Explain and describe the principles underlying acid-base chemistry

including the
Gibbs-Donnan effect and the physical chemical approach (Stewart
approach)

pH = - log10 aH+ (or: aH+ = 10

(-pH)

The advantages of pH compared to [H+] are:

It is the traditional symbol and remains in wide use

It is related to the activity of H+ (rather than concentration) or more
specifically the log of H+ activity and this is what physiological systems
seem to respond to.
It is what is measured by the pH electrode (ie activity of H+)
The alternative [H+] is not correct because the activity coefficient is
ignored
Free H+ (ie bare protons) are not the form really present in solution
anyway.

The disadvantages of pH are:

It is a contrived symbol which represents a double non-linear

transformation of [H+] (ie the log of a reciprocal)
It is difficult to learn and understand
It disguises the magnitude of changes in [H+]

the mean intracellular pH of man is 6.8 at 37C which is indeed the pH of

neutrality (pN) at that temperature
Definition of neutrality is the pH when [H+] = [OH-]. At a temperature of 25C,
this condition does indeed occur in pure water when pH is 7.0. This condition of
[H+] = [OH-] occurs when pH = 0.5 x pKw. This pH (the pN) is dependent only
on the ion product of water (pKw')- and this term is very temperature dependent.
So the required condition of [H+] = [OH-] occurs at different pH values at
different temperatures. Effectively, this means that the dissociation of water is
temperature dependent.
Two reasons why pH is so important for metabolism

Effect on small molecules: Intracellular trapping of intermediary

metabolites (ie the Davis hypothesis)
Effect on large molecules (proteins): Maintaining optimal protein function
(enzymes) both intracellularly and extracellularly.

Acids:
Types of acids:

Volatile respiratory acids

Fixed metabolic acids

Respiratory Acid

The acid is more correctly carbonic acid (H2CO3) but the term 'respiratory
acid' is usually used to mean carbon dioxide.
CO2 can be thought of as representing a potential to create an equivalent
amount of carbonic acid
CO2 is the end-product of complete oxidation of carbohydrates and fatty
acids.
It is called a volatile acid meaning in this context it can be excreted via the
lungs
Basal CO2 production is typically quoted at 12,000 to 13,000 mmols/day.
Basal Carbon Dioxide Production
o Consider a resting adult with an oxygen consumption of 250
mls/min and a CO2 production of 200 mls/min (Respiratory quotient
0.8):
o Daily CO2 production = 0.2 x 60 x 24 litres/day divided by 22.4
litres/mole = 12,857 mmoles/day.

Metabolic Acids

Because they are not excreted by the lungs they are said to be fixed in
the body and hence the alternative term fixed acids.
These acids are usually referred to by their anion (eg lactate, phosphate,
sulphate, acetoacetate or b-hydroxybutyrate).
Net production of fixed acids is about 70 to 100 mmoles of H+ per day in
an adult.
This non-volatile acid load is excreted by the kidney.
Fixed acids are produced due to incomplete metabolism of carbohydrates
(eg lactate), fats (eg ketones) and protein (eg sulphate, phosphate).

Henderson-Hasselbalch Equation
The starting point is the Henderson Equation, which is based on application of
law of mass action on reaction of CO2 with water,
[H+] x [HCO3-] = K x [CO2] x [H2O]
Hasselbalch modified Henderson's elegant idea by regarding the water
concentration as constant and taking logarithms of the remaining components.
This resulted in the Henderson-Hasselbalch Equation:
-

pH = pK + log ( [HCO3 ] / [CO2] )

The relationship between HCO3 and CO2 in the system can be described by the
Kassirer-Bleich equation, derived from the Henderson-Hasselbalch equation:
H+ = 24 Pco2/HCO3

This equation illustrates that acid-base balance depends on the ratio of Pco2 and
HCO3 , not on the absolute value of either one alone.
Existing approaches to acid base balance:

Copenhagen approach

Boston approach

Stewart approach

Copenhagen approach:

Acid-base disorders are classified as being of respiratory origin (primary

change in pCO2) or of metabolic origin (primary change in fixed acids).

Respiratory disorders are quantified by the amount of change in pCO2 in

the arterial blood.

