Biochemistry Final

BIOCHEMISTRY
Dr. Ambayehu (MD)
07/22/2024 Dr Ambayehu 1
Course outline
• Chapter One- Introduction
• 1.1.The scope of biochemistry
• 1.2.Significance to clinical medicine
• Chapter Two- The major chemical constituents of cells
• 2.1. General chemical composition
• 2.2. Water
• Chapter Three- Proteins
• 3.1 amino acids
• 3.2. Classification of proteins
• 3.3. Conformation of proteins
• 3.4. Myoglobin and hemoglobin
• 3.5. Enzymes
• Chapter Four- Carbohydrates
• 4.1. Monosaccharaides
• 4.2. Polysaccharides
• Chapter- Five- Lipids
• 5.1. Fatty acids
• 5.2. Acylglycerols
• 5.3. Phospho-glycerides
• 5.4. Sphingolipids
• 5.5. Sterols (steroids)
• Chapter Six- Intermediary metabolism
• 6.1. Definition
• 6.2. Carbohydrate metabolism
• Chapter Seven- Lipid metabolism
• 7.1. Digestion and absorption
• 7.2. Fatty acid oxidation and generation of energy
• 7.3. Deposition of fat (synthesis of triacyl glycerol)
• 7.4. Mobilization of fat (catabolism of triacyl glycerol)
• 7.5. Phospholipid metabolism
• 7.6. Lipid transport
• 7.7.Clinical correlates
• Chapter Eight- Protein Metabolism
• 8.1. Digestion and absorption of amino acids
• 8.2. Essential and non-essential amino acids
• 8.3. Catabolism of amino acid nitrogen
• 8.4. Catabolism of amino acid carbon skeleton
• 8.5. Amino acids as a source of special peptide
• Chapter Nine- Integration of metabolism
• 9.1. Metabolic profile of major organs
• 9.2. Hormonal regulators of fuel metabolism
• 9.3. Metabolic adaptation to prolonged starvation
• 9.4. Diabetes mellitus and disturbances in intermediary metabolism
• Chapter Ten- Porphyrin and bile pigments
• 10.1. A description on the synthesis of porphyrin and heme
• 10.2. Catabolism of Heme and formation of bilirubin
• 10.3. Transport, conjugation, excretion of bilirubin and bile pigments
• 10.4. Clinical correlates
• Chapter Eleven- Nucleotide
• 11.1. Nomenclature
• 11.2. Metabolism of purine and pyrimidine
• 11.3. Clinical correlates
• Chapter Twelve- Genetics
• Chapter Thirteen -Vitamins
• Chapter Fourteen - Minerals
• Chapter Fifteen- Blood
• Chapter Sixteen- Blood group antigens and antibodies
Evaluation:
• Attendance: 100%
• Quiz: 10%
• Mid exam: on 15-07-2015 ec. 40%
• Final exam: 50%. On 25-07-2015 ec.
• "Today's world needs people who express goodness in their words and
deeds.
If such noble role models set the example for their fellow beings, the
darkness
prevailing in today's society will be dispelled, and the light of peace
and nonviolence will once again illumine this earth. Let us work
together towards this goal".
—Mata Amritanandamayi Devi
CHAPTER ONE
INTRODUCTION
Dr. Ambayehu (MD)
Objectives:
• At the end of the session students will be able to:
Define biochemistry
Discuss the scopes and importance of biochemistry
What is biochemistry?
• Biochemistry is the language of biology. “Chemistry of living cells”
• Biochemistry seeks to describe the structure, organization &
function of living organisms in molecular terms.
• Biochemistry is the application of chemistry to the study of biological
processes at the cellular and molecular level.
Why Should We Study Biochemistry?
• The study of biochemistry is essential to understand basic functions of

the body.
• This study will give information regarding the functioning of cells at
the molecular level.
• How the food that we eat is digested, absorbed, and used to make
ingredients of the body?
• How does the body derive energy for the normal day to day work?
• How are the various metabolic processes interrelated?
• What is the function of genes?
Cont…
• What is the molecular basis for immunological resistance against
invading organisms?
• How do organisms store and transfer information necessary to
reproduce themselves?
• What primary molecules and processes were involved in the origin of
life?
• The demarcation of abnormal from normal constituents of the body is
another aim of the study of biochemistry.
• Biochemical study results have led to a molecular understanding of
diseases such as Diabetes, sickle-cell anemia, phenylketonuria, cystic
fibrosis, hypercholesterolemia, and some forms of cancer.
• Sequencing of the human genome will help in search off cures for
Alzheimer’s disease, West Nile virus, depression, influenza, and other
disease conditions.
• Recombinant DNA technology will play a major role in the diagnosis
and treatment of diseases (gene therapy).
• The study of enzymes and metabolism provides a foundation for the
rational design of drugs and for the detailed understanding of nutrition.
The Scope of biochemistry
• Structural and functional biochemistry; focuses on:
• Discovering the chemical structures
• Three-dimensional arrangements of biomolecules.
• Informational biochemistry; Defines the language(s) for storing
biological data and for transmitting that data in to cells and organisms.
• This area includes molecular genetics, which describes the molecular
processes in heredity and expression of genetic information
• Processes that communicate molecular signals to regulate cellular activities
(i.e., hormone action).
• Bioenergetics describes the flow of energy in living organisms and
how it may be transferred from one process to another.
Chapter two
The major chemical constituents of cells
Dr. Ambayehu (MD)
Objectives:
• The basic chemical constituents of cells.
• Definition and properties of water.
2.1. General chemical composition
• Only 31 chemical elements occur naturally in plants and animals
1. Elements found in bulk form and essential for life: C, H, O, N, P,
and S.
2. Elements in trace quantities in most organisms and very likely
essential for life, such as calcium, manganese, iron, and iodine.
3. Trace elements that are present in some organisms and may be
essential for life, such as arsenic, bromine, molybdenum, and
vanadium.
• More than 99% of the human body is composed of 6 elements, i.e.
oxygen, carbon, hydrogen, nitrogen, calcium and phosphorus.
• Molecular structures in organisms are built from 30 small precursors,
sometimes called the alphabets of biochemistry. These are 20 amino
acids, 2 purines, 3 pyrimidines, sugars (glucose and ribose), palmitate,
glycerol and choline.
• Human body is composed of about 60% water, 15% proteins, 15%
lipids, 2% carbohydrates and 8% minerals.
• In living organisms, biomolecules are ordered into a hierarchy of
increasing molecular complexity.
• These biomolecules are covalently linked to each other to form
macromolecules of the cell, e.g. glucose to glycogen, amino acids to
proteins, etc.
• Major complex biomolecules are proteins, polysaccharides, lipids and
nucleic acids. The macromolecules associate with each other by
noncovalent forces to form supramolecular systems, e.g. ribosomes,
lipoproteins.
• Finally at the highest level of organization in the hierarchy of cell
structure, various supramolecular complexes are further assembled
into cell organelle.
• In prokaryotes (e.g. bacteria; Greek word "pro" = before; karyon =
nucleus), these macromolecules are seen in a homogeneous matrix;
• But in eukaryotic cells (e.g. higher organisms; Greek word "eu" =
true), the cytoplasm contains various subcellular organelles.
2.2. Water
• Total body water depends on age, size and body composition
• In the average healthy subject, 60% of body weight in an adult, 70-
80% neonate and premature infant respectively
• Total body water varies inversely with fat content of the body
• Water is an inorganic compound with the chemical formula of H 2O.
• All known forms of life depends on water.
• Water is vital both as a solvent and as an essential part of many
metabolic processes with in the body.
• In anabolism, water is removed from molecules through energy
requiring enzymatic chemical reactions.
• In catabolism, water is used to break bonds in order to generate
smaller molecules.
• Water is fundamental to photosynthesis and respiration.
• Water is also central to acid-base neutrality and enzyme function.
• The hydrogen bond network of water molecules confers special
properties on water that are important for sustaining life.
• Cohesion
• Adhesion
• High Specific Heat
• High Heat of Vaporization
Cohesion
• Attraction between particles of the same substance

• Results in Surface tension (a measure of the strength of water’s surface)
• Produces a surface film on water that allows insects to walk on the surface of
water.
Adhesion
• Attraction between two different substances.
• Water will make hydrogen bonds with other surfaces such as glass,
soil, plant tissues, and cotton.
• Capillary action-water molecules will “tow” each other
along in a thin glass tube.
High Heat of Vaporization
• Water has a high heat of vaporization, ie, the amount of heat needed to
convert from liquid to gas phase.
• In conjunction with its high heat capacity, this property allows water
to carry away heat efficiently as it evaporates, which accounts for the
cooling effects of perspiration.
Chapter three
proteins
Dr. Ambayehu (MD)
Objectives:
• Define amino acids.
• Discuss structure and classification of amino acids.
• Discuss structure, classification and function of proteins.
• Discuss Structure and function of hemoglobin and myoglobin.
• Discuss enzymology.
3.1 amino acids
• Amino acid are monomer unit (building blocks) of proteins.
• Although about 300 amino acids occur in nature, only 20 of them are
seen in human body.
• Each amino acid has :
• A basic amino group (-NH2)
• An acidic carboxyl group ( -COOH)
• A hydrogen atom (-H)
• A distinctive side chain (-R)
• All except glycine have L- and D- configurations (chiral)
• Only L-amino acids used in human proteins
• D- isomers -found only in microbes
• Most of the amino acids (except proline) are alpha amino acids, which
means that the amino group is attached to the same carbon atom to
which the carboxyl group is attached.
Classification of amino acids
• Amino Acids Can Be Classified based on :
1. Structure
2. Side chine
3. Metabolism
4. Nutritional requirement
1. Based on structure
A. Aliphatic amino acids:
1. Monoamino monocarboxylic acids:
• Simple amino acids: Glycine, Alanine
• Branched chain amino acids: Valine, Leucine, Isoleucine Hydroxyamino acids: Serine, Threonine
• Sulfur-containing amino acids: Cysteine, Methionine
• Amino acids with amide group: Asparagine, Glutamine
2. Monoamino dicarboxylic acids: Aspartic acid, Glutamic acid
3. Dibasic monocarboxylic acids: Lysine, Arginine
B. Aromatic amino acids: Phenylalanine, Tyrosine
C. Heterocyclic amino acids: Tryptophan, Histidine
D. Imino acid: Proline: Proline differs from other amino acids in that its side chain and α-
amino N form a rigid, five-membered ring structure. contributes to the formation of the
fibrous structure of collagen
E. Derived amino acids:
2. Based on Side Chain
A. Amino acids having non-polar side chains: Each of these amino acids has a
nonpolar side chain that does not gain or lose protons or participate in
hydrogen or ionic bonds.
• These include Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine
and Tryptophan.
• These groups are hydrophobic (water repellant) and lipophilic. Therefore, the parts of
proteins made up of these amino acids will be hydrophobic in nature.
B. Amino acids having uncharged or non-ionic polar side chains: Glycine,
Serine, Threonine, Cysteine, Tyrosine, Glutamine and Asparagine belong to
this group.
• These amino acids are hydrophilic in nature.
• (Tyrosine and Cysteine may show hydrophobic character when present in the interior of
the protein).
C. Amino acids having charged or ionic polar side chains
(hydrophilic):
a) Acidic amino acids: They have a negative charge on the R group at
physiologic ph: Aspartic acid and Glutamic acid. (Tyrosine is mildly
acidic).
b) Basic amino acids: They have a positive charge on the R group:
Lysine, Arginine and Histidine.
3. Based on Metabolism
A. Purely ketogenic: Leucine is purely ketogenic because it is
converted to ketone bodies.
B. Ketogenic and glucogenic: Lysine, Isoleucine, Phenylalanine,
Tyrosine and Tryptophan are partially ketogenic and partially
glucogenic. During metabolism, part of the carbon skeleton of these
amino acids will enter the ketogenic pathway and the other part to
glucogenic pathway
C. Purely glucogenic: All the remaining 14 amino acids are purely
glucogenic as they enter only into the glucogenic pathway
4. Based on Nutritional Requirements
A. Essential or indispensable: Isoleucine, Leucine, Threonine, Lysine,
Methionine, Phenylalanine, Tryptophan, and Valine are essential
amino acids.
• Their carbon skeleton cannot be synthesized by human beings and so
preformed amino acids are to be taken in food for normal growth.
• Normal growth and optimal health will not occur, if one such amino acid is
deficient in the diet.
B. Partially essential or Semi essential: Histidine and Arginine.
• Growing children require them in food. But they are not essential for the adult
individual.
C. Non-essential or Dispensable: Alanine, Asparagine, Aspartic acid,
Cysteine, Glutamine, Glutamic Acid, Glycine, Proline, Serine and
Tyrosine.
• Their carbon skeleton can be synthesized by the body.
• So we need not have to ingest these amino acids as such. However, they are
also required for normal protein synthesis.
D. Conditionally essential amino acids: Arginine, Glycine, Cysteine,
Tyrosine, Proline, Glutamine and Taurine.
• These amino acids are normally non-essential, but become essential during
times of physiological stress.
Sources and Uses of Amino Acids
• Sources:
• Proteins in the diet
• Turnover of endogenous proteins
• De novo biosynthesis(non-essential amino acids)
• Use:
• Protein synthesis
• Energy source
• For hormone synthesis like T3/T4, EP/NEP..
• Purine and pyrimidine synthesis
3.2. Classification of proteins
• Protein means primary or first (Greek: “proteios”) and are necessary
for life.
• Out of the total dry body weight, 3/4ths are made up of proteins.
• Proteins are used for body building; all the major structural and
functional aspects of the body are carried out by protein molecules.
• Proteins contain Carbon, Hydrogen, Oxygen and Nitrogen as the
major components while Sulfur and Phosphorus are minor
constituents.
• Nitrogen is characteristic of proteins. On an average, the nitrogen
content of ordinary proteins is 16% by weight.
• Proteins are made by polymerization of amino acids through peptide
bonds.
• Alpha carboxyl group of one amino acid reacts with alpha amino
group of another amino acid to form a peptide bond or CO-NH bridge.
• Even though there is no universally accepted classification system,
proteins may be classified on the basis of their
• Composition
• Solubility
• Shape
• Biological function and
• Nutritional Value
1. Based on composition:
A. Simple protein: Yields only amino acids and no other major

organic or inorganic hydrolysis products.
• Eg: Albumins, Globulins, Glutelins, albuminoids, histones, and

protamines.
B. Conjugated Proteins: Yields amino acids and other organic

and inorganic components
• E.g. Nucleoprotein: For storage & transmission of genetic information

e.g. Ribosomes, Chromosomes
• Lipoprotein: Primarily in the transport of lipids.
• Phosphoprotein: Esterified with phosphates by ser, thr or tyr
• e.g. Casein: bring phosphorous to growing infant
• Metalloprotein: Metal storage proteins (e.g. ferritin) and Metal

containing enzymes e.g SOD
• Glycoprotein: - Mostly Proteins destined for an extracellular location.
• e.g. Fibronectin (adhesive glycoproteins) and proteoglycans for ECM.
• IgG: circulate in plasma.
• Many glycosylated membrane proteins on extracellular segment.
• Blood group antigens
• Hemoproteins: Subclass of metalloproteins. Having heme prosthetic
group.
• Flavoproteins - Containing flavin (vitamin B2 derivative) E.g.

oxidoreductases
2. Based on solubility:
• Albumins: These proteins such as egg albumin and serum albumin are
readily soluble in water and coagulated by heat.
• Globulins: these proteins are present in serum, muscle and other
tissues and are soluble in dilute salt solution but sparingly in water.
• Histones: Histones are present in glandular tissues (thymus, pancreas
etc.) soluble in water; they combine with nucleic acids in cells and on
hydrolysis yield basic amino acids
3. Based on shape:
A. Fibrous proteins: In these protein, are coiled cross-linked polypeptide
chains, they are insoluble in water and highly resistant to enzyme
digestion
I. Collagens: the major protein of the connective tissue, insoluble in water, acids
or alkalis. But they are convertible to water-soluble gelatin, easily digestible by
enzymes.
II. Elastins: present in tendons, arteries and other elastic tissues, not convertible
to gelatin.
III. Keratins: protein of hair, nails etc.
B. Globular proteins: These are globular or ovoid in shape, soluble in
water and constitute the enzymes, oxygen carrying proteins, hormones
etc.
4. Based on their Biological Functions
• Proteins are sometimes described as the "workhorses" of the cell
because they do So many things Like:
• Enzymes:- kinases, transaminases etc.
• Storage proteins:- myoglobin, ferretin
• Regulatory proteins:- peptide hormones, DNA binding proteins
• Structural protein:- collagen, proteoglycan
• Protective proteins:- blood clotting factors, Immunoglobins,
• Transport protein:- Hemoglobin, plasma lipoproteins
• Contractile or motile Proteins:- Actin, tubulins
5. Based on Nutritional Value
• Nutritionally Rich Proteins: They are also called as complete proteins or first class
proteins.
• They contain all the essential amino acids in the required proportion.
• On supplying these proteins in the diet, children will grow satisfactorily.
• A good example is casein of milk.
• Incomplete Proteins: They lack one essential amino acid.
• They cannot promote body growth in children; but may be able to sustain the body weight in
adults.
• Proteins from pulses are deficient in methionine, while proteins of cereals lack in lysine. If both
of them are combined in the diet, adequate growth may be obtained.
• Poor Proteins: They lack in many essential amino acids and a diet based on these
proteins will not even sustain the original body weight. Zein from corn lacks
tryptophan and lysine
3.3. Conformation of proteins
• The spatial arrangement of atoms in a proteins called its conformation.
• The possible conformations of a protein include any structural state it
can achieve without breaking covalent bonds.
• Proteins have different levels of structural organization; primary,
secondary, tertiary and quaternary.
A.Primary Structure of Proteins
• The primary structure of a protein is defined by the linear sequences of amino acid
residues.
• Protein contain between 50 and 2000 amino acid residues.
• The amino acid composition of a peptide chain has a profound effect on its
physical and chemical properties of proteins.
• Protein rich in polar amino acids are more water soluble.
• Proteins rich in aliphatic or aromatic amino groups are relatively insoluble in
water and more soluble in cell membranes.
Primary structure of human insulin
B. Secondary Structure
• The secondary structure of a protein refers to the local structure of a
polypeptide chain, which is determined by Hydrogen bond.
• The Interactions are between the carbonyl oxygen group and the
amide hydrogen of near by peptide bond.
• There are two types of secondary structure: The ∝-helix and The β-
pleated sheet
∝-helix
• The α-helix is a rod like structure with peptide chains tightly coiled.
• The side chains of the component amino acids extend outward from
the central axis.
• Each amide carbonyl group is hydrogen bonded to the amide hydrogen
of a peptide bond that is 4 - residues away along the same chain.
• There are 3.6 amino acids residues per turn of the helix.
• A very diverse group of proteins contains α-helices. For example,
keratin, myoglobin etc.
• The α-helix is disrupted by proline residues, because its secondary
amino group is not geometrically compatible with the right-handed
spiral of the α-helix.
• Large numbers of charged amino acids (like, glutamate, aspartate,
histidine, lysine, or arginine) also disrupt the helix by forming ionic
bonds, or by electrostatically repelling each other.
• Amino acids with bulky side chains, such as tryptophan, or branched
chain amino acids, such as valine or isoleucine, can interfere with
formation of the α-helix if they are present in large numbers.
ß- pleated sheet
• β-Sheet structures are made from highly extended polypeptide chains
that link together by hydrogen bonds between the neighboring strands.
• The backbone of the polypeptide chain is extended into a zigzag rather
than helical structure.
• The zigzag polypeptide chains can be arranged side by side to form a
structure resembling a series of pleats.
• In this arrangement, called a sheet, hydrogen bonds are formed
between adjacent segments of polypeptide chain.
• The adjacent polypeptide chains in a sheet can be either parallel or anti-parallel.
• Unlike the α-helix, β-sheets are composed of two or more peptide chains.
• In β-sheets the hydrogen bonds formed are interchain and perpendicular to the
polypeptide backbone.
c. Tertiary structure
• Is formed by combinations of secondary structural elements into a
three-dimensional organization.
• It is mainly stabilized by non-covalent interactions.
• Formed and stabilized by :
• Hydrophobic and hydrophilic interactions.
• Salt bridges.
• Hydrogen bonds.
• Disulfide bonds.
D. Quaternary structure
• Quaternary structure refers to a complex or an assembly of two or
more separate peptide chains.
• In most cases, as in hemoglobin, the subunits are held together by non-
covalent interactions.
• In some multi subunit proteins, such as immunoglobulins, the subunits
are held together by disulfide bonds or other covalent interactions.
• If the subunits are identical, it is a homogeneous quaternary structure;
but if there are dissimilarities, it is heterogeneous.
3.4. Myoglobin and hemoglobin
• Hemeproteins are a group of specialized proteins that contain heme as

a tightly bound prosthetic group.
• The role of the heme group is dictated by the environment created by
the three dimensional structure of the protein.
• For example, the heme group of a cytochrome functions as an electron
carrier that is alternately oxidized and reduced.
• In contrast, the heme group of the enzyme catalase is part of the active
site of the enzyme that catalyzes the breakdown of hydrogen peroxide.
• In hemoglobin and myoglobin, the two most abundant hemeproteins
in humans, the heme group serves to reversibly bind oxygen.
Structure and function of myoglobin
• Myoglobin, a hemeprotein present in heart and skeletal muscle,
functions both as a reservoir for oxygen and as an oxygen carrier that
increases the rate of transport of
oxygen within the muscle cell.
• Myoglobin consists of a single polypeptide chain that is structurally
similar to the individual polypeptide chains of the tetrameric
hemoglobin molecule.
• This homology makes myoglobin a useful model for interpreting some
of the more complex properties of hemoglobin.
• α-Helical content: Myoglobin is a compact molecule, with
approximately 80% of its polypeptide chain folded into eight stretches
of α-helix.
• Location of polar and nonpolar amino acid residues: The interior
of the myoglobin molecule is composed almost entirely of nonpolar
amino acids.
• They are packed closely together, forming a structure stabilized by
hydrophobic interactions between these clustered residues.
• In contrast, polar amino acids are located almost exclusively on the surface,
where they can form hydrogen bonds, both with each other and with water.
• Binding of the heme group: The heme group of the myoglobin
molecule sits in a crevice, which is lined with nonpolar amino
acids.
• Notable exceptions are two histidine residues. One, the proximal
histidine (F8), binds directly to the iron of heme.
• The second, or distal histidine (E7), does not directly interact with the
heme group but helps stabilize the binding of oxygen to the ferrous
iron.
• The protein, or globin, portion of myoglobin thus creates a special
microenvironment for the heme that permits the reversible binding of
one oxygen molecule (oxygenation).
• The simultaneous loss of electrons by the ferrous iron (oxidation to the
07/22/2024
ferric form) occurs only rarely.
Dr Ambayehu 76
A. Model of myoglobin showing helices A to H. B. Schematic diagram of the
oxygen-binding site of myoglobin.
Structure and function of hemoglobin
• Hemoglobin is found exclusively in red blood cells (RBC), where its main function is
to transport oxygen (O2) from the lungs to the capillaries of the tissues.
• Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide
chains (two α chains and two β chains) held together by noncovalent interactions.
• Each chain (subunit) has stretches of α-helical structure and a hydrophobic heme-
binding pocket similar to that described for myoglobin.
• However, the tetrameric hemoglobin molecule is structurally and functionally more
complex than myoglobin.
• For example, hemoglobin can transport H+ and CO2 from the tissues to the lungs and
can carry four molecules of O2 from the lungs to the cells of the body.
• Furthermore, the oxygen binding properties of hemoglobin are regulated by
interaction with allosteric effectors.
A. Structure of hemoglobin showing the polypeptide backbone. B. Simplified
drawing showing the helices.
• Quaternary structure of hemoglobin: The hemoglobin tetramer can be
envisioned as being composed of two identical dimers, (αβ)1 and (αβ)2.
• The two polypeptide chains within each dimer are held tightly together primarily by
hydrophobic interactions. In this instance, hydrophobic amino acid residues are
localized not only in the interior of the molecule, but also in a region on the surface of
each subunit.
• Multiple interchain hydrophobic interactions form strong associations between α-
subunits and β-subunits in the dimers. In contrast, the two dimers are held together
primarily by polar bonds.
• The weaker interactions between the dimers allows them to move with respect to one
other.
• This movement results in the two dimers occupying different relative positions in
deoxyhemoglobin as compared with oxyhemoglobin. The binding of O2 to the heme
iron pulls the iron into the plane of the heme. Because the iron is also linked to the
07/22/2024 proximal histidine (F8), there is movement
Dr Ambayehu of the globin chains that alters the interface
80
between the αβ dimers.
a) T form: The deoxy form of hemoglobin is called the “T,” or taut
(tense) form.
• In the T form, the two αβ dimers interact through a network of ionic bonds and
hydrogen bonds that constrain the movement of the polypeptide chains.
• The T conformation is the low-oxygen-affinity form of hemoglobin.
b) R form: The binding of O2 to hemoglobin causes the rupture of
some of the polar bonds between the αβ dimers, allowing movement.
• This leads to a structure called the “R,” or relaxed form.
• The R conformation is the high-oxygen-affinity form of hemoglobin.
Binding of oxygen to myoglobin and
hemoglobin
• Myoglobin can bind only one molecule of O2, because it contains
only one heme group.
• In contrast, hemoglobin can bind four O2 molecules, one at each of
its four heme groups.
• The degree of saturation (Y) of these oxygen-binding sites on all
myoglobin or hemoglobin molecules can vary between zero (all
sites are empty) and 100% (all sites are full.
• Pulse oximetry is a noninvasive, indirect method of measuring the
O2 saturation of arterial blood based on differences in light
absorption by oxyhemoglobin and deoxyhemoglobin.
• Oxygen-dissociation curve: A plot of Y measured at different partial
pressures of oxygen (pO2) is called the oxygen-dissociation curve.
• This graph illustrates that myoglobin has a higher oxygen
affinity at all pO2 values than does hemoglobin.
• The partial pressure of oxygen needed to achieve half-saturation
of the binding sites (P50) is approximately 1 mm Hg for
myoglobin and 26 mm Hg for hemoglobin.
• The higher the oxygen affinity (that is, the more tightly oxygen
binds), the lower the P50.
• The oxygen-dissociation curve for myoglobin has a hyperbolic
shape. This reflects the fact that myoglobin reversibly binds a
single molecule of oxygen.
• Myoglobin is designed to bind oxygen released by hemoglobin at the
low pO2 found in muscle.
• Myoglobin, in turn, releases oxygen within the muscle cell in response
to oxygen demand.
• The oxygen-dissociation curve for hemoglobin is sigmoidal in shape,
indicating that the subunits cooperate in binding oxygen.
Allosteric effects
• The ability of hemoglobin to reversibly bind oxygen is affected by the
pO2(through heme–heme interactions), the pH of the environment, the
partial pressure of carbon dioxide (pCO2) and the availability of 2,3-
bisphosphoglycerate.
• These are collectively called allosteric (“other site”) effectors, because
their interaction at one site on the hemoglobin molecule affects the
binding of oxygen to heme groups at other sites on the molecule.
• Heme–heme interactions: The sigmoidal oxygen-dissociation curve
reflects specific structural changes that are initiated at one heme group
and transmitted to other heme groups in the hemoglobin tetramer.
• The net effect is that the affinity of hemoglobin for the last oxygen
bound is approximately 300 times greater than its affinity for the first
oxygen bound.
• The cooperative binding of oxygen allows hemoglobin to deliver more
oxygen to the tissues in response to relatively small changes in the
partial pressure of oxygen.
• Bohr effect: The release of oxygen from hemoglobin is enhanced
when the pH is lowered or when the hemoglobin is in the presence of
an increased pCO2. NB: lungs having a higher pH and tissues a lower
pH.
• Both result in a decreased oxygen affinity of hemoglobin and,
therefore, a shift to the right in the oxygen-dissociation curve and
both, then, stabilize the T (deoxy) form.
• The concentration of both H+ and CO2 in the capillaries of
metabolically active tissues is higher than that observed in alveolar
capillaries of the lungs, where CO2 is released into the expired air.
• In the tissues, CO2 is converted by carbonic anhydrase to carbonic
acid:
CO2 + H2O H2CO3
• Which spontaneously loses a proton, becoming bicarbonate (the major
blood buffer): H2CO3 HCO3– + H+
• The H+ produced by this pair of reactions contributes to the lowering
of pH.
• The Bohr effect reflects the fact that the deoxy form of hemoglobin
has a greater affinity for protons than does oxyhemoglobin.
• An increase in the concentration of protons (resulting in a decrease in
pH) or a lower pO2 causes these groups to become protonated
(charged) and able to form ionic bonds (salt bridges).
• These bonds preferentially stabilize the deoxy form of hemoglobin,
producing a decrease in oxygen affinity. [Note: Hemoglobin, then, is
an important blood buffer.]
• Effect of 2,3-bisphosphoglycerate on oxygen affinity: It is the most
abundant organic phosphate in the RBC, where its concentration is
approximately that of hemoglobin.
• 2,3-BPG decreases the O2 affinity of hemoglobin by binding to
deoxyhemoglobin but not to oxyhemoglobin. This preferential
binding stabilizes the T conformation of deoxyhemoglobin.
• The concentration of 2,3-BPG in the RBC increases in response to
chronic hypoxia. Which lower the oxygen affinity of hemoglobin,
permitting greater unloading of oxygen in the capillaries of the tissues.
• Under physiologic conditions, HbF has a higher affinity for oxygen than
does HbA as a result of HbF only weakly binding 2,3-BPG.
• The γ-globin chains of HbF lack some of the positively charged amino
acids that are responsible for binding 2,3-BPG in the β-globin chains.
• The higher oxygen affinity of HbF facilitates the transfer of oxygen from
the maternal circulation across the placenta to the RBC of the fetus.
• Binding of CO2: Most of the CO2 produced in metabolism is
hydrated and transported as bicarbonate ion. However, some CO 2 is
carried as carbamate bound to the N-terminal amino groups of
hemoglobin forming carbaminohemoglobin.
• The binding of CO2 stabilizes the T or deoxy form of hemoglobin,

resulting in a decrease in its affinity for oxygen and a right shift in the
oxygen dissociation curve.
Carbon Monoxide poisoning
• Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin
iron, forming carboxyhemoglobin.
• When CO binds to one or more of the four heme sites, hemoglobin shifts
to the R conformation, causing the remaining heme sites to bind oxygen
with high affinity. This shifts the oxygen-dissociation curve to the left and
changes the normal sigmoidal shape toward a hyperbola. As a result, the
affected hemoglobin is unable to release oxygen to the tissues.
• The affinity of hemoglobin for CO is 220 times greater than for oxygen.
Consequently, even minute concentrations of CO in the environment can
produce toxic concentrations of carboxyhemoglobin in the blood.
• CO toxicity appears to result from a combination of tissue hypoxia and
direct CO-mediated damage at the cellular level.
• When 30-50% of Hb is saturated with CO, throbbing headache,
confusion and fainting are seen, and when the saturation reaches 80%,
the condition is rapidly fatal.
• CO poisoning is treated with 100% oxygen at high pressure
(hyperbaric oxygen therapy), which facilitates the dissociation of CO
from the hemoglobin.
HEMOGLOBINOPATHIES
• Defined as a group of genetic disorders caused by production of

hemoglobin with an altered amino acid sequence (qualitative
hemoglobinopathy: eg; Sickle cell anemia (HbS), hemoglobin C
disease (HbC), hemoglobin SC disease) or decreased production of
normal hemoglobin (quantitative hemoglobinopathy; eg: thalassemias).
• Sickle cell anemia (hemoglobin S disease): is a genetic disorder of
the blood caused by a single nucleotide substitution in the gene for β-
globin.
• A molecule of HbS contains two normal α-globin chains and two
mutant β-globin chains (βS), in which charged glutamate at position six
has been replaced with nonpolar valine.
• Therefore, during electrophoresis
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at alkaline pH, HbS migrates more
Dr Ambayehu 98
slowly toward the anode (positive electrode) than does HbA.
• At low oxygen tension, deoxyhemoglobin S polymerizes inside
the RBC, forming a network of insoluble fibrous polymers that
stiffen and distort the cell, producing rigid, misshapen RBC.
• Sickle cell anemia is characterized by lifelong episodes of pain
(“crises”); chronic hemolytic anemia with associated
hyperbilirubinemia. and increased susceptibility to infections,
usually beginning in infancy.
• The lifetime of a RBC in sickle cell anemia is less than 20 days,
compared with 120 days for normal RBC, hence, the anemia.
• Other symptoms include acute chest syndrome, stroke, splenic and
renal dysfunction, and bone changes due to marrow hyperplasia.
• Thalassemias: The thalassemias are hereditary hemolytic diseases
in which an imbalance occurs in the synthesis of globin chains.
• In the thalassemias, the synthesis of either the α- or the β-globin
chain is defective. A thalassemia can be caused by a variety of
mutations, including entire gene deletions, or substitutions or
deletions of one to many nucleotides in the DNA.
• β-Thalassemias: Synthesis of β-globin chains is decreased or absent.
• Excess α-globin chains cannot form stable tetramers and so precipitate,
causing the premature death of cells initially destined to become mature
RBC.
• α-Thalassemias: In these disorders, synthesis of α-globin chains is
decreased or absent, typically as a result of deletional mutations.
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Enzymes:
• Enzymes are protein catalysts that increase the rate of reactions
without being changed in the overall process.
• Although chemical process in the body (biological systems) can be
thermodynamically favorable i.e. can take place spontaneously, the
rate is very slow to meet the cellular demand and hence enzymes are
very crucial in accelerating cellular reactions.
• A bag of sugar can be stored for years, but it releases its chemical
energy in seconds in human body.
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• substrate active site products
enzyme
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PROPERTIES:
• Enzyme molecules contain a special pocket or cleft called the active site.
• The active site, formed by folding of the protein, contains amino acid
side chains that participate in substrate binding and catalysis.
• Binding is thought to cause a conformational change in the enzyme
(induced fit model) that allows catalysis.
• Enzyme-catalyzed reactions are highly efficient, proceeding from 103–
108 times faster than uncatalyzed reactions.
• Enzymes are highly specific, interacting with one or a few substrates and
catalyzing only one type of chemical reaction. The set of enzymes made
in a cell determines which reactions occur in that cell.
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• Holoenzyme refers to the active enzyme with its nonprotein component.
• Apoenzyme is enzyme without its nonprotein moiety and is inactive.
• Cofactor is an enzyme with non protein moiety is a metal ion, such as
Zn2+ or Fe2+.
• Coenzyme is an enzyme with non protein moiety is small organic
molecule.
• Coenzymes that only transiently associate with the enzyme are called
cosubstrates.
• Enzyme activity can be regulated, that is, increased or decreased, so that
the rate of product formation responds to cellular need.
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• Do not consume themselves: no changes in quantity and quality
before and after the reactions.
• Do not change the equilibrium points /Have no effect on Keq/ : only

enhance the reaction rates.
• Apply to the thermodynamically allowable reactions/ do not change

free energy (∆G) of a reaction/.
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• Reduce the activation energy .
General mechanisms of action
• Can be viewed from two different perspectives:

A. Energy changes occurring during the reaction: Virtually all
chemical reactions have an energy barrier separating the reactants
and the products. This barrier, called the free energy of activation,
is the energy difference between that of the reactants and a high-
energy intermediate that occurs during the formation of product.
• The lower the free energy of activation, the more molecules have
sufficient energy to pass through the transition state, and, therefore,
the faster the rate of the reaction.
• An enzyme allows a reaction to proceed rapidly under conditions
prevailing in the cell by providing an alternate reaction pathway with
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B. Chemistry of the active site: The active site is not a passive
receptacle for binding the substrate but, rather, is a complex molecular
machine employing a diversity of chemical mechanisms to facilitate the
conversion of substrate to product.
• The active site often acts as a flexible molecular template that binds
the substrate and initiates its conversion to the transition state, a
structure in which the bonds are not like those in the substrate or the
product.
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• Three models have been proposed to describe the manner in which
specific binding occurs:
• Lock-and-key model: the enzyme is considered to have an active
center complementary to the shape of the substrate it acts upon; and
thus part or all of the substrate fits in to the active site in the same
manner as the key matches a lock; a process that confers the
specificity to the enzyme.
• Gives no account for how enzymes increase the rate of a reactios.
KE
Y
LOC
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• Induced-fit model (Daniel kosland, in 1958): The enzyme changes the
shape of its active center during the substrate binding process to assume
a shape that correctly fits the substrate molecule structure; i.e., after the
binding is accomplished the enzyme takes on a complimentary shape to
that of the substrate.
• Even though this model explains well about the interaction of enzyme
and its substrate which is still acceptable it again fails to explain how
enzymes increase the rate of a given reaction
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• Michaelis and menten model (1913): Supports structural
modification of the enzymes active site and also tries to explain the
relationship between enzymes and activation energy of a reaction.
E-S complex
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FACTORS AFFECTING REACTION
VELOCITY
• Substrate concentration: The rate of an enzyme-catalyzed reaction increases
with substrate concentration until a maximal velocity (Vmax) is reached.
• The leveling off of the reaction rate at high substrate concentrations reflects
the saturation with substrate of all available binding sites on the enzyme
molecules present.
• Temperature: The reaction velocity increases with temperature until a peak
velocity is reached.
• Further elevation of the temperature causes a decrease in reaction velocity as
a result of temperature induced denaturation of the enzyme.
• The optimum temperature for most human enzymes is between 35°C and
40°C.
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• pH: The catalytic process usually requires that the enzyme and substrate have
specific chemical groups in either an ionized or un-ionized state in order to
interact.
• For example, catalytic activity may require that an amino group of the enzyme
be in the protonated form (–NH3+). At alkaline pH, this group is deprotonated,
and the rate of the reaction, therefore, declines.
• Extremes of pH can also lead to denaturation of the enzyme, because the
structure of the catalytically active protein molecule depends on the ionic
character of the amino acid side chains.
• The pH at which maximal enzyme activity is achieved is different for different
enzymes and often reflects the [H+] at which the enzyme functions in the body.
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INHIBITION OF ENZYME ACTIVITY
• Any substance that can decrease the velocity of an enzyme-catalyzed reaction is
called an inhibitor. It can be reversible or irreversible.
• Irreversible inhibitors bind to enzymes through covalent bonds.
• An important group of irreversible inhibitors are the mechanism-based
inhibitors that are converted by the enzyme itself to a form that covalently links
to the enzyme, thereby inhibiting it. They also are referred to as “suicide”
inhibitors.
• Reversible inhibitors bind to enzymes through noncovalent bonds and, thus,
dilution of the enzyme–inhibitor complex results in dissociation of the
reversibly bound inhibitor and recovery of enzyme activity.
• The two most commonly encountered types of reversible inhibition are
competitive and noncompetitive.
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• Competitive inhibition: This type of inhibition occurs when the inhibitor
binds reversibly to the same site that the substrate would normally occupy
and, therefore, competes with the substrate for that site.
• The effect of a competitive inhibitor is reversed by increasing [S]. At a
sufficiently high substrate concentration, the reaction velocity reaches the
Vmax observed in the absence of inhibitor.
• A competitive inhibitor increases the apparent Km for a given substrate. This
means that, in the presence of a competitive inhibitor, more substrate is
needed to achieve 1⁄2V max.
• Statin drugs as examples of competitive inhibitors. This group of
antihyperlipidemic agents competitively inhibits the rate-limiting (slowest)
step in cholesterol biosynthesis.
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• Noncompetitive inhibition: Occurs when the inhibitor and substrate
bind at different sites on the enzyme.
• The noncompetitive inhibitor can bind either free enzyme or the
enzyme-substrate complex, thereby preventing the reaction from
occurring.
• Noncompetitive inhibition cannot be overcome by increasing the
concentration of substrate. Therefore, noncompetitive inhibitors
decrease the apparent Vmax of the reaction.
• Noncompetitive inhibitors do not interfere with the binding of
substrate to enzyme. Therefore, the enzyme shows the same Km in the
presence or absence of the noncompetitive inhibitor.
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REGULATION OF ENZYME ACTIVITY
• The regulation of the reaction velocity of enzymes is essential if

an organism is to coordinate its numerous metabolic processes.
• Regulation of allosteric enzymes: Allosteric enzymes are
regulated by molecules called effectors that bind noncovalently at
a site other than the active site.
• Homotropic effectors: The presence of a substrate molecule at one site
on the enzyme enhances the catalytic properties of the other substrate-
binding sites.
• Most often, an allosteric substrate functions as a positive effector.
• Heterotropic effectors: The effector may be different from the substrate.
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• Regulation of enzymes by covalent modification: Many enzymes are regulated
by covalent modification, most often by the addition or removal of phosphate
groups from specific serine, threonine, or tyrosine residues of the enzyme.
• Phosphorylation reactions are catalyzed by a family of enzymes called protein
kinases that use ATP as the phosphate donor.
• Phosphate groups are cleaved from phosphorylated enzymes by the action of
phosphoprotein phosphatases.
• Depending on the specific enzyme, the phosphorylated form may be more or less
active than the unphosphorylated enzyme.
• For example, phosphorylation of glycogen phosphorylase (an enzyme that degrades
glycogen) increases activity, whereas phosphorylation of glycogen synthase (an
enzyme that synthesizes glycogen) decreases activity
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• Induction and repression of enzyme synthesis: Cells can also regulate the
amount of enzyme present by altering the rate of enzyme degradation or, more
typically, the rate of enzyme synthesis. The increase (induction) or decrease
(repression) of enzyme synthesis leads to an alteration in the total population
of active sites.
• Enzymes subject to regulation of synthesis are often those that are needed at
only one stage of development or under selected physiologic conditions.
• In contrast, enzymes that are in constant use are usually not regulated by
altering the rate of enzyme synthesis.
• Alterations in enzyme levels as a result of induction or repression of protein
synthesis are slow (hours to days), compared with allosterically or covalently
regulated changes in enzyme activity, which occur in seconds to minutes.
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The end!
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Chapter Four
Carbohydrates
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Objectives:
• Define carbohydrates in chemical terms
• Identify structural & functional roles of carbohydrates
• Classify carbohydrates in to their major classes with examples of each group
• Describe the structural & biochemical properties and functions of starch,
glycogen, cellulose and heteropolysaccarides.
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Introduction
• Carbohydrates are polyhydroxy aldehydes or ketones, or substances that
yield such compounds on hydrolysis.
• Many, but not all, have the empirical formula (C n(H 2O)n; n≥ 3
• However, only the simple sugars (monosaccharides) like glucose
(C6H1206 ), fit this formula exactly.
• Some also contain N, P, or S
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Carbohydrates have important Biological
roles:
• Energy/storage
• Structural and protective elements
• Cellulose of plants; exoskeleton of insects, cell wall of
microorganisms, mucopolysaccharides as ground substance
in higher organisms
• Other carbohydrate polymers
• Lubricate skeletal joints
• Recognition & adhesion b/n cells
• Glycoconjugates: Act as signals that determine the intracellular
location or metabolic fate of these hybrid.
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• Structural components of nucleic acids
• Metabolic precursors of all other biomolecules, proteins , nucleotides,
coenzymes, fatty acids..
• Glycerole can be considered the parent cpd, although it is not a CHO.
• It can be oxidized in to:
• Aldose, R-CH=O (glyceraldehyde) or
• Ketose, -C=O- (dihydroxy acetone)b
• These form the basis for all CHO.
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Classification of Carbohydrates
 According to the number of sugar units and the size of the molecule:
• Monosaccharides (simple sugars):
• Simplest form of CHO, which cannot be further hydrolyzed.
• They represent the building units and hydrolytic end products of the more
complex carbohydrates.
• Oligosaccharides:
• These are the conjugates of CHO where 2-10 monosaccharide units are linked
to each other.
• Polysaccharides:
• These are higher polymers of carbohydrates and contain more than 10
monosaccharide units per molecule
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Monosaccharides
• Monosaccharides vary from trioses to heptoses.

