n/V

Determination of plasma osmolarity can be achieved by calculation, measurement of

cryoscopic point or determination of electric resistivity.

a) Calculation congruent with concentration of osmotic active substances

Osmotic particles of body fluids are represented by ions (93%) and organic un-dissociated
components (glucose, urea, proteins), which represent 7%.
Total osmotic concentration can be calculated as sum of osmotic concentration of plasma
electrolytes and organic un-dissociated molecules.
Osmolarity of both electrolytes and organic un-dissociated molecules can be determined
according to the following formula:
mEq/l mg%  10
Osm electrolytes = Osm org. subst=
el.charge MW

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In practical applications, osmotic concentration of plasma can be estimated knowing plasma

concentration of Na+, urea and glucose.

Glucose (mg%) Urea (mg%)

mOsm/l = Na+ (mEq/l) ×2 + +
18 6

The osmolarity given by electrolytes is called corrected osmotic concentration.

Note. In normal circumstances, glucose and urea have the following normal values in plasma:
glucose = 70 – 110 mg% and urea = 15 – 45 mg%. Taking into account a value of 90 mg% for
glucose and 30 mg% for urea, each of these substances will have a contribution of 5 mOsm/l to
the osmotic concentration.

b) Measurement of cryoscopic point as an indirect method of determining the plasma

osmotic concentration
Cryoscopy represents measurement of a solution freezing temperature (cryoscopic point). The
freezing point of a solution decreases directly proportional to the number of particles solved in
a volume unit; for each 5,4 mOsm/l, the cryoscopic point decreases with 0.01 o C. Thus, an
osmolar solution (1 Osm/l) will freeze at - 1.85o C.
Freezing point of a solution is measured using the Beckman thermometer, and osmotic
concentration is calculated using the following formula:

cryosc. point
Osmolarity (mOsm/l) = × 1000
 1.85

Cryoscopic point of plasma with normal osmolarity = - 0.56ºC ± 0.01ºC.

Using cryoscopy method, we can determine total osmotic concentration. If glucose and urea
osmolarity are subtracted from this value, the result will be the osmolarity given by
electrolytes, which is called corrected osmotic concentration.

c) Measurement of electric resistivity as an indirect method of determining the plasma

osmotic concentration
There is an inversely proportional relationship between plasma electric resistivity and the
amount of ions present in a solution.
Electric resistivity is measured using Kohlrausch bridge.
Normal value of plasma electric resistivity = 0.70 – 0.74 Ω×m
Using electric resistivity measurement, we can only estimate the osmotic concentration given
by plasma electrolytes, so that this formula must be completed by glycemia and plasma urea
level measurement.

Normal value for osmotic concentration of plasma = 285 - 295 mOsm/l, which corresponds to
an osmotic pressure of 7.6 atm or 5776 mm Hg (at 37 o C), to a plasma resistivity of 0.70 - 0.74
Ω×m, and a cryoscopic point of – 0.56o C.

Pathological changes
 Increases of plasma osmolarity = hyperosmotic state. Causes:
 in hypertonic water and electrolytes disorders (hypertonic dehydration or
hyperhydration)

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 hyperaldosteronism
 hyperglycemia (diabetes mellitus)
 uremia (increased plasma urea concentration, e.g. renal failure).
 Decreases of plasma osmolarity = hypoosmotic state. Causes:
– in hypotonic water and electrolytes disorders (hypotonic dehydration or
hyperhydration)
Importance of knowing the osmotic concentration
Depending on osmotic pressure value, solution can be classified as follows:
 hypotonic solution – osmotic pressure < 7.6 atm; osmotic concentration  285 mOsm/l;
 isotonic solutions – osmotic pressure  7.6 atm; osmotic concentration  285 - 295
mOsm/l;
 hypertonic solutions – osmotic pressure  7.6 atm; osmotic concentration  295 mOsm/l.
In medical practice, isotonic solutions are used (e.g.: NaCl 0.9 %, glucose 5%) as intravenous
infusion, without creating osmotic imbalance between intra- and extracellular compartments.

3. Red blood cells behavior in solutions of NaCl of different osmolarities

Principle: Erythrocytes’ behavior depends on direction and amount of water transport through
their semi-permeable membrane.
Materials:
 blood
 test tubes
 NaCl solutions: isotonic 9‰, hypotonic < 9‰ and hypertonic >9‰
 Pasteur pipettes
Technique: In 3 test tubes, NaCl solutions are added with the following concentrations:
 isotonic 9‰
 hypotonic 3‰
 hypertonic 15‰
In each test tube, a drop of venous blood is introduced using a dry Pasteur pipette. The blood is
carefully mixed with NaCl solution. The results are evaluated after 30 minutes. A drop taken
from each tube is placed between the two slides and examined by light microscopy.