Metabolic disorders are quantified by the amount of excess fixed acids

(the metabolic acids) present in the blood. Often there may be more than
one type involved. It is difficult to predict which one it is and it is not
feasible to measure all of them.

The magnitude of the metabolic disorder (in the ECF) can be quantified
indirectly by the amount of change in the [HCO3]. Because,
o

Buffering of fixed acids in the extracellular fluid is predominantly by

bicarbonate.

One bicarbonate molecule will react with one H + molecule produced

by one molecule of fixed acid.

So [HCO3] will decrease by one molecule for every molecule of fixed

acid present.

The total amount of excess fixed acids should therefore be equal to

the amount by which the bicarbonate concentration drops from its
usual value.

Disadvantages of Copenhagen approach:

The implicit assumption so far that pCO2 and HCO3 are independent of
one another is not correct, because these 2 compounds are in chemical
equilibrium.

The buffering by the HCO3 in the blood sample is not representative of the
buffering by the ECF as a whole

The assumption that all buffering of metabolic acids is by HCO3 and not
other unmeasured ECF buffers is not totally correct.

Buffering by intracellular buffers is ignored

The system assesses compensation as another primary disorder

For the first mentioned disadvantage, several pCO2-independent indices have

been proposed as being suitable for this purpose:

Standard bicarbonate

Buffer Base

Base Excess

Standard bicarbonate:
Standard bicarbonate is the bicarbonate concentration of a sample when the
pCO2 has been adjusted (or standardised) to 40 mmHg at a temperature of
37C.
Buffer base is a measure of the concentration of all the buffers present in either
plasma or blood.
Base Excess (BE) is a measure of how far Buffer Base has changed from its
normal value & was introduced by Astrup and Siggaard-Andersen in 1958. BE in
whole blood is independent of pCO2 in the sample when measured in the blood
gas machine. BE is proposed as a measure of the magnitude of the metabolic
disorder because it assesses all the extracellular buffers (in the blood sample)
and is independent of pCO 2 (in vitro). Unfortunately, there are several problems
with the use of BE in this way. For example: (1) It is not independent of pCO2 in
vivo (This is because blood -which contains haemoglobin - is a better buffer than
the total ECF) (2) It does not distinguish compensation for a respiratory disorder
from the presence of a primary metabolic disorder

Boston Approach
This approach is based on actual experimental work in humans (eg whole body
titrations) rather than on blood samples in a machine.
The aim has been to determine the magnitude of the compensation that occurs
to graded degrees of acid-base disturbance.

These results are based on buffering and compensatory processes that affect the
whole body rather than just the blood. Additionally, appropriate compensation for
both acute and chronic disorders can be determined and corrected for when
interpreting the blood gas results.
Involves 6 rules of compensation (discussed later)

Within the traditional approach to acid-base analysis, the Boston 'bicarbonate

method' is preferable to the Copenhagen 'base excess method' because:

it is simpler to understand and to teach

it is based on whole body experiments rather than on test tube results on

a blood sample

it emphasises the need for clinical assessment and interpretation rather

than being driven by laboratory based derived quantities

Stewarts physicochemical approach

The traditional approach to acidbase balance using the Henderson
Hasselbalch equation fails to consider all the factors influencing hydrogen ion
concentration and is insufficient to explain complex metabolic abnormalities of
acidbase physiology and was challenged by Peter Stewart in the late 1970s.
Stewarts approach is based on 3 principles:
Electrochemical neutrality: In an aqueous solution, positively charged ions
must be balanced by an equal number of negatively charged ions. Net
charge within a compartment is zero.

Conservation of mass: For substances that may simultaneously exist in

different forms within a solution, such as dissociated and undissociated
forms, the total amount remains constant unless added to or removed
from a system.

Law of mass action: Equilibrium constraints on dissociation reactions must

be satisfied.

Essential features of stewart approach:

The concentration of H+ in any aqueous solution depends upon the

degree of dissociation of water into H+ and OH-.
Only three variables influence the dissociation of water, even in complex
solutions such as plasma. These independent variables are pCO2, total
concentration of weak acids [ATOT] and strong ion difference (SID).

