MBC 211

PHYSICAL AND ANALYTICAL

BIOCHEMISTRY

Dr. E. U. HENRY

DEPT. OF MEDICAL
BIOCHEMISTRY
 INTRODUCTION(1)
 A solution is a homogeneous mixture created by dissolving one or more
solutes in a solvent. The chemical present in a smaller amount, the
solute, is soluble in the solvent (the chemical present in a larger amount)

 standard (stock) solutions: Solutions with accurately known

concentrations. It is prepared by dissolving the desired amount of solute
into a volumetric flask of a specific volume

 Stock solutions are frequently diluted to solutions of lesser concentration

for experimental use in the laboratory

 Solution concentration = how much solute dissolved in solvent

INTRODUCTION(2)

 SOLUTE- substance which dissolves in a solution

 SOLVENT- substance which dissolves another to form a solution

 SATURATION- saturation is the point at which a solution of a substance can

dissolve no more of that substance and additional amounts of it will appear as
a precipitate.

 SUPER SATURATION-refers to a solution that contains more of the dissolved

material than could be dissolved in a solvent under the circumstances
Ways to Express Concentration
• Per cent concentration-This represents parts per 100. The most frequently used is
weight per volume (w/v) e.g. 9% saline (9 g/100 ml solution). For expressing
smaller concentration, mg (10–3g), μg (10−6 g), ng (10−9 g) and pg (10−12 g) are used.

• Parts per million (ppm)-This refers to the number of parts of a substance in one
million parts of the solution. Thus 10 ppm chlorine means 10 μg of chlorine in 1 g
of water.
• Molarity (M)-It is defined as the number of moles of solute per liter solution. NaCl
has a molecular weight of 58.5.
• To get one molar (1 M) or one mole solution of NaCl, one gram molecular weight
(58.5 g) of it should be dissolved in the solvent (H2O) to make to a final total
volume of 1 liter. For smaller concentrations, millimole and micromole are used.
• Molality-It represents the number of moles of solute per 1,000 g of solvent. One molal
solution can be prepared by dissolving 1 mole of solute in 1,000 g of solvent.

• Normality-Molarity is based on molecular weight while normality is based on equivalent

weight. One gram equivalent weight of an element or compound represents its capacity
to combine or replace 1 mole of hydrogen.

• In general, the gram equivalent weight of an element or a compound is equal to its

molecular weight divided by the total positive valence of the constituent ions.

• Thus, for NaOH and KOH, the molecular and equivalent weights are the same, while,
for H2SO4, equivalent weight is half of the molecular weight. The term milliequivalent
per liter (mEq/l) is used for smaller concentrations.
 Ways to Express Concentration
 Percent by mass (%) = (mass solute/ mass of solution) x 100

 Mole Fraction X

XA = nA/ ntotal = moles of some solute A/ total moles in solution. And if mixture of

multiple components A, B, C, … then 1= XA + XB + XC + …

 Molality (mol/kg) = m moles of solute/ kg of solvent = m

 Molarity (mol/L) = M moles of solute/ liter of solution = M

 Normality (equiv/ L) = N equivalents of solute/ liter of solution = N (Normality

used in acid base or redox reactions)
PERCENT SOLUTIONS

 W/W – 10% w/w solution contains 10g of solute in 100gm of solution

 W/V – 10% w/v solution contains 10g of solute in 100ml of solution

 V/V- 10% v/v solution contains 10ml of concentrate per 100ml of solution

 To make 3% w/v NaCl, dissolve 3.0g of NaCl in 100ml of water

 To make up 350ml of 12% w/v NaCl in water,

12g x (350/100)= 12 x 3.5g = 42g

So, place 42g in a beaker and add water up to 350ml

PERCENT SOLUTIONS CONTD.
 To make 70% v/v ethanol, mix 70ml of 100% concentrated ethanol in 30ml of
water

 To make up 912ml of 21.5% v/v TritonX-100,

21.5ml x(912ml/100ml)= 196.1ml,

Mix 196.1 mil of TritonX-100 with 715.9ml of water to get 21,5% v/v solution
Part per million

The number of parts by weights (or volume) of a solute per million parts by
weight( or volume) of the solution. Part per million or ppm is a unit of concentration
often used and denotes one part per 1,000,000 parts, one part in 106

1mg/L=1 ppm=1microgram/ml

Part per million is essential mass ratios (solute to solution) x 1,000,000

Ppm = mass or vol of solute/mass or vol of solution x 106

Part per billion = mass or volume of solute/mass or vol of solution x 109

molarity

Number of mole of solute in 1L of solution

To prepare 2M solution of NaCl(58)

1 M =58g, 2M=116g

116g of NaCl in 1L of solution

Prepare 100ml of 2M solution of NaCl

W1/V1= W2/V2 === 116g/1000ml=x/100ml ; W2 = 11.6g

11.6g of NaCl in 100ml of solution

molarity
To prepare a specific volume of a specific molar solution from a dry
reagent

A chemical has a formular weight (FW) of 180g/mol, and you did to make 25ml
of 0.15M solution. How many grams of chemical must be desolved in 25ml of
water to make this solution ?

