MD Rafi Biochemistry
MD Rafi Biochemistry
MD Rafi Biochemistry
Section I
Prelude – Molecular and
Functional Organisation of
Life
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Rafi MD: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 1
Chapter 1
Learning Biochemistry
What is Biochemistry?
• Water
Section I
Prelude
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Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 2
Chapter 2
Composition of Membranes
Lipids
The m ajor mem brane lipids are phospholipids. These
are amphipathic m olecules containing both hydrophilic
or polar (water soluble) and hydrophobic or non- polar
(water insoluble) groups.
The cell mem brane also contains equal amounts of
cholesterol.
Proteins
●
Proteins in m embranes occur in two different ways.
Some proteins are located in the interior of the
m embrane and are called integral m embrane
proteins.
●
Others are located in the outer surface of the
m embranes and are known as peripheral membrane
proteins.
●
Transm embrane proteins serve as:
a) Channels b) Carriers
c) Pumps d) Receptors
Carbohydrates
Membrane Function
Passive Transport
Here, substances move across the cell m embrane
without any energy ex penditure by the cell. It includes
diffusion and osm osis.
1 . Diffusion
Simple Diffusion
Facilitated Diffusion
2 . Osmosis
Active Transport
When substances are transported against their
chemical and electrical gradient requiring energy,
the process is called active transport. The main
types are
1. Primary active transport
2. Secondary active transport
3. Carrier transport
4. Vesicular transport
ii) Exocytosis
Section II
Molecules of Life
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Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 3
Chapter 3
Carbohydrates
Carbohydrates
Carbohydrates are aldehyde or ketone compounds
with multiple hydroxyl groups.
Carbohydrates can be defined as aldehyde or ketone
derivatives of polyhydric alcohols. Most
carbohydrates taste sweet and hence they are
popularly known as sugars.
They are the chief source of energy for all
organisms including humans.
1. Monosaccharides
2. Oligosaccharides
3. Polysaccharides
Monosaccharides
Classification of Monosaccharides
D and L Isomers
D- and L- Isomerism
A carbon atom which is attached to four different
groups is known as an 'asymmetric' or 'chiral' carbon.
Optical Isomerism
Optical isomerism is ex hibited by those compounds
which possess asymmetric carbon(s).
Optical isomers are capable of rotating the plane of
polarised light either to the right or to the left.
The optical isomer which rotates the plane of
polarised light to the right (clockwise) is said to
be dextrorotatory [represented by ‘d’ or (+ )] and
the substance which rotates the plane of
polarised light to the same ex tent but to the left
(counter clockwise) is termed as levorotatory
[represented by ‘ l’ or (–)].
A solution having equal concentrations of ‘d ’ and ‘l ’
forms, known as racemic mixture, cannot rotate the
plane of polarised light and hence shows zero
optical rotation.
EPIMERS
Monosaccharides that differ only in their
configuration around one carbon atom (other
than anom eric) are known as epimers of one
another.
Thus, D- glucose and D- mannose are epimers
with respect to C2 and D- glucose and D-
galactose are epimers with regard to C4 , that is,
they differ from each other in the arrangement of
–OH group around that particular carbon atom.
The pair of stereoisom ers that differ in
configuration around the carbonyl carbon are
called anomers.
The hemiacetal / hemiketal or carbonyl carbon is
called anomeric carbon.
In case of - anomer, the - OH group of the anomeric
carbon is on the opposite side of the terminal
primary alcohol group (C6 ) of the sugar ring.
Mutarotation
Formation of a common enediol from glucose, fructose and mannose. ‘R’ represents
t he common structure of all the three sugars C3 – C6 .
Osazone Formation
Only the first two carbons are involved in osazone
form ation.
The sugars which differ in configuration around the
first two carbons give identical osazones, since the
difference is masked by binding with phenyl
hydrazine.
This is the reason why glucose, fructose and mannose
give similar needle- shaped osazones.
Glucosazone and fructosazone – long needle-
shaped or broom stick- shaped crystals.
Maltosazone – Yellow coloured ‘petals of sun
flower’- shaped crystals.
Lactosazone – ‘Hedgehog- shaped’ or powder- puff-
shaped crystals.
Disaccharides
Maltose
Lactose
Sucrose
Sucrose is mostly present in sugarcane and fruits.
Hence it is also known as cane sugar. Sucrose is
synthesised by plants through photosynthesis.
Sucrose is composed of glucose and fructose linked
together through glycosidic linkage (1 –2 ). The
aldehyde group of glucose and the keto group of
fructose are engaged in bond formation leaving no
free functional group thus making sucrose
essentially non- reducing.
Formation of sucrose
2 . Heteropolysaccharides
They are composed of different types of
monosaccharides or their derivatives, e.g.,
glycosaminoglycans.
Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 3
Starch
●
Starch is the high energy nutrient synthesised from
carbon diox ide and water by photosynthesis in
plants.
●
It is the chief carbohydrate fuel source for higher
animals, including humans. The dietary sources of
starch include cereals, tubers, roots, legumes and
vegetables.
●
Starch is composed of two com ponents:
Amylose and Amylopectin.
●
Starch is digested by pancreatic amylase in the
intestine.
Structure of Starch
Cont inues. . .
Structure of Starch
Glycogen
Structure of glycogen
Glucose residues taking part in α - 1- 6- glycosidic linkages are shown in pink colour.
Glycogen is the instant storage form of energy in animals, including humans.
Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 3
Glycogenin in the gylcogen structure
Cellulose
●
Cellulose is the most abundant carbohydrate in
nature. To be m ore precise, it is the m ost
abundant organic substance on earth. Cellulose
m akes up most of the plant framework. It is absent
in animals.
●
Cellulose is the unbranched, linear polymer
com posed of - D- glucose units linked through -
(1–4)- glycosidic bonds.
Heteropolysaccharides
Glycosaminoglycans
1. Hyaluronic acid
2. Chondroitin sulphates
3. Keratan sulphates
4. Heparin and heparan sulphate
5. Dermatan sulphate
Hyaluronic acid
Chondroitin 4 - sulphate
(Continues…
Keratan sulphate
Heparin
(Continues…
Dermatan sulphate
Section II
Molecules of Life
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Rafi M D: Textbook of Biochemistry (4th Edition) Universities Press
CHAPTER 4
Chapter 4
Lipids
Lipids
Lipids can be defined as heterogeneous
substances grouped together under one class
based on their common property, of being
insoluble in water and soluble in organic solvents
(ether, benzene, chloroform ).
They are predom inantly composed of carbon and
hydrogen atoms. These atoms are linked together
through neutral covalent bonds making lipids
essentially non- polar.
FUNCTIONS OF LIPIDS
…Continued)
Classification of Lipids
a) Phospholipids
i) Glycerophospholipids
ii) Sphingophospholipids
b) Glycolipids
c) Lipoproteins
3 . Derived Lipids
4 . Miscellaneous Lipids
Fatty Acids
Fatty acids are components of the majority of lipids.
They are carbox ylic acids with a hydrocarbon side
chain.
The hydrocarbon chain accounts for the non- polar
nature of fatty acids.
Free and esterified fatty acids
Length of hydrocarbon chain
Even- and odd- number carbon fatty acids
Fatty acids which possess double bonds in their
structure are called 'Unsaturated fatty acids' (UFA)
and fatty acids with no double bonds are 'Saturated
fatty acids' (SFA).
Unsaturated fatty acids are further classified into
two subgroups based on the num ber of double
bonds.
Only linoleic acid and linolenic acid are essential
because hum ans lack the enzym es that can
introduce double bonds beyond carbon no. 9.
Eicosanoids
Eicosanoids are a group of biologically active m olecules
synthesised from eicosaenoic acids (C20 PUFA).
Eicosanoids are a group of five types of molecules.
1. Prostaglandins (PG)
2. Prostacyclins (PI)
3. Thrombax anes (TX)
4. Leukotrienes (LT)
5. Lipox ins (LX)
Most eicosanoids are synthesised from arachidonate.
1 . Inflammation
Eicosanoids are the natural mediators of
inflammation. This is the basis of the use of
corticosteroids and various non- steroidal
antiinflammatory drugs (NSAID) (which inhibit
the eicosanoid synthesis) in the treatment of
various inflammatory disorders (e.g., rheum atoid
arthritis).
4 . Effects on Reproduction
Prostaglandins are used in medical termination of
pregnancy (MTP).
Triacylglycerols
Saponification number
Saponification num ber is defined as the mg of KOH
required to saponify 1 g of fat or oil completely.
Saponification num ber is a m easure of the average
m olecular weight and chain length of the fatty acids
present.
Iodine Number
It is defined as the num ber of gram s of iodine absorbed
by 100 g of fat or oil. Iodine num ber is used to assess
the degree of unsaturation of fat.
Phospholipids
They are the structural components of biological
membranes and are responsible for the selective
perm eability of the m embrane.
General structure of
phospholipids
Phospholipids are divided into two classes based on
the type of alcohol present.
I. Glycerophospholipids: They contain alcohol glycerol
(C3 )
II. Sphingophospholipids: They contain alcohol
sphingosine (C18 )
Cephalin
Cephalin (Phosphatidyl
ethanolamine)
Lecithin
It is the phosphatidic acid with choline as the
base.
Lung alveoli are coated with a surfactant which is
predom inantly made up of dipalmitoyl lecithin.
Prem ature babies without sufficient
surfactant lining the alveolar walls can have
respiratory difficulties. This results in alveolar
collapse causing respiratory distress syndrome.
Phosphatidyl Inositol
Phosphatidyl inositol
Cardiolipin
Plasmalogens
Plasm alogens are predominantly phophatidal
ethanolamines:
The brain and m uscle tissue are rich in
plasm alogens.
Plasmalogen
Sphingomyelin
●
Lecithin- Sphingom yelin ratio (L/ S ratio) in am niotic
fluid is an indicator frequently used to evaluate fetal
lung m aturity.
Glycolipids are high in nervous tissues
Types of glycolipids:
1. Cerebrosides
2. Globosides
3. Gangliosides
Structure of cholesterol
Functions of Cholesterol
Section II
Molecules of Life
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Rafi M D: Textbook of Biochemistry (4 th Edition ) Universities
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CHAPTER 5
Chapter 5
Amino Acids and Proteins
Proteins
Proteins are the building material for body
structure.
Proteins are com posed of carbon, ox ygen,
hydrogen, nitrogen and sm all am ounts of other
elements, notably sulphur.
Proteins play the role of ‘ brick and mortar’ of the
body.
Proteins perform a number of roles including
biocatalysis, defence, hormonal function, transport,
storage and muscle contraction.
Proteins are polymers of amino acids
Standard amino acids
Amino Acids
Am ino acids are the structural units (monomers) of
proteins.
An amino acid is made up of two functional groups—
amino (–NH2 ), carbox yl (–COOH). They also contain a
hydrogen atom and a side chain (R) linked to the carbon
atom.
Am ino acids differ from each other in their side chains.
1. Glycine 2. Alanine
3. Valine 4. Leucine
5. Isoleucine
6. Serine
7. Threonine
8. Cysteine
9. Methionine
11. Asparagine
13. Gultamine
14. Lysine
15. Arginine
16. Histidine
17. Phenylalanine
18. Tyrosine
19. Tryptophan
20. Proline
The amino acids which cannot be synthesised by the
body and hence need to be supplied through diet are
called essential amino acids. Some are
conditionally essential.
The ten essential am ino acids in ascending order of
dietary requirement:
1. Arginine 6. Isoleucine
2. Tryptophan 7. Valine
3. Histidine 8. Phenylalanine
4. Methionine 9. Lysine
5. Threonine 10. Leucine
Ketogenic amino acids
Leucine is the only ex clusive ketogenic am ino acid.
Ketogenic and glucogenic amino acids
Isoleucine, lysine, phenylalanine, tyrosine and
tryptophan.
Glucogenic amino acids
The rem aining 14 amino acids are used for the
synthesis of glucose or glycogen.
Polar (hydrophilic) amino acids
glycine, serine, threonine, cysteine, asparagine,
glutam ine and tyrosine.
Non- polar (hydrophobic) amino acids
Phenyl alanine, tryptophan and proline.
