Control of Cell Volume

Dr. Simon Harrison

Professor of Physiology

Email: simonharrison@rossu.edu
Reading around this lecture: Any standard Physiology text book e.g. Chapter 1
from Ganong available on Access Medicine:

http://accessmedicine.mhmedical.com/content.aspx?bookid=393&sectioni
d=39736739&jumpsectionID=39736840
Total body water is around 60% of body weight in men and slightly less (50%)
in women. The volume of body water is determined by adipose tissue which
has a lower water content (about 10%) compared to e.g. muscle (about 75%).
New born infants have a larger water content, due to a large ECF volume and
comparatively small amounts of adipose. As we age, we tend to lose lean
body mass and gain adipose tissue, which is why TBW declines as grow older.

• You should remember that for a 70 kg man, TBW is around 60% of body
weight or around 42L.
• You should, given the body weight of a healthy newborn, man or woman
be able to calculate the approximate TBW.
Of the volume of total body water (TBW) of 42 Liters (60% of body weight), most is
contained within cells – and is called the intracellular fluid (ICF). The ICF is roughly
40% of body weight, or a little over half of the TBW. The ICF is separated from other
body fluids by the plasma membrane of cells.
The fluid "bathing” cells (actually a thin film of fluid coating cells) is the interstitial
fluid, which is contiguous with the lymphatic system, and together are equal to
approximately 20% of body weight. Interstitial fluid is separated from blood plasma
by the capillary walls. Together, the interstitial fluid, and plasma constitute the bulk
of the volume of the Extracellular Fluid (ECF) which is it self around 20 - 25% of body
weight.
The remaining component of the ECF is fluid known as transcellular fluid.
Transcellular fluid is actually separated from the ECF by an epithelial layer of cells in
addition to capillary wall. Transcellular fluid is found mostly in joints (synovial fluid),
the eye (aqueous and vitreous humors) and CNS (cerebrospinal fluid)
• You should be able to describe the general subdivisions of body fluids and their
approximate volumes.
• The “60 = 40 + 20” rule is also worth remembering.
SUPPLEMENTARY:
The ECF is essentially the “internal environment” this was first termed by the French
physiologist: Claude Bernard. Maintenance of the constancy of the volume and
composition of ECF (or homeostasis: described by the American Physiologist: Walter
B. Cannon) is ensured by the function of the kidneys and lungs. If homeostasis is
maintained, this ensures that cells can perform their functions to support the
physiological functions of the body.
The volume of any container of unknown volume can be determined by the indicator (or dye) dilution
method. A known quantity of a dye is introduced into the container and allowed to mix and come to
equilibrium. Then a sample is removed and the concentration measured (lots of different techniques for
doing this). The volume of the compartment can be calculated by dividing the amount of dye introduced
by the final concentration. In other words to what extent has the indicator been diluted (the greater the
volume of the fluid, the greater the dilution of the dye and vice versa).

In reality, all indicators behave in a less than ideal way. Particularly, many indicators are excreted or
metabolized to varying degrees. Therefore the equation is modified accordingly to:
V = M – (M excreted)/C.

• You should be able to remember and use the equation for calculating volume of a compartment
and use it to calculate any of the three variables if given the other two.
Any indicator must:
1. Be nontoxic.
2. Rapidly and evenly distribute throughout the relevant compartment.
3. Be restricted to the compartment.
4. Not be metabolized.
6. Be conveniently measured.
7. Not change body fluid distribution.

In reality, all indicators behave in a less than ideal way. Particularly, many indicators are
excreted or metabolized to varying degrees. Therefore the equation is modified accordingly
to:
V = M – (M excreted)/C.

If told that an amount of a dye is excreted or metabolized, you should be able to use the
modified equation.
This table shows examples of the most commonly used substances to measure body fluid
compartments.

To determine TBW the substance must distribute into all body fluid compartments.
Commonly used substances include the two isotopes of water: (tritiated water or deuterium
oxide), or the drug antipyrine (also known as Phenazone – a non steroidal anti-inflammatory)
.

Extracellular fluid volume (ECFV) is very difficult to determine accurately, because it is

composed of several different “sub-compartments” (plasma, interstitial and transcellular
fluids). Indicators must cross the capillary endothelium, but be excluded from cells i.e. unable
to cross plasma membranes. Radiosulfate, inulin (a polymer of fructose), mannitol (a sugar
alcohol) or thiosulfate, which is probably the most widely used marker for ECFV, giving a
volume of distribution of around 15 Liters.

An indicator of Plasma volume must remain confined to the vascular space, i.e. not cross the
capillary endothelium. Plasma proteins behave in this way, so radio-iodinated human serum
albumin is often used, or a dye that binds to plasma proteins such as Evan’s blue.

There are no suitable markers for transcellular fluid, so it is excluded from measurements of
ECFV. Likewise there are no markers that determine either just the interstitial fluid volume, or
the intracellular volume, and these are determined by calculation.