• Can be an aldose (-CHO) or a ketose (-C=O).
• All monosaccharides contain at least one asymmetrical (chiral)
carbon, (except DHA).– therefore, optically active
• Monosaccharides exist in either of the two conformations, D or L.
• Monosaccharides shows reducing property.
• Most have a sweet taste
• Most monosaccharides of >4 C’s tend to have Cyclic structures
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• Important monosaccharides:
1. D-glyceraldehyde and dihydroxyacetone: are aldo- and keto-trioses,
respectively.
• Intermediary compounds in carbohydrate and lipid metabolism.
D-Glyceraldehyde Dihydroxyacetone
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2. D-Ribose: is the most important pentose
• It is a component of the structures of RNA, DNA and important free
nucleotides (ATP).
• It is a reducing aldo-sugar synthesized in the body from glucose.
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3. Glucose (dextrose ): is the grape and blood sugar.
• It is fermentable and is a reducing aldo-sugar.
• It is the building unit and end product of hydrolysis of starch, dextrin, glycogen, sucrose,
maltose and lactose.
• It is the major body sugar; all monosaccharides ingested may be converted into glucose in the
body
• Inborn and acquired impairment of glucose metabolism is associated with a number of human
diseases, such as diabetes mellitus.
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4. Mannose: is reducing aldo-sugar that is a subunit in in
glycoproteins and neuraminic acid.
5. Fructose : is reducing keto-sugar and is the sweetest sugar known.
• It is the main sugar in bee's honey and fruits.
• It is obtained from inulin and sucrose hydrolysis.
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Physical and chemical Properties of
monosaccharides
• Isomers and epimers
• Compounds that have the same chemical formula but have different structures
are called isomers. For example, fructose, glucose, mannose, and galactose
are all isomers of each other, having the same chemical formula, C 6H12O6.
• Carbohydrate isomers that differ in configuration around only one specific
carbon atom (with the exception of the carbonyl carbon) are defined as
epimers of each other. For example, glucose and galactose are C-4 epimers
because their structures differ only in the position of the –OH group at carbon
4.
• Enantiomers
• A special type of isomerism is found in the pairs of structures that are mirror
images of each other. These mirror images are called enantiomers, and the
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two members of the pair are designated
Dr Ambayehu
as a D- and an L-sugar. 133
• Cyclization of monosaccharides: Less than 1% of each of the
monosaccharides with five or more carbons exists in the open-chain
(acyclic) form in solution. Rather, they are predominantly found in a ring
(cyclic) form, in which the aldehyde (or keto) group has reacted with an
alcohol group on the same sugar, making the carbonyl carbon (carbon 1 for
an aldose, carbon 2 for a ketose) asymmetric.
• This asymmetric carbon is referred to as the anomeric carbon.
• Anomers: Creation of an anomeric carbon (the former carbonyl carbon), generates a
new pair of isomers, the α and β configurations of the sugar (for example, α-
Dglucopyranose and β-D-glucopyranose), that are anomers of each other. [Note: In
the α configuration, the –OH group on the anomeric carbon projects to the same side
as the ring in a modified Fischer projection formula and is trans to the CH2OH group
in a Haworth projection formula.
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• The α and β forms are not mirror images, and they are referred to as diastereomers.
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• Reducing sugar: If the hydroxyl group on the anomeric carbon of a cyclized
sugar is not linked to another compound by a glycosidic bond, the ring can open.
• The sugar can act as a reducing agent and is termed a reducing sugar.
• Such sugars can react with chromogenic agents (for example, the Benedict
reagent) causing the reagent to be reduced and colored, with the aldehyde
group of the acyclic sugar becoming oxidized.
• All monosaccharides, but not all disaccharides, are reducing sugars.
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Oligosaccharides
• Disaccharides contain two monosaccharides joined together by O-
glycosidic linkage.
• The most common glycosidic linkage is between C1 of one and C4 of
the other monosaccharide.
• The linkage can be a- or β- depending upon the type of the anomeric
carbon participating in the linkage.
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• Maltose (malt sugar): Consists of an a-glucose units connected by a-
1,4-glucosidic linkage.
• It is produced by partial acid or enzymatic (amylase) hydrolysis of dietary
starch and glycogen.
• It is hydrolyzed into two glucose molecules by HCl or by the intestine maltase
enzyme.
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• Lactose (milk sugar): It is formed of ß-galactose and glucose
molecule linked by ß- 1,4-glycosidic linkage.
• It is hydrolyzed by HCl or by intestine enzyme, lactase, into galactose and
glucose.
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• Lactose is the most suitable sugar as a milk sweetener for baby
feeding because:
• It has the lowest degree of sweetness that delays loss of the baby appetite.
• It is a mild laxative and helps preventing constipation
• It is not an irritant to the stomach and helps preventing vomiting
• It facilitates absorption of milk minerals
• The unabsorbed sugar is used as a fuel for large intestinal bacteria that
produces some vitamins
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• Sucrose: It is the major cane and beet sugar, commonly named as table sugar.
• It is formed of a-glucose linked to ß-fructose by a-ß-1,2-glycosidic linkage.
• Hydrolyzed by the intestinal sucrase enzyme or by HCl, to produce fructose
and glucose.
• Although used as sweetener in most of the food preparations, sucrose is not
the sweetest of them all.
• Fructose is almost twice as sweet as sucrose.
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• DM patients and people on weight reduction protocols avoid sucrose
as sweetener.
• Most of the artificial sweeteners commonly known as ‘Sugar Free’
contain aspartame, which is a dipeptide L aspartyl-L-phenylalanine
methyl ester.
• Aspartame is also added to the beverages marketed as ‘low caloric’ or
‘Diet drinks’.
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Polysaccharides
• These are high molecular weight polymers of monosaccharides and
are the major form of carbohydrates occurring in nature.
• Classified into two categories according to the type of the building
units:
• Homopolysaccharides that are composed of one type of D monosaccharide or
D monosaccharide derivatives, and
• Heteropolysaccharides that are composed of more than one type of D
monosaccharides and their derivatives.
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• A) Homopolysaccharides
• Starch: It is the major storage and nutritional form of plant
carbohydrates.
• The starch granules are composed of two types of polysaccharides; amylase
and amylopectin.
I. Amylose - composed of a-glucose units linked through a-1,4- glucosidic
linkages and represents 20% of the granule.
II. Amylopectin - The outer shell (representing 80% of the granule) is made up
of the amylopectin .
• Is a glucose polymer with mainly a-1,4- glucosidic linkages, but it also has
branches formed by a-1,6-glucosidic linkages at the branch points .
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• Glycogen: It is the major storage form of CHO in most of the
animal cells, particularly in muscles and liver.
• Since it is the starch equivalent in animals, glycogen is also called
‘animal starch’.
• It is a branched chain glucosan formed by a-glucose units linked
through a-1,4 and a-1,6-glucosidic linkages resembling amylopectin.
• But glycogen has more a-1,6-glucosidic branches; on average, every 8-
12 residues & more compact than starch
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• Cellulose: a major constituent of plant cell walls.
• Consists of long linear chains of ß-glucose with ß-1,4- glucosidic linkage.
• It is water-insoluble straight chain glucosan composed of ß glucose units linked
by ß-1,4-glucosidic linkage.
• Although it is indigestible in human, it offers very important benefits as dietary
fibers:
• Dietary fibers prevents constipation by giving stool its bulkiness and increasing peristalsis
• Adsorbs endogenous and exogenous toxins and prevents their absorption into the body
including bile acids and cholesterol.
• This help lowering blood cholesterol.
• Its fermentation by large intestinal bacteria gives volatile fatty acids (particularly butyric acid)
that are strong anticancer agents against colorectal cancer.
• These large intestinal bacteria also synthesize some water soluble vitamins.
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• Dextran: It is a highly branched meshwork glucosan composed of a-
glucose units linked by a- 1,4, a- 1,3-and a-1,6-glucosidic linkages.
• Therapeutic Applications of Dextran:
• Dextran forms a colloidal solution in water and is used as a plasma substitute
(expander) to restore blood pressure in cases of hemorrhagic shock.
• It stays in plasma for a longer time and retains intravascular water to maintain
plasma volume.
• Dextran ferrous sulfate is a suitable form for intramuscular injection
of iron for treatment of iron deficiency anemia.
• Sodium dextran sulfate (SDS) is an anticoagulant.
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B. Heteropolysaccharides: In contrast to the homopolysaccharides,
heteropolysaccharides are composed of a mix of several types of
monosacchrides and/or their derivatives.
• Could be unbranched (when contain 2 types subunits) or branched (when
composed of more than 2 types of branched monosaccharide subunits).
• They provide protection, shape, and extracellular support for cells, tissues, and
organs for organisms starting from bacteria to the extracellular matrix of
different tissues.
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• They are of two types:
• Non-nitrogenous heteropolysaccharides, that do not contain
sugaramines. Eg. Plant gums and mucilages, Pectins, Agar
• Nitrogenous heteropolysaccharides, that contain sugaramines. Eg.
Glycoproteins, Glycosaminoglycans
• Blood group substances: The glycoprotein on the surface of red
cells are used for the classification of the blood groups.
• Glucosamine derivative is part of many structural polymers,
including those of the bacterial cell wall.
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The end!
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Chapter Five
Lipids
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Objectives:
• Define and classify fatty acids.
• Define and classify lipids.
• Explain the importance of lipids.
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Fatty acids
• Fatty acids are carboxylic acids with hydrocarbon chains ranging from 4
to 36 carbons long, usually C12- C20 have the general formula, R—CO—
OH, where COOH (carboxylic group) represents the functional group.
• Fatty acids, are included in the group of derived lipids. It is the most
common component of lipids in the body.
• They are generally found in ester linkage in different classes of lipids.
• In the human body, free fatty acids are formed only during metabolism.
• Fatty acids occur mainly as esters
• Free FA (unesterified ) are few in nature
• At physiological pH = FA are ionized
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• With a few exceptions, natural fatty acids:
• Contain an even number of carbon atoms
• Arranged in an un-branched line
• Have a carboxyl group (-COOH) at one end and Have a methyl group (CH3)
at the other end
• Odd chain fatty acids have 3, 5, 7, etc carbon atoms, are seen in
microbial cell walls and in milk.
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Classification of fatty acids
• Depending on the length of the hydrocarbon chain, there are
• Short chain fatty acids with 2 to 6 carbon atoms,
• Medium chain fatty acids, with 8 to 12 carbon atoms and
• Long chain fatty acids with 14 to 20 carbon atoms.
• Very-long chain fatty acid - VLCFA > 20 carbon atoms
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• Based on The presence of double bonds in the chain:
• Saturated: They have the general formula CH 3-(CH 2) n-COOH
• They are named by adding the suffi ‘anoic' after the hydrocarbon.
• The two carbon acetic acid and 4 carbon butyric acid are important metabolic
intermediates.
• The C16 (palmitic acid) and C18 (stearic acid) are most abundant in body fat.
• Each animal species will have characteristic pattern of fatty acid composition.
Thus, human body fat contains 50% oleic acid, 25% palmitic acid, 10%
linoleic and 5% stearic acid.
• The carbon atoms of fatty acids are numbered as C1, C2, etc. starting from the
COOH group. Or, starting from the methyl end, the carbon atoms may be
numbered as omega (w)—1,2,3, etc.
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• Unsaturated (double bonds): Contain One or More Double Bonds
• They are named by adding the suffi ‘enoic' after the systematic name.
• They are similar to saturated fatty acids in the reaction of the carboxylic group
but also show properties due to presence of the double bond.
• Unsaturated fatty acids exhibit geometrical isomerism at the double bonds .
• All the naturally occurring fatty acids have the cis configuration. However, in
the body during metabolism trans fatty acids are formed.
• The polyunsaturated fatty acids (PUFA) exist in cis configuration in naturally
occurring lipids.
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• Monounsaturated (MUFA) acids, containing one double bond.
• Polyunsaturated (PUFA) acids, containing two or more double
bonds
• Eicosanoids: These compounds, derived from eicosa- (20-
carbon) polyenoic fatty acids, comprise the prostanoids,
leukotrienes (LTs), and lipoxins (LXs).
• Prostanoids include prostaglandins (PGs), prostacyclins (PGIs),
and thromboxanes (TXs).
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Nomenclature
• SATURATED: parent hydrocarbon + oic e.g. C18: Octadecanoic acid
• UNSATURATED: with one double bond: + enoic e.g. C18:1 Octadecenoic acid
• With two double bonds: + dienoic e.g.C18:2 Octadecadienoic acid
• With three double bonds: + trienoic e.g. C18:3 Octadecatrienoic acid
• Standard nomenclature: Carbon atoms are numbered as 1,2,3… and α, β, γ the
terminal methyl carbon is known as the n or ω-carbon
• Delta system: y:z ∆x y = number of C, z = number of double bonds and x =
location of double bonds
• n system – defined with respect to C18:2 ∆ 9,12 (n-6)
• For polyunsaturated fatty acids (PUFAs) assigning the number 1 to the methyl
carbon three series of fatty acids known as ω 9 , ω 6 and ω 3 series families.
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Physical Properties
• Chain Length and Degree of Unsaturation
• ↑chain length = ↑ Boiling point (BP) and melting point (MP)
• fully saturated compounds - pack tightly
• ↑ unsaturation = ↓MP
• unsaturated fatty acids – kink – weak interaction - lower melting points
• ↑ chain length = ↓ solubility
• The membrane lipids are more unsaturated than storage lipids.
• Fully saturated fatty acids in the extended form pack into nearly crystalline arrays
• The presence of one or more cis double bonds interferes with this tight packing and results in less
stable aggregates
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• Melting Point: The short and medium chain fatty acids are liquids,
whereas long chain fatty acids are solids at 25oC.
• The solubility in water decreases, while melting and boiling points increase
with increase in chain length.
• The unsaturated fatty acids have lower melting point compared to saturated
fatty acids with the same chain length. For example, stearic acid (C18 fatty
acid, no double bond) has the melting point 69oC, oleic acid (C18, 1 double
bond) has 13oC; linoleic acid (C18, 2 double bonds) has –5oC and linolenic
(C18, 3 double bonds) has –10oC
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• Salt Formation: Saturated and unsaturated fatty acids form salts with
alkali.
CH3—COOH + NaOH → CH 3—COONa + H 2O
• Sodium and potassium salts of long chain fatty acids are called soaps.
Calcium and magnesium soaps are insoluble.
• Calcium soaps are used in grease.
• Alkyl sulfate (R—CH 2—O—SO 2—ONa) and alkyl sulfonate (R—CH
2—SO 2—O—Na) are not precipitated by hard water and are used as
detergents.
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• Ester Formation; Both saturated and unsaturated fatty acids form
esters with alcohols, especially with glycerol. Fatty acids can form
mono-, di- or tri- esters with alcohol groups of glycerol.
• Triglycerides or triacylglycerols are also known as neutral fat.
Glycerol + fatty acid → Monoacylglycerol
Monoglyceride + fatty acid → Diacylglycerol
Diglyceride + fatty acid → Triglyceride or triacylglycerol
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• Hydrogenation: Unsaturated fatty acids may be converted to the corresponding
saturated fatty acids by hydrogenation of the double bond.
(+) 2H (+) 2H (+) 2H

• Linolenic → Linoleic → Oleic → Stearic
• Hydrogenation of oils can lead to solidification and saturation, e.g. Vanaspathi.
• Halogenation: When treated with halogens under mild conditions, the
unsaturated fatty acids can take up two halogen atoms, at each double bond to
form the halogenated derivative of the fatty acid.
• For example, Oleic acid + I 2 → Di-iodo oleic acid
• The number of halogen atoms taken up will depend on the number of double
bonds and is an index of the degree of unsaturation.
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Nutritionally essential fatty acids
• Mammals are unable to introduce double bonds beyond Δ 9 in the fatty acid
chain. Hence, they cannot synthesize either linoleic acid (18:2c Δ9, 12), or
α- linolenic acid (18:3c Δ9, 12, 15).These are called, nutritionally- essential
polyunsaturated fatty acids, because they are essential lipid components that
must be provided in diet.
• After ingestion in mammals, they are in turn substrates for further
desaturation and elongation reactions. Particularly important, is the pathway
which leads from linoleic acid to arachidonic acid (20: 4c Δ5, 8, 11, 14).
• Note: A deficiency of essential fatty acids is characterized by scaly
dermatitis, hair loss, and poor wound healing.
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• PUFAs (n-6 and n-3) are essential for animals
• In higher animals, only the desaturases are known which
generate double bonds at carbons 9, 6, 5, and 4.
• Fatty acids containing double bonds beyond C-9 (acids n-6 and
n-3) are synthesized by plants. They are essential dietary
constituents for animals and serve as precursors of eicosanoids
• Providing the dietary intake is sufficient (vegetable seed oils,
fish), linoleate and α-linolenate act as precursors of other
essential polyenoic acids such as arachidonate (n-6) and
eicosapentaenoate (n-3), from which eicosanoids are formed.
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Omega-3 Fatty Acids
• Group of polyunsaturated fatty acids
• Essential–must be obtained in the diet
• Component of cell membranes
• Mediate inflammation, regulate blood clotting and contraction/relaxation of
arterial walls
• May be helpful in relieving symptoms in rheumatoid arthritis and age related
macular -degeneration
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Omega-6 fatty acids
• Essential polyunsaturated fatty acid.
• Lower LDL cholesterol.
• Reduce inflammation .
• Protect against heart disease.
• Intake usually adequate due to vegetable oil intake.
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lipids
• Lipids are chemically heterogeneous group of substances having a
common property of:
• Insolubility in water,
• Solubility in non-aqueous solvents such as chloroform and ether
• The lipids include fats, oils, steroids, waxes, and related compounds,
that are related more by their physical than by their chemical
properties
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Major functions of lipid:
• Provide energy reserves, predominantly in the form of

triacylglycerols.
• Serve as structural components of biological membranes
(phospholipids and cholesterol)
• Metabolic regulators (steroid hormones and prostaglandins)
• Both lipids and lipid derivatives serve as vitamins and hormones
• Lipophilic bile acids aid in lipid solubilization.
• Nonpolar lipids act as electrical insulators in neurons.
• lipoproteins serve as the means of transporting lipids in the blood.
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• Act as surfactants, detergents and emulsifying agents (amphipathic
lipids)
• Provide insulation against changes in external temperature
(subcutaneous fat)
• Give shape and contour to the body.
• Protect internal organs by providing a cushioning effect (pads of fat)
• Help in absorption of fat soluble vitamins (A, D, E and K)
• Improve taste and palatability of food.
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CLASSIFICATION of LIPIDS
• Based on composition:
1. Simple lipid:- ester of fatty acids with various alcohols
I. Natural fats and oils (triglycerides)
II. Waxes
a) True waxes: acetyl alcohol esters of fatty acids
b) Cholesterol esters
c) Vitamin A esters
d) Vitamin D esters
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2. Compound lipids:- esters of fatty acids with alcohol plus other
groups
i. Phospholipids: contains phosphoric acid and often a nitrogenous base
ii. Glycerophospholipids:
iii. Sphingophospholipids:
iv. Sphingolipids (also include glycolipids and cerebrosides): contains amino-
alcohol sphingosine, carbohydrate, N-base; glycolipids contains no
phosphate
v. Sulfolipids:- contains sulfate group
vi. Lipoproteins:- lipids attached to proteins
vii. Lipopolysaccharides:- lipids attached to polysaccharides
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3. Precursor and Derived lipids: hydrolytic products of simple &
complex lipids, with lipid characters
i. Saturated & unsaturated fatty acids
ii. Monoglycerides and diglycerides
iii. Lipid-soluble vitamins, and hormones
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• Based on functional organization
1. Storage Lipids (80%)
2. Structural lipids in membranes (5-10%) Read
ing a
ssign
3. Lipids as signals, cofactors, and pigments m ent
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1. Storage Lipids
• The fats and oils used almost universally as stored forms of energy in
living organisms are derivatives of fatty acids.
• Two types of fatty acid–containing compounds, triacylglycerols and
waxes.
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1.1 Triacylglycerol
• The triacylglycerols are the storage form of lipids in the adipose tissue.
• In a 70 kg normal person, body stores contain about 11 kg of triacylglycerol,
which is roughly equivalent to 100,000 kcal.
• If the same calories were stored as hydrated glycogen, the total weight of this
alone would have been 65 kg! When stored as TAG, water molecules are repelled
and space requirement is minimal. Excess fat in the body leads to obesity
• Is Fatty Acid Esters of Glycerol.
• The simplest lipids constructed from fatty acids
• Three fatty acids each in ester linkage with a single glycerol
• Simple - containing the same kind of fatty acid e.g. tripalmitin
• Mixed - contain two or three different fatty acids
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Physical Properties of Triacylglycerols
• TAGs are nonpolar, hydrophobic molecules, essentially insoluble in water
• Lipids have lower specific gravities than water
• Solid / semisolid at room temp = fats (neutral fats); in animals;
• liquid = oils (neutral oils), in plants, fish
• Plant triglycerides = ↓MP, liquid at room temp(RT) due to ↑unsaturated FA
• Animal triglycerides = ↑saturated FA, ↑MP
• When the constituent fatty acids have a higher chain length and are
predominantly saturated, ‘hard fat' is formed, e.g. pig fat
• Fats containing medium chain triacylglycerols or unsaturated fatty acids are soft
fats, e.g. butter, coconut oil. Coconut oil contains mainly medium chain TAG,
e.g. Lauric and Myristic acids.
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Foods Containing Triacylglycerols
• Most natural fats, such as vegetable oils, dairy products, and animal
fat, are complex mixtures of simple and mixed TAG.
• These contain a variety of fatty acids differing in chain length and
degree of saturation
• Vegetable oils such as corn (maize) and olive oil are composed of
TAGs with unsaturated fatty acids and thus are liquids at room T.
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1.2 Waxes
• They form the secretions of insects, leaves and fruits of plants,

e.g. Lanolin or wool fat, beeswax, whalesperm oil, etc.
• They are esters of higher fatty acids with higher monohydroxy
aliphatic alcohols and so have very long straight chains of 60–
100 carbon atoms.
• They are used as the base for the preparation of cosmetics,
ointments, polishes, lubricants and candles.
• Generally Higher MP than TAGs
• Serve as Energy Stores and Water Repellents
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2. Structural Lipids in Membranes
• General types of membrane lipids
i. Glycerophospholipids
ii. Galactolipids and sulfolipids
iii. Archaeal tetraether lipids
iv. Sphingolipids and
v. Sterols
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2.1 Glycerophospholipids/ Phosphoglycerides
• Phospholipid is a more general term; Any lipid containing phosphorus
• Phosphoglycerides contain:
• Glycerol
• Fatty acid
• Phosphoric acid with an amino alcohol
• Have hydrophobic and hydrophilic domains
• Suspended in water, they spontaneously rearrange into ordered
structures
• Hydrophobic group to center
• Hydrophilic group to water
• Basis of membrane structure
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• Glycerophospholipids are named as derivatives of the parent
compound, phosphatidic acid, according to the polar alcohol in the
head group.
• The phospho-amino-alcohol is highly hydrophilic
• They are used in:
– Cell membranes
– Emulsifying
– Micelle-forming agents in the blood
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Types of Phosphoglycerides
1. Ones made with choline are called lecithin

• Phosphatidylcholines (Lecithins) Occur in Cell Membranes
• Dipalmitoyl lecithin is a very effective surface active agent and a
major constituent of the surfactant preventing adherence, due to
surface tension, of the inner surfaces of the lungs.
2. Those made with ethanolamine are called cephalins
• Phosphatidylethanolamine (cephalin) and phosphatidylserine
(found in most tissues) differ from phosphatidylcholine only in that
ethanolamine or serine, respectively, replaces, choline.
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• Ether Glycerophospholipids: Some animal tissues and some
unicellular organisms are rich in ether lipids, in which one of the
two acyl chains is attached to glycerol in ether linkage.
• Platelet activating factor, or PAF
• Plasmalogens
• Common plasmalogen head groups include choline, ethanolamine, and
serine.
• These lipids are referred to as phosphatidal choline, phosphatidal
ethanolamine, and phosphatidal serine.
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Glycerophospholipid Degradation:
• Effects of Snake Venoms

• Contain PLA2
• Lysolecithin, acts as a detergent and dissolves the membranes of
RBCs, causing them to rupture
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2.2 Sphingolipids
• Are derivatives of Sphingosine

• Sphingolipids are composed of
1. one molecule of the long-chain amino alcohol sphingosine
2. one molecule of a long-chain fatty acid, and
3. a polar head group that is joined by a glycosidic linkage in some cases and a
phospho-diester in others
• Ceramide is the structural parent of all sphingolipids
• There are three subclasses of sphingolipids:
1. sphingomyelins,
2. neutral (uncharged) glycolipids, and
3. gangliosides.
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• Sphingomyelins
• Contain phosphocholine or phosphoethanolamine
• Especially prominent in myelin
• Glycosphingolipids
• occur largely in the outer face of PM
• they do not contain phosphate
• Cerebrosides
• have a single sugar linked to ceramide
• Galactose – PM of cells in neural tissue
• Glucose – PM of cells in non neural tissue
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• Globosides: are glycosphingolipids with two or more sugars usually
D-glucose, D-galactose, or N-acetyl-Dgalactoseamine.
• Gangliosides: have oligosaccharides as their polar head groups
• one or more residues of sialic acid
• Sialic acid gives gangliosides the negative charge at pH 7 that distinguishes
them from globosides.
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• Sphingolipids at cell Surfaces are sites of biological recognition.
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2.3 Sterols
• Have Four Fused Carbon Rings
• Sterols are structural lipids present in the membranes of most
eukaryotic cells.
• The characteristic structure of this group of membrane lipids is the
steroid nucleus, consisting of four fused rings, three with six carbons
and one with five.
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PHYSIOLOGICALLY IMPORTANT
ROLES of steroids
• Cholesterol is probably the best known steroid
• It is the precursor of a large number of equally important steroids
that include the: bile acids, adrenocortical hormones, sex
hormones, D vitamins, cardiac glycosides, sitosterols of the plant
kingdom, and some alkaloids.
• All of the steroids have a similar cyclic nucleus resembling
phenanthrene (rings A, B, and C) to which a cyclopentane ring
(D) is attached
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3. Lipids as Signals, Cofactors, and Pigments
• Phosphatidylinositols and Sphingosine Derivatives Act as Intracellular
Signals
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• Eicosanoids Carry Messages to Nearby Cells
• Classes of eicosanoids:
• Prostaglandins
• thromboxanes, and
• Leukotrienes
• Steroid hormones carry messages between tissues
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The end!
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Chapter six
Intermediary metabolism
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Objectives
• Define metabolism
• Types of metabolism
• Definition and steps of gluconeogenesis, glycolysis, Glycogen metabolism
• Storage disease…
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Introduction
• Thousands of chemical reactions are taking place inside a cell in an
organized, well coordinated, and purposeful manner; all these
reactions are collectively called as Metabolism .
• Intermediary metabolism refers to the sum of all intracellular chemical
process by which nutritive material is converted into cellular
components.
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Metabolism serves the following purposes:
• Chemical energy is obtained from the degradation of energy-

rich nutrients.
• Food materials are converted into the building block precursors
of cellular macromolecules. These building blocks are later
made into macromolecules, such as proteins, nucleic acids,
polysaccharides, etc. Biomolecules required for specialized
functions of the cell are synthesized.
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• Metabolic pathways are taking place with the help of sequential
enzyme systems. These pathways are regulated at three levels:
• Regulation through the action of allosteric enzymes, which increase or
decrease the activity under the influence of effector molecules.
• Hormonal regulation. Hormones are chemical messengers secreted by
different endocrine glands.
• Regulation at the DNA level; the concentration of the enzyme is changed by
regulation at the level of synthesis of the enzyme
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Types of Metabolic Pathways
a) Catabolic (degradation) pathways, where energy rich complex
macromolecules are degraded into smaller molecules. Energy
released during this process is trapped as chemical energy, usually as
ATP.
b) Anabolic (biosynthesis) pathways. The cells synthesize complex
molecules from simple precursors. This needs energy.
c) Amphibolic pathways are seen at cross-roads of metabolism, where
both anabolic and catabolic pathways are linked.
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Carbohydrate metabolism
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DIGESTION OF CARBOHYDRATES:
• In the diet, carbohydrates are present as complex polysaccharides (starch,
glycogen), and to a minor extent, as disaccharides (sucrose and lactose).
• They are hydrolyzed to monosaccharide units in the gastrointestinal tract.
Cooking makes the digestion process easier.
• The process of digestion starts in mouth by the salivary alpha-amylase.
However, the time available for digestion in the mouth is limited, because the
gastric hydrochloric acid will inhibit the action of salivary amylase.
• In the pancreatic juice another alpha-amylase is available, which will hydrolyze
the alpha-1,4 glycosidic linkages randomly, so as to produce smaller subunits like
maltose, isomaltose, dextrins and branched or unbranched oligosaccharides.
• The cells of brush border of intestine contain the enzymes, sucrase, maltase,
isomaltase and lactase.
• They hydrolyze the corresponding disaccharides into component
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monosaccharides, which are then absorbed.
Dr Ambayehu 208
ABSORPTION OF CARBOHYDRATES:
• Only monosaccharides are absorbed by the intestine.
• Absorption rate is maximum for galactose; moderate for glucose; and
minimum for fructose.
• The duodenum and upper jejunum absorb the bulk of the monosaccharide
products of digestion.
• However, different sugars have different mechanisms of absorption. For
example, galactose and glucose are transported into the mucosal cells by
an active, energy-dependent process that requires a concurrent uptake of
sodium ions, and the transport protein is the sodium-dependent glucose
cotransporter 1 (SGLT-1).
• Fructose utilizes an energy- and sodium-independent monosaccharide
transporter (GLUT-5) for its absorption.
• All three monosaccharides are transported from the intestinal mucosal cell
into the portal circulation by yet another
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transporter, GLUT-2. 209
Glycolysis
Anaerobic Oxidation of Glucose.
Glycolysis is cleavage of glucose or other hexoses into pyruvate/lactate.
It aims at producing ATP and other intermediates in every cell type.
However, because it can run independent of mitochondria/O2, it is mainly

called anaerobic oxidation of glucose.
The enzymes of glycolysis are also utilized in the opposite direction to
synthesize glucose in gluconeogenesis. Therefore, glycolysis is an
Amphipathic pathway.
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Intracellular site and tissue distribution:
Glycolysis occurs in the cell cytoplasm of all tissues of the body. It is especially
important in the tissues with un utilizability or short supply of oxygen.
Mammalian erythrocyte (RBCs): The red cells are devoid of mitochondria and
depend on glycolysis as the main source of energy. RBCs are unique in that about
90% of its total energy requirement is provided by glycolysis.
Contracting muscles: Most of the energy of the rapidly contracting muscles comes
from the anaerobic oxidation of glucose through glycolysis.
Cornea, lens and some parts of retina: They have a limited blood supply and lack
of mitochondria. Therefore, needed energy is derived from glycolysis.
Kidney (medulla), testicles, leukocytes and white muscle fibers: These tissues
along with Brain (glial cells and astrocytes), skin, and intestinal mucosa with
relatively few mitochondria normally derive most of their energy from glycolysis.
Tissues with relatively low mitochondria utilize simultaneous aerobic and
anaerobic glycolysis.
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Steps in glycolysis:
• Oxidation of glucose (or glycogen in muscles), when oxygen is available for
respiratory chain, leads to formation of pyruvate, whereas in the absence of
oxygen it is converted into lactate.
• Glucose is transported into cells by a glucose transporter that is insulin-
dependent in muscles and adipose tissue but not in other vital tissues, e.g.,
brain, heart, kidney and RBCs.
• Glucose Uptake into Liver is Not dependent of Insulin
• Liver is freely permeable to glucose and, therefore, uptake of glucose into
hepatocytes does not require insulin, but insulin activates glucose
utilization in liver by activating glucokinase.
• Classified as priming stage, splitting stage and energy stage.
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1. Activation of glucose: Circulating blood glucose metabolically inert unless
it is activated to glucose-6-phosphate (Glu-6-P) inside the cells.
• Once phosphorylated, glucose is trapped inside the cells because cell membrane is
impermeable to it.
• The activation takes place with the help of tissue-specific isoenzymes of

hexokinase.
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• Most of the metabolic pathways of carbohydrates are linked to
Glucose-6-P.
• It is a branch-point in carbohydrate metabolism for its utilization
(through glycolysis, pentose phosphate pathway, glucuronic acid
pathway, glycogen synthesis and synthesis of different sugar
derivatives and glycosylations) and generation (through
glycogenolysis, pentose phosphate pathway and gluconeogenesis).
• Glucose-6-P can also be called as the intracellular ‘Active Form’ of
glucose.
• The circulating in blood glucose has to be activated into Glucose-6-P to
begin any of its metabolic reactions.
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Hexokinase Glucokinase
Site: All tissues. Liver parenchymal and pancreatic β-cells.
Substrate: - and -anomers glucose mainly and Glucose only.
.
activates fructose and galactose at a slower rate.
Affinity for glucose is high (i.e., low Km, ~0.2 mM) Low affinity (i.e., high Km, 5-10 mM) and traps glucose
.
and traps glucose by phosphorylation for energy by phosphorylation for storage in the liver and so it acts
production even with a large glucose concentration optimally at postprandial high blood glucose concentrations
gradient difference between blood and cells. (>5 mM/L) and restores fasting glucose level.
Induction: It is a non-inducible enzyme. It is an inducible enzyme, i.e., affected by glucose and

insulin concentration.
Hormonal Regulation: Is not affected by antiinsulins, Is inhibited by anti-insulins.
e.g., epinephrine, glucocorticoids and growth hormone.
Allosteric inhibitor: glucose-6-phosphate. Glucose-6-phosphate does not inhibit it.
Feeding & insulin do not affect its activity They Increase rate of activity (induction).
Fasting & diabetes do not affect its activity. They decrease rate of activity (repressed).
07/22/2024 Low glucose and high F6P inactivate the enzyme.216
Dr Ambayehu
2. Isomerization of glucose-6-phosphate into fructose-6-phosphate:
• It is done by phosphohexose isomerase that is an aldo-keto-
isomerase.
• Only the -anomer of Glu-6-P is isomerized into fructose-6-phosphate
• It is a freely reversible reaction.
CHO CH OH 2
CHO H OH O
H OH HO H HO H
Glucokinase/Hexokinase Phosphohexose isomerase
HO H H OH H OH
Mg2+
H OH H OH O H OH O
ATP ADP
H OH H2C O P OH H2C O P OH
CH2OH OH OH
D-Glucose D-Glucose-6-phosphate D-Fructose-6-phosphate
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3. Activation of fructose-6-phosphate into fructose-1,6-diphosphate :
• The reaction is catalyzed by phosphofructokinase-1 (PFK-1), using ATP and Mg2+ ions.
• This is the most important, rate-limiting and irreversible reaction (i.e., the pace
maker) of glycolysis.
• The enzyme PFK-1 is activated by AMP and accumulation of fructose-2,6-diphosphate
(Fru-2,6-diP), which is produced by the bifunctional phosphofructokinase-2 (PFK-2)
and inhibited by ATP and citrate.
• The bifunctional enzyme carries out two enzyme activities, the synthesis and hydrolysis
of Fru-2,6-diP, and is regulated by the availability of Glu-6-P.
• When Gl-6-P concentration is high, its kinase activity is stimulated and it
phosphorylates Fru-6-P to Fru-2,6-DiP.
• When
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Glu-6-P is low, it works as an Fru-2,6-diP
Dr Ambayehu
phosphatase to produce Fru-6-P. 218
4. Cleavage of fructose-1,6-diphosphate: The six-carbon Fru-1,6-
DiP is split into two triose phosphates by aldolase. This reaction is freely
reversible. The enzyme, Aldolase exists as a number of tissue-specific
isoenzymes, all of which contain four subunits.
• Aldolase A occurs in most tissues and acts on Fru-1,6-DiP.
• Aldolase B occurs in liver and kidney and acts on fructose-1-phosphate
O
and Fru-1,6-DiP O H C O P OH 2
CH2OH H2 C O P OH O OH
OH CH2OH
O O
Dihydroxyacetone-phosphate
HO H HO H
Phosphofructokinase-1
H OH H OH Aldolase A/B Phosphotriose isomerase
Mg2+ CHO
H OH O H OH O
ATP ADP H OH O
H2C O P OH H2 C O P OH
H2C O P OH
OH OH
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Dr Ambayehu OH 219
D-Glyceraldehyde-3-phosphate (2)
5. Oxidation of glyceraldehyde-3-phosphate into 1,3-
diphosphoglycerate:
• The oxidation of glyceraldehyde-3-phosphate is catalyzed by
glyceraldehyde-3-phosphate dehydrogenase, an SH-containing
enzyme, in the presence of NAD+ as a coenzyme and inorganic
phosphate (Pi).
O O
C O P OH
CHO OH
H OH O D-Glyceraldehyde-3-phosphate dehydrogenase H OH O
O
H2 C O P OH H2 C O P OH
HO P OH 2 NAD+ 2 NADH.H+
OH OH
D-Glyceraldehyde-3-phosphate (2) OH 1,3-Diphospho-Glycerate (2)
Inorganic phosphate (2)
O
HO P OH O
O O
OH C O P OH
CHO HOHC S Enzyme C S Enzyme Inorganic
H OH
Enzyme-SH H OH H OH phosphate Enzyme-SH OH
O O O
H2 C O P OH H OH O
H2 C O P OH H2 C O P OH
NAD+ NADH.H
+ H2 C O P OH
OH OH OH
07/22/2024 D-Glyceraldehyde-3- D-Glyceraldehyde-3- Dr Ambayehu D-Glyceraldehyde-3- OH 220
phosphate phosphate phosphate 1,3-Diphospho-Glycerate
6. Conversion of 1,3-diphosphoglycerate into 3-phosphoglycerate:
• This reaction involves harvest of the high-energy phosphate at C1 to generate
an ATP from an ADP because the energy of the acyl-phosphate bond is high
enough (~13 kcal/mole) to make the reaction energetically favorable.
• The reaction is an example of substrate level phosphorylation,
• It is one of the very rare reactions that utilize ATP and yet are reversible under
normal cell conditions.
O O
C O P OH
COOH
OH COOH O
H OH O H OH O
Phosphoglycerate kinase Phosphoglyceromutase H O P OH
H2C O P OH H2C O P OH
Mg2+ CH2OH OH
07/22/2024
OH 2 ADP 2 ATP Dr Ambayehu
OH 2-Phospho-Glycerate
221(2)
1,3-Diphospho-Glycerate (2) 3-Phospho-Glycerate (2)
7. Conversion of 2-phosphoglycerate into phosphoenol pyruvate:
• Subsequent removal of water enolase enzyme in presence of Mg2+ or Mn2+ redistributes energy
within the molecule to form phosphoenol pyruvate.
• The redistribution of energy results in conversion of the phosphoester linkage into a high-energy
phosphate bond at C2.
8. Conversion Of Phosphoenol Pyruvate Into Pyruvate:
• The reaction is catalyzed by pyruvate kinase.. Thus, the overall reaction is irreversible and
energetically favorable because phosphoenol pyruvate is at a higher energy level than pyruvate
through releasing ~14 kcal/mole.
• This is the example of second substrate level phosphorylation in glycolytic pathway. It utilizes
the high-energy bond at C2 of phosphoenol pyruvate to convert an ADP into an ATP
COOH O COOH O COOH COOH
Enolase Pyruvate kinase Sponteneous
H O P OH O P OH OH O
Mg2+ Mg2+
CH2OH OH 2 H2O CH2 OH CH2 CH3
07/22/2024 2 ADPDr Ambayehu
2 ATP 222
2-Phospho-Glycerate (2) 2-Phospho-enol- Enol-pyruvate (2) Pyruvate (2)
• Under aerobic conditions (i.e., presence of oxygen and functional mitochondria), steps
of glycolysis end at this point and pyruvate is actively transported into mitochondria.
Inside the mitochondria it is oxidatively decarboxylated into acetyl-CoA that enters Krebs'
cycle for further oxidation and larger energy production.
• Under anaerobic conditions (i.e., in absence of O2 and/or mitochondria): The electron
transport chain can not operate in the absence of oxygen.
• The NADH.H+ generated in the glycolytic reactions cannot deliver its hydrogen (plus
electrons) to the electron transport chain.
• The unavailability of NAD+ might block the glycolysis; therefore, NADH is reoxidized
into NAD+ through transfer of its hydrogen into pyruvate to form L-lactate.
• The reaction is catalyzed reversibly by lactate dehydrogenase (LDH).
• Lactate and H+ are both transported out of the cell into interstitial fluid by a plasma
membrane transporter and then diffuse into the blood. After exceeding the buffering
capacity of the blood, lactic acidosis occur
07/22/2024 as a result.
Dr Ambayehu 223
Significance of Glycolysis Pathway
• It is the only pathway that is taking place in all the cells of the body.
• Glycolysis is the only source of energy in erythrocytes.
• In strenuous exercise, when muscle tissue lacks enough oxygen,
anaerobic glycolysis forms the major source of energy for
muscles.
• The glycolytic pathway may be considered as the preliminary step
before complete oxidation.
• The glycolytic pathway provides carbon skeletons for synthesis of
non-essential amino acids as well as glycerol part of fat.
• Most of the reactions of the glycolytic pathway are reversible, which
are also used for gluconeogenesis.
07/22/2024 Dr Ambayehu 224
TCA CYCLE
• Also called the Krebs cycle or the citric acid cycle)
• It is the final pathway where the oxidative metabolism of
carbohydrates, amino acids, and fatty acids converge, their carbon
skeletons being converted to CO2. This oxidation provides energy for
the production of the majority of ATP in most animals, including
humans.
• The cycle occurs totally in the mitochondria and is, therefore, in close
proximity to the reactions of electron transport ,which oxidize the
reduced coenzymes produced by the cycle.
07/22/2024 Dr Ambayehu 225