Isoelectric pH
D- amino acids
Non- Protein am ino acids
Am ino acid derivatives
1. Salting out
2. Isoelectric precipitation
5. Precipitation by alcohols
6. Heat coagulation
Classification of Proteins
Determ ination of amino acid com position in a
protein
Determ ination of the number of polypeptides in a
protein
Determ ination of amino acid sequence in a protein
Sanger's reagent
Edm an's reagent
●
Autom atic sequencing
1. Electron microscopy
2. X- ray crystallography
various levels
Covalent bonds
Peptide and disulphide linkages
Non- covalent bonds
Hydrogen bonds
Hydrophobic interactions
Electrostatic interactions
Van der Waals interactions
Features of - helix
(Continues…
- Helix
(Continues…
- Pleated Sheets
Anti parallel
Parallel
Hydrogen bonds are form ed between adjacent st rands wit hin a single polypept ide. Note t hat in bet a
sheet s running ant iparallel, hydrogen bonds bet ween NH and CO groups connect each am ino acid t o
a single am ino acid on an adjacent st rand. On t he contrary, in a parallel bet a sheet, hydrogen bonds
connect each am ino acid on one st rand wit h t wo different am ino acids on t he adjacent strand.
Proteins consisting of more than one polypeptide
chain display quaternary structure.
The protein is known as a multimeric protein or
oligomeric protein. For ex ample, hem oglobin, the
protein that transports ox ygen in the blood is an
oligomeric protein with four polypeptide chains (2
and 2).
Denaturation
Levinthal’s paradox
2. Molten globule
4. Chaperone proteins
Prion diseases
Creutzfeldt- Jakob disease
Kuru
Mad cow disease
Alzheimer's disease
Section II
Molecules of Life
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CHAPTER 6
Chapter 6
Nucleotides
Nomenclature of Nucleotides
Functions of Nucleotides
(cont inued…
nucleotides
Synthetic Nucleotides
Therapeutic applications
1. Synthetic nucleosides, cytarabine and vidarabine in
which ribose is replaced by arabinose are used in
chemotherapy to treat cancers.
(cont inues…
Structure of DNA
The double helical structure of DNA was first proposed by
Jam es Watson and Francis Crick in 1953.
…continued )
…continues)
…continued )
(continues…
…continued )
…cont inues)
…continued )
…continued )
…continues)
…continued )
…continued )
Chargaff's Rules
• B- DNA
• A- DNA
• Z- DNA
Unusual structures in DNA
• Hoogsteen pairing
• Triplex DNAs
• Tetraplex
• Palindromes
Denaturation of DNA
Organisation of DNA
• Supercoiling of DNA
• Topoisomerases
• Nucleoproteins
Histones
Chrom atin
Covalent modifications
Nucleosomes
Mitochondrial DNA
Genes
Introns
Ex ons
• Transposons
(continues…
Nucleases
Types of RNA
(Cont inues…
Messenger RNA is synthesised as the prim ary
transcript from the template strand of DNA and
later processed to m - RNA.
5 capping
Poly ‘A’ tail
Heterogeneous nuclear RNA (hnRNA)
Structure of t- RNA
Section II
Molecules of Life
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CHAPTER 7
Chapter 7
Enzymes
Enzymes
Enzymes are highly specialised proteins that act as
catalysts in biological systems (biocatalysts).
Enzym es are highly specific for their substrates
A four- digit Enzyme Com mission (E.C.) number is
assigned to each enzym e. The first digit represents the
class, the second digit indicates the subclass, the third
digit represents the sub- subclass and the fourth digit
specifies the individual enzym e.
Enzymes are classified into six major classes as per
the IUB system of enzyme classification.
Classification of Enzymes
(Continues…
Classification of Enzymes
Enzyme Structure
• Apoenzym e
• Coenzym e
• Holoenzym e
Active Site
A diagrammatic representation of
an enzyme and its active site
Coenzymes
Coenzymes of B- complex vitamins
Enzyme Specificity
• Transition state
• Ground state
• Activation energy
• Binding energy
S = substrate
P = product
Mechanism of Catalysis
• Covalent catalysis
1. Concentration of enzym e
2. Concentration of substrate
3. Concentration of product
4. Temperature
5. pH
The rate of an enzym e catalysed reaction increases
as the substrate concentration increases until it
reaches a m ax imal rate, which remains constant
despite further increases in substrate concentration.
The substrate concentration (ex pressed in m oles/ L)
required to produce half- m ax imum velocity (1/ 2
Vm ax ) is known as Km or Michaelis–Menten constant.
(Continues…
(Continues…
Enzyme Inhibition
1. Competitive inhibition
3. Allosteric inhibition
Competitive Inhibition
The inhibitor binds with the enzyme through non-
covalent interactions and hence the pro cess is
readily reversible.
inhibitors
The inhibitor binds to the enzyme covalently and
inactivates it making the process essentially
irreversible.
1. Allosteric regulation
2. Covalent modulation
4. Compartmentation
Allosteric Regulation
Allosteric enzymes
Allosteric m odulators
Cooperativity of allosteric enzym es
Classification of allosteric enzymes
K class allosteric enzymes
V class allosteric enzymes
Feedback regulation
Covalent Modulation
Regulatory enzym es or rate- limiting enzymes
Reversible protein phosphorylation
Protein kinases
Protein phosphatases
Isoenzymes
Isoenzymes of LDH
Clinical Enzymology
Section II
Molecules of Life
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Rafi M D: Textbook of Biochemistry (4thEdition) Universities
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CHAPTER 8
Chapter 8
Vitamins
Classification of Vitamins
Vitamin A
• Chemistry
Retinol
Retinal
Retinoic acid
- Carotene
(Continues …
…Cont inued)
(Continues…
…Continued)
(Continues…
1 RE = 1 g retinol
= 6 g - carotene
1 IU = 0.3 g retinol
Dietary Sources
(Continues…
Deficiency Manifestations
Night blindness
Night blindness (Nyctalopia) or the inability to see in
dim light is the earliest sym ptom of vitam in A
deficiency. It occurs due to im pairment of dark
adaptation.
(Continues…
Deficiency Manifestations
Xerophthalmia
Deficiency Manifestations
Bitot's spots
Bitot's spots are triangular, white patches on the
conjunctiva on either side of the cornea. They are usually
bilateral.
Keratomalacia
If the condition progresses beyond x erophthalmia, there
is ulceration and necrosis of the cornea (keratomalacia)
which results in total blindness. Keratom alacia is one of
the major causes of blindness in developing countries
and is usually associated with protein energy
malnutrition (PEM).
(Continues…
Vitamin D
(Cont inues…
The recom mended dietary allowance (RDA) is about
400 international units (IU) or 10 g. However, in
places with good sunlight (like the Indian
subcontinent), the daily requirem ent is about 200 IU
or 5 g. In growing children, during pregnancy and
lactation, it should be 10 g (400 IU).
Vitamin D can be derived both from sunlight and
food. The human body m anufactures vitam in D with
the help of sunlight. It also occurs m ainly in foods of
anim al origin.
(Continues…
Breastmilk contains relatively sm all am ounts of
vitam in D and infants are at risk of developing
vitam in D deficiency, particularly prem ature infants
(since the vitamin is transported across the placenta
mainly in the last trim ester of pregnancy).
Deficiency Manifestations
Rickets
• improper mineralisation
• bow- legs, knock- knees, pigeon chest,
kyphoscoliosis.
• ('rickety rosary')
• (Harrison's sulcus)
Osteomalacia
• Renal rickets
• Vitami- D resistant rickets
Functions of Vitamin E
Functions of Vitamin E
Men – 10 mg
Women – 8 mg
Biochemical Functions
Since vitam in K is synthesised by the intestinal
bacteria, its requirem ent in the diet is very low. A
recommended daily allowance (RDA) of 70 –140
mg/ day would suffice for adult hum an needs.
Green leafy vegetables are an ex cellent source of
vitam in K. It is also present in eggs and diary
products.
Deficiency Manifestations
●
Vitamin K deficiency leads to prolonged
coagulation and bleeding. Newborns, especially
preterm infants are m ore susceptible to vitamin K
deficiency
(Cont inues…
Deficiency manifestations
●
Vitam in K deficiency is seen in adults suffering from
obstructive jaundice and other diseases causing
severe fat malabsorption. Patients suffering from
small bowel diseases such as celiac disease, Crohn's
disease suffer from vitam in K deficiency.
Vitamin C
Biochemical Role
Collagen formation
Vitamin C plays a role in the maintenance of
normal connective tissue and is essential for
wound healing.
(Cont inues…
Biochemical Role
Antioxidant: Vitam in C plays a role in elim inating
potentially dangerous free- radicals from the
biological system.
Role in metabolism
Vitamin C plays a role in the metabolism of amino
acids
Tyrosine; Tryptophan
(Cont inues…
…Continued)
It spares vitamins A, E and some B- com plex
vitam ins from ox idation.
Phagocytosis
Recomm ended daily allowance is about 60 –75
mg/ day. About 100 m g/ day is required during
pregnancy and lactation. Sm okers, chronic
alcoholics and wom en on oral contraceptives
require up to 125 mg/ day.
The m ain dietary sources of vitamin C are fresh
fruits and green leafy vegetables. Citrus fruits,
Indian gooseberry (amla), guava, cabbage, spinach
are very high in vitamin C content. Germ inating
pulses contain large am ounts of vitam in C.
Deficiency Manifestations
Capillary fragility: Petechiae (sm all, pinpoint
hemorrhages under the skin), ecchymoses or
even hematomas. Internal bleeding into the
gastrointestinal tract, peritoneal cavity,
pericardium and the adrenal glands m ay occur.
(Cont inues…
Deficiency Manifestations
• Osteoporosis
• Barlow's disease
Thiamine (Vitamin B1 )
Biochemical Functions
Thiam ine ex erts its biochemical functions
through its coenzym e form , thiam ine
pyrophosphate (TPP).
Pyruvate dehydrogenase requires TPP as one of
the coenzym es.
- ketoglutarate dehydrogenase is also dependent
on TPP a a coenzyme.
Ox idative decarbox ylation reaction involving
branched chain am ino acids requires TPP.
(Cont inues…
Biochemical Functions
Enzyme transketolase of hex ose m onophosphate
shunt (HMP shunt) utilises TPP as the coenzym e.
TPP plays a role in the transmission of nerve
im pulses.
The recom mended dietary allowance (RDA) for
adults is 1–1.5 m g/ day.
Whole grain cereals, yeast, legum es, oilseeds and
nuts, especially groundnut, are im portant sources
of thiam ine. Animal foods such as pork, beef and
sheep liver are also good sources.
Deficiency Manifestations
Riboflavin (Vitamin B2 )
Coenzymes of Riboflavin
Riboflavin ex ists in two active coenzyme forms
Biochemical Functions
Milk and other dairy products, lean meat, fish, eggs
and legum es are good sources.
The recom mended dietary allowance (RDA) for an
adult is about 1.5 mg, with slightly higher amounts
(by 0.2 to 0.5 m g/ day) recomm ended for pregnant
and lactating women.
Deficiency Manifestations
Its deficiency usually occurs in association with
deficiencies of other Bcomplex vitamins. It usually
affects the tongue and lips; characterised by
glossitis (magenta tongue), angular stomatitis,
cheilosis (fissure- like lesions at the corners of the
m outh), pharyngitis and genital derm atitis.
Laboratory diagnosis done by measurement of
FAD- dependent glutathione reductase activity in
RBC.
Niacin
Coenzymes of niacin
1. Nicotinamide adenine dinucleotide (NAD + )
Biochemical Functions
Several enzym es require either NAD + or NADP+ in
ox idation–reduction reactions.
There is a fundamental distinction between NADH
and NADPH
- NADH generated in various catabolic pathways is
ox idised in the respiratory chain to generate ATP.
NADH thus plays a vital role in cellular respiration.
(Continues…
(Continues…
(Continues…
Deficiency Manifestations
Pellagra
Pellagra is a classic disease caused by the deficiency
of niacin.
Pyridoxine (Vitamin B6 )
Vitam in B6 and its coenzyme [PLP]
(Continues…
Biochemical Functions of B6
• Transamination
Transamination reaction
(Continues…
• Decarboxylation
Histam ine
(Continued…
Catecholam ines
(Continues…
…Continued)
• Heme synthesis
• Phosphorylase
• Deamination
Plants contain B6 as pyridox ine, whereas anim al
tissues contain PLP and pyridox amine phosphate.
Rich sources of B6 include wheatbran, rice bran,
dried yeast, legum es, nuts, meat, fish, milk, eggs
and leafy vegetables.
Requirem ents are related to protein intake as it is
chiefly concerned with am ino acid metabolism . The
RDA for adults is 2 mg/ day. As usual, during
pregnancy and lactation it is slightly higher.
Deficiency Manifestations
Neurological symptom s include irritability,
generalised weakness, peripheral neuropathy,
personality changes including confusion and
depression.