Grams /desired vol = desired molarity(M) X FW

Grams=0.025LX 180g/mol X 0.15mol/L=0.675g

Therefore, to make 25ml of 0.15M solution ; you need to place 0.675g of solute
in a container and add water until the final vol is 25ml
Molality (m)
Molality is the number of moles of solute dissolved per 1kg (1000g) of solvent

Molality = moles of solute/ kg of solution

Molality = mass of solute/ Molar mass of solute x kg of solution

A solution is prepared by mixing 1.00g of ethanol (C2H50H) with 100g of water.

Calculate Molality of the solution.

Soln ; mass of ethanol= 1g; mol wt of ethanol= 46g/mol

Moles of ethanol = 1g/46g/mol= 2.17 x10-2 mol

Mass of water= 0.1kg; molality of solution = moles of ethanol/kg of water

=0.217m
NORMALITY
 NORMALITY: Number of gram equivalents of solute dissolved in IL of solution.

 A NORMAL SOLUTION is a solution that contains 1 gram equivalent weight in 1L of

solution

It is based on chemical reaction

 Normality represent the molar concentration only for the acid component(H +) or base
component( OH-)

 Therefore, Normality N =M x Number of H+ OR OH-

2M H2SO4 = 4N ; 2M HCl= 2N

2M H3PO4 = 6N ; 0.3M H3PO4 =0.3x3=0.9N H3PO4

Normality contd.

The unit of normal concentration is Eq/L.This unit is often denoted by capital letter N (normal). mEq/L=
0.001Eq/L

EQUIVALENT WEIGHT OF ACIDS AND BASES

Eq. wt=Molecular of acid or base/(no. of H+ in acids or OH- in base)

EXAMPLE

HCL the MW= 36.5; EW= 36.5

H2SO4 the MW=98 EW= 49

H3PO4 the MW=98 EW=32.7

NaOH the MW=40

Normality contd

EQUIVALENT WEIGHT FOR SALTS

Eq wt = Mwt of salt/ (No. of cation or anion x valency)

EXAMPLE

KCl the MW=74.5 Eqwt= 74.5/1= 74.5

CaCl2 the MW =110.98 EW=110.98/2=55.49

Na2CO3 the MW 105.98 EW 105.98/2=52.99

To prepare IN NaOH solution, dissolve 40g of NaOH in 1000mL (L) of water to prepare 1N NaOH solution

To prepare 1N Ca(OH)2 solution, dissolve 37g OF Ca(OH) 2 in 1000mL (1L) to prepare IN Ca(OH) 2 Solution
DILUTIONS OF SOLUTIONS(1)

SIMPLE DILUTION:

 A simple dilution is one in which a unit of a liquid material of interest is combined

with an appropriate volume of a solvent liquid to achieve the desired concentration

 The dilution factor is the total number of unit volumes in which your material will
be dissolved. The diluted material must then be thoroughly mixed to achieve the
true dilution

 For example, 1:5 dilution entails combining 1 unit of diluent with 4 units volume
of solvent medium( dilution factor=1+4=5)

 TO dilute a streptavidin solution 1:300, mix 1 unit of Streptavidin solution with 299
volume of solvent medium
DILUTIONS OF SOLUTIONS(2)

SERIAL DILUTION

A serial dilution is simply a series of simple dilutions which amplifies the dilution
factor quickly beginning with a small initial quantity of material.

The source of dilution material for each step comes from the diluted material of
the previous

In serial dilution, the total dilution factor at any point is the product of
individual dilution factors in each step up to it. Total dilution factor(DF) = DF1
X DF2 X DF3 etc
 DILUTIONS OF SOLUTIONS(3)

MIXING F IX VOLUMES OF SPECIFIC CONCENTRATIONS FROM LIQUIDE REAGENTS :

Use the formula C1V1=C2V

Where C1= initial concentration; V1 =initial Volume; C2=concentration of the new solution;
V2= volume of the new solution

EXAMPLE: suppose you have 3ml of stock solution of 100mg/ml ampicillin and you want to
make 200ul of solution having 25mg/ml.