• The TCA cycle is an aerobic pathway, because O2 is required as the final
electron acceptor. Most of the body's catabolic pathways converge on the
TCA cycle. Reactions such as the catabolism of some amino acids generate
intermediates of the cycle and are called anaplerotic reactions.
• The citric acid cycle also participates in a number of important synthetic
reactions. For example, the cycle functions in the formation of glucose
from the carbon skeletons of some amino acids, and it provides building
blocks for the synthesis of some amino acids and heme. Therefore, this
cycle should not be viewed as a closed circle, but instead as a traffic
circle with compounds entering and leaving as required.
07/22/2024 Dr Ambayehu 226

07/22/2024 Dr Ambayehu 227
Conversion of pyruvate to Acetyl CoA
• Pyruvate, the end product of aerobic glycolysis, must be transported into the
mitochondrion before it can enter the TCA cycle.
• This is accomplished by a specific pyruvate transporter .
• Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate
dehydrogenase complex, which is a multienzyme complex
• Acetyl CoA is the fuel for Krebs Cycle to take place in the matrix
07/22/2024 Dr Ambayehu 228

* In the presence of CHO, energy being used,
• Metabolized to CO2, NADH, FADH2,GTP and, ultimately ATP
Θ If energy not being used (Lots of ATP present)
• Made into fat
 If energy being used, but no CHO present
• Starvation
• Forms ketone bodies
07/22/2024 Dr Ambayehu 229
Citrate Synthase Reaction (First)
O O O
C O
C
C O H2 O CoASH
CH2 O
H3C SCoA CH2
C C HO C C O
O + O
O
citrate synthase CH2
C
O O
acetyl CoA oxaloacetate
citrate
The condensation of acetyl CoA and oxaloacetate to form citrate (a

tricarboxylic acid) is catalyzed by citrate synthase .
 Citrate synthase is inhibited by its product, citrate, and by NADH
and succinyl CoA.
 Substrate availability is a key means of regulation for citrate synthase
07/22/2024 Dr Ambayehu 230

Aconitase Reaction
O O O O
C C
CH2 O
HO CH O
HO C C O HC C O
CH2 CH2
aconitase
C C
O O
O O
citrate isocitrate
Citrate, in addition to being an intermediate in the TCA cycle, provides

a source of acetyl CoA for the cytosolic synthesis of fatty acid.
Citrate also inhibits phosphofructokinase, the rate-setting enzyme of
glycolysis ,and activates acetyl CoA carboxylase (the rate-limiting
enzyme of fatty acid synthesis.
Citrate is isomerized to isocitrate by aconitase, an Fe-S protein.
 Aconitase is inhibited by fluoroacetate, a compound that is used as
a rat poison.
07/22/2024 Dr Ambayehu 231

Isocitrate Dehydrogenase
Isocitrate dehydrogenase catalyzes the irreversible oxidative

decarboxylation of isocitrate, yielding the first of three NADH
molecules produced by the cycle, and the first release of CO2. .
This is one of the rate-limiting steps of the TCA cycle.
The enzyme is allosterically activated by adenosine diphosphate
(ADP, a low-energy signal) and Ca2+, and is inhibited by adenosine
07/22/2024
triphosphate (ATP) and NADH,Dr Ambayehu
whose levels are elevated when the 232
α-ketoglutarate dehydrogenase
is catalyzed by the α-ketoglutarate dehydrogenase complex, which consists

of three enzymatic activities .
 The reaction releases the second CO2 and produces the second NADH of
the cycle.
Coenzymes are thiamine pyrophosphate, lipoic acid, FAD, NAD +, and CoA.
 α-Ketoglutarate dehydrogenase complex is inhibited by ATP, GTP, NADH,
and succinyl CoA, and activated by Ca++ .
07/22/2024 Dr Ambayehu 233
Succinyl CoA synthetase
SCoA O O
O
C C
CH2 GTP CoASH CH2
GDP
CH2 CH2
C C
O O succinyl CoA O O
succinyl CoA synthetase
succinate
Succinate thiokinase (also called succinyl CoA synthetase—named for the

reverse reaction) cleaves the high-energy thioester bond of succinyl CoA .
This reaction is coupled to phosphorylation of guanosine diphosphate (GDP)

to guanosine triphosphate (GTP). GTP and ATP are energetically
interconvertible by the nucleoside diphosphate kinase reaction.
07/22/2024 Dr Ambayehu 234

Succinate dehydrogenase
O O O
O
C FADH2 C
CH2 FAD C H
CH2 H C
C succinyl CoA
C
O O
O O dehydrogenase
succinate fumarate
Succinate is oxidized to fumarate by succinate dehydrogenase,

producing the reduced coenzyme FADH2.
Succinate dehydrogenase is the only enzyme of the TCA cycle that is

embedded in the inner mitochondrial membrane. As such, it functions
as Complex II of the electron transport chain.
07/22/2024 Dr Ambayehu 235
Fumarase
O O O O
C H2 O C
C H HC OH
H C CH2
C fumarase
C
O O O
O
fumarate
malate
Fumarate is hydrated to malate in a freely reversible reaction

catalyzed by fumarase (also called fumarate hydratase).
 Fumarate is also produced by the urea cycle , in purine synthesis ,
and during catabolism of the amino acids, phenylalanine and tyrosine.
07/22/2024 Dr Ambayehu 236

Malate Dehydrogenase
O O
C O O
HC OH C
NAD NADH C O
CH2
CH2
C C
O O malate O
dehydrogenase O
malate
oxaloacetate
Malate is oxidized to oxaloacetate by malate dehydrogenase .

This reaction produces the third and final NADH of the cycle.
 Oxaloacetate is also produced by the transamination of the amino
acid, aspartic acid.
07/22/2024 Dr Ambayehu 237

General Reaction Pathway of TCA
 Number of ATP molecules produced from the oxidation of one

molecule of acetyl CoA (using both substrate-level and oxidative
07/22/2024phosphorylation is 12-ATP Dr Ambayehu 238
Regulation of the TCA Cycle
 Availability of acetyl CoA from pyruvate is controlled by PDH activity,
which is regulated by the concentration of NADH and the ADP/ATP ratio.
 The rate-limiting step of the TCA cycle is the synthesis of α-ketoglutarate
from citrate, catalyzed by isocitrate dehydrogenase .
1. Isocitrate dehydrogenase is allosterically inhibited by NADH, an
indicator of the availability of high levels of energy.
2. The enzyme is activated by ADP and Ca2+, which signal a need for
energy in the cell.
 Conversion of α-ketoglutarate to succinyl CoA, catalyzed by α-
ketoglutarate dehydrogenase, is inhibited by NADH and ATP.
07/22/2024 Dr Ambayehu 239
Major regulatory interactions in the TCA cycle
07/22/2024 Dr Ambayehu 240

Importance of Krebs Cycle
Kreb cycle (Citric acid cycle or TCA cycle) is a amphibolic pathway: oxidative
catabolism and provide precursor molecules for anabolism, particularly
gluconeogenesis.
A. Acetyl CoA and the TCA cycle intermediates are involved in many cellular
reactions
 Acetyl CoA is the precursor for fatty acid and sterol biosynthesis .
The interconversion of α-ketoglutarate and glutamate are important for nitrogen
Metabolism.
The catalytic degradation of amino acids and pyrimidines yields pyruvate and
several TCA cycle intermediates, which can then be metabolized in this way
to yield energy.
 Pyruvate and TCA cycle intermediates serve as precursors for the biosynthesis
of amino acids.
Energy (2 ATPs per cycle) will be produced from succinyl Co A. Other compounds
produce NADH and FADH2 for oxidative
07/22/2024 Dr Ambayehu
phosphorylation in the mitochondria
241
to
Net From Kreb’s
Oxidative process
• 3 NADH
• FADH2
• GTP
X 2 per glucose
• 6 NADH
• 2 FADH2
• 2 GTP
All ultimately turned into ATP (oxidative phosphorylation…
later).
07/22/2024 Dr Ambayehu 242

Total Energy per glucose
Cytosol
• Glycolysis
• 2 NADH
• 2 ATP
Net ATP production is 2 ATP from making glucose to pyruvate
without using oxygen (anerobic).
Mitochondrion
• Pyruvate dehydrogenase
• 2 NADH
Krebs
• 6 NADH
• 2 FADH2
• 2 GTP
07/22/2024 Dr Ambayehu 243

In mitochondrion:
• Each NADH makes 2.5 ATP
• Each FADH2 makes 1.5 ATP
• GTP makes ATP
So…
• From in mitochondrion
• 8 NADH X 2.5 ATP/NADH = 20 ATP
• 2 FADH2 X 1.5 ATP/FADH2= 3 ATP
• 2 GTP X 1 ATP / GTP = 2 ATP
• TOTAL in mitochondrion 25 ATP
07/22/2024 Dr Ambayehu 244

Cytosol
• 2 ATP
• 2 NADH
• NADH can’t get into mitochondrion
• In eukaryotes two pathways,
• transferred to FADH2
• get 1.5 ATP/ FADH2
• Or transferred to NADH
• Get 2.5 ATP/ NADH
• 2 NADH X 1.5 ATP = 3 ATP

• Or 2 NADH X 2.5 ATP = 5 ATP
• + =2 ATP
• Total 3+ 2 or 5 + 2 so either 5 or 7
07/22/2024 Dr Ambayehu 245

ATP/glucose
Eukaryotes
Mitochondrial: 25 ATP
Cytosolic: 5 or 7 ATP
Total 30 or 32 ATP/glucose
30 ATP X 7.3kcal X 4.18 kJ = 915 kJ
ATP kcal
If 32 ATP = 976 kJ
Prokaryotes
32 ATP X 7.3kcal X 4.18 kJ = 976 kJ
ATP kcal
07/22/2024 Dr Ambayehu 246

GLUCONEOGENESIS
Gluconeogenesis , the synthesis of ‘NEW’ glucose, from non-
carbohydrates.
• Non-carbohydrate Sources of Glucose
 Lactate
 Pyruvate
 Propionate
 Glycerol (from diet and lipolysis)
 Glucogenic Amino Acids
07/22/2024 Dr Ambayehu 247

• The daily glucose consumption of the adult brain is approximately 75% of
the whole body requirements, i.e., 120 - 150 grams, whereas, the whole
body requires 160 - 190 grams.
• The body stores are 210 grams (190 grams from liver glycogen and 20
grams in body fluids) enough for a day.
• In a longer period of starvation, glucose must be formed from non-
carbohydrate sources for survival.
07/22/2024 Dr Ambayehu 248

Sources of blood glucose:
• Diet  Glycogenolysis  Gluconeogenesis

• Glycogenolysis can be turned on rapidly in response to
Hormonal stimulation
Gluconeogenesis is turned on with a slower response, Reaching maximal activity
over a period of hours
It becomes the primary source of our blood glucose, At about 8 hr’s into the post-
absorptive state
07/22/2024 Dr Ambayehu 249

• Tissues distribution
A) Liver (80%)
The major organ contributing to blood glucose.
 Liver is unique in that it has the capacity to convert three
carbon precursors (such as lactate, glycerol, pyruvate and
propionate) along with glucogenic amino acids into glucose.
B) Kidney Cortex (20%)

• Kidney medulla consumes most of glucose produced by the kidney
cortex.
• Subcellular location of enzymes
• pyruvate carboxylase: mitochondrial
• glucose-6-phosphatase: ER
07/22/2024 Dr Ambayehu 250
Biological Importance of
Gluconeogenesis
 There is always a basal requirement of glucose
• -Even if fatty acid oxidation is supplying enough energy in the tissues
 Gluconeogenesis supplies body cells with glucose
• -After 4 - 6 hours of last meal
 Becomes fully active as stores of glycogen are depleted
 Glc produced by gluconeogenesis has the following functions:
I. It is used to meet the needs for Glc by other cells:
• -Brain & nervous system, RBCs, renal medulla, Lens, etc.
II. Glucose is especially needed for:
• -Mammary gland as a precursor of lactose
07/22/2024 Dr Ambayehu 251
III. Glucose produces intermediates of TCA cycle in many tissues
• e.g., oxaloacetate
IV. Gluconeogenesis also has roles in:
• -Control of acid-base balance
• -Maintenance of amino acid balance
07/22/2024 Dr Ambayehu 252

• Gluconeogenesis requires both a source of:
Energy & Carbons  for formation of glucose
 Energy  provided by metabolism of fatty acids
 Carbon skeletons  provided from three primary sources:
o Lactate produced in tissues such as:
• -Red cell & Exercising muscle by anaerobic glycolysis
o Glucogenic amino acids derived from muscle protein
o Glycerol released from triacylglycerol (TAG)
• -During lipolysis in adipose tissue
o Others  Pyruvate & Propionic acid
07/22/2024 Dr Ambayehu 253
 Source of pyruvate & oxaloacetate for gluconeogenesis
 During fasting or carbohydrate starvation
 Is mainly amino acid catabolism
 Muscle proteins may break down to supply amino acids
 These are transported to liver where they are:
 Deaminated & converted to gluconeogenesis inputs
 Glycerol, derived from hydrolysis of TAGs in fat cells,
 also a significant input to gluconeogenesis
07/22/2024 Dr Ambayehu 254
Pathway of Gluconeogenesis
 It is essentially the reverse of glycolysis, except
At the three irreversible rxn’s, that by different enzyme(s) to be reversed.
Seven of the steps in gluconeogenesis are reversible, catalyzed by the same

enzymes used in glycolysis
Involving both mitochondrial & cytosolic enzymes
Starting point of the pathway will be differ based on its precursors: Lactate,
pyruvate, Propionic acid, Glucogenic amino acids, Glycerol.
07/22/2024 Dr Ambayehu 255

 Three of the reactions of glycolysis
are irreversible and must be
.
circumvented by four alternative
reaction
a)PK => PC/PEPCK
b)PFKI => F-1-6-bis phosphatase
c) HK/GK => G-6-phosphatase
07/22/2024 Dr Ambayehu 256

Conversion of
Pyruvate to PEP
07/22/2024 Dr Ambayehu 257

07/22/2024 Dr Ambayehu 258
 Gluconeogenesis is fairly efficient –
The liver can make a kilogram of glucose per day by
gluconeogenesis, &
Actually does so in poorly controlled, hyperglycemic diabetic
patients
 Gluconeogenesis from pyruvate is also moderately expensive,
Requiring a net expenditure of 6 moles of ATP & (2NADH)
This ATP is provided by oxidation of fatty acids
07/22/2024 Dr Ambayehu 259
Pyruvate Carboxylase PEP Carboxykinase
O O
C
O O O O
C ATP ADP + Pi C O GTP GDP C
C O CH2 C OPO32
HCO3 C CO2
CH3 CH2
O O
pyruvate oxaloacetate PEP
•Bypass of Pyruvate Kinase (2 enzymes):
•Pyruvate Carboxylase catalyzes:
pyruvate + HCO3- + ATP  oxaloacetate + ADP + Pi
•PEP Carboxykinase catalyzes:
07/22/2024
oxaloacetate + GTP  PEP + GDP + CO2
Dr Ambayehu 260
• Avidin:
• A protein in egg whites with a b barrel
structure, tightly binds biotin
• The strong avidin-to-biotin affinity

 Excess consumption of raw eggs can cause:
• Nutritional deficiency of biotin

• Fasting hypoglycemic
avidin
07/22/2024 Dr Ambayehu with bound biotin
261
O O
P E P C a rb o x y k in a s e R e a c tio n
C O O O
GTP GDP O
C O C C
CH2 C O C O P O 32
CO2
C CH2 CH2
O O
o x a lo a c e ta te PEP
•PEP Carboxykinase catalyzes GTP-dependent:

• Oxaloacetate  PEP; It is thought to proceed in 2 steps:
 Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate
 Phosphate transfer from GTP then yields PEP
07/22/2024 Dr Ambayehu 262

Phosphofructokinase 
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
263
5 H HO 2 5 H HO 2
H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase
•Phosphofructokinase (Glycolysis) catalyzes:
Fructose-6-P + ATP  Fructose-1,6-bisP + ADP
•Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes:
Fructose-1,6-bisP + H2O  Fructose-6-P + Pi
Dr Ambayehu 263
07/22/2024
Glucose-6-phosphatase
6 CH OPO 2 CH2OH
2 3
5 O O
H
264 H H H
H H2O H
4
OH H 1
OH H + Pi
OH OH OH OH
3 2
H OH H OH
glucose-6-phosphate glucose
•Hexokinase or Glucokinase (Glycolysis) catalyzes:
Glucose + ATP  Glucose-6-phosphate + ADP
•Glucose-6-Phosphatase (Gluconeogenesis) catalyzes:
Glucose-6-phosphate + H2O Glucose + Pi
07/22/2024 Dr Ambayehu 264
Glucose-6-phosphatase
6 CH OPO 2 CH2OH
2 3
5 O O
H
265 H H H
H H2O H
4
OH H 1
OH H + Pi
OH OH OH OH
3 2
H OH H OH
glucose-6-phosphate glucose
•Glucose-6-phosphatase: is embedded in the ER membrane in liver and kidney cells
•The catalytic site is found to be exposed to the ER lumen
•Another subunit may function as a translocase;
•Providing access of substrate to the active site
07/22/2024 Dr Ambayehu 265

Location & function of
Glc 6-phosphatase
• Glucose 6-phosphate
• Travels on a transporter
into the ER,
• Where it is hydrolyzed
Several ER proteins play a role in the generation
• by Glc 6-phosphatase
of glucose from glucose 6-phosphate.
• to Glucose & Pi –T1 transports glucose 6-phosphate into the lumen
of the ER.
• These products –T2 and T3 transport Pi and glucose, respectively,
back into the cytosol.
• Travel back to the cytosol on transporters –Glucose 6-phosphatase is stabilized by a Ca2+-
07/22/2024 binding
Dr Ambayehu protein (SP). 266
Gluconeogenesis From Amino acids
• Most amino acids are glucogenic, i.e.
• Following deamination their carbon skeletons can be converted into Glc
• Alanine & Glutamine:

• The major amino acids exported from muscle for gluconeogenesis
• Alanine is converted directly into pyruvate;
• By alanine aminotransferase (ALT), &
• Then gluconeogenesis proceeds as described for lactate
07/22/2024 Dr Ambayehu 267

 Other amino acids are converted into TCA cycle intermediates
• then to malate for gluconeogenesis ,for example:
• Aspartate is converted into oxaloacetate
 by AST
• Glutamate into α-ketoglutarate
 by glutamate dehydrogenase
07/22/2024 Dr Ambayehu 268

The amino groups of these
amino acids are converted
into urea
via the urea cycle, &
The urea is excreted in urine
07/22/2024 Dr Ambayehu 269

Gluconeogenesis From Glycerol
• Glycerol enters gluconeogenesis:
• At the level of triose phosphates
• Following release of glycerol & fatty acids
from adipose tissue into plasma
• The glycerol is taken up into liver &
phosphorylated by glycerol kinase
• Then enters the gluconeogenic pathway
as dihydroxyacetone phosphate
07/22/2024 Dr Ambayehu 270

Only the glycerol component of fats can be converted into glucose:
• Acetyl CoA & even c-chain number fatty acids:
• Can’t serve as substrates for net gluconeogenesis
• Odd C-chain number & branched-chain fatty acids

• Yield propionyl CoA,
• W/c can serve as a minor precursor for gluconeogenesis
07/22/2024 Dr Ambayehu 271

• Propionyl CoA is first carboxylated to methylmalonyl CoA;
• W/c undergoes racemase & mutase rxn’s to form succinyl CoA

• Succinyl CoA is converted into malate,
• Exits the mitochondrion & is oxidized to oxaloacetate
• Following decarboxylation by PEPCK,
• The 3-C of propionate appear intact in PEP for
gluconeogenesis
07/22/2024 Dr Ambayehu 272

07/22/2024 Metabolism of Propionate
Dr Ambayehu 273
Energetics of Gluconeogenesis
• Pyruvate Carboxylase
• 2 ATPs
• PEP Carboxykinase
• 2 GTPs
• 3-P-glycerate kinase
• 2 ATPs
• G-3-P DHN
• 2NADH
07/22/2024 Dr Ambayehu 274

Regulation of Gluconeogenesis
• Although gluconeogenesis occurs during fasting,
• it is also stimulated:
• During prolonged exercise
• By a high-protein diet
• Under conditions of stress
• The factors that promote the overall flow of carbon from
pyruvate to glucose include:
• The availability of substrate &
• Changes in the activity or amount of certain key enzymes
07/22/2024 Dr Ambayehu 275

•Glycolysis & Gluconeogenesis are both spontaneous;
•If both pathways were simultaneously active in a cell,
•It would constitute a "futile cycle" that would waste energy
Glycolysis:
glucose + 2 NAD+ + 2 ADP + 2 Pi  2 pyruvate + 2 NADH + 2 ATP
Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP 
glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
07/22/2024 Dr Ambayehu 276

Phosphofructokinase 
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5
277 H HO 2 5 H HO 2
H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
 Fructose-1,6-biosphosphatase
•Reciprocally regulated:
• To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis
•Local Control includes
• Reciprocal allosteric regulation by adenine nucleotides:
• Phosphofructokinase-1 (Glycolysis) is
• Inhibited by ATP and stimulated by AMP
• Fructose-1,6-bisphosphatase (Gluconeogenesis) is
07/22/2024 Dr Ambayehu 277
• Inhibited by AMP
Global Control
•Global Control in liver cells includes:
• Reciprocal effects of a cyclic AMP cascade,
• Triggered by the hormone glucagon when blood Glc is low
• Phosphorylation of enzymes & regulatory proteins in liver;
• by Protein Kinase A (cAMP Dependent Protein Kinase) results in
• Inhibition of glycolysis
• Stimulation of gluconeogenesis
• Making glucose available for release to the blood

07/22/2024 Dr Ambayehu 278
• Enzymes relevant to these pathways that are phosphorylated by Protein Kinase
A include:
 Pyruvate Kinase is inhibited when phosphorylated
 CREB (cAMP response element binding protein) w/c activates:
• Transcription of the gene for PEP Carboxykinase
• Leading to increased gluconeogenesis
 A bifunctional enzyme (PFK-2/FBPase-2) that makes & degrades
• An allosteric regulator Fructose-2,6-bisphosphate
07/22/2024 Dr Ambayehu 279

Reciprocal Regulation
•Reciprocal regulation by fructose-2,6-bisphosphate
 Fructose-2,6-bisphosphate stimulates Glycolysis:
• Allosterically activates Phosphofructokinase-1
• Also activates transcription of the gene for Glucokinase,
 Fructose-2,6-bisphosphate:
• Allosterically inhibits Fructose-1,6-bisphosphatase

07/22/2024 Dr Ambayehu 280
Enzymes Involved
in Regulating the
Substrate Cycles of
Glycolysis &
Gluconeogenesis
07/22/2024 Dr Ambayehu 281

Effect of Elevated
Glucagon on the IC [Fru
2,6-BP] in the Liver
07/22/2024 Dr Ambayehu 282

Glycogen Pyruvate
X Gluconeogenesis
Glucose-1-P Glucose-6-P Glucose + P i

Glucose-6-Pase
X
Glycolysis
Pathway
• Summary of effects of glucagon-cAMP cascade in liver:

 Gluconeogenesis is stimulated
 Glycolysis is inhibited
 Glycogen breakdown is stimulated
 Glycogen synthesis is inhibited
283
07/22/2024 Dr Ambayehu 283
 Free glucose is formed for release to the blood
Regulatory Enzymes of Glycolysis in Liver
Enzymes Mechanism (Allosteric Effectors)
Activated by Fru-1,6BP
Inhibited by ATP, Alanine
Pyruvate Kinase Inhibited by phosphorylation;
 Glucagon & Epinephrine lead to an se in cAMP levels,
w/c activates protein kinase A
Activated by Fru-2,6BP, AMP

PFK-1 Inhibited by ATP, Citrate
High Km for Glucose

Glucokinase Induced Drby Insulin
07/22/2024 Ambayehu 284
Regulatory Enzymes of Gluconeogenesis in Liver
Enzymes Mechanism (Allosteric Effectors)
Pyruvate carboxylase Activated by acetyl CoA
Phosphoenolpyruvate Induced by glucagon, epinephrine,

carboxykinase glucocorticoids Repressed by insulin
Inhibited by F2,6P, AMP

Fructose1,6-bisphosphatase Induced during fasting
Glucose 6-phosphatase
07/22/2024
Induced
Dr Ambayehu
during fasting 285
285
Metabolic Cooperation b/n Skeletal Muscle & Liver
The Cori CycleLactic Acid Cycle

• operates during exercise
• For a brief burst of ATP utilization, muscle cells utilize
• ~P stored as phosphocreatine
• Once phosphocreatine is exhausted, ATP is provided mainly by
• Glycolysis, with the input coming from glycogen breakdown and
• from glucose uptake from the blood
Aerobic fat metabolism is
• more significant during a lengthy period of exertion
07/22/2024 Dr Ambayehu 286
• Between meals
• blood Glc is derived primarily
from hepatic glycogen
• During the day
• depending on the frequency of
snacking,
• Glycogenolysis &
• Gluconeogenesis
• may be more or less active
• Late in the night or in early morning
gluconeogenesis
• becomes the primary source of
blood Glc
07/22/2024 Dr Ambayehu 287
Sources of Blood Glucose in: Fed, Fasting, & Starved States.
• Note that the scale changes from hours to days.
07/22/2024 Dr Ambayehu 288
07/22/2024 Dr Ambayehu 289
The Cori cycle
07/22/2024 Dr Ambayehu 290

Fates of lactate
Lactate pouring out from muscles, red cells and adiposities in
blood can have the following fates:
Conversion into glucose during starvation in the liver (80%).
Conversion into pyruvate and joins Krebs' cycle in heart and
muscles (20%).
Excretion of small amounts in sweat and urine as sodium
lactate.
Accumulation in the blood causes pH shifting and lactemia and
lactic acidosis.
07/22/2024 Dr Ambayehu 291

Glycogen Metabolism
• Glycogen:
• A highly branched homopolysaccharide
• Formed of a-glucose units linked by:
• a1-4-glucosidic linkage along the main chain
• a1-6-glucosidic linkage at the branching point
• In vertebrates, glycogen is found primarily in the:
• Liver & skeletal muscle;
• It may represent up to:
– 10% of the weight of liver
– 1% of the weight of muscle
07/22/2024 Dr Ambayehu 292

Function of Glycogen
• In muscle:
• A quick source of energy for anaerobic metabolism
• Can be exhausted in less than an hour during vigorous activity
• In Liver:
• Serves as a reservoir of glucose for other tissues;
• When dietary glucose is not available (b/n meals or during a fast) liver
release glucose to maintain blood glucose conc.
• Liver glycogen can be depleted in 12 to 24 hr’
07/22/2024 Dr Ambayehu 293

• Mechanisms for storing & mobilizing glycogen is The same in muscle
& liver; but the enzymes differ in subtle yet important ways which
Reflect the different roles of glycogen in the two tissues
• In humans, the total amount of energy stored as glycogen is Far less
than the amount stored as fat (TAGs),
• Glucose cannot be stored as such within the cells, b/c: It is osmotically
active, at equivalent amounts to glycogen it will induce osmotic lysis
07/22/2024 Dr Ambayehu 294

• Carbohydrates are stored mainly in the form of glycogen For the
following reasons:
1.Dietary intake of glucose and its precursors are sporadic
2.Storage of glucose in the form of fat is not suitable
Because Fat cannot be:
– Mobilized as rapidly in muscles as glycogen
– Used as a source of energy in the absence of oxygen
– Transformed directly to glucose by any pathway in human body
07/22/2024 Dr Ambayehu 295

Glycogenesis
• Synthesis of glycogen
• Takes place in virtually all animal tissues
• But is especially prominent in the liver & skeletal muscles
• Very small amount of glycogen synthesis & storage in the CNS, This
is why it is completely dependent on blood glucose
• Sources of glucose
• For liver glycogen: Blood glucose, Other hexoses, glucose from
gluconeogenesis
• For muscle glycogen: Blood glucose only
07/22/2024 Dr Ambayehu 296

 Site:
–Cytoplasm of all cells  particularly liver & muscles
 Steps:
–The pathway involves the following steps:
1. Conversion of Glc-6-P into Glc-1-P;
• Phosphoglucomutase
2. Activation of Glc-1-P to the sugar
nucleotide, UDP-Glc;
• UDP-glucose
pyrophosphorylase &
pyrophosphatase
• Primes the initial sugar residues in glycogen 2
07/22/2024 3. Glycogen primer formationDr Ambayehu 9
7 297
4.Transfer of glucose to glycogen chain in α1→4 linkage;
– Glycogen synthase (regulatory enzyme for glycogenesis)
5. When the α1→4 chain exceeds eight residues in length,
– Synthesis of branches by “branching enzyme”, also called
• amylo-a(14)  a(16)-transglucosidase (-transglycosylase)
or
• Glycosyl-(4:6)-transferase
 This branching enzyme :
• Transfers some of the α1→4-linked sugars to an α1→6 branch,

• Setting the stage for continued elongation of both α1→4 chains
• Until they, in turn, become long enough for transfer by branching enzyme
29
8
07/22/2024 Dr Ambayehu 298
How is a New Glycogen Molecule Initiated?
 Glycogen primer:
– usually a preformed (a14) polyglucose chain, or branch having at
least eight glucose residues,
– Glycogenin
» This intriguing protein is both:
• Primer & Enzyme that catalyzes their assembly
» The 1st step in the synthesis of a new glycogen molecule is the transfer of
a Glc residue from:
• UDP-Glc to the -OH group of serine/Tyrosine194 of glycogenin,
• Catalyzed by the intrinsic Glucosyltransferase
29
9
07/22/2024 Dr Ambayehu 299
O
C H 2O H HN
Glycogen H
H
O H
O N
synthesis OH H O
P O
O
P O CH
OH O 2
O
O- H H
H O O-
H H H
O OH
H
U D P-glucose
Uridine diphosphate glucose (UDP-Glc)
•Is active form of Glc for glycogen synthesis
•As glucose residue precursors are added to glycogen,
–UDP-Glc is the substrate & UDP is released as a rxn product
11
07/22/2024 Dr Ambayehu 300
Activation of Glc-1-P to UDP-Glc
 UDP-glucose is formed from glucose-1-phosphate:
• by UDP-Glc pyrophosphorylase & pyrophosphatase
 Glucose-1-phosphate + UTP  UDP-glucose + PPi
 PPi + H2O  2 Pi
• Overall:
 Glucose-1-phosphate + UTP  UDP-glucose + 2 Pi
12
07/22/2024 Dr Ambayehu 301
Glycogenin:
– Initiates glycogen synthesis
– Is also an enzyme that catalyzes attachment of a glucose molecule
to one of its own tyrosine residues.
– Is a dimer, and evidence indicates that the 2 copies of the enzyme
glucosylate one another.
13
07/22/2024 Dr Ambayehu 302
 Glycogen Synthase catalyzes:
• Transfer of the glucose moiety of UDP-glucose to the hydroxyl at C4 of

the terminal residue of a glycogen chain
» to form an a(1 4) glycosidic linkage:
Glycogen(n residues) + UDP-glucose Glycogen(n +1 residues) + UDP
• cannot make the (a16) bonds found at the branch points of
glycogen
 A branching enzyme:
 Transfers a segment from the end of a glycogen chain to the C6
hydroxyl of a glucose residue of glycogen
 to
07/22/2024
yield a branch with an a(16) linkage
Dr Ambayehu
14
303
Glycogen-branching Enzyme
• Also called:
• Amylo (14) to (16) transglycosylase or
• Glycosyl-(46)-transferase
• Makes branch points of glycogen
• Catalyzes transfer of a terminal fragment of 6 or 7 Glc residues
» from the none reducing end of a glycogen branch having at
least 11 residues
» to the C6 -OH group of a Glc residue at a more interior
position of the same or another glycogen chain
• Branch point should be at least 4 residues from the previous 15
07/22/2024 branch point in the sameDrchain
Ambayehu 304
O
1,4
O
1,4 O
1 ,4
O
1 ,6
O O O O O O O O O O
C o re
1,4 1 ,4 1 ,4 1,4 1,4 1 ,4 1 ,4 1 ,4 1,4 1,4
G l y c o g e n p r i m e r (glucose n )
O O O O O O O O
8 UDP-Glucose
1 ,4 1 ,4 1 ,4 1 ,4 1,4 1,4 1,4
O O O O O O O O
O
G l y c o g e n sy nt hase
1,4 1,4 1 ,4 1 ,4 1 ,4 1,4 1,4 1 ,4 1 ,4
O
1,4
O
1,4 8 UDP
O
1 ,6
O O O O O O O O O O
C o re
1,4 1,4 1 ,4 1,4 1,4 1 ,4 1,4 1,4 1,4
1,4
O O
G l y c o g e n p r i m e r (glucose n + 8 )
O O
O 1,4 1 ,4 1,4
O
1 ,4 O
O
1 ,4 Branching enzyme
1 ,4 O O
1,4
1, 4 O O
1 ,4
1 ,4 1 ,6 1,6
O O O O O O O O O O
C o re
1,4 1 ,4 1,4 1,4 1,4 1 ,4 1,4 1,4 1,4 1 ,4
O
O G l y c o g e n p r i m e r (glucose n + 8 and n ew branch)
1 ,4 O
1 ,4 O O O O O
1 ,4 O
U D P -G lu c o se n
1 ,4 1,4 1,4
O
1 ,4
O O G l y c o g e n sy nt hase
O 1 ,4 1,4
O O
1 ,4 1 ,4
O
1 ,4 O
1 ,4 O 1,4
1, 4 O O UDPn
1 ,4 1 ,6 1,6
O O O O O O O O O O
C o re
1,4 1 ,4 1,4 1,4 1,4 1 ,4 1,4 1,4 1,4
161,4
G l y c o g e n p r i m e r (glucose n + 1 2 )
07/22/2024 Dr Ambayehu 305
Branching of glycogen serves two major roles;
 Increased sites for synthesis &degradation,
• Permitting rapid release of glucose 1-phosphate for muscle activity
 Enhancing the solubility of the molecule
Endurance athletes require a slower, more sustained release of Glc 1-P;

• Formation of branch points in glycogen is slower than the addition of Glc units
to a linear chain.
• Some endurance athletes practice carbohydrate loading:
– Exercise to exhaustion (when muscle glycogen in largely depleted)
– Followed by a high carbohydrate meal,
which results in:
17
07/22/2024
Rapid glycogen synthesis, with fewer branch points than normal.
Dr Ambayehu 306
Glycogen Breakdown
 Glycogenolysis:
–It is the process of glycogen catabolism (or breakdown) into:
– Glucose  to blood, in the liver Or,
– Glucose-6-phosphate  in the skeletal muscles
–It is Not the reverse of glycogenesis; But it is a separate pathway
–In skeletal muscle and liver,
• Glucose units of the outer branches of glycogen enter;
– Glycolytic pathway or to blood through the action of three enzymes:
• Glycogen phosphorylase
• Glycogen debranching enzyme
• Phosphoglucomutase
07/22/2024 Dr Ambayehu 307
Pathway of Glycogenolysis
Site:
– Cytoplasm of all cells but highly activity in liver and muscles
Steps:
– Sequential removal of terminal glucose residues
– By glycogen phosphorylase
– Debranching
– By Bifunctional debranching enzyme
» Transferase activity &(a16) glucosidase activity
– Once branches are transferred & the glucosyl residue at C-6 is hydrolyzed
» Glycogen phosphorylase activity can continue
– Glucose 1-phosphate is converted to glucose 6-phosphate
» By Phosphoglucomutase
07/22/2024 Dr Ambayehu 308
CH 2 OH
O
Glycogen catabolism
H H
H
OH H
2-
(breakdown): OH O P O 3
H OH
g lu c o s e -1 -p h
o s p h a te
 Glycogen Phosphorylase catalyzes:

 Phosphorolytic cleavage of the a(14) glycosidic linkages of
glycogen
 Releasing glucose-1-phosphate as reaction product
Glycogen(n residues) + Pi 
Glycogen (n–1 residues) + Glucose-1-phosphate
20
07/22/2024 Dr Ambayehu 309
 Glycogen Phosphorylase:
 Homodimeric enzyme
 Subject to allosteric control;
 Its transitions between
 Relaxed (active) &
 Tense (inactive) conformations
 A glucose analog, N-acetylglucosamine (GlcNAc):

 is adjacent to PLP at the active site in the crystal structure shown above
21
07/22/2024 Dr Ambayehu 310
 A class of drugs developed
• For treating the hyperglycemia of
diabetes:
 Chloroindole-carboxamides;
 Inhibit liver Phosphorylase
allosterically
 These inhibitors bind at the dimer
interface
 Stabilizing the inactive
(tense)
conformation
 Question:
for diabetes?
 Why would an inhibitor 22
07/22/2024 of Glycogen Dr Ambayehu 311
Steps in glycogenolysis
 A glycogen storage site
 On the surface of the Phosphorylase enzyme
 Binds the glycogen particle
 Given the distance between storage & active sites,
 Phosphorylase can cleave a(14) linkages only to within 4 residues
of an a(16) branch point
 This is called a "limit branch“
23
07/22/2024 Dr Ambayehu 312
24
07/22/2024 Dr Ambayehu 313
Debranching
– By bifunctional enzyme:
• known as oligo (a16) to (a14) glucantransferase
» Transferase activity & (a16) glucosidase activity
– Has 2 independent active sites
 Transferase activity:
– Transfers 3 Glc residues from a 4-residue limit branch to the end of
another branch
– Diminishing the limit branch to a single Glc residue
 a(16) Glucosidase activity:
– Catalyzes hydrolysis of the a(16) linkage, yielding free glucose
The major product of glycogen breakdown is;
 Glucose-1-phosphate, from Phosphorylase activity 25
07/22/2024 Dr Ambayehu 314
G ly c o g e n G luc o s e
H e x o k in a s e o r G lu c o k
in a s eG luc o s e -6
G lu c o s e -1 -P -P a s e i
G l u c Go sl ye c- o6 l- yP s i s G lu c o s e + P
P a th w
a y Py ruva
G l u c o s tee m e t a b o l i s m in liv e r.
 Glucose-6-phosphate:
 May enter Glycolysis or
 Mainly in liver be dephosphorylated for release to the blood
 Liver Glucose-6-phosphatase catalyzes the following,
Glc-6-phosphate + H2O  Glucose + Pi

 Essential to the liver's role in maintaining blood glucose:
26
07/22/2024
 Most other tissues lack Drthis enzyme
Ambayehu 315
G ly c o g e n S y n th e sis
U T P U D P + 2 P i
g ly c o g e n (n ) + g lu c o se -1 -P g ly c o g e n (n + 1)
G ly c o g e n P h o sp h o ry la se P i
 Both synthesis & breakdown of glycogen are spontaneous

–If both pathways were active simultaneously in a cell, there would be a
–Futile Cycle
–With cleavage of one ~P bond per cycle (in forming UDP-glucose).
–To prevent such a futile cycle,
–Glycogen Synthase and Glycogen Phosphorylase are
–Reciprocally regulated by:
•Allosteric effectors & Phosphorylation 27
07/22/2024 Dr Ambayehu 316
Regulation of Glycogenolysis & Glycogenesis
 The principal enzymes controlling glycogen metabolism:
• Glycogen phosphorylase & Glycogen synthase
• Regulated by:
• Allosteric mechanisms
• Covalent modifications
 due to reversible:
Phosphorylation & dephosphorylation of
enzyme protein
• In response to hormone action
28
07/22/2024 Dr Ambayehu 317
Glycogen Phosphorylase in muscle is subject to
Allosteric regulation by AMP, ATP, & Glc-6-phosphate

 A separate isozyme of Phosphorylase expressed in liver is
 Less sensitive to these allosteric controls
 AMP activates Phosphorylase,
 Promoting the relaxed conformation
 ATP & glucose-6-phosphate, which both have
 Binding sites that overlap with that of AMP, inhibit Phosphorylase,
 Promoting the tense conformation
Thus glycogen breakdown is inhibited when
 ATP & Glc-6-phosphate are plentiful

29
07/22/2024 Dr Ambayehu 318
G ly c o g e n G luc o s e
H e x o k in a s e o r G lu c o k
in a s e G lu c o s e -6
G lu c o s e -1 -P -P a s e i
G l u c Go sl ye c- 6o -l yP s i s G lu c o s e + P
P a th w
a y P y ruv
G l u c o s ae t em e t a b o l i s m in live r.
 Glycogen Synthase is allosterically activated by;

– Glc-6-P (opposite of effect on Phosphorylase)
 Thus Glycogen Synthase is active
– When high blood Glc leads to elevated IC Glc-6-P
 It is useful to a cell to store glucose as glycogen
– When the input to Glycolysis (Glc-6-P), and the main product of Glycolysis (ATP),
are
adequate 30
07/22/2024 Dr Ambayehu 319
Allosteric mechanisms
31
07/22/2024 Dr Ambayehu 320
 Regulation by covalent modification (phosphorylation):
The hormones Glucagon & Epinephrine
Activate G-protein coupled receptors to trigger cAMP
cascades
Both hormones are produced in response to low blood Glc
 Glucagon:
 Which is synthesized by a-cells of the pancreas,