Carpal tunnel syndrome
Hematological m anifestations include microcytic,
hypochrom ic anem ia due to diminished hemoglobin
synthesis. Platelet dysfunction m ay also occur.
Dermatological manifestations include seborrheic
derm atitis, stom atitis, glossitis and cheilosis.
Drug interactions
Biotin
Biotin is more popularly known as anti- egg white
injury factor.
Structure of
biotin
Biochemical Functions
Role in gluconeogenesis and Krebs cycle
Conversion of pyruvate to ox aloacetate is catalysed
by biotin- dependent pyruvate carbox ylase.
Role in fatty acid synthesis
Role in the metabolism of odd- numbered fatty acids
and branched- chain am ino acids
RDA of 200–300 mg/ day.
Liver, soyabean, yeast, egg yolk, peanut, grains are
all rich sources of biotin.
Deficiency manifestations
Biotin deficiency is uncomm on since it is synthesised
by the intestinal microflora and its ubiquitous
occurence in nature.
However, prolonged use of antibiotics that kill the
gut bacteria and ex cess consumption of raw eggs
result in biotin deficiency.
Pantothenic Acid
(Continues…
Biochemical Functions
More than 70 enzymes have been identified till date that
are dependent on CoA making it the central molecule in
the metabolism of lipids, proteins and carbohydrates. A
few ex amples of enzymes involving coenzyme A are
given below.
Folic Acid
Folic acid reduced to tetrahydrofolic acid
(Continues…
Folic Acid
Biochemical Functions
Daily Requirement
The RDA of folic acid in adults is about
200 μ g/ day.
During pregnancy (400 μ g) and lactation
(300 μ g).
An additional supplement is recomm ended during
pregnancy because its deficiency is linked to neural
tube defects and other congenital malform ations.
Dietary Sources
Folic acid is widely distributed in natural foods.
Deficiency Manifestations
• Macrocytic anemia
• Hyperhomocysteinem ia
Folate Antagonists
Vitamin B12
[R = substituted by
different groups]
HC – haptocorrin
IF – intrinsic fact or
TC – t ranscobalam ins
Biochemical Functions
Synthesis of m ethionine from hom ocysteine
Biochemical Functions
Conversion of m ethylm alonyl CoA to succinyl CoA
Liver, m eat, fish, eggs, milk, curd and cheese are good
sources of vitam in B12 .
Deficiency Manifestations
• Megaloblastic anemia
• Demyelination
• Pernicious anemia
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CHAPTER 9
Chapter 9
Biochemistry of Blood
Plasma Proteins
2. Electrophoresis
3. Ultracentrifugation technique
4. Cohn's fractionation
Functions of Albumin
2. Transport function
3. Buffering action
4. Nutritional function
Album in, by binding to certain com pounds in
blood, prevents them from crossing the blood–
brain barrier, e.g., albumin–bilirubin complex ,
albumin–fatty acid com plex .
• Hypoalbum inem ia
• Tissue edem a
• Kwashiorkor
• Cirrhosis of liver
• Nephrotic syndrom e
(Continues…
• paucialbum inuria or
• m icroalbuminuria
The normal albumin–globulin ratio (A/ G ratio) is
about 1.2:1 to 2:1. The A/ G ratio is lowered or
reversed either when album in is decreased or
globulins are increased, usually observed in
cirrhosis of the liver, nephrotic syndrome and
m ultiple m yelom a.
Globulins
(Continues
V - Variable region, C - const ant region; each heavy chain is com posed of four unit s - V H ,
CH1 , CH2 , CH3 , while light chain consist s of t wo unit s - V L and CL.
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CHAPTER 9
Classes of Immunoglobulins
2. IgM
3. IgA
4. IgD
5. IgE
Multiple myeloma
• Bradshaw’s test
• Amyloidosis
RBC
• Structure
• RBC count
– Men 4.6–6.2 million/ L
– Women 4.2–5.4 million/ L
• Metabolism in RBC
• Hereditary spherocytosis
• hereditary elliptocytosis
• Hemoglobinopathies
• - thalassem ia
• - thalassem ia
WBC
Respiratory burst
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CHAPTER 10
Chapter 1 0
Hemoglobin - Chemistry of
Respiration
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CHAPTER 10
Structure of Hemoglobin
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Structure of Heme
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Types of Hemoglobin
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Hemoglobin Derivatives
• Met- hemoglobin
• Carbox y- hem oglobin (CO- Hb)
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CHAPTER 10
Myoglobin
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Oxygen Transport from Lungs to
the Tissues
Movement of inspired air into the alveoli is by bulk flow; all other
movements of ox ygen across membranes are by diffusion gradient.
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CHAPTER 10
Bohr Effect
2 , 3 BPG
– High altitude
– Anem ia
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CHAPTER 10
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CHAPTER 10
Carbon dioxide transport from
tissues to the lungs
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Biochemical basis of the diagnosis and
management of sickle cell disease
1. Sickling test
2. Electrophoresis
3. RDT
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CHAPTER 10
Thalassemias
Types of thalassem ias
(Continues…
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(Continued… CHAPTER 10
Types of thalassemias
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Section III
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CHAPTER 11
Chapter 1 1
(Continues…
Biosynthesis of heme
(Continues…
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CHAPTER 12
Chapter 1 2
Digestion in the mouth
Digestion in the small intestine
Non- digestible carbohydrates
Absorption of m onosaccharides
Mechanism of Absorption
Lactose intolerance
(Continues…
…Cont inued)
Digestion in intestine
• Em ulsiffication of lipids
• Role of bile salts
• Role of phospholipids as surfactant
(Continues…
…Continued)
(Continues…
Digestion of Phospholipids
Enzymatic cleavage of phospholipids
(Continues…
…Continued)
Absorption of lipids
Bergstrom theory
(Continues …
• Hydrochloric acid
• Pepsin
Meister cycle
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CHAPTER 13
Chapter 1 3
(Contiunes…
…Continued)
Outline of different phases of catabolism [All t he m ajor biom olecules – carbohydrat es, lipids
and prot eins undergo cat abolism t o finally yield acet yl CoA which is furt her ox idised in t he
citric acid (Krebs) cycle releasing carbon diox ide and reducing equivalent s (NADH + H + and
FADH2 ). Reducing equivalent s t hen ent er int o t he respirat ory chain, ult im at ely producing ATP
t hrough t he process of ox idat ive phosphorylation.]
Metabolic inhibitors
Genetic mutations
Genetic manipulations
Radioactive isotopes
Laws of Thermodynamics
First law of thermodynamics states that the total
amount of energy in the universe remains constant.
However, the form of the energy m ay change, but
within the total system , energy is neither destroyed
nor created during a physical or chemical change.
The second law of thermodynamics states that the
total entropy of a system must increase if a process
is to occur spontaneously.
Free Energy
G = H – TS
where
A list of high- energy and low- energy compounds along with their standard
free energy (G°) on hydrolysis.
Structure of ATP
ATP- ADP Cycle: [ATP links the two major components of metabolism –
catabolism (breakdown of biomolecules) and anabolism (synthesis of
biomolecules)].
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Chapter 1 4
Chapter 1 4
Central Metabolism:
Biological Oxidation
Mitochondrion
Krebs Cycle
1. Synthesis of citrate
2. Isomerisation of citrate to isocitrate
3. Ox idative decarbox ylation of isocitrate to -
ketoglutarate (- KG)
4. Ox idative decarbox ylation of - KG to succinyl CoA:
5. Formation of succinate
6. Ox idation of succinate to fum arate
7. Formation of malate
8. Conversion of malate to ox aloacetate
Krebs Cycle
(Continued…
…Continues)
Krebs cycle
Redox couples
Redox Potential
Redox potentials (Eo) of some ox idation–reduction systems
I. Nicotinamide nucleotides
II. Flavin nucleotides
III. Iron–sulphur proteins
IV. Coenzym e Q
V. Cytochromes
P:O Ratio
●
The P:O ratio can be defined as the number of
inorganic phosphate (Pi) molecules utilised for ATP
production for every atom of ox ygen consumed.
●
The ox idation of one m olecule of NADH through
ETC and ox idative phosphorylation yields about 2.5
ATP and the ox idation of one m olecule of FADH 2
yields 1.5 ATP.
Oxidative Phosphorylation
Chemiosmotic hypothesis
●
The electrochem ical gradient or proton gradient thus
developed, acts as the power that drives the
mechanism operating the synthesis of ATP
●
The essence of chem iosmotic theory is, the free energy
generated from the electron flow through ETC is
conserved by pumping protons out of the matrix to
create an electrochemical proton gradient across the
inner mitochondrial mem brane. The electrochemical
potential of this proton gradient is used to synthesise
ATP.
Chemiosmotic Theory
Diagram ex plaining
the chemiosmotic
theory of ox idative
phosphorylation
• Oligom ycin
• Atractyloside
• Ionophores
• Valinom ycin
• Nigercin
• Uncouplers
• 2, 4- dinitrophenol (DNP)
Oxidases
• Cytochrome ox idase
• Flavoproteins
Dehydrogenases
Hydroperoxidases
Perox idase
Catalase
Oxygenases
• Diox ygenases
• Monoox ygenases
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CHAPTER
15
Chapter 1 5
Metabolism of
Carbohydrates
in different tissues
Note that each GLUT occurs in m ore t han one t issue. Only t he predom inant
t issue/ organ is shown in t he t able.
Glycolysis 15
(Continues…
(Continues…
Reactions of Glycolysis
The reaction m echanism of conversion of pyruvate t o acet yl CoA cat alysed by PDH com plex
*This calculation is based on the assumption that NADH produced in glycolysis is transport ed from the cytosol t o mitochondria by t he
glycerol- phosphate shuttle which yields only 1.5 ATP per NADH. If the NADH is t ransported by the malate- aspartat e shut tle, t he yield is
2.5 ATP per mole of NADH and t he total ATP per mole of glucose will be 32. However, the shutt le operative is tissue- specific (see
Chapt er 14, Mitochondrial Shuttles).
** There is a gain of an additional ATP during anaerobic glycolysis if t he start ing point of glycolysis is glycogen inst ead of free glucose,
since step I catalysed by hex okinase that utilises an ATP is bypassed.
(Continues…
[The substrates for gluconeogenesis are shown in . The key enzym es of gluconeogenesis are shown in .
The enzym es catalysing the irreversible steps in glycolysis are shown in . The num bers represent the entry of
glucogenic am ino acids. 1 Alanine, glycine, serine, cysteine and tryptophan; 2 Aspartate and asparagine; 3
Glutam ate, glutam ine, arginine, histidine and proline; 4 Valine, isoleucine, threonine and m ethionine; 5
phenylalanine and tyrosine.
Note that a few reactions of gluconeogenesis occur in m itochondria which are shown in a separate com partm ent —
area shaded in blue. The rem aining of the pathway occurs in cytosol.]
glucose )
(Continues…
Steps in glycogenolysis
Degradation of
glycogen
(Continues…
Covalent Modification
Modification
(Continues…
In norm al healthy individuals, fasting plasma
glucose levels are 7 0 –1 1 0 mg/ dl.
When the concentration ex ceeds the norm al range,
it is called hyperglycemia. When the values are
below the normal limits it is known as
hypoglycemia .
• Diabetes mellitus
• Hypoglycemia
Glucose Regulation
Hypoglycemic effect
Insulin
• Prom otes tissue uptake of glucose
• Enhances glycolysis
• Favours glycogen synthesis
• Depresses glycogen breakdown
• Inhibits gluconeogenesis
• Inhibits glucagon release from the pancreas and
depresses glucagon gene ex pression
• Increases HMP shunt
Glucose Regulation
Hyperglycemic effect
Glucagon
• Promotes glycogen breakdown in liver
• Enhances gluconeogenesis
• Depresses glycogen synthesis
• Inhibits glycolysis
(Continues…
Epinephrine
Stim ulates glycogenolysis
Decreases glucose use
Increases gluconeogenesis
Stim ulates glucagon secretion and inhibits
insulin secretion by the pancreas
(Continues…
Cortisol
• Stim ulates gluconeogenesis
Growth hormone
• Stim ulates gluconeogenesis
• Decreases glycolysis
• Antagonises insulin prom oted glucose uptake
(Continues…
(Continues…
(Continues…
(Continues…
• Antioxidant role
(Continues…
Section IV
Metabolism
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CHAPTER
16
Chapter 1 6
Metabolism of Lipids
Lipids, apart from their prim e function as a
storage form of energy, also act as structural
elem ents. Being hydrophobic, lipids are
unique am ong biomolecules.