Results: Red blood cells behave like osmometers (Fig. 1).

Fig. 1. Red blood cells behavior in solutions of NaCl of different osmolarities

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 In the first test tube, where the isotonic NaCl solution was added, erythrocytes sediment
on bottom of test tube, the supernatant is clear. Water passes in both ways through the
membrane and the red blood cells remain unmodified (maintain their shape and volume).
 In the second test tube, where the hypotonic NaCl solution was added, the supernatant is
colored in red, the erythrocytes sediment on the bottom, but they change their shape and
volume (swell and increased volume). In order to equalize osmotic pressure, water passes
through the erythrocytes’ membrane from low osmotic pressure to high osmotic pressure.
Thus, because the red blood cells have a higher osmotic pressure inside compared to the
outside solution, water enters into the erythrocytes, the membrane brakes and osmotic
hemolysis occurs.
 In the third test tube, where the hypertonic NaCl solution was added, the erythrocytes
sediment on the bottom, and the supernatant is uncolored, clear. In order to equalize the
osmotic pressures, the water passes through the cell membrane from inside the cells
towards the solution, and the cells become smaller in size and change shape (crenel).

4. Osmotic hemolysis
Principle: This method is based on red blood cells’ behavior in hypotonic NaCl solutions,
assessing the concentrations to which hemolysis begins and to which the hemolysis is
complete.
 Initial hemolysis → the first test tube presented sediment of erythrocytes and pink
supernatant. Concentration of the corresponding NaCl solution indicates minimum osmotic
resistance.
 Total hemolysis → the first test tube which has no sediment on the bottom, all erythrocytes
being hemolysed, and the supernatant is red. Concentration of the corresponding NaCl
solution indicates maximum osmotic resistance.
Materials:
 NaCl solutions 1g% ;
 distilled water;
 hemolysis test tubes;
 1ml pipettes;
 Pasteur pipettes;
 fresh blood mixed with anticoagulant

Dilution technique: In 21 tubes of hemolysis are achieved concentrations of NaCl which

decreasing for 0.7 to 0.3 g%, using a NaCl solution concentration of 1 g% (Table I). We take 1ml
1 g% NaCl solution in a graded pipette, from which 0.7 ml are introduced into the first tube and
the remaining 0.3 ml in tube 21; the second time the we take 1ml of 1 g% NaCl solution, 0.68 ml
are places in the second test tube, while the remaining 0.32 ml in tube 20, and so on. When the
distilled water is pipetted, the reverse process is used.

Table I. Dilution technique with graded pipette for determine osmotic resistance.
Test tube 1 2 3 4 5 6 7 8 9 10 11
1g% solution 0.70 0.68 0.66 0.64 0.62 0.60 0.58 0.56 0.54 0.52 0.50
NaCl (ml)
Distilled 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50
water (ml)
NaCl solution 0.70 0.68 0.66 0.64 0.62 0.60 0.58 0.56 0.54 0.52 0.50
Conc. (g %)

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Test tube 12 13 14 15 16 17 18 19 20 21
1g% solution 0.48 0.46 0.44 0.42 0,40 0.38 0.36 0.34 0.32 0.30
NaCl (ml)
Distilled water 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70
(ml)
NaCl solution 0.48 0.46 0.44 0.42 0.40 0.38 0.36 0.34 0.32 0.30
conc. (g %)

Venous blood is harvested from human subjects, and one drop of venous blood is added in
each test tube using a Pasteur pipette, and is gently mixed.

Results evaluation: Results are evaluated after 30 minutes, if the test tubes were centrifuged,
at room temperature.
 In the first tubes, erythrocytes are completely sedimented and the supernatant is clear
 The concentration of the first tube showing erythrocytes sediment and pink supernatant is
noted – initial hemolysis
 The first tube showing no erythrocytes sediment and red supernatant represents total
hemolysis
Evaluation of the results can be performed after 3 hours, when the sedimentation process is
very advanced, without centrifugation, or after 24 hours if the test tubes were stored at 4o C.

NORMAL VALUES
 minimum osmotic resistance: 0.46 – 0.42 g%;
 maximum osmotic resistance: 0.38 – 0.34 g%.