 Activates cAMP formation in liver
 Epinephrine:
32
07/22/2024
 Activates cAMP formation in muscle
Dr Ambayehu 321
07/22/2024 Dr Ambayehu 322
 Glycogenolysis is activated in response to:
• Both acute & chronic stress

–The stress may be:
• Physiologic e.g. in response to:
» Increased blood Glc utilization during prolonged exercise
• Pathologic e.g. as a result of:

» Blood loss
• Psychological e.g. in response to:
» Acute or chronic threats
07/22/2024 Dr Ambayehu 323
• In skeletal muscles glycogen:
–a reservoir of glucosyl units for the generation of ATP
»From glycolysis & glucose oxidation
–Muscle glycogenolysis is regulated principally by;

» AMP  which signals a lack of ATP
» Ca2+ released during contraction
» Epinephrine which is released in response to:

• Exercise & other stress situations
07/22/2024 Dr Ambayehu 324

– Epinephrine through the β-adrenergic receptor (cAMP-mediated),
» Providing a supply of carbohydrate for the energy needs of muscle
– This occurs not only during 'fight or flight' situations,
– but also during prolonged exercise
 There are also two important hormone-independent mechanisms:
» for activation of glycogenolysis in muscle
07/22/2024 Dr Ambayehu 325

– First, the influx of Ca2+ into the muscle cytoplasm;
– In response to nerve stimulation
– Activates the basal, unphosphorylated form of phosphorylase kinase
– By action of the Ca2+-calmodulin complex
– A second mechanism for activation of muscle glycogenolysis involves;
– Direct allosteric activation of phosphorylase by AMP
 Activation of muscle glycogen phosphorylase during exercise
07/22/2024 Dr Ambayehu 326

P h o s p h o ry la s e K in a s e in a c tiv e
P h o s p h o ry la s e K in a s e -C a + +
p a rtly a c tiv e
P -P h o s p h o ry la s e K in a s e -C a + +
fu lly a c tiv e
 Ca++ regulates glycogen breakdown in muscle:

• During activation of contraction in skeletal muscle,
» Ca++ is released from the sarcoplasmic reticulum to cytosol;
• to promote actin/myosin interactions
• The released Ca++ also activates Phosphorylase Kinase,
» which in muscle includes calmodulin as its d-subunit
• Phosphorylase Kinase is partly activated by
» binding of Ca++ to this subunit
07/22/2024 Dr Ambayehu 327
Activation of muscle glycogen phosphorylase during exercise
07/22/2024 Dr Ambayehu 328
Hormones involved in control of Glycogenolysis
Effect on
Hormone Source Initiator Glycogenolysis
Glucagon pancreatic α-cells Hypoglycemia Rapid activation
Acute stress,
Epinephrine adrenal medulla
Hypoglycemia
Rapid activation
Cortisol adrenal cortex Chronic stress Chronic activation
Insulin pancreatic β-cells Hyperglycemia Inhibition

40
07/22/2024 Dr Ambayehu 329

Regulation of Liver Glycogen
Stores
State Regulators Response of Tissue
Blood: Glucagon 
Fasting Glycogen degradation 
Insulin 
Glycogen synthesis 
Tissue: cAMP 
Blood: Glucagon 
Insulin  Glycogen degradation 
Carbohydrate meal Glucose  Glycogen synthesis 
Tissue: cAMP 
Glucose 
Blood: Epinephrine  Glycogen degradation 

Exercise and stress Tissue: cAMP  Ca2+ - Glycogen synthesis 
calmodulin 
41
07/22/2024 Dr Ambayehu 330

Regulation of Muscle Glycogen Stores
State Regulators Response of Tissue
Fasting (rest) Glycogen synthesis 

Blood: Insulin 
Glucose transport 
Carbohydrate meal (rest) Glycogen synthesis 

Blood: Insulin 
Glucose transport 
Blood: Epinephrine 
Glycogen synthesis 
Exercise Tissue: AMP 
Glycogen degradation 
Ca2+ -calmodulin 
Glycolysis 
cAMP 
42
07/22/2024 Dr Ambayehu 331

Glycogen Storage Diseases
• “Glycogen storage disease”
• is a generic term to describe a group of inherited disorders
• characterized by deposition of an abnormal type or quantity of

glycogen in the tissues.
–Glycogen storage diseases are genetic enzyme deficiencies.
07/22/2024 Dr Ambayehu 332

Glycogen Storage Diseases (GSD)
 Type 0
 deficiency of liver glycogen synthase
• fasting hypoglycemia
• ketosis and
• early death
07/22/2024 Dr Ambayehu 333

 Question:
 How would you nutritionally treat deficiency of liver Glycogen
Synthase?
 Frequent meals of complex carbohydrates
 Avoiding simple sugars that would lead to a rapid rise in blood
glucose
 Meals high in protein to provide substrates

 For gluconeogenesis
45
07/22/2024 Dr Ambayehu 334
 Type I (von Gierke’s disease)
 Deficiency:
 G-6-phosphatase (Type Ia)
 G-6-P translocase (Type Ib)
 G-6-P = high :  GS
 GP
• Accumulation of glycogen=>Hepatomegaly
 Low blood glucose= Glucagon => Gluconeogesis end with high amount of G-
6-P, but unable to be converted in to glucose
 Renomrgally
 Failure: Glycogenolysis and Gluconeogenesis
 Sevier fasting hypoglycemia
 Dislipdemia
 Lactic acidosis
 hyperuricemia
07/22/2024 Dr Ambayehu 335
 Type II (Pompe’s disease)
• an autosomal recessive disorder
• lysosomal storage disease
• deficiency of lysosomal a-1,4 and a-1,6-glycosidase (acid
maltase).
• Glycogen accumulates in lysosomes of all tissues, mianly:
 Skeletal muscle; myopathy (muscle weakness), muscular
dystrophy
 Cardiac muscle; cardiomegaly, heart failure
07/22/2024 Dr Ambayehu 336

 Type III (Limit Dextrinosis, Cori’s or Forbes' disease)
• deficiency of liver glycogen debranching enzyme.
• stored glycogen is abnormal “Limit Dextrin Type” with short and

missing outer chains.
 Hepatomegaly
 Miled hypoglycemia
07/22/2024 Dr Ambayehu 337

 Type IV (Andersen’s disease)
• deficiency of the Glycogen branching enzyme

• accumulation of abnormal glycogen with fewer
branching points and longer outer branches
• Activation Immune system
 Cirrhosis
 Liver transplantation seems to be the only successful

treatment
07/22/2024 Dr Ambayehu 338

 Type V (McArdle’s disease)
• partial or total lack of muscle glycogen phosphorylase:
• Glycogen is accumulated in the skeletal muscles in an

abnormally high amount (2.5-4.1%)
• Blood lactate did not increase after exercise
• muscle cramps and damage occurs leading to

myoglobinuria during exercise
07/22/2024 Dr Ambayehu 339
 Type VI (Hers'disease)
• partial or total lack of liver glycogen phosphorylase:
– moderate hypoglycemia
– trigger oxidation of fatty acids causing ketosis
– prominent Hepatomegaly
07/22/2024 Dr Ambayehu 340

52
07/22/2024 Dr Ambayehu 341
Glycogen Storage Diseases
Glycogenosis &
Name Cause of Disorder Characteristics
Liver cells and renal tubule cells loaded with

Type I- Von Gierke’s Deficiency of Glc-6-phosphatase glycogen. Hypoglycemia, lactic acidemia, ketosis,
disease hyperlipemia
Type II- Pompe’s Deficiency of lysosomal α-1→4- & Fatal, accumulation of glycogen in lysosomes,
disease 1→6-glucosidase heart failure.
Type III- Limit Accumulation of a characteristic branched

dextrinosis, Forbes’ or Absence of debranching enzyme
Cori’s disease polysaccharide.
Accumulation of a polysaccharide having few

Type IV-Amylopectinosis, branch points. Death due to cardiac or liver failure
Andersen’s disease Absence of branching enzyme
in first year of life. 53
07/22/2024 Dr Ambayehu 342

Glycogen Storage…(Cont’d)
Glycogenosis &
Name Cause of Disorder Characteristics
Type V- Myophosphorylase Diminished exercise tolerance; muscles have

deficiency, McArdle’s Absence of muscle phosphorylase abnormally high glycogen content (2.5–4.1%).
syndrome Little or no lactate in blood after exercise.
High glycogen content in liver, tendency toward

Type VI- Hers’ disease Deficiency of liver phosphorylase hypoglycemia.
Deficiency of phosphofructokinase As for type V but also possibility of hemolytic

Type VII- Tarui’s disease in anemia.
muscle and erythrocytes
Deficiency of liver
Type VIII phosphorylasekinase As for type VI.
54
07/22/2024 Dr Ambayehu 343

The end!
07/22/2024 Dr Ambayehu 344

Chapter Seven
Lipid metabolism
07/22/2024 Dr Ambayehu 345

Objectives
• Define lipid metabolism.
• Lipogenesis, and lipolysis
• Ketone body metabolism.
07/22/2024 Dr Ambayehu 346

Dietary lipids
• 90% triacylglycerol (TAG)
• 10%: cholesterol (most dietary cholesterol is unesterified)
• + cholesteryl esters
• + Phospholipids + non-esterified fatty acids (NEFAs).
07/22/2024 Dr Ambayehu 347

Digestion and absorption of lipids.
• The digestion of lipids begins in the stomach, catalyzed by a lipase
(lingual lipase) that originates from glands at the back of the tongue.
• TAG molecules, particularly those containing fatty acids of short- or
medium-chain length (fewer than 12 carbons such as are found in milk
fat), are the primary target of this enzyme.
• These same TAGs are also degraded by a separate gastric lipase, secreted
by the gastric mucosa.
• Both enzymes are relatively acid stable, with pH optimums of pH 4 to pH
6. These “acid lipases” play a particularly important role in lipid digestion
in neonates, for whom milk fat is the primary source of calories.
07/22/2024 Dr Ambayehu 348
Digestion in Intestines
• Emulsification is a pre-requisite for digestion of lipids.
• The lipids are dispersed into smaller droplets; surface tension is reduced;
and surface area of droplets is increased.
• This process is favored by:
1. Bile salts (detergent action)
2. Peristalsis (mechanical mixing)
3. Phospholipids.
• The bile salts present in the bile (sodium glyco cholate and sodium
taurocholate) lower surface tension. They emulsify the fat droplets.
• The emulsifiation increases the surface area of the particles for enhanced
activity of enzymes.
07/22/2024 Dr Ambayehu 349
Digestion of TAG
• In mouth: no digestion
• In stomach: gastric lipase is active only in infant stomach (pH is 4-5)
and is not functioning in adult stomach (pH is 2)
• lingual lipase – for TAG molecules containing short or medium chain Fatty
acids
• In the duodenum: The chyme (meal contents) is exposed to:
1. Bile that contains bile salts - Emulsification
2. Pancreatic juice that contain sodium bicarbonate making the
contents alkaline (favorable for the action of pancreatic lipase).
07/22/2024 Dr Ambayehu 350

• Pancreatic lipase:
• It splits two ester linkages (C1 and C3) in the TAG molecule to 2 moles of
Fatty acid and 2- monoacylglycerol (2MG).
• Co-lipase: The binding of co-lipase to the triacylglycerol molecules at the oil
water interface is obligatory for the action of lipase.
• The 2-monoacylglycerol may be
• absorbed as such
• isomerized to 1-monoacylglycerol
• further digested by pancreatic lipase to yield free glycerol and the third fatty acid of TG.
07/22/2024 Dr Ambayehu 351

07/22/2024 Dr Ambayehu 352
Hydrolysis of Phospholipids
• Phospholipids (PL) is hydrolyzed by pancreatic phospholipase
A2(PLA2) that splits off fatty acid in C2 leaving lysophospholipid.
• FA (in the lysophospholipid) is removed by lysophospholipase
leaving glycerophospho-base.
• glycerophospho-base is either
• further split to its components
• absorbed as such (being water soluble) or
• pass out with stools
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07/22/2024 Dr Ambayehu 354
Digestion of Cholesterol ester
• CE undergo hydrolysis by pancreatic cholesterol ester
hydrolase (cholesterol esterase) to free cholesterol and
fatty acid.
07/22/2024 Dr Ambayehu 355

07/22/2024 Dr Ambayehu 356
Absorption of lipids
• Formation of mixed micelles
• Long chain FAs, 2-MG, cholesterol, lysophospholipid + bile salts form complex
molecules (micelles)
• Micelle is heterogeneous mixture of hydrophilic and hydrophobic components. The
micelles are spherical particles with a hydrophilic exterior and hydrophobic interior core.
• Micellar formation is essential for the absorption of fat-soluble vitamins such as vitamin
A, D and K
• The micelles get access into enterocytes. In these cells, long chain fatty acids are
activated into acyl CoA( re-esterified to form TAG).
• Short- and medium-chain fatty acids and glycerol are pass through the basal border into
the portal vein.
• Utilization of Acyl CoA
a) Reformation of triacylglycerol (TAG) from MAG
b) Cholesterol ester from free cholesterol
07/22/2024c) Phospholipid from lysophospholipid. Dr Ambayehu 357
Chylomicrons
• They are composed of core of TG, cholesterol esters that are
enveloped by phospholipids and free cholesterol, and the
outer-most layer is hydrophilic coat of apolipoprotein B48.
• They pass out of enterocytes by exocytosis through side
borders of the cells and taken up by lymph vessel terminals
(lacteals).
07/22/2024 Dr Ambayehu 358

Lipid malabsorption (steatorrhea):
• Is the loss of > 6g of fat together with fat soluble vitamins (A,D,E,K) and essential fatty
acids.
• Causes of steatorrhea:
 Defective digestion: due to deficiency of pancreatic lipase as a result of chronic
pancreatitis, cystic fibrosis and severe gastric hyperacidity (Zollinger-Ellison syndrome,
obstruction of pancreatic duct by tumors and pancreatectomy.
 Fecal fat is mostly undigested TAGs.
 Defective absorption: due to deficiency of bile salts as a result of bile duct obstruction
as in tumors or stones of the bile duct and in some cases of hepatitis and liver cirrhosis.
 Fecal fat is in the form of 2-monoacylglycerol.
07/22/2024 Dr Ambayehu 359

07/22/2024 Dr Ambayehu 360
Fate of absorbed fat
 TAG content of chylomicrons is hydrolyzed by lipoprotein
lipase, LPL (bound to capillary endothelium).
 LPL is activated by Apo-CII and insulin
 TAG is splitted to:
 Glycerol (utilized by the liver for gluconeogenesis)
 Fatty acids enter the cells for further metabolism
07/22/2024 Dr Ambayehu 361

Fate of glycerol
• Glycerol is absorbed by the liver and converted to glycolytic
intermediates.
07/22/2024 Dr Ambayehu 362

Fate of free-fatty acids
 Directly enter adjacent muscle cells or adipocytes
 Alternatively, free fatty acids may be transported in blood in association with

serum albumin, until they are taken by cells
 Most cells can oxidize fatty acids to obtain energy (ATP)

 However, brain and other nervous tissues, erythrocytes and the adrenal
medulla cannot use plasma free fatty acids for fuel
 Adipocytes can also re-esterify free-fatty acids to produce triacylglycerol

molecules, which are stored until the fatty acids are needed
07/22/2024 Dr Ambayehu 363
Fate of chylomicron remnants
 After removal of most of its triacylglycerol (TAG) content they are taken up
by the liver where, they are hydrolyzed to their component parts.
This is due to the presence of apolipoprotein E on the surface of chylomicrons

which has apolipoprotein E receptors in hepatic cells plasma membranes.
Deficiency of apolipoprotein E, leads to familial type III hyperlipoproteinemia

(familial dys-betalipoproteinemia), which leads to defective removal of
chylomicron-remnants from the plasma, and they accumulate in plasma.
07/22/2024 Dr Ambayehu 364
Fatty Acid Metabolism
• FFAs have to be activated in form of acyl CoA by making high-
energy thioester bond with CoA SH.
• Thioester bond conserves energy content higher than high- energy
phosphate bond.
07/22/2024 Dr Ambayehu 365

Fatty Acid Oxidation
 β -oxidation is the main pathway that occurs in mitochondria (all
tissues).
 Preparatory steps of β - oxidation :
 Formation of acyl CoA , consuming two high-energy phosphate bonds
 Entry of acyl CoA into the mitochondria
 This necessitates the presence of carnitine. It is either provided in diet
or synthesized by the liver or kidney.
 The fatty acid gets access into the mitochondria utilizing carnitine and
carnitine-palmitoyl transferase I (CPT- I) in outer mitochondrial
membrane.
07/22/2024 Dr Ambayehu 366
Cont…
 Acyl carnitine passes into the mitochondrial

matrix via carnitine acyl-carnitine translocase.
 In the matrix acyl part is transferred to CoA to
form acyl CoA catalyzed by carnitine palmitoyl
carnitine transferase-II (CPT-II) located in the
inner-mitochondrial membrane.
 This sets Carnitine free.
 It is ejected out to the inter-membrane-space
using carnitine acyl-carnitine translocase
07/22/2024 Dr Ambayehu 367

FIGURE: Fatty acid entry into mitochondria via the acyl-carnitine/carnitine
transporter
07/22/2024 Dr Ambayehu 368
Dr Ambayehu
Carnitine clinical aspects
 Carnitine deficiency may be
 Inherited or primary causes due to deficiency of
 CPT-I(carnitine palmitoyltransferase I) in liver and is presented
as fasting hypoglycemia due to failure of gluconeogenesis and
increased glucose consumption.
 It is associated with Fatty liver.
 CPT II affects skeletal and cardiac muscles causing
cardiomyopathy and muscle weakness. Fat vacuoles
accumulate in the muscle due to lack of long chain FA
oxidation.
 Prolonged exercise is accompanied with myoglobinemia
07/22/2024 36 369
9
 Acquired or secondary causes are:
 Hemodialysis (for renal failure) due to escape of carnitine in the
dialysate
 Chronic liver disease (lack of synthesis)
 Malnutrition (also in vegetarians)
 pregnancy
 Sever trauma, burns or sever infections
 The presenting symptoms are:
 muscle fatigability
 muscle weakness and wasting
 myoglobinemia with prolonged exercise
 Improving the condition
 Intake of milk-derived fat (rich in medium-
chain fatty acids, they do not need carnitine as
transporter.)
 Adequate carbohydrates in diet
07/22/2024
 Avoidance
8/29 /2 016
of fastingwithDr Ambayehu
s u pplementation 83 370
Dr Ambayehu  β-Oxidation of Fatty acids
 The reactions are rather cyclic

 In each cycle two-carbon units (acetyl CoA) is lost from
the hydrocarbon chain of acyl CoA running of the
cycle.
 The shortened acyl CoA is recycled, loosing other
C2- units
 In each cycle, FADH2 and NADH are produced
generating 5ATP(?) at the level of electron-transport
chain.
07/22/2024 371
07/22/2024 Dr Ambayehu 372
07/22/2024 Dr Ambayehu 373
Energy Production
07/22/2024 8/29/2016 87
Dr Ambayehu 374
 Oxidation of odd-chain fatty acids
Dr Ambayehu
 Final step of β-oxidation produces:

 propionyl CoA + acetyl CoA
1. Synthesis of D-methylmalonyl CoA:
 propionyl CoA is carboxylated, forming D-methylmalonyl
CoA
 Enzyme propionyl CoA carboxylase: (biotin-dependent)
 propionyl CoA + ATP + CO2 + biotin +Mn2+ →
D- methylmalonyl CoA + ADP+ Pi
07/22/2024 375
2. Formation of L-methylmalonyl CoA:
 the D-isomer is converted to the L-form by methylmalonyl-
CoA
epimerase
3. Formation of succinyl CoA

 The L-methylmalonyl-CoA then undergoes an
intramolecular rearrangement to form succinyl-CoA
 L-methylmalonyl CoA mutase: (adenosyl cobalamin-
dependent)
 methylmalonyl CoA → succinyl CoA
07/22/2024 Dr Ambayehu 376

Dr Ambayehu
Q
• When propionyl CoA enters TCA cycle how
many ATP will be harvested?
• What is the total ATP yield for the oxidation of 1

mole of stearic acid to carbon dioxide and water?
07/22/2024 377
Dr Ambayehu
 oxidation of unsaturated fatty acids
07/22/2024 378
 Alternate routes of FA
oxidation
A. Peroxisomal Oxidation of Fatty Acids
VERY-LONG-CHAIN FATTY ACIDS
07/22/2024 Dr Ambayehu 92 379

The differences of FA oxidation in Peroxisomes and
mitochondria
1. In peroxisomes, the flavoprotein acyl-CoA oxidase that
introduces the double bond passes electrons directly to O2,
producing H2O2.
What happen in mitochondria?
2. the specificity for fatty acyl–CoAs;
 the peroxisomal system is much more active on very-long
chain fatty acids of 24 to 26 C atoms and
 on branched-chain fatty acids such as phytanic acid and
pristanic acid
• Their catabolism in the peroxisome involves several
auxiliary enzymes unique to this organelle.
07/22/2024 Dr Ambayehu 380
B. ω-Oxidation of Fatty Acids
07/22/2024 Dr Ambayehu 381

C. α- Oxidation of fatty acids
• Branched chain fatty acid (phytanic acid) that contains
four side-chain methyl groups is of plant origin undergoes
α- oxidation.
• These acids cannot be substrate of acylCoA dh because of
the β-methyl group.
• The α -carbon is hydroxylated (by α- hydroxylase) to α-

hydroxy-acid, then, dehydrogenated to α- ketoacid that
undergoes oxidative decarboxylation to α-methyl fatty acid
• Then, β-oxidation proceeds giving propionyl CoA instead
of acetyl CoA.
07/22/2024 Dr Ambayehu 382

 Phytanic acid is present in:
– plants
– milk and dairy products in association with milk fat.
Lack of α-hydroxylase leads to accumulation of
phytanic acid in the blood and tissues. This
results in:
 Neurological disturbances in form of cerebellar ataxia
 Polyneuropathy
 Blindness and deafness
 Treatment:
• Restriction of dairy products ingestion
(withdrawal of phytanic acid).
07/22/2024 Dr Ambayehu 383
07/22/2024 Dr Ambayehu 384
 Regulation of β-Oxidation
• Regulation of β–oxidation. (1) Hormones control the supply of fatty

acids in the blood. (2) Carnitine:palmitoyl transferase I is inhibited
by malonyl CoA, which is synthesized by acetyl CoA carboxylase
(ACC). AMP-PK is the AMP-dependent protein kinase. (3) The rate
of ATP utilization controls the rate of the electron transport chain,
which regulates the oxidative enzymes of β -oxidation and the TCA
07/22/2024 Dr Ambayehu 98 385
c8y/2c9/
 Genetic Deficiencies in the Acyl CoA Dehydrogenases
• Impair β-oxidation of fatty acids at different stages of the
chain shortening process.
 The best characterized is the medium chain acyl CoA
dehydrogenase (MCAD), deficiency
 usually manifests itself within the first 2 years of life after a
fasting period of 12 h or more.
• Typical symptoms include vomiting, lethargy, and
frequently coma, accompanied by hypoketotic
hypoglycemia and dicarboxylic aciduria.
• Management: low-fat, high carbohydrate diet; including
avoiding long intervals between meals.
07/22/2024 Dr Ambayehu 386 386

 Metabolism of Ketone Bodies
 The precursor of KB is acetyl CoA.
 Ketone bodies are water and fat soluble.
 They are:
 aceto-acetate (the parent)
 3-hydroxybutyrate
 acetone (volatile non-metabolizable substance)
 Excretion:
– In urine.
– in expired air (ketotic breathing of acetone)
07/22/2024 Dr Ambayehu 387 387

Ketone body synthesis
Definition: It is formation of ketone bodies
Site:
 Occurs in hepatic mitochondria
 It occurs during states in which fat is the major energy
substrate
 Starvation
 uncontrolled diabetes mellitus
 high-fat, low-carbohydrate diet
 There is insulin deficiency and anti-insulin hormones
predominates.
 Flux of fatty acids from adipose tissue increases
 β-oxidation in the liver makes acetyl CoA to
accumulate.
07/22/2024 Dr Ambayehu 102 388
Figure: Formation of
ketone bodies from acetyl-
CoA.
Healthy, well-nourished
individuals produce ketone
bodies at a relatively low rate.
When acetyl-CoA
accumulates (as in starvation
or untreated diabetes), thiolase
catalyzes the condensation of
two acetyl- CoA molecules to
acetoacetyl-CoA
07/22/2024 Dr Ambayehu 103 389

Cont…
 Acetyl CoA inhibits pyruvate DH and activates pyruvate

carboxylase
 Oxaloacetate production increases, favoring
gluconeogenesis
 Acetoacetyl CoA (the beginner of ketogenesis) can be
produced:
 During β-oxidation
By condensation of two moles of acetyl CoA, in a
reaction catalyzed by thiolase
 A third mole of acetyl CoA is added to acetoacetyl CoA
to form 3-hydroxy-3- methylgluteryl CoA (HMGCoA) in
presence of HMG-CoA synthase.
07/22/2024 Dr Ambayehu 390 390
 Rearrangement of HMG CoA occurs by HMG-CoA lyase
and acetyl CoA is released with formation of acetoacetate.
 Fate of Acetoacetate:
 released as such, from liver
reduced to 3-hydroxybuterate if NADH/NAD+ ratio
is raised
decarboxylated to acetone.
 Ketosis: The three ketone bodies rises in blood (ketonemia)
and they are excreted in urine (ketonuria).
 This leads to acidosis particularly in uncontrolled diabetes
mellitus (diabetic ketoacidosis).
07/22/2024 Dr Ambayehu 391 391

 Ketolysis
• Definition: It is oxidation of ketone bodies
• Site: Occurs in extra hepatic mitochondria
(particularly skeletal and cardiac muscles and brain)
• 3-hydroxybutyrate is oxidized in reversible reaction to that
of ketogenesis, to acetoacetate.
• Acetoacetate is activated to acetoacetyl CoA by either of
two mechanisms:
 Reacting with succinyl CoA by enzyme acetoacetate CoA
transferase (thiopherase). It is the main pathway. This enzyme is
lacking in the liver.
 Activation with specific acetoacetate thiokinase (of
less importance).
• Acetoacetyl CoA undergoes thiolysis into two moles of
07/22/2024 acetyl CoA by thiolase (analogous
Dr Ambayehu to that is used in 106 392
8/2 9/ 20 16
FIGURE D-β-
Hydroxybutyrate as a fuel.
D-β-Hydroxybutyrate,
synthesized in the liver,
passes into the blood and
thus to other tissues, where
it is converted in to
acetyl- CoA.
07/22/2024 Dr Ambayehu 107 393

Dr Ambayehu
FIGURE: Ketone body formation and export from the liver.

07/22/2024 394 394
Dr Ambayehu
07/22/2024 395 395

Q
Dr Ambayehu
• How ketone bodies are overproduced in

Diabetes and during Starvation?
• Why Ketogenic diets are used to treat children
with pyruvate dehydrogenase deficiency?
• Why can’t red blood cells use ketone bodies
for energy?
• Oxidation of Ketone Bodies as Fuels
 β-hydroxybutyrate – 21.5 ATP
 Acetoacetate – 19 ATP
07/22/2024 396 396

 Lipogenesis
A triacylglycerol with an unsaturated

fatty acid on carbon 2
07/22/2024 Dr Ambayehu 397

Steps of synthesis of triacylglycerols(TAGs)
i. Synthesis of glycerol-3-phosphate
ii. Conversion of a free-fatty acid to its activated form fatty
acyl CoA, catalyzed by Fatty acyl CoA synthetase
(thiokinase)
iii. Synthesis of a molecule of TAG from glycerol-3-p’hate

and fatty acyl- CoA
07/22/2024 Dr Ambayehu 398

Sources of glycerol-3-phosphate for synthesis of TAG in the liver and
adipose tissue
Figure: Pathways for production of glycerol phosphate in liver and

adipose tissue
07/22/2024 Dr Ambayehu 399
 The first stage in the biosynthesis
of triacylglycerols is
 the acylation of the two free
hydroxyl groups of L-glycerol 3-
phosphate by two molecules of fatty
acyl–CoA to yield diacyl-glycerol 3-
phosphate, more commonly called
phosphatidic acid
 Why People with severe

diabetes mellitus - lose weight?
07/22/2024 Figure
Dr Ambayehu Phosphatidic acid in lipid biosynthesis 400
Regulation of glyceroneogenesis
07/22/2024 Dr Ambayehu 401

Regulation of triacylglycerol synthesis by insulin
07/22/2024 Dr Ambayehu 402

 Adipose tissue
stores TAG as depot fat.
Liver
TAG is derived from locally synthesized FA, or other extra hepatic
sources.
It is exported to extra hepatic tissues in form of VLDL.
This process necessitates lipotropic factors.
These factors are important for synthesis of phospholipids,
particularly lecithin required for synthesis of VLDL.
07/22/2024 Dr Ambayehu 403

 Types of body fat
Depot fat (stored fat)
 It is a fat stored in the fat cells of adipose tissues. The amount and
composition of depot fat varies according to the nutritional state of the
individuals so it is called variable elements.
 Source: The origin of depot fat is dietary fat and lipogenesis.
 Composition: Triglycerides mainly
 Fate: Source of energy for body by first lipolysis (release of fatty
acids from fats) by hormone sensitive lipase (HSL) which is inhibited
by insulin and activated by adrenaline and glucagon.
 Tissue fat (constant element)

Def. It is the fat present in each cell. It is the lipids that enter in the
structure of body cells as cell membrane and mitochondria
It is not affected by hormones
It is never used as source of energy i.e. never oxidized to give energy
07/22/2024 Dr Ambayehu 404
LIPOLYSIS
It is breakdown of TAG stored in adipose tissue

It is under control of catecholamines
The key enzyme is hormone-sensitive lipase that releases a
terminal FA (in C1 and C3) in TAG molecule
The remaining fatty acids are released by monoacylglycerol
lipase
Hormone-sensitive lipase is active in phosphorylated form.
Phosphorylation is mediated by cAMP that is synthesized
by adenylyl cyclase under control of epinephrine
07/22/2024 Dr Ambayehu 405

 Mobilization of
triacylglycerols
stored in adipose
tissue
07/22/2024 Dr Ambayehu 406

• cAMP is degraded by phosphodiesterase that is activated by
insulin. That is why insulin inhibits lipolysis.
• FFA circulates bound to plasma albumin.
• Its entry through cell membrane, particularly in muscle and
heart, is mediated by cell membrane fatty acid binding
protein.
• Blood brain barrier is impermeable to long chain fatty acid
but not ketone bodies.
07/22/2024 Dr Ambayehu 407

Hormone Sensitive Lipase
07/22/2024 Dr Ambayehu 408

 Fatty liver
• Imbalance in the Rate of TAG Formation & Export
• large vacuoles of fat accumulate in the liver, resulting from:
• Overfeeding of carbohydrates
• Increased fat intake
• Over-mobilization of fat from depot to liver
• Impaired fatty acid oxidation
• Deficiency of pantothenic acid
• Abnormality in Carnitine shuttle
• Alcoholism
• Decreased mobilization of fat from liver to blood
• Decreased protein synthesis
• Decreased phospholipids synthesis
07/22/2024 Dr Ambayehu 409

 Fatty livers fall into two main categories
1. The first type is associated with raised levels of plasma FFAs
resulting from
 Mobilization of fat from adipose tissue or
 From the hydrolysis of lipoprotein TAG by LPL in extra-
hepatic tissues
 The production of VLDL does not keep pace with the increasing
influx and esterification of FFAs, allowing TAG to accumulate,
07/22/2024 Dr Ambayehu 410

2. The second type of fatty liver is usually due to
A metabolic block in the production of plasma
lipoproteins (VLDL),thus allowing TAG to
accumulate in the liver cells.
 Theoretically, the lesion may be due to

i. a block in apo-lipoprotein synthesis,
ii. a block in the synthesis of the lipoprotein
from lipid and apolipoprotein
iii. a failure in provision of phospholipids that
are found in lipoproteins, or
iv. a failure in the secretory mechanism itself.
07/22/2024 Dr Ambayehu 411
 One type of fatty liver that has been studied extensively in
rats is due to a deficiency of choline, which has therefore been
called a lipotropic factor.
 The antibiotic puromycin, ethionine (α-amino-γ-
mercaptobutyricacid), CCl4, lead, and arsenic all cause fatty
liver and a marked reduction in concentration of VLDL in rats.
 The action of CCl4 probably involves formation of free

radicals causing lipid peroxidation.
 The action of ethionine is thought to be due to a reduction

in availability of ATP
07/22/2024 Dr Ambayehu 412

 Lipotropic factors
Factors help mobilization of TAG from liver. Thus helping
the formation of VLDL. They include:
substance essential for biosynthesis of phospholipids:
Essential FA, Inositol, Choline
Substance required for choline biosynthesis
methionine, glycine, folic acid and vit. B12.
substance essential for biosynthesis of proteins;
Proteins of high biological value
07/22/2024 Dr Ambayehu 413

 Ethanol Also Causes Fatty Liver
 Alcoholism leads to fat accumulation in the liver, hyperlipidemia,
and ultimately cirrhosis.
 Ethanol consumption over a long period leads to the accumulation
of fatty acids in the liver that are derived from endogenous synthesis.
 Oxidation of ethanol by alcohol dehydrogenase leads to the
production of acetaldehyde and excess NADH
 Acetaldehyde, which is oxidized by aldehyde
dehydrogenase, produces acetate.
 The NADH generated inhibiting β oxidation, and
decreasing activity of the citric acid cycle
 The net effect is to increased esterification of fatty acids in
TAG, resulting in the fatty liver.
07/22/2024 Dr Ambayehu 414

 Plasma lipoproteins
a. Chylomicrons (CM).
b. Very low density lipoproteins (VLDL). (Pre β-lipoprotein)
c. Low density lipoproteins (LDL). (β-lipoprotein)
d. High density lipoproteins (HDL). (α-lipoprotein)
07/22/2024 Dr Ambayehu 415

Lipoproteins
• Lipoproteins (LP) are particles
synthesized by the liver or intestine.
• LP are composed of apolipoproteins,
polar lipid envelope [phospholipids (PL)
and free cholesterol], Non polar core of
triacylglycerol (TG) and cholesterol ester.
• They are spherical whose diameter 10-
1200 nm.
07/22/2024 Dr Ambayehu 416

Function of Lipoproteins
 keep lipids soluble as they transport them in plasma,&
 To provide an efficient mechanism for delivering their
lipid contents to the tissues.
 In humans the delivery system is less perfect than in other

animals and as a result, humans experience a gradual
deposition of lipid , especially cholesterol in tissues.
 This is a potentially life-threatening occurrence when
cholesterol deposition contributes to plaque formation,
causing narrowing of blood vessels, a condition known as
atherosclerosis !!
07/22/2024 Dr Ambayehu 417

Composition of the plasma lipoproteins. Keys
Note the high concentration of
cholesterol and cholesteryl esters in
LDL.
07/22/2024 Dr Ambayehu 418
 Electrophoresis of lipoproteins
LPs move in the electric field according to the charges they
carry and their molecular size.
 HDL being rich in phospholipids and apoproteins, they are more
polar and escape away from the site of sample application than
other types of LP.
They are α-lipoprotein.
 Chylomicrons are the most non-polar, they fail to move in the
electric field.
LDL is slightly more polar than chylomicrons. So, they make β-
lipoprotein.
 VLDL moves faster than LDL. VLDL is then, pre β lipoprotein.
07/22/2024 Dr Ambayehu 419

 Apolipoproteins (Apoproteins)
 The apolipoproteins associated with lipoprotein particles
have a number of diverse functions including:
1. Serving as structural components of the particles.

2. Providing recognition sites for cell – surface receptors.
3. Serving as activators or coenzymes involved in
lipoprotein metabolism.
 Apolipoproteins are divided by structure and function

into classes A to H, with most classes having subclasses,
for example, apo A- I and apo C- II.
07/22/2024 Dr Ambayehu 420

Table; Major Apolipoproteins
Apolipoprotein Primary Source Lipoprotein Association Function
ApoA-I Intestine, liver HDL, CMs Structural protein for HDL

Activates LCAT
ApoA-II Liver HDL, CMs Structural protein for HDL
ApoA-IV Intestine HDL, CMs Unknown

ApoA-V Liver VLDL, CMs Promotes LPL-mediated
triglyceride lipolysis
ApoB-48 Intestine CMs Structural protein for chylomicrons
ApoB-100 Liver VLDL, IDL, LDL, Lp(a) Structural protein for VLDL, LDL,
IDL, Lp(a)
Ligand for binding to LDL receptor
07/22/2024 Dr Ambayehu 421

Cont…
ApoC-I Liver CMs, VLDL, HDL Unknown
ApoC-II Liver CMs, VLDL, HDL Cofactor for LPL
ApoC-III Liver CMs, VLDL, HDL Inhibits lipoprotein

binding to receptors
ApoD Spleen, brain, testes, HDL Unknown

adrenals
ApoE Liver CM remnants, IDL, Ligand for binding to

HDL LDL receptor
ApoH Liver CMs, VLDL, LDL, B2 glycoprotein I

HDL
07/22/2024 Dr Ambayehu 422
I. Metabolism of chylomicrons
 Chylomicrons are assembled in intestinal mucosal cells and carry

dietary TAG, C and CE (plus additional lipids made in these cells) to
peripheral tissues.
 Assembly of chylomicrons
 Apolipoprotein synthesis begins on the rough endoplasmic

reticulum
 apolipoproteins are glycosylated as they move through the
ER and Golgi.
 The enzymes involved in TAG, cholesterol and
phospholipid synthesis are located in the smooth ER.
07/22/2024 Dr Ambayehu 423
07/22/2024
Metabolism of chylomicrons.
Dr Ambayehu 424
 This animation shows how chylomicrons are metabolised once they enter the
circulation from the lymphatic system
B48 Tissues
CM CII
E Lipoprotein
B48 lipase
Taken up by CII
E CMR
liver (via LDL receptors)
E CII
B48
HDL
E CMR CII Capillary wall
(endothelial surface)
07/22/2024 Dr Ambayehu 425

 In tissues, TAG content is hydrolyzed by LPL that is
activated by apo C-II and insulin and remnant
chylomicrons are left.
 Remnant chylomicrons are taken up by the liver mediated by
remnant receptors and are hydrolyzed by liver lipase.
 Therefore, dietary fat are consumed by peripheral tissues and

liver.
 In the liver, the FFA derived from remnant chylomicrons are
incorporated in TG to be carried by VLDL out of the liver.
 Deficiency of LPL results in hyperlipidemia type I.
07/22/2024 Dr Ambayehu 426

II. Metabolism of VLDL
• hepatic in origin.
• carry TG synthesized by the liver from FFA of the remnant
chylomicron and from lipogenesis to peripheral tissues.
• Carry apo-lipoproteins (Apo-B 100, Apo-C, and Apo-E).
• In tissues, VLDL is partially hydrolyzed by LPL leaving
remnant VLDL and LDL.
• Remnant VLDL is taken up by the liver.
07/22/2024 Dr Ambayehu 427

07/22/2024  Metabolism of VLDL and LDL.
Dr Ambayehu 428
This animation shows how VLDL are metabolised once they enter the
circulation from the liver
B100 Tissues
VLDL B100
LDL Lipoprotein
lipase
Some LDL taken up E CII
by liver (LDL receptors)
Having lost TAG to tissues
LDL contains a large
proportion of
cholesterol/cholesterol esters
Capillary wall
Some LDL taken up by (endothelial surface)
other tissues (LDL receptors).

