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Chapter- 1 General Introduction
1
Section 1.1
Biomolecules
1.1 Introduction
1.1.1 Introduction to biomolecules
Biomolecule are those molecules that are produced by living organisms and
they include large polymeric molecules like proteins, polysaccharides, lipids, and
nucleic acids as well as small molecules such as primary and secondary metabolites
and natural products. Biomolecules form the bodies of all living beings and they are
the causes and products of all chemical processes that keep them alive [1].
Biomolecules play extremely important roles in the functioning of all body tissues,
organs, organ systems, and the organism as a whole. Study of these biomolecules in
terms of their structure, functions, properties and many more is subjected as bio-
molecular chemistry.
The fields of research covering chemistry, biochemistry and molecular biology
have been one of the most active areas of scientific inquiry in recent decades. At the
core of the life science disciplines, the area of biochemistry and molecular biology
seeks to understand fundamental processes of life, because they provide a detailed
theoretical understanding of the chemistry of biomolecules.
Living organisms generate and sustain an enormous variety of organic
compounds [2], some of which are particularly relevant as structural and functional
components, whereas others are present in very minute quantities and still act as
regulators, messengers, or defense compounds. Particular attention is bestowed to
such organic compounds of living organisms, as carbohydrates, amino acids and
proteins, nucleic acids and lipids as they perform all structural and/or function related
roles that allow an organism to survive and reproduce.
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Chemists are more familiar with such biomolecules as carbohydrates, proteins,
lipids, enzymes and such others. However, many may not have clear in depth
understanding of biology related aspects of chemistry. Without the development and
improvement of bioanalytical methods over the recent decades, the enormous
progress in genomics and proteomics would have been impossible.
1.1.2 Classification of biomolecules
The term biomolecule refers to naturally occurring chemical compounds
present in living organisms, invariably all of which contain carbon. The study of
carbon-containing molecules is the subject of organic chemistry. This involves the
study of characteristic and reactions of chemical compounds that primarily involve
carbon and hydrogen, but may also contain other chemical elements. The field of
organic chemistry emerged with the false impression by chemists that all organic
molecules were related to life processes and that a ‘vital force’ [3] was necessary to
make such molecules. This thinking was blown out of the water when organic
molecules such as soaps and urea were synthesised in laboratory without any ‘vital
force’. Thus, it was concluded that all organic molecules are not biomolecules. Life
processes depend not only on organic molecule but inorganic molecules, also play
important-roles.
There are many methods of classifying biomolecules, and this very often leads
to some confusion. The most simple way of division of biomolecules is on the basis of
their size, that is, small (micromolecules) and large (macromolecules). Smaller
molecules are most often referred to by their actual names (e.g. amino acid) or the
more popular term small molecule.
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It is not easy to classify biomolecules depending on their role, hence they are
classified based on their structure and function. A diverse range of biomolecules exist,
including large polymeric molecules comprising proteins, polysaccharides, and
nucleic acids as well as smaller biomolecules such as metabolites and natural
products.
The polymeric biomolecules are made of small biomolecules (monomeric
units) called as building blocks. Carbohydrates are the building blocks of poly
saccharides. Like wise all polymeric biomolecules are the products of monomeric
biomolecules. A few biomolecules of importance are explained.
1.1.2.1 Polysaccharides:
Polysaccharides polymer chains are made of carbohydrate joined together by
glycosidic bonds with the general formula Cx(H2O)y. If the monomeric unit is of same
carbohydrate then these are termed as homopolysaccharide, if more than one type of
monomeric unit is present then it is referred as heteropolysaccharide.
When polysaccharides are made of large number of glucose units are joined
together by glycosidic bonds then it becomes starch. This polysaccharide is
synthesized by plant which becomes the energy storage source. The same
polysaccharide of glucose when present in humans and fungus is called as glycogen
which is synthesized in human body system and acts as secondary source for energy
storage.
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1.1.2.2 Proteins
Proteins are the high molecular mass complex biopolymers of amino acids
linked together by peptide bonds. They are the most abundant organic molecules of
the living system and form the fundamental basis for structure and function of life.
They invariably occur in every part of the body and take part in the entire structure
and functions of life. The amino acids required for the synthesis of protein and those
synthesized within the body are referred to as non-essential amino acids, those which
are obtained from outside source are considered as essential amino acids. They are
very much needed for growth and maintenance of the body system and to carry out all
functions. Enzymes, hemoglobin and such others also form part of protein.
