COURSE UNIT 4: CLINICAL CHEMISTRY

UNIT 1: INTRODUCTION TO CLINICAL CHEMISTRY

1. TOPICS
2. Fundamentals of chemistry
 Atomic structure and atomic mass
 Molecules and molecular mass equivalent weight and valency
 Acids and bases
 PH
 Indicators
 Buffers
3. S.I units
4. Water purification
 Distillation
 De-ionization
 Reverse osmosis
5. Concentration of solution
Preparation of:
 Molar solution
 Normal solution
 Percentage solution
 Dilutions
6. Standards
 Primary
 secondary
 Characteristics of an ideal standard
7. Basic introduction to organic and inorganic compound
a. Organic
 Proteins
 Carbohydrates
 Fats
 Enzymes
b. Inorganic
Electrolytes

TOPICS
a. Colorimetry
a. Principles of absorption spectroscopy
b. Beer - lamberts law and formulae
c. Component of colorimeter
d. Components of spectrophotometer
a. Electromagnetic spectrum
a) Comparison using lovibond comparator

2. Use and care of cuvettes

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 a) Shape
b) Size
c) Types of glass, quartz, plastic
d) Use
e) Care
Flame photometry
a) Principle of flame emission spectroscopy
b) Diagram of basic components of a flame photometer

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INTRODUCTION TO CLINICAL CHEMISTRY

Definition
Chemistry is a branch of science which deals with the composition of all forms of matter and
change of matter from one form to another.

Biochemistry is a branch of chemistry which is concerned with various molecules that exist
in the living cells and their chemical reactions.

By definition, biochemistry is a chemistry of life i.e. a science concerned with the chemical
basis of life. Biochemistry describes and explains in molecular terms all the chemical
processes of a living cell.

The cell is a structural unit of a living system. This concept of a cell leads to a functional
definition of biochemistry as a science concerned with chemical constituents of a living cell
and reactions that take place in these living cells.
Importance of clinical chemistry in medicine
 To reveal the cause and mechanism of the disease
 To suggest treatment of the disease
 To help in diagnosis
 To help in research
 To help in monitoring of the treatment of the disease
 To help in surveillance of the disease
 T o check for the levels of drugs in the body fluid (distribution and metabolism of the
drug)
 Study purpose
 Preparation of reagents
 Reference
RELATIONSHIP BETWEEN BIOCHEMISTRY AND MEDICINE
Health is a state of complete physical, mental and social well being and not just the
absence of disease. It is a situation in which all the chemical constituents of the body are in
normal values and all of the many thousands of extra and intra cellular reactions occur in the
body at rates maximal to the physiological state.

Most chemical substances in the body are in equilibrium if their rates of production and
loss (by excretion or degradation) are equal. Diseases may change this equilibrium in
several ways e.g plasma proteins increase in chronic renal disease. Diseases are
manifestations of abnormalities of molecules and chemical reactions in the body

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Major causes of disease which influence various biochemical mechanisms in the body or
human cells are;
1. Physical agents: - accidents (mechanical trauma) or extreme temperature or
atmospheric pressure, radiation, electrical shock among others.
2. Chemical agents:- toxic compounds, therapeutic drugs etc.
3. Biochemical agents:- viruses, bacteria, fungi, and higher forms of parasites.
4. Oxygen:- loss of blood, poisoning of the oxidative enzymes.
5. Genetic disorder:- congenital, molecular.
6. Immunological reactions e.g autoimmune diseases.
7. Nutritional imbalances: - deficiencies or excesses.
8. Endocrine imbalances: - hormonal deficiencies or excesses.

Clinical chemistry is concerned with analysis/measurement of chemical constituents of

body fluids and their techniques in the laboratory. It basically consists of biochemistry and
physiology which is used and applied in clinical medicine.

In disease condition, inorganic or organic or both components are affected.

When changes occur in any of the components, the physician should reflect on the organ
affected e.g. change in glucose which is organic demands that attention be made to the
organ responsible for its metabolism-pancreases / liver , Increased values of urea reflect on
excretory function where the kidneys play a major role, gas changes reflect the excretory
function of the lungs. These are used for diagnosis and treatment.

Analytical methods employed in analysis of analysts

1. Qualitative method: This detects presence or absence of an analyte in a mixture e.g.
fouchet’s reaction detects presence or absence of bilirubin in urine, occult blood test by
Aminophenozone method Bence jones test by using conc . HCL . These qualitative
analytical methods are used for detecting substances in samples where their presence is
not expected at normal condition e.g. sugar in urine.

2. Semi-quantitative method: This gives a rough estimate of the amount of substance in

particular spaceman. The results are reported as: trace, +, ++, +++ e.g. use of uristix for
urine protein can be reported as protein nil, trace, +, ++, +++ etc

3. Quantitative methods: this determines the actual amount of the analyte / substance in
the sample being analyzed/ measured. Results are reported in mg/dl, g/l, mmol /l, u/l etc.
Here, the quantity of an analyte is measured after colour development or reaction rate (in
kinetic methods) e.g. using a spectrophotometer (for enzyme assays) or colorimeter or
flame photometer for electrolyte assay.

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NB Principle of analysis of analyte depends on chemical reactions involved. In clinical
chemistry, it is important to have knowledge in basic chemical reactions, atoms, atomic
weights, isotopes, molecular weights, valence, oxidation-reduction reactions, effect of
electrolytes on some of the reactions, equivalent weight, radicals, acids, bases, acid-base
reactions, PH, PKa, buffer solutions, concentration of solutions(% solution, normal solution,
molar solution) e.t.c

Analytes in clinical chemistry.

These can be organic or in organic;
1. Organic compounds. The great classes of organic compounds associated with life are;
fats, proteins and carbohydrates plus the associated enzymes or hormones.

Proteins Are made of nitrogen, carbon, oxygen, sulphur and phosphorus on drastic
hydrolysis. But on lesser hydrolysis, they are composed of amino acids. Each one of the
proteins contains a diversity of amino acids. This confers on proteins a diversity of properties
e.g. haemoglobin made of amino acids confers on respiratory function. Enzymes because of
their composition and nature become functional, Nucleo proteins deal with genetic
inheritance. Therefore, if proteins are assayed, protein levels will reflect a certain disease e.g
kwashiorkor (low protein levels), multiple myeloma (high protein levels).

Fats On hydrolysis are made up of carbon, hydrogen and oxygen. They mainly supply
energy and acts as a food reserve for cells. Some combine phosphorus to form
phospholipids. Phospholipids are important for cell wall composition so as to allow fat soluble
substances to enter the cell. Important fats are cholesterol, glycosides, high density and low
density lipids. When these are assayed, the levels reflect on the organ affected, majorly the
heart.

Carbohydrates Are made of carbon, hydrogen and oxygen. Are mainly required for fuel.
Hexoses are first preferred for energy followed by pentoses. Are metabolized through the
glucose path way (Krebs cycle) to produce energy in form of ATP. Glucose assay can give a
clue disease condition e.g. diabetes mellitus.

Enzymes Is protein in nature and catalyse reactions within the body. Their actions are
localized and are classified differently according to their action e.g. Hydrolases, oxidases,
Assay of enzyme levels reflect disease on a particular organ producing it e.g creatine kinase
in cardiac disease.

Hormones Control body functions from centres elsewhere other than where they are
produced. They are mainly protein in nature but a few have steroidal structure with

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cholesterol as their raw material. By assaying the hormone, we can pin point the diseased
organ
Examples of hormones include :

2. In organic compounds.
Are mainly electrolytes e.g. cations and anions which migrate to the electrode of the opposite
charge to it e.g. Na+, K+ , Mg2+ ,Ca2+ which constitute cations and anions ions, sulphate ions,
phosphate ions, constitute anions. The two ions must balance each other to make the body
neutral. They are functionally important for the movement water and acid base-balance.
Assay of electrolytes reflects on renal function and acid-base balance of the body.

Vitamins: Are mainly proteins but majorly flavoproteins (proteins to which some active
molecule is attached). Are in minute quantities which are necessary for body functions e.g vit
C, B and B complexes, A, K, D and vit E.

Water: The greater percentage of the body weight is water. It is divided into compartments
i.e. Extra cellular and intracellular water is found in cells. Water is obtained by direct drinking
but 300mls is made from metabolic process. Water in the body caters for osmotic pressure
and this is regulated by hormones, electrolytes and proteins.

In conclusion, due to minute quantities of body cell constituents, most of the analysis entails
chemical reactions to produce colour where by the colour produced is measured

FUNDAMENTALS OF CLINICAL CHEMISTRY

Definitions
Matter- is anything or is a substance that has mass and occupies space (has volume).
Mass- Is the measurement of matter’s ability to respond to gravitational force.
Volume- Is a three dimensional (length by width by height) space occupied by matter

Matter is composed of very small particles called molecules which themselves are composed
of atoms. The fundamental unit of matter is an atom. Matter has three forms i. e solid, liquid
and gas.

An element- Is a substance made up of same atoms

An atom- Is a smallest particle of an element that can take part in a chemical reaction.

Atoms cannot be broken down to smaller particles without losing their chemical properties.

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Composition of Atoms.
An atom is made up of two basic parts; the nucleus and orbital electrons. There are three
principle types of sub atomic particles and these are; protons, neutrons and electrons. The
nucleus is composed of two types of these particles i.e. Protons and neutrons.
The nucleus is a cluster of protons and neutrons and gives the atom most of its mass. The
region outside the nucleus is almost an empty space with electrons that surround or orbit the
nucleus like planets revolving round the sun. These outer most electrons are responsible for
all chemical reactions with other atoms.

Atomic structure or configulation of the atom

Properties of sub-atomic particles

Electrons;
Are the smallest of the sub-atomic particles and reside on the outside of the cell with
enormous amount of space between the nucleus and electrons. The electrons are negatively
charged and are given the symbol e or e-

They have negative one charge (-1) and mass of 0.000549(mass 0) and travel at about 1/10
the velocity of light. Electrons are held in their orbits around the nucleus by binding energy. In
an electrically neutral atom, there are equal numbers of protons and electrons. Chemical
reactions of an atom are directly dependent on its number of electrons.
Protons;
Are found within the nucleus of an atom and is symbolized p or p+. They have positive one
(+1) charge, which is equal and opposite to the charge of electrons. The proton has a mass
of 1.0759(mass 1). The total number of protons is the atomic number, symbolized by the
letter Z.
Neutron;
Are also found within the nucleus of the atom. They have no charge and mass of
1.00898(mass 1). They are symbolized by the letter n.
Atomic mass;
Is the number of protons which is also equal to the number of electrons in an atom. It is
symbolized by letter A.

Atomic structure or configuration of the atom

Electron

Neutron nucleus

proton Orbital shall

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 CHEMICAL REACTIONS
 The electrons are on the periphery of the atom, and therefore undergo changes during
chemical reactions. The nucleus is not penetrated and therefore not affected during
chemical reactions.

 Ions are charged atoms or group of atoms possessing an electrical charge. Atoms are
neutral possessing equal number of negative electrons and positive protons. However
atoms have a tendency of losing one or more electrons during a chemical reaction. An
atom looses electrons to become positively charged meaning it now has more protons
than electrons. Positively charged species are called cations. And metallic elements tend
to lose electrons and become cations. Other atoms gain one or more electrons which
make these atoms to have more protons than electrons hence become negatively
charged. They are referred to as anions. Non metallic elements have atoms tend to gain
electrons and become anions.

 Elements are substances that are made up of atoms of the same kind. These
substances have a unique set of properties and cannot be split by any chemical means
into simpler substances. There are 82 naturally occurring elements and about 31 are
artificially made. Each element has different atoms from other elements and the number
of sub-atomic particles (p, n and e-). Each atom is symbolically distinguished by a
specific symbol to represent the element. A symbol is a letter or picture used to
represent something. Elements have been given letters taken from a principle letter or
letters of the name of the element. One or two letters are used to represent the element
e.g Magnessium-Mg, Manganese-Mn, Phosphorous-P, Aluminium-Al, and Calcium-Ca.

 Other elements are given symbols of their outdated latin names e.g. Iron (ferrum)-Fe,
Copper(Cuprium)-Cu, Lead(Plumbum)-Pb, Gold (Aurium)-Au, Sodium(Natrium),
Potassium(Kallium)-K

 A molecule is the smallest unit of an element that can exist on its own (independently)
and retains all the chemical properties(x-tics) of that substance. Two or more atoms may
combine to form a molecule. Molecules are clusters of atoms that are held together by
strong electrical forces called bonds. They can have as few as two or as many as
thousands of atoms and therefore thousands of compounds may be created by changing
the number of atoms within the molecule.

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 A molecule is symbolized by the symbols of the atom’s elements that make up the
molecule or compound. These symbols are called formulae. A formula is used as a quick
way to show the composition of molecules or compounds. The subscript that appears
right after the symbol is the number of the atomic particles making up a single unit of the
molecule or the compound.

E.g. H + H--------------------------------H 2
1 atom 1 atom 1 molecule (same atoms)
Cl + Cl--------------------------------Cl 2
1 atom 1 atom 1 molecule (same atoms)
Cu + O ------------------------------- CuO
1 atom 1 atom 1 molecule (different atoms)

 Molecular mass is the sum of atomic mass of the elements forming the molecule.
 Molecular weight is the weight of the molecule compared to the weight of a carbon atom
weighing 12 units. Mathematically, molecular weight is the sum of the atomic weights of
the elements forming the molecule.
E.g. molecular weight of water,

H2O----------------------------------2H + O
Water Hydrogen Oxygen
2 16
Therefore, the molecular weight of water is 2+16=18

 Equivalent weight of an element or compound is chemically defined as that weight of

an element or compound which displaces or combines with one part by weight of
hydrogen or 12 parts by weight of carbon.
 Valence of an element is the number of hydrogen atoms which will combine with or
displace one atom of the element. Valence can be defined as the combining capacity
of an element. Elements that have a valence of one are termed monovalent e.g
Sodium, potassium, Chloride, Iodine etc. Elements that have two are termed divalent
e.g. Calcium, Zinc, Magnessium etc and those having three are termed trivalent e.g.
Aluminium, Nitrogen

Mathematically, equivalent weight of an element or compound is

Atomic weight for an element. OR molecular weight for a compound

Valence valence

e.g 1 equivalent weight for an element. E.g 2 Equivalent weights of compounds.

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 Sodium; Atomic weight=23, valence=1 Sodium Chloride (Nacl)
Therefore, Equivalent weight =23/1=23. Molecular weight = 58.5
Valence of Nacl = 1
Calcium: Atomic weight=40, valence=2 Therefore equivalent weight
=58.5=58.5
Therefore, Equivalent weight =40/2=20. 1
Sodium carbornate:
Molecular weight = 106
Valence = 2
Equivalent weight = 106/2 =53

Sulphuric acid (H2SO4)

Molecular weight = 98
Valence = 2
Equivalent weight =98/2 = 49
Radicles
Certain groups of atoms when combined together in a compound behave as a single atom.
Such units are called Radicles. Each radical has an electrical charge and can combine with
another atom or another radical to form a compound.
Examples: Symbol Valence
+
Ammonium NH4 1
-
Acetate CH3COO 1
-
Bicarbonate HCO3 1
-2
Carbonate CO3 2
-
Hydroxide OH 1
-
Nitrate NO3 1
-
Nitrite NO2 1
-
Permanganate MnO4 1
-2
Sulphate SO4 2
-3
Phosphate PO4 3

Compounds
Elements electrically combine their atoms to produce compounds. Compounds are
substances that have only one kind of molecules. They are made of two or more elements
that are electrically combined. E.g sulphuric acid (H2SO4) has three elements; hydrogen,
sulphur and oxygen combined in different proportions. The compound HCl-hydrochloric acid
has two elements; hydrogen (H) and Chloride (Cl) And water has two elements; Hydrogen
and Oxygen

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When elements are joined, they lose their individual properties and have properties different
from those of the elements they are composed of.

Mixtures
When we physically combine elements or compounds, we get a mixture. Mixtures by
definition are substances which have more than one molecule not chemically combined
together. They are physical combinations of two or more elements that are not in definite
proportions. Being physical combinations, mixtures can be separated by use of the difference
in physical properties between the pure substances in the mixture e.g. common salt and
sand.

SYSTEME INTERNATIONAL (SI) UNITS

The SI unit is the modern metric system of measurement which is based on the meter-
kilogram-second system (MKS).

Base and derived units;

There are seven base units i.e Metre, kilogram, second, ampere, mole, Kelvin and candela.
The base units are the seven well defined units that are regarded dimensionally independent.
Other units are derived from a combination of some of the seven base units. Derived units
are those formed by combining base units according to their algebraic relations linking
quantities.

Base units
Physical quantity measured Symbol SI base unit
Length (L) m meter
Mass (m) kg kilogram.
Time (t) s second
Amount of substance mol mole.
Electrical current (I) A Ampere
Temperature (T) k Kelvin
Luminous intensity cd Candela

Derived units
Physical quantity measured Symbol SI base unit
Area (A) m2 square metre.
Volume (v) l litre.
Velocity (v) m/s meter per second.
Density kg/m3 kilogram per cubic metre
Other units
Physical quantity measured Symbol SI base unit

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Wavelength nm nanometer.
International unit u/l international unit

Submultiples of units
Prefix symbol multiplication factor divided by
Deci d 10-1 10
Centi c 10-2 100
Mili m 10-3 1000
Micro u 10-6 1000000
Nano n 10-9 1000000000
Pico p 10-12 1000000000000
Fento f 10-15 1000000000000000
Multiplied by;
Deca da 101 10
Hecto h 102 100
Kilo k 103 1000
Mega M 106 1000000
Giga G 109 1000000000
Tetra T 1012 1000000000000
Peta p 1015 1000000000000000

Commonly used units in the laboratory

 Litre
The SI unit of volume is meter cubed (m3). This is a very large unit hence a militre has
been recommended in the laboratory. One litre is therefore equivalent to 10 decilitres.
1000 militres or 1000000 microlitres. One deciliter is equivalent to 100 militres and one
militre is equivalent to 1000 microlitres.
 Gram
A kilogram is the SI unit for mass and a gram is a working unit. Mass measurements
are made in grams or multiples of a gram e.g milligram, microgram, nanogram,
picogram. One gram is therefore equivalent to 1000 milligrams, 1000000 micrograms,
or 1000000000 nanograms. One milligram is equivalent to 1000 micrograms.
 Mole
The mole is the SI unit for the amount of substance and the measurement of the
amount of substance are made in moles or millimoles (mmol), micromoles nanomoles.
One mole is equivalent to 1000mmol or 1000000000nmol.
Earlier, the results of the tests mmol/l were expressed in mg/100ml or
microgram/100ml.
Therefore the formula used to convert mg/100ml(mg%) to mmol/l is as follows;

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 Mg/100ml × 10 = mmol/l
Molecular weight of substance.

International unit
This unit is used to express enzyme activity of certain substances such as hormones.
Definition. An international unit is the amount of enzyme which under defined assay
conditions will catalyze the conversion of one micromole of substrate per minute. And a mole
is the amount of substance of a system which contains as many elementary units (atoms,
ions) as there are carbon atoms in 0.012kg of pure nuclide of carbon 12.

ACIDS AND BASES

An acid is a substance that liberates hydrogen ions in solution and acts as a proton
donor(donates H ions)
Properties of acids
 Have a sour taste.
 Turn dump litmus paper red.
 React with carbonates and liberate carbon dioxide gas.
 React with bases to produce salt and water.
 Are corrosive.
Acids are divided into two; strong and weak acids.
Strong acids
Are acids which ionize almost completely in solution with very few intact molecules of the
acid remaining. Examples include; Sulphuric acid, hydrochloric acid and nitric acid.
NB: Ionization is the dissociation of a substance in solution into electrically charged atoms
called ions.
Weak acids
Are acids that ionize very little in solution with many intact molecules of the acid remaining
E.g Lactic acid, Glacial acetic acid.
Commonly used acids in the laboratory
1. Sulphuric acid:
Has a relative density of 1.84, molecular weight of 98.08 and percent concentration of
95-97% by weight. It si very corrosive, viscous, oily and reacts with water very
violently.
NB: Never add water to acid but add acid to water. This is because acids react
violently with water.
2. Hydrochloric acid.
Has a relative density of 1.190, molecular mass of 36.46, percent concentration of
37% by weight of the gas hydrogen chloride. It fumes and has irritating vapour and
therefore, it is injurious. Store it in a well stoppered bottle.

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3. Nitric acid.Has a relative density of 1.510, molecular mass of 63.01, and concentration of
about 99% by weight, it fumes, stains the skin yellow-brown and is a powerful oxidizing
agent. It should be handled with great care.
4. Acetic acid.
Concentrated acetic acid is a colourless liquid which freezes at -170 degrees celcious
to form crystals that resemble glacius(a river of ice). This freezing property earned it a
name glacial acetic acid which should be applied to undiluted acetic acid. It is not
corrosive.
BASES
Are substances that liberate hydroxyl ions in solution and can accept a proton.
Properties
 Have a soapy feel.
 Turn dump litmus paper blue.
 React with acids to form salt and water.
Common bases include; Oxides and Hydroxides of metals such as sodium, potassium, lead,
copper and ion. Oxides of sodium, potassium, and calcium dissolve in water to form alkalis.
Sodium hydroxide and potassium

Potassium hydroxide is referred as strong alkalis because they dissociate completely into
cations and hydroxyl ions in water.

StrongAcids StrongBases WeakAcids WeakBases

HCl NaOH Acetic acid Ammonia

Hydrocyanic
HNO3 KOH Magnesium hydroxide
acid

HBr etc HF Pyridine

H2SO4 Oxalic acid Sodium carbonate

HI Ethanoic acid Potassium carbonate

HClO4 etc etc

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ACID-BASE REACTIONS
Acids react with bases to form salt and water. The process of adding an acid to a base or a
base to an acid to give a neutral solution consisting of a salt and water is known as
Neutralisation.

ACID BASE SALT WATER

HCL + NaOH = NaCl + H2O
HCl + KOH = KCl + H2O
H2SO4 + 2NaOH = Na2SO4 + H2O
HNO3 + KOH = KNO3 + H2O

HYDROGEN ION CONCENTRATION OF SOLUTIONS

For a convenient way of expressing hydrogen ion concentration, the symbol PH was
introduced. PH is defined as a negative logarithm to the base 10 of hydrogen ion
concentration { PH = -log10[H] }. Most solutions have H+ and OH- ions. Neutral solutions
have equal numbers of H+ and OH- ions.
Pure water is neutral because it ionizes to give one H+ ion and one OH- ion for each molecule
of water ionized.

H2O-------------------------------------------H+ + OH-
Applying the law of mass action.

K = [H+] [OH-]
[H2O]
Since the degree of dissociation of water is very small, H2O can be considered to be
constant. Therefore we, we have a constant KW which is the ionic product of water
expressed as;
KW = [H+] [OH-]
Pure water at 25 degrees Celsius contains 0.0000001g or 10-7 of hydrogen ions per litre. The
molecular mass of H+ is 1. The hydrogen ion concentration of water can be expressed as;

[H+] (pure water) = 1 × 10-7 moles/l

Since KW = [H+] [OH-], then 1 × 10-7 × 1 × 10-7 = 10-14

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In a solution, the predominance of one type of ion (H+ or OH-) over the other determines the
degree of acidity or alkalinity of a solution. The acidity or alkalinity of a solution. The acidity or
alkalinity of a solution only refers to hydrogen ion concentration.