LDL delivers cholesterol and
TAG to the extra hepatic tissues.
07/22/2024 Dr Ambayehu 429

 Modification of circulating VLDL
 As VLDLs pass through the circulation their structure is altered.
 TAG is removed by lipoprotein lipase, causing the VLDL to decrease
in size and become more dense.
 Surface components, including the apoC-II and apo E are
transferred to HDL.
 Finally , CE are transferred from HDL to VLDL in an exchange
reaction that concomitantly transfers TAG or phospholipid from VLDL
to HDL.
 This exchange is accomplished by cholesteryl ester transfer protein.
07/22/2024 Dr Ambayehu 430

 Modification of circulating VLDL
Production of LDL from VLDL in
plasma
• After these modifications, the

VLDL has been converted in the
plasma to LDL.
• An intermediate- sized particle,
the intermediate density
lipoprotein ( IDL) is observed
during transition from VLDL to
LDL in the plasma.
• IDLs can also be taken up by
cells through receptor-mediated
endocytosis.
07/22/2024 Dr Ambayehu 431
 Low-density lipoproteins (LDL)
• Derived from remnant VLDL by removal of TG and loss of
some apo-lipoproteins (Apo-C and Apo-E) that are delivered
to HDL.
LDL particles retain apo B -100
They contain much less TAG than their VLDL predecessors
• They are the most cholesterol-rich particles.
• LDL receptors are widely distributed in tissues and liver.
• They bind LDL that is engulfed into the cells by endocytosis
to be broken down by lysosomal enzymes.
07/22/2024 Dr Ambayehu 432

 Metabolism of low density lipoproteins
1. Receptor- mediated endocytosis:
 The primary function of LDL particles is to provide cholesterol to

peripheral tissues.
 They do so both by depositing free cholesterol on the membranes
of cells as they come in contact with the cell surface, and
 by binding to receptors on the cell- surface membranes that
recognize apo B-100.
 A similar mechanism is used for the cellular uptake and

degradation of chylomicron remnants and HDLs by the liver.
07/22/2024 Dr Ambayehu 433

07/22/2024 Dr Ambayehu 434
 LDL receptors are negatively charged glycoprotein molecules.
 A deficiency of functional LDL receptors causes a significant elevation in
plasma LDL, and therefore of plasma cholesterol, but plasma TAG levels
remain normal, for example, as in type II hyperlipidemia (familial –hyperbeta-
lipoproteinemia). This can greatly accelerate the progress of atherosclerosis.
 After binding, the LDLs are internalized as intact particles by endocytosis.
 The vesicle containing the LDL rapidly loses its clathrin coat and fuses with
other similar vesicles, forming larger vesicles called endosomes.
 The pH of the contents of the endosome falls, allowing the separation of the LDL
from its receptor. The receptors then migrate to one side of the endosome,
whereas, the LDLs stay free within the lumen of the vesicle.
This structure is called CURL – the compartment for uncoupling of receptor and
ligand.
07/22/2024 Dr Ambayehu 435

 The receptors can be recycled, whereas, the lipoprotein
remnants in the vesicle are degraded by lysosomal enzymes,
releasing cholesterol, amino acids, fatty acids and
phospholipids. These compounds can be recycled by the cell.
 The number of receptors for lipoproteins(LDL–receptors)

varies according to the availability of these lipoprotein
particles and according to the needs of the cell.
For example, if there is a large amount of a particular
circulating plasma lipoprotein (LDL), the number of cell-
surface receptors for it decreases, frequently termed “ down –
regulation”. Conversely, if cells are starved for cholesterol,
they increase the number of cell- surface receptors “ up-
regulation”.
07/22/2024 Dr Ambayehu 436
 Effect of endocytosed cholesterol on cell cholesterol
content.
 The chylomicron remnant, HDL and LDL –derived cholesterol affects
cellular cholesterol content in several ways:
1. HMG CoA reductase activity is inhibited by cholesterol
2. If the cholesterol is not required immediately for some structural
or synthetic purpose, it is esterified by acyl CoA : cholesterol
acyltransferase (ACAT).
 ACAT transfers a fatty acid from a fatty acyl CoA derivative to
cholesterol, producing a cholesteryl ester that can be stored in the cell.
 The activity of this enzyme is enhanced in the presence of increased
intracellular cholesterol.
3. Synthesis of new LDL –receptor protein is lowered by decreasing
transcription of the LDL-receptor gene.
07/22/2024 Dr Ambayehu 437

 High-density lipoproteins
• Synthesized by liver and intestine.
• The main lipid component is phospholipids (PL), particularly lecithin
and apoproteins
• They have Apo-A, Apo –C and Apo-E.
• They are present in two forms:

• Discoid
• Spherical.
• The discoid is the newly synthesized, more active, after transferring
cholesterol from peripheral tissues they become spherical HDL.
07/22/2024 HDL is the predominant form in plasma.
Dr Ambayehu 438
 Metabolism HDLs
They perform a number of important functions:
1. Serve as a circulating reservoir of apo C-II, an activator of lipoprotein
lipase. Also it acts as a reservoir for apo –E, necessary for the recognition of
chylomicron remnants by the liver receptors.
2. Removes free (Unesterified) cholesterol from extrahepatic tissues and
esterifying it , using the plasma enzyme phosphatidylcholine : cholesterol
acyltransferase, (PCAT—also known as LCAT, where” L “stands for
lecithin).
3. Transfers cholesteryl to VLDL and LDL in exchange for TAG.
4. Carries cholesteryl esters to the liver, where the HDL is degraded and
cholesterol released.
07/22/2024 Dr Ambayehu 439

Metabolism of HDL
07/22/2024 Dr Ambayehu 440
 HDL uptake of free cholesterol
 Newly secreted HDLs are disc shaped particles containing predominantly
unesterified cholesterol, phospholipid (largely phosphatidylcholine) , and a number
of apolipoproteins including apoE, apoA-I and apoC-II.
 They are rapidly converted to spherical particles as they accumulate cholesterol.
HDL particles are excellent acceptors of unesterified cholesterol.
 As the nacent HDL accumulates cholesteryl esters it first becomes classified as
HDL3 and eventually becomes a round, micellar –like particle HDL 2.
 Esterification of free cholesterol
 Once free cholesterol is taken up by the HDL particle, it is immediately esterified
by PCAT, a plasma enzyme synthesized by the liver, which is activated by apoA-1
of the HDL.
 The fatty acid from carbon 2 of phosphatidylcholine is transferred directly to
cholesterol leaving lysophosphatidylcholine.
07/22/2024 Dr Ambayehu 441

 Reverse cholesterol transport
 The selective transfer of cholesterol from peripheral cells to HDLs and

from HDLs to the liver for bile acids synthesis or disposal via the bile and to
steroidogenic cells for hormone synthesis is a key component of cholesterol
homeostasis.
This is in part the basis for the inverse relationship seen between plasma
HDL concentration and atherosclerosis and for HDL’s designation as the
“good cholesterol carrier”.
 Reverse cholesterol transport involves efflux of cholesterol from

peripheral cells to HDL, esterification of cholesterol by PCAT, binding of the
cholesterol ester-rich HDL (HDL2) to the liver and steroidogenic cells, the
selective transfer of cholesteryl esters into these cells and the release of lipid-
depleted HDL (HDL3). The process is thought to be mediated by a cell-surface
receptor (scavenger receptor class B, SR-B 4) that binds HDL.
07/22/2024 Dr Ambayehu 442

 Lipoproteinemias
Lipoproteinemias may be :
 Hyperlipoproteinemia (hyperlipidemia) - Increased plasma lipids
Synthesis, clearance or transport of lipoproteins is defective.
It is either a combination of a genetic and environmental factors or

secondary to other diseases.
 Primary
Secondary
Hypolipoproteinemia
• Primary
07/22/2024 Dr Ambayehu 443
Primary hypolipoproteinemia
1) Tangier disease
2) Abetalipoproteinemia
3) Hypobetalipoproteinemia
 Secondary hypolipidemia
• It occurs when liver fails to synthesized adequate amount of
proteins as malnutrition chronic liver failure malabsorption
syndrome
07/22/2024 Dr Ambayehu 444

 Primary Hyperlipoproteinemias
 Type I, Familial Hyperlipoproteinemia
 lipoprotein lipase deficiency
• Plasma TG, VLDL are raised
• LDL is normal or even reduced, HDL is decreased.
 Eruptive xanthomas are the most characteristic skin
manifestation of this disorder
07/22/2024 Dr Ambayehu 445

 Type II, Familial hypercholesterolemia,
It is due to combined genetic and dietary factors
There is a genetic defect in either number or activity of
tissue apo-B100 receptors which results in variable degrees
of hypercholesterolemia.
The disease is inherited whether homo- or heterozygous.
The more severe form is the homozygous that presents with
CHD in teens of age.
Plasma cholesterol levels are severely elevated, but plasma
TAG levels are typically normal
have severe atherosclerosis and may present with
xanthomas
07/22/2024 Dr Ambayehu 446

 Type III hyperlipidemia
(Familial dysbetalipoproteinemia)
is characterized by the accumulation of IDL, which is
manifested by increases in both TAG and cholesterol levels
in the plasma
Various mutations of apoprotein E impair its ability to bind
to the IDL receptor
premature atherosclerosis and xanthomas
07/22/2024 Dr Ambayehu 447

 Type IV hyperlipidemia (Familial hypertriglyceridemia)
over production of VLDL

severe elevations of plasma TAG levels. Plasma cholesterol
levels are typically normal
eruptive xanthomas
commonly associated with CHD, type II DM, obesity,
alcoholism
 Type V
• Genetic defects of the apolipoprotein C-II
• result in the accumulation of chylomicrons and VLDL
• pancreatitis and eruptive xanthomas
07/22/2024 Dr Ambayehu 448

 Secondary hyperlipidemia
Secondary hypercholesterolemia
o in pregnancy, hypothyroidism, cholestasis, and acute intermittent
porphyria.
Secondary hypertriglyceridemia
o associated with oral contraceptive use, DM, alcoholism,
pancreatitis, gout, and type I glycogen storage disease
Combined hypercholesterolemia and hypertriglyceridemia

o in nephrotic syndrome, chronic renal failure, and steroid
immunosuppressive therapy.
07/22/2024 Dr Ambayehu 449

 Hypolipoproteinemia
Abetalipoproteinemia
• Is a rare autosomal recessive disease caused by loss-of-
function, mutations in the gene encoding MTP, a protein that
transfers lipids to nascent chylomicrons and VLDLs in the
intestine and liver, respectively
• Plasma levels of cholesterol and triglyceride are extremely
low
• chylomicrons, VLDLs, LDLs, and apoB are undetectable in
plasma
• Abetalipoproteinemia usually presents in early childhood
with diarrhea and failure to thrive and is characterized
clinically by fat malabsorption, spinocerebellar degeneration,
07/22/2024
pigmented retinopathy, andDracanthocytosis
Ambayehu 450
 Tangier Disease
• Low plasma HDL-C caused by mutations in the gene
encoding ABCA1, a cellular transporter that facilitates efflux
of unesterified cholesterol and phospholipids from cells to
apoA-I
• Patients with Tangier disease have low plasma HDL-C levels
and extremely low circulating levels of apoA-I.
• The disease is associated with cholesterol accumulation in
the reticuloendothelial system, resulting in
hepatosplenomegaly and pathognomonic enlarged, grayish
yellow or orange tonsils
07/22/2024 Dr Ambayehu 451

 Coronary artery disease (CAD)
• Occurs when the inside (the lumen) of one or more coronary
arteries narrows, limiting the flow of oxygen-rich blood to
surrounding heart muscle tissue.
• Atherosclerosis is the process that causes the artery wall to
get thick and stiff.
• It can lead to complete blockage of the artery, which can
cause a heart attack.
07/22/2024 Dr Ambayehu 452

• The disease process begins when LDL (“bad” cholesterol)
deposits cholesterol in the artery wall.
• The body has an immune response to protect itself and sends
white blood cells called macrophages to engulf the invading
cholesterol in the artery wall.
• When the macrophages are full of cholesterol, they are
called foam cells because of their appearance.
• As more foam cells collect in the artery wall, a fatty
streak develops between the intima and the media.
• If the process is not stopped, the fatty streak becomes a
plaque, which pushes the intima into the lumen, narrowing
the blood flow.
07/22/2024 Dr Ambayehu 453

• The plaque develops a fibrous coating on its outer edges.
• But if cholesterol continues to collect in foam cells inside the
plaque, the fibrous outer coating can weaken and eventually
rupture.
• Smaller arteries downstream from the rupture can quickly

become blocked.
• Over time, a clot may develop at the rupture site and
completely block the artery.
• A myocardial infarction (heart attack) occurs when the
heart muscle tissue does not receive vital oxygen
07/22/2024 Dr Ambayehu 454

07/22/2024 Dr Ambayehu 455
The end!
07/22/2024 Dr Ambayehu 456

Chapter Eight
Protein Metabolism
07/22/2024 Dr Ambayehu 457

Objectives
• Understand the digestion and absorption of proteins.
• Disorders of Protein Malnutrition, Digestion and Amino Acid Absorption.
• Protein Turnover.
• The Urea Cycle
07/22/2024 Dr Ambayehu 458

Introduction
Dietary protein is a vital source of amino acids. Proteins ingested
in the diet are digested into amino acids or small peptides that
can be absorbed by the intestine and transported in the blood.
Another source of amino acids is the degradation of defective or
unneeded cellular proteins.
07/22/2024 Dr Ambayehu 459

The digestion & absorption of dietary proteins
Proteolytic enzymes (also called proteases) break down dietary proteins into
their constituent amino acids in the stomach and the intestine.
Many of these digestive proteases are synthesized as larger, inactive forms
known as zymogens.
After zymogens are secreted into the digestive tract, they are cleaved to
produce the active proteases by removal of a peptide fragment in the lumen
of the digestive tract.
Proteases can be endopeptidases or exopeptidases.
No single enzyme can completely digest a protein since proteases have
varied specificities. However, by acting in concert, they can digest dietary
proteins to amino acids and small peptides, which are cleaved by peptidases
associated with intestinal epithelial cells.
The process of protein digestion can be divided, depending on the sources of
peptidases/proteases.
07/22/2024 Dr Ambayehu 460
A. Gastric Digestion
Entry of a protein in to stomach stimulates the gastric mucosa cell to secrete a

hormone gastrin which in turn stimulates the secretion of HCl by the parietal cells
of the gastric glands and pepsinogen by the chief cells.
Functions of HCl:
Dietary proteins are denatured by the acid in the stomach. This serves to
inactivate the proteins and partially unfolds them such that they are better
substrates for proteases.
Destroys most microorganisms that enter in to GIT.
Creates optimum pH required for gastric proteolytic enzymes.
Alters the conformation of pepsinogen so that it can cleave itself, producing the
active protease pepsin. Thus, the activation of pepsinogen is autocatalytic.
However, at the low pH of the stomach, pepsin is not denatured and acts as an
endopeptidase, cleaving peptide bonds at various points within the protein chain.
07/22/2024 Dr Ambayehu 461
07/22/2024 Dr Ambayehu 462
Gastric digestion…
A proteolytic enzyme secreted by gastric mucosa cell of infants is
chymosin (rennin)
It digests casein (a milk protein) into paracaseinate which combines
with Ca++ to form insoluble Calcium-paracaseinate (milk clot),
which is then acted upon by pepsin to release smaller peptides
(peptones).
Important for infants because it coagulates milk in the stomach to
prevent its rapid passage to the intestine. This gives the baby the
satiety and sensation of fullness.
07/22/2024 Dr Ambayehu 463

B. Pancreatic Digestion
Pancreatic zymogens proceed digestion as the acidic stomach
contents pass in to the small intestine
A low pH triggers the secretion of a hormone secretin in the
duodenum.
Secretin stimulates pancreas to secrete HCO-3 (bicarbonate),
which in the small intestine neutralizes the acid content from the
stomach.
The entry of large peptide fragments in to duodenum excites the
release of a hormone cholecystokinin (CCK).
CCK:
Stimulates gall bladder contraction.
Stimulates secretion of several pancreatic proenzymes whose
activity is between pH 7and 8. These proenzymes include
trypsinogen, chymotrypsinogen, proelastase, and
07/22/2024 procarboxypeptidase, localized
Dr Ambayehu in the exocrine cells of 464
Pancreatic Digestion…
Synthesis of these enzymes as inactive precursors protects:
The exocrine cells from destructive proteolytic attack.
These enzymes from digesting each other.
When the proenzyme reach the lumen of the small intestine,
initially the enteropeptidase (old name Enterokinase) a protease
produced by duodenal epithelial cells, activates pancreatic
trypsinogen to trypsin by the removal of a hexapeptide from NH2
– terminus.
Trypsin in turn auto catalytically activates more trypsinogen to
trypsin and other proenzymes and liberating chymotrypsin,
elastase, and carboxypeptidase.
Thus, trypsin plays a central role in digestion because it both
cleaves dietary proteins and activates other digestive proteases
produced by the pancreas.
07/22/2024 Dr Ambayehu 465
Activation of the gastric and pancreatic zymogens. Pepsinogen
catalyzes its own cleavage as the pH of the stomach drops.
Trypsinogen is cleaved by enteropeptidase in the intestine to
form the active protease trypsin. Trypsin then plays a key role
by catalyzing the cleavage and activation of the other pancreatic
zymogens.
07/22/2024 Dr Ambayehu 466
The pancreas also synthesizes a secretory trypsin inhibitor.
The need for the inhibitor is to block any trypsin activity that
may occur from accidental trypsinogen activation.
Recall that the exocrine pancreas, in addition to secreting
proteolytic zymogens, also secretes amylase for starch
digestion and lipase and co-lipase for dietary triacylglycerol
digestion.
Elastase is also found in neutrophils, white blood cells that
have the job of engulfing and destroying invading bacteria.
Neutrophils frequently act in the lung, and elastase is
sometimes released into the lung as the neutrophils work.
In normal individuals, the released elastase is blocked from
destroying lung cells by the action of circulating 1α-
antitrypsin, a protease inhibitor synthesized and secreted by
07/22/2024
the liver. Dr Ambayehu 467
C. Intestinal Digestion
Since pancreatic juice does not contain appreciable
aminopeptidase activity, final digestion of dipeptides and
oligopeptides depends on the small intestinal enzymes.
Exopeptidases produced by intestinal epithelial cells act
within the brush border and also within the cell.
Aminopeptidases, located on the brush border, cleave one
amino acid at a time from the amino end of peptides.
Intracellular peptidases act on small peptides that are
absorbed by the cells.
The concerted action of the proteolytic enzymes produced by
cells of the stomach, pancreas, and intestine cleaves dietary
proteins to amino acids.
The digestive enzymes also digest themselves as well as
07/22/2024 intestinal cells that are regularly
Dr Ambayehusloughed off into the lumen. 468
07/22/2024
Protein Digestion
Dr Ambayehu
Action of digestive proteases

469
Absorption of Amino Acids
1. Co-transport of Na+ and Amino Acids

Amino acids are absorbed from the lumen of the small intestine principally by
semi-specific Na+ -dependent transport proteins in the luminal membrane of the
intestinal cell brush border, similar to that already seen for carbohydrate
transport.
The co-transport of Na+ and the amino acid from the outside of the apical
membrane to the inside of the cell is driven by the low intracellular Na+
concentration.
Low intracellular Na+ results from the pumping of Na+ out of the cell by Na+ ,
K+-ATPase on the serosal membrane.
Thus, the primary transport mechanism is the creation of a sodium gradient; the
secondary transport process is the coupling of amino acids to the influx of
sodium.
This mechanism allows the cells to concentrate amino acids from the intestinal
lumen.
The amino acids are then transported out of the cell into the interstitial fluid
07/22/2024principally by facilitated transporters inDr
the serosal membrane
Ambayehu 470
Amino acids absorbed by intestinal epithelial cells enter the
portal vein by facilitated transport.
These amino acids are either metabolized by the liver or released

into the general circulation. [Note: Branched-chain amino acids are
important examples of amino acids that are not metabolized by the
liver, but instead are sent from the liver into the blood].
07/22/2024 Dr Ambayehu 471
2. Glutathione transport system (-glutamyl cycle)
Occurs during absorption of amino acids from

intestine, re-absorption from kidney and transport into
brain, epididymis and seminal vesicle.
Amino acid in the lumen reacts with glutathione (-
glutamyl-cysteinyl-glycine) in the cell membrane,
forming a -glutamyl amino acid and the dipeptide
cysteinyl glycine.
The amino acid is carried across the cell membrane
attached to -glutamate and released in to the
cytoplasm. The -glutamyl moiety is used in the re-
synthesis of glutathione.
However, the major role of this cycle is glutathione
07/22/2024 synthesis, and many tissues lack the transpeptidase and 5-
Dr Ambayehu 472
The -glutamyl cycle
07/22/2024 Dr Ambayehu 473

3. Passive transport of D amino acids: D-amino acids are
absorbed very slowly by passive diffusion that needs no
energy.
4. Amino acids transport from blood to cells
Amino acids from the blood are transported across cell
membranes of the various tissues principally by Na+-
dependent co-transporters and, to a lesser extent, by
facilitated transporters.
In this respect, amino acid transport differs from glucose
transport, which is Na+-dependent transport in the intestinal
and renal epithelium but facilitated transport in other
cell types.
The Na+ dependence of amino acid transport in liver,
muscle, and other tissues allows these cells to concentrate
amino acids from the blood.
07/22/2024 Dr Ambayehu 474
Disorders of Protein Malnutrition,
Digestion and Amino Acid Absorption
The principal causes of protein mal-digestion and mal-absorption are
diseases of the exocrine pancreas (such as cystic fibrosis) and small
intestine.
1. Hartnup disease
Hartnup disease is genetically determined and relatively rare
autosomal recessive disorder
Due to defects in neutral amino acid transporter in intestine, kidneys
and also in brain.
This leads to defective transport of neutral amino acids, particularly,
07/22/2024 Dr Ambayehu 475
Trp, Ala, Thr, Gln, and Phe.
Symptoms: mental retardation, intermittent cerebellar ataxia, other
neurological symptoms, Pellagra-like skin rash (niacin deficiency) and
hypersensitivity to sunlight. Most of these symptoms are caused by
the reduced synthesis of serotonin and nicotinic acids due to
excessive loss of trp.
Blood levels of trp and other neutral amino acids are lower than normal.
Fecal and urine content is 5-10 times higher than normal (neutral
aciduria)= hyperaminoaciduria.
NB: The small intestine and the proximal tubule of the kidney have
common transport systems for amino acid uptake; therefore, a defect in
any one of these systems results in an inability to absorb particular amino
acids into the gut and into the kidney tubules.
07/22/2024 Dr Ambayehu 476
2. Cystinuria:
Due to genetically determined
defect in the transport of
cystine and the basic amino
acids, lysine, arginine, and
ornithine, across the brush-
border membranes of cells in
both their small intestine and
renal tubules.
The most serious problem for
these patients is the
insolubility of cystine, which
can form kidney stones
(calculi) that may lodge in the
ureter, causing bleeding and
severe pain.
07/22/2024 Dr Ambayehu cystine urolithiasis 477
3. Kwashiorkor
Kwashiorkor, a common problem of
children in Third World countries, is
caused by a deficiency of protein in a diet
that is adequate in calories. Children with
kwashiorkor suffer from muscle wasting
and a decreased concentration of
plasma proteins, particularly albumin.
The result is an increase in interstitial fluid
that causes edema and a distended
abdomen that make the children appear
“plump”
The muscle wasting is caused by the lack
of essential amino acids in the diet;
existing proteins must be broken down to
produce these amino acids for new protein
07/22/2024 synthesis. Dr Ambayehu 478
Protein Turnover
Most proteins in the body are constantly being synthesized and then
degraded. The concentration of protein in the cell is maintained
through:
Regulation of their synthesis (inducible proteins).
Regulation of their degradation (constitutive proteins).
In healthy adults, the total amount of protein in the body remains
constant, because the rate of protein synthesis is just equivalent to the
rate of degradation.
Protein turnover involves the hydrolysis and re-synthesis of 300–400
g of body protein each day.
07/22/2024 Dr Ambayehu 479

The rate of protein turnover varies widely for d/t proteins.
Short-lived proteins (eg., many regulatory enzymes and
misfolded or defective proteins. Long-lived proteins, with half-
lives of days to years, constitute the majority of proteins in the
cell e.g. Collagen, crystallins....
A protein's half-life correlates with its N-terminal residue
 Proteins with N-terminal Met, Ser, Ala, Thr, Val or Gly have
half lives greater than 20 hours.
 Proteins with N-terminal Phe, Leu, Asp, Lys, Ile, Glu, Tyr or
Arg have half lives of 3 min or less.
07/22/2024 Dr Ambayehu 480

Protein Degradation
There are two major enzyme systems responsible for degrading damaged or
unneeded proteins:
1. lysosomal degradation pathway involves non-energy-dependent
degradative enzymes (acid hydrolases).
Lysosomal enzymes degrade primarily extracellular proteins, such as
plasma proteins that are taken into the cell by endocytosis, long-lived
intracellular proteins, and cell-surface membrane proteins that are used in
receptor-mediated endocytosis.
Circulating proteins in plasma are mostly glycoproteins; carry
oligosaccharides on their surfaces.
07/22/2024 Dr Ambayehu 481

Removal of one sialic acid from these oligosaccharides marks the
proteins for degradation.
The asialated protein is internalized through the ‘asialoglycoprotein
receptor’ present on hepatocytes.
The internalized proteins are degraded in lysosomes by proteases
termed cathepsins.
This pathway helps in removal of a number of hormones from plasma
and hence keeps their plasma concentration in check.
07/22/2024 Dr Ambayehu 482

2. Ubiquitin-proteasome degradation pathway
Is ATP-dependent cytosolic degradation.

Mainly degrades intracellular and regulatory proteins (enzymes)
with short half-lives, and abnormal proteins.
A chain of ubiquitin (76 AA protein) gets covalently attached to
proteins destined for degradation
The proteasome (garbage disposal) is a cylindrical macromolecular,
protein complex with multiple internal proteolytic enzymes.
Before the proteasome can break down proteins, the protein must
first be ‘tagged’ by complexing with ubiquitin; since ubiquitin marks
proteins for death.
The protein substrate has amino groups in the side chains of its Lys
residues
07/22/2024 Dr Ambayehu 483
Ubiquitin has a C-terminal Gly. The carboxyl group of this Gly forms an
isopeptide bond with the amino group of Lys
Furthermore, the target protein is polyubiquitinylated, in which additional
ubiquitin molecules are added to previous ubiquitin molecules
The AA residue present at its amino terminal affects whether a protein is
ubiquitinated. Amino terminal Met or Ser retards, whereas Asp or Arg
accelerates ubiquitination
In addition, many proteins that contain regions rich in the amino acids
proline (P), glutamate (E), serine (S), and threonine (T) have short half-
lives. These regions are known as PEST sequences.
The‘ubiquitination’ of proteins is mediated by three enzymes: E1
(Ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme),
07/22/2024
and E3 (Ubiquitin-protein Drligase)
Ambayehu 484
07/22/2024 Dr Ambayehu 485
The proteasome is a complex of multiple proteases.
It contains two main types of sub complexes: a core particle (20S), also
called catalytic unit and regulatory 19S particles on both ends of the barrel
 The catalytic core particle and the regulatory particles make up the
functional 26S proteasome
The 19S particles may unfold proteins and translocate the unfolded
proteins into the 20S particle; energy from ATP is consumed in the process
The 19S particles also cleave isopeptide bonds and free the ubiquitin.
The protein is degraded by the 20S particle and free amino acids are
released into the cellular space
ATP hydrolysis is used both to unfold the tagged protein and to push the
protein into the core of the cylinder.
07/22/2024 Dr Ambayehu 486

Ubiquitin attached to the protein
interacts with the proteasome and opens
the regulatory unit.
The targeted proteins are pushed
through the catalytic subunit to be cut
into smaller peptides and AAs.
After the target protein is degraded,
the resultant amino acids join the
intracellular pool of free amino acids.
The released intact ubiquitin is
recycled again to mark other proteins
for degradation.
07/22/2024 Dr Ambayehu 487

Proteasomal degradation and cancer
Proteasomal degradation of particular proteins is an essential mechanism by which cellular
processes (cell division, apoptosis, differentiation and development) are regulated.
The proteins that are required only during a specific stage of the cell cycle are rapidly degraded by
the ubiquitin-dependent pathway after completing their function.
Inability to degrade proteins that activate cell division (or rapid degradation of those proteins
that suppress tumor formation) can lead to cancer
 Progression through the cell cycle is controlled in part through regulated degradation of
proteins called cyclins that activate cyclin-dependent kinases which inturn activate TFs & the
expression of genes.
Some pathological conditions clearly illustrate the importance of the regulation of protein turnover.
For example, human papilloma virus (HPV) encodes a protein that activates a specific E3 enzyme.
The enzyme ubiquitinates the tumor suppressor p53 and other proteins that control DNA repair,
which are then destroyed. The activation of this E3 enzyme is observed in more than 90% of
cervical
07/22/2024
carcinomas. Dr Ambayehu 488
The catabolism of amino acids
Fig. Fate of amino acid carbons & nitrogen

07/22/2024 Dr Ambayehu 489
The fate of amino acid nitrogen
α-amino groups removed from amino acids mostly through
reversible transamination reactions:
 The amino acid becomes a keto acid when it donates the
amino group to α-ketoglutarate (changing it to glutamate)
 Occurs mainly in liver, kidney, brain ,heart.
 First step in the catabolism of AAs.
 All amino acids except lys, thr, pro and hydroxyproline
undergo transamination reactions.
 The enzymes involved are known as transaminases or
aminotransferases
 Pyridoxal phosphate (PLP) is the cofactor
The glutamate thus collects the amino groups and gives them
off for biosynthesis or excretion.
07/22/2024 Dr Ambayehu 490
 For example, the amino acid aspartate can be transaminated to form its
corresponding α-keto acid, oxaloacetate
 In the process, the amino group is transferred to α-keto-glutarate, which is
converted to its corresponding amino acid, glutamate
Aminotransferases are intracellular enzymes, that have both cytosolic and

mitochondrial isoforms.
Their appearance in the blood indicates presence of cellular damage
somewhere in the body.
 ALT and AST are abundant in the liver.
 Elevated plasma levels of ALT and AST (as occurs with cirrhosis or viral
hepatitis) are diagnostic tests of liver disease or injury.
07/22/2024 Dr Ambayehu 491
Oxidative deamination:
Reversible removal of –NH2 as
NH3 from AA.
Transdeamination:
The process of transamination plus
oxidative deamination of glu :
Transamination of most of AAs
channels their amino nitrogen into
L-glu.
GDH catalyzes oxidative
deamination of glu into -KGA and
liberates NH3that enters urea cycle.
Glu links metabolism of AAs to
synthesis of urea.
07/22/2024 Dr Ambayehu 492

Generation of Ammonium ions from Amino Acids
Free ammonium can be released in different ways:

A. The oxidative deamination of glutamate by glutamate dehydrogenase - the
only enzyme that can use NAD+ or NADP+ as electron acceptor
The reaction is reversible and takes place in the mitochondria.
B. Serine and threonine dehydratases release NH4+; histidine can be directly
deaminated to give NH4+
C. Intestinal bacteria produce NH4+ from amino acids or urea; the ammonia
enters the portal vein
D. Glutamine and asparagine lose their side chain amino groups through
deamidation
E. The purine nucleotide cycle in the brain & muscle – aspartate is used as a
substrate and fumarate and ammonium are released
07/22/2024 Dr Ambayehu 493
07/22/2024 Dr Ambayehu 494
Blood Ammonia
Processing of the amino groups of the amino acids produces
ammonia, which is toxic in its free form, especially to nerve
cells. So, its metabolism is designed to keep blood levels low
(i.e, < 40 μM).
For this reason, it is rapidly removed from circulation by the
liver (mainly) and converted either to Glu, Gln or urea.
In liver disorders like alcoholic liver disease or cirrhosis, NH3
is not converted into urea and rises in blood, a toxicity
condition known as hyperammonemia.
NH3 passes directly to systemic blood through collateral
arteries and exposes brain tissue to the toxic concentrations.
The brain detoxifies NH3 into gln that depletes -KGA of
Krebs' cycle. This leads to energy failure in the brain cells.
07/22/2024 Dr Ambayehu 495
Disposal of blood NH3 :Anabolic fates
Free NH3 removed by deamination enters the NH3 pool of the body and may be
used for :
Synthesis of urea via urea cycle
Synthesis of non-essential AAs
Synthesis of purines, pyrimidines, porphyrins and sugaramines
Glutamine synthesis (glutamine cycle): Gln acts as a carrier of N and provides a
mechanism for detoxification of NH3 in extra-hepatic tissues particularly brain,
kidney, retina and muscles.
In these tissues, glu reacts with NH3 in presence of ATP, Mg2+ and the
mitochondrial glutamine synthetase to produce gln.
Gln is transported through blood from various tissues to the kidney and liver
where it is hydrolyzed by glutaminase into glu and NH3.
Glutaminase is present in liver, kidneys, brain as well as retina.
07/22/2024 Dr Ambayehu 496

Glutamine cycle: Glutamine serves as a transport vehicle
for ammonia from brain to kidneys or liver
The glutaminase reaction is
particularly important in the
kidney, where the ammonium
ion produced is excreted
directly into the urine as a
buffer.
07/22/2024 Dr Ambayehu 497

Synthesis of glutamine in peripheral tissues and its transport
to the liver.
Glucose/alanine cycle
07/22/2024 Dr Ambayehu 498

In muscle, transamination of
pyruvate forms alanine, which is
transported to the liver, where the
reaction is reversed (the alanine
cycle).
In general, glutamate is used as a

reservoir for amino groups while
alanine and glutamine are the major
transport forms of nitrogen in the
blood.
Figure: Transport of ammonia

from peripheral tissues to the
liver.
07/22/2024 Dr Ambayehu 499
Conversion of alanine to glucose
& urea
Role of glutamate in urea formation
07/22/2024 Dr Ambayehu 500

Major nitrogen wastes
Different animals excrete excess nitrogen as ammonia, uric
acid, or urea.
 Most terrestrial vertebrates including humans convert NH4+
into urea, which is then excreted in urine: they are ureotelic
 Terrestrial reptiles and birds convert NH4+ into uric acid for
excretion (considered uricotelic).
 Aquatic animals excrete NH4+ as it is (considered
ammonotelic)
The urine of humans also contains nitrogenous compounds
other than urea, including uric acid (from purine catabolism),
creatinine (from degradation of muscle creatine phosphate),
and ammonia, (which serves to buffer acidic products like
ketone bodies).
07/22/2024 Dr Ambayehu 501

Ammonia Toxicity (encephalopathy)
The presence of NH3 in conc. > 80 g/dL in blood:
hyperammonemia.
The reasons for the toxic symptoms of hyperammonemia
include:
1. Depletion of -KGA of Krebs' cycle in brain leading to failure
in energy production.
2. Depletion of brain glutamate leading to deficiency of GABA (an
inhibitory neurotransmitter) that is a decarboxylation product of
glutamate.
3. Increased entrance of tryptophan to the brain (in exchange with
glutamine) leading to increased level of serotonin (stimulatory
neurotransmitter) synthesized from tryptophan.
4. Increased synthesis of the toxic -ketoglutaramate.
07/22/2024 Dr Ambayehu 502
The Urea Cycle (Krebs-Henseleit Cycle)
Elucidated by Krebs and Henseleit (1932).

A five-reaction cyclic cascade that utilizes two amino groups and a
CO2 to synthesize urea, a nontoxic nitrogenous waste of our body.
One of the nitrogen atoms for urea synthesis comes from ammonia
(NH4+) and the other is donated by aspartate.
The carbon and oxygen of urea are derived from CO2.
Sites of urea synthesis
Liver is the only tissue that synthesizes urea from NH3 and CO2.
The cycle operates partly in mitochondria and partly in the
cytoplasm of liver cells.
Other tissues that have activity of part of the urea cycle enzymes
include kidney and brain.
07/22/2024 Dr Ambayehu 503
Kidneys operate urea cycle up to the formation of arg.
but enzyme arginase is absent, so urea is not
synthesized but the cycle provides a source of arginine
in the kidneys.
Brain tissue lack the enzyme ornithine
transcarbamoylase, i.e., can not form citrulline. Rest of
the enzymes are present, hence brain tissue can
synthesize urea if citrulline is provided.
Urea formed in the liver is highly water-soluble and is
carried by the blood to the kidneys where it is filtered
and excreted in the urine.
07/22/2024 Dr Ambayehu 504

Steps of urea cycle
The reactions of the urea cycle are catalyzed by five enzymes.
The first two reactions occur in the mitochondria.
1. Synthesis of carbamoyl-phosphate:
The rate-limiting step, formation of carbamoyl phosphate, is catalyzed
by the key enzyme, carbamoyl phosphate synthetase I (CPS-I).
Allosteric activator : N-acetyl glutamate (NAG).
NAG increases the affinity of CPS-I for ATP.
The second type of this enzyme – the cytosolar liver CPS-II is utilized
in pyrimidine biosynthesis and does not require NAG as an allosteric
regulator.
07/22/2024 Dr Ambayehu 505

2. Synthesis of citrulline:
Transfer of the carbamoyl residue from carbamoyl phosphate into
ornithine to form citrulline with liberation of phosphate.
Enzyme: Mitochondrial ornithine carbamoyl transferase (ornithine
transcarbamoylase).
Citrulline goes to cytosol (facilitated by a specific transporter) to
complete the cytosolar component of the urea cycle, where, the rest
of the reactions take place.
Ornithine carbamoyl transferase is clinically utilized to check for
liver function.
The level of this enzyme in blood is increased in many diseases,
e.g., liver metastasis.
07/22/2024 Dr Ambayehu 506

3. Synthesis of argininosuccinate:
Citrulline is condensed into the
amino group of aspartate to form
argininosuccinate with liberation of
H2O.
Enzyme : argininosuccinate
synthetase
Cofactors: ATP and Mg2+.
4. Cleavage of Argininosuccinate:
Enzyme: Argininosuccinase found in liver and
kidney tissues.
Fumarate formed by this cleavage joins Krebs'
cycle.
Fumarate is also converted into oxaloacetate that
could be transaminated into asp to return the N
into the urea cycle.
07/22/2024 Dr Ambayehu 507

5. Cleavage of arginine into urea and ornithine
Liberation of urea is brought about by cleavage of arginine
catalyzed by arginase.
The ornithine returns to mitochondria (facilitated by a
specific transporter).
Arginase is present mainly in the liver of all ureotellic
organisms.
07/22/2024 Dr Ambayehu 508

Fig. Summary for rxns of Urea cycle
The overall reaction of the urea cycle indicates that handling of ammonia requires
expenditure of significant energy.
Aspartate + NH4+ + CO2 + 3ATP → Urea + Fumarate +
07/22/2024
2ADP + AMP + 2Pi + PPi + 3H2O Dr Ambayehu 509
07/22/2024 Dr Ambayehu 510
Regulation of urea synthesis
Urea formation increases during increased rate of protein
breakdown under conditions like prolonged starvation and lack of
energy or after eating high protein diet.
In general, the urea cycle is regulated by:
1. Availability of substrates: aspartate, ammonia, and CO2.
2. Allosteric activation of CPSI by N-acetylglutamate (NAG).
3. Induction/repression of the synthesis of urea cycle enzymes.
07/22/2024 Dr Ambayehu 511

Urea cycle abnormalities: Hyperammonemia
When the liver function is compromised, due either to

genetic defects of the urea cycle, or liver disease, blood
levels of ammonia can rise highly.
Such hyperammonemia is a medical emergency, because
ammonia has a direct neurotoxic effect on the CNS.
For example, elevated concentrations of ammonia in the
blood cause the symptoms of ammonia intoxication, which
include tremors, slurring of speech, somnolence, vomiting,
cerebral edema, and blurring of vision.
At high concentrations, ammonia can cause coma and death.
The two major types of hyperammonemia are:
07/22/2024 Dr Ambayehu 512

1. Acquired hyperammonemia:
Liver disease is a common cause of hyperammonemia in
adults.
It may be a result of an acute process, for example, viral
hepatitis, ischemia, or hepatotoxins.
Cirrhosis of the liver caused by alcoholism, hepatitis, or
biliary obstruction may result in formation of collateral
circulation around the liver.
As a result, portal blood is shunted directly into the systemic
circulation and does not have access to the liver.
The detoxification of ammonia (that is, its conversion to
urea) is, therefore, severely impaired, leading to elevated
levels of circulating ammonia.
07/22/2024 Dr Ambayehu 513

2. Hereditary hyperammonemia:
Genetic deficiencies of each of the five enzymes of the urea cycle
have been described, with an overall prevalence estimated to be 1 in
30,000 live births.
Ornithine transcarbamoylase (OTC) deficiency, which is X-linked, is
the most common of these disorders, affecting males predominantly,
although female carriers have been clinically affected.
All of the other urea cycle disorders follow an autosomal recessive
inheritance pattern.
In each case, the failure to synthesize urea leads to hyperammonemia
during the first weeks following birth.
Symptoms include many of the neurologic manifestations of acquired
hyperammonemia, but they are seen mainly in infants and frequently
lead to mental retardation
07/22/2024 Dr Ambayehu 514
07/22/2024 Dr Ambayehu 515
Treatment includes:
Limiting protein in the diet to minimize nitrogen burden.

Liver transplantation in cases where liver damage is severe.
Administration of benzoate, phenylbutyrate, or phenylacetate
which bind covalently to amino acids, producing nitrogen-
containing molecules that are excreted in the urine.
Benzoate combines with glycine to form hippurate, which is
excreted in the urine and decreases the overall nitrogen burden.
Phenylbutyrate given orally is converted to phenylacetate. This
sequesters free ammonia by combining with glutamine to form
phenylacetylglutamine, which is cleared by the kidneys.
When other measures fail, hemodialysis should be considered.
07/22/2024 Dr Ambayehu 516

The Metabolism of
Benzoic acid (A)&
phenylbutyrate (B) two
agents used to reduce
nitrogen levels in patients
with urea cycle defect
07/22/2024 Dr Ambayehu 517

Degradation of carbon skeleton of amino acids
The catabolism of the amino acids found in proteins involves the
removal of α-amino groups, followed by the breakdown of the
resulting carbon skeletons.
These pathways converge to form seven intermediate products:
oxaloacetate, α-ketoglutarate, pyruvate, fumarate, succinyl CoA, acetyl
CoA, and acetoacetyl CoA.
These products directly enter the pathways of intermediary
metabolism, resulting either in the synthesis of glucose or lipid, or in
the production of energy through their oxidation to CO2 and H2O.
There is no storage form of protein, thus excess dietary protein causes
metabolic burden.
Amino acids can be classified as glucogenic, ketogenic, or both based
on which of the seven intermediates are produced during their
catabolism.
07/22/2024 Dr Ambayehu 518

A. Glucogenic amino acids
Amino acids whose catabolism yields pyruvate or one of the intermediates of
TCA cycle are termed as glucogenic.
These intermediates are substrates for gluconeogenesis and, therefore, can give
rise to the net formation of glucose or glycogen in the liver and glycogen in the
muscle.
B. Ketogenic amino acids
Amino acids whose carbon skeleton yields either acetoacetate or one of its
precursor, (acetyl CoA or acetoacetyl CoA) are termed as ketogenic.
Acetoacetate is one of the ketone bodies which, also include 3-hydroxybutyrate
and acetone.
Leucine and lysine are the only exclusively ketogenic amino acids found in
proteins.
Their carbon skeletons are not substrates for gluconeogenesis and, therefore,
07/22/2024 cannot give rise to the net formationDrof glucose or glycogen.
Ambayehu 519
C. Glucogenic and ketogenic amino acids
The carbon skeletons of isoleucine (Ile), phenylalanine
(Phe), tyrosine (Tyr), and tryptophan (Trp) are both
glucogenic and ketogenic.
07/22/2024 Dr Ambayehu 520

07/22/2024 Dr Ambayehu 521
Fates of the carbon
skeletons upon catabolism
of amino acids
07/22/2024 Dr Ambayehu 522
The end!
07/22/2024 Dr Ambayehu 523

Chapter Nine
Integration of metabolism
07/22/2024 Dr Ambayehu 524

Introduction
• In the body, individual pathways of carbohydrate, lipid

and protein metabolism on tissues or organs do not
function in isolation.
• They are interrelated and they interact with each other.
• They form a community in which one pathway (organ)

produce substrate to another pathway (organ).
07/22/2024 Dr Ambayehu 525
• The integration of these pathways to generate energy is
largely controlled by hormones like insulin, glucagon and
catecholamines.
• Changes in the levels of these hormones in plasma allow

body to store energy and grow when food is available
in plenty or to make stored energy available for
utilization when food is not available.
07/22/2024 Dr Ambayehu 526

07/22/2024 Dr Ambayehu 527
5
2
07/22/2024 Dr Ambayehu 8 528
Major energy metabolism
pathways
07/22/2024 Dr Ambayehu 529

Compartmentalization of the Major Pathway of Metabolism
1. Glycolysis
Glycolysis
Pentose phosphate pathway
Fatty acids synthesis
2. Gluconeogenesis
TCA cycle,
3. Glycogen Metabolism ETC & Oxidative
phosphorylation
b-oxidation of FAs
Ketone body formation
4. Fatty Acid Metabolism
5. Citric Acid Cycle

Gluconeogenesis
6. Oxidative PhosphorylationDr Ambayehu Urea synthesis
07/22/2024 530
• Only the liver can carry out all of the reaction the major
pathways.
• The key junction points are Glucose-6- phosphate, Pyruvate and
Acetyl CoA.
07/22/2024 Dr Ambayehu 531

How do organs obtain energy?
 Brain: Glucose is primary fuel, switched to ketone bodies under
duress.
 Muscle: uses FAs when resting, glucose when exercising, AAs

when fasting.
• Heart muscle is unique in that it prefers FAs and ketone bodies over
glucose.
 Kidney: Glucose and FAs are the source of energy

 Liver: Prefers FAs and alpha-ketone acids over glucose ketone
and bodies
-Produce ketone bodies from FAs when fasting
07/22/2024 Dr Ambayehu 532
Liver:
Primarily depends on β-
oxidation of FAs for its own
energy needs.
07/22/2024 Dr Ambayehu 533

Metabolism of amino acids in the
liver
•Amino acids go directly to the
liver through the portal vein
after absorption.
•Uses them to make proteins, for
gluconeogenesis, for
biosynthesis of nitrogen-
containing molecules, or for
fuel.
07/22/2024 Dr Ambayehu 534
 Cardiac muscle is aerobic
only with circulating fats
the preferred fuel
 Lack of O2 leads to tissue

death (myocardial
infarction).
07/22/2024 Dr Ambayehu 535

Cont…
• Blood glucose concentrations fluctuate around an average value
of 5 mmol/L (90mg/dl)
• The absorptive state, during which ingested nutrients
are entering the blood from the gastrointestinal tract.
• On feeding, blood glucose concentrations rise and this stimulates

insulin release from pancreas.
• The post-absorptive state, during which the gastrointestinal

tract is empty of nutrients and energy must be supplied by the
body’s own stores.
12
07/22/2024 Dr Ambayehu 536
Cont…..
 Normal Blood glucose levels
 Fasting levels: 70-100 mg/dL
Postprandial : up to 140 mg/dL
Maintained with in physiological limits
by
1. Rate of glucose entrance into blood

circulation
2. Rate of its removal from the blood
stream.
 Glucose enters in to the blood by:

1. Absorption from intestine
2.07/22/2024
Hepatic glycogenolysis and Dr Ambayehu 537
Cont…
 Rate of removal of glucose from blood depends on:
1. Oxidation of glucose by tissue to supply energy
2. Hepatic and muscle glycogenesis
3. Conversion of glucose to fats in adipose tissues
4. Synthesis/formation of fructose in seminal fluid, lactose

in mammary gland, synthesis of glycoproteins.
5. Formation of ribose sugars and nucleic acid synthesis.