1.1.2.3 Nucleic acid:
Nucleic acids, RNA and DNA, are biomolecular polymers made by the
sequence of monomeric entities called nucleotides, which are composed of three
different parts: a sugar, a base and a phosphate. These biomolecules play an essential
role in transmitting hereditary characters and also biosynthesis of specific proteins
which are required by the organism. The backbone of nucleic acids is essentially
formed by the sugar, ribose in RNA and 2- deoxyribose in DNA, both linked to each
other through a phosphate bridge.
1.1.2.4 Metabolites
Metabolites are the products of enzyme-catalyzed reactions that occur
naturally within cells. Metabolites are classified as primary and secondary [4].
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1.1.2.4.1 Primary metabolites
Primary metabolites are those which are directly involved in normal growth,
development, and reproduction [5]. Amino acids, nucleotides, proteins, carbohydrates,
lipids and vitamins are the primary metabolites needed for the growth of the body,
whereas acetone, ethanol, butanol, organic acids, and others are required for deriving
energy. The quantam of some of these metabolites produced exceeds much more than
that actually needed by the body. Absence of these primary metabolites may lead to
some serious disorders, sometimes even to death.
1.1.2.4.2 Secondary metabolites
Secondary metabolites are not directly involved in the normal growth,
development, or reproduction and do not result in to immediate death, but rather had
to in long-term some kind of impairment to the organisms [6]. Some examples of
secondary metabolites are alkanoids, terpenoids, glycosides and phenolics.
1.1.2.5 Natural compounds
Natural products are those chemical compounds or substances which are
produced by any living organism – existing in nature [7]. The end products of
secondary metabolism are often termed as natural products; each compound is unique
for a particular organism or classes of organisms. Natural products possess some or
other kinds of pharmacological or biological activities and are the major source for
most of the active ingredients of medicines [8].
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1.1.3 Metabolism:
Metabolism encompasses the entire process of chemical reactions that take
place in a living organism that not only allow it to reproduce, develop and maintain its
structure but also respond to the environment [9]. These chemical reactions form an
intricate network of pathways and cycles in which the flow of reaction products
(metabolites) is determined by many regulatory mechanisms. Metabolism includes
each and every cellular process, ranging from DNA replication to transcription and
translation to enzyme function, and also involves the chemistry of small molecules in
the cell.
Enzymes are crucial to metabolism [10] because they allow organisms to drive
desirable reactions for which energy is required and the reaction will not occur by
themselves, but this is achieved by coupling them to spontaneous reactions that
release energy. Enzymes act as catalysts and allow these reactions to proceed quickly
and efficiently. Enzymes also allow the regulation of metabolic pathways in response
to changes in the cell's environment or signals from other cells. Traditionally,
metabolism is subdivided into two aspects, catabolism and anabolism.
1.1.3.1 Catabolism
Catabolism is otherwise a destructive type of metabolism. The process
involves a series of degradative chemical reactions that break down complex
molecules into smaller units, and release energy in the process. Carbohydrates are
broken down into simpler molecules that release energy in the form of adenosine
triphosphate. Carbohydrates are primarily broken into sugar molecules known as
monosaccharides and then catabolized to glucose, which enters the bloodstream.
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1.1.3.2 Anabolism
Anabolism is on the otherhand a constructive phase of metabolism. The
process involves a sequence of chemical reactions to construct or synthesize
molecules from smaller units by consuming energy in the process. Proteins are
constructed by anabolic metabolism of amino acids joined together by peptide
linkages. Anabolic metabolism of glucose gives polysaccharide which is stored as
starch or glycogen.
1.1.4 Significance
1.1.4.1 Significance of biomolecules in clinical diagnosis
Each living system is composed of networks of interacting biopolymers, ions
and metabolites. These components drive a complex array of cellular processes, many
of which cannot be observed when the biomolecules are examined in their purified,
isolated forms [11]. Every action of ours requires energy, from sitting to walking and
running, even sleeping. Our energy requirement at any particular point of time
depends on the level of activity in which we are engaged. Our required energy is
derived from the metabolism of the food we consume. Some molecules in the food
that we consume are converted into polymeric biomolecules and are stored as energy
source, which could be reutilized when the need arises.
Metabolism of these biomolecules is carried out in compartmentalizations
(Cells) as per specific metabolic pathways, which are nothing but a series of chemical
reactions to produce specific products. These reactions are catalysed by complex
biomolecules called as enzymes. Metabolism process is interconvertible and
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irreversible. By the regulation of this metabolism process the flow or flux of
metabolites, reactants and products could be controlled through a given pathway.