PH OF SOLUTIONS
PH is defined as a negative logarithm to the base 10 of hydrogen ion concentration
PH = -log10 [H+]
POH = -log10[OH-]
KW = [H+][OH-] for pure water.
If [H+] = 1×10-7, then PH = log101/102
PH = 2.0
It follows that;
 A neutral solution has PH 7.0
 Acidic solution has PH less than 7.0
 An alkaline solution has a Ph > 7.0
Strong acids and alkalis are completely dissociated in solution. Therefore, their PH can be
calculated directly.
Example;
1. To calculate the PH of a 0.1M HCl
Number of H+ ions 1× 10-1mol/l
From PH = -log1010-1 = log101/101 PH = 1.0

2. To calculate the PH of a 0.1M NaOH

[OH-] = 1× 10-1mol/l
Since [H+][OH-] =10-14
1× 10-1[H+] = 10-14
[H+] = 10-14+1
+
[H ] = 10-13
From PH = -log10 [H+], PH = log101/1013
Therefore, PH = 13.0
3. To calculate the PH of a 0.15M HCl solution
[H+] = 1.5 × 10-1mol/l
PH = -log101.5 × 10-1
PH = log 15

PH = 1.2

Weak acids and bases are not completely dissociated in solution. Therefore, it is important
that the degree of dissociation be calculated first.
E,g to calculate the PH of 0.1M acetic acid when the degree of dissociation is 20%
[H+] =20% × 0.1 =0.2 × 0.1 = 0.002mol/l
PH =-log100.002 same as -log102 × 10-3

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Measurement of PH
In the Laboratory, measurement of PH is done on;
i. Body fluids; Urine, gastric secretions to rule out any abnormalities in the body’s
metabolic process.
ii. Some laboratory solutions such as reagents and buffers before using them for the
analysis of body substances(analytes)
PH of solutions can be measured using the following;
a) Litmus paper: This turns blue if the solution is alkaline and red if the solution is acidic.
b) PH indicators: These give approximate PH values. These PH paper strips are
composed of indicators such as methyl orange and bromothymol blue. These strips
are are sensitive to PH range of 5-9.
c) PH meter; this provides the most accurate method for determining PH

Measurement of PH using PH indicator papers.

The coloured PH papers into the solution whose PH is to be determined, Colour changes
according to PH of solution, and colour changes of the paper are then compared with the
standard control chat giving the corresponding figure.
Method;
 Dip the whole strip into solution.
 Remove the strip immediately.
 Drain off excess fluid by touching the side of the container.
 Wait for the period of time specified by the manufacturer to read the results.
 Compare the colour formed with the standard chart on the bottle.
 Record the PH value.
Measurement of PH using PH meters
The PH meter is an instrument used for measuring the reaction (PH) of solution. The PH
metre determines the degree of acidity or alkalinity of the solution.
There are a wide range of PH meters including battery powered and mains electrically
operated models, Modern PH meters are digital electronic type. When using a PH meter,
Always follow the manufacturer, instruction.

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Diagram;

The determination of PH of solutions is based on the glass bulb electrode principle. I.e
“When two solutions of different hydrogen ion concentration are separated by a thin glass
membrane, an electrical potential is generated between them and varies with the hydrogen
ion concentration(PH) of the solution and can be measured by a voltmeter”
When a glass bulb electrode is immersed into a solution of unknown PH(solution under test),
an electrical potential develops develops across a thin glass separating the two solutions as
a result of movement of H+ ions through the lattice of the glass that is selective to hydrogen
ions.
The potential developed depends on the H+ ion concentration of the test of the test solution is
compared with the fixed H+ ion in the glass bulb that is measured with the aid of the
reference electrode and registered by a meter.

PROCEDURE FOR OPERATING A PH METER

This procedure is a WPA PH CD60 meter.

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  Measure the temperature of the test solution. Adjust the temperature dial so that it
reads the temperature of the solution.
 Switch on the PH meter to warm for a long time(as specified by the manufacturer)
before use.
 Turn on the PH scale.
 Using a wash bottle, was the electrode using distilled water or de-ionised water.
Note: When the meter is not in use, the electrode must always be stored in an acid
solution e.g PH 4 buffer.
 Transfer the electrode into a beaker containing a standard buffer i.e buffer of known
PH. The PH of the standard buffer should be closed to the expected PH value of the
test solution(buffers of PH 4,7, 9 are available for standardization). Adjust the buffer
control knob to give the reading of the standard buffer.
 Wash the electrode using distilled or de-ionised water and transfer it to the test
solution and record its PH reading.
 Turn off the PH scale.
 Rinse the electrode with distilled or de-ionsed water and put it back in a container of
PH 4 buffer or 3% HCl solution.

Care and maintenance of a PH meter.

For the detailed use, care and maintenance of a PH meter, always consult the
manufacturer’s instruction operation manual. However, the general guidelines regarding care
and maintenance of a PH are important.
 Handle the electrode with great care. If the glass bulb is cracked or even scratched,
the electrode must be replaced.
 Use plastic containers to hold the buffer and test solution. When measuring PH, do not
allow the electrodes to touch the sides of the container.
 When an electrode is transferred from one solution to another, rinse it with distilled or
de-ionised or with the test solution.
 When measuring the PH of solution, allow adequate time for electrodes to reach
equilibrium before taking the reading.
 Always adjust the temperature calibration Knob to the temperature of the test solution
being measured(unless the meter adjusts automatically for temperature changes).
 Do not remove the electrode from a solution while the measuring circuit is switched
on.
 When an electrode is not in use, store it in a buffer solution of around PH 4(acid
solution will prolong the life of the electrode).
 If the PH meter is battery operated, it is important to ensure that the battery is
sufficiently charged to give reliable readings.

19
 INDICATORS

Indicators are substances that give different colour shades at different PH values. They are
used to determine the PH of liquids and the end point of acid-base titrations. Indicators are
either weak acids or weak bases.

Action of indicators.
Weak acid indicators
Consider a weak acid indicator represented as HIn. This indicator will ionize in water as
follows;
HIn + H2O................H+ + In-(at equilibrium), where HIn and In- have different colours.
If an acid is added to the indicator solution, the effect of the added H+ ions will be to decrease
the concentration of In-ions and increase the concentration of HIn i.e. the added H+ ions will
react In- ions to almost completely the HIn coloured form.
HIn + H2O………………… H+ + In-
H+ + In-…………………….HIn.
If an alkali is added to the indicator, the H+ ions itself will neutralize the alkali (OH-); i.e more
of the In – will be produced, leading to change in In- form.
HIn + H2O ………………………H+ + In-
H+ + In- ………………………….H2O
Weak base indicator.
Consider a weak base indicator represented as In. This indicator will ionize in water as
follows;
In + H2O ………………………HIn+ + OH- (at equilibrium), where HIn+ and In have different
colours.
If an alkali is added, force the point of equilibrium to the left and the In colour will be
predominant.
HIn+If an acid is added, the base OH- is neutralized by the added H+ ions and the dissociation
equilibrium left to right is favoured, with HIn+ colour predominating

HIn + H2O ………………………HIn+ + OH-

H+ + OH- ………………………….H2O

Colour change intervals

20
Each indicator has a PH range over which a visible colour change occurs. Examples of
indecators and their colour change intervals are shown below;
Indicator colour change PH range
Methyl orange red to orange 3.1 to 4.6
Methyl red red to yellow 4.2 to 6.3
Bromothymol blue yellow to blue 6.0 to 7.6
Phenol red yellow to red 6.8 to 8.4
Phenolphthalein colourless to red 8.0 to 9.8

SOLOUTIONS
Definition:
A solution is a homogenous mixture of one or more substances (solute) in a sufficient
quantity of of the dissolving medium (solvent).
A solute is a dissolved substance and a solvent is a substance in which a solute dissolves.
Many measurements in a clinical laboratory concern the determination of the dissolved
solutes in a solvent.
In dealing with solutions, our primary concern is usually the concentration of the solute.
Standard solution:
Is a solution whose exact concentration or strength is known and is used for comparing the
strengths of other similar solutions e.g comparing the strength of test solutions
Concentrations are always the ratio of the solute to the solution or the solvent.

Concentration = amount of solute in moles or grams

Amount of solution or solvent.

Expressing the concentration of solutions.

The strength of a solution can be expressed in four ways:
i. Percent solutions.
ii. Part dilutions.
iii. Molar solutions.
iv. Normal solutions.
i. Percent solutions:
Percent implies per one hundred. A 30% contains 30 parts of the solute per 100 parts of the
whole solution.
Percent solution can be expressed in the following ways;
a) Weight per weight (w/w).
This implies that the weight of both the solute and the solvent add up to 100
regardless of the final volume produced.
21
 Example: 25% w/w solution.
Means 25gms solute dissolved in 75gms of solvent. The resultant solution may not be
100 but the total weight must be 100gms.

b) Weight per volume (w/v).

This is a common method of preparing laboratory solutions. In this method, the weight
of the solute is dissolved in a final volume of 100mls
NB: it is wrong to dissolve a weight of the solute in 100ml of the solvent but to
dissolve in a lesser volume of the solvent then make or top the volume to 100mls.
Preparation:
Assemble the requirements
 Measuring cylinder.
 Volumetric flask.
 Weighing scale.
 Solvent.
 Solute.
 Stering rod.
 Storage container.
 Labels.
Procedure
 Weigh the solute
 Place the weighed solute in a volumetric flask.
 Dissolve in a little of the solvent.
 Make up the total volume with the solvent.
 Transfer the solution to a storage container.
 Label the container.
The label carries the following information;
o Name or concentration of the solution.
o Date of preparation
o Expiration date.
o Name of person who prepared the solution.
o The lot number
o The batch number.
o Any special storage condition e.g 2-80c, store in a deep freezer or store in at
room temperature.
c) Volume per volume (v/v)
This is applicable if the solution is to be prepared from reagents which are both liquids.
A volume of a liquid solute is made up to a final volume e.g 100ml with the solvent. A
volumetric flask is used and a final volume is made by adding a solvent.
22
 Preparation:
 Measure the volume of the liquid solute e.g 30mls in a cabrated container
(volumetric flask)
 Add the solvent to the flask to bring the solution to the calibrated mark.
 Mix and transfer to a suitable storage bottle and stopper.
 Store as recommended.
ii. Part dilution:
In this method, the dilution is expressed as that part of the whole volume in to which
one part (or more parts) of the solute is dissolved e.g WBC total count dilution is 1 in
20 (1:19) implying that there is one volume of dissolved solute in 20 volumes of the
final solution. Though actually mixed with 19 volumes of diluent.
iii. Molar solutions:
A molar solution contains a molecular mass of solute. The molecular mass is found by
adding the atomic masses of the different atoms present in the compound.
E.g The molecular mass of sodium chloride (NaCl) is 58.454. Hence one molar
solution of the salt contains 58.454gms of NaCl in one litre of solution (g/l).

To convert a percent solution in to mmol/l solution, the formula below can be used;

Mmol/l solution = g%(w/v) solution × 10

Molecular mass of substance

g% can be expressed in g/100ml or g/dl or gdl-1

Mol/l is the unit of concentration of solutions. It is also known as molarity which is
abbreviated as M (capital M)

Molarity = number of moles of solute

Amount of solution in litres
A mole is the amount of substance which contains as many elementary units, entities
or particles as there are in 12g of C12. 12g of this carbon contains the Avogadro’s
number (6.022 × 1023) in 12g of C12. Any substance which contains 6.022 × 1023 is
equal to one mole
Mass and amount of substance (mole) are related by the equation below;
Number of moles = Mass (g)
Molecular mass
Therefore, mass = number of moles × molecular mass.
Example;
Sodium hydroxide has a molecular mass of 40. This means that 40g 0f NaOH dissolved in a
litre of solution forms a molar solution. Calculate the concentration per litre of solution
prepared from 4g of NaOH.
23
Solution
Number of moles of NaOH = Mass = 4/40 = 0.1
Molecular mass
Concentration of NaOH = 0.1 moles in one litre of solution = 0.1mol/l =0.1M
Molarity

Example 2
Preparation of 10M solution of NaOH.
40g…………………………………….1M
x……………………………………….10M
40 × 10 = 400g
So weigh 400g of NaOH and dissolve in one litre of solution.
Molar concentrations are expressed as mol/l, or M and smaller units are;
Mmol/l mM 10-3
µmol/l µM 10-6
nmol/l nM 10-9
pmol/l pM 10-12
Some calculations on molarity.
Molarity of solution (M) = Moles of solute
Litres of solution
M = Number of moles of solute(n)
Volume of solution in litres (V)
Example
Calculate the concentration of NaCl solution containing 0.125moles of NaCl in 0.5 litres.
Given, n = 0.125 moles.
Volume (V) = 0.5 litres
M = n = 0.125/0.5 = 0.25M.
V

Example;
Given that the morality of copper sulphate is 0.025. Calculate the number of moles of copper
sulphate in 250 mls of copper sulphate.
M = 0.02M,
V = 250/1000 = 0.25litres.
n = M × v =0.002 × 0.25 = 0.005 moles.
Diluting solutions
A weaker solution can be made from a stronger one using the following formula;
CV/O or CV/S where; C = concentration of the solution required.
V = volume of the solution required

24
 S = strength of a stronger solution.
Take a stronger solution and dilute it. When a solution is diluted, more solvent is added to it.
The concentration changes upon dilution, but number of solute particles does not change.
Example;
To make 500ml of sodium hydroxide (NaOH) 0.25mmol/l, from 0.4mmol/l solution.
Solution:
C = 0.25mmol/l, V = 500mls, S = 0.4 mmol/l
From the above, CV/S, Volume (mls) required = 0.25 × 500
0.4
V = 312.5ml
Therefore, measure 312.5ml of the NaOH and make the volume to 500ml
iv. Normal solution:
A normal solution is one which contains the gram equivalent weight (equivalent weight
in grams) of substance per litre of solution.
a) The equivalent weight of an element is calculated by dividing the atomic weight of the
element by the valence. Valence is the number of hydrogen atoms one atom of an
element will combine with or displace.
b) The equivalent weight of an acid is the weight of it which contains, 1.008g (one atomic
weight) of replaceable hydrogen. It is calculated by dividing the molecular weight of
acid by the number of the replaceable hydrogen atoms in the molecule.

Example
Calculate the equivalent weight of sulphuric acid (H2SO4).
Solution
Molecular weight of sulphuric = 98.082
Number of replaceable hydrogen atoms = 2.
Equivalent weight of H2SO4 = 98.082/2 = 49.041
c) Equivalent weight of an alkali.
The equivalent weight of an alkali is that weight of it which will neutralize the
equivalent weight of an acid. It is calculated by dividing the molecular weight of the
alkali by the number of hydroxyl radicles in the molecule.
Example
Calculate the equivalent weight of Ca(OH)2
Solution;
Molecular weight of Ca(OH)2 = 91.042
Number of OH- radicles = 2
Equivalent weight =91.041/2 =45.552g
d) Equivalent weight of a salt:

25
 The equivalent weight of a salt is calculated by dividing its molecular weight by the
number of metal ions (cations) per molecule, Multiplied by the valence of the ion
(cation)
Example:
Calculate the equivalent weight of sodium sulphate (Na2SO4).
Solution
Molecular mass of Na2SO4 = 142.060
Number of metal ions = 2
Valence = 1
Equivalent weight = 142.060 × 1 = 71.030
2
Note When the above calculated equivalent weights are dissolved in one liter of solution, the
solutions produced are called Normal solutions. Hence the normality of solution
concentration expressed in gram equivalents solute per liter

BUFFER SOLUTIONS AND THEIR ACTIONS

Definition:
A buffer is defined as a chemical system that prevents change in the concentration of
another chemical substance when little amount acid or base is added . In clinical
laboratories, proton donor and acceptor systems are used as buffers to prevent change in
hydrogen ion concentration.

Example of buffers (In the body)

 Blood (haemoglobin)
 Plasma protein
 Bicarbonate
 Carbonate
 Phosphates

Others (not in human body)

 Acetate
 2,3 diphsphoglyerate
 Boric acid
All weak acids or bases, in the presence of their salts, form buffer systems. The action of
buffers and their role in the maintenance of a solution’s PH is explained with the equation of
Henderson and Hasselbalch equation.
The Henderson Hasselbalch equation.
Consider the ionization of a weak acid, HA and of a salt of that acid, BA represented as:
HA ---------------------------------------------H+ + A-

26
 BA ---------------------------------------------B+ + A-
The dissociation constant, Ka, for a weak acid may be calculated from the following equation;
Ka = [H+][A-]
[HA]
Thus [H+] = Ka × [HA]
[A-]
+
OR Log10[H ] = Log10Ka + Log10[HA]
[A-]
Where the brackets indicate the concentration of the compound contained within.
Now multiplying through out by -1
-Log[H+] = -LogKa - Log[H+] and pKa = -LogKa;

PH = pKa + Log10 + [HA]

[A-]
-
Because A is derived principally from the salt, may for practical purposes be written as :
PH = pKa + Log10 [salt]
[Undissociated acid]
Or simply, PH = pKa + logka + log[salt]
[acid]
-
Where [salt] = [A ] = Concentration of dissociated salt and [acid] = [HA] =
concentration of undissociated acid.
Consequently, the PH of the system is determined by the pKa of the acid and the ratio of [A -]
to [HA].
The buffer has its greatest buffer capacity at its pKa, that is that PH at which the [A -][HA].
This factor when entered in the preceding equation gives;
PH = pKa + log 1
PH = pKa + 0
PH = pKa
The capacity of the buffer decreases as the ratio deviates from 1 (one). In general, a buffer
should not be used at a PH greater than 1 (one).

MODE OF ACTION OF BUFFER SOLUTON

The chemical mechanism by which buffers exert their effect may be seen through the
considerations of the reactions involved when a base is added to a buffer solution containing
acetate ions (CH3COO-) and acetic (CH3COOH) molecules.
When NaOH is added;
CH3COOH CH3COONa

27
 + NaOH → + H2O
CH3COONa CH3COONa
-
The OH ions added are removed through combination with hydrogen ion dissociated from
acetic acid, thus minimizing PH changes.
The electron of alkali decreases the CH3COOH in the buffer and increases the CH3COONa.
The PH of the solution increases in proportion to the changes in the ratio of salt to the acid in
the buffer solution.
When acid is added e.g HCl;
CH3COOH CH3COOH
+ HCl → + NaCl
CH3COONa CH3COOH
The added H+ ions are removed through combination with acetate ions to form poorly
dissociated acetic acid In this case, the addition of HCl acts to decrease CH3COONa and
increase CH3COOH in the buffer. The PH of the solution drops in proportion to the change
in ratio of salt to acid in the solution. However, because the PH is related to the logarithm of
the A-/HA ratio, only a small change in PH occurs.

CHROMOGENS
A chromogen is a substance that change colour when oxidised examples include the
following

Examples .

 2,6 dichlorophenol .
 Indophenol
 Benziine
 4-aminophenol
 Ortho-tulidine

STANDARDS AND STARDARD SOLUTIONS

Standards are substances (solutes or analytes) of known concentration. A standard solution
is a solution which contains a known amount of solute in a definite volume of solution and is
used for comparing the strength of other similar solutions e.g comparing with the test
solutions.
Primary standards:
A primary standard is a substance available in pure and dried form, can easily be weighed
and the concentration does not change with time e.g sodium carbonate, potassium
dichromate, succinic acid among others.
Properties of an ideal standard.

28
Properties or characteristics of an ideal standard include the following:
i. Must be stable, easy to obtain, to dry and to preserve in a pure state.
ii. It should have a large molecular weight to minimize the effect of errors in weighing.
iii. Must not be altered by air e.g absorb moisture during weighing.
iv. It must be readily soluble in a solvent.
v. Should not give rise to any product likely to interfere with the procedure e.g titration.
vi. During titration, reactions with the standard should give intact end points.
Preparation of primary standard solutions:
Assemble requirements such as;
Solute, solvent, volumetric flask, weighing scale, a rod for stiring and storage container.
Procedure;
o Weigh the solute accurately.
o Dissolve in a little of the solvent in a volumetric flask.
o When all the crystals or the powder has dissolved, make the volume to required level
e.g top up to 1000mls.
o Transfer the solution to the storage bottle.
o Label the bottle with following information; Name of solution, lot number, Name of
person who prepared the solution, expiration date, concentration of solution,
precautionary measures if any and any special storage conditions.
Secondary standards:
A secondary standard is a substance which cannot be accurately weighed, its concentration
changes with time and cannot be made into a solution of a known concentration. Examples
include; Hydrochloric acid, acetic acid, sodium hydroxide, calcium hydroxide and ammonium
hydroxide.
Preparation of secondary standard solutions:
The solution is not prepared directly as for primary standards. It is prepared arbitrarily and
then its concentration calculated by titration against a suitable primary standard.
Preparation of acids
e.g preparation of approximately 0.1M HCl.
i. Direct measurement.
o Measure 500mls of water into a volumetric flask.
o Add 9ml of pure HCl into the flask.
o Make a final volume to 1000mls.
o This will give an approximate of 0.1M HCl
ii. Standardization of HCl against sodium carbonate solution
Equation of the reaction;
NaCO3 + HCl → 2NaCl + H2O +CO2
From the equation above, calculate the volume of HCl.
o Measure out this volume and place in a biuret

29
 o Measure out the volume the carbonate solution
o Titrate the HCl against the sodium carbonate .
o Obtain the standardized concentration of the HCl from the equation of
reaction/titration e.g (m1v1 = m2 v2)
NB In case of strong acids, a known volume of the acid is usually diluted to a given volume to
give an approximate concentration of the solution. The volume of the acid required can
readily be calculated using; specific gravity, percent composition and molecular mass.
Example:
The specific gravity of concentrated HCl is 1.18, the percentage composition is 35.4% and
the molecular weight is 36.5
Volume of conc HCl to be diluted to make approximately 1.1M HCl = 36.5 × 1.0 × 100
1.18 × 35.4
= 87.2ml
Thus if 87.2ml of conc HCl are diluted to one litre, an approximate molar solution is obtained.
Example:
The specific gravity of conc H2SO4 = 1.83
The % composition is 96.0
The molecular weight = 98
Therefore, the volume of the acid to be to a litre to prepare approximate molar solution is:
Mls of acid = 98 × 1.0 × 100 = 55.8
1.83 × 96
MI of the acid to make a molar solution = molecular weight × 1.0 × 100
Specific gravity × % composition
PHOTOMETRY
At the end of this topic, students are expected to;
 Define photometry.
 List the photometric techniques used for measurement of analytes in body fluids.
 Explain the principles of photometric analysis.
 Discus the theory of absorptiometry.
 Describe the use, care and maintenance of the equipment used in photometric
analysis.
ELECTROMAGNETIC RADIATION
Definition
Electromagnetic radiation(EMR) is energy emitted in form of waves or particulates and has
no mass and charge. EMR at very high frequencies are packets of energy called photons
(light, X and Y radiation)
Photons are chargeless and zero mass bundles of energy that travel in a vacuum at a
velocity of light which is 3 × 1010cm/sec
Components of the electronic radiation.