Soln; C1=100mg/ml; V1= UNKNOWN; C2= 25mg/ml, V2= 0.2ml,

V1= C2V2/CI =0.05ml= 50uL

• So you will take 50uL of stock solution and dilute it with 150uL of solvent to have 200uL
of of 25mg/ml solution needed

CONVERSION OF CONCENTRATION UNITS

Conversion of wt% to molarity
Molarity(M) = (% x10)/Molecular wt

Conversion of Molarity to wt%

Wt.% =(M x Molecular wt)/10

Conversion of wt% to normality

Normality(N)=(% X 10)/ Eq wt
CONVERSION OF CONCENTRATION UNITS CONTD.

Conversion of Normality to Molarity

Normality(N)= Molarity(M) x n

Conversion of Molarity to Normality

Molarity (M) = Normality (N)/n (where n= no. of H+ or OH- or valency of salt)

Conversion of Molarity to g/l

g/l = M x Mol.wt
ACID-BASE BALANCE(1)
Dr. HENRY EKORIKO

DEPT OF MEDICAL BIOCHEMISTRY

UNIUYO.
INTRODUCTION (1)

 The acid-base balance or pH of the body fluids is maintained by a closely regulated

mechanism.

 This involves the body buffers, the respiratory system and the kidney.

 Hydrogen ion concentration decides the ionization of weak acids and thus affects their
physiological functions

 According to Bronsted, acids are substances that are capable of donating protons

 bases are those that accept protons.

 Acids are proton donors and bases are proton acceptors.

INTRODUCTION (2)
HA H + + A-

HCl H+ + CL-

H2CO3 H+ +HCO3-

BASE

H+ + NH3 NH4

H+ + HCO3- H2CO3
STRONG AND WEAK ACIDS
 Strong acids dissociate completely in solution e.g HCL

 In a solution of HCl, almost all the molecules dissociate and exist as H+ and Cl–
ions. Hence, the concentration of H+ is very high and it is a strong acid

 weak acids ionize incompletely, for example H2CO3

 weak acid (e.g. acetic acid), it will ionize only partially. So, the number of acid
molecules existing in the ionized state is much less, may be only 50%.

 the dissociation of an acid is a freely reversible reaction

 at equilibrium the ratio between dissociated and undissociated particle is a

constant.
HYDROGEN ION CONCENTRATION (pH)

 pH is defined as the negative logarithm of hydrogen ion concentration to the base 10. The
hydrogen ion concentration [H+] is expressed in moles/litre. pH = –log [H+] = log 1/[H+]

 The acidity of a solution is measured by noting the hydrogen ion concentration in the
solution

 pH scale ranges from 0 to 14. pH = 7 is neutral, while pH > 7 is alkaline and pH < 7 is
acidic

 Pure water or neutral aqueous solutions have [H+] = 1 × 10–7 mol/litre. Therefore their pH
according to definition can be calculated to be equal to 7.

 A rise or fall in pH by 1 signifies a tenfold fall or rise in the H+ conc. respectively. pOH is
the negative logarithm or [OH–] to the base 10
 pH value is inversely proportional to the acidity. Lower the pH, higher the
acidity or hydrogen ion concentration while higher the pH, the acidity is lower

 At a pH of 1, the hydrogen ion concentration is 10 times that of a solution with a

pH 2 and 100 times that of a solution with a pH of 3 and so on. The pH 7
indicates the neutral pH, when the hydrogen ion concentration is 100
nanomoles/liter.

 Ionic product of water = [H+][ OH–] = 10–14 i.e ; pKw = pH+ pOH. pKw of water
at room temp is 14.
Henderson-Hasselbalch Equation
HA = H+ + A–

 Equilibrium constant K can be written as, k= [H+] + [A–]/ [HA]

 pH = pKa + log[A-]/[HA] ; this is Henderson-Hasselbalch Equation where Ka = acid

dissociation constance; [A-] = conjugate base, [HA] = acid

 pH = pKa + log [base]/acid When [base] = [acid]; then pH = pKa

 pKa of an acid group is that pH at which the protonated and unprotonated species are
present at equal concentrations. pKa is the pH at which the acid is half ionised; Salt : Acid=
1 : 1.

 Therefore, when the concentration of base and acid are the same, then pH is equal to pKa.
Applications of Henderson-Hasselbalch
Equation
 The Henderson-Hasselbalch’s equation, has great practical application in clinical
practice in assessing the acid-base status, and predicting the limits of the
compensation of body buffers.