Bio-macromolecules in food cannot be utilised by our body system in their
native form. They have to be broken down and converted into simpler substances,
which is acheived in the digestive system. This process of conversion of complex
food substances to simple absorbable forms is called digestion and is carried out by
our digestive system through mechanical and biochemical methods.
Cellular metabolic activity, or the many vital biochemical processes executed
by different cell types in various tissues and organs, also gives rise to a variety of
unnecessary metabolites or end products, which must be eliminated from the system.
Excessive amounts of nutrients, such as water, electrolytes, and minerals produced
must also be eliminated in order to maintain the physiological equilibrium of body
systems, such as neural activity, cardiac function, and blood pressure. Through the
process of respiration, the lungs deliver oxygen into the blood circulation and collect
carbon dioxide from the venous vessels, to be eliminated through exhalation. The
process of digestion requires the elimination of non-absorbable substances and other
particles that are excreted from the body in the form of feces. One-fourth of the fecal
matter that is eliminated from the intestine is constituted by solid matter and the
remainder is water.
Urine excreted through kidney contains several ion solutes, and many
metabolites including urea, uric acid, creatinine, bilirubin, and a variety of toxins that
have been the outcome from either endogenous or exogenous metabolized products
by the cellular enzymatic detoxification process. These metabolites are ultimately get
eliminated from the body mostly through the urine.
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Enzymatic metabolic processes and detoxification as well as metabolic waste
removal are important for several vital purposes, such as maintenance of blood
pressure and appropriate levels of body fluid, protection of cells and organs against
oxidative stress, electrolyte balance, DNA integrity, control of hormonal levels, and
protection from environmental pollutants and solar radiation by detoxification.
1.1.4.2 Significance of metabolites
The global pool of all metabolites in a cell or metabolome, is a reflection of all
the metabolic functions of an organism under any particular growth condition. In the
absence of any in situ methods capable of universally measuring metabolite pools,
intracellular metabolite measurements need to be performed in vitro after extraction
[12]. The study of these metabolites is termed as metabolomics. Blood serum and
plasma are the biofluids that are increasingly important in metabolomics.
Metabolomics of fluids from the circulatory system will provide a view of the
metabolic status of an organism. Urine analysis provides information about an
organism’s waste products, whereas serum or plasma analysis measures homeostatic
levels of metabolites throughout the organism. The differences observed in metabolite
status may be either correlated to the disease being studied in clinical biomarker
discovery or changes in metabolic output in toxicology studies.
1.1.5 Analytical tools in metabolomics
The metabolome has been defined as the qualitative and quantitative collection
of data of all low molecular weight biomolecules (metabolites) present in a cell that
are participant’s in general metabolic reactions and that are essential for the
maintenance, growth and normal functioning of a cell.
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Metabolomics combines strategies to identify and quantify cellular metabolites
using sophisticated analytical techniques with the application of statistical and multi-
variant methods for information extraction data analysis, and data interpretation.
Metabolites are considered to “act as spoken language, broadcasting signals from the
genetic architecture and the environment” [13], and therefore, metabolomics is
considered to provide a direct “functional readout of the physiological state” of an
organism [14]. A range of analytical technologies and tools has been employed to
collect and analyze metabolites related data in different organisms, their tissues, or
body fluids.
Due to the huge diversity of chemical structures and the large differences in
abundance of analytical methods, there is no single technology fit to analyze the entire
metabolome. Therefore, a number of complementary approaches have to be resorted
to for extraction, detection, quantification, and identification of as many metabolites
as possible [15]
However, metabolites, especially secondary metabolites, are extremely
important for most organisms to defend themselves from stressful environments or
predators. Although primary metabolites involved in central metabolism can be used
to determine nutritional and growth status, secondary metabolite profiles may better
reflect the differentiation between species and their complex response to
environmental factors and other organisms.
1.1.5.1 Mass spectrometry (MS)
Mass spectometry is the most widely applied technology in metabolomics, as it
encompasses the blend of rapidity, sensitivity and selectivity coupled with qualitative
and quantitative analyses and has the ability to identify specific metabolites. Mass
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spectrometers operate by ion formation, separation of ions according to their mass-
tocharge (m/z) ratio and detection of separated ions. There are combined mass
spectrometric methods also available.
