Chapter- 1 General Introduction - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10686/8/08_chapter 1.pdf · Chapter- 1 General Introduction 2 Chemists are more familiar with

Chapter- 1 General Introduction

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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

[1] J. C. Jewetta, C. R. Bertozzi, Chem. Soc. Rev., 39 (2010) 1272–1279.

[2] L. E. Orgel, Trends in Biochem. Anal., 23 (1998) 491-495.

[3] G. R. Desiraju, News and views feature, Macmillan Magazines Ltd, (2001)

397-400.

[4] A. L. Demain, Biomed and Life., 67 (1980) 582-587

[5] S. W. Drew, A. L. Demain, Ann. Rev. of Microbio., 31 (1977) 343-356.

[6] A. L. Demain, Appl. Microbiol. Biotechnol., 52 (1999) 455-463.

[7] D. H. Williams, M. J. Stone, P. R. Hauck, S. K. Rahman, J. Nat. Prod., 52

(1989) 1189–1208.

[8] A. L. Harvey, Drug Discovery Today, 13 (2008) 894-901.

[9] J. A. Harris, F. G. Benedict, Proc. Natl. Acad. Sci. USA., 4 (1918) 370–373.

[10] M. J. Wolfgang. M. D. Lane, Annu. Rev. Nutr., 26 (2006) 23-44.

[11] J. A. Prescher, C. R. Bertozzi1, Nature, 1 (2005) 13-21.

[12] R. P. Maharjan, T. Ferenci, Anal Biochem., 13 (2003) 145-154.

[13] M. C. Jewett, G. Hofmann, J. Nielsen. F, Curr. Opin. Biotechnol. 17 (2006)

191-197.

[14] U. Roessner, J. Bowne, BioTechniques 46 (2009) 363-365.

[15] S. G. Villas-Boas, U. Roessner, M. Hansen, J. Smedsgaard, J. Nielsen.

Metabolome Analysis: An Introduction. John Wiley & Sons, Inc., Hoboken,

NJ (2007).

[16] R. Bakhtiar, L. Ramos, F.L.S. Tse, J. Liq. Chromatogr. Relat. Technol., 25

(2002) 507.

[17] J.C. Lindon, E. Holmes, J.K. Nicholson, Anal. Chem. 75 (2003) 384A.

[18] N.V. Reo, Drug Chem. Toxicol. 25 (2002) 375.

Chapter-1 General Introduction

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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.

http://en.wikipedia.org/wiki/Glomerular_filtration_rate


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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


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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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22

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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


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)



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


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.


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.



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

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