30
EMR is the energy wave made by up of oscillating electric(E) and magnetic(M) fields and
propagated with the speed of light(C).
Diagram.

The electric field E varies in magnitude in the direction perpendicular to the direction in which
the wave is travelling and the magnetic field M oriented at right angles to the electric field E.
Both E and M travel at the speed of light C.
Relationship between wavelength, speed, frequency and energy of EMR.
Heat, radio waves, microwaves, infrared, visible light, ultraviolet light and X-rays are all forms
of EMRS. These forms of radiation differ from each other in wave length, frequency and
energy.
Diagram

Wave length is the distance between the adjacent peaks in a series of periods waves. It is
measured in metres or fractions of a metre.(nm-10-9m)
Frequency(v) is equivalent to one cycle per second and is measured in Hertz (Hz). Wave
length and frequency are related by the formula;
C = ʎ × ᵞ ------------------------------- 1

Where;
C is the speed of light (3 × 10m/s)
ʎ is the wave length in metres.
ᵞ is the frequency – cycles per second in Hertz(Hz)
From the formula above, wave length and frequency are inversely related to each other.
Long wave length corresponds to low frequency and short wavelength to high frequency. The
relationship between the energy of light photons and their frequencies is;
E = h × ᵞ --------------------------------- 2
Where; E is energy in ergs, ᵞ is frequency of light given in cycles per second , h is plank’s
constant (6.62 × 10-27 ergs/sec).
From equation 1, ᵞ = C/ʎ ------------------3
By combining 2 and 3, we get E = hC/ʎ
This equation shows that the energy of light is inversely proportional to the wavelength
 The longer the wave length, the lower the frequency (heat, radiowaves) = the less the
energy.
 The shorter the wavelength, the higher the frequency (visible light x and ᵞ rays) = the
higher the energy.

31
A full range of wave lengths, frequencies and energies of the EMR is called the
electromagnetic spectrum (EMS).
VISIBLE LIGHT SPECTRUM:
The visible light spectrum refers to the narrow portion of EMS that can be sensed by the
human eye. The visible light waves or portions are the only electromagnetic waves we can
see. We see these waves as colours of the rainbow .
Diagram;

Each color that we perceive corresponds to a certain wave length band in the 400 – 700 nm
region. Wave lengths about 700 nm are seen by the eye as red light while those of shorter
wave lengths give rise to orange, yellow, green, blue, indigo and violet which is produced by
short wave length range of 400 – 700 nm. The human eye cannot see wave lengths above
700 nm and below 400 nm.
Wave length colours;
Ultra violet None
Violet 400
Indigo 445
Blue 475
Green 510
Yellow 570
Orange 590
Red 650
Infrared None

White light.
Is a mixture of colours of the visible spectrum. It is composed of all the wave lengths of the
visible colors i.e the red, orange, blue, indigo and violet (ROYGBIV), the colors of the
rainbow. Black is a total absence of light’s

CARBOHYDRATES
These are the most abundant type of food in nature and can be starch, sucrose, glucose etc
they are composed of the elements Carbon (C), Hydrogen (H) and oxygen (O) upon on
heating they lose H2O molecule causing a black residue of carbon hence the name carbon
hydrated or hydrated carbon
They are also referred to as saccharides a Greek name meaning Sugar. They have a
general formula of Cn H2n On implying the ratio of C: H: O is 1: 2: 1

NOMENCLATURE OF SUGARS
They are named basing on the No of the carbon atoms they poses in their chains

32
No of the carbon Name Empirical formulae
atoms
3 Triose C3H6O3
4 Tetrose C4H8O4
5 Pentose C5H10O5
6 Hexose C6H12O6
7 Heptose C7H14O7

The commonest simple sugars are tetroses, pentose and hexoses Sugars can be referred to
as aldesoses if they possess an aldehyde group or ketoses if they possess a ketone group

CLASSIFICATION OF CARBOHYDRATES
They are categorized under 3
- Monosaccharide
- Oligosaccharides
- Polysaccharides

Monosaccharide Are the simplest unites of carbohydrates that often can not hydrated into
much simpler units under mild conditions
They are usually sweet and soluble in water the most important monosaccharide of the body
are I . Pentose like D- ribose & D- deoxribose usual in synthesis of RNA and DNA
Ii. Hexose like D Glucose
Oligosaccharides
These are composed of few Monosaccharide’s units usually 2-10 linked together through
glycosidic linkages
Disaccharides
Are made up of two monosaccharide units linked through by glycosiolic bonds. Upon
hydrolysis they yield two Monosaccharide’s which may be similar or different they include
Maltose – made up of 2 glucose units (D. glucose)
Lactose – made up of glucose and galactose units
Sucrose – Glucose and fructose
Polysaccharides
Are polymers of several Monosaccharide’s joined though glycosidics. On hydrolysis with
acids or enzymes the yield monosaccharides or they are denatured. Polysaccharides which
on hydrolysis yield one type of monosaccharide are refereed as homopolysaccharide while
these one that yield more than one type of Monosaccharides is referred to as
heteropolysaccharides
Glycogen is a homopolysaccharide that only yield glucose on complete hydrolysis and is
storage polysaccharide of animal stored mainly in the liver

33
 CARBOHYDRATE METABOLISM

DIGESTION PROCESS OF CARBOHYDRATE.

Carbohydrates are of major components of diet and before they are absorbed, they must be broken
down to monosaccharide. This process is called digestion.
34
 Digestion begins in the mouth where salivary amylase hydrolysis starch to form intermediate dextrins
and maltose.

In the stomach salivary amylase is inactivated by acid PH or gastric juice. The PH of small intestines
is more alkaline, therefore the digestion of starch and glycogen to maltose is completed by pancreatic
amylase. The maltoses together with any digested disaccharides are then hydrolyzed by the enzymes
from the intestinal mucosa to form a monosaccharide.

The monosaccharides are absorbed through the intestinal wall into the blood stream.

Maltose Glucose + glucose

sucrose glucose + fructose

lactose galactose + glucose

The monosaccharides are transported to the liver through the portal circulation.

Since glucose is the only monosaccharides of interest utilized by the body galactose and fructose are
converted by the liver enzymes to glucose..

In the first step the glucose metabolism , the glucose in the liver react with adenosine triphosphate
(ATP) which is the energy molecule in the presence of hexokinase to form glucose 6-phosphate. The
glucose 6-phosphate serve as pivot for several degradation process for 3 possible pathways through
which glucose can be metabolized .

If the body needs energy, glucose is metabolized completely to carbondioxide, water and energy

The main process in the metabolism of glucose

Glucose 6 phosphate

Glycolysis (Glycolytic path way pentose phosphate pathways glycogen

 Glycolytic path way
 Hexose monophosphate shunt (pentose phosphate pathway)

In the glycolytic pathway, glucose 6- phosphate is cleaved through a series of steps to triose phosphate
and finally to two molecules of pyruvate.
The conversion of glucose to pyruvate or lactate is anaerobic and takes place in cytoplasm of the cell
and provides two of ATP per mole of glucose metabolized.
35
The pyruvate can then be converted back to glucose – 6- phosphate a different pathway or can be
converted to lactate by the enzyme dehydrogenase (LDH)
Under aerobic conditions pyruvate under goes oxidative decarboxylation to form acetyl Co A which is
yet another pivotal molecule.
The degradation of acetyl-CoA provides the cells most of the energy potentially available from the
oxidation of glucose.
Acetyl co A enters the TCA- Tricarboxycyclic acid cycle and this is the aerobic cycle of glucose
metabolism and occurs in the mitochondria of the cell. It consists of a sequence of redox reactions in
which acetyl coA is oxidized to carbondioxide, water and 24ATPs- 12 form each mole of acetyl coA
are liberated
This ATP formation is coupled to the mitochondrial electron transfer system. The complete oxidation
of one mole of provides 38 moles of ATP(2 moles from glycolysis,24 moles from TCA, and the
remaining 12 moles are from the oxidation of NADH2 ) and other phosphorylation steps.
In the hexose monophosphate shunt, glucose 6- phosphate is converted to ribose-5 phosphate which is
pentose sugar with the production of NADPH which generates the reducing power and it is important
as energy source for anabolic processes such as fatty acids and steroids synthesis. It also plays a key
role for glycolysis in red blood cells which lack miton chondria and thus are not capable of oxidative
phosphorylation of the TCA cycle.
The ribose- 5 phosphate can further be converted to a triose phosohate and the triose is an intermediate
in carbohydrate metabolism.
If glucose is not needed for immediate energy release (provision) can be stored as glycogen. Here
glucose-6-phosphate is enzymatically polymerized by a series of steps

REGULATION OF GLUCOSE IN THE BODY

a. Process that remove glucose from the body

i. Oxidation of glucose to carbon dioxide and water in the tissue via the glycolytic pathway or the
hexose monophosphate shunt and tricarboxylic acid cycle with the production of carbon dioxide
and water and liberation of energy which is used in the formation of ATP.

ii. Glucose conversion to fatty acids in the adipose tissue. Much of the absorbed glucose is
converted into fatty acid the a adipose tissue via acetyl co-enzyme A

iii. Glycogen synthesis and utilization in the muscles. Some blood glucose is converted into
glycogen (glycogenesis) in the muscles which lack glucose 6-phosphate.

iv. Renal excretion when blood glucose concentration is high.

The glucose in the glomerulus filtrate is almost completely re-absorbed, urinary loss of glucose is
high so that the amount of glucose passing into the glomerula filtrate exceeds the maximum

36
 tubule re-absorption capacity or if tubule re-absorption is lowers in patients with low renal
threshold for glucose.

(a) Process that add glucose to the body

1. Gluconeogenesis.

Is a pathway that is important in maintaining blood glucose level especially during long term fasting.

Gluconeogenesis is the formation of glucose from non-carbohydrate sources such as amino acids,
proteins, glycoprotein of lipids. It’s not the reverse of glycolysis but rather is an oxidative process
involved in TCA cycle where glucose is form from non-carbohydrate source.

The blood glucose level is markedly constant under normal condition, under brief fasting condition;
glycogenolysis is replaced by Gluconeogenesis to maintain blood glucose level.

Glycolysis - the oxidation metabolism of glucose molecules to obtain ATP and pyruvatePyruvate from
glycolysis enters the Krebs cycle, also known as the citric acid cycle,

Glycogenesis - the conversion of excess glucose into glycogen as a cellular storage mechanism; this
prevents excessive osmotic pressure buildup inside the cell

Gluconeogenesis synthesis of glucose molecules from simple organic compounds. an example in

humans is the conversion of a few amino acids in cellular protein to glucose. (derivation of glucose
from non carbohydrates)
(a) Process that reduce glucose to the body

Glycolysis - the oxidation metabolism of glucose molecules to obtain ATP and pyruvatePyruvate from
glycolysis enters the Krebs cycle, also known as the citric acid cycle,

Glycogenesis - the conversion of excess glucose into glycogen as a cellular storage mechanism; this
prevents excessive osmotic pressure buildup inside the cell
Lipogenesis- process which involves formation of fats from excess glucose. These entire pathways
have delicate control mechanism with feedback inhibition or hormonal control mechanism despite the
changes of feeding and fasting.

Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain,
and red blood cells

- Glycogenolysis It is the breakdown of glycogen into glucose, which provides glucose supply for
glucose-dependent tissues. (Conversion of glycogen back to glucose) or

37
Is a process of breaking down glycogen in the liver to glucose in brief condition or fasting. This
process raises the blood glucose level.

The glycogen in the muscles do not has glucose 6-phosphate enzymes necessary for glycogen
breakdown to glucose.

(b) Hormones involved in glucose metabolism. (regulation)

1. Insulin.

Is the only hormone capable of lowering or preventing excessive rise in blood glucose,
therefore presence of insulin hormone in an inadequate amount will lead to one in increased
concentration of glucose in blood (hyperglycemia) and if it persists for long (chronic ), then it
is referred to as diabetes mellitus.

It is synthesized in beta cells of islets’ of langerhanns in pancreas.

Insulin increases carbohydrates utilization by all metabolic pathways and may also
diminish hepatic output of glucose. Insulin primarily increases cell permeability to glucose.

The secretion of insulin is stimulated by rise in blood glucose level and directly or indirectly
by gastral intestinal hormone certain sugars, fatty acids e.t.c

Absolute of relative deficiency of insulin as it occurs in diabetes mellitus causes rise in blood
glucose, depletion of glucose metabolism in the tissue.

Insulin increases glucose uptake and utilization in the muscles (glycogen) and lactic formation
and catabolism to carbon dioxide and water.

2. Glucogen
Is a polypeptide which is synthesis is in the pancreatic upper cells and gut. It increases
glycogenolysis in the liver and stimulates Gluconeogenesis.
3. Adrenaline
It is produced in the adrenal medullar. It increases glycogenesis and inhibits the uptake of
glucose by the tissue.
4. Growth hormone
It is synthesized in the interior pituitary gland. It blocks the uptake of glucose by muscles and
assists in Gluconeogenesis of fats.
5. Corticosteroid hormone e.g. hydro cortisone

38
 This is produced by the adrenal cortex. They are insulin antagonists, inhibiting carbohydrates
utilization by the tissues and promote the synthesis of protein instead of carbohydrates for the
production of heat and energy.
6. Thyroxin hormone
It is produced by thyroid glands. Its effect on glucose metabolisms are complex because its
influences the speed up many metabolic processes in the body as well as increasing the
sensitivity of many tissues
7. Somatosatim
It I produced by delta cells of the pancreas. Its effect on glucose level is minor however it
inhibits release of insulin and glucagon.
8. Epine phrine.
It is produced by the adrenal medulla or causes glycogenolysis thereby increasing glucose level
in the blood.
9. Somatomelins
It is produced in the liver. It has insulin like activity.

METHODS FOR GLUCOSE DETERMINATION.

Procedures commonly used for glucose determination can be classified as;
1- Chemical methods
2- Enzymatic methods
The enzymatic methods show increased specificity as compared to the chemical ones.
Most of the chemical methods are obsolete, because of lack of specifity and
cumbersomeness. However, they may be described briefly here because of their historical
interest.
1- Chemical methods:
a) Oxidation- reduction method; in the theory, in hot alkaline solutions glucose reduces
copper 11 ions to copper 1 ions. The enol form of glucose is favored in alkaline
solutions.
Diagram
The double bond and the negative charges on the enol form make an active reducing
agent. Since glucose, fructose only on the second carbon atom they all form the same
enodiol and are measured by any method based on reducing properties of glucose.
Pther reducing substances are also measured in this method. Examples of such
substances include: creatinine, ascorbic acid, uric acid etc

39
 Difference in the various methods is in their degree of specificity which in turn depends
on how effectively interfering substances have been removed during deproteinisation;
and the nature of the color producing reactions.
The methods include Somogyi Nelson method, Folin and Wu method, and alkaline
ferricyanide method.
b) Ortho- Toluidine method:
Is the most specific of the chemical methods. However, it uses ortho- toluidine, which is a
health hazard now classified as carcinogenic,
Principle:
Ortho-toluidine, an aromatic amine condenses with the aldehyde groups of aldohexoses
such as glucose in acid solutions to form a glucosylamine and the corresponding Schiff
base. By further re-arrangement produce a green chromogen whose absorbance is
measured at 630nm.
2- Enzymatic methods:
Are of two types. They use enzyme systems as reagents and they have specificity for
glucose. They are simple and rapid to use, they can easily be automated and they use
small volumes of samples.
Two enzyme systems are used; hexokinase and glucose oxidase. The generally accepted
method is the one based on hexokinase systems.
The hexokinase method:
This involves two coupled reactions.
Glucose + ATP glucose-6 phosphate + ADP
hexokinase

Glucose-6-phosphate dehydrogenase 6-phosphogluconate + NADPH +H

NADP (Nicotinamide Adenosine Dinucleotide Phosphate)

The increased in absorbance due to the formation NADPH is monitored at 340

spectrophotometrically and is propotional to the amount glucose present.
NADP is required as a co-factor when the enzyme glucose-6-phosphate dehydrogenase
is obtained from yeast and NAD is used when the enzyme comes from a bacterial source.
The hexokinase system can also be coupled to an indicator reaction using Phenasone
methosulphate and iodonitrotetrazolium salt so that absorbance can be measured in the
visible range.
The iodonitrotetrazolium is reduced by NADPH to form a colored product (pink)

Glucose oxidize method

40
 Glucose oxidase catalyses the oxidation of glucose to D-gluconolactone and hydrogen
peroxide. Glucose oxidase is highly specific for beta D-glucose. But in the aqueous
solutions glucose contains approximately 1/3 alpha D –glucose and 2/3 beta D-glucose,
in equilibrium . as the beta –form is oxidized by the glucose oxidase , the alpha –form
converts to the beta form by law of mass action.
Glucose oxidase preparations sometimes contain an enzyme mutorotase which hastens
the conversion from alpha to beta form of glucose, a precaution which is not necessary
for hexokinase method.
Measurement of blood/plasma glucose (glucose-oxidase – peroxidase method)
Purpose/value of test:
Plasma or blood glucose is measured mainly in the diagnosis and management of
diabetes mellitus
Principle:
Glucose oxidase (GOD) catalyses the oxidation of glucose to give hydrogen peroxide
(H2O2) and gluconic acid. In the presence of the enzyme peroxidase (POD), the hydrogen
peroxide is broken down and the oxygen released reacts with a chromogen, eg. 4-
aminophenazone, or perid and phenol to give a pink colour. The absorbance of the colour
produced is measured in a colorimeter using a green filter or in a spectrophotometer at
520-540nm
Specimen:
 Plasma from sodium fluoride-oxalate anticoagulated blood
 Serum can be used the assay is carried out within 30 minutes of collecting the blood
(without fluoride preservative the glucose is broken down by glycolysis).
NOTE. There are three different types of blood specimens you can collect for blood
glucose measurement namely:
-Fasting specimen
This is blood collected after a period of no food intake. For adults the fasting time is 10-
16hours and for children its 6hours

-Post- prandial specimen

This is blood taken after a meal has been taken. The specime is taken after 2 hours.
-Random specimen
The blood specimen is taken at anytime, regardless of food intake.
Equipment:
Colorimeter, spectrophotometer
Timer
Water bath
Reaction tubes
50µl,100µl, 10ml pipette.
Reagents:

41
Protein precipitant reagent
Sodium tungestate, dehydrate…………………………….5g
Di-sodium hydrogen………………………………………5g
Sodium chlgoride…………………………………………...4.5g
Phenol………………………………………………………0.5
Hydrochloric acid, 1mol…………………………………..62ml
Distilled water……………………………………………..to 500ml
1.weigh sodium tungstate, dehydrate, Di-sodium hydrogen phosphate and sodium chloride,
and transfer these to a 500ml volumetric flask.
2.half fill the flask with and mix until the chemicals are completely dissolved
3. add 62mlof 1mol/l of HCl and mix
4. weigh the phenol and to the flask, mix to dissolve .
5. Make up to 500ml with distilled water and mix well.
6. Transfer to reagent bottle, label and store at room temperature. The reagent is stable
indefinitely.
Coloring reagent:
1.4-aminophenozone, 5g/l…………………………….16ml
2.Fermcoczyme……………………………………….4ml
3.Phosphate buffer……………………………………150ml
- Di-sodium hydrogen orthophosphate……………….10g
- Potassium dihydrogen orthophosphate……………..10g
- Sodium azide………………………………………….2g
- Distilled water………………………………….to 500ml
-weigh the chemicals and transfer to 500ml flask
- Half fill the flask with the water and mix to dissolve the chemicals. Make up to 500ml with
water and mix well.
- Transfer to a resgent bottle, label and store at room temperature.
Stock glucose standard 100mmol/l
1.weigh accurately 1.8g of anhydrous glucose (analytical reagent grade)
To ensure that the glucose you are using is dry, heat in it in an open container in an oven at
60-800c for 4hours. Remove and close the container immediately. When cool, weigh the
glucose.
2.Transfer the glucose to a 100ml volumetric flask. Half fill the flask with 1g/l benzoic acid
and mix until the glucose is fully dissolved. Make up to the 100ml mark with the benzoic acid
reagent and mix well.
3.Transfer to a storage bottle and label. You can store the reagent frozen in atightly
stoppered container and it will be stable for 1year.
Working glucose standard 10mml/l
Add 10ml of stock glucose solution to 100ml volumetric flask and make up to 100ml mark
with distilled water.

42
Procedure:
Pipette into tubes as Blank Standard test
follows

Protein precipitant 1.5ml 1.5ml 1.5ml

Distilled water 0.o5ml - -

Standard 10mmol/l - 0.05ml -

Patients - - 0.05ml
serum/plasma

Mix well and centrifuge the tests to obtain clear supernatant fluids.
Take a second set of tubes and label them blank, standard, test
Transfer the 0.5ml 0.5ml 0.5ml
supernatant fluids
to the respective
tubes
Colour reagent 1.5ml 1.5ml 1.5ml
0
Mix well, incubate at 37 c for 10minutes
Read absorbance’s at 515nm
Calculate the concentration of glucose as follows
Glucose mol/l Ab of Test X conc of STD
Ab of std

Interpretation of results.
NORMAL VALUES
Serum, plasma (fasting) 64.8-115.2mg/dl or 3.6-6.4mmol/l
Serum, plasma (random) 59.4-133.2mg/dl or 3.3-7.4mmol/l
Having described the glucose oxidase method which uses the locally prepared
reagents you can also use the commercially prepared reagents you can also use the
commercially prepared reagents (kits) readily available on the market but always
remember to follow the instructions of the kit manufacturer how to use it when
performing the test.
Interpretation of blood glucose results
Increase (hyperglycemia):
A raised blood sugar level is called hyperglycemia. When there is significant hyperglycemia,
a diagnosis of diabetes mellitus is made.

43
- In an adult with symptoms, a random venous plasma glucose value of 11.1mmol/l or more
on two occasions is diagnostic of diabetes mellitus. The fasting blood glucose values of
7.8mmol/l or more on two occasions is also diagnostic of diabetes mellitus.
-Random blood sugar values of below 7.8mmol/l and fasting blood sugar value below
6.4mmol/l excludes a diagnosis of diabetes mellitus
- A mild hyperglycemia may accompany or occur in pancreatic disease or pituitary and
adrenal disorders. Steroid therapy may also cause hyperglycemia due to reduced
carbohydrate metabolism.
- Transient hyperglycemia often occur occurs following severe stress e.g. in surgery, injury,
shock, infections or severe burns.
Decreases (hypoglycaemia):
A low blood glucose level is called hypoglycemia. Persistent occurrence of hypoglycaemia
with glucose levels of less than 2.2mmol/l accompanied by symptoms such as fainting,
confusion or violence should be investigated.
Causes of hypoglycaemia
-kwashiorkor
-severe liver disease
-alcoholism
-insulin secreting tumors.
-Addison’s disease.
-certain drugs e.g. propanolol, salicylates and disopyramide.
-over treatment of diabetes mellitus. Treatment of D.M without appropriate monitoring can
result in hypoglycemia.
Neonatal hypoglycemia:
Newborn infants may suffer hypoglycemia when blood glucose levels fall below
1.1mmol/l.
Infant particularly at risk are;
-under weight poorly nourished babies.
-Twins
-Premature babies.
-Babies born of diabetic mothers.
DIABETES MELIITUS.
Objectives.
By the end of this session, the students should be able to:
1. Define diabetes mellitus.
2. State the different classes of diabetes mellitus.
3. Differentia between diabetes mellitus and Diabetes. inspidus
4. Make diagnosis of diabetes mellitus.