 It can be used to determine pH of blood, if the concentration of salt, i.e.

bicarbonate and acid (carbonic acid) is known

 To determine the pH of a buffer solution if pKa of the buffer acid and the molar
ratio of salt to acid in the solution are known

 Example; if approx. concentration of bicarbonate in healthy individual is 0.025

M, approx. concentration of carbonic acid is 0.00125 M and pKa of carbonic acid
= 6.1. Calculate the pH of blood.
BIOLOGICAL IMPORTANCE OF pH
 Enzymes function at optimum pH. Various body fluids and cell organelles, etc. maintain a
specific pH in order to allow the activity of the enzymes located there

 Amino acids and proteins exist as Zwitterions at isoelectric pH. The magnitude of the
charge depends on the pH

 Specific tautomeric form of nucleic acid bases exist at pH 7.4. This helps in proper
hydrogen bonding between the complementary base pairs

 pH is responsible for Gibbs-Donnan membrane equilibrium

 pH and Keq : pH influences the Keq product yield and spontaneity of metabolic oxidation-
reduction and some nonenzymatic acid-base catalysis.
Donnan membrane equilibrium
• When membrane is freely permeable to ions (say Na+, Cl−) and if the
concentration of ions on both the sides is different, the ions freely diffuse to
attain equal concentration
• Gibbs-Donnan observed that the presence of a nondiffusible ion on one side
of the membrane alters the diffusion of diffusible ions.
• In the molecule sodium proteinate (Na+Pr−), the protein (Pr−) ion is
nondiffusable through the membrane. Let us consider two sides of a
compartment separated by a membrane. Initially, sodium proteinate is on side
I while sodium chloride is on side II
• Diffusible ions (Na+, Cl−) can freely pass through the membrane.
• On side I, Na+ ions will balance the incoming Cl− ions besides Pr− ions, while on side II Na+ ions have
to balance only Cl− ions.
• Therefore, the concentration of Na+ on side I is greater than on side II. However, from the
thermodynamical point of view, at equilibrium, the concentration of Na+ Cl− on both the sides should be
the same.

• Consequently, the concentration of Cl− ions should be greater on side II.

• Further, the total concentration of ions on side I is higher than on side II.
• The salient features of Donnan membrane equilibrium include;
1. The presence of a non-diffusible ion influences the concentration of diffusible ions across the
membrane.
2. The concentration of oppositely charged ions (Na+), is greater on the side of membrane containing non-
diffusible ions (Pr−).
3. The concentration of similarly charged ions (Cl−) is higher on the side of the membrane not containing
non-diffusible ions (Pr−).
4. The net concentration of total ions will be greater on the side of the membrane containing non-diffusible
 Applications of Donnan membrane
equilibrium
• 1. Difference in the ionic concentrations of biological fluids : The lymph and
interstitial fluids have lower concentration of inorganic cations (Na+, K+) and
higher concentration of anions (Cl−) compared to plasma. This is due to the
higher protein (Pr−) content in plasma.

2. Membrane hydrolysis : The relative strength of H+ and OH− ions and, therefore, the
acidic or alkaline nature on either side of a membrane, is influenced by the presence of
non-diffusible ions. This phenomenon is referred to as membrane hydrolysis. Donnan
membrane equilibrium explains the greater concentration of H+ ions in the gastric juice.

3. Lower pH in RBC : Hemoglobin of RBC is negatively charged and, this causes

accumulation of positively charged ions including H+. Therefore, the pH of RBC is
slightly lower (7.25) than that of plasma (7.4).
• 4. Osmotic imbalance : Donnan membrane equilibrium—which results in the
differential distribution of ions in different compartments of the body—partly
explains the osmotic pressure differences.

5. Dialysis in renal failure : Donnan membrane equilibrium is the basic principle

involved in the artificial means of purifying blood by dialysis in the patients of
renal failure.
 BUFFERS

 Buffers are solutions which can resist changes in pH when acid or alkali is added

 Buffers consist of mixtures of weak acids and their corresponding salts, alternatively, weak bases and their
salts. The former type is the more important and common in human body

 The process by which the added H+ or OH– are removed is called as buffering action.

 The extent of resistance of change in pH by buffer is called its buffering capacity

 the buffer capacity is determined by the actual concentrations of salt and acid present, as well as by their
ratio

 The pH of a buffer solution is determined by two factors:

 The value of pK: The lower the value of pK, the lower is the pH of the solution.
 A few examples are : H2CO3/NaHCO3 (Bicarbonate buffer) (carbonic acid and

sodium bicarbonate); CH3COOH/CH3COONa (Acetate buffer) (acetic acid and

sodium acetate); Na2HPO4/NaH2PO4 (Phosphate buffer)

MECHANISM OF ACTION OF A BUFFER
 MECHANISM OF ACTION OF A BUFFER
(2)
The sketchy diagram above shows the weak acid HA and its completely ionised salt B+A–.