07/22/2024 Dr Ambayehu 538
07/22/2024 Dr Ambayehu 539
Integration of Metabolism in well fed state and
interrelation among major organs
• In the well-fed condition, transient increases in
plasma glucose, amino acids, and triacylglycerols
(TAG) occur.
• Islet tissue of the pancreas responds to the elevated

levels of glucose and amino acids with an
increased secretion of insulin and a drop in the
release of glucagon.
16
07/22/2024 Dr Ambayehu 540
07/22/2024 Dr Ambayehu 541
• The elevated insulin to glucagon ratio
– increase synthesis of glycogen, TAG and protein.
• During this absorptive period, virtually all tissues

use glucose as a fuel.
• Metabolic response of the body is dominated by

alterations in the metabolism of liver, adipose tissue,
muscle, and brain.
07/22/2024 Dr Ambayehu 542
1. Metabolic changes in liver in well fed state
Carbohydrate Metabolism
1. Uptake of glucose (GLUT2)
2. More glucose is converted to acetyl – CoA
3. Glucose utilization through HMP shunt increases. This results

in more NADPH production.
4. Gluconeogenesis is decreased
5. Glycogenesis increases
07/22/2024 Dr Ambayehu 543
Lipid Metabolism
1. FA biosynthesis increases
2. TAG formation increases
Amino acid Metabolism

3. New protein synthesis occurs to replace old proteins
and to support growth.
4. Excess amino acids are degraded to acetyl-CoA
(pyruvate) or intermediates of TCA cycle.
-The acetyl-CoA is used for FA biosynthesis.
07/22/2024 Dr Ambayehu 544
21
Major metabolic pathways ofDrliver
07/22/2024 Ambayehu in the absorptive 545
2. Metabolic changes in adipose tissue in well fed state
• Glucose uptake (GLUT4) increases

• This glucose is used to generate NADPH through HMP shunt
and glycerol-3-phosphate through glycolysis.
Lipid Metabolism
1. More of TAG are produced

2. TAG break down is slowed down
07/22/2024 Dr Ambayehu 546
3. Metabolic changes in skeletal muscle in well-fed state
 Glucose uptake (GLUT4) increases
 Increases glycogenesis
 Amino acid uptake increases
 New protein is synthesized to replace old proteins by

using the amino acids.
07/22/2024 Dr Ambayehu 547
07/22/2024 Dr Ambayehu 548
4. Metabolic changes in Brain in well-fed state
 The brain uses glucose exclusively as a fuel
 The brain contains no significant stores of glycogen and is,

therefore, completely dependent on the availability of blood
glucose.
Fat metabolism
 The brain has no significant stores of TAG, and the oxidation
of fatty acids.
 FA do not cross the BBB.
07/22/2024 Dr Ambayehu 549
07/22/2024 Dr Ambayehu 550
27
07/22/2024 . Dr Ambayehu 551
Integration of Metabolism in fasting state and inter-organ relations
• Fasting is a period of going without food, e.g. to lose weight or

for religious reasons.
• In the absence of food, plasma levels of glucose, amino acids, and FA

fall, triggering a decline in insulin secretion and an increase in
glucagon release.
– make the period a catabolic period characterized by degradation
of TAG, glycogen, and protein.
28
07/22/2024 Dr Ambayehu 552
Fuel stores
• The metabolic fuels available in a normal 70–kg man at the
beginning of a fast are
– Fat: 15 kg = 135,000 kcal
– Protein: 6 kg = 24,000 kcal
– Glycogen: 0.2 kg = 800 kcal
 Energy needed for a 24 h period → 1600 kcal - 6000 kcal
 Sufficient reserves for starvation up to 1 – 3 months
 However glucose reserves are exhausted in 1 day
• Even under starvation → blood-glucose level must be above 40
mg/dl
07/22/2024 Dr Ambayehu 553
Liver in Fasting Carbohydrate metabolism
• Increased glycogen degradation
 rapid mobilization of liver glycogen stores (which contain about 80 g of
glycogen in the well-fed state).
– glycogen phosphorylase
 liver glycogen is nearly exhausted after 10–18 hours of fasting;
therefore, hepatic glycogenolysis is a transient response to early fasting.
• Increased gluconeogenesis
– Gluconeogenesis begins four to six hours after the last meal and
becomes fully active as stores of liver glycogen are depleted
07/22/2024 Dr Ambayehu 554

• Gluconeogenesis plays an essential role in maintaining
blood glucose during both overnight and prolonged
fasting.
B. Fat metabolism
– Increased FA oxidation
– Increased synthesis of ketone bodies
07/22/2024 Dr Ambayehu 555

Major metabolic pathways
Cont…
in liver
during starvation.
07/22/2024 Dr Ambayehu 556

Adipose Tissue in Fasting
 Adipose tissue is second to liver in its ability to distribute fuel molecules.
• Carbohydrate metabolism
• Glucose transport (GLUT-4 ) into the adipocyte and its subsequent
metabolism are depressed
• This leads to a decrease in fatty acid and TAG synthesis.
• Fat metabolism
• Increased degradation of TAG (hormone sensitive lipase)
• Increased release of FA
• Decreased uptake of FA
07/22/2024 Dr Ambayehu 557

Major Metabolic Pathways in Adipose Tissue During Starvation
07/22/2024 Dr Ambayehu 558

Resting Skeletal Muscle in
Fasting
– Resting muscle uses FA as its major fuel source.
– By contrast, exercising muscle initially uses its glycogen stores

as a source of energy.
– As these glycogen reserves are depleted, free fatty acids

provided by the mobilization of TAG from adipose tissue
become the dominant energy source.
07/22/2024 Dr Ambayehu 559

• Glucose transport (GLUT-4) into skeletal muscle cells and
its subsequent metabolism are depressed
Lipid metabolism
• During the first two weeks of fasting, muscle uses FAs from
adipose tissue and ketone bodies from the liver as fuels.
• After about three weeks of fasting, muscle decreases its
use of ketone bodies and oxidizes FAs almost exclusively.
• This leads to a further increase in the already elevated
level of circulating ketone bodies.
07/22/2024 Dr Ambayehu 560
Protein metabolism
• During the first few days of fasting, there is a
rapid breakdown of muscle protein.
• By several weeks of fasting, the rate of muscle proteolysis

decreases because there is a decline in the need for glucose
as a fuel for the brain, which has begun using ketone
bodies as a source of energy.
07/22/2024 Dr Ambayehu 561

Major Metabolic Pathways in Skeletal Muscle During Starvation
07/22/2024 Dr Ambayehu 562

Brain in Fasting
• During the first days of fasting, the brain continues to
use glucose exclusively as a fuel
• In prolonged fasting (greater than two to three weeks),

plasma ketone bodies reach significantly elevated
levels, and replace glucose as the primary fuel for the
brain.
• This reduces the need for protein catabolism

for gluconeogenesis.
07/22/2024 Dr Ambayehu 563
Major Metabolic Pathways in Brain During Starvation
07/22/2024 Dr Ambayehu 564

Prolonged Fasting
• The utilization of FA by peripheral tissues and ketone body
utilization by brain continues till the body fat is depleted.
• In the final phase of starvation, i.e., when the body fat is
exhausted, the energy demand of body must be met
entirely from muscle protein.
• So for the survival of individual muscle protein
breakdown occurs at increased rate and the individual
becomes physically inactive, which ultimately leads to
death.
07/22/2024 Dr Ambayehu 565
Kidney in Long-Term Fasting
• As fasting continues into early starvation and beyond,
the kidneys play important roles.
• Kidney expresses the enzymes of gluconeogenesis,

including glucose 6-phosphatase, and in late fasting
about 50% of gluconeogenesis occurs here.
• Kidney also provides compensation for the acidosis

that accompanies the increased production of ketone
bodies (organic acids).
07/22/2024 Dr Ambayehu 566
• The glutamine released from the muscle is taken up by the
kidney and acted upon by renal glutaminase and glutamate
dehydrogenase , producing α-ketoglutarate that can enter
the TCA cycle, plus ammonia.
• The ammonia picks up H+ from ketone body dissociation,

and is excreted in the urine as NH4 +, decreasing the acid
load in the body.
• In long-term fasting, then, there is a switch from

nitrogen disposal in the form of urea to disposal in the
ammonia.
form of 43
07/22/2024 Dr Ambayehu 567
44
07/22/2024 Dr Ambayehu 568
Metabolic Adaptation: summary
Fed State Blood glucose ~ 6 mM. Liver and muscle make

glycogen. Liver uses amino acids and fatty acids.
Triaclyglycerols stored in adipose cells.
6 -12 hrs Blood glucose ~ 4.5 mM. Liver uses muscle amino acids to
make and export glucose. Triacylglycerols split and the glycerol
is used by the liver to make glucose. Fatty acids used by liver
and muscle.
1-3 days Carbohydrate reserves depleted. Muscle rapidly degraded

12/6/2016
to amino acids. Triacylglycerols used. 45
07/22/2024 Dr Ambayehu 569
Why do neurons not use fatty acids asfuel?
1) Fatty acids may be more prone to causing ROS damage because

more of their products are processed through the FADH2 entry
point into the electron transport chain (due to more FADH2
production), which generates a lot of ROS.
2) Neurons have low levels of anti-oxidant enzymes (catalase,
SOD, glutathione peroxidase), so are prone to ROS damage.
3) They have poor regenerative capacity and are vulnerable to
irreversible ROS damage and death.
07/22/2024 Dr Ambayehu 570
4) Fatty acid metabolism requires more oxygen per ATP molecule
produced than glucose or ketone body metabolism, with
possible increased risk of hypoxia.
5) Ketone bodies and glucose are metabolized more rapidly than FA

and therefore can better keep up with high neuron demand for ATP,
which is needed especially for synaptic signaling.
6) Neurons have low levels of enzymes for beta-oxidation of FA.
07/22/2024 Dr Ambayehu 571

Impairment of metabolic integration
1. Obesity
Is a disorder of body weight regulatory systems characterized by an accumulation of
excess body fat.
Is the result of taking in more calories in the diet than are expended by the body’s
energy-consuming activities.
When calorie intake exceeds calorie usage, the excess fuel is stored as fat.
Is defined in terms of body mass index (BMI) as:
BMI = weight in kg/(height in m)2
BMI < 25 is considered as normal
= 25 to 30 is overweight (35% of adults in USA)
> 30 is obese (30% of adults in USA).
07/22/2024 Dr Ambayehu 572
Obesity increases a risk for developing T2DM, hypertension, heart
disease, cancers, fatty liver and gallstones, arthritis and gout, with
reduction in life expectancy.
Leptin (Greek leptos, “thin”): is a small protein hormone that is
produced in adipocytes and moves through the blood to the brain,
where it acts on receptors in the hypothalamus to control appetite.
Leptin carries the message that fat reserves are sufficient, and it
promotes a reduction in fuel intake and increases expenditure of
energy.
In humans, leptin increases the metabolic rate and decreases appetite.
Obese humans have leptin deficiency
07/22/2024 Dr Ambayehu or resistance to its action. 573
2. Diabetes Mellitus
Diabetes mellitus (DM) describes a complex metabolic disorder of multiple
aetiologies
Characterized by chronic hyperglycemia with disturbances of carbohydrate,
fat and protein metabolism
Results from defects in insulin secretion, insulin action, or both.
It is the commonest endocrine disorder
It highly reduces quality of life and needs enormous societal costs.
Is the fourth leading cause of death in most developed countries and a prime
cause of excess cardiovascular morbidity and mortality in Western populations
IDF projects that worldwide diabetes prevalence will increase from 3.43%
(2010) to 5.3% (2030).
07/22/2024 Dr Ambayehu 574
In the fed-state, insulin is responsible for the following
metabolic effects:
a. Glucose transport into the skeletal muscles and the
adipose tissues through GLUT4
b. Increase in the levels of glucokinase synthesis that traps
glucose (in the form of glucose-6-phosphate) in the liver
c. Inhibition of gluconeogenesis in the liver.

d. Inhibition of protein and TAG degradation.
If the actions of insulin are impaired, hyperglycemia,
hyperlipidemia, and their complications are gradually
caused.
07/22/2024 Dr Ambayehu 575

Types of Diabetes Mellitus:
 Classified into four clinical cases on the basis of the pathogenic
process that leads to hyperglycemia.
Type I Diabetes Mellitus (TIDM):
Circulating insulin is very low or absent.
Pancreatic β-cells fail to respond to all insulin-secretory
stimuli.
Accounts for only 5-10% of those with diabetes.
Results from an autoimmune disease in which the body's
own immune system destroys β-cells of the pancreas
Patients exhibit diabetic ketoacidosis (DKA), weight loss,
deep and rapid breathing.
Patients rely on exogenous insulin for survival.
07/22/2024 Dr Ambayehu 576

07/22/2024 Dr Ambayehu 577
Type II Diabetes mellitus (T2DM):
Characterized by progressive development of insulin
resistance or insulin-secretory defect.
Most prevalent, 90-95% of those with diabetes.
About 90% of patients who develop T2DM are obese.
Since the patients have ability to secrete some
endogenous insulin, they may not show DKA.
Patients are considered to require insulin but not to
depend on insulin treatment to survive.
The risk of developing type 2DM can be influenced by
ethnicity, obesity, dyslipidema, hypertension, sedentary
lifestyle, and aging.
07/22/2024 Dr Ambayehu 578

07/22/2024 Dr Ambayehu 579
Gestational diabetes mellitus (GDM)
Defined as any degree of glucose intolerance with first
onset or recognition during pregnancy
Caused due to significant hormonal change/increase.
Associated with insulin resistance
Usually disappears when pregnancy is over
But women have a higher risk of developing type 2
diabetes later in their lives
Other specific types of diabetes
These are resulted due to other causes that include:
Genetic defects of β-cell function, e.g. MODY (Maturity
Onset Diabetes in the Young) syndromes
Infections, e.g. congenital rubella
07/22/2024 Dr Ambayehu 580
Stress diabetes: due to increased secretion of
catecholamines as in emotional states
Secondary to Cushing's syndrome, pheochromocytoma
Pituitary diabetes: due to over-secretion of growth
hormone, as in acromegaly.
Third-World Diabetes: due to eating cheap high
carbohydrate diet over a long period of time which can
lead to gradual exhaustion of the β-cells.
Note: Fasting and glucose-stimulated C-peptide levels
have been used to distinguish type 1 from type 2 diabetes
07/22/2024 Dr Ambayehu 581

Metabolic changes in diabetes mellitus
07/22/2024 Dr Ambayehu 582

Clinical features of diabetes mellitus
Glucose accumulates in blood (hyperglycemia) that exceeds the renal
reabsorption limit (renal threshold) and hence is excreted in urine in large
amounts (glucosuria).
Glucose is osmotically active and, hence, draws large amount of water into
the plasma (hyperosmosis) causing frequent urination (polyuria) that leads to
excessive thirst (polydypsia) and intracellular glucose starvation causes
hunger (polyphagia).
Weakness, irritability, muscle wasting and weight loss occur due to inability
of muscles to take up glucose and tissue protein catabolism that provides
amino acids for gluconeogenesis.
Impaired immunity, frequent skin infections, sexual problems, and fruity odor
on breath
07/22/2024 Dr Ambayehu 583

The end!
07/22/2024 Dr Ambayehu 584

Chapter Ten
Porphyrin and bile pigments
07/22/2024 Dr Ambayehu 585

Heme synthesis
Both myoglobin and haemoglobin are hemoproteins
Heme is the prosthetic group of heme proteins (prosthetic group is
organic ligand that firmly or tightly attached to proteins)
Heme is a member of a family of compounds called porphyrins.
Many important proteins contain heme as a prosthetic group.
Examples of Some Important Human and Animal Hemoproteins:
a. Hemoglobin (Transport of oxygen in blood)
b. Myoglobin (Storage of oxygen in muscle)
c. Cytochrome c (Involvement in electron transport chain)
d. Cytochrome P450 (Hydroxylation of xenobiotics)
e. Catalase (Degradation of hydrogen peroxide)
f. Tryptophan pyrrolase (Oxidation of tryptophan)
07/22/2024 Dr Ambayehu 586
Heme synthesis…
The major sites of heme biosynthesis are the liver, which
synthesizes a number of heme proteins (particularly
cytochrome P450), and the erythrocyte-producing cells of the
bone marrow, which are active in hemoglobin synthesis.
Heme synthesis occurs in all cells due to the requirement for
heme as a prosthetic group on enzymes and electron transport
chain.
The initial reaction and the last three steps in the formation of
porphyrins occur in mitochondria, whereas the intermediate
steps of the biosynthetic pathway occur in the cytosol.
Note: Mature red blood cells lack mitochondria and are
unable to synthesize heme.
07/22/2024 Dr Ambayehu 587

Heme synthesis…
Reactions:
1) Delta-aminolevulinic acid synthase (ALA synthase)
The substrates are succinyl-CoA and glycine
The product is delta-aminolevulinic acid (ALA).
An essential cofactor is pyridoxal phosphate (vitamin B 6).
This is the rate-limiting reaction of heme synthesis in all tissues, and it is
therefore tightly regulated.
2) ALA dehydratase /porphobilinogen Synthase
The substrates are two molecules of ALA.
The product is porphobilinogen, the first pyrrole.
ALA dehydratase is a -SH containing enzyme.
It is very susceptible to inhibition by lead.
ALA synthase occurs in the mitochondria, whereas ALA dehydratase is
present in the cytosol
07/22/2024 Dr Ambayehu 588
Reactions 1 & 2 Biosynthesis of porphobilinogen
07/22/2024 Dr Ambayehu 589

Heme synthesis…
3) Uroporphyrinogen I synthase and uroporphyrinogen III synthase
They catalyze Conversion of porphobilinogen to uroporphyrinogens.
The porphyrin ring is formed by condensation of 4 molecules of
porphobilinogen.
Porphyrins are cyclic compounds formed by the linkage of four pyrrole rings
through methyne (==HC—) bridges having different substituents.
Uroporphyrinogen I synthase is also called porphobilinogen (PBG)deaminase or
hydroxymethylbilane (HMB) synthase.
Porphobilinogen Deaminase catalyzes successive porphobilinogen
condensations, initiated in each case by elimination of the amino group to
hydroxymethylbilane (linear tetrapyrrole)
Unlike uroporphyrin I, Production of uroporphyrin III requires two enzymes.
Uroporphyrinogen III synthase catalyze cyclization of hydroxymethylbilane to
Uroporphyrinogen III but cyclization of hydroxymethylbilane to Uroporphyrinogen
I is spontaneous
Uroporphyrinogen III is the precursor for synthesis of vitamin B12, chlorophyll,
and heme, in organisms that produce these compounds.
07/22/2024 Dr Ambayehu 590
Uroporphyrinogen I
synthase and
uroporphyrinogen III
synthase
07/22/2024 Dr Ambayehu 591

Heme synthesis…
4) Uroporphyrinogen decarboxylase
Decarboxylates the acetic acid groups on Uroporphyrinogen III ,
converting them to methyl groups (Coproporphyrinogen III is
produced)
5) Coproporphyrinogen III oxidase
Catalyzes the conversion of two propionic acid groups to vinyl groups
(Protoporphyrinogen III/IX is produced)
6) Protoporphyrinogen IX oxidase
Protoporphyrinogen IX oxidase converts the methylene bridges between
the pyrrole rings to methenyl bridges (Protoporphyrin IX is a product).
7) Ferrochelatase
Ferrochelatase adds Fe++ to protoporphyrin IX, forming heme.
The enzyme requires Fe++, ascorbic acid and cysteine (reducing agents).
Ferrochelatase is inhibited by lead.
07/22/2024 Dr Ambayehu 592
Fig. Uroporphyrinogen decarboxylase reactions
07/22/2024 Dr Ambayehu 593

Fig. Uroporphyrins and coproporphyrins. (A, acetate; P, propionate; M, methyl.)
07/22/2024 Dr Ambayehu 594

Fig. Ferrochelatase catalyzed iron addition
CH2 protoporphyrin IX CH2 heme
CH CH3 CH CH3
H3C CH CH2 H3C CH CH2

NH N N N
Fe++ 2H+
Fe
N HN N N
H3C CH3 H3C CH3
CH2 CH2 Ferrochelatase CH2 CH2
CH2 CH2 CH2 CH2

- - - -
COO COO COO COO
07/22/2024 Dr Ambayehu 595

Names of Porphyrins
The names of the porphyrins of interest consist of a word and a number,
e.g., uroporphyrin III. The word denotes the kinds of substituents found
on the ring, and the number denotes how they are arranged.
There are three important words:
1. Uroporphyrin contains A and P only
2. Coproporphyrin contains M and P only (A has been changed to M)
3. Protoporphyrin contains M and P and V (some P has been changed to
V)
There are two important numbered series, I and III.
1. In series I the substituents repeat in a regular/symmetric manner.
2. In series III the order of substituents repeats in irregular/asymmetric
manner.
A= acetate, P= Propionate , M= Methyl & V=Vinyl
07/22/2024 Dr Ambayehu 596

Summary of Heme
synthesis
07/22/2024 Dr Ambayehu 597

Fig. Lead inhibition on heme biosynthesis
High levels of lead can

affect heme metabolism
by combining with SH
groups in enzymes such
as ferrochelatase
and ALA dehydratase
07/22/2024 Dr Ambayehu 598

Effects of Lead poisoning
High ALA is thought to cause some of the neurological effects of
lead poisoning, although Pb++ also may directly affect the nervous
system.
ALA is toxic to the brain, perhaps due to:
• Similar ALA & neurotransmitter GABA (γ-aminobutyric acid)
structures.
• ALA autoxidation generates reactive oxygen species (oxygen
radicals).
COO  COO
CH2 CH2
CH2 CH2
C O CH2
CH2 NH3+
NH3+
ALA GABA
07/22/2024 Dr Ambayehu 599
Regulation of heme synthesis
Substrate availability: Fe++ must be available for ferrochelatase.

Regulation of transcription or post-translational processing of enzymes
of the heme synthesis pathways differs between erythrocyte forming
cells & other tissues.
 In erythrocyte-forming cells there is steady production of pathway enzymes,
limited only by iron availability.
 In other tissues expression of pathway enzymes is more variable & subject to
feedback inhibition by heme.
Effects of drugs and steroids: Certain drugs and steroids can increase
heme synthesis via increased production of the rate limiting enzyme,
ALA synthase.
Most of these drugs are metabolized by a system in the liver that utilizes
a specific hemoprotein, cytochrome P450.
During their metabolism, the utilization of heme by cytochrome P450 is
greatly increased, which in turn diminishes the intracellular heme
concentration.
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Porphyrias
Porphyrias are rare, inherited (or occasionally acquired) defects in

heme synthesis, resulting in the accumulation and increased excretion
of porphyrins or porphyrin precursors (purple color pigment in urine).
Porphyrias may be divided into two major types.
1. Erythropoietic porphyria is a defect of porphyrin metabolism of
blood-producing tissues.
2. Hepatic porphyria is a defect in porphyrin metabolism of the liver.
Either type may be hereditary (caused by a gene defect) or acquired
I. Acute intermittent porphyria (defect of hepatic
uroporphyrinogen I synthase activity)
Delta-aminolevulinic acid and Porphobilinogen (the substrate)
accumulates, and is excreted in the urine.
Heme synthesis is reduced. ALA synthase activity therefore
increases.
07/22/2024 There are neurological symptoms
Dr Ambayehu 601
Porphyrias…
II. Congenital erythropoietic porphyria ( defect of uroroporphyrinogen
III synthase)
Large amounts of type I porphyrins
Skin photosensitivity
The porphyrins, when exposed to light of 400nm wavelength, are thought
to become "excited" and then react with molecular oxygen to form
oxygen radicals.
This Oxygen radicals injure lysosomes and other organelles. Damaged
lysosomes release their degradative enzymes, causing variable degrees of
skin damage, including scarring.
III. Variegate Porphyria
It is an acute disease caused by deficiency of Protoporphyrinogen oxidase
in the liver.
Protoporphyrinogen IX and other intermidiates prior to the block appear
in the urine
Patients are Photosensitive
07/22/2024 Dr Ambayehu 602
Porphyrias…
IV. Porphyria cutanea tarda
It is a chronic disease of the liver and erythroid tissues.
The disease is associated with a deficiency in uroporphyrinogen
decarboxylase.
Uroporphyrin accumulates in urine
Clinical expression of the enzyme deficiency is influenced by
various factors, such as hepatic iron overload, exposure to sunlight,
and the presence of hepatitis B or C, or HIV infections.
V. Hereditary coproporphyria
Coproporphyrinogen oxidase deficiency in the liver with high
urinary and/or fecal levels of coproporphyrins and intermediates
prior to block
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07/22/2024 Dr Ambayehu 604
Drugs and Porphyrias
As described above, ALA Synthase is the key regulatory enzyme of the

heme biosynthetic pathway in liver.
A large number of drugs (eg, barbiturates, griseofulvin) induce the enzyme.
Most of these drugs do so by inducing cytochrome P450, which uses up
heme and thus derepresses (induces) ALA Synthase
Thus, taking drugs that cause induction of cytochrome P450 (so-called
microsomal inducers) can precipitate attacks of porphyria.
It is important for patients to avoid drugs that cause induction of
cytochrome P450.
Ingestion of large amounts of carbohydrates (glucose loading) or
administration of hematin (a hydroxide of heme) may repress ALA
Synthase , resulting in diminished production of harmful heme precursors.
Patients exhibiting photosensitivity may benefit from administration of β-
carotene; this compound appears to lessen production of free radicals, thus
diminishing photosensitivity. Sunscreens that filter out visible light can
also be helpful to such patients.
07/22/2024 Dr Ambayehu 605
Fig. Porphyrias
07/22/2024 Dr Ambayehu 606

Heme Degradation
Catabolism of heme produces bilirubin

Most of the heme which is degraded comes from
hemoglobin in red blood cells, which have a life span of
about 120 days.
There is thus a turnover of about 6 g/day of hemoglobin.
Normally, senescent red blood cells and heme from other
sources are engulfed by cells of the reticuloendothelial
system. The globin is recycled or converted into amino
acids, which in turn are recycled or catabolized as
required. Heme is oxidized.
The iron-free porphyrin portion of heme is also degraded,
mainly in the reticuloendothelial cells of the liver, spleen,
and bone marrow.
07/22/2024 Dr Ambayehu 607
Heme Degradation …
1)Conversion of heme to bilirubin (cells of the

reticuloendothelial system in spleen, liver and bone
marrow)
Heme ring is cleaved by a microsomal heme oxygenase
between the I and II pyrrole rings.
Biliverdin reductase reduces the central methene bridge of
biliverdin, producing bilirubin.
The high lipid solublity of bilirubin determines its behavior
and its further metabolism that it must be transported in the
blood by a carrier; the physiological carrier is serum albumin
that it is soluble in the lipid bilayers of cell membranes.
07/22/2024 Dr Ambayehu 608

Fig. Conversion of heme to bilirubin
The green pigment biliverdin is

produced as ferric iron and CO are
released
Biliverdin is reduced, forming the
red-orange bilirubin.
Bilirubin and its derivatives are
collectively termed bile pigments.
07/22/2024 Dr Ambayehu 609

2) Conjugation of bilirubin with glucuronic acid: (hepatocytes)
This increased its water solubility, decreases its lipid

solubility and eases its excretion. Conjugation is
accomplished by attaching two molecules of glucuronic
acid to it in a two step process by UDP glucuronyl
transferase .
The reaction is a transfer of two glucuronic acid groups
sequentially to the propionic acid groups of the bilirubin.
The major product is bilirubin diglucuronide .
Bilirubin diglucuronide is excreted in the bile. It is subject
to subsequent transformations to other species by the
intestinal bacteria.
07/22/2024 Dr Ambayehu 610
Fig. Conjugation of bilirubin
07/22/2024 Dr Ambayehu 611

Heme
Reticuloendothelial
Biliverdin
system
Unconjugated bilirubin
Unconj.bilirubin/
Systemic circulation
albumin complex
Kidney
Unconj. bilirubin
Hepatocytes
Bilirubin
diglucuronide
urine
Bilirubin diglucuronide
Stercobilins Urobilinogen Bilirubin
Large intestine Small intestine
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07/22/2024 Dr Ambayehu 613
Hyperbilirubinemia causes jaundice
When bilirubin in the blood exceeds 1 mg/dL (17.1 mol/L),
hyperbilirubinemia exists.
Hyperbilirubinemia may be due to the production of more bilirubin
than the normal liver can excrete, or it may result from the failure of
a damaged liver to excrete bilirubin produced in normal amounts. In
the absence of hepatic damage, obstruction of the excretory ducts of
the liver—by preventing the excretion of bilirubin—will also cause
hyperbilirubinemia.
In all these situations, bilirubin accumulates in the blood, and when it
reaches a certain concentration (approximately 2–2.5 mg/dL), it
diffuses into the tissues, which then become yellow. That condition is
called jaundice or icterus.
In clinical studies of jaundice, measurement of bilirubin in the serum
is of great value.
The clinical determination of plasma bilirubin distinguishes between
conjugated (direct) and unconjugated (indirect) bilirubin.
07/22/2024 Dr Ambayehu 614
Hyperbilirubinemia causes jaundice…
A method for quantitatively assaying the bilirubin content of the
serum was first devised by van den Bergh by application of Ehrlich's
test for bilirubin in urine. The Ehrlich reaction is based on the
coupling of diazotized sulfanilic acid (Ehrlich's diazo reagent) and
bilirubin to produce a reddish-purple azo compound. Conjugated
bilirubin is water soluble and reacts directly. This is called the
DIRECT bilirubin.
Unconjugated bilirubin bound to albumin, alcohol is added to release
it into solution, where it can now react. This is called the INDIRECT
bilirubin.
Depending on the type of bilirubin present in plasma—ie,
unconjugated or conjugated—hyperbilirubinemia may be classified
as retention hyperbilirubinemia, due to overproduction, or
regurgitation hyperbilirubinemia, due to reflux into the bloodstream
because of biliary obstruction.
07/22/2024 Dr Ambayehu 615
Hyperbilirubinemia causes jaundice…
Because of its hydrophobicity, only unconjugated bilirubin can
cross the blood-brain barrier into the central nervous system; thus,
encephalopathy due to hyperbilirubinemia (kernicterus) can occur
only in connection with unconjugated bilirubin, as found in
retention hyperbilirubinemia.
On the other hand, because of its water-solubility, only conjugated
bilirubin can appear in urine. Accordingly, choluric jaundice
(choluria is the presence of bile pigments in the urine) occurs only
in regurgitation hyperbilirubinemia, and acholuric jaundice occurs
only in the presence of an excess of unconjugated bilirubin.
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07/22/2024 Dr Ambayehu 617
Causes of Jaundice
1) Hemolytic anemia
--  destruction of erythrocytes
2) Hepatitis or cirrhosis
--  conjugation and excretion of bilirubin
3) Bile duct obstruction
-- conjugated bilirubin not delivered to intestine;
4) Neonatal “physiological jaundice”
-- immature hepatic system of the newborn:
 uptake, conjugation, excretion of bilirubin
it backs up, spills over into the blood
07/22/2024 Dr Ambayehu 618
Fig. Diagrammatic representation of some major causes of jaundice
07/22/2024 Dr Ambayehu 619

Fig. Diagrammatic representation of the three major processes (uptake, conjugation,
and secretion) involved in the transfer of bilirubin from blood to bile.
07/22/2024 Dr Ambayehu 620

07/22/2024 Dr Ambayehu 621
Chapter Eleven
Overview To Nucleotides And
Molecular Genetics
07/22/2024 Dr Ambayehu 622

Topic Outline
Þ Purine and pyrimidine bases
ÞNucleotide and its biological importance .
ÞPurine synthesis Denovo
ÞPurine salvage pathway. And Purines degradation.
ÞPyrimidine synthesis and Pyrimidines degradation.
ÞThymine nucleotide synthesis.

07/22/2024 Dr Ambayehu 623
Learning objectives
At the end of this section students will be able to:
ÞMention functions of nucleotides with examples
ÞIdentify precursors for de novo synthesis of purines/
pyrimidines
ÞDescribe the “Salvage” pathway for synthesis of purines and
pyrimidines.
ÞDescribe the regulation of Purine/pyrimidine biosynthesis
ÞExplain how purines/pyrimidines are degraded.
ÞAnalyze the biochemical basis of disorders related to
Purine/pyrimidine metabolism (Gout, Lesch- Nyhan syndrome,
Orotic aciduria, SCID)
07/22/2024 ÞApply biochemistry of nucleotides
Dr Ambayehu to drug therapy 624
Overview
Important amino acids is the synthesis of purine and pyrimidine
ATP is the most commonly but GTP is used in protein synthesis
UTP is the source of energy for activating glucose & Galactose.
CTP is an energy source in lipid metabolism.
AMP is as coenzymes like NAD and Coenzyme A.
DNA /RNA
07/22/2024 Dr Ambayehu 625
Nomenclature
Nitrogen Bases
ÞThere are two kinds of nitrogen-containing bases - Purines
and Pyrimidines.
ÞPurines consist of a Six-Membered and a Five-Membered

nitrogen-containing ring, fused together.
ÞPyrimidines have only a Six-Membered N-containing ring.
ÞThere are 4 purines and 4 Pyrimidines

07/22/2024 Dr Ambayehu 626
Pyrimidines Purines
07/22/2024 Dr Ambayehu 627

Nomenclature of Nucleic Acid Components
Base Nucleoside Nucleotide Nucleic acid
 Pyrimidines
Cytosine Cytidine Cytidylate RNA
Deoxycytidine Deoxycytidylate DNA
Thymine Thymidine Thymidylate DNA

Deoxythymidine Deoxythymidylate)
Uracil Uridine Uridylate RNA

07/22/2024 Dr Ambayehu 628
Nomenclature of Nucleic Acid Components
Base Nucleoside Nucleotide Nucleic
acid
 Purines
Adenine Adenosine Adenylate
RNA
Deoxyadenosine Deoxyadenylate DNA
Guanine Guanosine Guanylate

07/22/2024 Dr Ambayehu 629
Nucleoside and Nucleotide
 Sugar +Base = Nucleoside
B
S
 Phosphate+ Sugar + Base =

Nucleotide
P
B
S
07/22/2024 Dr Ambayehu 630
Ribonucleoside Deoxyribonucleoside
N-glycosidic
Bond
Purine linkage with sugar N9-C1

07/22/2024 Dr Ambayehu 631
Pyrimidine linkage with sugar N1-C1
07/22/2024 Dr Ambayehu 632

These are some important
1. ATP, GTP, CTP, UTP, NAD, FAD and CoA are important
Ribonucleotides which act as Coenzymes.
2. Deoxyribonucleotides are required for DNA replication and
repair.
3. Ribonucleotides are required for RNA Synthesis .
4. They are used in Biosynthetic reactions like UDP-glucose, in

07/22/2024 Dr Ambayehu 633
glycogen synthesis and UDP-galactose in lactose synthesis.
These are some important
4. ATP acts as Currency of free energy for all cellular
activities.
5. Act as intracellular messenger’s .eg. c AMP, c GMP
6. GTP is used in Protein synthesis.
7. SAM participates in Transmethylation reactions
07/22/2024 Dr Ambayehu 634

Nucleotide Synthesis
and
Degradation
07/22/2024 Dr Ambayehu 635

Degradation of nucleic acids
Nucleoprotein
In stomach Gastric acid and pepsin
Nucleic acid Protein

In small Endonucleases: RNase and DNase
intestine
Nucleotide
Nucleotidase
Phosphate Nucleoside
Nucleosidase
07/22/2024
Base Dr Ambayehu
Ribose 636
Absorption of nucleotides
Nucleoprotein
Protein Nucleic acid

Nucleases
Nucleotide
Nucleotidase
Absorption
Phosphate Nucleoside Blood
Nucleosidase
Base Ribose
07/22/2024 Dr Ambayehu 637

Purine Synthesis
07/22/2024 Dr Ambayehu 638

Metabolism of Purine nucleotides
Biosynthesis of purine nucleotides
De novo synthesis
Salvage pathway
AMP GMP
07/22/2024 Dr Ambayehu 639
De Novo Synthesis of Purine Nucleotides
Sources of individual atoms in the Purine ring
CO2
07/22/2024 Dr Ambayehu 640

PP-1-R-5-P （ 5’- AMP ATP
R-5-P
phosphoribose 1’-pyrophosphate, （ 5’-phosphoribose ）
PRPP ） PRPPK
Gln
Glutamine PRPP
amidotransferase
(GPRT) Glu
H2N-1-R-5´-P
（ 5´-phosphoribosyl-amine ）
Gly, one carbon
units, Gln, CO2, Asp
involved step by step
AMP
IMP
GMP
07/22/2024 Dr Ambayehu 641
AMP ATP
PP-1-R-5-P （ 5’-phosphoribose 1’- R-5-P
pyrophosphate, PRPP ）（ 5’-phosphoribose ）
PRPPK
Gln
Glutamine PRPP
amidotransferase Glu
(GPRT)
H2N-1-R-5´-P
（ 5´-phosphoribosyl-amine ）
Gly, one carbon
units, Gln, CO2, Asp
involved step by
step
AMP
IMP
07/22/2024
GMP
Dr Ambayehu 642
5-Phosphoribosyl-1-pyrophosphate (PRPP).
Ribose 5-phosphate+ATP = 5-Phosphoribosyl-1-

pyrophosphate
• Synthesis is catalyzed by PRPP synthetase .
• The Enz. is activated by (Pi) and inhibited by end-product
• Sugar in PRPP is ribose

ATP AMP
PP-1-R-5-P
R-5-P （ 5’-phosphoribose 1’-
（ 5’-phosphoribose ）
PRPPK pyrophosphate, PRPP ）
Activator -Pi Inhibitor Purine ribonucleotides

07/22/2024 Dr Ambayehu 643
Regulation of de novo synthesis of purine nucleotides
—
— —
+ + Adenyl-
AMP ADP ATP
PRPPK GPAT succinate
R-5-P
PRA IMP
PRPP
ATP — XMP GMP GDP GTP
—
_
Adenyl-
GTP AMP ADP ATP
succinate
+
IMP
XMP + GTP
ATP GMP GDP
_
07/22/2024 Dr Ambayehu 644
APRT
Adenine+ PRPP AMP + PPi
HGPRT
Hypoxanthine+ PRPP IMP + PPi
HGPRT
Guanine+ PRPP GMP + PPi
Adenylate
kinase
Adenosine AMP
ATP
ADP
APRT: adenine phosphoribosyltransferase

HGPRT: inosine-guanine phosphoribosyl transferase
07/22/2024 Dr Ambayehu 645
Cont…..
HGPRT
Hypoxanthine+ PRPP IMP + PPi

HGPRT
Guanine+ PRPP GMP + PPi
HGPRT: inosine-guanine phosphoribosyl transferase
Lesch-Nyhan Syndrome
07/22/2024 Dr Ambayehu 646

Symptoms of patients with Lesch-Nyhan syndrome
including :
Self-mutilation,
Spastic movements, and
Mental retardation.
Gout
 Allopurinol treatment alleviate the symptoms of uric acid over-
production but does not remedy the neurologic problems.
07/22/2024 Dr Ambayehu 647

Purine Catabolism
The end product of purine catabolism in man is uric acid.
Uric acid is formed primarily in the liver and excreted by

the kidney into the urine.
07/22/2024 Dr Ambayehu 648

Cont….
Inosine Adenosin
Only T-cell e
Both T&B-cell
Hypoxanthine
Xanthine Urate
07/22/2024 Dr Ambayehu 649
Urate in the blood could accumulate either through an
overproduction and/or an under excretion of uric acid.
07/22/2024 Dr Ambayehu 650