44
Definition:
Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which
glucose is underutilized, producing hyperglycemia.
It can be defined as a state of diminished insulin action due to its decreased availability or
effectiveness, resulting in chronic hyperglycemia, usually accompanied by glucosuria and
other biochemical abnormalities, expressed as a wide range of clinical presentations ranging
from asymptomatic patients with relatively mild abnormalities to patients admitted to hospital
with severe metabolic decomposition if rapid onset that has lead to coma. E.g. acidosis and
hyperosmolar coma.
As the disease progresses individuals are at increased risk for development of specific
complications, including Retinopathy leading to blindness, renal failure, Neuropathy (nerve
damage), Atherosclerosis-which may result in stroke, Gangrene, or coronary artery disease.
Classification of diabetes mellitus:
1. Primary (idiopathic) diabetes; caused by:
-In ability of the pancreas to produce sufficient insulin (from the earliest stages of the
disease)
-Substances in the blood which inhibit or interfere with the action of insulin.
-Production of an abnormal form of insulin.
Types of primary diabetes;
a) Type 1 diabetes:
Type 1 diabetes mellitus was formally called insulin Dependent Diabetes Mellitus (IDDM) or
Juvenile onset Diabetes Mellitus. It is an acute and severe form of the disease in which
insulin is lacking following the destruction of islet cells in the pancreas possibly by viruses,
production of autoimmune antibodies which damage islet cells and or genetic factors. Human
leucocyte antibodies have also been associated with development of this type of diabetes.
Symptoms such as polydipsia and rapid weight loss usually present acutely.
Diabetes individuals have insulinopenia (a deficiency of insulin) because of loss of pancreatic
islet beta cells and depend on insulin to sustain life and prevent ketosis.
The peak incidence of this disease occurs in child hood and adolescence. About 75% of the
cases occur before the age 30years, but the remaining percentage occurs at any age.

b).Type 11 diabetes mellitus:

This type was formally known as Non-Insulin Dependent Diabetes Mellitus (NIDDM) or
Maturity onset Diabetes mellitus, it comprises about 90% of the all the individuals with
diabetes mellitus. This disease is usually less acute than the juvenile type. The individuals
have minimal symptoms and are not prone to ketosis and do not depend on insulin to
prevent ketonuria.
The disease usually develops after 40years of age, but type 11 diabetes may also occur in
young people particularly those who are obese.
2-Secondary diabetes mellitus

45
Is caused as a result of;
-Pancreatic disease such as pancreatitis in which insulin secretion is reduced
-Endocrine disorder in which there is an abnormal secretion of hormones that make the
action of insulin ineffective e.g. Cushing’s syndrome, acromegaly and phaeochromocytoma.
-Drugs that interfere with carbohydrate metabolism e.g. beta blockers, corticosteroids
oestrogen containing contraceptives and thiazide diuretics.
-Occasionally genetic disorders such as Down’s syndrome or Turner’s syndrome.
Summary of the general characteristics
Characteristics Type 1 DM Type 11 DM
Age at on set Mostly common below 30 Common above 30 years
years
Associated with obesity Yes No
propensity to keto acidosis
requires insulin for control
Endogenous insulin Extremely low to Significant relatively to plasma
secretion undetectable plasma level glucose accompanied by
resistance
Islet cell antibodies Yes No
diagnosis
Islet pathology Selective of most B- cell Smaller than appearing islets
cells
Associated risk of Yes Yes
retinopathy vascular
disease

LABORATORY DIAGNOSIS
The diagnosis of diabetes mellitus may be suggested on the basis of the patient’s history or
by the results of side tests for glucose on urine specimens. However, urine glucose
measurements are inadequate by for themselves diagnostic. Although convenient and
sensitive; they yield many false positive results, and in fasting diabetes, may yield false
negative results. A provisional diagnosis of diabetes mellitus must always be confirmed by
glucose measurement on blood specimens.
Laboratory diagnosis of type 1 diabetes is straight forward based on clinical symptoms and
history, confirmed by significant hyperglycemia on more than one occasion.
Type 11 may be more difficult and early diagnosis is important to avoid development of micro
vascular diseases.
The following tests are important in diagnosing both types:
1)- Fasting blood glucose:

46
Normal fasting plasma glucose after 10-16 hour fasting is given as 60-90mg/dl (it may
depend on the analytical method). Values tend to increase with age. Elevation of fasting
plasma glucose indicates diabetes mellitus. Plasma glucose levels of greater or equal to
140mg/dl (7.8mmol/l) on more than one occasion is almost diagnostic of diabetes mellitus.
2) Urine glucose
In diabetes mellitus, glucose levels exceed the renal threshold for glucose which is about
160-180mg/dl (8.9-10mmol/l). When this occurs glucose is usually excreted in urine. The
measurement of urine glucose is normally used as a screening test for diabetes mellitus and
as a guide to insulin therapy.
Note: positive urine test results must be confirmed by measurement of blood glucose. This
takes care of renal glucosuria.
3)- Two hour post prandial plasma glucose is measured two hours after a patient has
consumed a meal of approximately 100g of glucose mixed with other food stuffs.
Plasma glucose levels of greater than 200mg/dl (11.1mmol/l) is indicative of diabetes
mellitus. A plasma glucose value less than 140mg/dl is normal.
Although the test is very simple, it is difficult to control the meal content and the time of
consumption of the meal.
4) Oral glucose tolerance test (OGTT):
This is used to establish the diagnosis of diabetes mellitus in patients who have plasma
fasting glucose of less than 140mg/dl.
It consists of serial measurements of plasma glucose and after an oral administration of
glucose dose.
GLUCOSE TOLERANCE TEST
Objectives:
By the end of this session, the students should be able to;
1-Define glucose tolerance test.
2-Explain the significance of glucose tolerance test.
3-State the principle of glucose tolerance test.
4-Prepare a patient for glucose tolerance test.
5-Perform glucose tolerance test.
6-Interpret glucose tolerance test results.
Definition:
Glucose tolerance a test that measures the ability of the body to cope with a standard dose
of glucose introduced orally or intravenously into the body. As the glucose is introduced into
the body, it is absorbed into the tissues leading to a rise of glucose levels in circulation. The
pancreatic cells are stimulated to produce insulin hormone that tries to lower the glucose
levels through stimulation or inhibition of metabolic processes.
Value of the test:
The main value of glucose tolerance test is that it may help to diagnosis of diabetes mellitus
or impaired glucose tolerance at a time when the metabolic abnormality is mild.

47
The glucose tolerance is particularly valuable in this diagnosis of impaired glucose tolerance
in pregnancy.
Glucose tolerance test may also be used to evaluate a patient with unexplained nephropathy,
retinopathy and neuropathy with random glucose concentration of less than 140mg/dl.
It may also be used for epidemiological studies.
Note:
It has recommended that diabetes mellitus may be diagnosed with either a raised fasting or
2-hour blood glucose value. However, some patients may not have overnight properly or may
have alarmed by the venepuncture and this may have false fasting blood glucose.
Diagnosis of diabetes mellitus should not be made from a single blood glucose
measurement, unless this is greater than 11.1mmol/l and the clinical history unequivocal.
Routine glucose tolerance tests are unnecessary if the fasting plasma glucose is greater than
8.0mmol/l. Random plasma of 11.1mmol/l or more in children and adults is evidence of
diabetes mellitus and a GTT is usually unnecessary if there are classic symptoms o f this
disease.
Plasma glucose value greater than 11.1mmol/l at 1 or 2hours after the oral dose confirms
diabetes mellitus.
In the absence of classic things of diabetes, fasting values between 6.1mmol/l and 7.8mmol/l
and 2hours values between 7.8mmol/l and 11.1mmol/l suggest impaired glucose tolerance
which may be due to hypothyroidism, severe liver disease, Clushing’s syndrome, active
acromegally and sometimes sepsis and carbohydrate starvation.

Principle of the test:

Standard dose of 75g (1.75g/kg body weight) of glucose is in about 250-300ml of clean water
is to a patient orally after 10-16hours of fasting and the patient’s body response will be
monitored at 1/2hour interval for 2hours.
Preparation of a patient for Oral Glucose Tolerance Test (OGTT)
1. The patient is advised to be on an unrestricted diet containing about 150-300ml of clean
water is to a patient orally after 10-16hours of fasting and the patient’s body response will be
monitored at 1/2 hour interval for 2hours.
2. Test is performed in the morning after an overnight fast. Therefore, the patient is instructed
t fast for 10-16 hours if adult or 6hours if a child. The patient must not smoke on the test
(before or during the test). The must not drink or eat anything during the test other than the
glucose dose.
3. The patient must not be suffering from the effects of trauma or be recovering from a
serious illness.
4. Drugs such as corticosteroids and diuretics should be stopped or discontinued when
preparing for OGTT since they may impair glucose tolerance.

48
5. The patient should not indulge him or herself in an unaccustomed or discontinued when
preparing for OGTT.
6. First counsel the patient just before the test and give the patient enough time to rest after
arrival at the health unit.
7. Instruct the patient to sit in an upright position during the test. Or if the patient is lying
down, should be lying over on the right side so as to facilitate rapid emptying of the stomach.
Steps followed when performing OGTT;
-Prepare a GTT chart on which to record the collection times and the test results.
-Collect the fasting blood specimen just before administering the glucose.
-Administer a standard dose of glucose (75g anhydrous glucose or 82.5g of glucose
monohydrate) dissolved in about 250-350ml of water, flavored with lemon or chilled or both
to avoid nausea, is given by mouth.
Note: small amounts of glucose e.g.1.75g/kg body weight of glucose monohydrate not
exceeding 75g or 1.92g/kg body weight of glucose monohydrate not exceeding 82.5g is
given to children or small adults.
-Blood specimens are collected at 30 minute interval for up to 2hours, into a fluoride bottle.
-Urine specimens are also collected (fasting and after the glucose dose). Every time blood
specimen is collected, urine must also be collected.
-Measure the blood glucose concentrations and estimate the glucose levels in all the urine
specimens collected, using uristix. Urine sugar measurements will help to identify patients
with renal glucosuria.
-Record the results on the chart and interpret the results

Glucose tolerance responses:

Graphical representation.

Time (minutes)
Key.

49
× × Normal response
Diabetic response
-------------- lag storage response
×------------× flat response
Normal response;
In this type of response, the glucose fasting levels are in the normal reference range of about
70-90mg/dl. Following the oral glucose intake, the glucose blood concentration will rise within
the first hour, but will not exceed its renal threshold of about 150mg/dl. After the 3hours, the
glucose levels will go back to the normal random blood glucose levels.
There will be no glucose passed out in the urine.
Diminished (Diabetic) responses:
In this type of response, the glucose fasting levels are above the normal fasting reference
range that is between 70-90mg/dl. Following the oral glucose intake, the glucose blood
concentration will rise higher than in normal individuals. Values will remain higher for a longer
period of time than for normal individuals.
After the 2hours, values will not go back to the normal random blood glucose levels.
Glucose will be passed out in the urine specimens collected.
This phenomenon implies that the body cannot tolerate the glucose that has been introduced
into the circulation.
Flat response to OGTT:
This describes the response in which the plasma glucose concentration fails to rise
significantly. If the fasting blood glucose concentration was e.g. 5.0mmol/l, the 30min. and
the 60min. and the 90min….,or all specimens would normally have a value of about
7.0mmol/l or more in a GTT.
A flat response is often caused by incorrect positioning of the patient during the test, resulting
in delayed gastric emptying.
It is occasionally due to intestinal malabsorptive disease, it may also occur in hypothyroidism
and in adrenal hyposecretory states.
Lag storage response:
Lag storage response to GTT describes the pattern results in which there is a sharp rise in
plasma glucose concentration with the peak value appearing and sometimes exceeding
11mmol/l. there is also a tendency for the 2hour value to be much lower than the fasting
value. There may be glucosuria. This pattern of response is due to rapid absorption of
glucose from the intestine; the tissues (mainly liver) are unable to attain sufficiently rapid
uptake to match the rapid absorption.
It may be seen after gastric surgery (e.g. gastroenterostomy) or in patients with severe liver
disease, and is sometimes seen in apparently healthy individuals.
Renal glucosuria:
This is applied to patients who exhibit glucosuria at some point during the GTT although the
plasma glucose concentrations at all times remains below the renal threshold for glucose.

50
It is due to lowered renal threshold for glucose and usually has no pathological significance.
However, it may be associated with other renal tubular defects such as Fanconi syndrome.

RENAL FUNCTION TESTS

Introduction

Renal function tests (kidney function tests) are used to assess the status of the kidney and
rule out any renal dysfunction. The commonly used tests to monitor kidney function are;
urinalysis, measurements of urea, creatinine and serum electrolytes. It is important that when
testing for kidney function, both creatinine and urea are measured. This is useful to
determine whether the condition is pre-renal, renal or post renal.

Urine analysis (urinalysis)

Introduction

Renal function tests as already highlighted are important in assessing the function of the
kidney and other disease conditions. Urine examination is one of the tests that used in the
assessing the functional status of the Nephron that include the glomerular filtrate, rate,
secretory capacity of particular exogenous and endogenous compounds and its reabsorptive
capacity. The composition of urine not only reflects the state of the kidneys physiologic or
pathogenic but also changes in the acid-base and electrolyte balance and in the metabolism
of hormones, carbohydrates, proteins, salts, and many other physiologic and non physiologic
substances and drugs.

Learning objectives

By the end of this session you should be able to:

I. Define the following terms

 Nephron
 Urine
 Urinalysis
II. Describe the formation of urine by functional unit of the kidney

Definitions

 Nephrons:
51
 A Nephron is a functional unit of the kidney; it is composed of different structures
namely: Bowman’s capsule that houses the glomerulus, the proximal, loop of henle,
distal and collecting duct tubules. It is the one that is involved in blood filtration leading
to the formation of urine.
 Urine:
This is a liquid containing water and water soluble waste products removed from the
blood stream via the kidneys i.e. Organic and inorganic products e.g. urea, uric acid,
creatinine, salts (sodium, potassium, chloride, phosphates, ammonium). The urinary
system consists of the kidneys, bladder and the urethra.

The formation of urine takes place in the functional units (Nephrons) then it passes to the
bladder for temporal storage by way of the urethra and eliminated from the body through the
urethra.

 Urinalysis:

Urinalysis is a physical, chemical, and microscopic analysis (investigation) of a urine

sample.

Formation of urine by the nephrons

Urine formation is done by the functional units of the kidney called the nephrons. The kidney
performs its role in order to maintain the internal body constituents constant. The regulating
of the internal environment is known as homeostasis. Before we describe the process of
urine formation, let us first review the structure of the kidney and the functions of the kidney

THE STRUCTURE OF THE KIDNEY

Renal artery

Medulla renal vein

Medullary pyramid Pelvis

52
 Urethra

Cortex

In the body there are two kidneys that are flattered bean shaped capsulated organs divided
into an outer region called the cortex and an inner region called the medulla.

FUNCTIONS OF A KIDNEY

1. Maintenance of water and electrolyte balance of the body. The amount of water
and electrolyte in the body regulated by the kidney by the help of Anti-diuretic
(ADH) and aldosterone hormones so that the body fluids are restored to the normal
composition and volume.
2. Maintenance of PH of blood
i. The normal PH of blood is between 7.36-7.45 and the kidneys are responsible
for removing substances that cause it to become acid or more alkaline.
ii. Substance of acid reaction are waste products of protein metabolism e.g. urea,
uric acid, and creatinine that are constantly being formed and therefore
reaction, likewise they should be excreted for in both cases if their
concentration is allowed to rise, the PH of blood will be altered leading to rise,
the PH of blood will be altered leading to physiological process upset
3. Excretion of drugs and toxins:
Drugs after completing their action in the body leave waste products that are
excreted by the kidneys. If the drugs are not metabolized, them selves are
excreted. Toxins are rendered harmless by detoxification in the liver and the
conjugated compounds so formed excreted in urine.
4. Production of erythropoietin substance necessary for the normal production of red
cells in the born marrows
5. Production of rennin that is proteolytic enzymes that stimulates the causes of the
constriction of arterioles
6. Re absorption of useful filtered compounds back to the body e.g. glucose and
amino acids.

Urine formation is done by the functional units of the kidney called the nephrons.
The kidney plays its role in order to maintain the internal body constituents
constant. The regulating of the body internal environments is known as
homeostasis:
53
 The following processes achieve this homeostasis:
1. Filtration of body plasma by the glomerulus
2. By selective re-absorption by tubules of the essential materials e.g. salts,
glucose, water.
3. By secretion of certain substances into the filtrate e.g. drugs (penicillin), H+,
NH4, Uric acid

NEPHRON

Afferent arteriole efferent nerve

Glomerulus

Bowman’s capsule

To other nephrons

P.C.T

Proximal convulated

Tubule

Descending limb
Ascending limb

Collecting duct
to urethra

Loop of henle

The Nephron consists of the glomerulus that is made up of tuffs of arteriolar capillaries that
are encapsulated in a bowman’s capsule. The Bowman’s capsule is a double walled
epithelial sac surrounding the glomerulus. This then leads to the tubules, which finally join to
the collecting duct. Along the duct there is proximal, distal and loop of henle tubules.

54
There are about 1 million nephrons in a single kidney but not all function at the same time
some act as reserves.

GLOMERULAR-FILTRATION

Blood enters the glomerulus through the afferent arteriole and leaves it through the efferent
arteriole. The process of filtration takes place between the glomerulus and the glomerulus
and the glomerular capsule.

This is possible by the hydrostatic net pressure of 25mmHg which is as a result of higher
(75mmHg) pressure being exerted in the glomerulus by about 1200mls(25% hearts out put
per minute) of blood circulating in the kidney overcoming the opposing pressure of 50mmHg.

The diameter of the efferent vessel is smaller compared to the afferent arteriole this creates
in pressure that leads to glomerular filtration. The hydrostatic net pressure of 25mmHg is the
energy used in the filtration.

Substances with a large molecular weight (above 70,000 MW e.g. cells and plasma proteins
are not filtered through the semi-permeable membrane of the capsule. The glomerular
filtration rate is controlled by a number of factors, plasma flow, blood pressure, anatomic
changes with in the arteriole, number of glomeruli functioning at a given time and resistance
to tubular flow. Therefore any change in any of the above factors leads to a fall of the filtrate
volume. The glomerular filtrate has the same composition like the blood plasma except it is
protein free except for less than 1% albumin. It contains about 5.5mmol/l glucose, 150mmol/l
sodium, 15mg/l urea, and 1mg/dl creatinine.

NOTE:

Of the 1 liter of blood passing through the capsule approximately 120-130ml is filtered per
minute (glomerular filtration rate). After filtration in the glomerulus, water, glucose,
electrolytes, amino acids, and waste products of metabolism (urea, uric acid, and creatinine)
pass from the blood into the Bowman’s capsule.

SELECTION RE-ABSORPTION

From the Bowman’s capsule the filtrate moves to the proximal convulated tubule (PCT). Here
about 85% water is re-absorbed passively together with the electrolytes absorbed actively
(Na+, K+, Cl-, HCO3). All threshold substances such as glucose, amino acid, lactose, ascorbic
acid, which are normally present in the blood and not in the urine actively reabsorbed across
the cell membrane of PCT into circulation.

From the PCT, the modified filtrate moves down to the descending limb of the loop of henle
where both electrolytes and water continue to be reabsorbed, as the walls are impermeable
to water molecules are \taken in causing the filtrate to become hypotonic.
55
From the loop of henle the filtrate moves to the distal convulated tubules (DCT) where the
remainder of (Na+, K+, Cl-, HCO3). Salts are reabsorbed, sodium is reabsorbed under the
influence of (aldosterone hormone) produced by the adrenal cortex in case the plasma
electrolyte (sodium) is reduced. This hormone increases the reabsorption of sodium in the
tubules.

The filtrate then joins the collecting duct (C.D) where the remainder of water and the
electrolytes are reabsorbed.

NOTE:

”antidiuretic hormone” controls water reabsorption in the D.C.T and collecting duct. Its
release is controlled by the osmotic pressure of blood (osmo-recepters in the posterior
pituitary gland). If blood passing through the pituitary gland is concentrated e.g. in the
dehydration then the gland produces ADH that makes the DCT and D.C more permeable to
water.

However if the body needs to excrete more water, the production of ADH falls and the tubes
become more impermeable to water which results in more water being excreted in the urine.
At times in the decrease conditions when the ADH is produced in inadequate amounts more
water is passed in the urine in large volumes a condition known as ”diabetes inspidus”.

From the collecting duct the filtrate emerges as urine consisting of organic compounds i.e.
urea, uric acid, creatinine, water, salts (Na+, Cl-, K+, NH4, PO43, SO42-)

NOTE

Certain substances like drugs i.e. penicillin are secreted into the convulated tubule and
passed out in urine, H+, NH4, are also secreted into the filtrate to maintain to the correct acid
base balance in the body.

RENAL THRESHOLD

A threshold of a substance in the highest level at which the constituent is present in

the blood before it appears in urine.

The constituents of the glomerular filtrate have either a high, medium, low or no threshold
value. For example; glucose is a high threshold substance because it is completely
reabsorbed from the filtrate and only appears in urine when the blood level is about 150mg/dl
(8.33mmol/l) i.e. it is normal threshold value.

Urea has a threshold value of zero because only small amount of it is reabsorbed in the
tubules and it is always present in urine no matter what the blood level happens to be.

56
Creatinine is a no threshold of any substance as it is not reabsorbed and is always present in
urine. The threshold of any substance can be altered by impaired renal function.

Note: the urine analysis yields useful information in many abnormal conditions e.g. infections
of the kidney, urethra and the body as a whole. Normal urine consists of approximately 95%
water, urea, uric acid, creatinine, Na+ , s K+, Cl-, Ca2+, HCO3 . Surplus acids and alkalis to
maintain acid base balance in the body.

The composition of urine varies from day to day depending on the food and fluid in take.

Standard operation procedure of urine examination

Introduction:

Performing any test procedure in the laboratory requires that a standard format is followed for
the production of quality and reliable results. Therefore when examining microbiological
specimen’s up to date steps should be followed if good quality results are to be obtained

Objectives:

By the end of this session you should be able to:

 Define standard operating procedure (SOP)

 Outline the importance of SOPs
 Describe the components of SOPs
 Describe the SOPs for the examination of urine

Standard operating procedures (SOP)

Standard operating procedure (SOP) is a set of instructions or steps some one follows to
complete a job or a task safely and correctly producing reliable results. The SOP is reviewed
from time to time and kept up to date using appropriate valid technologies.