 Added H+ ions, in the form of strong acid, combine with anions A– to form the weakly dissociable HA, so
that pH does not become more acidic as it would be in the absence of the buffer. The capacity to
combine with added acid remains so long as there is a supply of the buffer salt in the medium

 Added OH– ions, in the form of a strong base, combine with H+ ions derived from the acid HA and form
the weakly dissociable H2O molecules and pH does not become as alkaline as would happen in absence
of the buffer.

 The buffer capacity is determined by the absolute concentration of the salt and acid. But the pH of the
buffer is dependent on the relative proportion of the salt and acid
MECHANISMS OF REGULATION OF pH

 “Front-line” defence: They are mainly:

 Buffer systems in the blood

 Respiratory mechanisms: Regulation of excretion of CO2 and hence, regulation

of H2CO3 concentration in EC fluid

 “Second-line” defence: This is achieved by kidneys(Renal mechanisms).

 Dilution factor: The acids introduced into and formed in the body are distributed
throughout the ECF volume
BUFFER SYSYTEMS OF THE BODY

Extracellular Intracellular Erythrocyte

fluid fluid fluid
1 NaHCO3/H2CO3 K2 HPO4/KH2 PO4 K+Hb/H+Hb
(phosphate) (hemoglobin)
(bicarbonate)
2 Na2HPO4/NaH2PO4 K+ Protein/H+Protein K2HPO4/KH2 PO4
(phosphate) (protein buffer) (phosphate)
3 Na+ Albumin/H+ Albumin KHCO3/H2CO3 KHCO3/H2CO3
Bicarbonate Buffer System
 The most important buffer system in the plasma is the bicarbonate-carbonic
acid system (NaHCO3/H2CO3). It accounts for 65% of buffering capacity in
plasma and 40% of buffering action in the whole body.

 Normal ratio in blood = NaHCO3/H2CO3 = 1/20, pH 7.4

 It accounts for 65% of buffering capacity in plasma and 40% of buffering action
in the whole body. The normal bicarbonate level of plasma is 24mmol/L.

 They are the chief buffers of blood and constitute the so called alkali reserve.
Neutralisation of strong and non-volatile acids entering the ECF is achieved by
the bicarbonate buffers.
 Alkali reserve : It is represented by the
The bicarbonate carbonic acid NaHCO3 concentration in the blood that has
buffer system is the most not yet combined with strong and non-
important for the following volatile acid.
reasons:
 Presence of bicarbonate in
relatively high concentrations.
 The components are under
physiological control, CO2 by
lungs and bicarbonate by
kidneys
 Produces H2CO3, which is a
weak acid and volatile and CO2
is exhaled out
 Phosphate Buffer System
 It is mainly an intracellular buffer. Its concentration in plasma is very low. The pKa value is 6.8. In
the body, Na2HPO4/NaH2PO4 is an effective buffer system, because its pKa value is nearest to
physiological pH.

 Na2HPO4 / NaH2PO4 = [Alk PO4]/[Acid PO4]) Normal ratio in plasma is 4:1. This ratio is kept
constant with the help of the kidneys. Thus, phosphate buffer system is directly linked up with the
kidneys. Below is the illustration of the action of phosphate buffer when an acid(HCl) and when
the base(NaOH) enter the bood
Protein Buffer System

 Buffering capacity of protein depends on the pKa value of ionizable side

chains. The most effective group is histidine imidazole group with a pKa
value of 6.1

 In acidic medium: protein acts as a base, NH2 group takes up H+ ions from
the medium forming NH+3, Proteins become +vely charged

 In alkaline medium: Proteins act as an acid. Acidic COOH gr dissociates and

gives H+, forming COO–. H+ combines with OH– to produce a molecule of
water, proteins become –vely charged.
Hemoglobin as a Buffering Agent

 Like other proteins, the buffering action of Hb depends on the following : acidic-

COOH gr, basic-NH2 gr, Guanidino group and most important is imidazole group,
which varies with the pH of the medium.