The treatment of gout is the drug Allopurinol, an isomer of
hypoxanthine
Allopurinol is a substrate for xanthine oxidase
07/22/2024 Dr Ambayehu 651

Metabolism of pyrimidine nucleotides
Biosynthesis of pyrimidine nucleotides
De novo synthesis
Salvage pathway
07/22/2024 Dr Ambayehu 652

De novo synthesis of pyrimidine
nucleotides
4
Glutamin 3 5
e Aspartat
2
CO2
1
6
e
07/22/2024 Dr Ambayehu 653

Process of de novo synthesis of UMP
1. Formation of Carbamoyl phosphate (CP)
CO2 + glutamine + H2O + 2ATP
Carbamoyl Phosphate Synthetase-

Ⅱ
O
(CPS-Ⅱ )
H2 N C O ~ PO32- + 2ADP + Pi
Carbamoyl
phosphate
07/22/2024 Dr Ambayehu 654
2. Formation of UMP
O
O ~ PO32-
H2 N C
+ Aspartate
Carbamoyl
phosphate
Carbamoyl
Aspartate
Orotate
PRPP
UMP
07/22/2024 Dr Ambayehu 655

Pyrimidine Synthesis
2 ATP + HCO3- + Glutamine + H2O
O
C
HN CH
2 ADP +
Glutamate + Carbamoyl O
Phosphate C C
Pi
Synthetase II
C O N
HN CH PRPP PPi COO
2-
O3P O CH2
O
NH2
C C Orotate Phosphoribosyl H H 
N Transferase
O C O H H
H COO OH OH
O PO3-2 Orotidine-5'-monophosphate
Orotate
(OMP)
Carbamoyl Phosphate
Reduced
Aspartate Quinone OMP
Aspartate Dihydroorotate
Transcarbamoylase Decarboxylase
Dehydrogenase CO2
(ATCase)
Pi Quinone
O
O
O
C
C HN CH
HO C
HN CH2
CH2 C CH
NH2 H2O
O N
C CH
C CH O N 2-
O3P O CH2
Dihydroorotase O
O N H COO H H 
H COO
H H
Dihydroorotate OH
Carbamoyl Aspartate OH
Uridine Monophosphate
07/22/2024 Dr Ambayehu (UMP) 656
Synthesis of CTP, dTMP or TMP
UMPK NDK CTP

UDP CTP
UTP synthase
ATP ADP Gln
ATP ADP Glu
UMP ATP ADP
dUDP
dCMP
dTMP
dUMP
TMP synthase
07/22/2024 Dr Ambayehu 657

Uracil to Thymine: 1-Carbon fragment on folate DNA
Dihydrofolate: Dead-end metabolite Enzymology
Fudr
Cytoplasm (fluorodeoxyuridate)
Suicide Inhibitor
TMP
TMP Synthase
Synthase
dUMP 11
dTMP
N FH
FH22
N55,N
,N1010-CH
-CH22-FH
-FH44 NADPH
NADPH ++ H+
H+
Glycine
Glycine
2.
2. Dihydrofolate
Dihydrofolate Rib5P
Rib5P
reductase
reductase
3.
3. Serine
Serine Hydroxymethyl
Hydroxymethyl transferase
transferase PPP
FH
FH44
NADP
NADP++
Serine
Serine
G6P
G6P
Aminopterin
Methotrexate
07/22/2024 Trimethoprim
Dr Ambayehu 658
Regulation of de novo synthesis of pyrimidine nucleotides
+ ATP + CO2+ Glutamine 1. Activated by substrates

2. Inhibited by products
Carbamoyl Phosphate
Aspartate
- -
Carbamoyl Aspartate
+
- Purine Nucleotides
PRPP ATP + 5-phosphate ribose
UMP
- Pyrimidine Nucleotides
UTP CTP
-
07/22/2024 Dr Ambayehu 659
Ribose to Deoxyribose Nucleic Acid Metabolism
Ribonucleotide reductase Cytoplasm
H
H++ ++ NADPH
NADPH NADP
NADP++
FAD
FAD FADH
FADH22
Thioredoxin Reductase Glutathione Reductase
Glutathione
Thioredoxin
Glutaredoxin
Ribonucleotide
Ribonucleotide reductase
reductase
Base Base
PPO O PPO O
2 2
OH OH OH
H
07/22/2024 Dr Ambayehu 660

Salvage pathway of pyrimidine nucleotides
Uracil Phosphate Ribosyl
transferase
Uracil + PRPP UMP + PPi
Uridine Phosphorylase
Uracil + 1-phosphoribose UMP +ADP
Uridine Kinase
UMP +ADP
Uracil ribonucleoside + ATP
07/22/2024 Dr Ambayehu 661

Degradation of pyrimidine nucleotides
Nucleotide
Nucleotidase
Nucleosides
Nucleoside Phosphorylase
Phosphoribose Pyrimidine
07/22/2024 Dr Ambayehu 662

Cytosine Thymine
NH3
Uracil
β-Ureidoisobutyrate
Dihydrouracil
H2 O H2 O
+ CO2 + NH3 +
β-aminoisobutyrare
β-alanine
liver Excreted
Succinyl CoA in urine
Acetyl CoA Urea
TAC Glucose
TAC
07/22/2024 Dr Ambayehu 663
Regulation of synthesis of pyrimidine nucleotides
07/22/2024 Dr Ambayehu 664

The End !!
07/22/2024 Dr Ambayehu 665

Molecular Biology
07/22/2024 Dr Ambayehu 666

Key point on DNA replication
— What is DNA
— Importance of replication
— The replication factory
— Mode of replication : semi conservative
— Phase of replication and Place of replication
— Direction of replication
— Initiation of replication: factors involved –prokaryotes
— Eukaryotic initiation of replication : factors involved
— Proteins and enzymes involved in replication
07/22/2024 Dr Ambayehu 667
— Replication fork and Mechanism of replication : steps
— Leading and lagging strands
— Okazaki fragments
— Dna polymerases : Prokaryotes
— Dna polymerase; Eukaryotic
— Difference in prokaryotic and eukaryotic replication
— Speed of replication
— Telomerase
07/22/2024 Dr Ambayehu 668

Comparative Genome Sizes Of Organisms
Gene Chrom.
Organism Size (bp) Average gene density
number number
Human 3.2 billion ~25,000 1 gene /100,000 bases 46
Mus musculus
2.6 billion ~25,000 1 gene /100,000 bases 40
(mouse)
(Yeast) 12.1 million 6000 1 gene / 2000 bases 32
Escherichia coli
4.6 million 3200 1 gene / 1400 bases 1
(bacteria)
H. influenzae
1.8 million 1700 1 gene /1000 bases 1
(bacteria)
07/22/2024 Dr Ambayehu 669

Components involve in molecular biology
DNA RNA Protein
07/22/2024 Dr Ambayehu 670

 The flow of information from DNA to RNA to protein is
termed the “Central dogma” of molecular biology
 With the exception of some viruses that have RNA as the

storehouse of their genetic information.
07/22/2024 Dr Ambayehu 671

DNA Structure, Functions and
Properties
07/22/2024 Dr Ambayehu 672

The primary structure of DNA is the sequence
5’ end
5’
3’
3,5-Phosphodiester linkage 3’ end

07/22/2024 Dr Ambayehu 673
Each nucleotide consists of:
1. Phosphate group
2. Pentose sugar
3. Nitrogenous base
Phosphate
Nitrogenous
Base
Pentose
Sugar
X=H: DNA
07/22/2024 X
Dr Ambayehu X=OH: RNA 674
 Sugar +Base = Nucleoside
B
S
 Phosphate+ Sugar + Base =

Nucleotide
P
B
S
07/22/2024 Dr Ambayehu 675
Basic Structure Of
Pyrimidine And Purine
bases
07/22/2024 Dr Ambayehu 676

Pyrimidines Purines
07/22/2024 Dr Ambayehu 677

The Secondary structure of DNA is the double helix
07/22/2024 Dr Ambayehu 678

DNA Double helix
Watson - Crick have proposed a double helical model of DNA,

having the following Important Characteristic Features ??
07/22/2024 Dr Ambayehu 679
Normally hydrated DNA: B-form DNA
The major groove where majority of the regulatory proteins interact with DNA.
The minor groove where Histones and very few regulatory proteins interact
with DNA.
Minor groove: 12 Å across
Major groove: 22 Å across
07/22/2024 Dr Ambayehu 680

Background: Chromatine structure
07/22/2024 Dr Ambayehu 681

DNA Replication
DNA
07/22/2024 Dr Ambayehu 682
Pictorial Representation Of Basic Components
07/22/2024 Dr Ambayehu 683

Synthesis of the Leading Strand
07/22/2024 Dr Ambayehu 684

Synthesis of the Lagging Strand
07/22/2024 Dr Ambayehu 685

Eukaryotic Cell Cycle
 The cell cycle of eukaryotes consists of four phases .
 The first three phases (G1, S, and G2) constitute
interphase.
07/22/2024 Dr Ambayehu 686

Transcription
07/22/2024 Dr Ambayehu 687

Transcription products :
Þ mRNAs
Þ rRNA,
Þ tRNAs, and
Þ snRNA molecules
that perform specialized
structural, catalytic, and
regulatory functions .
07/22/2024 Dr Ambayehu 688

Cont…
07/22/2024 Dr Ambayehu 689

Structures of tRNA
07/22/2024 Dr Ambayehu 690

The general structure of ribosomes in Prokaryotic.
rRNA Proteins Subunits Assembled

ribosomes
Prokaryotic
07/22/2024 Dr Ambayehu 691

Lodish 132pg
The general structure of ribosomes in Eukaryotes.
rRNA Proteins Subunits Assembled
ribosomes
Eukaryotes
 The S number given each type of rRNA: the rate at

which the molecules sediment in the Ultracentrifuge.
 The larger the number, the larger the molecule (but not
07/22/2024 proportionally). Dr Ambayehu 692
Transcription
DNA
1st steps of gene expression/

The Central dogma
mRNA
07/22/2024 Dr Ambayehu 693
Holoenzyme
The holoenzyme of RNA-pol in E.coli consists of 5
different subunits: 2   .
subunit MW Function
Determine the DNA to be

 36512
transcribed
 150618 Catalyze polymerization


 155613 Bind & open DNA template  
Recognize the promoter
 70263  
for synthesis initiation 
07/22/2024 Dr Ambayehu 694

Pictorial representation of promoter in Prokaryotic
Transcription Unit
Coding 5' 3'
-50 -40 -30 -20 -10 1 10
Template 3' 5'
-35
region -10 start
region
TTGACA
AACTGT
T A T A A T Transcribed region
ATATTA
(Pribnow box)
Consensus sequence
07/22/2024 Dr Ambayehu 695
Pictorial representation of promoter in Eukaryotic
cis-acting element
structural gene
GCGC CAAT TATA
exon intron exon
start
TATA box (Hogness box)
-25
enhancer CAAT box
-70
GC box
07/22/2024 Dr Ambayehu 696

Cont….
07/22/2024 Dr Ambayehu 697

Transcription products of all three RNAP are processed
 RNAP I transcribes the 28S, 18S, and 5.8S rRNA genes,

an activity that is localized to the nucleolus, a region of high
nucleoprotein density in the cell’s nucleus.
07/22/2024 Dr Ambayehu 698

 RNA pol II is responsible for Transcription of snRNA genes
and of structural genes encoding mRNAs leading to protein

synthesis.
07/22/2024 Dr Ambayehu 699

 RNA pol III transcribes the tRNA genes &the 5S rRNA gene.
07/22/2024 Dr Ambayehu 700

Summery Of Transcription Cycle
07/22/2024 Dr Ambayehu 701

Mechanism of RNA Splicing
07/22/2024 Dr Ambayehu 702

Summary of mRNA synthesis modification .
07/22/2024 Dr Ambayehu 703

mRNA
Translation
Protein
07/22/2024 Dr Ambayehu 704

Genetic Code- Table
Genetic Code
07/22/2024
Notice Tryptophan and Met are onlyDrcoded
Ambayehu
for by UGG and AUG Respectively 705
Summery Of Point Mutation
n
Nonsense
io
at
Mutation
ut
tM
len
Si
Missense Mutation
07/22/2024 Dr Ambayehu 706

Translation
What are the required components for
Translation?
07/22/2024 Dr Ambayehu 707

Components Required For Translation
 All 20 amino acids  Enzymes:
Essential aminoacids Aminoacyl tRNA synthetase

 mRNA, Peptidyl transferase
 Transfer RNA (tRNA),  Protein Factors needed for
 Functional Ribosome's
Initiation,
Small subunits
Elongation, &
large subunits
 Energy sources: Termination.
GTP and
07/22/2024 ATP Dr Ambayehu 708
Initiator tRNA :
Overview of Translation
tRNA with first
amino acid aa1
aa1
Ribosomal Initiation E
P A
UAC subunits
Anticodon AUG
Start codon
mRNA
UAG
3′
Stop codon 5′
3′ Elongation
5′ AUG
Recycling of translational (occurs many times.)
Start codon aa1
components aa2
aa3
Release aa4
Factor aa5
Completed E
P A
E
P A
Polypeptide
UAG Termination
Stop
07/22/2024 5′ Codon Dr3′Ambayehu 3′ 709
5′
Steps In Prokaryotic Cont.. IF3 IF1 30S subunit
1st step:
IF1 and IF3 bind to the 30S subunit.
IF3 IF1
Portion of
16S rRNA
Start Codon
5′ SDS ( 9 nt long) 3′
2nd step :
The mRNA binds to the 30S subunit.
SD sequence is complementary to a portion of the 16S rRNA.
07/22/2024 Dr Ambayehu 710
Summery of Prokaryotic initiation complex
IF-3
IF-1
30S Subunit
IF-3
mRNA Template
IF-3
IF-2
tRNAi fMet
come to the P-Site
GTP
IF-3
IF-2 GTP
50S Subunit IF-2

+ GDP + Pi
IF-3
07/22/2024 Dr Ambayehu 711

Cont…
Pre-initiation complex
2
1st
3nd
eIF5
4th
Initiation Complex
Complete Initiation Complex
07/22/2024 Dr Ambayehu 712

2.Elongation
Þ Elongation of the polypeptide chain involves the Addition of AA
to the carboxyl end of the growing chain.
Þ During elongation, the Ribosome Moves from the 5′to 3′-end of \
the mRNA that is being translated.
07/22/2024 Dr Ambayehu 713

ProkaryotesPolypeptide A site
Amino acid
EF-Tu
GTP
P site
Anticodon
mRNA
1 tRNA a.a to the A-site =EF-TU
Stop codon
mRNA Movement 2
Peptide bond formation By Peptidyl
Transferase
3
GTP
07/22/2024 Translocation is activated by EF-G

Dr Ambayehu 714
Elongation cycle in Eukaryotes. GTP
eEF1a
 eEF1 and GTP are bound to

1
aminoacylated tRNA to A-site
 Ribosome catalyzes formation

of a peptide bond by peptidyl
transferase
 Translocation will be done by

GTP 2
eEF2 and GTP hydrolysis eEF2
07/22/2024 Dr Ambayehu 3 715

Cont…
1 2 3
07/22/2024 Dr Ambayehu 716

Inhibitors Of Translation
07/22/2024 Dr Ambayehu 717

Summery of antibiotics they Inhibit protein synthesis
Antibiotics Action
Choramphenicol Inhibit Prokaryotic Peptidyl
Transferase
Erythromycin Inhibit prokaryotic peptide chain
Elongation 50S plus Translocation
Tetracyclines Binding to 30S subunits interfere

with aminoacyl-tRNA binding "A-
site”
Streptomycin Binding to 30S subunits causes
mRNA misreading
Cycloheximide Inhibit Eukaryotic Peptidyl
Transferase
07/22/2024 Dr Ambayehu 718

Posttranslational Modification
07/22/2024 Dr Ambayehu 719

Matching
Antibiotics Action
Choramphenicol A. Inhibit Eukaryotic Peptidyl
Transferase
Erythromycin B. Binding to 30S subunits interfere
with aminoacyl-tRNA binding
Tetracyclines C. Inhibit prokaryotic peptide chain

Elongation 50S plus Translocation/
Cycloheximide D. Inhibit Prokaryotic Peptidyl

Transferase
07/22/2024 Dr Ambayehu 720
Regulation Of Gene
Expression
Part A: Control In
Prokaryotes
07/22/2024 Dr Ambayehu 721

Negative Regulation
Initially Gene –ON State
Then Off
07/22/2024 A repressor stops RNA polymerase

Dr Ambayehufrom initiating Transcription 722
Positive Regulation
Initially Gene –Off State
Then ON
Transcription factors enable RNA polymerase to bind to the

07/22/2024
promoter Dr Ambayehu 723
Induction can be positive or negative control
Negative Control Positive Control
Induction Negative Inducible,
Positive Inducible,
ON
07/22/2024
OFF Dr Ambayehu 724
Model-I
Lac Operon Regulation
Pcrp crp Pi I P O Lac Z Lac Y Lac A
07/22/2024 Dr Ambayehu 725

Regulation of the Lac Operon
Pcrp crp Pi I P O Lac Z Lac Y Lac A
DNA Function Protein Function

Pcrp Promoter for crp gene
Crp Gene for CAP protein Positive regulator

Pi Promoter for I gene
I Gene for Lac Repressor Negative regulator

P Promoter for Structural Genes
O Operator Target for I

Structural
Genes
Lac Z Gene for B-galactosidase Cleaves lactose

Lac Y Gene for Permease Lactose transport
Lac A Gene for Acetylase Unknown
07/22/2024 Dr Ambayehu 726
Summar
CHO Activator Repressor RNA poly Lac Operon
y
protein protein
+ Not bound to Lifted off Keeps falling
ON
GLUCOSE DNA operator site off promoter
site low frequency
+ LACTOSE of
Transcription
+
GLUCOSE
Not bound to
DNA
Bound to
operator site
Blocked by the
repressor Off
Transcription
- LACTOSE
- GLUCOSE
- LACTOSE
Bound to
DNA
Bound to
operator site
Blocked by the
repressor Off
Transcription
- GLUCOSE
+ LACTOSE
Bound to
DNA
Lifted off
operator site
Sits on the
promoter site ON
high frequency
of
Transcription
07/22/2024 Dr Ambayehu 727
Model-II
Tryptophan Operon
Regulation
trpA
07/22/2024 Dr Ambayehu 728

 Feedback Inhibition : This results in a temporary Shutoff of
Trp biosynthesis until the levels of Trp have decreased.
07/22/2024 Dr Ambayehu 729

Tryptophan Operon
trpA
DNA Function RNA/Protein Function

Trp R Gene for Binds to operator to inhibit transcription
Repressor
P Promoter
O Operator
Trp E, Structural Enzymes acting in pathway to produce
D, C, B, genes Trp.
A
Gene order correlates with order of
reactions in pathway.
5’ UTR Premature termination of transcription when
Leader Trp levels are high
07/22/2024 Dr Ambayehu 730
Control of Trp Operon Transcription
Trp Repressor is Inactive  Initial State: ON
 In the absence of trp, the repressor is inactive, allowing

RNA polymerase to bind and transcribe structural genes. Leads
07/22/2024
to synthesis of tryptophan. Dr Ambayehu 731
Control of Trp Operon Transcription
Trp binding activates Repressor  Final State: OFF
When tryptophan High
No Transcription
 In the presence of tryptophan (Corepressor), the repressor

binds Trp & changes conformation.
 Repressor / Corepressor complex binds to operator DNA,
changes conformation, and prevents binding of RNA pol to
07/22/2024
the promoter , therefore our trp Operon will not be transcribe .
Dr Ambayehu 732
Summary of Trp Operon
Level of Trp Operon
Tryptophan
On:
Trp repressor inactive
Low Leads to high rate of mRNA production
Lack of attenuation
Off
Tryptophan + repressor = Active repressor
High Reduction of mRNA production
By Attenuation
07/22/2024 Dr Ambayehu 733
Part-B :Regulation of Gene In
Eukaryotes Cells
07/22/2024 Dr Ambayehu 734

 Gene expression can be regulated at many stages of the
Central Dogma, but the Initiation of transcription is the
main one, and the first problem in initiation is getting into
the DNA !
 DNA is wrapped around Histones octamers to form nucleosomes
Chromatin may be tightly packed1 into Heterochromatin, or loosely

packed into Euchromatin
Heterochromatin Euchromatin
07/22/2024 Dr Ambayehu 735
Eukaryote gene expression is regulated at 7 levels:
1. Chromatin remodeling
2. Transcription
3. RNA processing
4. mRNA transport
5. mRNA degradation
6. Translational regulation
07/22/2024
7. Protein modificationsDr Ambayehu 736
The Function Of Histones Tails
07/22/2024 Dr Ambayehu 737

Cont..
HAT (Histone Acetyltransferase)
DNA
H DNA H
O O
-
O-P-O O -
+ NH-C O-P-O
NH3
O CH3
O
Unacetylated Acetylated
HDAC (Histone Deacetylase)

Closed –No space for RNA Pol
Open – RNA Pol Bind to DNA
Transcription Off
Transcription On
07/22/2024 Dr Ambayehu 738

 Methylation of CpG islands can block transcription by
two distinct pathways: Direct blocking of TFIID binding .
Methyl
group
absent
Promoter Exon-1 Exon-2
Transcription Methyl
group
present
Promoter Exon-1 Exon-2
No Transcription
07/22/2024 Dr Ambayehu 739

Coactivators
Enhancers:
 Cis regulators that interact
with regulatory proteins &
TFs to increase the efficiency of
transcription initiation.
Silencers –
 Cis-acting, bound by
repressors, or cause the
chromatin to condense and
become inactive.
07/22/2024 Dr Ambayehu 740

Cont…
07/22/2024 Dr Ambayehu 741

The end!
07/22/2024 Dr Ambayehu 742

Chapter Twelve
Vitamins and Minerals
07/22/2024 Dr Ambayehu 743

Vitamins
07/22/2024 Dr Ambayehu 744

Introduction
Vitamins are Organic cpds that are required in the diet in small
amounts for the maintenance of normal metabolic integrity
 Exceptions – Vitamin D & Niacin
 Classification – Based on solubility
 Lipid-soluble
 Water-soluble
07/22/2024 Dr Ambayehu 745

Lipid-Soluble Vitamins: Sources and Deficiency
Symptoms
07/22/2024 Dr Ambayehu 746

Vitamin A
Precursors
1. Retinoids – Animal origin
 Retinol: Found in animal tissues as a retinyl ester with long-
chain fatty acids.
 Retinaldehyde: Derived from the oxidation of retinol.
 Retinal and retinol can readily be interconverted.
 Retinoic acid: Derived from the oxidation of retinal.

 Can’t be reduced but is active in the maintenance of epithelial cells.
07/22/2024 Dr Ambayehu 747

Vitamin A
2. Carotenoids – Plant origin
 Provitamin A - Carotene & related cpds (eg.cryptoxanthin)
 Can be cleaved to yield retinaldehyde, then retinol & retinoic acid
 Cleaved by carotene dioxygenase (intestinal mucosa) to retinaldehyde, which is
reduced to retinol, esterified, and secreted in chylomicron together with esters formed
from dietary retinol
 If assymmetric cleavage occur, the cleavage products (8′-, 10′-, and 12′-apo-
carotenals) cannot be used as sources of retinol/ retinaldehyde
 Can also circulate without cleavage.
07/22/2024 Dr Ambayehu 748

Vitamin A
 Carotenoids in foods
bring colors to meals
 Retinoids in our eyes

allow us to see them.
07/22/2024 Dr Ambayehu 749

Functions of Vitamin A
 Vision
 Retinaldehyde funs as prosthetic grp the light-sensitive opsin proteins,
forming rhodopsin (in rods) & iodopsin (in cones).
 One cone cell contain one type of opsin (sensitive to one color)
 Regulation of gene transcription & tissue differenciation

 Retinoic acid – Act like steroid hormones
 Acts as a differentiation factor in growth & dev’t process.
 Bind to nuclear receptors & then to response elements of DNA
 Retinol and retinal are essential for normal reproduction:

spermatogenesis and preventing fetal resorption in the female.
07/22/2024 Dr Ambayehu 750

Retinaldehyde’s Role in Vision
07/22/2024 Dr Ambayehu 751

Role of Retinaldehyde
in vision
- In pigment epithelium of retina
- Isomerization of trans-retinol
to 11-cis-retinol in liver
Guanine nucleotide amplification

cascade and then a nerve impulse
07/22/2024 Dr Ambayehu 752

 Deficiency
 Night blindness & Abnormal epithelial cell growth (tend to be
keratinized)
 Visual impairment if prolonged- xeroptathalmia (progressive
drying and wrinkling of the conjunctiva of the eye)
 Growth disturbances
 Impaired differentiation of immune cells – susceptibility to
disease
 Toxicity
 If beyond capacity to metabolize & bind to binding capacity of
proteins
 E.g. Affect CNS – associated with increased CSF pressure
 Affect liver – hepatomegaly & hyperlipidemia
07/22/2024 Dr Ambayehu 753
VITAMIN D – really it is a hormone (!)
Bioactive form:
 Calcitriol/ 1,25-(OH)2D3
 3 sources
• Ergocalciferol (vitamin D2) from plants
• Cholecalciferol (vitamin D3) from animals
• 7-dehydrocholesterol (intermediate in cholesterol synthesis) by the skin
Cholecalciferol/ Egrocalciferol are absorbed from the intestine and

transported to the liver bound to a specific vitamin D-binding protein.
07/22/2024 Dr Ambayehu 754

Vitamin D synthesis
& activation
07/22/2024 Dr Ambayehu 755

Function of Vitamin D
 Main function Vitamin D: as a steroid hormone
 for Ca2+ absorption & homeostasis
 Mechanism of Ca2+ homeostasis (plasma concentration)

 1- increasing intestinal Ca2+ absorption
 Inducing expression of calbindinD28K ( a protein involved in intestinal Ca2+
absorption)
 Increased absorption of Ca2+ requires concomitant absorption of –ve charge

predominantly Pi
 2- Reduce excretion by stimulating re-absorption in

distal tubule
 3- Bone mineral mobilization
07/22/2024 Dr Ambayehu 756

 Low Ca2+
Calcium causes
balance increase in
PTH
Calcitonin Vitamin D
07/22/2024 Dr Ambayehu 757

 Regulation by inhibition of Vitamin D activation
 By plasma concentration of Ca2+
 Calcitriol acts to reduce its biosynthesis by inducing 24-hydroxylase
and repressing 1-hydroxylase in kidney.
07/22/2024 Dr Ambayehu 758

 Additional functions
 Through binding of calcitriol to nuclear receptors for
 Insulin secretion
 Differentiation of monocyte precursor cells
 Modulation of cell proliferation.
 Deficiency :
 Children – Rickets
 Adults - Osteomalacia (demineralization of bone)
 Tocicity – if dietary
 Contraction of blood vessel (Ca2+) – high blood pressure
 Calcinosis – calcification of soft tissues

07/22/2024 Dr Ambayehu 759
Calcium and Osteoporosis
07/22/2024 Dr Ambayehu 760

Vitamin E
 Synthesized only by plants, so plant oils are the main dietary sources
 Mixture of several related compounds known as tocopherols & tocotrienols
 Absorbed in the form of chylomicron to tissues
 Accumulates in cellular membrane, fat deposits & circulating chylomicrons
07/22/2024 Dr Ambayehu 761

Major function of Vitamin E – as antioxidant
 Scavenging free radicals & molecular oxygen

 Preventing peroxidation of PUFAs.
 The tocopheroxyl free radical product is relatively unreactive
 VE is interrelated with vitamin C in its fun.
 Oxidized vitamin C is poorly reactive

 -tocopherol can also scavenge two peroxy free radicals and then be
conjugated to glucuronate for excretion in the bile
 Defficiencies of vitamin E – not common
 Severe fat malabsorption - Hemolytic anemia, nerve & muscle
memb. Damage
07/22/2024 Dr Ambayehu 762
 Toxicity: not common
Anti-oxidant Protection Against Free Radicals:
Association of Vitamin E & Vitamin C
e- e- 2 H+ e- H+
O2
.-
O2 H 2O2 OH
. + H 2O
protein
.
protein
or OH
. or
+
lipids lipids .
(PUFA) (PUFA)
protein
. protein
.
or + Vit. E or + Vit. E
lipids . (active)
lipids
(inactive)
(PUFA) (PUFA)
.
Vit. E + Vit. C Vit. E + Vit. C
07/22/2024 (inactive)
Dr Ambayehu (active) 763
Vitamin K
 Natural
 Vitamin K1: Phylloquinone
 2-methyl-3-phytyl-1,4-naphthoquinone/ Phytomenadione/
Phytoandione/ Phytylmenadione
 the normal dietary source, found in green vegetables
 Vitamin K2: Menaquinones
 Menatetratone/ Menaquinone K4 Vitamin MK-4
 synthesized by intestinal bacteria (gram +ve), with d/t side-chain
 Synthetic: compounds that can be metabolized to phylloquinone
 Vitamin K3/ Menadione: 2-methyl-1,4-Naphthoquinone
 Vitamin K4/ Menadiol: Menaquinol 2-methyl-1,4 naphthoquinol / 2-
methyl-1,4-naphthohydroquinone/ reduced menadione
 Menadiolodiacetate
07/22/2024 Dr Ambayehu 764

07/22/2024 Dr Ambayehu 765
Notable food
sources of
vitamin K
include:
milk, eggs,
liver, cabbage
07/22/2024 Dr Ambayehu 766

Role of Vitamin K
 Maintenance of levels of blood clotting proteins, (factors II, VII, IX, X,
etc which are synthesized in the liver as inactive precursor proteins)
 Mechanism
 As Cofactor for the carboxylation of glutamate residues to form

γ-carboxyglutamate (Gla), which chelates the calcium ion.
 e.g. Preprothrombin (factor II) is modified to prothrombin (chelate
Ca2+)
 Upon chelation of Ca2+, prothrombin interacts with phospholipids in
membranes and is proteolysed to thrombin through the action of activated factor
X
 Other clotting factors also have Gla that chelate Ca2+ which permit
the factors to bind to membrane
07/22/2024 Dr Ambayehu 767
Vitamin K & biosynthesis of -carboxyglutamate
 Additional role:
 Synthesis of Bone Gla Proteins
 Kidney Gla proteins: may be for Ca2+ re-absorption
 Deficiency – Not common but hemorrhagic syndrome during deficiency
 Toxicity: Rare but cause cramp-like pains, tachycardia, cardiac
irregularity, chest pain,
07/22/2024 Dr Ambayehu 768
Summary of Lipid-soluble vitamins
07/22/2024 Dr Ambayehu 769

WATER-SOLUBLE VITAMINS
07/22/2024 Dr Ambayehu 770

Vitamin B1 (Thiamin)
Structure
 Dimethyl 6-aminopyrimidine + Methyl hydroxyethyl thiazole
 linked by methylene
 Converted to active form (Thiamine pyrophosphate/ TPP) in brain & liver by

thiamin diphosphotransferase
07/22/2024 Dr Ambayehu 771

Key role – Carbohydrate metabolism
 In decarboxylation rxns
 Pyruvate dehydrogenase: a component of

PDH complex
 -ketoglutarate dehydrogenase:
Oxidative decarboxylation in TCA
 -keto-acid dehydrogenase:
 decarboxylation steps involved in

degradation of - ketobutyrate and -
ketoderivatives of branched chain aas
(leucine, isoleucine, and valine)
Coenzyme for Transketolase catalyzed reactions of the HMP
 TPP provides a reactive carbon on the thiazole moiety that forms a carbanion,
07/22/2024 Dr Ambayehu 772
which then adds to the carbonyl group
Deficiency
 Cause degeneration of myelin sheaths of nerve fibers in
both the peripheral nerves and the CNS
 “polyneuritis” characterized by pain radiating along the course of
one or many peripheral nerves.
 Muscles atrophy, resulting in severe weakness.
 Weakens the heart and causes peripheral vasodilation
 Additional effect - In high carbohydrate diet, it may lead to

lactic acidosis.
07/22/2024 Dr Ambayehu 773

VITAMIN B2 (RIBOFLAVIN)
Structure: Dimethyl isoalloxazine ring + ribitol

 Converted to active coenzyme forms (FMN or FAD) using ATP.
 Two bioactive forms
 FMN = ATP-dependent phosphorylation of riboflavin
 FAD = FMN + AMP moiety of ATP
 Main dietary source – milk & dairy products
07/22/2024 Dr Ambayehu FAD 774

Metabolic role of VB2
 Electron Carriers in Redox Reactions

 The two nitrogen atoms of isoallxazine nucleus accept H atoms
 E.g. Xanthine oxidase – during Xanthine to uric acid (uses NAD+)
Deficiency
- Digestive system disturbances
- Burning sensations of the skin and eyes, cracking at the

07/22/2024
corners of the mouth, headaches,Drmental
Ambayehu
depression 775
VB2 -Dependant Enzymes
FMN-dependent Enzymes
 In Mitochondrial ETC : NADH dehase contains FMN.
 NADH → FMN → CoQ
 During the amino acid oxidation, FMN is reduced.
 FMN is also re-oxidized by O2 to produce H2O2.
 FAD-dependent enzymes
 Succinate to fumerate by succinate dehydrogenase
 Acyl CoA to --unsaturated acyl CoA by acyl CoA dehydrogenase
 Erythrocyte glutathione reductase
 Dihydrolipoate dehydrogenase reaction
 pyruvate to acetyl CoA
 -ketoglutarate to succinyl CoA

07/22/2024 Dr Ambayehu 776
Niacin - vitamin B3
 Structure: Pyridine-3-carboxylic
acid
 Dietary sources: Nicotinic acid &
Nicotinamide in tissues
NAD+
• Can be synthesized from Tryptophan
(insufficient)
 Active forms: NAD+ & NADP+

• Niacin + Ribose → Mononucleotide
• Mononucleotide + AMP →
Dineucleotide
• Dineucleotide + Glutamine to form its

amide
NADH
07/22/2024 Dr Ambayehu 777
Biological roles
Examples
 NAD+ dependent enzymes
a. Lactate dehase (lactate → pyruvate)
b. Glyceraldehyde-3-phosphate dehydrogenase
(Glyceraldehyde-3-phosphate → 1,3-BPG)
c. Pyruvate dehase (Pyruvate → acetyl CoA)
d. -hydroxyl acyl CoA dehase (-oxidation)
e. Mitochondrial isocitrate dehydrogenasease
07/22/2024 Dr Ambayehu 778

2. NADPH
 NADPH generating reactions
 Glucose-6-phosphate dehydrogenase
 Glucose-6-phosphate → 6-phosphogluconolactone
 6-phosphogluconate dehydrogenase
 6-phosphogluconolactone → 3-keto6-phosphogluconate
 Malic enzyme (malate to pyruvate)

 NADPH utilizing reactions
 -keto acyl ACP to -hydrxy acyl ACP
 HMG CoA to Mevalonate
 Folate to dihydrofolate then to tetrahydrofolate
07/22/2024 Dr Ambayehu 779

 Additional role of NAD - as source of ADP-Ribose
 For the ADP-ribosylation of proteins and polyADP-ribosylation of
nucleoproteins involved in the DNA repair mechanism
Deficiency - either because of niacin or tryptophan deficiency

Causes 1. Hartnup disease
Defect of the membrane transport mechanism for aas like
07/22/2024
tryptophan, resulting in large losses due to intestinal
Dr Ambayehu 780
 2. In Carcinoid syndrome
 There is metastasis of a primary liver tumor of enterochromaffin cells which
synthesize 5 -hydroxytryptamine from tryptophan (upto 60% of tryptophan
metabolism).
 Causing pellagra because of diversion from NAD synthesis
 Pellagra – characterized by photosensitive dermatitis, diarrhea, dementia &

finally death
 Women are more affected probably b/se of inhibition of tryptophan

metabolism by estrogen metabolites
 Toxicity > 500 mg/d can cause liver damage
07/22/2024 Dr Ambayehu 781

VITAMIN B6
 6 types
 Pyridoxine, pyridoxal, pyridoxamine, and their 5′-phosphates
 Active form – Pyridoxal phosphate/ PLP
 80% of VB6 is available as PLP in muscle, mostly associated with glycogen phoaphorylase
 Released during starvation and avilable in kidney & liver to meet increased requirement
for gluconeogenesis from aminoacids
07/22/2024 Dr Ambayehu 782

Biological importance
 Coenzyme in aminoacid metabolism -
transamination & decarboxylation
 Cofactor of glycogen phosphorylase, where the

phosphate grp is catalytically important.
 Important in steroid hormone action where it removes

the hormone-receptor complex from DNA binding,
terminating the action of the hormones.
07/22/2024 Dr Ambayehu 783
PLP and Methionine for Gluconeogenesis PLP and Phenylalanine catabolism
07/22/2024 Dr Ambayehu 784

 Deficiency – Rare
• Moderate defficiency causes abnormalities of

tryptophan & methionine metabolism
• Increased sensitivity to steroid hormone may cause

dev’t of hormone- dependant cancer of breast, uterus &
prostate
 Toxicity - Causes sensory neuropathy
• Intakes in excess of 200mg/d are associated with

neurologic damage
07/22/2024 Dr Ambayehu 785

Vitamin B12 (Cobalamin)
 Chemistry
• Complex corrin ring structure and
a Co3+ in the center
• two of the 4 pyrrole rings are

directly linked.
 X may be
 X = CN–in cyanocobalamin
 X = OH–in hydroxocobalamin
 X= 5′-deoxyadenosyl in
5′deoxyadenosylcobalamin
 X = H2O in aquocobalamin
 X = CH3 in methylcobalamin
07/22/2024 Dr Ambayehu 786
Vitamin B12
 Source: Only in foods of animal origin. Vegans???