1. Volatile and thermally stable compounds are first separated by gas
chromatography followed by detection of eluting compounds traditionally by
electron-impact mass spectrometer. This method is known as gas
chromatography- mass spectrometry (GC-MS).
2. Separation of biomolecules by liquid chromatography followed by
electrospray ionization or atmospheric pressure chemical ionisation [16]. This
technique differs from GC-MS in distinct ways (lower analysis temperature,
and volatility of the sample not required) and this simplifies sample
preparation. This method is termed as liquid chromatography- mass
spectrometry (LC-MS).
1.1.5.2 FT-IR
FT-IR spectroscopy is a well established, constantly improving analytical
technique that enables rapid, nondestructive, reagentless and high-throughput analysis
of a diverse range of sample types. The principle of FT-IR lies in the fact that, when a
sample is interrogated with light (or electromagnetic (EM) radiation), chemical bonds
at specific wavelengths absorb this light and vibrate in either any one of the number
of ways, such as stretching or bending vibrations. These absorptions/vibrations can be
correlated to single bonds or functional groups of a molecule for the identification of
unknown compounds.
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1.1.5.3 NMR Spectroscopy
In the analysis of biomolecules, NMR spectroscopy is considered to be a
rapid, non-destructive, high-throughput method that requires minimal sample
preparation [17, 18]. NMR spectroscopy is a high-throughput fingerprinting
technique. NMR spectroscopy functions by the application of strong magnetic fields
and radio frequency pulses to the nuclei of atoms. Samples need to be mixed with a
reference compound solution (e.g., tetramethylsilane dissolved in D2O for 1H NMR).
The spectra are complex, containing a number of signals relating to metabolites. The
chemical shifts can be assigned to specific metabolites and by adding pure metabolite
further clarification can be obtained. The spectrum pattern is generally useful in the
classification of samples.
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Literature cited
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Section 1.2
Scope of research work
1.2.1 Introduction
Analysis of biomolecules provides an excellent remark on the working
condition of the organisms. The metabolome is the total complement of metabolites in
a cell under any given growth condition [1]. To define and follow all cellular
metabolites are no less important than to determine the proteins in proteomes or
RNAs in transcriptomes. Global metabolite profiling is beginning to provide deeper
insights not only into metabolism but also into cellular physiology and functional
genomics [2]. Even though the strategies and methodologies of metabolome analysis
are still in development, the metabolome approach has been employed in
differentiating unique aspects of metabolism under several environmental stress states
[3].
In course of our research work we have selected three major biomolecules,
which are considered for routine blood and urine tests. The merits and demerits of the
analytical methods for those biomolecules are briefly explained.
1.2.1.1 Bilirubin
Bilirubin level in serum gets enhanced under a variety of clinical conditions
such as dyserythopoiesis and hemolysis [4], total bilirubin concentration includes its
conjugated and unconjugated forms, which represent bilirubin before and after hepatic
processing. The normal total bilirubin concentration ranges from 5-19 µmol/L in
blood. Increase in its concentration in blood leads to hyperbilirubinemia, which may
result in serious pathological disorders especially in neonates since their brain tissues
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are sensitive to its toxic effect, which can lead to kernicterus [5], with impairment, of
auditory, motor or mental functioning. Other disorders of excess bilirubin include
Dubin-Johnson syndrome [6] and Rotor syndrome [7]. Hyperbilirubinemia cannot be
totally prevented but early diagnosis and treatment are important in controlling
bilirubin levels. This is also currently being used as a reference in the management of
jaundice [8].
Some of the recently reported techniques for estimation of bilirubin include
enzymatic [9], fluorometric [10], chemiluminescence [11], and HPLC [12]. But still,
spectrophotometry, is the most extensively used technique. Most of the
spectrophotometric methods for the determination of bilirubin in serum are based on
Ehrlich [13], where bilirubin in urine reacts with 1-diazobenzenesulfonic acid to form
a chromophore. Van den Bergh and Snapper applied this method for the quantitation
of bilirubin in serum. Later, van den Bergh and Muller [14], described the accelerator
effect of ethanol on this reaction. Malloy and Evelyn [15], proposed a method in
which a lower alcohol concentration was used to avoid loss of bilirubin by protein
precipitation. Adler and Strauss [16], found caffeine-sodium benzoate could be used
to replace alcohol. Jendrassik-Grof [17, 18], combined caffeine-sodium benzoate with
sodium acetate as an accelerator at pH 13.4 to couple bilirubin with diazo reagent to
form alkaline azobilirubin. The Jendrassik-Grof method, involving the use of
diazotized sulfanilic acid is the currently used method and has been recommended as
the procedure of choice for total bilirubin estimation by the U.S. National Committee
for Clinical Laboratory Standards (NCCLS) [19]. This Candidate Reference Method
for total bilirubin was further developed and validated by the Committee on Standards
of the American Association for Clinical Chemistry and is now being used world
wide. Most of these methods have serious limitations with respect to their sensitivity
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and linearity. In most instances, the conditions required for best sensitivity,
stoichiometry (linearity or range), and for the elimination of interferences have not
been completely outlined.