Importance of SOPs

SOPs, some times referred to as the local laboratory bench manual are required for the
following reasons:

 To improve and maintain the quality of laboratory service to patients and identify
problems associated with poor work performance.
57
  To provide laboratory staff with written instructions on how to perform tests consistently
to an acceptable standard in their laboratory
 To prevent changes in the performance of tests which may occur when new members
of staff are appointed. SOPs also help to avoid short-cuts being taken when
performing tests.
 To make clinical and epidemiological interpretations of test results easer by
standardizing specimen collecting techniques, test methods and test reporting.
 To provide written standardized techniques for use in training of laboratory personnel
and potential publication in scientific journals.
 To facilitate the preparation of a list and inventory of essential reagents, chemicals and
equipment.
 To promote safe laboratory practice.

Important features of SOPs

SOPs must be:

 Applicable and achievable in the laboratory in which they will be used

 Clearly written and easy to understand and follow
 Kept up to date using appropriate valid technologies

Components of a SOP

SOPs must be written and implemented by a qualified, experienced laboratory officer, and
followed exactly by all members of staff.

For each SOP it is best to follow a similar format with the information present under separate
headings. Each SOP must be given a title and identification number, and be dated and
signed by an authorized person.

A list of staff able to perform the test( unsupervised and supervised) should be identified in
the SOP. There should also be an indication of the cost of the test.

Here below is a suggested layout for district laboratory SOPs.

Title of SOP

Authorized signature: Number:

Date:

58
Staff able to perform test:

 Unsupervised: list names

 Under supervision: list names

Value of test
Principle of a test
Specimen
Equipment
Reagents/stains
Controls
Method of test
Reporting results/interpretation
Safety measures
Sources of error
References

Contents of each of the components

Purpose of the test

State the main objectives/overall reasons of carrying out the test

Value of test

State the reasons/clinical significance for performing a test, i.e. clinical and or public health
reasons. Example: to detect, identify, and quantify malaria parasites in a person with
suspected malaria (malaria test)

Indicate any relevant limitations of the test.

Principle of the test:

State briefly the technology used. Examples: microscopical examination of fields stained
thick blood film for malaria parasites(malaria test) or chemical reagent strip test to detect
glucose in urine based on glucose oxidase reaction(urine glucose test)

Specimen:

State the specimen required and how to collect it including:

- volume required

59
 - container and its preparation
- use of any anticoagulant or stabilizer/preservative
- collection procedure(for adult and child) with health and safety notes
- how container should be labeled
- stability of specimen and requirement for storage and transport
- time within which the specimen should reach the laboratory

Describe the checks to be made when the specimen and request form reach the laboratory
and criteria which may make it necessary to reject the specimen.

State if the specimen requires priority attention (e.g. c.s.f)

Equipment

List the items of the equipment needed to perform the test. Main items of equipment such as
a microscope, centrifuge, colorimeter, incubator, water bath/heat block, should be listed in a
separate appendix (e.g. appendix B) at the back of the sop manual and given a number
which can be referenced in each sop (e.g. B/1, B/2).

Reagents/stains

List reagents, stains, reagent strips needed to perform the test. Include the reagents and
stains in a separate appendix at the back of the SOP manual.

Controls

List controls and source(s) to be used in the test e.g. positive and negative controls in
serological tests, control sera in clinical chemistry assays, positive and negative controls in
urine chemical tests.

Method/procedure steps to be followed while performing the test

Describe in a numbered sequence how to perform the test. For quantitative tests include
details of calibration, use a graph of factor, and calculations. Describe how to control the
procedure and also the health and safety measures which apply. Full details of safely
procedures should be included in a separate health and safety appendix. E.g. appendix C
with each procedure being given a number, (C/1, C/2) which can be referenced in the SOPs.

Reporting results/interpretation

State how the test should be reported, including:

60
 - units to be used and format of reporting(explain any abbreviations)
- accepted reference ranges for a quantitative test
- action to make if a result is seriously abnormal or un expected e.g. need for
verification, additional testing and all immediate notification of the result
- give target turn-around time for issuing the report(routine and urgent)
- interpretation comments that should accompany the test result

Sources or errors

Summarize the important and the commonest causes of an incorrect test result. Examples;
samples not well mixed, smear too thick for staining, inaccurate measurement (pipetting) of a
blood or serum sample dots in anticoagulated blood sample, air bubbles in the solution when
using a colorimeter or the sides of the cuvette not being clean and dry.

References

List the main sources of the information contained in the SOP, e.g. journal, published paper,
and manufacturer’s leaflets, WHO guide lines or documents, a test book.

Having described the components of the SOPs, now let us discuss the SOP for urine
examination.

Standard operating procedures of urine examination

Purpose:

Urine is examined to detect diseases of the kidney and urinary tract (renal pelvis, urethra,
bladder) as well as metabolic diseases of the body for example diabetes mellitus.

Principle:

Urine examination involves assessing the physical, chemical properties and urine
microscopy. The physical properties include; appearance of urine for colour and turbidity,
volume, and odour. The chemical properties include; PH, specific gravity, protein, glucose,
ketones, blood, nitrites bilirubin, urobilnogen and microscopic examination done after
centrifugation for cells, casts, crystals, amorphous salts, bacteria, and parasites affecting the
urinary tract.

Specimen

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There are six types of urine specimens collected in a health laboratory for diagnosis of
different disease conditions namely:

Random or spot urine specimen

This is urine collected at any time of the day into a clean universal bottle. It is used for
qualitative chemical testing for PH, proteins, glucose, ketones, bilirubin, urobilinogen, blood
nitrites and specific gravity.

Mid-stream urine specimen

 To collect mid-stream urine (MSU), instruct the patient to pass a small amount of urine
into the toilet or latrine to ensure that bacteria, cells or parasites that have entered the
urethra from the vagina or perinea area are flushed away.
 Collect about 20ml of urine in a sterile universal bottle and then pass the remaining urine
in the bladder into the toilet. This sample is used for microscopy and culture to
investigate bacterial infections of the urinary tract.

First or early morning urine specimen

 Provide the patient with a clean container and tell the patient to empty the bladder into
the toilet or latrine before retiring for the night and on rising in the morning to collect all
urine into the container.
 The urine sample is used to detect mycobacterium infection of the urinary tract and
pregnancy.

Terminal urine specimen

 Provide the patient with a clean container and tell the patient to empty the bladder into
the toilet and collect the last portion of urine in the container.
 This sample is used to demonstrate ova of schistosoma haematobium

First voided urine specimen

 Provide the patient with a clean container and instruct him to collect the first drops of
early morning urine
 It is used to demonstrate trichomonas vaginalis in males

24 hour urine specimen

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  Provide the patient with a clean container containing a specific preservative
depending on the analyte to be detected in the urine specimen or it can be
refrigerated. For example:
o 40% formalin is used for preserving samples for qualitative chemical testing, 10ml
for 24 hour urine.
o 10ml concentrated hydrochloric acid is excellent for preserving urine for hormones
such as adrenaline, nore-adrenaline, catecholamine, vanillylmandelic acid and
steroids. It lowers the PH, destroys formed elements and may precipitate uric acid.
o Thymol one 5mm crystal/dl of urine inhibits bacteria and fungal growth. An excess
may give false positive results for precipitation tests for albumin.
o Sodium carbonate 5-10gm is used to preserve samples for porphyrins
determination and samples must be protected from light by wrapping the container
in foil.
o 5ml hibitaine (chlorhexidine gluconate 200g/l) is used to preserve samples for 6
weeks at room temperature for glucose measurement.

Note:

 In infants and babies, a random urine sample collected as cleanly as possible may be
used for all types of urine investigation, including microscopy and culture.
Clean the perineal area carefully with soap and water and rinse thoroughly with clean
water. Dry and leave the perineal area uncovered. Ask the mother to sit with the child
after feed if possible.
Provide the mother with a sterile bottle and ask her to catch a few drops of urine as
soon as the child urinates.

Reagents and materials

1. 25% sulphosalicylic acid

2. Benedict’s solution
3. Fouchets reagent
4. Ehrlichs reagent
5. Absolute methanol
6. Zinc acetate (power)
7. Tincture of iodine
8. Rotheras powder
9. Urine dipsticks commercially available-singly or in combination for detection of PH,
specific gravity, protein, glucose, ketones, blood, bilirubin, urobilinogen.
10. Universal bottle
11. Centrifuge tubes
12. Glass slides
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 13. Cover slips
14. Pasteur pipettes
15. Gloves
16. Laboratory register

Procedure

Urine examination involves three stages that is assessing its physical and chemical
properties and urine deposit microscopy

1. Physical properties
Microscopic examination for:-
- Colour
- Turbidity
- Foam
- Volume
- Odour
1. Qualitative chemical testing for:
- PH
- Protein
- Glucose
- Ketones
- Blood
- Bilirubin
- Urobilinogen
- Specific gravity
- Nitrite
2. Microscopic examination of urine for:
- Cells
- Casts, crystals and amorphous salts
- Bacteria parasites

Steps followed for urine examination

 Check the clinical history on the laboratory request form and instruct the patient
how to collect the required specimen of urine.
 On receiving the specimen, label the container with the patient laboratory number
using a maker. Secure the lid of the container and gently shake the urine sample
 Observe the appearance of the urine and record the findings as follows
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Technique of using Albustix
Dip the test end into freshly passed uncentrifuged urine for about a second.
Remove excess urine and compare the colour of the impregnated part with the colour chart
within a recommended time by the manufacturer e.g.30-60sec. if the test end remains yellow,
the urine contains no proteins.
General precautions of using Test strips.
1. The urine sample container should be cleaned and free from contaminants e.g.
disinfectants and detergents.
2. Do not touch the test end of the strip.
3. Re-cap the strip container as soon as the strip is removed to avoid the trip from the
moisture
4. Do not leave the strip in urine for a long time as the impregnated part might dissolve.
5. Read the strip in bright white light.
NB
For the strips;
(a) Positive results are obtained with globulin, Hb, albumin and Bence Jones protein when
using test strips.
(b) Alkaline urines give false negative results while false positive results are obtained if
the urine is acidified.
(c) The strips are not affected by urine turbidity, x-ray contrast media, drugs and their
metabolism.
Quantity estimation of proteins in 24hr urine sample.
This test is used to assess the renal function of the kidney. Urine is collected for 24hrs and a
preservative and the concentration of proteins passed out within that period is determined by
use of the formula.
Conc. Of protein (mgdl-1) × total vol. of urine passed out
100
Ref. range: 25-150mg protein/24hrs
Procedure
Determine total volume of urine passed out
Determine the protein concentration by use of a test strip
Calculate the concentration of protein per 24hrs by use of formula above
Quantitative method using 3% SSA
QUALITATIVE ESTIMATION OF REDUCING SUBSTANCES IN URINE
A reducing substance is one which reduces a blue alkaline CuSO4 solution to red cuprous
oxide. The most important reducing substances are;
(a) The carbohydrate
65
e.g. glucose, fructose, galactose and pentoses (ribose, xylose and Arabinose), Lactose most
of disaccharides are reducing sugars however sucrose and trehalose are non reducing
sugars
NB.
Other substances apart for the carbohydrates can also reduce the CuSO4 to red cuprous
oxide e.g. homogentisic acid
This is excited in a condition called alkaptonuria a congenital metabolic disease where the
metabolism of tyrosine and phenyl Alanine is defective. A greenish brown or black colour
results from boiling such a urine sample with benedicts. The precipitate appears brown forms
but when it settles to the bottom of the tube, it is seen as cuprous oxide.
(b) Uric acid and creatinine
Are protein waste products formed during protein metabolism. However , they cause a slight
reduction when found high concentration.
(c) Glucoronic acid
Is present in traces in normal urine but excreted in high concentration when drugs are being
taken e.g. salicylic acid aspirin, Morphine, Menthol, Penicilin.
(d) Preservatives e.g. Formaldehyde
NB.
The alkaline CuSO4 solution is reduced by all sugars however a part from glucose, other
sugars even if they are encountered in urine samples, they are harmless.
The reducing sugars in the lab are detected by use of
(i) Benedict’s qualitative test
(ii) Clinitest method (Tablets)
(iii) Glucose reagent strip
Benedict’s reagent
Composition
Na citrate…………………………. 173g
Top up 1L after mixing of Distilled water
Anhydrous Na2CO3 - 100g
CuSO4 .5H2O - 17.3gm
Principle:
Soluble blue cupric ions of CuSO4 when heated strongly in alkaline medium are reduced to
yellowish red insoluble cuprous ions of cupper 11 ion (Cu2O) by urine reducing agent.
or
The aldehyde or ketone group of carbohydrates reduces blue cupric hydroxide to an
insoluble yellow or red cuprous oxide. if no carbohydrates is present in the urine the cupric
hydroxide when heated is converted to an insoluble black cupric oxide, but the presence of
sodium citrate in the reagent prevents this spontaneous reduction.

66
 OH
Cu CuO + H 2O
Cupric Oxide (black)
OH
Cupric hydroxide
(blue)
Reaction in absence of reducing agent.

OH
2CO Cu2O + 2H2O
Cuprous Oxide
OH (yellow red)

Reaction in presence of reducing agent.

Method:
Mix 5ml of benedicts + 0.5mls of sample
Heat until it boils. Allow to cool
Observe the colour change.
Results:
Greenish blue precipitate ……………………Trace
Yellow tinge ……………………………………… +
Orange precipitate ……………………………. ++
Brick red …………………………………………+++
Glucose is not excreted and detected in urine because the amount filtrated is almost
completely absorbed by the tubules leaving 20mg/dl glucose which is not detected by clinical
methods However in disease conditions, glucose appears in urine when its renal threshold
goes above 10mmol /l (180mg/dl) in blood This is Glycosuria (passing glucose in urine )
Conditions leading to Glucosuria
1. When blood glucose levels is elevated above the renal threshold as a result of the
deficiency like incase of diabetes mellitus
2. When excessive glycogenolysis fallows a emotional and physical stress
3. An increase in the rate of glomerular filtrate as may sometimes occur in
pregnancy
4. When the glucose transport mechanism of the renal tubules is defectives because
of the congenital or acquired tabular disease (nephrosis) that allow glucose to spill
into urine at normal blood glucose level (renal glycosuria)

67
 Renal blood glucosuria is the condition in which glucose is passed out in urine when
blood glucose is normal but due to tubular diseases (nephrosis)
(ii) Clinitest method (Tablets
This is a modification of Benedicts tubes method 1 tablets form. Clinitest tablets
contain CuSO4, NaCO3, NaOH and citric acid
Method
- Add diluted urine (2 drops of distilled water + 2 drops of the urine ) to tablet on white
tile
- As diluted urine is added to the tabulate the citric acid is neutralized by Na 2 CO3 to
NAOH , an intense heat is produced and CO2 produced and is released
- The heat produced brings the mixture to boil and CU2 + are reduced by the glucose
- An orange colour develops after 15 seconds which is compared to reference chart
NB
This method is not specific for the glucose
(iii) Glucose reagent strip test method
This method used plastic reagent strips impregnated with a chemical specific for
glucose qualitative and semi qualitative
Principle
Glucose in urine is oxidized to gluconic acid with release of H2O2 by atmospheric
oxygen in the presence of glucose oxidase enzyme (specific for the glucose). The
H2O2 In presence of a peroxidase enzyme oxidizes a chromogen from a reduced
state to coloured oxidized shades of colour (brown)
Glucose + O2 Gluconic acid + H 2O release H2O2
(From air) Glucose oxidized

H2O2 + chromogen (O2 acceptor) Oxidised chromogen (coloured)

Peroxidase
Method
1) Dip the test end of the reagent strip into fresh urine and remove immediately
2) Remove the excess urine / sample
3) Compare colour of the test area with the colour chart in good light after a
specified period on the container
NB
The reagent strips have different names depending on the manufacture eg some call the
clinistix , glucotest others glucostix
During use the glucose reagent strips, there may be false results due the following
1. Disinfectants eg bleach will oxidize the chromogen directly therefore cause false
positive results
2. Catalase enzyme in high concentration in E- Coli infections can destroy H2O2 and
cause false negative results

68
 3. Oxygen receptors in urine when present in high concentration can be oxidized by the
H2O2 to preference to chromogen leading to loss of sensitivity of the strip e.g.
ascorbic acid and drug metabolites
4. Presence of acetate , acetone and ascorbic acid

KETONES BODIES

Acetoacetate, beta-hydroxybutyrate and acetone are collectively referred to as ketone bodies

or simply ketones. The excretion of more than a trace of these substances in the urine is
called ketonuria.

Formation of ketones:

The metabolism of glucose normally provides the body with its energy requirements. If,
however, the intake of glucose is insufficient as in starvation, or glucose metabolism is
defective due to a lack of insulin as it occurs in untreated or uncontrolled diabetes, the
body obtains its energy by breaking down fats. It is this increase in fat metabolism that leads
to a buildup of ketones in the body. An accumulation of ketones in the body is called ketosis.

After being formed, they can be used as a source of energy in the muscles. During fat
metabolism. aceto acetic acid is first from access acetyl CoA then acetone is formed by
loss of CO2 from aceto acetic acid , β- hydroxybutyric acid is formed by addition of hydrogen
atoms (reduction of aceto acetic acid )

However, if more fats are metabolized , the muscles are unable to utilize all the ketones
bodies being formed. This result in increase ketone bodies in blood a condition called
Ketosis which later leads to ketones being passed in urine a condition called Ketonuria

Ketones are toxic to the brain and if present in sufficiently high concentration in the blood
they can contribute to the coma found in diabetic ketoacidosis. Metabolic acidosis is
Caused by a Variety of non-volatile acids accumulating in the plasma of patients with diabetic
coma due to ketoacidosis, ketonuria is
always present.
……………………………………………………………………………
Increased levels of ketones bodies in the body occur in the following conditions:

1. Diabetes mellitus (chronic hyperglycemia )

In this condition, glucose metabolism is defective due to lack of enough insulin for the
controlling glucose levels in the body as result, the body resort to using fats as source
of the energy resulting into production of the in high contractions
2. Starvation
This is when the body uses all the stored carbohydrates and resorts to fats as source
of the energy
3. Ketogenic diet

69
 This is a diet rich in fats with low carbohydrates. It will also lead to accumulation of the
ketones bodies in the body
4. Severe liver diseases
Most carbohydrates are stored in liver in form of glycogen. However, in the diseases
conditions the liver fail to store glucose in form of glycogen and the body resort to the
using the fats as source of the energy leading to increased ketones bodies
Physiological effects of ketones in the body
Aceto acetic and β-hydroxbutyric acid contribute excess H+ in the blood resulting in
acidosis eliminating excess acids un urine. Urine with ketone is therefore associated
with low PH
Acetone and aceto acetic acid is toxic to the brain when present in increased amounts
TESTS FOR KETONES IN URINE
1. Rothera’s Nitroprusside Test
2. Gerhardt’s ferric chloride Test
3. Modified Rothera’s Nitroprusside Test
4. Acetest tablets
5. Ketostix

Rothera’s Nitroprusside Test

Principle
Ketones (Acetone and aceto acetic acid) react with Sodium Nitroprusside in an alkaline
medium to give a mauve purple colour (a violet dye complex)

Procedure
- Saturete 5 ml of the urine with solid ammonium sulphate (few crystals )to give aline
medium
- Shake to dissolve
- Add 0.5 of 2% sodium Nitroprusside
- Add 0.5 mls of silver conc NH3

Results
- A purple colour will be given by acetone and aceto acetic acid within 15 minutes

Gerhardt’s ferric chloride

Reagents
Add 10% ferric chloride (2-3 drops) to few mls of urine (5-10mls) in a reaction tube
Results

If ketones bodies (aceto acetic acid) are present the mixture will turn purplish

70
A similar colour is also given by salicylates (from acetylsalicylic acid) however , this can be
differented from aceto acetic acid by their behavior on heating

If the purple colour formed is due to ketones , on heating the colour disappears because
aceto acetic acid is converted to acetone but if its due to salicylates , the colour persists

Modified Rothera’s Nitroprusside test

Preparation of Rothera’s powder

- Na Nitroprossude ………………..1g
- (NH4)2SO4 …………………………20g
- Anhydrous Na2CO3……….…….20g
- Grind and mix form the powder
Procedure
- Place a pinch of powder on a filler paper or white tile
- Add a drop of urine to the powder
Results
If ketones bodies are present , a voleit purple colour develops with in 30-60 seconds
5. Acetest tablets
This is Rothera’s test in tablets form. The tablet contains sodium Nitroprusside amino
acetic acid, lactose and disodium phosphates

Principle
Ketones react with Na Nitroprusside at optimum alkaline PH provided a buffer system
to give a purple colour. Lactose enhances the development of the colour.
Procedure

Put a table on a white tile and add a drop of urine

Results
Purple colour is given by aceto acetic acid and acetone

6. Ketostix

These are strips made of stiff absorbent cellulose one end of which is impregnated
with a buffered mixture of glycine and sodium nitroprusside

Procedure
- Dip test end into a fresh specimen of urine. Remove immediately and compare the
colour chart 15 sec later.

71
 Results
- The test is positive if a purple colour develop

NB
There is no routine test β-hydroxybutyric acid, although it occurs with acetone and aceto
acetic acid. To test for this substance, it is first oxidized to aceto acetic acid by of H 2O2

Procedure
- Add a few drop of acetic acid to urine dilute 1: 1 with distilled water Boil for a few
minutes to remove to remove acetone and aceto acetic acid.
- Add about 1ml of H2O2.
- Warm gently and apply Rothera’s test
Results
- It is positive if β-hydrobutyric acid was originally present in given urine sample

Quantitative estimation of bile pigment in urine.

1. Fouchets method.

Principle.

The sulphates in urine are precipitated by Bacl which is seen as a white precipite.
Bilirubin if present get attached to precipitated Ba2so4. On additition of
fouchets Reagent to the precitate, iron 111 chloride oxidizes the precipitate to green
blue biliverdin. The intensity of the colour is propotional to the concentration of
bilirubin in the sample.

Reagents.

10% Bacl solution

Fouchets solution.
Disosolve 25g of trichloro acetic acid in 50mls distilled water.
Add 10mls 10% ferric chloride.
Make up to 100mls with distilled water.

Procedure.

Add 5mls of 10% Bacl to 10mls of urine and mix well.

72
 If the white ppt formed is insufficient, a drop of dilute H2SO4is then filtered or
centrifuged to otain the deposit.

Add 1 drop of fouchet`s reagent 0n the filter paper or on the sedment.

Results.

An immediate blue green colour is formed if bilirubin is present.

If bilirubin is absent, white colur remains.

2. ICTO test.
This is the tablet reagent test.

Principle.

It is based on joining of of bilirubin to a stable diazonium solid salt to produce a purple

Azo-bilirubin.This methord is specific for bilirubin.

Reagentes
0.2mg P-Nitrobenzene diazonium P-toluene sulphonate salt.
100mg sulphosalicylic acid.
20mg NaHCO3.
25mg boric acid.

Procedure
Add 5 drops of urine to a white tile.
Place the icto test on the sample.
Add 2 drops of water to the tablet.

Results

A blue purple colour in 30 seconds indicates a positive test for bilirubin.

3. Bilirubin strip.

Principle.

Bilirubin estimation by use of the strips is based on the coupling of bilirubin with 2,6
dichlorobenzene diazonium tetrafluoroborate in acid medium to give a violet redish
azo-dye.