PATHOLOGIC VARIATIONS
 when hemolysis occurs at higher than normal concentrations of NaCl solution, osmotic
resistance is considered decreased. Causes:
- hemolytic anemia
- hereditary spherocytosis (congenital hemolytic jaundice)
- benzene intoxications
 when hemolysis occurs at lower than normal concentrations of NaCl solution, osmotic
resistance is considered increased. Causes:
- thalassemia
- after acute hemorrhages
- after splenectomy

5. CLINICAL CASES
Case 1
A person ingest a large amount of water. What modifications occur?

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• Consequences:
– EC volume 
– EC osmolarity 
– The water moves from EC to IC
– IC volume 
– osm IC  until osm EC = osm IC
• Other consequences:
– Signs of haemodillution in EC
•  plasma protein concentration
•  haematocrit
• Conclusion: hipotonic hyperhidration

CASE 2
A person is under high temperature, perspirate a lot and loses a high quantity of fluids. What
modifications occur?

• Consequences:
– EC volume 
– EC osmolarity 
– The water moves from IC to EC
– IC volume 
– osm IC  until osm EC = osm IC
• Other consequences:
– Signs of haemoconcentration in EC
•  haematocrit
•  plasma protein concentration
• Conclusion: hipertonic dehidration

CASE 3
O person has hypoaldosteronism loses a lot of Na. What modifications occur?

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• Consequences:
– EC osmolarity 
– The water moves from EC to IC
– IC volume 
– EC volume 
–  osm EC and IC until osm EC = osm IC
• Other consequences:
– Signs of haemoconcentration in EC
•  haematocrit
•  plasma protein concentration
• Conclusion: hypotonic dehidration

6. Interpretation bulletins

1. Calculate and interpret total plasma osmotic concentration knowing the following:
Na+ = 140 mEq/l, glycemia = 2.52 g/l, urea = 0.6 g/l.

2. Calculate osmotic concentration given by electrolytes (corrected osmotic concentration)

knowing the following: cryoscopic point = -0.62ºC, glycemia = 1.8 g/l, urea = 2.4 g/l.

3. Calculate and estimate corrected osmotic concentration knowing: cryoscopic point = -0.67ºC,
glycemia = 90 mg%, urea = 30 mg%.

4. Which is the corrected osmotic concentration of plasma if: cryoscopic point = - 0.62ºC,
glycemia = 1.8 g/l, urea = 2.4 g/l.

5. Which is the corrected osmotic concentration of plasma if: cryoscopic point = - 0.58ºC,
glycemia = 1.4 g/l, urea = 0.3 g/l.

6. Calculate total plasma osmotic concentration knowing the following: Na+ = 129 mEq/l,
glycemia = 1.1 g/l, urea = 0.3 g/l.

7. SCQs (one correct answer)

1. Osmotic hemolysis occurs in:

A. Isotonic solutions
B. Hypotonic solutions
C. Hypertonic solutions
D. All these statements are true

2. Red blood cells behavior in hypertonic solution is represented by:

A. Hemolysis
B. Change their shape (crenel)
C. Remain unmodified in shape and volume
D. Agglutination

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3. Red blood cells behavior in hypotonic solution is represented by:
A. Agglutination
B. Remain unmodified in shape and volume
C. Change their shape (crenel)
D. Hemolysis

4. Red blood cells behavior in isotonic solution is represented by:

A. Hemolysis
B. Change their shape (crenel)
C. Remain unmodified in shape and volume
D. Agglutination

5. Maximum osmotic resistance is indicated by:

A. The first test tube showing erythrocytes sediment and pink supernatant
B. The test tubes showing erythrocytes sediment and clear supernatant
C. The first test tube without erythrocytes sediment and red supernatant
D. The second test tube without erythrocyte sediment and red supernatant

6. Minimum osmotic resistance is indicated by:

A. The first test tube showing erythrocytes sediment and pink supernatant
B. The test tubes showing erythrocytes sediment and clear supernatant
C. The first test tube without erythrocytes sediment and red supernatant
D. The second test tube without erythrocyte sediment and red supernatant

7. Decreased osmotic resistance could be found in:

A. Thalassemia
B. Intoxication with benzene
C. After splenectomy
D. After acute hemorrhages

8. Increased osmotic resistance could be found in:

A. Hemolytic anemia
B. Hereditary spherocytosis
C. Thalassemia
D. Intoxication with benzene

9. The hypotonic solutions have:

A. Osmotic concentration between 285-295 mOsm/l
B. Osmotic concentration  285 mOsm/l
C. Osmotic pressure  7.6 atm
D. Osmotic concentration > 295 mOsm/l

10. The isotonic solutions have:

A. Osmotic pressure < 7.6 atm
B. Osmotic concentration between 285-295 mOsm/l
C. Osmotic concentration > 295 mOsm/l
D. No answer is correct

Answers: 1-B, 2-B, 3-D, 4-C, 5-C, 6-A, 7-B, 8-C, 9-B, 10-B