1.2.1.2 Creatinine
Creatinine, the break-down product of creatine phosphate present in muscle is
normally produced at a fairly constant rate in the body depending on age, muscle
mass, sex and other factors. Clinically creatinine itself has no notable toxicity, but
determination of its concentration in biological fluids is necessary for diagnosis of
renal, muscular, and thyroid [20] abnormalities. Creatinine level is mainly needed to
calculate the creatinine clearance, which reflects the glomerular filtration rate (GFR)
[21], the marker of renal function. Estimation of GFR is the most widely used test for
renal function in clinical practice.
Many analytical methods have been proposed for the estimation of creatinine
concentration in biological fluids most of them is based on Jaffe’s picric acid method.
Picric acid method is widely accepted for creatinine measurement involving alkaline
sodium picrate; but it is normally affected by some endogenous species present in
biological samples. Several modifications were effected to Jaffe’s method to eliminate
or reduce interferences. These included specific adsorption of creatinine, removal of
interfering compounds, dialysis, varying the pH, and kinetic measurements. But, none
of these modifications could successfully eliminate the interferants present with
varying concentrations in biological matrix.
Completely enzymatic based methods have been developed to improve the
specificity of creatinine determination. Although such methods under optimum
conditions give accurate results but they involve high cost and the precision is low.
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The use of multi-enzyme system requires caution, as the risk of interference by
enzymes increases with the use of more number of enzymes [22, 23]. A coupled-
enzyme assay for creatinine may involve creatinine amidohydrolase, creatine kinase,
pyruvate kinase, lactate dehydrogenase, NADH, and the change in absorbance at 340
nm [24, 25].
There are other methods for the assay of creatinine which include 3,5-
dinitrobenzoic acid [26, 27], 3,5-dinitrobenzoyl chloride [28, 29], methyl-3,5-
dinitrobenzoate in a mixture of dimethyl sulfoxide, methanol, and tetramethyl
ammonium hydroxide [30], 1,4-naphthoquinone-2-sulfonate [31-33], Sakaguchi’s
color reaction of creatinine with o-nitrobenzaldehyde [34, 35] and mass
fragmentography [36].
1.2.1.3 Hemoglobin
Hemoglobin, the main component of the red blood cell, functions in the transportation
of oxygen and CO2. Hemoglobin consists of 1 molecule of globin and 4 molecules of
heme (each containing 1 molecule of iron in the ferrous state). Globin consists of 2
pairs of polypeptide chains. In the hemoglobin molecule, each polypeptide chain is
associated with 1 heme group; each heme group can combine with 1 molecule of
oxygen or CO2.
Drabkin’s method of haemoglobin estimation has been used since long [37]. In
spite of the availability of newer techniques, which give more reliable and accurate
result, this method is still in vogue. The principle of this method is that when blood is
mixed with a solution containing potassium ferricyanide and potassium cyanide, the
potassium ferricyanide oxidizes iron to form methemoglobin. The potassium cyanide
then combines with methemoglobin to form cyanmethemoglobin, which is a stable
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color pigment read photometrically at a wave length of 540nm.
The quantitative determination of plasma haemoglobin is of clinical
importance in haemolytic disorders, which occur either in vivo [38, 39] or in vitro
[40]. However, the carcinogenity of many commonly used reagents is undesirable for
routine laboratories. Benzidine, 0-tolidine [41] and dicarboxidine [42], used in
previous studies, are all carcinogens. Of the suggested alternative non-carcinogenic
chromogens, tetramethylbenzidine [43, 44] aminophenazone [45], and 2,2'- azino-di-
(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) [46] have proved suitable for the
quantitative determination of plasma haemoglobin. The reaction of these materials
with haemoglobin is based on the peroxidase activity of the haemoprotein.