 the buffering capacity of Hb is due to the presence of “Imidazole” nitrogen

group which remains dissociated in acidic medium and conjugate base forms
• Hemoglobin as a buffer
• Hemoglobin of erythrocytes is also important in the respiratory
regulation of pH.
• At the tissue level, hemoglobin binds to H+ ions and helps to
transport CO2 as HCO3- with a minimum change in pH (referred to as
isohydric transport).
• In the lungs, as hemoglobin combines with O2, H+ ions are removed
which combine with HCO3- to form H2CO3. The latter dissociates to
release CO2 to be exhaled
• Generation of HCO3- by RBC
• Due to lack of aerobic metabolic pathways, RBC produce very little CO 2. The
• plasma CO2 diffuses into the RBC along the concentration gradient where it
combines with water to form H2CO3. This reaction is catalysed by carbonic
anhydrase (also called carbonate dehydratase). In the RBC, H 2CO3
dissociates to produce H+ and HCO3- . The H+ ions are trapped and buffered by
hemoglobin.
• As the concentration of HCO3- increases in the RBC, it diffuses into plasma
along with the concentration gradient, in exchange for Cl − ions, to maintain
electrical neutrality. This phenomenon, referred to as chloride shift, helps to
generate HCO3-
Generation of bicarbonate by the erythrocyte (CA–Carbonic
anhydrase; Hb–Hemoglobin).
ACID-BASE
BALANCE(2)
Dr. HENRY EKORIKO
DEPT. OF MEDICAL BIOCHEMISTRY
UNIUYO
DISTURBANCES IN ACID-BASE
BALANCE
The body cells can tolerate only a narrow pH change
Acidosis is the clinical state, where acids accumulate or
bases are lost.
A loss of acid or accumulation of base leads to alkalosis
Respiratory acidosis: Primary excess of carbonic acid.
Metabolic acidosis: Primary deficit of bicarbonate
Respiratory alkalosis: Primary deficit of carbonic acid.
Metabolic alkalosis: Primary excess of bicarbonate
Metabolic Acidosis(primary alkali
deficit)
It is caused when there is a reduction in the plasma HCO3– ↓(B. HCO3
↓) with either no or little change in the H2CO3 fraction.
pH is decreased, (primary bicarbonate deficit).
In uncompensated phase
• Disproportionate decrease in [HCO3-] ↓
• [H2CO3] ↓pCO2 ↓Ratio ↓
compensatory mechanism:
Primary: Respiratory-low pH stimulates respiratory centre producing
hyperventilation( Kausmaul breathing) and decrease in [H2CO3] ↓
Metabolic Acidosis
• Secondary: Renal
• H+ -Na exchange ↑ increased
• HCO3- reabsorption ↑ increased
• NH3 formation ↑ increased
Urinary findings
pH: Acidic
Increase excretion of NH4Cl and NaH2PO4
Increase in titratable acidity
CAUSES OF Metabolic Acidosis
• Abnormal increase in anions other than HCO3- (acid gain acidosis)
• Endogenous production of acid ions when excessive
Diabetic acidosis
Starvation
Lactic acidosis
Ingestion of acidifying salts
• Renal insufficiency: Retention of acids normally produced
• Ingestion of acidifying salts
• Abnormal loss of HCO3–, e.g. in severe diarrhoea, fistulas
 Respiratory acidosis (carbonic
acid excess).
• The underlying abnormality here is increase in H2CO3 ↑ in the blood,
which follows decreased elimination of CO2 (pCO2 ↑) in the
pulmonary alveoli
• [HCO3-]/[H2CO3] is low, resulting lowering in pH ↓
• In uncompensated phase;
Disproportionate increase in [H2CO3] ↑, Ratio ↓