73
 Procedure.

Dip the strip into a fresh urine sample.

Remove the excess sample by lap
Wait for the recommendable time by the manufacturer and compare with colour chart.

UROBILINOGEN.

Formed as the result of bacterial action on the conjugated bilirubin in the intestine. The
urine sample for bilirubin estimation should be fresh and analysed immediately as it is
easly oxidised to urobilin (orange yellow pigment) on exposure to light and air.

NB
If delay in urine analysis for urobilinogen is un avoidable, then the technique for
detecting urobilinogen (Schlesinger`s test) is employed, the result is related to the
concentration of urobilinogen present in the sample.

If a 24 hour urine sample is to be used, then it should be collected in a brown bottle

containg 5g of anhydrous Na2CO3(1-2litres).

Normal urine contains urobilinogen in small amounts , its concentration depends on

the amount of bilirubin being produced and entering the intestine and the ability of the
liver to excrete.

Increase in urobilinogen is found in liver disorders and conditions involving

haemolysis.

Decrease of urobilinogen occurs in the following conditions;

 Obstruction of the bile duct preventing flow of bilirubin to intestines for conversion of
bilirubin to urobilinogen.
 Hepatocellular damage preventing conjugation and excretion of bilibin for conversion
to urobilinogen.
 Absence or reduction of intestinal bacterial flora which leads to little or no urobilinogen
being produced.

74
 METHODS OF DETECTION.

Ehrlichs test/ Wallance and Diamond Reaction.

Principle.

Urobilinogen reacts with P.dimethylaminobenzaldehyde to form a red condensation

product. The colour intensity being proportional to the concentration of urobilinogen in
the sample.

Reagents (Ehrlichs reagent).

P-dimehtylaminobenzaldehyde.
Conc HCl
Distilled water
20% v/v HCl.

NB
If urine sample contains bilirubin, it should first be removed by precipitation by BaCl or
CaCl and then supernatant be used for analysis.

Procedure.

- To 10mls of the sample, add 1ml of Ehrlichs reagent.

- To another 10mls of urine, add 1ml of 20% HCl.
- Mix and stand for 3 minutes at room temperature.
- Observe colour by looking down the tube held over a white surface.

Results.

Faint red colour (pink)…………….Urobilinogen in normal amounts.

Distinct cherry red colour……….. Urobilinogen in excess.
No red colour development……….Urobilinogen absent.

NB
A similar cherry red colour is given by porphobilinogen ( an intermediate compound in
haem synthesis) which is produced in urine of patients with acute iodopathetic
porphyria (inherited disease that affects muscles and nerves) therefore the colour due

75
 to urobilinogen is differentiated from that of porphobilinogen by use of sodium acetate,
amyl alcohol or chloform.

After developing the red colour with Ehrlichs reagent, saturated sodium acetate
solution is added which alters the PH, intensifying the colour given by urobilinogen
and making it more soluble in extracting soluvents (Amyl alcohol or chloroform) than
porphobilinogen.

Reagents for detecting porphobilinogen.

1 Ehrlichs reagent.
2 saturated sodium acetate.
-Hydrated sodium acetate---100g
-Distilled water
3 Amyl alcohol or chloroform.

Method.

- To 5mls of fresh urine samples, add 1ml of Ehrlichs reagent, mix and allow to stand
for 3-5 minutes.
- Add 10mls of saturated sodium acetate solution and mix. Leave for 2-3 minutes.
- Add 1-2 mls of chloroform/ Amyl alcohol.
- Mix and allow to settle.

Results.

Urobilinogen is soluble in organic solvents so any red color remaining in aqueous phase after
extraction indicates positive test for porphobilinogen.

Urobilistix strip test.

These are plastic strips whose end is imprignated with 4-dimthyl aminobenzaldehyde in acid
buffer which turns in the presence of urobilinogen.

NB
The strips are not specific for urobilinogen. Other substances like P- amino salicylic acid
reacts in a similar way like urobilinogen therefore, a positive urobirinogen result should be
combined with other results before diagnosis is made.

76
HEAMATURIA AND HEAMOGLOBINUIA.

Heamaturia is a condition when intact red blood cells are passed out in urine. Even there
might be Hb in urine. The presences of red blood cells are often visible to the naked eyes but
a slight decrease is only magnified by microscopic examination or by chemical means. This
condition is recognized as significant when red blood cells are present in fresh urine sample.

Heamoglobinuria is a condition when free Hb is passed in urine. It occurs when there is

excess red blood cell breakdown intravascularly Hb being released into plasma more than
that can be taken by haptoglobin (plasma proteins that binds free Hb to prevent it from being
lost from the body).

Both these conditions can be investigated macroscopically, microscopically and by use of

chemical tests for occult blood.

Causes of heamaturia.
- Acute glomerular nephrites.
- Lesions in the urinary tract.
- Schistosoma Heamatobium infection.

Causes of heamoglobinuria.

This condition is caused by conditions associated with;

-Intravascular heamolysis.
-Severe infections eg malaria, small pox and yellow fever.
-Reactions following incompatible blood transfusion.
-Sickle cell disease crisis.
-Septiceamia –bacterial infection eg Eschelicia coli.
-Snake bites.

CHEMICAL TESTS.

1 Ortho-tolidine test.
Principle
The peroxide enzyme in Hb reduces H2O2 to H2O and O is liberated. The liberated O2 is
accepted by Ortho-tolidine to give a blue or green oxidized product

Hb + H202 2H2O+O2

77
O2 + Ortho – tolidine (chromogen) Blue or green oxidation product

Reagents

- 4% ortho- tolidine in glacial acetic acid

- 10% H2O2
Procedure

- Add 2mls of 4% Ortho-tolidine to 2mls 10% of H202

- Leave it to stand for 1-2 min while observing for colour development
- If it changes colour, discard and prepare another one
- If there is no colour development, then add urine sample to the mixture
- A blue- green colour indicated presence of blood. The colour intensity being
proportional to blood concentration

2. Haemastix and chemo strip

These are to test for Hb and red blood in urine. The test area is impregnated with
mixture of organic peroxide and ortho- tolidine. This test is more sensitive to fresh urine
(Hb) or red blood cells

Dip the test area and remove it immediately and compare the colour development after 30
second
NB
False positive reaction occurs when urine is contaminated with oxidizing detergents eg
bleach heavy proteinaemia false negative Vit C or tetracycline

Occult blood testing in stool

This is a test used to detect of blood in faecal samples. Detection of blood in the faecal is
important in determining the cause of the following

1. Hypochromic anaemia (Fe deficiency where Hb is reduced in red blood cells) resulting
in chronic loss of the blood
2. Detection of ulceration or neoplastic diseases of the gastro- intestinal system
3. Detect cancer of the stomach

Occult blood detection can be macroscopic, microscopic or by use of chemical tests.

The chemical tests is based on the same principle like that occult blood in
A good number of chromogen can be used eg
- Benzidine
- Ortho-tolidine

78
 - O-dianisidine
- Gum guaiac

Note: Benzidine is no long used because is carcinogenic and O- dianisidine is less

sensitive

Precaution to be undertaken before collection of stool sample

1. The patient should be on a diet free from meat or fish 2-3 days before the sample is
taken
2. Patient should stop taking iron salts eg FeSO4 tablets and vitamin C which interfere
with the oxidation of the chromogen
3. Faecal samples which are contaminated with toilet water as cleaning agent may can
cause false positive results
4. The container should be chemically cleaned free from formalin which affects oxidation
and bacteria or white blood cells capable of produceding Peroxidase enzyme giving
false result
5. Patients should exclude tooth brushing and avoid ASA drug (Asprin)
6. Three daily specimen should be examined before the presence or absence of the
occult blood is conformed for three consecutive days
Test methods for occult blood in stool (Haematest method)

Principle
The test is based on the Peroxidase like activity of Hb in catalyzing the oxidation by peroxide
of a chromogen tetra methyl benzidine to form a blue colour

Method
a) Apply the sample on filter paper with an applicator stick
b) Place the tablet on the spacemen so that part of it is touching the clean filter paper
c) Add a drop of distilled water on to a tablet and allow 5-10 sec for water to penetrate
them
d) Add a second drop so that water runs on sides of tablet on to spacemen and filter
paper
e) Observe filter paper for any appearance of any traces of blue colour surrounding the
tablet for up to 2 min
f) Ignore colour that develop on or directly, wilt the tablet and that which develop after 2
min

Quality control of this test

Prepare a 1: 15000 dilution of blood in water and place a drop on tablet on filter paper.
Appearance of blue colour on paper with in 2 min indicates that the tablet is sensitive

79
 BILE PIGMENT METABOLISMS
Bile is an excretory product from the liver and contains mucus , bile salts pigments , inorganic
salts and lipids.

Bilirubin pigment is formed after red blood cells are broken in reticulo endotherial system
(spleen, liver, thymus gland and bone marrow of the short bones) After their life span of 120
days. The Hb is split into Haem (iron porphryrin) and globin a basic protein

The globin is transferred to the protein pool for new amino acids synthesis which are later
used in Hb synthesis

The haem (iron porphrin complex ) is split into iron which transferred to iron pool where its
stored in 2 forms
- Ferritin
- Haemosiderin
Ferritin is a sold iron protein complex which can be re-utilised for Hb synthesis. The porphylin
part which remains after iron removal is converted to agreen substance called biliverdin
which is reduced by tissue enzymes to a yellow pigment called bilirubin.
This bilirubin circulates in blood while attached to plasma albumin and in this form is water
insoluble but alcohol soluble i.e. un conjugated or indirect birilubin while attached to plasma
albumin its transported to the liver whereit is conjugated by glocuronic acid under the action
of glucoronyl transferase enzymes forming birilubin diglucurunides(conjugated or direct
birilubin) which is water soluble but alcohol insoluble
The direct birilubin passes into the bile duct up to the intestines where its degraded (reduced
by bacterial actions to compounds collectively known as urobilinogen). 99% of urobilinogen is
passed out in faeces as stercobilinogen which is oxidized to stercobilin on exposure to air
giving stool its brown colour.
1% is re-absorbed into the portal circulation passed into systemic circulation up to the
kidneys where its excreted as urobilinogen
NB.
When urobilinogen is exposed to air, its converted to urobilin. Accumulation of birilubin in
blood leads to yellowing of the skin, sclera of the eyes and even the brain tissue leading to
acondition called Juandice or Icterus

BILE PIGMENT METABOLISM

RBC eticilo- endothelial system Haemoglobin

80
 (Iron porphyrin) Haem Globin Amino acid stores
(protein stores)

Porphyrin iron iron stores

Biliverdin Bilirubin unconjugated (indirect bilirubin )

Liver (conjugated by gluconic acid )

Direct conjugated bilirubin

Intestines (direct or conjugated bilirubin converted to

Urobilirubin under glucurony/transeferase enzyme)

Stercobilinogen passed out in faeces

Kidney (uronbilinogen passed out in urine)

TYPES OF JUANDICE
1. Heamolytic (Prehapatic Juandice)
Occurs when these is excessive red blood cell breakdown so that the liver cells are
unable to conjugate all the bilirubin here is un conjugated not secreted in urine and
can only be detected in blood. However, small amounts of urobilinogen is passed
out in urine. This can be caused by malaria infection, HDNB, incompatible blood
transfusion, drugs e.g. sulphonamides (Fancidar, sulphurdimidine or quinine
sulphates/auto immune haemolytic anaemia, toxins form bacteria, snake venoms
and herbs).
Laboratory findings

Total serum bilirubin:High

Direct serum bilirubin:Normal

81
 Indirect serum bilirubin:High

Urine bilirubin:Absent

Urine urobilinogen:High

Hepatic jaundice

2. This is when birilubin is built up in plasma because its not transported conjugated or
excreted by the cells because they are damaged. This is caused by;
(i) Chronic hepatitis
(ii) Drugs e.g. chloropromazine (lagactyl)
(iii) Chemicals
(iv) Plant toxins
(v) Brucellosis (Brucella abortus)
Here bilirubin is both conjugated and the former can be detected both in urine and blood.
There will be increased amounts of urobilinogen in urine
Laboratory findings

Total serum bilirubin:High

Direct serum bilirubin:High
Indirect serum bilirubin:High
Urine bilirubin:Present
Urine urobilinogen:High

3. Post hepatic (Obstructive juandice)

This occurs when there is obstruct to the flow of the bile in the extrahepatic ducts
which will lead to build up of conjugated bilirubin/blood. This is ‘‘choloestasis’’. It is
caused by;
- Gall stones, tumour of the head, of the pancrease
- Chlangitis (inflammation of the biliary ducts)
- Helminthes infection e.g. Fasciola ssp opisthorchis, ascaris
- Pressure on the small bile ducts or as in hepatitis or side effects of the
drug.
Here bilirubin is conjugated and therefore found in urine but no urobilinogen is passed
in urine.
Laboratory findings

Total serum bilirubin:High

Direct serum bilirubin:High
Indirect serum bilirubin:Normal

82
 Urine bilirubin:Present
Urine urobilinogen:low &Absent

4. Neonatal jaundice/physiological jaundice

Is commonly known as physiological jaundice and found in new born babies due to
the following;
-ABO HDNB

-Septicemia

-Congenital syphilis

In this type of jaundice its mainly the unconjugated type & when in high level can
be deposited in the brain & a condition is known as kernicterus-cause permanent
brain damage.

Laboratory findings

Total serum bilirubin:High

Direct serum bilirubin: Slight High
Indirect serum bilirubin: To High
Urine bilirubin:Present
Urine urobilinogen:High

QUALITY ASSURANCE AND QUALITY CONTROL

Quality assurance Involves all the steps taken both in and outsides the laboratory to
achieve reliable results, starting with the preparation of the patient and collection of a
specimen and ending with the correct Interpretation of results.

It involves all action also takes to ensure that quality results are produced. These
include:

-Vilification of quality of sample collection.

-Sample processing.

-Test method selection.

-Reforming of results

-Interpretation of the final report by the clinician,

83
- It also achieves prompt reporting and better use of their results as well as the compliance
and adequacy of laboratory staffs.

ELEMENTD OF QUALITY ASSURANCE

(1) Technical compliance. The service provides must have the right knowledge, skills
and attitude to perform lab.

(11) Effectiveness. The services provide and the lab in general should follow the norms
and guidelines of all the procedures.

(111) Efficiency: An efficient service provides school produce the last results within
available remark

(iv) Contritely: It means providing arrange of service with in the means of the laborites

(v) Safety: should prevent hazards in the laborites, the service provider, patients, other
health workers and any others person who enters the laboratory

(vi) Validation: It is checking whether the test procedure all equipment satisfies the set
stands.

(VII) Facility: The lab should be of a suitable size, Construction and location to meet
the requirements of arrange of lists offered i.e. the lab should have data storage facilities,
sample storage facilities, a place when sample are processed.

(viii) A system of communication of results, should have storage for reagents, the
reagents should be labeled when to expire, preparations date, identification togs and their
conclusion.

QUALITY CONTROL
This describes the steps taken by the laboratory to ensure that tests are performed
contently. It involves establishing performance standard for each test procedure that
ensures percussion and accuracy, reporting result s clearly and in minimum delay.

 Use of controls. A control samples to check for bail. This involve procedures like
clarity results regularity to check for scatter and taking action when results are
becoming unreliable

84
  Training Lab worker to perform tests correctly and continuously updating the
knowledge and skill

 Quality assurance. Can be divided into Pre-analytical and analytical and post
analytical phases.

1. The pre- analytical phase of quality accuracy ensures quality in everything before
testing process both within and outside the lab. These include:

o Proper selection and evaluation of the listing procedures

o Collect ordering of the tests.

o Preparation of the patient should be informed a bout any patient preparation e.g.
flashing or metrication retractions

o Proper Identification of the patient, the name and must Identification of the collected
sample, request form must match each own and must also match the collect patient.

o Proper collection of samples, i.e. in a collect contains, and under conditions specified
by the test procedure.

o Timely transportation of the samples to the lab, the time between collection and the
stay should be with limits defined by the laboratory

o Proper handling of the sample from the time of transportation to the time of analysis.
This many include, avoiding exposures to light a keeping the sample in ice and all
samples must be handled as though as infectious.

- Proper handling of the sample in the lab. This includes proper documentation of the
sample, Identification with in the lab collect centrifugation technique and if necessary
timely separation of the serum and scrum plasma from cells

Analytical Phase
In addition to Internal and external quality control programmes, analytical quality assurance
includes:

- Proper labeling and use of reagents, Reagents must be labeled with concentration,
data of preparation, expiry date, lot number, Initials of the person prepared.
- Periodic calibration of pippetting devices and careful maintenance of Instruments.
- Using control samples to check for bias

85
 - Training lab workers
- Establishing performance stands for each test
- Periodically checking that temperature of refrigerators or a heating units are collect
and all other equipments.

NOTE External quality assurance is a system of checking the performance of your lab in
comparison with the standard labs

POST ANALYTICAL
This is the process verifying quality once the sample has been an analyzed. These include:-

- Verification of calculation on final report.

- Pre view of list results for possible errors
- Writing reports that one easy to read and Interpreted.
- Procedures for informing the physicians results for the test that requires immediate
attention.
- Verification of collect Interpretation of lab tests by the physician.

ERRORS THAT INFLUENCE THE COLLECTIONESS OF THE TEST RESULTS

These are two typses of errors that can occur when performing clinical chemistry tests ie.

- Errors of scatter or random error s ( Imprecision)

- Errors of bias (systematic errors) leading to Inaccuracy

ERRORS OF SCATTER / impression (RANDOM)

This is when the results different from the collect results by varying amount s. The
common cause of scatter in clinical chemistry ware Include:
- Incorrect and variable pippetting.
- Inadequate mixing of samples with reagents
- Incubation of tests at Inconsistent temperature or for incorrect length of time
- Dirty tubes, pipettes and other glassware used in the test
- Too heavy workload resulting in faulty technique and mistakes being made by using
shortcuts.
- Too low workload resulting in loss of concentration and errors being made.
- Fluctuating or erratic colometers readings due to unreliable mains voltage supply.
- Use of dirty or finger marked curettes or reading samples when they contain air
bubbles.
86
 - Incomplete removal of Interfering substances in serum e.g. red blood cells or protein.

2. ERRORS OF BIAS (SYSTEMATIC) OR INACCURACY

These are consistent or regular errors. All results differ from the collect results
by approximately the same amount.
The common cause of error of bias are:-
- Use of unsatisfactory reagent because they contain Impure chemicals, having been
prepared wrongly, stored incorrectly or used after the stated expiry date
- Incorrect or Infrequent calibration of the test method.
- Use of control or serum that has been wrongly prepared or has expired.
- Tests being read at incorrect wavelength or wrong filter being used
- An automatic pipettor is used that has been set to measure an incorrect amount

ELEMENTARY STATISTICS TERMINOLOGIES

- Mean it is a term used often in laboratory measurement s. It is mathematical average
calculated by dividing the sum of all individuals’ values by the number of values.

- Median. It is the middle value of in body data in a body of data. If all the variables
are arranged in order of increasing magnitude, the median is that variable which falls.
Half way between the highest and the lowest.

- Mode. It is the value that occurs most commonly in amass of data.

How the means, the median and the mode are used is explained in the example
below:.
Series of results reported for a laboratory test done on 7 different specimen is 7, 2,
3,6,5,4 and 2.
Oq calculate the mean, mode, and median

ESTABLISHING PERFORMANCE STANDAARDS (OCV AND RCV)

Procedures should be established to determine the acceptable and test performance

should for each test method.

87
 Before any test is used for any patient the lab must first ensure that method is reliably
and that it can be performed with acceptable limits of variability. I.e. must be constantly and
adequately controlled.

Therefore establishing OCV and RCV can be used to assess reliability and
performance of the test method.

OPTIMAL CONDITIONS VARIANCE (OCV)

METHOD:
- Obtain a sufficient quality of a control serum that contains a known a amount of the
substance being measured .i.e. the concentration of the substance should be similar
to the amounts expected in patients samples.
- Under optimal conditions, perform 20 measurement s on the same sample of control
serum.
- Read off the values from the calibrations graph
- List the value s for each of the 20 estimations
- Calculate the mean (Average value) by adding up all the results and dividing the total
by
20.
- For each result, work out the difference in value from the mean, entering the difference
in the second common of the chart.
- Multiply each difference by its self to give the squired difference values, entering those
in the 3rd column of the chart.
- Add up the figures in column three to give the sum of the squared differences
- Calculate the standard deviation (SD) using the formula.

SD= Sum of the squared difference

N-1

Where n=20

- Calculate the optimal conditions variance using the formula.

OCV= (SD x 100).
Mean

Note In correct statistical terms (SD x 100) is called coefficient of variation (CV).
Mean
- Chart the results. I.e.

- Take a sheet of graph paper and draw on it three horizontal lines corresponding to the
mean, +2SD and -2SD.

88
- Work out the values for +2SD and -25D I. e

+ 2SD = Mean + 2SD

- 2SD = mean - 2SD.

- Make the horizontal axis control and number it 1to 20.

- Examine the chart for the distribution of values around the mean. It should show a fairly
even distribution of values on each side of the mean within +25D AND -25D

+2SD

conc in mmol/l Mean

- 2SD

Control

Note: If the OCV is more than the maximum acceptable figures, there must not be put into
routine use .i.e. The standard, calibration graphs, reagents and other possible error must be
investigated.

ESTABLISHING ROUTINE CONDITIONS VARIANCE (RCV)

When at test is performed under routine conditions of work the degree of variability i.e.
routine conditions variance (RCV) will inevitably be higher than the OCV and therefore the
range of acceptable values will be a single

Although the OCV can be determined in a single experiment it takes some time to
accumulate enough Information from routine test to calculate the RCV.

When, therefore, a new test is first introduced it is usual to set up the first control chart
Using twice the optimum conditions SD and OCV,

89
HOW TO USE AQUALITY CONTROL CHART
After establishing that a test method is reliable and has an acceptable OCV; it can be put
into routine use producing it can be controlled adequately a control serum must be used
every batch of tests.
Control chart will enable most faults to be indentified and correct at an early stage
before patients’ results are affected and reporting has to be delayed.

SETTING UP ADAILY QUALITY CONTROL CHART

a. It is similar to the OCV control chart only that the honzorital axis is numbered
for the days of the month. + 25D are calculated from the routine conditions
stands deviations.

II. Calculate HO + 25D are and -25D valves for the a essay
III. Plot the values on the daily control chart using the same control serum for each batch
of the test
IV. Each day after charting the control values, check that it is with in the acceptable limits
of +25D and that there is no mined change in the distribution of results above or below
the mean or a mount (drift) towards the take action zone

Month……….. Mean value………………….

Analyst……….. SD……………………………
ASSAY……………..

Take action
+2SD

Mean

+2SD
Conc mmol/l Take action

Days

INTERPRETATION OF RESULTS
i. Control value outside+ SD limits.
It can be assumed that the patient’s results are reliable and therefore they can be
reported with confidence.
ii. Control value outside +2SD limits

90
 It is UN an acceptable and the patients results must not be reported.

 A fresh control serum should be measure together with a few of the patients sample if
the result of the fresh control serum is with In +25D limits and the results of the
repeated tests agree with those of the first testing all the patients results must be
reported.