1.2.2 Biological samples
Our research work is mainly based on biomolecules which have clinical
significance. The proposed method applications have been tested in human serum and
urine samples. It is very difficult to know whether the human blood sample is
infectious or not. Thus all human blood specimens are to be treated as infectious and
must be handled according to “standard precautions.”
Before conducting the applicability of the proposed methods we obtained the
necessary permission from Institutional Human Ethical Committee (IHEC-UOM
No.22/Ph.D/2008-09) of University of Mysore. With the help of clinicians, the blood
samples were withdrawn from the donors. The donors were well informed and their
consents were obtained before collecting the blood samples.
So obtained human blood samples were stored in tubes containing anti
coagulants and was preserved at 4 °C for use. Serum sample was obtained by
centrifuging the blood sample at 16,000 rpm using Remi Desktop centrifuge, heavier
Chapter-1 General Introduction
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particles like RBC’s settles at the bottom and serum samples remains on the surface.
The serum sample layer was pipetted using micropipette. The tubes used for storage
and the pipettes used for collecting serum samples are shown in figure
Heparinised tube used for sample
collection,
Micro pipettes used for pipetting serum
Human blood and blood products are classified and managed as medical waste
because of the possible presence of infectious agents that cause blood-borne disease.
Wastes in this category include bulk blood and blood products as well as smaller
quantities of blood samples drawn for testing or research. The used blood samples
were returned back to the clinical laboratory for disposal.
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Chapter-1 General Introduction
22
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Chapter-1 General Introduction
23
Section 1.3
Graphical abstract of the Research findings
1.3.1 Analytical probes for bilirubin estimation.
1. The basis of the present research is the cleavage of central methylene group of
bilirubin to formaldehyde and the latter reacting with azo group yielding to
coloured dye.
2. 3-Methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) is used as the
analytical probes for the assay of bilirubin.
3. The analytical probes have been characterized through absorbance, pH
response, temperature, stability, etc.
4. Linearity, sensitivity and effect of interferrants are studied broadly in this
method
5. Recovery and applicability of the method is tested in human serum samples.
6. The proposed method is compared with the standard kit method used in
clinical laboratories
Bilirubin
Formaldehyde
MBTH
Coloured dye
Formaldehyde
MBTH
Chapter-1 General Introduction
24
1.3.2 Analytical probes for creatinine estimation.
1. The basis of the present research involves the Pseudo enzyme activity of
copper creatinine complex.
2. The following are the analytical probes used in the assay of:
p-Phenylenediamine dihydrochloride with 3-dimethylaminobenzoic
acid (PPDD- DMAB)
p-Phenylenediamine dihydrochloride with Butylated hydroxyl Anisole
(PPDD- BHA)
3. The analytical probes have been characterized through absorbance, pH
response, temperature, stability, etc.
4. The Michaelis-Menten of each of the reactants was evaluated by the
Lineweaver-Burk plot.
5. Linearity, effect of interferrants and recovery studies are studied in this
proposed method
Cromogenic
probes
Coloured product
Pseudo
enzyme
Creatinine, Copper
Chapter-1 General Introduction
25
Catalytic parameters
Since it is a pseudo enzymatic reaction the following parameters were
analysed
Michaelis-Menten constant
Catalytic efficiency
Catalytic constant
Applications in the biological samples
The proposed methods were tested for their applicability in human blood and
urine sample and the result of the same was compared with that of the standard
method used in clinical laboratories.
Chapter-1 General Introduction
26
1.3.3 Analytical probes for hemoglobin estimation.
1. The basis of the present research is the oxidation of the reagent using
peroxidase activity of hemoglobin in presence of hydrogen peroxide.
2. 2,4-Dimethoxyaniline (DMA) is the analytical probes used in the assay of.
3. The analytical probes have been characterized through absorbance, pH
response, temperature, stability, etc.
4. The Michaelis-Menten of each of the reactants was evaluated by the
Lineweaver-Burk plot.
5. Linearity, effect of interferrants and recovery studies are studied in this
proposed method.
Catalytic parameters
Since it is a pseudo enzymatic reaction the following parameters were
analysed
Michaelis-Menten constant
Catalytic efficiency
Catalytic constant
Applications in the biological samples
The proposed methods were tested for their applicability in human blood and
urine sample and the result of the same was compared with that of the standard
method used in clinical laboratories.
Hydrogen Peroxide,
Cromogenic probe
Self coupled
coloured product.
Hemoglobin