The lipids in the body ex ist in three forms.
1. Structural lipids
2. Storage lipids
3. Plasm a lipids and transport lipids
(Continues…
…Continued)
(Continues…
(Continues…
Phospholipases on a Phospholipid
Substrate
Degradation of Sphingomyelin
Biosynthesis of cerebrosides
16
Biosynthesis of cerebrosides
inborn errors
A subscript numeral indicat es t he num ber of double bonds (e.g., PGE2 with t wo double
bonds),which also represent s t he group which t hey belong to. All t he above compounds are
Group 2 eicosanoids.
Malonyl CoA
The complex is a dim er with two ident ical polypept ides 1 and 2 running ant iparallel t o each
other, each consist ing of seven enzym e act ivit ies and an acyl carrier prot ein– ACP).
(Continues…
(Continues…
(Continues…
acyl CoA
(Continues…
Further processing of acetyl CoA through ETC and ox idative phosphorylation is briefly shown.
palmitic acid
(Continues…
(Continues…
(Continues…
(Continues…
Synthesis
by LCAT
of Bile Acids
• Plasma cholesterol
In healthy persons, the levels of total plasma
cholesterol vary between 1 5 0 –2 0 0 mg/ dl.
Principle of estimation
• HDL- C and LDL- C ratio
• Hypercholesterolemia
• Cholesterol and heart disease
• Mevastatin (compactin)
• Lovastatin (mevinolin)
• Provastatin
• Simvastatin
• Starvostatin
• Cholestyram ine
• Cholestipol
1. Chylomicrons
2. Very low density lipoproteins (VLDL)
3. Low density lipoproteins (LDL)
4. High density lipoproteins (LDL)
5. FFA–Albumin com plex
plasm a lipoproteins
chylomicrons
different m echanisms
The sm all red- balls represent protein clat hrin, sm all yellow- balls represent free cholest erol.
cholesterol transport
(Continues…
Causes
• Increased synthesis of TAG: High fat intake
• Reduced utilisation and rem oval of fats
• In protein- energy malnutrition (PEM)
To summ arise:
• Choline
• Inositol
• Methionine
• Betaine
• Vitamin E and selenium
• Om ega- 3 fatty acids
Disease
• Avoid smoking
• Ex ercise regularly and maintain norm al body
weight
• Dietary m easures
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CHAPTER
17
Chapter 1 7
Body protein content is not static but ex ists in
a dynamic steady state.
It is estimated that about 300–400 g of
protein is degraded and synthesised a day,
which represents the body protein turnover.
How does a cell recognise proteins that are
to be degraded?
• Ubiquitin
• Proteosome
Amino Group
Transmination
The transfer of an - amino group from an -
am ino acid to an - keto acid is known as
transam ination. The reaction involves the
interconversion of a pair of am ino acids and
a pair of keto acids. The overall process is
catalysed by a group of enzym es known as
transaminases or aminotransferases. The
derivative of vitamin B6 - pyridoxal
phosphate (PLP) acts as the coenzym e.
• Mechanism of transamination
• Significance of transm ination
Transamination reaction
(Continues…
A and B : Reactions catalysed by ALT and AST, respectively. C : Formation of Schiff base
• Deamination of histidine
Ammonia toxicity
Removal of ammonia
Amm oniotelics
Uricotelic
Ureotelic
(Continues…
…Continued)
Reactions of the urea cycle
This is represented
in different colours.
The first two
reactions of the cycle
occurs in the
mitochondria.
TCA Cycle
Urea levels in the blood are estim ated to assess
kidney function. In healthy individuals, the normal
blood urea concentration ranges between 15 –40
m g/ dL.
The term uremia is used to refer to elevated blood
urea due to renal disease. The conditions in which
blood urea levels are elevated are classified into
three categories.
1 . Pre- renal
Severe dehydration, fevers, diabetic coma,
thyrotox icosis, severe burns and so on are
pre- renal causes of elevated urea levels.
2 . Renal
Blood urea is increased in kidney diseases such as,
acute glomerulonephritis, pyelonephritis, polycystic
kidney, nephrotic syndrom e and so on.
3 . Post- renal
When there is an obstruction to the flow of urine
(e.g. stones, tumours, stricture urethra,
enlargm ent of prostate and so on) blood urea is
elevated, due to the increased reabsorption in the
renal tubules.
2 . Synthesis of heme
3 . Formation of glutathione
4 . Glycine as conjugating agent
5 . Glycine as a neurotransmitter
(Continues…
...Continued…
Creatinine
(Continues….
…Continued)
3 . Hypervalinemia
amino acids
Formation of Cystine
(Continues…
Taurine
PAPS (3’- Phosphoadenosine- 5-
phosphosulphate)
Glutathione
Cystinuria
Cystinosis
Hom ocystinurias
asparagine metabolism
metabolism
…Continued)
Therapeutic effects of NO
tyrosine metabolism
(Continues…
…Continued)
from Tyrosine
Not e that t yrosine residues occur as a part of t hyroglobulin, a huge glycoprot ein t hat
occurs in t he lum en of t hyroid follicles.
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CHAPTER
17
Synthesis of Melanin from Tyrosine
Tyrosine
Phenylketonuria
Enzyme defect in PKU: Phenylketonuria results from a
deficiency of the enzym e, phenylalanine hydroxylase.
Altered metabolism in PKU
Clinical features of PKU
Diagnosis of PKU
– Phenylalanine levels in blood
– Ferric chloride test
– Guthrie test
– DNA probes
Management of PKU
Alkaptonuria
Enzym e defect
Biochemical changes in alkaptonuria
Diagnosis and treatm ent
Albinism
Tyrosinem ias
Tyrosinem ia type I (hepatorenal tyrosinemia)
Tyrosinem ia type II (Occulocutaneous tyrosinem ia)
I.Kynurenin–Nicotinate pathway
(Continues…
…Continued)
(Continues…
…Continued)
(Continues…
Synthesis of serotonin
Degradation of serotonin
Biological role of serotonin
1. Serotonin is a potent vasoconstrictor and
causes sm ooth muscle contraction in
arterioles and bronchioles.
2. It serves as an ex citatory neurotransmitter in
the brain
(Continues…
…Continued)
Degradation of tryptophan
to indoleacetate
(Continues…
…Continued)
Biosynthesis of polyamines
Metabolism
of Amino Acids
Note that some amino acids appear more than once, reflecting different fates for different parts of their
carbon skeletons. Glucogenic amino acids depicted in black, ketogenic in red and both glucogenic and
ketogenic in green.
20 Amino acids
(Continues…
…Continued)
Out line of biosynt hesis of 20 am inoacids in plants and microorganism s. Not e that t he
precursors from glycolysis (red), HMP pat hway (blue) and t he citric acid cycle (green) are
shaded, and am ino acids derived from t hem are shown in box es of corresponding colours).
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CHAPTER
18
Chapter 1 8
Metabolism of Nucleotides
(Continued…
(Continues…
(Continued…
(Continues…
(Continued…
(Continues…
(Continued…
(Continues…
(Continues…
Not e that purine ribonucleot ides and deox yribonucleotides are degraded by t he same
pat hway and enzymes).
Gout 18
• Primary gout
• Hyperactivity of PRPP synthetase
• Superactivity of PRPP glutamyl am idotransferase
• HGPRT deficiency
• Glucose- 6- phosphatase deficincy
• Secondary gout
• Treatment of gout
This genetic disorder is characterised by spasticity,
m ental retardation, self- injurious behaviour and
gout. It was first described in 1964 by a m edical
student, Michael Lesch, and his professor, William
Nyhan.
Inherited deficiency of the purine salvage enzyme,
hypoxanthine- guanine
phosphoribosyltransferase (HGPRT), is
responsible for the Lesch–Nyhan syndrom e.
purine metabolism
Severe combined imm unodeficiency (SCID)
Gene therapy
nucleotides
(Continues…
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CHAPTER
19
Chapter 1 9
Integration of Metabolism
The reactions occurring in various m etabolic
pathways do not occur in isolation; they occur in an
integrated, well- coordinated manner based on the
overall requirements of the organism.
The pathways of all three classes of biomolecules
are interlinked through som e common
intermediates.
All these biom olecules are catabolised by the Krebs
cycle, electron transport chain and generate energy
(ATP) through ox idative phosphorylation.
Each class of these biomolecules provides the raw
materials for the synthesis of the components of
other classes.
various metabolisms
(Continues…
(Cont inues…
The biochemical changes following the
consum ption of food constitute the well- fed or
post- prandial state.
The carbohydrate content of the diet after
digestion is mostly absorbed as glucose and enters
the blood. The rise in blood glucose levels induce
the beta cells of the islets of Langerhans in the
pancreas to secrete insulin.
(Cont inues…
Within the cells, the glucose is converted to pyruvate
or lactate by glycolysis or it is used in the HMP
shunt for the formation of NADPH and pentose
sugars.
Availability of a plentiful supply of glucose in the
well- fed state coupled with the high insulin levels
result in the storage of glucose as glycogen
(glycogenesis) in the liver and m uscle. The liver, by
thus storing glucose as glycogen during the fed
state, releases glucose into blood from its stores
(glycogenolysis) in between meals.
(Cont inues…
Dietary proteins are digested and absorbed as
amino acids in the intestine. Very small amounts
are used by the intestinal cells and the rest is
transported through portal blood to the liver and all
other tissues where they are prim arily used for
protein synthesis.
(Cont inues…
The dietary lipids are packaged into chylomicrons
in the intestinal epithelial cells and are transported
to the blood through the lym phatic system.
(Cont inues…
The TAG thus produced from dietary fat in the liver
along with sm all am ounts of TAG produced from
glucose and amino acids by de novo synthesis
(endogenous fat) are packaged into very low density
lipoproteins (VLDL) and secreted by the liver into the
blood.
The fasting state constitutes the period after an
evening m eal, ex tending into the fasting state
during the night until breakfast the nex t m orning.
The glucose levels in the blood start dropping after
a few hours during the post absorptive period. The
fall in blood glucose decreases insulin secretion and
stim ulates glucagon secretion. Glucagon stimulates
glycogen breakdown and inhibits glycogen
synthesis.
(Cont inues…
As a result, glycogenolysis in the liver contributes
to blood glucose in the early fasting state.
In addition, glucagon also inhibits glycolysis
Glucagon also inhibits fatty acid synthesis
The liver glycogen levels are depleted by late night
and the early hours of the morning (8–10 hours
after supper), and the body switches over to hepatic
gluconeogenesis to maintain the blood glucose
levels.
(Cont inues…
Since the fatty acids cannot be used for the
synthesis of glucose, the net glucose synthesis by
gluconeogenesis derives its carbon source from
glycerol and m uscle proteins.
A substantial amount of acetyl CoA produced by
fatty acid ox idation is used for the formation of
ketone bodies
After breakfast, fat is metabolised as described in
the normal fed state. But glucose is metabolised
differently. The liver does not use the glucose which
is available aplenty in the blood, but leaves it for
the peripheral tissues. Interestingly, the liver
continues to remain in the gluconeogenic mode for
3–4 hrs after feeding. The glucose synthesised by
gluconeogenesis is used to replenish the liver ’s
glycogen stores.
(Cont inues…
The gluconeogenic mode of the liver com es to an
end and the liver starts synthesising glycogen
directly from fresh blood glucose.
When the fasting state is prolonged, it results in
starvation. During starvation, the entire body
m etabolism is reorganised for the survival of the
organism.
Section IV
Metabolism
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CHAPTER
20
Chapter 2 0
Metabolism of Xenobiotics:
Detoxification
Human beings are ex posed to a number of foreign
chemicals in their day- to- day life. This includes food
additives, pesticides, cosmetics, environmental
pollutants and most important, drugs. All these
foreign chem icals are collectively called
'x enobiotics’.
When drugs and other x enobiotics gain access to the
hum an biological system, they undergo chemical
alteration, a process known as biotransformation.
Compounds?
Even though all tissues m etabolise x enobiotics, the
liver is the major organ involved.
The m etabolism of x enobiotics is considered in two
phases.
• Phase I reactions
Hydrox ylation, Reduction and Hydrolysis
• Phase II reactions
• Conjugation reactions
1 . Oxidation or Hydroxylation
Reduction
The nitro compounds are reduced to their am ines and
aldehydes, ketones are reduced to alcohols, e.g.,
Hydrolysis
Many of the tox ic m olecules in the body are broken
down into smaller molecules by hydrolysis. The
hydrox yl group and the hydrogen ion of H 2 O are
transferred to different fragm ents of the substrate.