 If however, the control results are still not acceptable, the patient’s results must be
reported. The error s must found and put right and the batch of test is repeated.
Check practically for:-
- Reagent deterioration or the incorrect preparation of wrong reagent.
- Faulty equipment.
- Wrong fitter long used or coulometer going fluctuating teachings

Control value moving towards TAKE ACTION ZONE

Patient’s results can be reported but a drift of values up ward s or down wards is a warning
that test is becoming un reliable and the cause must be investigated before the next batch of
tests is performed

REFERENCE RANGES
A lab which should known the accepted reference /Normal ranges and clinical significance of
routine biochemist ry chemical tests.

This will ensure that significantly abnormal results area detected, checked and reported as
soon a possible such as actions in the lab may prevent loss of life or leads to earlier and
more efficiency treatment of the patients.

BIOLOGICAL FACTORS
The tests results are affected by both biological and laboratory factors and the re need to
be consulted when deciding the reference range for a particular test.

- Age e.g. higher plasma urea conc. are found in the elderly or higher alkaline phosphate
activity in growing children compared with adults

- Sex e.g higher value s plasma creatinine, urate and urea are found in men compared with
woman during the reproductive phase of life

- Diet and nutritional slate e.g. plasma cholesterol and calcium are affected by diet.

- Time of the day (diurnal variation) e.g. serum Iron levels rise as the day progresses.
91
- Posture: e.g. plasma protein levels are lower in samples collected from patient s when they
are lying down.

- Muscular activity: e.g. the concentration of plasma creatrine is the following exercise.

Normal range may also affected by weight , phase of menstrual cycle, emotional
state, geographical location, rural or city life , climate, genetic factors, cultural habits and
Intrinsic homeostatic variation.

LABORATORY FACTORS
Among the analytical factors that influence reference range, the most significant are:

1. Type of sample, the concentration of glucose is 12-13% higher in plasma than in whole
blood small venations as occur between serum and plasma samples for potassium and
some other substances.
2. Test method. Glucose oxide enzyme method will give narrower reference range for
blood glucose then any other technique because it is specific for glucose while other
tests for reducing substances.

HOW TO ESTABLISH NORMAL RANGES

1. Test large number of healthy people.
2. Plot a graph of frequency of value against concentration of the individual analytics
3. The graph produced is symmetrical in shape showing the highest number of people
having values around the mean / average with gradual decrease in frequency an each
side of the mean.
- In statistical terms the distribution of values around the mean can be exposed as SD.

When the results of a particular test shows a symmetrical type of curve, the reference range
for the substance being measured is calculated by plus or minutes 2SD from the mean.

Frequency

92
 Test for individuals

END

RENAL FUCTION TESTS (RFTs)

The kidneys are primary organ involved in removal of the waste products though other
organs like lungs and intestinal do excretion as well other functions of kidney include :-

- Production of hormone like erythropoietin

- Regulation of the body water (water body balance )
- Elimination of soluble waste products of the metabolisms

Renal function tests refer to a group of the test used to assess the general functioning of the
kidney they include –

1. Creatinine test
Creatinine clearance
2. Urea test
BUN (Blood urea nitrogen)
3. Electrolytes (sodium , potassium , chloride bicarbonate e. t. c )
4. Uric acid test

CREATININE

Creatinine is a nitrogenous waste product formed from the metabolism of creatine in the
skeletal muscles, Creatinine diffuse freely throughout the body water it is filtered from the
extracellular fluid by the kidney and excreted in the urine

Creatinine formation

93
The excretion of the Creatinine is mainly by the renal and in the absence of the renal disease
it is relatively constant and has direct relationship with muscle- mass

It is less affected by diet , dehydration ,sepsis, internal bleeding, age, sex or exercise
sometimes serum concentration in female and children is low just because of the muscle –
mass

Serum Creatinine is a better indicator in renal function test than urea- nitrogen, urea or uric
acid because of its Constance in formation

Increasingly the measurement of the serum Creatinine is used to investigated HIV

associated renal diseases and monitor patients being treated with nephorotoxic antiretroviral
drug eg fenofovir

TESTING FOR THE SERUM/PLASMA CREATININE

Reference ranges

- Male 0.7 - 1.4 mg/dl (mg%) 60 – 130 µmol/l

- Female 0.4 – 1.3 mg/dl 40 - 120 µmol/l
- Children 0.4 - 1.2 mg/dl 40 - 110 µmol/l

Analytical techniques of Creatinine

Most of the commonly used method for the determination of Creatinine is based on Jaffe
reaction (Jaffe slot alkaline picrate Creatinine)

Principle

Creatinine reacts with picric acid in alkaline medium to give a reddish colour which when
read at wave length of 490nm is directly proportional to the concentration of the Creatinine.

The method is not specific for Creatinine it can be interfered with by a variety of substances
e.g. glucose , protein, acetate, pyruvate, uric acid, fructose and ascorbic acid

- The reaction is also sensitive to temperature and PH Changes

- To increase specificity aluminum silicate is used separate Creatinine from other
Chromogens

Preparation of Creatinine working reagent

Reagents
94
 - Sodium dodecyl sulphate…. 40g
- Phosphate borate buffer PH 12.8
- Picric acid
- Acetic acid 60% v/v

Method

N.B. Creatinine working reagent is not stable once prepared it can be used for one day only.

Creatinine method

Page 336 Monica

Interpretation/clinical significance

Increased concentration of serum Creatinine raises when there is impaired formation or

excretion of urine, when there is a disease which of either can cause the pre-renal, renal or
post renal. Creatinine is increased in:-

- Kidney insufficiency
- Sickle cell
- Muscle wasting

Any disease or condition that cause a fall in glomerular filtration rate (GFRT) will increase
plasma Creatinine levels. Such conditions/diseases include:-

- Glomerulonephrophritis (inflammation of glomerular)

- Pyelonephritis (inflammation of pelvis of the kidney)
- Renal tuberculosis

Diseases causing obstraction of urine outflow may cause kidney failure e.g.

- Urethra structures
- Prostatic enlargement
- Urine schistosomiasis

Acute renal failure is due to sudden reduce blood flow to the kidney occurring in:-

- Haemorrhages
- Obstestrical
- Surgical emergencies
- Malaria and septicemia

Non renal causes of plasma Creatinine level include:-

- Strenousins exercise
95
 - Effect of the drugs such as acetoacetate
- Ascorbic acid
- Cephalosporin antibiotics

CREATININE CLEARANCE

It is measured as an indicator of GFR, this blood and urine test is a specific measurement
ordered to determine kidney function, primary glomerular filtration. It measures the rate at
which Creatinine is cleared from the kidney. Clearance of a substance may be defined as the
imaginary volume [ml/min] of plasma from which the substance would have to be completely
extracted in order for kidney to excrete that amount in 1 min. Creatinine is a substance that,
in health, is easily excreted by the kidney. Because all the Creatinine that is filtered in a given
time interval appears in the urine, the Creatinine is equivalent to the GFR and thus a disorder
of the kidney function prevents excretion of Creatinine

Precautions taken when collecting samples for creatinine clearance

-it is performed on a 24hr urine sample (preserved with conc. Hcl), plus a random blood
sample.

* Urine sample preparation:

- dilute 1 in 50 with distilled water

Calculation
Creatinine clearance [ml/min] = urine Creatinine × 24hr urine [ml
Serum Creatinine × 1440

Urine Creatinine - M- <150mg/dl,

W-<250mg/dl
Creatinine clearance
RR Man 98 – 156 ml/min
Women 95 -160 ml/min
Clinical implications
1. A normal clearance cannot be used as a standard for who is known to have existing renal
disease.

2. A decreased clearance gives a reliable indication of impaired kidney function

96
Reduced Creatinine clearance
-acute shock
- Chronic pyelonephritis
-Malignant hypertension
-Glomerulonephritis

UREA (CO (NH2)2

Urea Is an end product of protein metabolism and other protein compound formed from de-
amination of amino acid in the liver

Urea is formed in the liver by ornithine –arginine (urea cycle)Its production is in the liver as
means of detoxifying ammonia generated by the breakdown of an acid resulting from
catabolism of protein both directly and endogenously

Because of the toxicity of ammonia, it is essential to be converted to urea and eliminated as

rapidly as possible. This is achieved by the urea cycle (ornithine cycle)

Urea is formed in the ornithine-arginine and is primarily controlled by the enzyme arginine
usually found in the liver (arginase)

The ornithine –arginine (urea cycle)

Urea is synthesized in the body of many organisms as part of the urea cycle, either from the
oxidation of amino acids or from ammonia.

97
In this cycle, amino groups donated by ammonia and L-aspartate are converted to urea,
while L-ornithine, citrulline, L-argininosuccinate, and L-arginine act as intermediates.
Urea production occurs in the liver and is regulated by N-acetylglutamate. Urea is then
dissolved into the blood (in the reference range of 2.5 to 6.7 mmol/liter) and further
transported and excreted by the kidney as a component of urine.

In addition, a small amount of urea is excreted (along with sodium chloride and water)
in sweat.
Amino acids from ingested food that are not used for the synthesis of proteins and other
biological substances are oxidized by the body, yielding urea and carbon dioxide, as an
alternative source of energy. The oxidation pathway starts with the removal of the amino
group by a transaminase; the amino group is then fed into the urea cycle.
In water, the amine groups undergo slow displacement by water molecules, producing
ammonia, ammonium ion, and bicarbonate ion. For this reason, old, stayed urine has a
stronger odor than fresh urine.

MEASUREMENT OF UREA

Mainly there are 2 different clinical approaches in determining serum urea.

 Enzymatic method
 Chemical method

1. Enzymatic methods

Enzymatic methods are indirect because they use enzyme to convert urea to the products
that are later quantified. The most enzyme used is Urease which hydrolysis urea to
ammonium ion and water

The first one employs the enzyme Urease that splits off ammonia from urea molecules in a
highly specific reaction.

(NH2)2C=O +2 H2O urease 2NH3 + CO2 g

(Urea)
The ammonia formed further react with phenolic chromogen, sodium hypochlorite and
sodium nitroprusside as a catalyst to produce a very stable solution

Sodium Nitroprusside

98
 NH3 + phenol + sodium hypochlorite indophenols (blue/ green
coloured complex)

SAMPLES FOR UREA DETERMINATION

Samples
 Serum /plasma sample
 except plasma from fluoride bottle and ammonium oxalate
I. This is because sodium fluoride is any enzyme inhibitor and therefore unsuitable
for urease method
II. Ammonium oxalate bottles contain ammonium and Berthelot’s method depends
upon the measurement of ammonium

Berthelot’s method

Principle

 Urea in the sample is hydrolyzed in the presence of urease enzyme (urea

amidohydrolase) enzyme to form ammonia and carbon dioxide

 The reaction is buffered with EDTA which also serves to chelate heavy metal ions
that may inactive urease

 The ammonia formed reacts with phenol and hypochlorite to form several
intermediate products that final produce blue coloured indophenol.

 Sodium nitroprusside acts as a catalyst in the reaction

 The intensity of the blue coloured developed is directly proportional to amount of urea
present in the sample and is measured calorimetrically at 540 nm

(NH2)2C=O +2 H2O urease 2NH3 + CO2 g

(Urea)

Sodium Nitroprusside
NH3 + phenol + sodium hypochlorite indophenols (blue/ green
coloured complex)

Method

Equipments and materials

Colorimeter or spectrophotometer
99
Reaction tubes
Water bath
Timer
Gloves
20µl , 200µl and 500µl pipettes

Reagents
1. Phenol – sodium nitroprusside solution
- Weigh 50gms phenol and 0.25gm sodium nitroprusside
- Dissolve the chemical in 1 litre of distilled water
- For the use dilute the solution 1 to 5

2. Sodium hydroxide – hypochlorite

- Weigh sodium hydroxide and 2.1 gm sodium hypochlorite
- Dissolve the chemical in 1 litre of distilled water
- For the use dilute the solution 1 to 5

3. Buffered urease solution

- weigh 100gm of urease powder
- weigh I gm of sodium ethylene diamine tetra acetic (EDTA) and dissolve it in 1000ml of the
distilled water the 100 gm urease powder in about 90 ml of a EDTA solution and adjust the
PH to 6.5 and dilute to 100ml with distilled water
- Store at 4 – 8 0C for up to four weeks

Test procedure:

Blank Standard Test

Urease buffer 0.2ml 0.2ml 0.2ml

Sample - - 0.02ml

Standard - 0.02ml -

Incubate at 37 o C for Minutes

Phenol nitroprusside 5ml 5ml 5ml

Sodium hypochlorite 5ml 5ml 5ml

Incubate at 37oC for 15 minutes

100
- Measure the absorbance of the standard and test against the blank at 630nm
- Read the urea concentration from the calibration curve or calculate using the formula:

Absorbance of the test × conc. of the standard = urea concentration

Absorbance of the standard

Approximate urea reference (normal) range

Adults: 3.3–7.7 mmol/l (20–46 mg%)

Infants: 1.3–5.8 mmol/l (8–35 mg%)

To convert from mmol/l to mg%, multiply by 6.

To convert from mg% to mmol/l, divide by 6.

Advantages of Berthelot’s method

- Complete and highly specific
- Blue colour developed is stable for several hours and beer s law is obeyed
- Interference of the reaction by any electrolyte in the reaction mixture is got rid of since we
use urease solution buffered with EDTA
- Precipitation of plasma protein is not required before urea measurement

2. Chemical method
In chemical method urea is measured directly.

Diacetyl monoxime method (Fearon method)

Principle:
Urea reacts with diacetyl monoxime at high temperature (100o) in an acid medium in the
presence of cadmium ions and thiosemicarbazide. The absorbance of the red colour
produced is measured in a colorimeter using a green filter 520 nm or in a spectrophotometer
at 530 nm wavelength.

Advantages of Diacetyl monoxime method

 It is specific

101
  Stock reagent are stable at room temperature

Disadvantages

 Red colour developed is unstable and sensitive to light

 Coloring reagent is corrosive
 Reaction is carried out at 1000C

BUN (BLOOD UREA NITROGEN)

RR- [urea]; 15 – 45 mg/dl,

BUN =0.466 (urea)

BUN; 7- 18mg/dl

This is part of blood urea sometimes asked by clinician , However it does not call for
specillaied testing instead one may just requires a conversion factor to convert BUN to urea
or urea to BUN

NH2

(NH2)2CO Urea C= O

NH2

RMM of urea from above chemical formulae is 60

The mass of nitrogen in the a molar of urea is = 28

There fore BUN = 28 = of urea and urea = 60 of BUN

60 28

Urea is synthesized in the liver from ammonia; as a result of deamination of aminoacids.This

biosynthetic pathway is the chief means of excretion of surplus nitrogen in the body by the
kidneys into the urine. The test for BUN, measuring the nitrogen portion of urea, is used as
a gross index of glomerular function and the production and excretion of urea. Rapid protein
catabolism and impairment of kidney function will result in an elevated BUN. The rate at
which the BUN rises is influenced by the degree of tissue necrosis, protein catabolism, and
the rate at which the kidneys excrete the urea nitrogen
The test BUN is used in the diagnosis of renal & metabolic disorders

Timed or random urine specimen can be used for the determination of urea concentration ,
only that urine is diluted 1in 20 before testing

102
A. INCREASED UREA [AZOTEMIA]
1. The most common cause of azotemia is inadequate excretion due to kidney disease or
urinary obstruction, frequently occurring in cases of prostate enlargement.

(a) An increased BUN of 50 to 150 mg/dl indicates serious impairment of renal function
(b) An increased BUN of 150 to 250 mg/dl is definitive for severely impaired renal function.
2. Increased BUN levels are associated with
(a) Impaired renal function
(b) Shock
(c) Dehydration
(d) Gastrointestinal hemorrhage
(e) Diabetes
(f) Acute MI
(g) Chronic gout
(h) Excessive protein intake or protein catabolism

B. REDUCED UREA (BUN)

-liver disease
-pregnancy because of physiologic hydremia
-over hydration
-low protein intake
-Nephritic syndrome (occasionally)
-Impaired absorption as in celiac disease
-Negative nitrogen balance, as may occur in malnutrition, excessive use of IV fluids.

Interfering factors:
The BUN is usually lower in children and women coz they have a smaller muscle mass than
adult men, older persons may have an increased BUN when their kidneys are not able to
concentrate urine adequately.
Increased BUN values normally occur in late pregnancy and infancy because of increased
use of protein
Decreased BUN values may occur normally earlier in pregnancy because of physiologic
hydremia
Older persons may have an increased BUN when their kidneys are not able to concentrate
urine adequately

URIC ACID

103
Reference ranges
Males 3.7-7.5mg/dl
Females2.5-6.5mg/dl

- Uric acid is a purine compound that circulates in plasma sodium urate and is excreted in
the urine.
- It is derived from break down of nucleic acid that are ingested or come from destruction of
tissue cells
- Urate is the end product of purine metabolism in man. Urate and uric acid are less soluble
in plasma and pose a danger by precipitating as uric acid crystals and it raise PH in
tissues.
- Although measurement of serum uric acid is as a primary test for evaluation of kidney
function. It may serve as a confirmatory(check) in the analysis of urea and creatinine
- The main value of serum uric acid test is in diagnosis or for following up treatment of
proteins with disease
- It is also sometimes used as an indicator or scale break down of nucleic acid e.g.

 In toxemia of pregnancy,
 After massive radiation of a tumor following administration of cytotoxic agents in
the treatment of malignancies
 Gout. This is characterized by precipitation of uric crystals urates in tissues
and joints.
 The deposition of urates in the kidney leads to renal failure which presents the
greatest danger to patients

MEASUREMENT OF URIC ACID

The classic chemical method for determination of serum urate is determined by reduction of
phosphor tungstic acid.

Principle
 Phospho tungstic acid is reduced to photo tungstic by uric acid in alkaline medium to
give a blue colour
 Uric acid itself is oxidized to allonation, the interaction of the colour produced is
proportional to amount of uric acid present and is read calorimetrically at length of
650-700nm (660nm)
NB. The presence of proteins during colour development, turbidity and quenching (reducing)
of absorbance. Other sequences present in serum (reducing agents) interfere with the test.

ENZYMATIC METHOD

104
Uricase catalyses the oxidation of urate to allonation , hydrogen peroxide and carbondioxide

Urate allonation + H2O2 + CO2

The serum urate may be determined by measurement of either the absorbance at 292nm
before and after treatment with Uricase since urate absorbs light a wavelength. OR. The
amount of oxygen consumed during Uricase reaction.

Clinical significance
Serum urate concentration is elevated in:
- Gout
- Renal disease
- After increased breakdown of nucleic acid or nucleo proteins (leukemia)
- Polycythaemia
- Toxemia of pregnancy
- After irradiation of
- X-ray sensitive carcinoma
NB. Some individuals have an elevated concentration of serum urates despite the absence
of disease that is called idiopathic hypervuricamia
Serum urates concentration is decreased in:
- After administration of cortical like steroids
- Certain drugs that decrease re-absorption of urates e.g. aspirin or by drugs that block
a step in the formation of uric acid

MEASUREMENT OF SERUM ELECTROLYTES

Electrolytes are compounds that dissociate in solution to form electrically charged particles or
ions. The positively charged ions are called cations and the negatively charged ions are
anions.

Electrolytes are important in helping to maintain the stability of the body’s internal
environment. To maintain the electrical neutrally in the body, the proportion of cations to
anions must balance.

The electrolytes found in the body’s fluid include:

Main cations: main anions

- Sodium - bicarbonate
- Potassium - chloride
105
 - Magnesium -phosphate, organic acid
- Calcium -sulphates, protein
The frequently measured electrolytes in the laboratories are sodium, potassium, calcium
chloride, and the bicarbonates.

Functions of electrolytes

 Electrolytes are important in helping to maintain the stability of the body’s internal
environment.
 Sodium with its associated anions chloride (Cl-) accounts for most of the osmotic
activity of the extracellular fluid
 It is important in determining the distribution of water across the cell membrane
 Bicarbonate is important in preserving the acid-base balance in the body
 K+, Na+, Mg2+ and Ca2+ are important for neuromuscular activity and cardiac function
 Plasma proteins are important in controlling plasma volume. Higher osmotic pressure
of plasma tends to draw fluids from tissues into plasma to counter balance the
hydrostatic blood pressure. In the capillaries that tend to force fluid out of plasma into
the tissues.
 Regulates the heart rhythm /heart beat
 Serves as co-factor for enzymes
 Maintains bone mineralization
 Assist in blood coagulation mechanisms
 Regulates electron transfer reaction

TECHNIQUES USED TO MEASURE SERUM ELECTROLYTES

106
STABILITY OF ANALYTES IN BLOOD SPECIMENS

SODIUM:

Normal values:

Adult: 135 - 148 mmol/l

Newborn: 134 – 144 mmol/l

Child: 138 – 144 mmol/l

-It’s the main extra cellular cation, sodium, a blood electrolyte, is the most abundant cation
and the chief base of the blood. Its primary functions in the body are to chemically maintain
osmotic pressure and acid-base balance and to transmit nerve impulses.

-Sodium concentration is control of the kidneys and the CNS acting through the endocrine
system. In health, the level of sodium is kept constant within narrow limits despite wide
fluctuations in dietary intake.

107
Determinations of plasma Na+ levels are useful in detecting gross changes in water and salt
balance. Urinary Na+ is a more sensitive indicator of altered Na+ balance. Mechanism
for maintaining a constant sodium level in plasma and extracellular fluid include;

1. Renal blood flow

(a) Increased renal blood flow to the glomeruli will result in increased sodium and chloride
excretions.

(b) Decreased renal blood flow to the glomeruli will result in sodium and chloride retension
and edema. This occurs in patients with reduced cardiac out

2. Carbonic anhydrase enzyme activity

(a) The level of activity of this system is an important factor in control of the rate of sodium
excretion

(b) Inhibition of carbonic anhydrase enzyme activity results in increased sodium reabsortion
in the tubules.

3. Aldosterone

(a) Aldosterone acts on the distal tubules and also affects sodium reabsorption

(b) Regulation of aldosterone secretion is

*primarily by the rennin-angiotensin system

*secondarily by ACTH, sodium, and potassium concentration

(c) In primary hyperaldosteronism, sodium will be retained and hypertension will result. In
exchange for sodium, potassium will often be excreted and decreased potassium may be
found in this condition.

4. Action of other steroids whose plasma level is controlled by the anterior pituitary gland

These steroids can cause salt and water retension. During the menstrual cycle,E2 and P4
cause salt and water retension before menstruation and dieresis if fertilization has not taken
place.

5. Renin is a potent stimulus to aldosterone secretion. It regulates renal blood flow, the
glomerular flitration rate, and salt and water excretion. In renal diseases, excessive amounts
of rennin secreted into the plasma result in water and salt retension and hypertension.

6. Antidiuretic hormone (ADH) ,vasopressin secretion

(a) ADH controls the reabsorption of water at the distal tubules of the kidneys
108
(b) Secretion of this hormone is responsive to changes in extracellular fluid volume.