• You should know typical indicators for each body fluid compartment.
• You should be able to calculate interstitial and intracellular fluid volumes if given
volume of distribution of indicators of other appropriate compartments.
You will remember from the membrane transport lecture that the
composition of the fluids inside a cell (ICF) and the fluid outside (interstitial
fluid) are markedly different in composition. However, the osmotic pressure
of both compartments are the same. For this to happen water must distribute
equally between the intracellular and extracellular compartments. Movement
of water across plasma membranes is through pores: the “Aquaporins” (see
Membrane Transport Lecture), Aquaporin 1 is expressed ubiquitously in the
plasma membranes of most cells in the body. Therefore, any osmotic
imbalance between these compartments will result in water movement
across plasma membranes.
The exchange of fluid across different compartmental barriers can be caused
by two forces: hydrostatic pressure and osmotic pressure. Osmotic pressure is
caused by the presence of dissolved solutes in the fluid. In the situation of
two compartments each containing equal volumes of pure water, separated
by a membrane permeable to water, water molecules will diffuse randomly
across the membrane. The movement of water in either direction is called a
unidirectional flux. In this situation the two unidirectional fluxes will be
equal, so net flux (the difference between the two unidirectional fluxes) will
be zero.

Now, if a solute that is impermeant to the membrane is dissolved in the water

on one side of the membrane, two things happen. Firstly, the concentration
of solute increases markedly in the compartment to which the solute has
been added. The presence of these solute particles reduces the diffusion of
water from the compartment containing the solute. Secondly, the
concentration of water in the compartment to which the solute has been
added is reduced. The diffusion of water to the side containing the solute
continues and so there will now be net movement of water from the side
containing only water to the side containing the solute. The volume of fluid
containing the solute will increase, causing the generation of a hydrostatic
pressure h measured in mmHg. This pressure is equal to the osmotic pressure
generated by the solute. We can also determine the osmotic pressure by
applying a hydrostatic pressure that prevents the net influx of water.
• You should be able explain the basis of an osmotic pressure and, how it could be
measured.
Summary of osmosis and its function in the body.
The approximate osmotic pressure of a solution can be calculated by using the Van’t
Hoff Equation which is given by:
π = nRTC, where
 = osmotic pressure (mmHg)
n = # of particles (e.g. NaCl = 2; CaCl2 = 3; glucose = 1, etc.)
R = universal gas constant (0.0821 L.atm/mol.K)
T = absolute temperature (K)
C = concentration of the solute (mol/L)
• So for any given temperature, the osmotic pressure = n.C
• You should be able to use the Van’t Hoff equation to calculate the theoretical
osmotic pressure of a given solution.
Supplementary:
The Van’t Hoff equation predicts the osmotic pressure of very dilute solutions, in
which the solute particles have no interaction with one another. This is not actually
the case in physiological solutions, where the interaction between solute particles
(particularly ions) cause the solution to behave as though its osmotic pressure is less
than the theoretical value (n.C). A correction factor known as the osmotic coefficient
( ) which is specific for each solute must be used to more accurately predict the
osmotic pressure of a given solution.
The  varies with the specific solute and its concentration and the values range from
0 to 1. For example, for NaCl at 0.15 mol/L is 0.93.
In medicine, it is more convenient to express osmotic pressure in quantities of
concentration of osmotically active particles (osmolytes) rather than mmHg
or atmospheres* (as is calculated by the Van’t Hoff equation).

• The units are called osmoles.

• For physiological solutions, we usually refer to the osmolality or

osmolarity of a solution, and express it as mOsmol/Kg H2O or mOsmol/L
of solution respectively.

• Osmolality is usually measured in the clinical lab by the technique of

freezing point depression.
• For simplicity, units are often abbreviated to mOsm/Kg H2O.

In practice there is little real difference between these different units.

* Osmotic pressures are significant! For example, the osmotic pressure of

plasma is normally 280 mOsm/Kg H2O. If this is calculated as atmospheres – it
is seven times that of atmospheric pressure!
Obviously, two or more solutions may have the same or different
osmolalities.

• Two solutions of the same osmolality are called isosmotic.

• A solution with a lower osmolality than another is called hypoosmotic. The
solution with the higher osmolality is called hyperosmotic.
Now, we have said that typical plasma osmolality is around 280
mOsmol/kg.H2O. So the osmolality of a typical cell e.g. an erythrocyte should
be the same.

So what will happen if red blood cells are transferred from a solution that is
isosmolar to the red blood cell to one that is either hypoosmotic or
hyperosmotic?

If RBCs are placed into a hypoosmotic solution they will swell as water is
drawn into the cells by the higher concentration of osmolytes within the cells.
If placed into a hyperosmotic solution cells will shrink as water is drawn out of
the cells into the more osmotically concentrated solution outside.
From the experiment in the last slide, it might be expected that red blood
cells suspended in two different but isosmotic solutions should behave the
same. But they don’t! What is going on here?
The reason that the experiment with the red blood cells didn’t go as expected
is because in terms of osmotic pressure and cell volume, the permeability
properties of the membrane are important. This means that different solutes
will exert different “effective” osmotic pressure on a cell (next slide). Plasma
membranes are highly selective (see membrane transport lecture) and the
ease with which a solute can permeate (cross) the membrane is measured by
what are called “reflection coefficients”.