Phase II Reactions
Glutathione
Glycine
As a part of norm al body m etabolism, glycine
conjugates bile acids. It also detox ifies m any other
substances, e.g. benzoic acid (a food preservative).
Glucuronic acid
Sulphation
Acetylation
Methylation
Section V
Nutrition
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CHAPTER
21
Chapter 2 1
Minerals
Dietary sources
Milk and m ilk products are rich sources for calcium .
Leafy vegetables, beans, egg and fish (eaten with
bones) also contain good amounts of calcium.
Cereals (wheat, rice) contain only small amounts of
calcium.
But cereals are the staple diet in India. Therefore,
cereals form the m ajor source of calcium in our diet.
Daily requirement
An adult needs 800 mg/ day and a child about 1.2
g/ day.
Requirem ent m ay increase to 1.5 g/ day during
pregnancy and lactation.
After the age of 50, there is a general tendency to
develop osteoporosis, especially in post- menopausal
wom en, which may be prevented by increased
calcium (1500 mg/ day) plus vitam in D (20 m g/ day).
1. Phytic acid
2. Ox alates
3. Malabsorption syndrome
4. Phosphate
5. Alkalinity
of circulating calcium
Vitamin D and absorption of calcium
Vitamin D and bone
Vitamin D and renal tubules
PTH on bones
PTH on kidney
PTH on intestine
Calcitonin
Calcitonin, calcitriol and PTH act together to
achieve calcium homeostasis
Hypercalcemia
Increased serum Ca level is associated with
hyperparathyroidism
Hypocalcemia
Hypocalcemia is m ostly due to hypoparathyroidism
Biochemical functions
1. Sulphur as a component of iron–sulphur (Fe–S)
proteins plays a crucial role in mitochondrial
cellular respiration.
(Continues…
…Continued)
Dietary requirements
Adult male – 10 mg/ day
Menstruating wom an – 20 mg/ day
Pregnant and lactating wom en – 40 mg/ day
Dietary sources
Jaggery, organ meats (e.g. liver, heart), leafy
vegetables, pulses, cereals, apples, dry fruits and fish.
Iron deficiency anemia
Hemosiderosis
Hemochromatosis
contain copper
(Continues…
…Continued)
Wilson’s disease
It is a genetic disease (autosom al recessive) which
occurs due to mutations similar to that of Menke's
disease but in a different gene (a gene encoding a
copper- binding ATPase ex pressed only in liver cells).
Hence, the condition is also known as hepatolenticular
degeneration.
Ceruloplasmin levels in blood are decreased .
(Continues…
…Continued)
Kayser–Fleischer ring
●
Protein- bound iodine (PBI)
(Continued…
…Continued) Zinc
Biochemical functions
1. Zinc is an integral component of m any enzym es
(over 300 metalloenzymes) in the body, e.g.
carbonic anhydrase, alkaline phosphatase, alcohol
dehydrogenase.
2. It plays a crucial role in the synthesis and
stabilisation of DNA, RNA and proteins. It is an
integral component of DNA and RNA polym erases.
It forms zinc fingers.
(Continues…
…Continued) Zinc
Biochemical functions
1. Mn serves as a cofactor for a number of enzymes,
e.g. pyruvate carbox ylase, hex okinase, superox ide
dism utase, glutamine synthetase, arginase.
(Continues…
…Continued)
Manganese
Biochemical functions
1. Selenium ex erts synergistic antiox idant function
along with vitamin E.
2. Selenium is a component of glutathione
perox idase.
3. As a constituent of iodothyronine deiodinase,
selenium is involved in the rem oval of iodine from
thyroid hormones.
4. Selenium is also a constituent of thioredox in
reductase
…Continued) Selenium
(Continues…
…Continued) Fluorine
Dietary sources
Drinking water is the m ain source of fluoride.
Disease states
Dental caries
Fluorosis
Biochemical functions
1. Chrom ium potentiates the action of insulin.
2. Com ponent of a functional complex termed glucose
tolerance factor (GTF).
3. Cr lowers cholesterol levels in blood.
COBALT
The only role of cobalt in the hum an body is as a
component of vitamin B12 .
MOLYBDENUM
Molybdenum is an essential cofactor of a number of
enzymes, in particular, x anthine ox idase which converts
the end products of purine metabolism to uric acid.
It participates in nitrogen fix ation reactions as a
component of nitrite reductase.
Section V
Nutrition
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CHAPTER
22
Chapter 2 2
Principles of Nutrition
Macronutrients
Micronutrients
●
More comm only, BMR is ex pressed as Cal/ day. For an
adult male, BMR is around 24 Cal/ kg body wt/ day
and for an adult fem ale, it is 22 Cal/ kg body wt/ day.
Age
Gender
Body surface area
Built
Clim ate
Race
Physical activity and ex ercise
Starvation
(Continues…
Fever
Hormones
Stress
Significance of BMR
Mechanism of SDA
(Continues…
Significance of SDA
Provided the BMR and SDA are more or less constant,
physical activity is highly variable in different
individuals.
Energy requirements in average adults are roughly as
follows (energy required for BMR, SDA and physical
activity).
(Continues…
TDEE …Continued)
expenditure in humans
(Continues…
NEAT . . . Continued)…
Thus, NEAT plays a vital role in keeping the body fit and healthy.
Carbohydrates
Provide 50%–60% of the total calories.
Sources of carbohydrates
(Continues…
(Continues…
(Continues…
Foods with high glycemic index (GI > 70) include white
bread, table sugar (sucrose), all tubers (e.g.,
potatoes), polished rice, free glucose and maltose
powders, etc.
●
Cereals, pulses and vegetables do not contain
cholesterol, which is obtained only from foods of
animal origin. It is well known that high
cholesterol levels (> 240 m g/ dl) are associated
with increased risk of coronary artery disease.
●
In norm al individuals, the cholesterol level in the
blood ranges between 150–200 mg/ dl (desirable
< 180 m g/ dl).
(Continued…
Negatie nitrogen balance
Positive nitrogen balance
Calorie requirem ent has to be assessed based on
age, gender, build, health status and physical
activity.
Protein requirem ent has to be assessed based on
age, gender, build, health status and physical
activity.
(Continues…
Protein requirement can be calculated as
0.8 g/ kg body weight/ day. During growing age,
pregnancy and lactation it can be m ore than 2 g/ kg
body wt. Protein can be obtained as anim al protein
or m ix ed vegetable protein. The calorie yield from a
protein diet should be 10%–15% of total calorie
requirem ents.
(Continues…
●
Fats can provide 20%–25% of total calorie requirem ents
of the individual. Visible (fats and oils) should be
restricted to not m ore than 20 g/ day for a healthy
adult.
●
Choose a diet with plenty of whole grains,
vegetables and fruits and green leafy foods which
contain both digestible carbohydrates and dietary
fibre. Ideally 55%–65% of calories can be obtained
from carbohydrate diet.
Food pyramid : A
guide to
balanced diet
(Continues…
(Continues…
(Continues…
Disease (CAD)
• Limit the cholesterol in the diet to less than 200 mg per
day.
• Take adequate amounts of PUFA. They are required for
the esterification of cholesterol and its subsequent
rem oval. ω - 3 fatty acids reduce LDL- C levels.
• Restrict the saturated fats and trans- unsaturated fatty
acids to less than 10% of the total calorie intake. Ex cess
consumption of these tends to interfere with the
lipoprotein metabolism. They increase the levels of
LDL- C and decrease HDL- C. They also reduce
mem brane fluidity.
(Continues…
(Continues…
(Continues…
(Continues…
(Continues…
Section VI
Clinical Biochemistry
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Water
Role of water
Distribution of water in the body
Water balance
Water input
Water output
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Distribution of Water in the Body
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Water Balance in Humans
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Electrolytes – Distribution and Balance
Electrolytes are well distributed in body fluids to
maintain osmotic equilibrium and water balance.
Na+ is the principal cation of ECF, while K+ is the
chief cation of ICF.
Osmolarity: Osmotic pressure ex erted by the
num ber of m oles per litre of solution.
Osmolality: Osmotic pressure ex erted by the
num ber of m oles per kg of solvent. Osm olality is
more comm only used than osmolarity in clinical
practice.
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…Continued Electrolytes – distribution and
balance
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…Continued) Electrolytes – distribution and
balance
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…Continued)
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…Continued)
Regulation of water and electrolyte balance
Aldosterone
Anti- diuretic hormone (ADH)
Renin- Angiotension system
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Dehydration
Causes of dehydration
Dehydration m ay occur as a sequel to diarrhea,
vom iting, ex cessive sweating, fluid loss in burns,
adrenocortical dysfunction, kidney diseases (e.g. renal
insufficiency), deficiency of ADH (diabetes insipidus).
Features of dehydration
The clinical m anifestations of severe dehydration include
increased pulse rate, low blood pressure, sunken
eyeballs, decreased skin turgor, lethargy, confusion and
eventually coma.
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Treatment of Dehydration
●
Intake of plenty of water is a simple and effective
method of treating dehydration. In persons who
cannot ingest it orally, water can be given via a
nasogastric tube.
●
If this is not possible, water should be given
intravenously (IV) in an isotonic solution, usually as
5% glucose (5% dex trose). If there is an associated
electrolyte depletion, dex trose- saline (4% dex trose,
0.18% NaCl) should be given intravenously.
(Continues…
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Treatment of dehydration
…Continued)
●
When sodium depletion alone is the problem , giving
intravenous isotonic fluid – normal saline (0.9% NaCl)
or plasma ex panders or album in is the first line of
managem ent of salt deficit.
●
After restoring the intravascular volume, the
underlying cause m ust be treated.
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(Continues…
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…Continued)
Oral Rehydration Therapy (ORT)
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Electrolytes - Sodium
Dietary sources
Comm on salt (NaCl) used in cooking medium is the
major source of sodium. Whole grains, nuts, eggs,
leafy vegetables, milk and bread are good sources of
Na+ .
(Continues…
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…Continued) Electrolytes - Sodium
Dietary requirements
The recom mended dietary allowance (RDA) of sodium
is about 5–10 g/ day.
(Continues…
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…Continued) Electrolytes - Sodium
Biochemical functions
1 . Sodium regulates the body's acid–base balance
along with chloride and bicarbonate. It is involved in
forming a bicarbonate buffer system (NaHCO3 –
H2 CO3 ) and a phosphate buffer system (NaH 2 PO4 –
Na2 HPO4 ). These buffer systems play an im portant
role in the acid–base balance.
(Continues…
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…Continued) Electrolytes - Sodium
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Diseases States
Hyponatremia
When the plasma sodium level falls below normal, the
condition is referred to as hyponatrem ia. The causes
include
1. Vomitting
2. Diarrhea
3. Burns
4. Addison's disease (adrenocortical insufficiency)
5. Renal tubular acidosis (tubular reabsorption of
sodium is defective)
6. Severe sweating
(Continues…
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…Continued)
Hypernatremia
This condition is marked by an elevation in the plasm a
sodium level.
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Potassium
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Biochemical Functions of Potassium
(Continues…
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…Continued) Biochemical functions of potassium
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Disease States
Hypokalemia
Hypokalem ia is manifested as muscular weakness,
impaired m yocardial contractility leading to cardiac
arrhythmias and even cardiac arrest.
(Continues…
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…Continued)
Hyperkalemia
Hyperkalem ia, leads to ventricular arrhythmia,
ventricular fibrillation, bradycardia and m ay lead to
cardiac arrest .
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Section VI
Clinical Biochemistry
Universities Press
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Hydrogen Ion Homeostasis
I. Buffer system s
II. Respiratory m echanism
III. Renal regulation
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Buffer Systems
Buffers are the solutions which resist change in pH
on the addition of sm all amounts of acids or bases.
Any substance that can reversibly bind hydrogen
ions can be labelled as a buffer.
Buffers can neither remove H + ions from the body
nor add them to it. They can only keep H + ions in a
temporarily suspended form. The H + ions have to be
ultimately eliminated by the kidneys.
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Major Buffer Systems
1. Bicarbonate buffer
2. Phosphate buffer
3. Protein buffer
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Bicarbonate Buffer
The concentration of the bicarbonate ion is not
directly m easured; it is calculated from the
Henderson–Hasselbalch equation.
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Handerson–Hasselbalch Equation
(Continues…
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Respiratory Regulation of pH
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Chloride Shift
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Renal Regulation of pH
1. Reabsorption of bicarbonate
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Reabsorption of Bicarbonate
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Excretion of Ammonium Ions
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Excretion of Free H + Ions
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Anion Gap
(Continues…
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Acid- Base Disorders
Metabolic acidosis
The primary abnormalities in m etabolic acidosis are:
Increased production or decreased ex cretion of H +
ions or both at once.