-An increase in plasma sodium normally results in 3 compensatory mechanisms coming into
play:

* Thirst prompts oral fluid intake

* ADH secretion from the pituitary is increase leading to renal water retention

* There is a shift of water from the intra-cellular to extra cellular.

Clinical implications

HYPERNATRAEMIA

1. Increased sodium levels are uncommon, but when they do occur they are associated with

(a) Dehydration and insufficient water intake

(b) Conn’s syndrome

(c) Primary aldosteronism

(d) Coma

(e) Cushing’s disease

(f) Diabetes insipidus

HYPONATRAEMIA:

1. Hyponatremia usually reflects a relative excess of body water rather than a low total body
sodium

2. Reduced levels are associated with

(a) Severe burns

(b) Severe diarrhea

(c) Vomiting

(d) Excessive IV induction of nonelectrolyte fluids

109
(e) Addison’s disease (lack of adrenal steroids impairs sodium reabsortion)

(f) Severe nephritis

(g) Pyloric obstruction

(h) Malabsorption syndrome

(i) Diabetic acidosis

(j) Drugs like mercurial diuretics and chlothiazide diuretics

(k) Edema

PANIC values < 120 or ≥155 mmol/l

POTASSIUM:

-It’s the principal intracellular cation

-98% of it is maintained within the cells by the ATP dependent mechanism [sodium pump] i.e
any sodium which diffuses in the cells is actively excreted in exchange for potassium

-it transimittes nerve conduction

-it has a role in intracellular osmolarity

-together with ca+ & Mg++, Ca++. K+ controls the rate & force of contraction of the heart &
thus the cardiac output

- K+ & Na+ ions are vital in the renal regulation of acid-base bal coz H+ are substituted 4
sodium & K+ in the renal tubule

* Insulin accelerates the cellular uptake of K+ thus elevated levels of plasma potassium
encourage secretion of insulin

Evaluation of test; this test is used to evaluate changes in body K+ and is helpful in
diagnosing disorders of acid-base and water balance in the body.

RR:
110
Adults: 3.5 – 5.0mmol/l

Newborn: 3.7 – 5.9mmol/l

Child: 3.4 -4.7mmol/l

HYPERKALAEMIA:

1. Renal failure

(a) Oliguria

(b) Anuria

2. Cell damage as in burns, accidents, surgery, chemotherapy, DIC {damaged cells will
release potassium into the blood)

3. Acidosis –drives potassium out of the cells

4. Addison’s disease

5. Selective hypoaldosteronism

6. Internal hemorrhage

7. Uncontrolled diabetes

8. Acidosis

Hypokalemia

1.Values ≤3.5 mmol/l are commonly associated with deficiency ,rather than normality

2. Decreased levels are associated with

* Diarrhea

*Pyloric obstruction

*starvation

* Malabsorption

*severe vomiting

111
 *severe burns

*primary aldosteronism

*Renal tubular acidosis

*Diuretic administration

*other drugs like steroids and E2

*liver disease with ascites

*chronic fever

Interfering factors

*Opening and closing the fist 10 times with a tourniquet in place results in an
increase of the potassium level by 10 – 20% thus its recommended that the blood
sample obtained without a tourniquet.

Panic values

<2.5 or >6.5 may cause heart problems leading to death

CHLORIDE

Normal value: 95 – 106mmol/l

-major extracellular anion, chemically it exists primarily in combinations of sodium chloride

and hydrochloric acid; its helpful in diagnosing disorders of acid-base and water balance

Chloride maintains cellular integrity through its influence on osmotic pressure. It is also
significant in monitoring acid –base balance and water balance.

Chloride has the reciprocal power of increasing or decreasing in concentration whenever

changes occur in the concentration of other anions. In metabolic acidosis, there is a
reciprocal rise in chloride concentration when the bicarbonate concentration drops.Similarily,
when aldosterone directly causes an increase in the reabsorption of sodium (which is a
positive ion), the indirect effect is an increase in the absorption of chloride (the negative ion)

112
Chlorides are excreted with cations during massive dieresis from any cause and are lost from
the gastrointestinal tract as a result of vomiting, diarrhea.

EXPLANATION OF TEST

The measurement of chloride is usually done for its inferential value and is helpful in
diagnosing disorders of acid-base and water balance. Because of the relatively high
concentration of chloride in the gastric juices, prolonged vomiting may lead to considerable
chloride loss and lowered serum level.

Clinical implications

1. Whenever the serum level is much lower than 100 mmol/l ,the urinary excretion of chloride
falls to a very low level.

2. Decreased chloride levels occur in

(a) Severe vomiting

(b) Severe diarrhea

(c) Ulcerative colitis

(d) Sever burns

(e) Diabetic acidosis

(f) Addison’s disease

(g) Acute infections such as pneumonia

(h) Fever

(i) Use of drugs as mercurial and chlorothiazide diuretics

3. Increased chloride levels occur in

(a) Dehydration
113
(b) Cushing’s syndrome

(c) Hyperventilation

(d) Anemia

(e) Cardiac decompensation

(f) Some kidney disorders.

*Interfering factors

The plasma concentration of chloride of infants is usually higher than of children and adults

**Panic value: <70 or >120 mmol/l

LIVER FUNCTION TESTS (LFTs)

FUNCTIONS OF THE LIVER

 The liver a very important organ in the body with very many functions and a few of
them are below:-
 Storage of the iron. The liver stores iron which is used in formation of red blood cells
 Bilirubin conjugation and removal. When Bilirubin accumulates in the blood, jaundice
or yellow pigmentation of the body fluids observed and the liver prevents this by
conjugating Bilirubin and removing it through the urine and other ways like in stool
 Storage of glycogen. Glycogen is an animal substance. This can be of use when the
body lacks or requires glucose. It is converted to glucose which will provide energy.
 Production of blood clotting factor. The liver also participates in formation of clotting
factors
 Manufacture/formation of bile .Bile is used in fat emulsification which is important for
the body
 Filter system of the body. The liver works as a filter for the body .Here it removes
bacteria which may cause harm to the body
 The liver works as a store for vitamin A & B12, iron
 Detoxication. Substances which may become poisonous if allowed to accumulate
such as urea is converted to ammonia in ornithrine cycle
 Hydrolysis. The liver hydrolyses alcohol and poisons to products which are non
poisonous and removes them.
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  Manufacture of plasma proteins such as fibrinogen
 Removal of worn out red blood cells
 Formation of red blood cells especially red blood cells.

PROTEINS

 These are the main nitro genous constituents of the body tissues and foods we eat
 They comprise of carbon, oxygen, nitrogen, hydrogen, phosphorus and sometime
sulphur
 They are obtained from foods like meat, eggs, milk, cheese, peas, fish, beans etc
 The building blocks of proteins are amino acids linked together through peptide bonds
to form polypeptides
 Plasma proteins can be obtained from diet, liver synthesis e.g. fibrinogen,
prothrombin, albumin, alpha and beta globulin. Plasma cells and lymphoid tissues can
produce gamma globulins just like the reticulo-endothelium system

BASIC STRUCTURE OF AMINO ACIDS

NH3/NH2 R-CH-NH3/NH2-COOH

H-C-COOH

PROPERTIES OF AMINO ACIDS

The are 20 naturally occurring amino acids which differ from one another depending on the
nature of R

they contain

 Amino acids become charged differently at different times .Some times they are
positively charged and at other times negatively charged.
 At PH of 7.4amino acids carry both positive and negative charges co-currently and
they are referred to as zwitter ions
NH3+

CH-COO- (ZWITTER ION)

 Amino acids can be designated as L or D just like Retto

 When the amino group on the second last carbon of the amino acid is on the right
hand side, it is a D-amino acid but if it’s on the left, it is an L-amino acid

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 COO-
COO-

H-C-NH3 H2N-C-H

CH3 CH3

D-alamine L-alamine

 Amino acids are broadly classified as essential or non-essential. Essential amino

acids are the type that cannot be synthesized in sufficient quantities by the body to
meet its needs.
 Whereas non-essential amino acids can be synthesized in the body at a rate that
matches the bodies metabolic needs. Therefore essential amino acids can only be
synthesized in sufficient quantities through diet

Examples of no essential

 Isoleucie, leicine, valine

Example of non-essential amino acids
 Alanine, Arginine, asparagines

HOW PROTEINS ARE FORMED

 Amino acids are joined to one another to form a dipeptid e, tripeptide and eventually a
polypeptide
 The bonding is usually between a carboxylic group of one amino acid and the amino
group of another amino acid
H3N-CH-COO- + H3N-CH-COO- H3N-CH-CH-C-NH-CH-COO-
 Depending on the length of the chain, extent of folding and nature of bonds holding
the polypeptide chain, the resulting protein structure formed can be referred to as
primary, secondary, tertiary and quaternary

PRIMARY STRUCTURE

Comprises of a linear polypeptide chain and a basic structure that gives rise to the other
protein levels. It only has covalent bonds.

SECONDARY STRUCTURE

Are x-terised by folding of the polypeptide bonds to form an alpha helix and it is a spiral form.
It has peptide bonds,hydrogen bonds and non polar hydrophobic bonds in its build up

TERTIARY STRUCTURE

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Are x-terised by extensive folding and coiling of the polypeptide chain to form a complex
globular 3-dimensional structure

It is usually a result of intramolecular forces like hydrogen bonds, hydrophobic bonds, dipole-
dipole attractions, electrostatic attraction and disulphide linkages.

QUATERNARY STRUCTURE

Are characterized by interaction of 2 or more tertiary pattern structures to form a biologically

functional protein e.g. Hemoglobin which is made of 2 alpha & 2 beta globulin patterns
(polypeptide chains)

Many biological enzymes have quaternary pattern structure which when altered renders
them non functional

Denaturation as is commonly used with regard to enzymes is a result of altering the

functional structure or the pattern making it up.

FUNCTIONS OF PROTEINS

 Act as enzymes or are used in synthesis of enzymes.

 May be used in synthesis of hormones in the body .
 Act as carrier molecules in the body e.g. Hemoglobin transports oxygen to tissues and
carbon dioxide back to the lungs. Albumin transports non water soluble Bilirubin to the
liver for conjugation
 Antibodies provide an immunological defence.
 Help in blood clotting by fibrinogen.
 Work as amphoteric compounds where they work as buffers
 Tendons and collagen in the body which offer mechanical support to various internal
organs are patterns in nature
 Can be used as a source of energy following deficiency of RCHO.
 Protein control colloid osmotic pressure of the body.
 Used for growth and repair of bodies

THE ROLES OF A LIVER IN PROTEIN METABOLISM

 The liver synthesizes albumin

 The liver is the site of synthesis of alpha 1 globulin, alpha 2 globulin.
 The liver also synthesizes Beta globulin.
 Also synthesis fibrinogen

When liver damage is chronic and severe, the ability of the liver to synthesizes patterns can
be impaired.

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Properties of protein

 They are denatured by heat through conjugation but can be also denatured by strong
acids, bases, u.v rays
 They can be precipitated by salts of heavy metals e.g. lead, mercury, copper and zinc
 They can be changed to form amphoteric molecules
 They are soluble in water to form colloidal solutions
 In their purified form, they odorless and tasteless

CLASSIFICATION OF PROTEINS

 Proteins are either simple, conjugated or derived

 Simple pattern is one which on hydrolysis yields only one type of amino acids e.g.
albumin
 Conjugated patterns are pattern groups e.g. glycoprotein.
 Derived proteins are usually obtained following the Denaturation or partial digestion

PLASMA PROTEINS

 These are mainly made up of albumin but also consists of alpha 1, 2, beta and gamma
globulins
 It is common to find serum total pattern and albumin analyzed in the laboratories
 The difference 1.1 2 concentrations usually gives a rough estimate of the plasma
globulin concentration. i.e.(serum total ptn)-(serum albumin)=(serum globulin)

TOTAL PROTEINS

 Total pattern concentration in plasma may vary because of changes in the volume of
plasma water or in amounts of individual proteins
 Hyper proteinaemia is seen in digestion occurring either to diabetic ketosis
 Haemo dilution causes a decrease in concentration of all pattern and occurs in water
intoxication or water retention syndromes during massive intravenous infusions and ot

MEASUREMENT OF PROTEINS

 Serum total pattern can be measured by biuret’s method

 Serum /plasma albumin can be determined by bromocresal green method
 Salting out can be used for determination of varions pattern fractions. Sodium
sulphate, ammonium sulphate at different concentrations precipitates out different
plasma patterns.

SPECIMEN REQUIREMENTS

 Serum, plasma or other body fluids may be used for determining total patterns.

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  Pattern concentration

Total pattern is stable in serum and plasma for up to 24hour at room temperature and for at
least 1month when refrigerated at 4 C

N.B. The most frequently used method for determining pattern is the biuret reaction. And is a
suitable method for quantitative determination of total patterns by spectrophotometry.

BIURET METHOD/BIURET REXN

“In this reaction, cupric ions (Cu2+) in biuret’s reagent react with peptide bonds of
ptn. In an alkaline condition form a purple colored solution whose intensity is directly
proportional to the concentration of patterns in the sample and is measured
calorimetrically at 540nm”

DETERMINATION OF TOTAL PROTEIN USING A REFRACTOMETER

 Refractometry is a quick method used in analysis of total serum proteins
 The refractive index of water is 1.33 at 20C
 When substances are dissolved in water, S.G increases
 The refractive index of a solution is affected by its temperature, however,
refractometers for clinical laboratory use have been designed to compensate for
effects of different temperature in a range 11-15-37C
 Clinical instruments are calibrated for total salts expressed either as concentrations of
patterns in g/dl of serum or as serum gravity(S.G)for urine but significant errors may
be caused by alterations in albumin, globulin ratio, Azotemia, hyperglycemia,
hyperbilirubinaemia and especially lipaemia.

Principle;
“The refractive index of a clear serum is viewed directly with the total ptn concentrator. Light
entering the instrument in a beam parallel to the prism is refracted by the solution and is
projected against the eye piece which is marked with scales the field in view is demarcated
into a light and dark area. The scale is read where boundary of light and dark demarcation
cuts it.”

 The water of S.G 1.000 for zeroing

 At a ptn concentration of <3.5g/dl, refractometric results are likely to be in accurate
 At a level of ptn >11.0g/dl, are valid results can be obtained by diluting the serum with
equal parts of water and then read

METHOD.
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  Spread a drop of serum on the plate , the prism and the cover
 Hold the end against bright light and read the ptn concentration where the
demarcation line between the bright and dark area crosses

Reference ranges

6.6-8.1g/l

CLINICAL SIGNIFICANCE

Total proteins can be increased in ;

 Multiple myeloma
 Wasting disease e.g. T.B, HIV
 Dehydration

Total protein can be reduced in:

 Kwashiorkor
 Hemolytic anemia
 Slightly reduced in pregnancy
 Reduced in kidney diseases more so nephritic syndrome

ELECTROPHORESIS
This refers to the migration of charged particle in an electrolyte solution when an electric
current is passed through it. The different components at a PH above or below their
isoelectric point will move at different rates. At the end of electrophoresis, they will appear as
separate from one another clearly seen after staining. Their bands on separation after
staining will be seen in order the heaviest to the lightest (least moving to fastest). As gamma
globulin, fibrinogen, beta globulin, alpha 1, 2 & albumin

After electrophoresis of normal plasma constitutes about 55.2% of the total plasma protein,
globulin constitutes 44.8%, alpha 1 5.3%, alpha 2 8.6%, B 13.4%, gamma 11% and
fibrinofen about 6.5%

ALBUMIN

The main biological function of albumin includes;

 Transport of various ligands in the body e.g. Bilirubin, long chain fatty acids, calcium,
thyroid horrrmones, drugs, cortisol, sex steroids etc
 Maintenance of plasma osmotic pressure
 Serves as a source of endogenous amino acids. Albumin is the main source abundant
protein in human plasma representing about 55-65% of the total ptns
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  Albumin is produced in the liver
 Because of it’ relatively small size, albumin is a useful indicator of the integrity of the
glomerular and other membranes
 Albumin qualification is useful in the management of degradation and other conditions
such as
I. Excessive protein albumin loss due to renal damage in
 Chronic glomerular nephritis
 Diabetes mellitus
 Severe hemorrhage or burns
II. Impaired synthesis as seen in hepatic disease
 Toxaemia of pregnancy and
 Genetic ddisorder
III. Increased catabolism as a result of tissue damage and inflammation
IV. Reduced absorption of amino acids caused by mal absorption syndrome or
malnutrition
V. Altered distribution as in where pressure in the partial circulation results in the
movement of albumin into the peritoneal fluid
 For analysis, serum is the specimen of choice but hepatinised plasma can also be
used if e precaution are taken

Precaution

Various stasis must be avoided during vein puncture as haemo concentration increases of
the concentration of plasma proteins

ANALYSIS OF ALBUMIN
a. Electrophoresis is considered by many to be the reference method for albumin
determination although it is labor intensive
b. Routinely, albumin estimation is based on its binding with anionic dye bromocresyl
green is usually use
BCG is preferred because;
 The dye specifically binds with albumin in the presence of other patterns
 There is high affinity of binding from the dye and albumin and small changes in
ionic strength and PH will not disrupt the dye-ptn complex
 Also bill and hemoglobin aren’t absorbed at absorption maximum of the dye-
albumin species which is 628nm
c. Albumin can be measured by reaction of anti-albumin antibody and monitored by
turbid metric nephlometric means
 Antibody-albumin complexes that form increases the absorption (turbidmetry) or the
scattering (nephlometry) of incident light which can be related to albumin
concentration and measured using an automated instrument
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INTERPRETATION

 The usual reference range serum albumin is 3.5-5g/dl

 The most severe albuminaemia is caused by protein has by way of urine or faeces
 When plasma albumin levels are below 2.5g/dl, edema results as the low plasma
osmotic pressure allows water to move out of the blood capillaries and into the tissues
 Albumin levels of <2g/dl may also be seen in situations of amino acid supply e.g.;
 Mal absorption syndrome due to chronic pancreatitis
 Alcoholic liver disease and
 Protein loosing enteropathy e.g. cancinomous of the stomach
 Because of its relatively small size, albumin is useful indicator of the integrity of
glomerular and the other membranes
 Anelbuminamia (congenital absence of albumin ). Is asymptomatic except for causing
occasional oedema
 The only known cause of hyperalbunaemia is that of haemoconcentration

BCG reagent (Bromo Cresyl Green)

 Dissolve 5.6g of succinic acid
5.8g of Bromo cresyl green
100mg of sodium azide in about 900ml of water
 Add 1g of NaOH and mix to dissolve
 Add 2.5mls of brij-35 (triton)
 Transfer slowly (avoid frothing) to 1litre volumetric flask and made up the volume to
1000mls with the water
 The solution is stable for several month at 2.8C

Principle
“Albumin has the ability to blind certain dyes. When BCG binds to albumin there is a shift in
dyes absorption wave length. Serum is diluted with buffered BCG at a PH 4.2. The
measurement of absorption at 630nm within 30second of mixing the serum and BCG.
Incubation is to avoid non specific reaction of BCG with globulins”.

Alternative principle
“Bromocresol green is an indicator which is a yellow in colour at PH 3.5-4.2 which binds with
albumin and changes colour from yellow to blue-green. The intensity of the colour
development is directly proportional to the concentration of albumin in the sample. And is
measured spectrophotometrically at 632nm

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FIBRINOGEN

Principle
“Fibrinogen is precipitated with freshly prepared 12.5% sodium sulphate solution. The
turbidity is compared with artificial pattern standard”

Reagents

 12.5% sodium sulphate solution

 Prepare before use 12.5g of anhydrous sodium sulphate and make up to 100ml

Method

 3.8mls of freshly prepared 12.5% sodium sulphate

 Use 0.2ml of oxalated plasma to add into the solution
 Mix and leave at room temperature for 10 minutes
 For the control, instead of oxalated plasma, use 0.2ml of serum. Normal range 200-
400mg/dl

Clinical significance

 Fibrinogen is increased in some disease or in infections such as : pneumonia,

rheumatoid fever, TB, slightly in premature separation of the placenta
 Severe liver disease
 Coagulation deficiency
 Typhoid fever
 Hemorrhage after child birth and feotal death in the uterus (intra uterine death)

CHANGES IN PLASMA PROTEIN CONCENTRATION DISEASE

1. Albumin concentration rarely becomes elated except in haemo-concentration or

dehydration. However, hypoalbuminaemia which refers to a decline in plasma albumin
concentration is a common finding in:-
 Prolonged malnutrition occurs in inadequate dietary intake of proteins, impaired ptn
digestion or absorption
 Chronic loss of proteins in urine as occurs in nephritic syndrome or in extra-vascular
e.g. in burns
 In ability to synthesize albumin like in liver arrhosis
 Familial dysproteinaemia
 Idiopathic hypoproteinaemia
2. Alpha globulins often increase in acute febrile disease, moderate to advanced
tuberculosis and

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C-RECTIVE PROTEINS (CRP)
 Is an abnormal constituent in the alpha globulin fraction of human serum? It is
produced in the body following an inflammatory reaction.
 It is called CRP because it forms a precipitate with the somatic c-polysaccharide of
pneumococcus.
 It is done as a serological test with precipitation with CRP antiserum or by kits that
show bands on reading

Note: beta globulin are elated following increase in plasma lipids.

ENZYMES
 Are biological catalysts(produced in living tissues) that will speed up the rate of
biochemical reactions but will remain unchanged at the end of the reaction
 Enzymes commonly exist in higher concentrations in cells producing them than in
serum/plasma
 In the event of cell damage (due to trama/disease),they release their contents into
plasma which leads to measureable increase in the concentrations in plasma which
reflected by the increase in their catalytic activity in plasma
 Because of this , blood samples for enzymes activity analysis should have serum
separated from cells as soon as possible to minimize changes in the activity after
sample collection
 Haemolysis should be kept minimal by taking precaution container for sample
collection

NB. Enzyme concentrations are usually small and would be difficult to measure instead,
their activity is measured by monitoring the rat

e of product formation or rate of substrate consumption.

CLASSIFICATION OF ENZYMES

There are 6 major classifications of enzymes

 Oxido reductases
 Transferases
 Hydrolases
 Lysases
 Isomerases
 Ligases

1.Oxidoreductases

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These are enzymes which catalyze a variety of oxidation reduction reaction and frequently
employ co-enzymes like NAD+, NADP+, FAD, hydrogen acceptors

CH3-CH-COO- + NAD+ CH3-C-COO- +NADH +H+

OH OH

Lactate pyruvate

They are commonly referred to as oxidases, peroxidases, reductases, dehydrogenases etc

2.Transferases

 These are enzymes which bring about exchange of amino groups between substrates
e.g. AB + CD AC + BD
these are divided into:-
I. Amino Transferases which bring about exchange of amino and keto groups
between amino acids and keto acids
II. Phospho Transferases that transfer a phosphate radical
III. Phosphorlyases

3.Hydrolases

These is a large group of enzymes containing all digestive enzymes. The enzymes are
specifically named after the substrate they work on. 0781385227

They catalyse cleavage of bonds by addition of a water molecule

NH2-C-NH2 + H2O CO2 + 2NH3

O urease

Other examples include proteolytic enzymes (hydrolyse peptide bonds) carbohydrates,

carboxy esterases, deaminases etc

4.Are enzymes which remove groups of substr

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