increase in [HCO3-] ↑
pCO2 ↑, Total CO2↑ increased
Respiratory acidosis contd
• In fully compensated phase:
Total CO2 content is high but the increase in [H2CO3] is proportionate
and ratio 20:1 and pH maintained.
Compensatory mechanism
Primary: Renal: Most important
Increase in H+: Na exchange, More HCO3- reabsorption, Increase NH3
formation
Secondary: Respiratory—partial as the pathogenesis involves
Lung disorders/or depression of respiratory centre
Urinary findings- same as metabolic acidosis
CAUSES OF RESPIRATORY ACIDOSIS
1.Conditions in which there is depression/or suppression of
respiration
 Damage to CNS
Brain damage (trauma, inflammation, compression),convulsions
Drug poisoning like morphine or barbiturates
Excessive anaesthesia
Bulbar polio
 Effects of pain like pleurisy
CAUSES OF RESPIRATORY ACIDOSIS
CONTD
2 Reduction of respiratory surface:
• Emphysema, pneumonia, Pulmonary fibrosis, pulmonary oedema,
etc.
3. Condition in which there is ‘obstruction’ to escape of CO2 from the
alveoli: Obstruction to respiratory tract, Rebreathing from a closed
space
4. Conditions in which pulmonary blood flow is insufficient, e.g. certain
congenital heart diseases
ALKALOSIS
• Metabolic Alkalosis (primary alkali excess).
• This condition results from an absolute or relative increase in [HCO3-].
The respiratory centre (RC) is inhibited by alkalosis causing shallow,
irregular breathing
In uncompensated phase: Disproportionate increase[HCO3-] ↑,
[H2CO3] ↑ or N, pCO2 ↑ or N, Total CO2 ↑, pH ↑
In fully compensated phase: Total CO2 is high ↑ but increase in
[HCO3-] and [H2CO3] are proportionate and ratio 20:1 and pH is
maintained
• Compensatory mechanisms
• Primary: Respiratory-Depression of RC and hypoventilation
leading to retention in CO2
• Secondary: Renal--H+ —Na exchange ↓,NH3 formation ↓,
bicarbonate (HCO–3) reabsorption ↓, K+ excretion ↑,Cl– retention
• Urinary findings--pH of urine: Alkaline, Decrease NH3 ↓, Decrease
titratable acidity ↓
Causes
Excessive loss of HCl: Protracted gastric lavage, Pyloric
obstruction, High intestinal obstruction
Alkali ingestion and alkali administration
Excessive loss of K+ leading to K+ deficiency
Respiratory alkalosis
• Primary H2CO3 deficit
• In uncompensated phase: Disproportionate decrease[H2CO3] ↓, [HCO3-] ↓
or N, pCO2 ↓ or N, Ratio ↑, Total CO2 ↓, pH ↑
• In fully compensated phase: Total CO2 content is low.↓decrease in [HCO3-]
and [H2CO3] are proportionate and ratio 20:1 and pH maintained
• Compensatory mechanisms
• Primary: Renal--Decreased H+ –Na+ exchange ↓, Decreased
excretion of acid ↓,Increased excretion of HCO3- ↑,Decreased
excretion of NH3 ↓,K+ excretion ↑,Cl– retention
• Secondary: Respiratory-High pH and low pCO2 produces hypoventilation and
increase in H2CO3
Causes
Stimulation of respiratory centre (RC)
• CNS disease: Meningitis, encephalitis
• Salicylate poisoning
• Hyperpyrexia
• Others e.g High altitude ascending, Injudicious use of respirator, Some
cases of hepatic coma
Acid-base disturbances
• pCO2 > 45 mm Hg = Respiratory acidosis
• pCO2 < 35 mm Hg = Respiratory alkalosis
• HCO3 > 33 mmol/L = Metabolic alkalosis
• HCO3 < 22 mmol/L = Metabolic acidosis
• H+ > 45 nmol/L = Acidosis
• H+ < 35 nmol/L = Alkalosis