Loss of HCO3 – from the body
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…Continued)
Management
Metabolic acidosis can be effectively m anaged by
identifying and treating the underlying cause. This
includes treatm ent of diabetes, control of diarrhea,
correction of shock and so on.
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Metabolic Alkalosis
(Continues…
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…Continued)
Mineralocorticoid ex cess (Cushing's syndrome,
Conn's syndrome), alkalosis occurs due to increased
H+ ion ex cretion in the urine
Principles of management include correcting the
underlying cause, adequate intravenous fluid
administration and replacem ent of K+ correcting
hypokalem ia.
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Respiratory Acidosis
Causes
• Airway obstruction and pulm onary disease – chronic
obstructive airway disease (emphysema, bronchitis
and so on)
– severe asthma
– bronchopneumonia
– respiratory distress syndrom e (RDS)
– flail chest
(Continues…
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Respiratory Alkalosis
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…Continued)
Hypox ia occurring in high altitudes, severe anaem ic
conditions and pulmonary disease
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Section VI
Clinical Biochemistry
Universities Press
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Chapter 2 5
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Liver Function Tests
Major Functions of the Liver
1 .Metabolic function
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2 . Synthesis function
Synthesis of plasm a proteins, clotting factors, cholesterol,
TAG and lipoproteins.
3 . Excretory function
Ex cretion of bile pigm ents and bile salts into the bile.
4 . Detoxification
Am m onia is detox ified to urea; drugs and other x enobiotics
are detox ified and ex creted.
6 . Storage function
Glycogen, vitam ins A, D, K, B12 and iron.
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Classification of Liver Function Tests
A. Biochemical classification
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…Continued
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…Continued
B. Clinical classification
I. Markers of liver dysfunction
i. Serum bilirubin: Total, conjugated
ii. Urine: Bile pigments, bile salts and
urobilinogen
iii. Total protein, serum albumin and
A/ G ratio
iv. Prothrom bin tim e
v. Blood ammonia
(Continued…
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Bilirubin
Serum bilirubin
Norm al serum bilirubin level varies from 0.2 to 1.0
mg/ dl. Of this, the unconjugated bilirubin (bilirubin –
albumin complex ) varies from 0.2 to 0.4 mg/ dl and
conjugated bilirubin varies from 0.2 to 0.6 m g/ dl.
(Continues…
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…Continued
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Causes of Different Types of Jaundice
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Tests for the differential
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diagnosis of jaundice
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Metabolic Function
Galactose tolerance test
Plasma amino acid test
Anti- pyrine breath tests
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Synthesis Function
Serum albumin
Ceruloplasmin
A1- Antitrypsin
Haptoglobin
A2- Macroglobin
Transferrin
A- Fetoprotein
Prothrom bin tim e (PT)
Electrophoretic separation of plasma proteins
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Tests Based on Enzyme Estimations
Aminotransferases or transaminases
alanine aminotransferase (ALT)
aspartate aminotransferase (AST)
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…Continued
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…Continued)
Nucleotidase
The activity of 5'- nucleotidase (norm al 2 –15 IU/ L) is
elevated in obstructive liver disease
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Tests Based on Detoxification Function
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Renal Function Tests
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…Continued
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Creatinine Clearance Test
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Estimated GFR (eGFR)
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Urea Clearance Test
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Inulin Clearance Test
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Analysis of Blood/ Serum
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Gastric Function Tests
Fractional test meal (FTM)
Pentagastrin stimulation test
Augm ented histam ine test
Alcohol test meal
Tubeless gastric analysis
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Pancreatic Function Tests
Measurement of pancreatic enzymes
Secretin–cholecystokinin test
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Section VI
Clinical Biochemistry
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Hyderabad 500 029 (A.P.), India
Phone: 040-2766 5446/5447
Email: info@universitiespress.com
marketing@universitiespress.com
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Chapter 2 6
Biochemistry of Cancer
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Etiology of Cancer
I. Predisposing factors
Age
Heredity
Environmental factors
– Lifestyle
– Diet
– Occupation
– Iatrogenic
Acquired pre- cancerous disorders
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Carcinogenic Agents
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Radiant Energy
i) Direct effect:
Radiation dam ages DNA by events such as single or
double strand breaks, elim ination of
purine/ pyrimidine bases, cross linking of strands
and formation of pyrim idine dim ers.
By inflicting injuries by the above m echanisms,
radiations like X- rays, - rays, UV- rays harm to the
DNA and are m utagenic.
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Chemical Carcinogens
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…Cont inued)
A selected list of chemical carcinogens
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Oncogenic Viruses
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Moecular Basis of Cancer
●
Certain genetic alterations in cells cause their
uncontrolled m ultiplication. Two types of regulatory
genes – oncogenes and antioncogenes have been
identified as crucial players in the development of
cancer.
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Oncogenes
●
The genes that can cause cancer are known as
oncogenes. They were first identified in tum our-
causing viruses (Rous sarcoma virus). These viral
oncogenes were later found to be intim ately similar
to certain genes present in normal host cells which
are called protooncogenes. These are the genes that
norm ally encode for growth- regulating proteins in
the human body. The conversion of protooncogenes
to oncogenes is the crucial factor in the causation of
cancer.
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Activation of Protooncogenes to 26
Oncogenes
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Enhancer insertion
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Chromosomal translocation
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Growth Factors
●
Growth factors norm ally stimulate the proliferation
of cells. They regulate cell division by signal
transduction across the cell mem brane to the
interior portions of the cell including the nucleus. It
is widely accepted that growth factors play a key
role in carcinogenesis.
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A selected list of polypeptide growth factors and their
functions in human tissues
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Mechanism of Action
●
Genes other than oncogenes (opposing their effects)
have been found to play a major role at least in som e
types of cancers. They are also called tum our
suppressor genes since they are m ostly involved in
the regulation of cell proliferation.
●
An im portant m odel for understanding their role is
retinoblastoma which occurs in children.
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…Continued)
●
Loss of antioncogenes producing cancer has been
incrim inated also in various other types of cancers
which include Wilm 's tum our of kidney, small cell
carcinoma of lung, and a specific type of breast
cancer.
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Diagrammatic representation of the role of m ultiple factors in 26
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Tumour Markers
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Cancer Therapy
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Section VII
Molecular Biology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Hyderabad 500 029 (A.P.), India
Phone: 040-2766 5446/5447
Email: info@universitiespress.com
marketing@universitiespress.com
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Chapter 2 7
DNA Metabolism
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Replication of DNA
Principles of Replication
1. Copying is accurate
2. Replication is semiconservative
3. Supercoils and DNA topoisom erases
4. Replication fork
5. Replication is sim ultaneous and bidirectional
6. DNA synthesis is catalysed by DNA polymerases
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Replication fork
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Replication in Prokaryotes
Most of the inform ation about the replication in
prokaryotes is obtained from the studies made on
the intestinal bacterium , E.coli.
The process of replication can be studied
conveniently in three stages.
1. Initiation
2. Elongation
3. Termination
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Initiation
Helicase enzym es
Replication fork
Topoisomerases (also called as DNA gyrases)
Single- stranded DNA binding (SSB) proteins
Primer
Primase
DNA polymerase III
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Elongation
Okazaki fragments
DNA polymerase I
Proof- reading ability
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Discovery of Okazaki fragm ents
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Termination
Terminus sequence
DNA polymerase II
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Replication in Eukaryotes
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…Continued)
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…Continued)
1. Initiation
2. Elongation
3. Termination
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Initiation
Helicases
Topoisomerases
Replication protein A (RPA)
Replication fork
Multiple sites of origin of replication
Primers
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Overview of DNA replication on the lagging strand in eukaryotes
(leading strand is not shown)
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Elongation
Replication factor C (REC)
Proliferating cell nuclear antigen (PCNA)
Sliding clamp
DNA polymerase
Okazaki fragments
RNase H
FENI (flap endonuclease I)
DNA polymerase
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Termination
Telomeres
Telomerases
RNA dependent DAN synthesis
Reverse transcriptase
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Inhibitors of Replication
●
The differences between the DNA replication in
bacteria and hum an cells give scope for developing
antibiotics that target bacterial replication but do not
affect hum an cells. Several drugs (e.g., ciproflox acin,
nalidix ic acid ) act by inhibiting the enzym es of
bacterial replication.
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Cell Cycle
• S (Synthetic) phase
• M (m itotic) phase
• Gap1 (G1 ) and gap2 (G2 )
• Dormant phase (G0)
• Cyclins
• Cyclin- dependent kinases (CDKs)
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Apoptosis
Apoptosis
A programm ed cell suicide is referred to as apoptosis
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Recombination
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Homologous Recombination
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Holliday model of homologous recombination (the areas shown in 27
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Non- homologous Recombination
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DNA Damage
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Types of DNA Damage
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Repair of DNA
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Mechanisms of DNA Repair
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Mismatch Repair
●
Even though replication occurs with high
fidelity, defects do occur during copying.
Mism atch repair corrects errors involving
single base pair or a sm all region of
unpaired DNA
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A diagrammatic representation of mismatch repair
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Base Excision Repair
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Nucleotide Excision Repair
●
Xeroderma pigmentosum
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Section VII
Molecular Biology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Hyderabad 500 029 (A.P.), India
Phone: 040-2766 5446/5447
Email: info@universitiespress.com
marketing@universitiespress.com
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Chapter 2 8
RNA Metabolism
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●
Cells contain three major types of RNA:
●
ribosom al RNA (rRNA) is the chief constituent of
ribosom es;
●
messenger RNA (m RNA) carries the information
required for protein biosynthesis in its
nucleotide sequences;
●
transfer RNA (tRNA) delivers amino acids to the
ribosom es for protein biosynthesis of rRNA.
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Principles of Transcription
●
Transcription is the synthesis of the RNA molecule
from DNA. Unlike replication where the entire
sequence of DNA is duplicated, during
transcription only certain segments of DNA are
ex pressed. These transcribable portions of the DNA
are referred to as genes.
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…Continued)
●
Only one strand of the duplex DNA takes part at a
tim e in the synthesis of RNA.
●
The sequence of ribonucleotides in the newly
synthesised RNA m olecule is com plementary to the
sequence of deox yribonucleotides in this strand of
DNA m olecule and is called the tem plate strand.
●
The other DNA strand which does not take part in
transcription is referred to as the coding strand.
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…Continued
Transcription initiation site (TIS)
Downstream
Upstream
The figure shows that the genes occur on both strands of the
DNA and are always transcribed in the 3' to 5' direction (note
the direction of the arrows)
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RNA polym erase (RNAP)
How does RNAP know where to start the process of
transcription in a stretch of DNA?
Promoters
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…Continued)
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Transcription in Prokaryotes
1. Initiation
2. Elongation
3. Termination
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…Continued)
Initiation
The process of RNA synthesis in bacteria begins with
the binding of RNA polymerase (RNAP) m olecule on the
DNA.
RNAP recognises the transcription start site on the
tem plate strand of DNA with the help of certain
specific regions on the DNA known as prom oter
elem ents (PE).
Pribnow box (TATA box )
The ‘–35’ sequence
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…Continued
Elongation
●
The elongation of the RNA molecule occurs from the
5' to its 3' end, antiparallel to the template strand.
RNAP assembles ribonucleotides in a
complem entary sequence to the tem plate strand by
Watson- Crick base pairing rules
Termination
• Rho () factor
• Palindrom es
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TIS = Transcription
initiation site
Sigma factor
Termination factor
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Transcription in Eukaryotes
Eukaryotic transcription is m ore complex com pared
to that of prokaryotes. There ex ist three distinct RNA
polymerases transcribing different types of RNAs
compared to the single RNAP in prokaryotes.
RNAP I catalyses the formation of large ribosom al
RNAs. RNAP II synthesises the prim ary transcript
form of m RNAs. RNAP III takes part in the synthesis
of tRNAs and small ribosomal RNAs.
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Initiation
Transcription begins with the RNAP recognising the
promoter site. Two types of sequence elements are
identified as eukaryotic promoters which occur
prox imal to the TIS.
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…Continued)
1. TATA box
This sequence is the Goldberg- Hogness box .
Determines where ex actly the transcription has to
begin along the template strand of DNA.
2 . CAAT box
This region determines how frequently the
transcription has to occur and at what speed .