• The reflection coefficient (σ, sigma), ranges from 0 – 1.

• A σ of 1 indicates that the solute is not permeant (it is completely reflected

by the membrane) to the membrane while a σ of 0 indicates that the
solute is readily permeable to the membrane. Values of σ between 0 and 1
represent varying degrees of permeability.
• An ineffective osmolyte (e.g. urea) will equilibrate across the membrane
very rapidy. So in the experiment in the previous slide, urea will have
rapidly equilibrated across the surface membrane and therefore with
respect to the extracellular solution following the equilibration of urea,
that in itself will not cause the movement of water molecules into the cells.
So what does attract water into the cells? The osmolytes trapped within
the cells (i.e. the constituents of the intracellular fluid) will attract water
into the cells to the extent that the cells burst.

• The same solute may have different values of σ for plasma membranes of
 different cell type.

The higher the σ for a solute, the more effective an osmolyte it will be, because its
presence constitutes an osmotic driving force that will cause water movement.

• You should be able to explain the term reflection coefficient.

• You should be able to explain the relationship between reflection coefficient
and the effectiveness of a solute as an osmolyte.
So from the previous slide, you should be able to see that the “real world” osmotic
pressure that cells experience is not only that predicted by the composition of the
solution (from the Van’t Hoff equation), but also the permeability properties of the
plasma membrane itself (see urea experiment). This leads to the concept of a
solution’s tonicity (next slide).

So for example, the osmolality of plasma is determined by all of the major effective
osmolytes dissolved in it.

• You should be able to calculate the effective osmotic pressure of any solution if
given the concentration and a value of σ.
• You should be able to predict the direction of movement of water between two
compartments containing solutions of different effective osmotic pressure.

In the clinic, plasma osmolarity can be estimated from the plasma concentrations of
Na+, glucose and blood urea nitrogen (BUN). Blood urea nitrogen is the amount of
nitrogen derived from the metabolism of urea.

The clinical formula used is: plasma osmolarity mOsm/L = 2 X [Na+] + [glucose]/18 +
[BUN]/2.8

The Na+ concentration is multiplied by 2 because Na+ must be balanced by an equal

concentration of anions (Cl− and HCO3−). The glucose concentration in mg/dL is
converted to mOsm/L when it is divided by 18. The BUN in mg/dL is converted to
mOsm/L when it is divided by 2.8.
In terms of the effect of a given solution on cell volume, it is the
concentration of non-penetrating solutes(effective osmolytes) that
determines what will happen to cell volume.

Isotonic: A solution containing 280 mOsmol/L of non-penetrating solutes,

regardless of the concentration of membrane-penetrating solutes that may
be present.

Hypertonic: A solution containing greater than 280 mOsmol/L of non-

penetrating solutes, regardless of the concentration of membrane-
penetrating solutes that may be present.

Hypotonic: A solution containing less than 280 mOsmol/L of non-penetrating

solutes, regardless of the concentration of membrane-penetrating solutes
that may be present.
When cell volume increases because of extracellular hypotonicity, there is
rapid activation of transport mechanisms that tend to decrease cell volume.
Different cells use different mechanisms of Regulatory Volume Decrease
(RVD) to move solutes out of the cytoplasm, many mechanisms involve efflux
of K+ ions (channels and transporters) as well as organic solutes such as amino
acids. The net result is a decrease in intracellular solute content and a
consequent efflux of water, reducing cell volume towards its original value.

When cells shrink when in a hypertonic solution, this triggers Regulatory

Volume Increase (RVI). Many cells activate transporters responsible for Na+
influx so as to increase intracellular [Na+]*, and increase cell volume towards
its original value.

• You should be able to explain the basic general mechanisms of both RVD
and RVI

Supplementary:

* Mechanisms involving increasing intracellular [Na+] are only effective for

short periods, because the Na+/K+-ATPase (membrane transport lecture) will
be activated and reduce intracellular [Na+]! In cells that regularly encounter
extracellular hypertonicity (e.g. cells in the medulla of the kidney or some
neurons in the Central Nervous System), an additional mechanism has evolved, that
relies on increased synthesis of intracellular organic effective osmolytes such as
sorbitol or inositol.
Supplementary:

The patient described at the beginning of the lecture was showing the
symptoms of a complication of treatment for hyponatremia (plasma [Na+] less
than 135 mmol/L) known as osmotic demyelination syndrome (ODS).
Correction of hyponatremia by intravenous saline therapy must be carefully
controlled to prevent a too-rapid rise in ECF [Na+] that may result in transient
hypernatremia. This creates a hypertonic ECF that causes water to move from
central neurons by osmosis too quickly for normal cell-volume regulation to
take place. This causes demyelination of central neurons particularly within
the pontine region of the brain resulting in a variety of symptoms including
coma. These changes may prove irreversible.

More information about ODS is available at:

http://www.uptodate.com/contents/osmotic-demyelination-syndrome-and-
overly-rapid-correction-of-hyponatremia