Other factors affecting water dissociation are dependent variables and

include: [H+], [OH-], [HA], [A-], [HCO 3-] and [CO32-]

PCO2: Manipulation of pCO2 by adjusting alveolar ventilation causes rapid

[H+] changes in aqueous solutions due to the reversible dissociation of
carbonic acid. Small size and high solubility allow CO2 to pass easily
between compartments and alter [H+] in all body fluids.

Total weak acid concentration [ATOT]: Weak acids do not fully

dissociate in biological solutions. Proteins are the main weak acids in
plasma and their concentration is largely controlled by the liver, with
changes occurring over days. Phosphates also contribute to [ATOT]
becoming particularly significant during hypoalbuminaemia.

Strong ion difference: Strong ions completely dissociate in aqueous

solutions. The most important strong ions are sodium, potassium,
magnesium, calcium, chloride and lactate. Strong ion difference is the
amount by which strong cations exceed strong anions, measured in
milliequivalents per litre. So, for plasma,

In plasma, there is normally SID ~ 4048 meq/L. Since electrochemical

neutrality must be maintained, SID has a powerful influence on the
dissociation of water, and therefore [H+].

As SID increases (becomes more positive), there is less dissociation of

water and hydrogen ion concentration is reduced (pH increases), in order
to maintain electrical neutrality. Conversely, as SID falls (becomes less
positive) H+ concentration increases (pH falls).

Apparent SID (SIDa): The above calculated value is the apparent SID and is
based upon easily measured strong ions.

Effective SID (SIDe): Calculation of SIDe makes no assumptions about

which strong ions constitute the SID in plasma. It is based upon the
concentration of bicarbonate and the charge contributions from inorganic
phosphate and albumin.

Strong ion gap (SIG): Any difference between SIDa and SIDe is termed the
strong ion gap (SIG) and indicates the presence of other, unmeasured,
strong anions (such as keto-acids, sulphates and urate). Normally value
about zero.

Though similar to anion gap, unlike the anion gap, SIG is not affected by
variations in albumin or lactate concentration and therefore may provide a
more precise representation of the mechanism underlying a metabolic
acidosis.

Stewarts model clarifies the role of the kidneys, liver and gut in acidbase
control. Renal control of plasma electrolytes, particularly chloride, allows
manipulation of SID and therefore plasma pH. Liver and gut function influence
[ATOT]. The metabolic alkalosis resulting from chronic hypoalbuminaemia in
critically ill patients is explained by a low [ATOT].

Gibbs donnan equilibrium

Definition: It is a state of equilibrium across a semi-permeable membrane
involving uneven distribution of diffusible charged particles across it secondary
to already existing potential difference across membrane resulting from uneven
distribution of charged particles to which membrane is either semi-permeable or
impermeable.

It is not stable. As there is difference in the particle concentration across the

membrane, osmotic gradient develops and causes the diffusion of water and
hence upsets the equilibrium.
The Gibbs-Donnan factor for univalent cations is 0.95. For example [Na"] in ISF is
0.95 X [Na+]in plasma water. The Gibbs-Donnan factor for anions is 1.05
The situation for Ca" and Mg+ is more complicated because these ions are
significantly protein bound so only the concentration of the free ion should be
used. The Gibbs-Donnan factor for divalent cations is 0.90 (ie 0.95 X 0.95) & for
divalent anions is 1.10.
Extremely important, in particular for:
Stability of cell volume: It is vitally important in maintenance of cell
volume. The Donnan effect due to the non-diffusible intracellular proteins
and organic phosphates is balanced by the Donnan effect of the effectively
non-diffusible Na+ in the ISF. This dynamic balance is responsible for the
stability of cell volume.

Plasma oncotic pressure: The equilibrium also causes alterations in the

distribution of other ions across the capillary membrane. This results in a
small net increase in ions present in the plasma. This is very important
because it causes a significant increase in the effective value of the
plasma oncotic pressure in the blood.

The Gibbs-Donnan effect also makes a small contribution to the value of

the resting membrane potential.