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…Cont inued)
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Elongation
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●
The mRNA in prokaryotes is fully functional as soon
as it is synthesised.
●
On the contrary, all types of eukaryotic RNA
produced by transcription as prim ary transcripts are
non- functional as such. They undergo ex tensive
structural alterations to produce the mature
functional molecules. The phenom ena of RNA
processing in eukaryotes are known as post-
transcriptional m odifications.
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Messenger RNA
• Heterogeneous nuclear RNA (hnRNA)
• Mature mRNA
• The cap
• The tail
• Splicing
• m RNA editing
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Inhibitors of Transcription
●
There are a number of drugs and tox ins capable of
inhibiting the process of transcription at various
stages, in bacteria.
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●
- Amanitin, a mushroom poison binds to the RNAP II
and inhibits the transcription in eukaryotes. Heparin,
a glycosaminoglycan binds to RNAP and inhibits the
transcription.
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Section VII
Molecular Biology
Universities Press
3-6-747/1/A & 3-6-754/1, Himayatnagar
Hyderabad 500 029 (A.P.), India
Phone: 040-2766 5446/5447
Email: info@universitiespress.com
marketing@universitiespress.com
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Chapter 2 9
Protein Biosynthesis
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Genetic Code
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Features of Genetic Code
•
Universality
•
Specificity
•
Non-overlapping
•
Degeneracy
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Genetic Code
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Codon–Anticodon Pairing
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Wobble Hypothesis
●
A single tRNA can recognise more than one codon. This is
possible because the third base (3' base) in the codon
sometimes fails to recognise its complementary base in
the anticodon (5' base).
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Mutations
Classification of mutations
Mutations can be classified into two major categories.
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…Continued
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…Continued
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Link between amino acids and CHAPTER 29
nucleic acids
The link between amino acids and nucleic acids is first made by
enzymes called aminoacyl tRNA synthetases.
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Translation in Prokaryotes
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Initiation
Figure
continued
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Elongation
•
Binding of an incoming aminoacyl-tRNA
•
Peptide bond formation
•
Translocation
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Figure
continues
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…Continued)
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Termination
•
Stop codons UAA, UAG, UGA
•
Release factors (RF-1, RF-2, and RF-3)
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Translation in Eukaryotes
1. Initiation
2. Elongation
3. Termination
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Initiation
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Initiation of Translation in Eukaryotes
(see textbook for abbreviations)
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Elongation
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Elongation and Termination in
Eukaryotic Translation
See textbook for
abbreviations.
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...Figure continued
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Termination
•
Stop codons (UAA, UAG, UGA)
•
Release factor (eRF)
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Post-translational Modifications
•
Trimming
•
Structural reorganisation
•
Modifications at the N-terminal and
C-terminal ends
•
Covalent modifications
Phosphorylation
Hydroxylation
Glycosylation
(Continued…
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Protein Targeting
•
Signal sequence
•
Signal recognition particle (SRP)
•
Endoplasmic reticulum (ER)
•
Transport vesicles
•
SNARE proteins
•
Receptor-mediated endocytosis
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Protein destination by different pathways
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Some conformational diseases due to
mutations affecting protein targeting
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Section VII
Molecular Biology
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Chapter 3 0
Regulation of Gene
Expression
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Principles of Gene Regulation
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DNA- Protein Interactions
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Diagrammatic representation of motifs mediating DNA–protein
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Regulation of gene expression in 30
prokaryotes
• LAC operon
• Structure of lac operon
• How does the lac operon operate?
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Lac operon
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Tryptophan Operon
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Gene Amplification
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Gene Rearrangement
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Chapter 3 1
Recombinant DNA
Technology
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Recombinant DNA Technology 31
Protocol
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Technology
• Enymes
• Vectors
• Hosts
• DNA to be cloned
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Enzymes
• Nucleases
• Ex onucleases
• Endonucleases
• Restriction endonucleases
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Restriction Endonucleases
• Nomeclature of RE
• Recognition sequences
• Restriction m ap
• Cleavage patterns
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Some restriction enzymes with their sources, recognition
sequences and the products formed
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Enzymes commonly used in rDNA technology/ genetic engineering
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Vectors – The Cloning Vehicles
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(. . . Continued)
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Different cloning vectors with the corresponding hosts and
max imum sizes of foreign DNA inserts
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1) Plasmids
2) Bacteriophages
3) Cosmids
4) Artificial chromosome vectors
5) Ex pression vectors
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Hosts
• Prokaryotic hosts
• Eukaryotic hosts
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Gene Cloning Strategies
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Gene Banks
●
Gene banks are the outcome of novel concepts
where certain centres have been developed for the
storage of an individual's DNA for future use (e.g.,
in the diagnosis of diseases)
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Applications of rDNA Technology
Insulin
Technique for production
of recombinant insulin
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• Hepatitis- B vaccine
• Growth hormone
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(Continued…
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DNA Vaccine
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DNA Finger Printing/ DNA Typing
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• Interferons
• Antibiotics
• Recom binant factor VIII
• Tissue plasm inogen activator (tPA)
• Monoclonal antiboides
• Diagnosis of HIV infection
• Gene therapy
• Transgenic animals
• In agriculture
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(. . .Continued)
(Continues…)
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(. . .Continued)
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Selected list of diseases for which recombinant vaccines have 31
(Continues . . .)
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(. . . Continued)
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Chapter 3 2
Molecular Biology
Techniques
DNA–DNA
hybridisation
Application
DNA libraries contain thousands or even m illions of
irrelevant DNA fragm ents. It is difficult to pick out a
specific gene (a DNA sequence of interest) from these
fragments. Probes are used to search and locate a
specific gene from DNA libraries.
Blotting techniques are com m only used analytical tools for the
identification of desired DNA or RNA fragm ents.
of DNA Fragments
(Continues…
…Continued)
(Continues…
(Continues…
…Cont inued)
Applications
It is an ideal technique for determ ining the number
of genes (through mRNA) present on a given DNA.
But this is not practically applicable since each gene
may give rise to two or more RNA transcripts.
It is used to size and quantitate specific RNA
molecules.
(Continues…
Application
It is currently being used as one of the tests for
detecting the AIDS virus. In this case, the presence of
viral proteins in the blood is detected by antibodies. In
Western blot analysis, antibodies against different
components of the virus are analysed so it is considered
to be the confirm atory test for diagnosis.
●
It is a modification of the Southern and Northern
blotting techniques. In this technique, the nucleic
acids (DNA/ RNA) are directly spotted on to the filters
(not subjected to electrophoresis). The hybridisation
procedure is the sam e as that of the blotting
techniques.
●
It is useful in accum ulating data for the evaluation of
gene ex pression.
(Continues…
(Continues…
Diagnosis
Cancer detection
Prenatal diagnosis
Medico- legal cases
Genetic disorders
PCR in DNA sequencing
Fossil studies
Com parative studies of genom es
●
Production of monoclonal antibodies in vitro is called
hybridom a technology.
●
The hybridoma cells formed retain the properties of
both the parent cells, that is, antibody secretion
property of B- lymphocytes and uncontrolled division
property of cancer cell.
(Continues…
…Continued)
Polymorphisms (RFLPS)
i) Southern hybridisation
ii) PCR
Repeats (VNTRS)
SSCP analysis
Electrophoresis (CSGE)
●
Enzyme linked imm une sorbent assay (ELISA) is
based on the imm unochem ical principles of the
antigen–antibody reaction.
●
Types of ELISA
●
Antibody detection by ELISA (Indirect ELISA)
(Continues…
(Continued…
(Sandwich ELISA)
Radioimmunoassay
32
Applications
Radioim munoassay is widely applied in the diagnosis of
cancers, hormonal disorders and also for therapeutic
m onitoring of drugs. It is an ex tensively used tool in
biomedical research.
(Continues…
…Continued)
(Continues…
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Chapter 3 3
Biochemical Techniques
●
Electrophoresis is the migration of charged
particles in an electric field.
●
Electrophoresis is widely used for the separation of
proteins in body fluids (mostly the plasma proteins
including immunoglobulins, lipoproteins,
hemoglobins, isoenzymes and nucleic acids).
• Zone electrophoresis
• Imm unoelectrophoresis
• Isoelectric focusing
• High voltage electrophoresis
• Capillary electrophoresis
(Continues…
…Continued)
●
In the stationary phase, there is a porous solid
medium through which the sample mix ture in the
moving solvent (m obile phase) percolates.
HPLC – High
performance liquid
chromatography;
(Continues…
…Continued)
(Continues…
Electromagnetic spectrum
Components of a colorimeter
(Continued…
Beer’s law
The amount of transm itted light diminishes
ex ponentially with an increase in the concentration of
absorbing m aterial.
Lambert’s law
The amount of transm itted light decreases
ex ponentially with the increase in the thickness of the
medium.
• Spectrophotom eter
• Flam e photometer
• Fluorimeter
• Auto Analyser
• Atomic absorption spectrophotom etry
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Chapter 3 4
Immunochemistry
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Types of immunity
Immune defences or imm unity of the body operates in
two different ways, which are interdependent to some
ex tent.
• Innate (or natural) imm unity
• Adaptive (or acquired) im munity
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Organisation of the Immune System
B-lymphocytes
B-lymphocytes specifically recognise each antigen and
produce antibodies (immunoglobulins) against each of them.
B-cells are involved in humoral immunity.
(Cont inues
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…Continued)
T-lymphocytes
T-lymphocytes identify viruses and microorganisms from the
antigens displayed on their surfaces. T-cells are involved in
cell-mediated immunity.
Cytokines
They regulate the host cell division (mitosis) and also
mediate communication between different cell types of the
immune system.
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Innate Immunity
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Adaptive Immunity
Adaptive imm unity is m ost specific in its action and
ex erts a m ore effective immune response.
It responds to m acrom olecules (mainly proteins), but not
to small foreign molecules unless these are attached to
large ones. These foreign macrom olecules are called
antigens, and antibodies are produced in response to
them . Lymphocytes mediate the adaptive imm une
response.
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Acquired Immunodeficiency 34
Syndrome (AIDS)
Mode of transmission
1. Majority of cases (about 80%) are through sex ual
contact. Men indulging in homosex ual relationships
are particularly at high risk. Heterosex ual
intercourse presents a relatively lower risk.
2. Intravenous drug abuse (by the sharing of needles).
3. Unprotected blood transfusions.
4. Vertical transm ission (mother to infant during
pregnancy)
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Structure of HIV
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Immunology of AIDS
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Cells of the imm une system
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Hematopoiesis
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Important cytokines and their functions
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Phases of adaptive immune
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In vivo immune response
(…Cont inues
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In vivo im m une response
. . . Continued)
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Auto- imm une diseases with corresponding
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auto- antibodies
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Section IX
Biochemical Perspective of
Endocrinology
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Chapter 3 5
Hormone Action
HRU = Hormone response unit; PIC = Pre- initiation complex ; RNAP = RNA polymerase; TFIID = Transcription factor
IID; HRE = Hormone response element, HR complex = Hormone- receptor complex , AF= Accessory factors, DE =
DNA element, ?= Unknown DNA regulatory elements
●
G- Proteins
●
Generation of second messengers – cyclic amp
●
Cyclic GMP
Horm one signal t ransducing t hrough HR com plex → G- prot eins → Adenylyl cyclase → cAMP
→ Prot ein kinase A → Prot ein phosphorylat ion, result ing in the ult im at e biochem ical response
(Continues…
…Continued)
as second messengers
phospholipase C
(Continues…
…Continued)
transduction cascades
●
Several receptors have enzyme activity (intrinsic protein
kinase activity) that is activated by the binding of the
hormone to the receptor.
These kinases autophosphorylate the tyrosine residues,
triggering a cascade of phosphorylation–dephosphorylation
reactions involving several other protein kinases and
phosphatases inside the cell.
(Continues…
…Continued)
●
Different signal transducing m olecules such as Jak,
STATs, Ras, MAP kinase SH2 domains are involved in
a com plex array of m olecular interactions in the
cytosol and inside the nucleus.
●
Horm ones such as insulin, growth hormone,
prolactin, erythropoietin, leptin and so on initiate
their action by activating intrinsic tyrosine kinase
activity.
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Chapter 3 6
functions of GH
• Effects on growth
• Effects on protein metabolism
• Effects on carbohydrate m etabolism
• Effects on lipid m etabolism
• Effects on mineral m etabolism
• Effects on lactation
Deficiency of GH
• Dwarfism GH-deficient dwarfs
• Laron type dwarfs and pygmies
Excess of GH
• Gigantism in children and acromegaly in adults
family
1. ACTH
2. - lipotropin (- LPH)
3. - MSH
●
–lipotropin (–LPH)
Endorphins and enkephalins
●
Melanocyte- stimulating horm one (MSH)
●
Two hormones, nam ely ox ytocin and antidiuretic
hormone (ADH), are released from the posterior
pituitary gland (neurohypophysis).