• pH (normal 7.4); pH <7.35 is acidemia and >7.45 is alkalemia

Anion Gap
• The “anion gap” is a mathematical approximation of the
difference between the anions and cations routinely measured in serum. Anion gap is
defined as the difference between the total concentration of measured cations (Na+ and
K+) and that of measured anion (Cl− and HCO3- ).
The anion gap (A−) in fact represents the unmeasured anions in the plasma
which may be calculated as follows, by substituting the normal concentration of
electrolytes (mEq/l).
Routine electrolyte measurements include Na+, K+,
Cl– and HCO3–
The unmeasured anions constitute the anion gap. This is due to the
presence of protein anions, sulphate, phosphate and organic acids
The anion gap is calculated as the difference between (Na+ + K+) and (HCO3– + Cl–).
Normally this is about 12 mmol/L.
Assessment of Acid-Base
Parameters
• Arterial blood is used to measure the acid-base parameters.
• Arterial blood gas (ABG) analyzer is used in assessing acid- base
status and it measures pH, pCO2 and pO2 directly, by means of
electrodes
• Bicarbonate is estimated by titration to pH 7.4. From the values of
Na+, K+, Cl– and HCO3–, the anion gap is calculated.
• In the absence of a blood gas analyzer, venous blood may be collected
under paraffin (to eliminate contact with air).
• Arterial Oxygen Saturation (SaO2) is measured by pulse oximeter
 Normal serum electrolyte and
arterial blood gas values
• pH = 7.35 – 7.45
• Bicarbonate = 22–26 mmol/L
• Chloride = 96–106 mmol/L
• Potassium = 3.5–5 mmol/L
• Sodium = 136–145 mmol/L
• PO2 = 95 (85–100) mm Hg
• PCO2 = 40 (35–45) mm Hg
 ROLE OF RESPIRATION IN ACID-
BASE REGULATION
An increase in blood pCO2 and of only 1.5 mmHg (0.2 per cent
increase in CO2) results in 100 per cent increase in pulmonary
ventilation, which increases also with slight increases in H+ ion
concentration of the blood (acidosis). The excess CO2 is thereby
promptly removed from the ECF in the expired air
A decrease in blood pCO2↓ or H+ ion concentration(alkalosis), causes
depression of respiratory centre, with consequent slow and
hypoventilation resulting to retention of CO2 in the blood until the
normal pCO2 and pH are restored.
• This respiratory mechanism, therefore, tends to maintain the normal
B-H CO3/H2CO3 ratio in the EC fluids
 RENAL MECHANISMS FOR
REGULATION
OF ACID-BASE BALANCE(1)
Normal urine has a pH around 6; this pH is lower than that of extracellular fluid (pH
= 7.4). This is called acidification of urine.
non-volatile acids viz. Lactic acid, H2SO4, ketone bodies, etc are buffered with cations
(principally Na+) are removed by glomerular filtration. The pH of the urine may vary
from as low as 4.5 to as high as 9.8, depending on the amount of acid excreted.
 Na+ is recovered in the renal tubules by reabsorption in exchange of H+ ions which
are secreted. It is recovered as NaHCO3 (“alkali reserve”).
Kidneys use three mechanisms to achieve acid base balance viz;
A. Bicarbonate mechanism
B. Phosphate mechanism
C. Ammonia mechanism
 RENAL MECHANISMS FOR
REGULATION
OF ACID-BASE BALANCE(2)
Bicarbonate Mechanism
Excretion of H+; Generation of Bicarbonate
Occurs in the proximal convoluted tubules
The CO2 combines with water to form carbonic acid, with the help of carbonic
anhydrase
The H2CO3 then ionizes to H+ and bicarbonate.
The hydrogen ions are secreted into the tubular lumen; in exchange for Na+
reabsorbed. These Na+ ions along with HCO3- will be reabsorbed into the
blood.
Excretion of hydrogen ions in the proximal
tubules; CA =
Carbonic anhydrase
Reabsorption of bicarbonate from the
tubular fluid; CA =
Carbonic anhydrase
• Above mechanism provides:
For complete reabsorption of all of NaHCO3,
Reduction of H+ ion load of plasma with little change in pH of urine
Phosphate Mechanisms
The pH of the urine is determined by the ratio both disodium hydrogen
phosphate (Na2HPO4, alkaline PO4) and monosodium dihydrogen phosphate
(NaH2PO4)
 In plasma, concentration of Na2HPO4 exceeds that of NaH2PO4 and the ratio is
maintained to 4:1.
But in urine, the concentration of NaH2PO4 exceeds that of Na2HPO4 and the
ratio becomes 9:1.
Due to the Na+ to H+ exchange occurring at the renal tubular cell boarder, the
Na2HPO4 (basic phosphate) is converted to NaH2PO4 (acid phosphate) The acid
and basic phosphate pair is considered as the urinary buffer.
The maximum limit of acidification of urine is pH 4.5.
Phosphate mechanisms operates in “distal tubule” and can be inhibited by
Phosphate Mechanisms
C. Ammonia Mechanism
This predominantly occurs at the distal convoluted tubules
H+ is excreted and HCO3– is reabsorbed
The Glutaminase present in the tubular cells hydrolyze glutamine to
ammonia and glutamic acid.
The NH3 (ammonia) diffuses into the luminal fluid and combines with
H+ to form NH4+(ammonium ion).
The glutaminase activity is increased in acidosis.
 large quantity of H+ ions are excreted as NH4+ in acidosis
Ammonia Mechanism
Mixed acid-base disorders
• Sometimes, the patient may have two or more acid-base disturbances
occurring
• simultaneously. In such instances, both HCO3- and H2CO3 are altered.
In general, if the biochemical data (of blood gas analysis) cannot be
explained by a specific acid-base disorder, it is assumed that a mixed
disturbance is occurring.
• Many a times, compensatory mechanisms may lead to
mixed acid-base disorders.
• Acid-base disorders and plasma potassium
• Plasma potassium concentration (normal 3.5–5.0 mEq/l) is very important as it affects the
contractility of the heart. Hyperkalemia (high plasma K+) or hypokalemia (low plasma K+)
can be life-threatening
Potassium and diabetic ketoacidosis
• The hormone insulin increases K+ uptake by cells (particularly from skeletal muscle). The
patient of severe uncontrolled diabetes (i.e. with metabolic acidosis) is usually with
hypokalemia.
• When such a patient is given insulin, it stimulates K+entry into cells. The result is that
plasma K+ level is further depleted. Hypokalemia affects heart functioning, and is life
threatening.
• Therefore, in the treatment of diabetic ketoacidosis, potassium has to be given (unless the
patients have high plasma K+ concentration).
• Potassium and alkalosis
• Low plasma concentration of K+ (hypokalemia) leads to an increased
excretion of hydrogen ions, and thus may cause metabolic alkalosis.
Conversely,metabolic alkalosis is associated with increased renal
excretion of K+.