Describe the chemistry of buffer mechanisms and to explain their relevant

roles in

the body
Definition: A buffer is a solution which has the ability to minimise changes in
[H+] when an acid or base is added to it. Buffering is a physicochemical process
that can occur in a test tube or in the body fluids.
Mechanism: A buffer typically consists of a solution which contains a weak acid
HA mixed with the salt of that acid and a strong base eg NaA. The principle is
that the salt provides a reservoir of A to
replenish [A] when A is removed by reaction with H +. For the majority of buffer
systems, buffering capacity is maximal at the pKa of the weak acid. The majority
of buffering occurs in the range pKa l.
Buffer power : The effectiveness of different buffers can be compared by
measuring how much base or acid needs to be added to cause a unit change in
pH. Buffer power is defined as d[B+] /dpH where d[B+] refers to the change in
concentration of base and dpH refers to change in pH.
Physicochemical buffering provides a powerful first defence against acid-base
perturbations because:
a HUGE buffering capacity, and
this system is essentially IMMEDIATE in effect.

The Major Body Buffer Systems

Site

Buffer
System

Comment

ISF

Bicarbonate

For metabolic acids

Phosphate

Not important because concentration too low

Protein

Not important because concentration too low

Bicarbonate

Important for metabolic acids

Haemoglobin

Important for carbon dioxide

Plasma protein

Minor buffer

Phosphate

Concentration too low

Proteins

Important buffer

Phosphates

Important buffer

Phosphate

Responsible for most of 'Titratable Acidity'

Blood

ICF

Urine

Bone

Ammonia

Important - formation of NH4+

Ca carbonate

In prolonged metabolic acidosis

Buffering of metabolic acidosis

ECF

43% (by bicarbonate & protein buffers)

ICF

57% (by protein phosphate and bicarbonate buffers) due to entry

of H+ by:

Na+-H+ exchange 36%

K+-H+ exchange 15%

Other 6%

Buffering of metabolic alkalosis: 32% of the buffering of a metabolic alkalosis occurs

intracellularly and Na+-H+ exchange is responsible for most of the transfer of H+.
Buffering of respiratory acidosis: 99% in ICF
Buffering of respiratory acidosis: 97% in ICF

The Bicarbonate Buffer System

The major buffer system in the ECF is the CO2-bicarbonate buffer system and is
responsible for about 80% of extracellular buffering.
Most important ECF buffer for metabolic acids only. Cannot buffer respiratory
acid-base disorders.
The components are easily measured and are related to each other by the
Henderson-Hasselbalch equation.
Henderson-Hasselbalch Equation
pH = pKa + log10 ( [HCO3] / 0.03 x pCO2)
The pKa value is dependent on the temperature, [H+] and the ionic
concentration of the solution. It has a value of 6.099 at a temperature of 37 C and
a plasma pH of 7.4.
The pK'a is derived from the Ka value of the following reaction:
CO2 + H2O <=> H2CO3 <=> H+ + HCO3(where CO2 refers to dissolved CO2)
The concentration of carbonic acid is very low compared to the other
components so the above equation is usually simplified to:
CO2 + H2O <=> H+ + HCO3-

By the Law of Mass Action:

Ka = [H+] . [HCO3-] / [CO2] . [H20]
(Ka is equilibrium dissociation constant for the dissociation of an acid (HA) to
produce H+ and the acid anion A-, in this case HCO3-).The concentration of H2O
is so large (55.5M) compared to the other components, the small loss of water
due to this reaction changes its concentration by only an extremely small
amount. This means that [H2O] is effectively constant. This allows further
simplification as the two constants (Ka and [H2O] ) can be combined into a new
constant Ka.
Ka = Ka x [H2O] = [H+] . [HCO3-] / [CO2]
Substituting:
K'a = 800 nmol/l (value for plasma at 37C)
[CO2] = 0.03 x pCO2 (by Henrys Law) [where 0.03 is the solubility coefficient]
[H+] = (800 x 0.03) x pCO2 / [HCO3-] = 24 x pCO2 / [HCO3-] nmol/l
Taking the logs (to base 10) of both sides yields the Henderson-Hasselbalch
equation:
pH = log10(800) - log (0.03 pCO2 / [HCO3-] )
pH = 6.1 + log ( [HCO3] / 0.03 pCO2 )
On chemical grounds, bicarbonate buffer with a pKa of 6.1 should not be a good
buffer at a pH of 7.4 if it were a simple buffer which is known to work best in the
range pKa 1. The system is more complex as it is open at both ends
(meaning both [HCO3] and pCO2 can be adjusted) and this greatly increases the
buffering effectiveness of this system. The excretion of CO2 via the lungs is
particularly important because of the rapidity of the response. The adjustment of
pCO2 by change in alveolar ventilation has been referred to as physiological
buffering. The bicarbonate buffer system is an effective buffer system despite
having a low pKa because the body also controls pCO2 and the changes in the
ratio [HCO3] / 0.03 pCO2 is minimized.
Other buffers in blood:
Protein and phosphate buffer systems only blood buffer systems capable
of buffering respiratory acid-base disturbances. Protein buffers in blood
include haemoglobin (150g/l) and plasma proteins (70g/l).
Phosphate buffer
The concentration of phosphate in the blood is so low that it is
quantitatively unimportant. Phosphates are important buffers
intracellularly and in urine where their concentration is higher.
Phosphoric acid is triprotic weak acid and has a pKa value for each of the three
dissociations:

The three pKa values are sufficiently different so that at any one pH only the
members of a single conjugate pair are present in significant concentrations.

At the prevailing pH values in most biological systems, monohydrogen phosphate

(HPO4-2) and dihydrogen phosphate (H2PO4-) are the two species present. The
pKa2 is 6.8 and this makes the closed phosphate buffer system a good buffer
intracellularly and in urine. The pH of glomerular ultrafiltrate is 7.4 and this
means that phosphate will initially be predominantly in the monohydrogen form
and so can combine with more H+ in the renal tubules. This makes the
phosphate buffer more effective in buffering against a drop in pH than a rise in
pH.
Haemoglobin buffer
Haemoglobin is an important blood buffer particularly for buffering CO2
Buffering is by the imidazole group of the 38 histidine residues which has a pKa
of about 6.8.
This is suitable for effective buffering at physiological pH.
Haemoglobin is quantitatively about 6 times more important than the plasma
proteins as it is present in about twice the concentration and contains about
three times the number of histidine residues per molecule. For example if blood
pH changed from 7.5 to 6.5, haemoglobin would buffer 27.5 mmol/l of H+ and
total plasma protein buffering would account for only 4.2 mmol/l of H+.
Deoxyhaemoglobin is a more effective buffer than oxyhaemoglobin and this
change in buffer capacity contributes about 30% of the Haldane effect. The
major factor accounting for the Haldane effect in CO2 transport is the much
greater ability of deoxyhaemoglobin to form carbamino compounds
Another factor which makes haemoglobin an important buffer is the phenomenon
of isohydric exchange. Oxygen unloading increases the amount of
deoxyhaemoglobin and this better buffer is produced at exactly the place where
additional H+ are being produced because of bicarbonate production for CO2
transport in the red cells.
Isohydric Principle
All buffer systems which participate in defence of acid-base changes are in
equilibrium with each other as there is after all only one value for [H+] at any
moment. This is known as the Isohydric Principle.
It means that an assessment of the concentrations of any one acid-base pair can
be utilised to provide a picture of overall acid-base balance in the body.
Conventionally, the components of the bicarbonate system (ie [HCO3] and pCO2)
alone are measured. They are accessible and easy to determine. Blood gas
machines measure pH and pCO2 directly and the [HCO3] is then calculated using
the Henderson-Hasselbalch equation.
Role of Bone Buffering

The carbonate and phosphate salts in bone act as a long term supply of
buffer especially during prolonged metabolic acidosis.

The hydroxyapatite crystals contain a large amount of carbonate (CO 3-2).

This anion can be substituted for both phosphate and hydroxyl in the
apatite crystals.

Bone is the major CO2 reservoir in the body and contains carbonate and
bicarbonate equivalent to 5 moles of CO2 out of a total body CO2 store of
6 moles.

The bicarbonate makes up a readily exchangeable pool. The carbonate is

present in the crystals and its release requires dissolution of the crystals.
This is a much slower process but the amounts of buffer involved are
much larger.

Mechanism: Two processes are involved:

o Ionic exchange
o Dissolution of bone crystal

In acute metabolic acidosis uptake of H+ by bone in exchange for Na+ and

K+ is involved in buffering as this can occur rapidly without any bone
breakdown.

In chronic metabolic acidosis, the major buffering mechanism by far is

release of calcium carbonate from bone. Occurs by two processes: direct
physicochemical breakdown of crystals in response to [H+] and
osteoclastic reabsorption of bone.