The amino acids that are different in ox ytocin and ADH are shown in red.
●
Abnormalities of ADH production or action cause diabetes
insipidus, a condition characterised by the excretion of
high volumes of dilute urine (polyuria).
●
Nephrogenic diabetes insipidus
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Chapter 3 7
Thyroid Hormones
• Thyroglobulin
• Organification
• Coupling
• Phagocytosis (Continues…
…Continued
• Hydrolysis of thyroglobulin
• Release of T3 and T4
Biosynthesis and
release of T 3 and
T4 in the thyroid
gland
(Continued…
functions
Goitre
Hyperthyroidism
• The m ost com mon cause of hyperthyroidism is
Grave's disease
Hypothyroidism
• Cretinism
• Myx oedema
thyroid function
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Chapter 3 8
Hormones of the
Adrenal Gland
• Glucocorticoids
• Mineralocorticoids
(Continues…
…Continued)
• Mineralocorticoid synthesis
• Glucocorticoid synthesis
• Androgen synthesis
glucocorticoids
(Continues…
…Cont inued
functions of mineralocorticoids
function
Synacthen test
(Continues…
Mechanism of action
The catecholamines act through two classes of
receptors.
2. - adrenergic receptors
catecholamines
• Response to stress
Fight-or-flight response
Hyperglycemic effect
Lipolysis
Production
Pheochromocytomas
●
The disease is manifested by signs and sym ptoms
characteristic of catecholam ine ex cess (e.g.,
hypertension, palpitations, severe headache,
ex cessive sweating).
●
The tum our cells produce more norepinephrine than
epinephrine.
●
The levels of vanillylmandelic acid (VMA) in urine
(normal < 8 mg/ day) are elevated in patients of
pheochromacytom a. This is m easured in clinical
chemistry laboratories.
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Chapter 3 9
Hormones of the
Gonads
• Androgens
• Estrogens
• Progestins
Biosynthesis of androgens
For the enzymes of the initial steps of the pathway see Fig. 37.2
(Continues…
of androgens
2. Metabolic effects
3. Androgens along with FSH induce Sertoli cells to
produce a hormone called inhibin.
Inhibin plays a role in promoting sperm atogenesis and
testosterone production by stim ulating the secretion
of FSH through a negative feedback loop.
(Continues…
…Continued)
androgen defects
• Hypogonadism
• Castration
progesterones
of estrogens
(Continues…
…Continued )
progesterone
(Continues…
…Continued )
●
Role of estrogens and progesterone in
maintaining the menstrual cycle
Role of estrogens and progesterone during
pregnancy
• Contraception
• Menopause
reproductive system
• Prim ary hypogonadism
• Secondary hypogonadism
• Turner’s syndrome
• Polycystic ovary disease (PCOD) (Stein- Levinthal
syndrom e)
• Congenital adenal hyperplasia
• Precocious puberty
• Amenorrhea
• Dysm enorrhea (painful m enstruation)
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Endocrinology
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Chapter 4 0
Hormones of the
Pancreas and Diabetes
Mellitus
The pancreas is unique in that it contains two
different organs within its structure; an ex ocrine
gland and an endocrine gland.
The ex ocrine part secretes enzymes that are used for
digestion.
The endocrine portion contains about 1–2 m illion of
the islets of pancreas containing different cell types
(, , and so on). They secrete different horm ones.
Biosynthesis of insulin
Proteolytic
processing of
proinsulin to
insulin
Diagrammatic
representation
of signalling
pathways
downstream
from the insulin
receptor .
For
abbreviations,
see the
tex tbook .
(Continues…
…Continued)
…Continued)
Glucagon 40
It is a metabolic disorder resulting either from deficiency of
insulin or resistance to its action causing increased blood
glucose levels (hyperglycemia) which lead to several
systemic complications.
Diabetes mellitus is broadly classified into two categories
based on etiology rather than treatment.
●
Metabolic syndrome
Diabetic ketoacidosis
• Diabetic ketoacidosis is characterised by hyperglycemia,
hyperketonemia and metabolic acidosis.
• Kussmaul’s respiration
• Sweet smell
• Hyperglycemia
• Ketosis
(Continues…
• Metabolic acidosis
• Severe dehydration
• Electrolyte loss
Hypersomolar nonketotic coma
Lactic acidosis
These include
• Atherosclerosis
• Retinopathy
• Nephropathy
• Neuropathy
Diabetes Mellitus
and Proteins
• Normal response
• Diabetes mellitus
• Impaired glucose tolerance (IGT)
• Renal glycosuria
• Alim entary glycosuria
• Gestational diabetes mellitus (GDM)
●
Glycated hemoglobin
The concentratio of HbA1 c reflects the mean
blood glucose concentration of the preceding 8 –
1 0 weeks.
●
Fructosamine
Reference values of
HbA1 c
• Whipple’s triad
• Reactive hypoglycemia
• Fasting hypoglycem ia
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Chapter 4 1
Extracellular Matrix
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Composition of extracellular matrix
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Functions of ECM
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Biomedical significance of ECM
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Functions of Collagen
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Types of collagen and their distribution
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2 . Ehlers–Danlos syndrome
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4 . Epidermolysis bullosa
This condition which shows blisters and cracks in the skin following
sm all injuries is caused by m utations in the gene encoding type VII
collagen.
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5 . Scurvy
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6 . Lathyrism
Like scurvy, lathyrism is also not a genetic disorder but a condition
characterised by lack of strength in or inability to m ove lower lim bs
observed in people who consum e pulse lathyrus sativa or sweet pea
(kesari dal) regularly as a part of their diet. This pulse contains a
tox in called β - ox alyl am inoalanine (BOAA) which inhibits the
enzym e lysyl ox idase, which adversely affects the cross- linking of
lysine in the collagen structure.
The result is weakening and brittleness of bones.
7 . Menkes disease
Deficiency of copper results in defective cross- linking of collagen
and elastin by the copper- dependent enzym e lysyl ox idase.
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Genetic Abnormalities of Elastin
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Marfan Syndrome
Mutations in the gene for fibrillin cause Marfan syndrome.
They are tall and have large body frames; even the digits of their
hand are extra-long.
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Structure of Aggrecan
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Functions of glycosaminoglycans
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Functions of
glycosaminoglycans
6. They are the components of synaptic vesicles.
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Heteropolysaccharides
Glycosaminoglycans (GAGs)
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The major GAGs include:
1. Hyaluronic acid
2. Chondroitin sulphates
3. Keratan sulphates
5. Dermatan sulphate
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Structures of Glycosaminoglycans
Hyaluronic acid
Chondroitin 4- sulphate
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Keratan sulphate
Heparin
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Dermatan sulphate
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Important Mucopolysaccharidoses (MPS)
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Types of Glycoproteins
O- linked glycoproteins
They are form ed by O-glycosidic linkage with the –OH of
serine or threonine.
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Types of glycoproteins
N- linked glycoproteins
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Disorders associated with glycoproteins
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. . . Continued)
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Chapter 4 2
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…Cont inued)
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Sources and generation of ROS
1 . Respiratory chain
About 2% of inhaled ox ygen undergoes partial
reduction liberating superox ide anion and perox ide
radical which are potentially destructive.
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…Cont inued)
2 . Respiratory burst
Macrophages and neutrophils engulf bacteria, utilising
large amounts of ox ygen in the process. This
phenomenon is called respiratory burst. As a result of
this phagocytic activity, about 10% of the consumed O 2
is diverted to the generation of ROS such as superox .ide
. and hypochlorite ion
(O2 –), H2 O2 , hydrox yl radical (OH)
(CIO–).
Interestingly, the generated ROS ex erts a potent
bactericidal action (killing the bacteria).
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…Cont inued)
3 . Lipid peroxidation
Initiation phase
Propagation phase
Termination phase
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Other sources of ROS
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Mechanism of action of ROS
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ROS and disease
• Ageing
• Cardiovascular disease
• Cancer
• Inflam matory diseases
• Respiratory diseases
• Diabetes
• Infertility
• Beneficial effects of ROS
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Antioxidant Enzymes
• Catalase
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Antioxidant Vitamins
• Vitamin E
• Vitamin C
• - Carotene
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Other Antioxidants
• Selenium
• Glutathione
• Uric acid
• Bilirubin
• Lipoic acid
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Antioxidant Foods
●
Unprocessed vegetable oils (like soyabean oil, rice
bran oil, cotton seed oil, sunflower oil), whole grains,
green leafy vegetables, legum es, fruits (like citrus
fruits, guava, am la), green vegetables (like spinach,
cabbage, cauliflower), carrots, papaya, waterm elon,
tom atoes have a high content of various antiox idants.
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…Cont inued)
●
Grapeseed, turm eric, walnuts, onions, beverages
such as green tea, red wine and organ meats contain
different polyphenols, catechins, curcuminoids,
proanthocyanidins, phenolic acids have antiox idant
properties. Certain food additives such as
propylgallate, butylated hydrox yl anisole, butylated
hydrox yl toulene are used to prevent lipid
perox idation in packed foods.
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Chapter 4 3
Biochemical Genetics
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Genetic diseases and modes of 43
inheritance
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Chapter 4 4
Environmental Pollutants,
Toxins and Biomedical
Waste Management
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Types of Pollution
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Properties and Classification of Pollutants
1. Physical pollutants
3. Biological pollutants
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Classification based degradability
1. Biodegradable pollutants
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Air Pollution
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Water Pollution
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• Soil pollution
• Noise pollution
• Therm al pollution
• Nuclear pollution
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Toxins and Poisons
• Lead
• Mercury
• Aluminium
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Toxins in Foods
• Enzyme inhibitors
• Goitrogens
• Biogenic am ines
Pesticides
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• Biochemical Waste Management
Colour- coded bags for biomedical waste segregation as per 2016 rules
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… Continued
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Chapter 4 5
human genome
• Mapping protocols
– Probes
– Hierarchical shotgun approach
– RFLP
– VNTR
– Cytogenetic maps
– FISH
(Continues…
…Continued)
• Sequencing protocols
– Cloning
– rDNA technology
– PCR
– Gel electrophoresis
– Max am - Gilbert m ultiplex technique
– Sanger sequencing
– Chromosome walking
– Chromosome jumping
– Number of genes
– Orphan genes
– Non- coding RNAs
– Junk DNA
– Sizes of genes
– Genes associated with diseases
– Single nucleotide polym orphisms
– Satellite DNAs
(Continues…
LINEs
LTRs
HGP
Applications in medicine
The hum an genome project, through its sequencing of
the DNA, helps in understanding the root causes of
diseases, enabling us to develop effective treatment
strategies.
• Applications in biotechnology
• Agriculture
• Environm ental cleanup
• ELSI of HGP
Scope of bioinformatics
• DNA sequencing data
• Gene ex pressions profiles
• RNA and protein sequences
• Protein 3- D structure analysis
• Microarrays (DNA chips)
• Data from patients’ clinical trials
• Drug designing
• Primary databases
• Secondary databases
Addresses
Applications of Bioinformatics
• Bioinform atics can be applied to the identification of
nucleotide sequences of protein- encoding genes. Since
amino acid sequencing is a tedious task, the base
sequence of the gene for a given protein can be
translated into an am ino acid sequence using the genetic
code.
…Continued)
Applications of Bioinformatics
Applications of Bioinformatics
…Continued)
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Chapter 4 6
Gene Therapy
Viral vectors
• Retroviruses
• Adenoviruses
• Herpes viruses
Transfer through liposomes
Receptor- mediated endocytosis
Artificial chromosome vectors
Antisense therapy
Target tissues for gene transfer
The retrovirus carries an RNA copy of the therapeutic gene into the cell. The endosome containing virus
dissolves and the viral RNA and reverse transcriptase are released into the cell. The enzyme copies the RNA into
a double- stranded DNA that eventually integrates into the host DNA. Transcription and translation of this DNA
(the therapeutic gene) produces the therapeutic protein. Note that the virus does not multiply since its genes
were removed and replaced by the RNA copy of the therapeutic gene.
A defect in the adenosine deaminase (ADA) gene
located on chrom osom e 20 causes severe com bined
im munodeficiency syndrom e (SCID).
Familial hypercholesterolem ia
Cystic fibrosis
• Antisense therapy