 Synthesis
 Exclusively by bacteria and is found in the liver of animals bound to

protein as methycobalamin or 5'-deoxyadenosylcobalamin
 Existence: in diets, either free or bound to proteins
 If free :
 Bind to proteins (R-binders/ haptocorrins/ transcobalamin I) secreted by salivary

gland or gastric mucosa
 If bound: released by proteases in stomach & intestine
 Haptotocorrins are also digested in small intestine
07/22/2024 Dr Ambayehu 787

Mechanism of absorption
• Free cobalamin bind to intrinsic factor (a
glycoprotein by parietal cell)
• Intrinsic facor-VB12 complex attach to

receptor on ileum & absorbed
• The B12 within the enterocyte complexes

with transcobalamin II and then is released
into circulation.
07/22/2024 Dr Ambayehu 788

Biological Roles: two major functions
 Rearrangement of the methyl group of L-

methylmalonyl CoA to form succinyl CoA
 A metabolic route for the conversion of carbons from
 Amino acids: valine, isoleucine, threonine, thymine
 Fatty acids: odd-chain FAs
 All of which form propionyl CoA and then to succinyl

CoA (the TCA cycle intermediate)
 Transfer of a methyl group from N5-methyl FH4

to homocysteine to form methionine
07/22/2024 Dr Ambayehu 789

Folic acid and Vitamin B12 in Methionine Synthesis
 If Vitamin B12 deficient,

 inhibition of methionine synthase activity
 causing homocysteinuria and the trapping of folate as FH4.CH3
07/22/2024 Dr Ambayehu 790
Deficiency
 Results in Pernicious anemia (PA)
 caused by failure of RBC maturation
 Arises when vitamin B12 deficiency blocks the metabolism of folic

acid, leading to functional folate deficiency.
 This impairs erythropoiesis, causing immature precursors of erythrocytes to
be released into the circulation (megaloblastic anemia).
 Commonest cause: of PA is failure of the absorption of VB12 rather

than dietary deficiency.
 This can be due to failure of intrinsic factor secretion caused by autoimmune
disease of parietal cells or to generation of anti-intrinsic factor antibodies.
07/22/2024 Dr Ambayehu 791

Folic acid
- Pteridine ring structure + PABA = Pteroic acid
- Pterioc acid + Glutamic acid = Folic acid (active form)
07/22/2024 Dr Ambayehu 792

Major Sources
Yeast, leafy vegtables (foliage), fruits, and legumes, Liver
Direct folate intake in diet
Dietary type: Polyglutamate form

Folate conjugases (Brush border, ileum)
Monoglutamate form of folate (less –ve charge)
Absorbtion
N5-methyl-folate (in intestinal cells) (circulatory form)
Reconjugated to polyglutamate (Liver)
Storage : In cells (mainly liver), reduced to THF by reductase, an

NADPH-requiring enzyme
07/22/2024 Dr Ambayehu 793

Metabolic Role of THF4
 Carrier of One-Carbon
 Forms
 N-5 (formyl, formimino, or methyl groups)
 N-10 (formyl group)
 Bridging N-5 to N-10(methylene or methenyl grp)
07/22/2024 Dr Ambayehu 794

 Major source of substituted
folate
 Serine – by serine-
hydroxymethyltransferase
Note: when one-carbon folates are not required, the oxidation of formyl
THF4 to yield CO2 provides a means of maintaining a pool of free folate
Deficiency - Megaloblastic Anemia

07/22/2024 Dr Ambayehu 795
Biotin (Vitamin H)
 Exsistence
 In foods as biocytin (ε-amino-biotinyl lysine) & released by proteolysis.
 Synthesized in excess by intestinal flora
 Deficiency – Unknown
07/22/2024
 Uncooked egg white contain avidin, a Drprotein that binds biotin and renders it unavailable
Ambayehu 796
for absorption.
 Metabolic Role
 Transfer CO2 in a small number of carboxylation reactions.
 React with the lysine residue of the apoenzymes of carboxylases
 Reactive intermediate: 1-N-carboxybiocytin

 Formed from bicarbonate in an ATP-dependent reaction
07/22/2024 Dr Ambayehu 797

Types of Carboxylases for biotin  Propionyl-CoA carboxylase
 Odd-chain Fatty Acid oxidation
 Acetyl-CoA carboxylase
 Fatty Acid synthesis
 Pyruvate carboxylase
 Anaplerotic reaction in
TCA cycle
07/22/2024 Dr Ambayehu 798

PANTOTHENIC ACID
(VB5)
-alanine + pantoic acid
Metabolic Role of VB5
1- Required for synthesis of Coenzyme A (CoA)
Takes part in
TCA cycle
Fatty acid synthesis and oxidation
Cholesterol synthesis
2- Component of acyl carrier protein (ACP) domain of fatty acid

synthase
So, it has central role in acyl group metabolism
07/22/2024 Dr Ambayehu 799

VB5 as component of CoA VB5 as component of ACP
07/22/2024 Dr Ambayehu 800

Vitamin C (Ascorbic Acid)
-Derived from glucose via the uronic acid pathway.
Gulonolactone L-gulonolactone oxidase Ascorbic acid
The enzyme is absent in primates
07/22/2024 Dr Ambayehu 801

Metabolic role
 Synthesis of many peptide hormones – hydroxylation of carboxyl terminal

(usually glycine residue)
 Redox coenzyme that functions in collagen synthesis and

others
 Free radical defense due to its action as a reducing agent and

oxygen radical quencher.
 Regeneration of reduced form of vitamin E
07/22/2024 Dr Ambayehu 802

Examples BH4 = Tetrahydrobiopterin
1. Formation of norepinephrine
Use of Ascorbic acid

 During hydroxylation, the Cu+ is oxidized to Cu2+
 Reduction of Cu2+ to Cu+ specifically requires ascorbate, which is

oxidized to monodehydroascorbate
07/22/2024 Dr Ambayehu 803

 2. Collagen synthesis
 Postsynthetic modification of procollagen to collagen
 Hydroxylation of proline and lysine residues in collagen
 Hydroxyproline - for H-bonding (stabilize triple helix)
 Hydroxylysine - sites of attachment of disaccharides (galactose-glucose)
07/22/2024 Dr Ambayehu 804

3. Free radical defense
07/22/2024 Dr Ambayehu 805

4. Formation of osteocalcin & C1q component of complement .
- for Proline hydroxylase
5. Synthesis of carnitine
Trimethyllysine and -butyrobetaine hydroxylases
6. Reduction of cytochrome (A & C) in oxidative phosphorylation
 Defficiency – Scurvy
 Defective collagen formation leading to
 Subcutaneous hemorrhage (skin changes), aching bones, joints, and muscle in

adults, rigid position and pain in infants.
 Fragility of blood capillaries, gum decay, tooth loss, & bone fracture
 Excess intake - Vitamin C enhances the absorption of iron, and this

07/22/2024 Dr Ambayehu 806
depends on the presence of the vitamin in the gut.
Summary: Water-Soluble Vitamins
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Water-Soluble Vitamins:
Summary:
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Minerals
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Introduction
• Minerals are Naturally occurring, inorganic, homogeneous
substances; chemical elements required for growth,
maintenance, reproduction and lactation.
• Major minerals
• Essential mineral nutrients found in the human body in amounts >
5 grams
• Trace minerals
• Essential mineral nutrients found in the human body in amounts <5
grams
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Minerals: Function
• Minerals serve three roles:
• Provide structure: As structural component
• Help in maintaining normal heart rhythm, muscle

contractility, neural conductivity, and acid-base balance
• Help in regulation cellular metabolism by becoming part

of enzymes and hormones
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Minerals in Human Body
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Calcium (Ca2+)
• The most abundant mineral in the body
• Most of calcium (99%) is found in the bones (some in teeth):
• The calcium bank for the body fluids
• The remaining calcium (1%) is found in the blood and has many functions.
• Major Dietary Sources: Milk & milk Products, Broccoli, Soybean curd, bread and
other baked goods made from calcium fortified flour
• Calcium Absorption
 Absorption rate for adults is 25% of calcium consumed.
 Calcium-binding protein is needed for calcium absorption.
 Factors that enhance absorption: Stomach acid, Vitamin D, Lactose, Growth hormones
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Calcium balance
 Bone and blood calcium are kept in balance with a system of
hormones and vitamin D.
 The skeleton serves as a bank from which the blood can borrow
and return Ca²⁺ as needed.
 Withdrawal and deposition of Ca²⁺ are regulated by hormones

sensitive to blood level of Ca²⁺.
 Parathyroid hormone (PTH)
 Calcitonin: ( Calcium lowering hormone)
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Functions of Calcium
 Calcification of teeth.
 Bone Mineralization: the process whereby minerals crystallize on the collagen

matrix of a growing bone, hardening of the bone.
 Blood clotting mechanism: E.g. thrombokinase- activator
 It is important for maintenance of the heart beat.
 Important in nerve transmission and muscle contraction.
 Enzyme cofactor: E.g. Lipase
 Act as intracellular signal – For many effects: cell aggregation, secretion,

transformation and cell division, as well as muscle protein degradation.
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Calcium
• Calcium Deficiency
• Osteoporosis is the disease where the bones become porous and fragile
• May be due to disease of: parathyroid, kidney and disturbed vitamin D
level.
• No obvious symptoms of mineral loss in bones appear. It is silent.
• Deficiency in children can present as stunted growth.
• Calcium tetany develops when there are low blood calcium levels and causes
uncontrolled muscle contractions.
• Toxicity symptoms:
• Include constipation, increased risk of urinary stone formation, kidney
dysfunction
• Calcium rigor develops when there are high blood Ca levels
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Phosphorous
• Distribution: Second most abundant mineral in the body
• Commonly found in nature in its pentavalent form in combination
with oxygen, as phosphate (PO43-).
• Absorption by passive and some by active transport which is
facilitated by 1,25-hydroxyvitamin D
• PTH is known to decrease serum phosphorus concentration by
an increase in urinary excretion.
• PTH secretion has been shown to be induced by a low calcium
and high phosphorus diet.
• Sources include all animal foods including meat, fish and
poultry, milk and eggs
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Functions of phosphorous
• Mineralization of bones and teeth (most part of body phosphorus)
 Energy transfer in metabolism
• Enters in the structure of nucleotides and nucleic acids.
• It is important for the biosynthesis of phospholipids of cell membrane.
• It is in carbohydrate metabolism as hexose ester (glc-6-P and Fr-6-P).

 Buffer systems that maintain intracellular acid-base balance
• Enter the formation of coenzymes as NADP.
• Regulation of enzyme activity
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Hydroxyapatite (crystal structure)
Ca10(PO4)6 OH2
Ca P O H
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Phosphorus
• Deficiency signs:
• Include anorexia, anemia, muscle weakness, bone pain, rickets, ataxia and even
death
• Phosphorus toxicity : Rare
• May include the calcification of non-skeletal tissues, especially the kidneys.
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Potassium
• It is found in foods mostly milk, fruits, vegetables, fish, beef, liver,
chcken
• Processed foods have less potassium.
• Absorption mainly passive via intestine
• Diuretics can deplete the body’s potassium
• It is associated with hypertension.

• Low potassium intakes increase blood pressure.
• High potassium intakes prevent and correct hypertension.
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Potassium (K)
• Potassium Roles in the Body
• Maintains normal fluid and electrolyte balance, major intracellular cation
• Facilitates many biochemical rxns

• Cofactor for a number of enzymes including glycerol dehydrogenase,
mitochondrial pyruvate carboxylase, pyruvate kinase, vitamin B12-dependent
diol dehydratase, L-threonine dehydratase, adenosine triphosphatase and
aminoacyl transferase.
• Required for the secretion of insulin by the pancreas

• Potassium is also involved in phosphorylation of creatine, in carbohydrate
metabolism and protein synthesis
• Supports cell integrity
• Assists in nerve impulse transmission and muscle contractions
• Maintains the heartbeat
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Potassium
• Potassium Deficiency - Hypokalemia
• Symptoms include muscular weakness, paralysis, confusion,
increased blood pressure, salt sensitivity, nausea and vomiting,
diarrhea, etc.
• Can induce growth retardation.
• Potassium Toxicity
• Results from supplements or overconsumption of potassium salts
• Symptoms include muscular weakness and vomiting.
• If given into a vein, potassium can cause the heart to stop

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Sulfate
• Sulfate requirements are met by consuming a
varied diet.
• It is found in essential nutrients including protein.
• No recommended intake and there are no known
deficiencies.
 Sulfur is found in thiamin and several amino
acids
• High sulfur content in
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Sodium (Na)
• Sodium Roles in the Body
• Maintains normal fluid and electrolyte balance along with
K+
• Main extratracellular cation
• As ion channel and gated channel
• Attracts water as osmotic control and volume of ECF
• Assists in nerve impulse transmission and muscle contraction
• Assits absorption of glucose and others
• Filtered out of the blood by the kidneys
• Dietary recommendations include a moderate intake of salt
and Na+
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Sodium
• Sodium Deficiency- Unusual
• Can lead to low blood pressure, dehydration and muscle cramps
• Sodium and water must be replaced after vomiting, diarrhea or
heavy sweating.
• Sodium and Hypertension
• Salt has a great impact on high blood pressure.
• Salt sensitivity: Individual’s response to a high salt intake with
high blood pressure.
• Dietary Approaches to Stop Hypertension (DASH) is a diet plan
that helps to lower blood pressure.
• Sodium and Bone Loss (Osteoporosis)
• High sodium intake is associated with calcium excretion.
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Chloride (Cl-)
• Chloride in an essential nutrient that plays a role in fluid balance, major body’s
negative ion
• It is associated with sodium and part of hydrochloric acid in the stomach.
• Chloride Roles in the Body
• Maintains normal fluid and electrolyte balance
• Part of hydrochloric acid found in the stomach
• Necessary for proper digestion
• Nerve & muscle activity
• Chloride Intakes
• Abundant in foods
• Absorbed actively with exchange of bicarbonate (make intestinal lumen alkaline)
• Abundant in processed foods as NaCl
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Chloride
Chloride Deficiency and Toxicity
◦ Deficiency is rare.
◦ Losses can occur with vomiting, diarrhea or heavy

sweating.
◦ Dehydration due to water deficiency can concentrate

chloride to high levels.
◦ The toxicity symptom is vomiting.

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Magnesium
• Approximately 50% of total body magnesium is found in bone.
• The other half is found predominantly inside cells of body tissues and
organs.
• Only 1% of magnesium is found in blood, but the body works very hard
to keep blood levels of magnesium constant.
• Dietary Sources: Wide spread in foods
• Nuts and legumes, whole grains (unrefined), dark green vegetables (spinach),
seafood, chocolate and cocoa
• Hard water and some mineral waters
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• Efflux of magnesium from the cell is coupled to sodium transport
and requires energy.
• Magnesium influx also appears to be linked to sodium and
bicarbonate transport
• Magnesium Intakes
• RDA Adult Men: 400 mg/day for 19-30 years of age
• RDA Adult Women: 310 mg/day for 19-30 years of age
• Upper level for adults: 350 mg nonfood magnesium/day
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Magnesium - Function
• Magnesium Roles in the Body
• Bone mineralization: can be drawn out for all the cells to use in
building protein
• Building of protein
• Enzyme action: more than 300 biochemical reactions.
• E.g. Protein and carbohydrate metabolism
• Normal muscle contraction and steady heart rhythm
• Nerve impulse transmission
• Maintenance of teeth
• Functioning of the immune system
• Blood clotting
• For normal function of the parathyroid gland, vitamin D metabolism
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Magnesium
• Magnesium Deficiency
• Deficiencies are rare.
• Symptoms
• Weakness and confusion
• Convulsions in extreme deficiency
• Bizarre muscle movements of the eye and face
• Cardiovascular problem
• Growth failure in children
• Causes of hypomagnesaemia are reduced intake, impaired

intestinal absorption, renal loss and genetic diseases.
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Trace Minerals
• Needed in very small (trace) quantities (<5 g)

• Toxic levels can easily be reached with the use of supplements.
• Can be obtained only by consuming a wide variety of foods.
 Food Sources: Wide variety of foods
• Depends on soil and water composition
• Depends on processing and Bioavailability
• Include: Iron, Zinc, Copper, Fluoride, Selenium, Iodine, Chromium, Manganese,

Molybdenum
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Trace…
Deficiencies
◦ Severe deficiencies of some minerals are easy to recognize,
while others can be difficult to diagnose.
◦ Deficiencies have wide-reaching effects.
◦ Deficiencies affect all ages, but can affect growth in children
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Iron
• Dietary sources:
• Liver, meat, beans, nuts, dried fruits, poultry, fish, whole grains or enriched
cereals, soybean flour and most dark green leafy vegetables.
• Main dietary forms: haem and non-haem (major).

 Haem from Hemoglobin and Myoglobin: from meat, poultry and fish
• Non-haem Fe (95 % dietary Fe) mainly of Fe salts: from plant and dairy products.
• Lactoferrin found in mother's milk.
• Most of the inorganic Fe in foods present as Fe3+ form
• Absorption is at upper small intestine
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Iron Absorption
• Dietary constituents that solubilise Fe enhance absorption, whereas compounds

that either precipitate or polymerise Fe decrease absorption.
• Vitamin C enhances non-haem iron absorption, and the presence of meat, fish or
poultry within a meal has been shown to increase absorption approximately 4-fold
• Ligands such as citric acid, fructose and amino acids also promote non-haem iron
absorption.
• Vitamin A and β-carotene are also reported to improve the bioavailability of non-
haem iron.
• Conversely, many dietary factors inhibit the absorption of non-haem iron: some
calcium salts (for example, calcium phosphate), bran, phytate, some animal and
vegetable protein digestion products, polyphenols (in tea and some vegetables), and
possibly components of coffee.
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Fe in the body
Found in the body in 2 forms: functional
(2/3rd ) and storage (in woman, store only
1/8th of total)
 Exist in bound form because free ion may
cause oxidative stress (Fenton reaction with
H2O2)
• Storage: liver, reticuloendothelial cells, and
bone marrow
• with storage proteins, ferritin and haemosiderin
• Ferritin- iron storage in cells, plasma
07/22/2024 • Transferrin – Ferric iron transport in blood
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Biochemical Iron
• Functions in respiration
• O2 carrier: Hemoglobin (85% of functional iron) and
Myoglobin
• Function in metabolism
• Cytochromes (haem-conaining): electron carriers in
membranes
• Cytochrome c oxidase in electron transport for mitochondrial ATP
synthesis
• Nonhaem iron-containing enzymes: Fe-S complexes

• NADH dehydrogenase and succinate dehydrogenase
• Cytochrome P450- detoxifying enzyme: haem-containing

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Biochemical Iron
• Aconitase of TCA cycle
• PEPCK in gluconegenic pathway
• Tyrosinehydroxylase: synthesize L-DOPA
• Phenylalanine hydroxylase-synthesizes Tyrosine
• Ribonucleotide reductase: synthesis of deoxyribose from ribose
• Other Fe- dependant enzymes include:
• Amino acid oxidases
• Fatty acid desaturases
• Nitric oxide synthetase
• Peroxidases: Use peroxide as substrate
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 Deficiency
◦ May not be clinically apparent until iron stores are exhausted
◦ Progressive stages of iron deficiency include depleted stores, deficient

erythropoiesis, and iron deficiency anaemia
◦ Include adverse effects on work capacity, intellectual performance,

behaviour, thermoregulation and reduced resistance to infection
◦ Caused by inhibitors of iron absorption, poor absorption; menstruating

women or individuals with pathological blood loss
 Toxicity include: multisystem damage, principally

 Hepatotoxicity (within 2 days of ingestion)
 Exacerbate oxidative stress as catalyst in free radical-mediated rxns.

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Zinc
• Dietary source: Protein containing foods
• Absorption is from the entire intestinal tract
• Transport: as free solute (Zn2+) and/or as a complex which can
liberate Zn2+ before or after membrane transport.
• Tissue distribution is ubiquitous with different concentration
• Mainly excreted via GI tract including pancreatic secretion
• No specific storage site, supplement when highly needed
• Recycling from RBC is similar to Fe.
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Zinc Function
• Three general functional classes:
• catalytic, structural, and regulatory
• Enzyme cofactor: over 300 enzymes
• Dehydrogenases
• RNA, DNA polymerase
• Carbonic anhydrase
• Carboxypeptidase
• Amino peptidase
• Potein structure – Zn-fingers (coordinate with Cys and His) for protein
interaction with others like RNA and also proteins
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Zn
Zn2+Polarizes
2+
PolarizesHH22O
O
and
andmaking
makingititaabetter
better
nucleophile
nucleophile
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Role of Zinc
• Involvement of Zn in regulating IGF binding to cell-
surface IGF receptors
• Genetic expression regulator:
• As transcription factors (zinc finger proteins)
• Extracellular Zn may activate phospholipase C and calcium

ion release from the endoplasmic reticulum
• hence, cellular signaling through a Zn-sensing membrane receptor
or through regulation of protein tyrosine kinases and phosphatases
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Zinc Deficiency
• Tissue damage including
• Peroxidation
• Cytotoxicity/cytoprotection, apoptosis (role in signalling)
• Decreased cell proliferation and growth (related to apoptosis)
• Immune deficiency
• Defect in reproduction (hypogonadism)
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Zinc Toxicity
• Acute Zn toxicity: causes gastric distress, dizziness, and nausea.
• Chronic toxicity
• Gastric problems are observed in chronic toxicity.
• Other effects include reduction in immune function (lymphocyte stimulation by

phytohemagglutinin) and decreased high-density cholesterol.
• Zn has been implicated in the pathogenesis of Alzheimer disease. Zn

may bind to the normal glycoprotein, amyloid protein, alter its
secondary structure.
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Copper (Cu+, Cu2+)
• Dietary sources: legumes, whole grains, nuts, shellfish, seeds
• absorbed through the intestinal mucosa,
• transported via the portal blood to the liver (by albumin), and
incorporated into ceruloplasmin (which deliver Cu to tissues)
• Albumin-bound copper exchanges with tissue copper
• Stored in liver (but small amount)
• Excreted mainly via GI with unabsorbed dietary Cu
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Biochemical roles of Cu
• Enzyme cofactor
• Superoxide dismutase (cytoplasmic and extracellular) – Form peroxide
• Tyrosinase (Tyrosine to dopamine in melanin formation)
• Cytochrome oxidase (with Fe)
• Lysyl oxidase – for cross-linking of collagen and elastin in connective
tissue
• Amine oxidases
• Dopamine beta hydroxylase: Dopamine to norepinephrine
• Iron metabolism
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Deficiency of Cu
• Deficiency is rare
• Excessive copper produces epigastric pain, nausea, vomiting, and
diarrhea, which usually prevents the more serious manifestations of
copper toxicity.
• Serious manifestations include coma, hepatic necrosis, vascular
collapse, and death.
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Manganese
 Dietary sources: nuts, whole grains, leafy veggies
 Its oxidation state ranges between -3 and +7, although the most
stable valence is +2 and the most abundant is 4+.
 Mn2+, main form absorbed by humans, is oxidized to Mn3+, the
oxidative state, over time in plasma.
 Bound to proteins like transferrin and albumin
 Taken up by liver, pancreas, kidney
 Bone is main storage site
 Excretion mainly via bile
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Function of Mn
 Mn is essential as a cofactor for the metalloenzymes
 superoxide dismutase (SOD)
 xanthine oxidase
 Arginase
 galactosyl transferase
 pyruvate carboxylase
 Also activates decarboxylases, glutamine synthetase,
hydrolases, kinases, and transferases such as glycosyl
transferases (in polysaccharide biosynthesis)
 Deficiency is rare
 Too much can affect the central nervous system
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Iodine
• Dietary source: dairy products, meat, grain, iodized salt
• A variety of dietary forms, broken down to iodide in the gut and is
quickly absorbed into the circulation
• Concentration mainly in thyroid (some in salivary, mammary gland)
• Excretion by the kidney.
• Essential component of thyroid hormone
• Homone response affected proteins vary among tissues; include growth
hormone, liver enzymes controlling lipogenesis, myosin, and various
brain enzymes.
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Deficiency of Iodine
Goiter: enlarged thyroid gland due to increase in TSH by
pitutary because of low circulating T4
◦ Cretinism: Congenital hypothyroidism showing

irreversible mental and physical retardation (in infants)
 World’s ocean and iodized salt are best sources
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Selenium
• Dietary sources: Seafood, meats, whole grains, vegetables
• Usually derived ultimately from plants, and selenocysteine, mainly from
animal selenoproteins.
• Transport in plasma by selenoproteins
• Functions
• Components of Glutathione Peroxidases – catabolize peroxides
• Iodothyronine Deiodinases – Regulate concentration of active T3
• Thioredoxin Reductases – in DNA synthesis
• Deficiency: associated with infection, GSH metabolic defect, etc
• Toxicity: Loss and brittleness of hair and nails
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• Fluoride
 Presence of fluoride makes
◦ bones stronger replace hydroxyl grp of hydroxyapatite
◦ teeth more resistant to tooth decay
 Fluoridated water is best source
◦ most bottled water is lacking
 Too much can damage teeth
 Chromium:
 Essential nutrient involved in carbohydrate and lipid metabolism
◦ Maintains glucose homeostasis and Potentiate insulin action
◦ beneficial effects on the blood lipid profiles.
 Deficiency
◦ Creates diabetic like symptoms
 Dietary sources include liver, whole grains, yeast
 Molybdenum
07/22/2024
 Cofactor for many enzymes eg. Xanthine oxidase
Dr Ambayehu 855
 Found in legumes, cereals, organ meat
Cation Function (as examples)
 Ca2+ Bone formation, Blood Coagulation
Neuromusclar activity, enzyme activator (eg.
Collagenase)
 Cu2+ Cofactor (Cytochrome a3), Dopamine –hydroxylase
 Co3+ Active center of Vitamin B12
 Fe2+ O2 transport & storage; Cofactor of enzymes:
Cytochromes
 Mg2+ Enzyme cofactors e.g. Kinases, etc.
 Mn2+ Cofactor of enzymes (Pyruvate carboxylase, xanthine
oxidase)
 Zn2+ Structural Component of metaloenzymes
 Na07/22/2024
+
& K+ Major electrolytes
Dr Ambayehu 856
Anion Function (as examples)
 Cl- Coenzyme of Amylase, synthesis of HCl, electrolyte
 F- Bone formation
 I- T3/T4 biosynthesis
 HPO4= Bone formation, Phosphorylation of metabolites, enzymes
Synthesis of organic cpds (DNA, ...)
 Selenium Biosynthesis of Glutathione Peroxidase
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The end!
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Chapter Thirteen
Plasma proteins
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Major Functions of Blood
The functions of blood except for specific cellular ones such as oxygen transport
and cell-mediated immunologic defense are carried out by plasma and its
constituents
1. Respiration —transport of oxygen from the lungs to the tissues and of CO2
from the tissues to the lungs
2. Nutrition —transport of absorbed food materials
3. Excretion —transport of metabolic waste to the kidneys, lungs, skin, and
intestines for removal
4. Maintenance of the normal acid-base balance in the body
5. Regulation of water balance through the effects of blood on the exchange of
water between the circulating fluid and the tissue fluid
6. Regulation of body temperature by the distribution of body heat
7. Defense against infection by the white blood cells and circulating antibodies
8. Transport of hormones and regulation of metabolism
9. Transport of metabolites
10. Coagulation
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Plasma proteins
Over 70% of plasma solids are plasma proteins with different
function
The concentration of total protein in human plasma is approximately
7.0–7.5 g/dL
Dehydration gives increased levels, in other cases, high values are
frequently due to increases in one or more globulin fractions.
A decrease in total plasma protein is usually associated with a low
[albumin].
The proteins of the plasma are actually a complex mixture that
includes not only simple proteins but also conjugated proteins such as
glycoproteins and various types of lipoproteins.
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Composition of Human Plasma
Electrolyte or Gas Normal (adult) Metabolite Normal (adult) level
Levels[mmol/L]
Glucose (Fasting) 70 – 100 mg/dl
Carbon dioxide 21 – 28
(Arterial) Total Lipids 400 – 800 mg/dl
Carbon dioxide 24 – 30
(venous) Total Proteins 6.0 - 8.0 g/dl
Bicarbonate 21 – 28 Urea 20 – 40 mg/dl
Chloride 95 – 103 Uric acid 2.7 – 7.0 mg/dl
Sodium 136 – 142 Creatinine 0.6 – 1.2 mg/dl
Bilirubin 0.1 – 1.2 mg/dl

Potassium 3.8 – 5.0
Bile acids 0.3 – 3.0 mg/dl
Calcium 2.3 – 2.74
Acetone 0.3 – 2.0 mg/dl
Magnesium 0.65 – 1.23
Acetoacetic acid 0.2 – 1.0 mg/dl
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General functions of plasma proteins
1. Intravascular osmotic effect : Contribute significantly to the intravascular

osmolarity , hence important in maintaining fluid and electrolyte balance.
2. Viscosity : Next major factor in maintaining viscosity of blood after the cellular
component.
3. Transport of materials through blood (such as apoliproteins involved in
transport of lipids; transferrin for iron transport)
4. Protein Reserve: Can be used as fuel under during starvation or malnutrition.
5. Clotting: Blood clotting is one of the most important defensive mechanisms of
our system and takes place with the help of clotting factors in plasma.
Fibrinogen (a clot forming protein) makes a major fraction of plasma proteins.
6. Fibrinolysis: Once the wound is healed, the fibrin clot is retracted by enzymatic
fibrinolysis.
7. Inflammatory Response: Play important role in the process of inflammation.
8. Protection from infection: (e.g. gamma globulins, complement system, Enzyme
inhibitors).
9. Enzymatic function (e.g. Lipase)
10. Maintenance of acid-base balance: The surface charges on proteins help in
07/22/2024 buffering the changes in pH by absorbing or releasing protons.
Dr Ambayehu 863
General characteristics of plasma proteins
1. Most plasma proteins are synthesized in the liver
2. Plasma proteins are generally synthesized on membrane-
bound polyribosomes
3. Almost all plasma proteins are glycoproteins.
4. Many plasma proteins exhibit polymorphism such as α1-
antitrypsin, transferrin, haptoglobin.
5. Each plasma protein has a characteristic half-life in the
circulation.
6. The levels of certain proteins in plasma increase during
acute inflammatory states or secondary to certain types of
tissue damage
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Fractionation of Plasma Proteins
May be separated by precipitation, gel filtration, chromatography , electrophoresis.
Precipitation: The plasma proteins fractions have be sequentially precipitated by two
methods:
1) Cohn’s fractionation: Alcohol used as precipitating agent – increasing
concentrations of alcohol result in different types of proteins being precipitated out
of plasma
2) Salt precipitation: Increasing salt concentrations extract water from proteins and
makes them insoluble resulting in their precipitation.
At increasing degrees of saturation, different types of proteins are precipitated from
plasma.
Cohn’s Fraction Proteins precipitate
Fraction I Rich in fibrinogen
Fraction II γ globulins
Fraction III α & β globulins and Prothrombin
Fraction IV α & β globulins

Fraction V Mainly Albumin
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Simple electrophoresis of a serum sample separates its proteins in to five bands
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Different types/classes of plasma proteins
Pre albumin and retinol binding protein

Albumin
Globulins
 α1- globulin : α1- antitrypsin
 α2- globulins : ceruloplasmin, haptoglobulin, α2-macroglobulin
 β - Globulins :Transferrin , Hemopexin, Plasminogen, Fibrinogen
 γ - Globulin: IgA , IgM, IgG , IgE, IgD
Enzymes (released from cells)
• Alkaline Phosphatase /AP,
• Lactate Dehydrogenase/ LDH,
• Creatine Kinase/ CK
• Alanine aminotransferase /ALT ;
• Aspartate aminotransferase /AST
• Serum glutamate oxaloacetate transaminase/ SGOT
others
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Prealbumin and Retinol binding protein
Small proteins that circulate in plasma as 1:1 dimer that migrate ahead of
albumin on electrophoresis.
Prealbumin : Homotetramer; (MW= 54 kD); synthesized in liver & brain;
half life about 48 hours; also known as transthyretin.
Retinol binding protein (RBP): Monomeric protein (MW 21= kD) ;
involved in transport of vitamin A (retinol), synthesized in liver, rate of
secretion from liver is influenced by binding of retinol to RBP in plasma.
Function of Prealbumin: Transport of thyroid hormones

(triiodothyronine and thyroxine); has two binding sites for the hormones
but mostly only one of them is occupied. About 10% of the total plasma
thyroid hormones are bound to transthyretin.
Function of Retinol Binding Protein: Involved in transport of all-trans
retinol. The formation of the hetero-dimer with transthyretin is kind of a
symbiotic interaction. It provides protection to transthyretin against being
excreted in urine due to its small MW & also enhances the binding
kinetics of retinol with RBP. Once retinol is delivered to the target cells,
the dimer dissociates. Zinc is required for the synthesis of RBP ad hence
RBP as well as vit. A deficiency is associated with zinc deficiency.
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Albumin
The major plasma protein and is synthesized and
secreted by the liver.
It accounts for about 50 % of the total hepatic protein production.
For this reason, measurement of serum albumin concentration is
used to assays liver function test.
Makes about half of total serum proteins.
Non-covalently binds as many as 10 fatty acids per
protein monomer and carries fatty acids to tissues.
Albumin makes the biggest contribution to the plasma
oncotic pressure. If albumin concentration falls very low,
edema is the result.
07/22/2024 Dr Ambayehu 869

Functions of albumin
1. Transport: It can bind and transport many diverse molecules and
serve as low-specificity transport protein, which include:
a. Metal ions: such as calcium and copper.
b. Free fatty acid: albumin binds to free fatty acid released by
adipose tissue and facilitates their transfer to other tissue.
c. Bilirubin: this protects from the toxic side effects of
unconjugated bilirubin.
d. Bile acid: albumin carries the bile acids that are recycle from
the intestine to the liver in the hepatic portal vein.
e. Hormones: such as thyroid hormones and the steroid
hormones.
07/22/2024 Dr Ambayehu 870

2. Maintenance of Colloid osmotic pressure
Colloid osmotic pressure, is a form of osmotic pressure exerted
by proteins in blood plasma that usually tends to pull water into
the circulatory system.
Because large plasma proteins cannot easily cross through the
capillary walls.
In conditions where plasma proteins are reduced,
e.g. from being lost in the urine (proteinuria) or from
malnutrition, there will be a reduction in osmotic pressure,
leading to enhanced fluid retention in tissue spaces (edema).
07/22/2024 Dr Ambayehu 871

Fig. Pathogenesis of edema in hypoalbuminaemia
07/22/2024 Dr Ambayehu 872

Clinical aspects
1. Albumin binds different drugs and strongly affects the
pharmacokinetics of these drugs.
 For example, sulfonamides can cause the release of unconjugated
bilirubin from albumin by competitive binding. If given to infants,
sulfonamides may lead to kernicterus.
2. In cases of liver disease or starvation, albumin synthesis decreases.
 This lead to edema.
3. Hypoalbuminemia
 lowered plasma albumin in malnutrition, nephrotic syndrome and
cirrhosis of liver.
4. Albuminuria
 albumin is excreted into urine in nephrotic syndrome and certain
inflammatory conditions of urinary tract.
5. Albumin is therapeutically useful for the treatment of burns and
hemorrhage.
07/22/2024 Dr Ambayehu 873
Globulins
(A) A small amount of serum or other fluid is applied to a cellulose acetate strip. (B)
Electrophoresis of sample in electrolyte buffer is performed. (C) Separated protein bands
are visualized in characteristic positions after being stained. (D) Densitometer scanning
from cellulose acetate strip converts bands to characteristic peaks
07/22/2024 Dr Ambayehu 874
α1–Antitrypsin /α1–Antiproteinase/ α1–AT
It (52 kDa) is a glycoprotein with 394 AAS.
It is a major constituent of α1 globulin fraction of plasma protein, normal
concentration about 200mg/dl.
It is a serine protease inhibitor and can combines with trypsin, elastase
and other protease and inhibits them.
About 5% Emphysema is due to the deficiency of α 1–AT. This is
associated with lung infection and increase the activity of macrophage to
release elastase that damage lungs tissue.
Smoking can cause oxidation of Met 358 to methionine sulfoxide and
inactivate α1–AT.
Due to mutant α1 –antitrypsin accumulates and aggregates to form
polymers, by unknown mechanism, cause liver damage followed by
07/22/2024 accumulation of collagen resulting in fibrosis (cirrhosis).
Dr Ambayehu 875
Intravenous administration of α1 -antitrypsin
(augmentation therapy) has been used as an adjunct in the
treatment of patients with emphysema due to α1 -
antitrypsin deficiency
(A) normal inactivation of elastase by α1 -antitrypsin and (B) situation in which

the amount of α1 -antitrypsin is substantially reduced, resulting in proteolysis by
elastase and leading to tissue damage.
07/22/2024 Dr Ambayehu 876

α2 –Macroglobulin (α2 –MG)
It (720 kDa) is a glycoprotein with 4 identical subunits, a major
constituent of α2 fraction.
It is a panprotease inhibitor and can combine and inhibit many
protease.
It can bind cytokines such as PDGF and TGFβ and target them to
particular cells to affect on cell growth or function.
Approximately 10% of the zinc in plasma is transported by α2 -
macroglobulin, the remainder being transported by albumin.
α-MG levels are increased in nephrotic syndrome a condition where
in the kidneys start to leak out some of the smaller blood proteins.
Because of its size, α2 -MG is retained in the bloodstream. This
increase has little adverse effect on the health, but is used as a
07/22/2024
diagnostic clue. Dr Ambayehu 877
Hepatoglobin (Hp)
It (90 kDa) is a glycoprotein.

It can bind with the free hemoglobin (extra-corpuscular Hb) in a
tight non-covalent complex Hp-Hb during hemolysis.
Hp-Hb(155 kDa) cannot pass through glomeruli of kidney while
free Hb(65kDa) can and Hp prevent the loss of free Hb into urine.
Low levels of plasma concentration of Hp can diagnose hemolytic
anemia.
Different fates of free

hemoglobin and of the
hemoglobin-haptoglobin
complex
07/22/2024 Dr Ambayehu 878

Hemopexin
Certain other plasma proteins bind heme but not
hemoglobin. Hemopexin is a β1 -globulin that binds free
heme.
Albumin will bind some metheme (ferric heme) to form
methemalbumin, which then transfers the metheme to
hemopexin.
07/22/2024 Dr Ambayehu 879

Fig. Function of Haptoglobin and Hemopexin.
07/22/2024 Dr Ambayehu 880

Ceruloplasmin
It (160 kDa) is a blue-coloured, copper-containing α2 fraction.
It can carry 90% of plasma copper tightly so that copper is not readily
exchangeable.
It processes copper-dependent oxidase activity but its physiologic significance has
not been clarified apart from possible involvement in the oxidation of Fe 2+ in
transferrin to Fe3+ .
Albumin carries the other 10% , which is the major supplier of copper to tissue.
Low level of ceruloplasmin is associated with Wilson’s disease (hepatolenticular
degeneration)
 Wilson's disease is an inherited disorder in which there is too much copper
in the body's tissues. The excess copper damages the liver and nervous
system .
Treatment: penicillamine is the first treatment used.
 This binds copper (chelation) and leads to excretion of copper in the urine.
07/22/2024 Dr Ambayehu 881

Transferrin (Tf)
Transferrin (Tf) is a β1 -globulin with a molecular mass of approximately 76 kDa.
It is a glycoprotein and is synthesized in the liver..
It can transport iron in plasma as ferric ions (Fe3+) and protect the body against
the toxic effects of free iron.
It plays a central role in the body's metabolism of iron because it transports iron
(2 mol of Fe3+ per mole of Tf) in the circulation to sites where iron is required, eg,
from the gut to the bone marrow and other organs.
Approximately 200 billion red blood cells (about 20 mL) are catabolized per day,
releasing about 25 mg of iron into the body—most of which will be transported by
transferrin
There are receptors (TfR1 and TfR2 ) on the surfaces of many cells for
transferrin. It binds to these receptors and is internalized by receptor-mediated
endocytosis
07/22/2024 Dr Ambayehu 882

Fig. Transport and delivery of iron by transferrin
07/22/2024 Dr Ambayehu 883

Major iron fate
07/22/2024 Dr Ambayehu 884

Acute Phase Proteins, APP
The levels of certain plasma proteins change during inflammation,
infection, injury, cancer etc. These proteins are “Acute Phase
Proteins, APP ”
 Include C-reactive protein(CRP), α1-acid glycoprotein,
fibrinogen, haptoglobin ,α1-antitrypsin, albumin and transferrin.
APP are believed to play a role in the body’s response to
inflammation, changes in their plasma concentrations are generally
regarded as being sensitive, although non-specific, indicators if
inflammation.
07/22/2024 Dr Ambayehu 885

C-reactive protein, CRP
A major component of acute phase protein.
It reacts with the C polysaccharide of
pneumococci.
Involved in the promotion of immune system
through the activation of complement cascade.
Estimation of CRP in serum is important for Complement
the evaluation of acute phase response. activation
CRP rises up to 50,000-fold in acute
inflammation, such as infection. It rises
above normal limits within 6 hours, and
peaks at 48 hours.
Complement: rupturing membranes of foreign cells, cell

swells and bursts.
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Summary of Different plasma proteins ,their function & reason for
their assay
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Immunoglobulins/γ-globulins
Ig is a functional term
γ-globulin is physical term
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Introduction
Living organisms live in environment made up of ‘foreign’ and often
‘hostile’ substances.
Continuously exposed to invasions by these foreign substances which may
be biological like bacteria or viruses; or non-biological like xenobiotics.
The immune system of the body consists of three major components: B
lymphocytes, T lymphocytes and the innate immune system.
The B lymphocytes are mainly derived from bone marrow cells in higher
animals and from the bursa of Fabricius in birds. The T lymphocytes are of
thymic origin.
The B cells are responsible for the synthesis of circulating, humoral
antibodies, also known as immunoglobulins.
The T cells are involved in a variety of important cell-mediated
immunologic processes such as graft rejection, hypersensitivity reactions,
and defense against malignant cells and many viruses. The innate immune
system defends against infection in a non-specific manner and unlike B cells
and T cells is not adaptive.
The B-cells differentiate into specialized cells called plasma cells, which,
when stimulated by specific foreign substances (antigens) synthesize and
07/22/2024 secrete immunoglobulins. Dr Ambayehu 889
Immunoglobulin(Ig)/antibody(Ab)
Glycoprotein molecules that are produced by plasma cells in response
to an immunogen and which function as antibodies, mostly associated
with γ fraction.
Functions
1. Antigen(Ag) binding
- Ig binds to a specific antigenic determinant
2. Effector functions
- Complement activation
- Binding to various cells such as phagocytic cells, lymphocytes,
mast cells: antibody-mediated phagocytosis or antibody-
dependent cell-mediated cytotoxicity (ADCC).
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Two Forms of Ig
1. Membrane Ig, mIg
It confers antigenic specificity on B cells.
2. Secreted Ig, SIg

mIg  It can circulate in the blood and serve as
the effectors of humoral immunity by
searching out and neutralizing antigens
or marking them for elimination.
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SIg
Structure of immunoglobulins
The molecule consists of two light (L) chains and two heavy (H)
chains.
Each light chain consists of avariable (VL ) and a constant (CL )
region. Each heavy chain consists of a variable region (VH ) and a
constant region that is divided into three domains (CH 1, CH 2, and
CH 3).
1. Variable region (V): VL&VH
2. Constant region (C): CL&CH
3. Hinge region: flexibility
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Fig. Structure of immunoglobulins
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Fig. Structure of immunoglobulins
07/22/2024 Dr Ambayehu 894

Fig. Functions of the domains on Ig
Ag Binding
Complement Binding Site

Binding to Fc
Receptors
Placental Transfer
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Fig. Hypervariable region: also called Complementarity
Determining Regions (CDRs)
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General Properties of Immunoglobulins
Are plasma proteins, synthesized and secreted by the specialized B-
lymphocytes called as plasma cells.
Are glycoproteins consisting of about 4 -18 % carbohydrates by weight.
On electrophoresis, they migrate very slowly towards the anode and hence
lag behind most the plasma proteins giving rise to a diffuse dense band
near the cathode known as ‘γ-band’.
On an average, human plasma contains 0.5 to 1.6 g/dl immunoglobulins.
07/22/2024 Dr Ambayehu 897

Immunoglobulin Classes and Subclasses
In terms of the differences in amino acid sequence of constant
region of heavy chain, immunglobulin molecules are divided
into 5 classes:
– IgG, IgA, IgM, IgD and IgE
Heavy chain:
– 5 types: γ,α,μ,δ and ε.
Light chains
– 2 types: κ and λ.
07/22/2024 Dr Ambayehu 898

Immunoglobulin Classes of Mammals
1. IgG - γ heavy chains

2. IgM - µ heavy chains
pentamer
3. IgA - α heavy chains
dimer
4. IgD - δ heavy chains
5. IgE - ε heavy chains
monomer dimer pentamer
07/22/2024 Dr Ambayehu 899

Immunoglobulin G (IgG)
Constitutes about 80% of the total serum Ig.
Most antibacterial and antiviral antibodies are IgG class.
The IgG molecule consists of 2 H chains and 2 L chains.
Based on AA sequence of the constant region of the H-chains, IgG
classified into 4 subclasses, encoded by different germ-line CH genes.
Numbered IgG1, IgG2, IgG3, and IgG4 according to their concentrations
in serum.
The structural characteristics that distinguish these subclasses from one
another are the size of the hinge region and the number and position of the
interchain disulfide bonds between the H chains.
The 4 subclasses also differ in their biological activities:
Passive immunity to the fetus: Although IgG is the only class of Ig that can
cross the placental barrier and hence transfer passive immunity to the
fetus, there are differences between the subclasses, e.g. IgG2, is very slow
or is unable to cross the placenta. IgG1, IgG3, and IgG4 readily cross the
placenta and play an important role in protecting the developing fetus.
07/22/2024 Dr Ambayehu 900

Immunoglobulin G (IgG)…
Complement fixation: IgG3 is the most effective complement activator, followed by
IgG1; IgG2 is less efficient, and IgG4 is not able to activate the complement at all.
Phagocytosis: The constant regions of IgG1 and IgG3 bind with high affinity to Fc
receptors on phagocytic cells (Macrophages, monocytes, polymorphonuclear
leukocyte, and some lymphocytes) and thus mediate opsonization. IgG4 has an
intermediate affinity for Fc receptors, whereas IgG2 has an extremely low affinity.
Examples :
 Blood Group Abs: Anti-A, Anti-B and Anti-Rh
 Antibacterial Abs: Anti-streptococcal and Anti-pneumococcal
 Anti-toxin Abs: Anti-Diphtheria toxin Ab, Anti-Streptolysin Ab, Anti-tetanus toxoid
Ab
 Anti-spirochaetal Ab
 Anti-viral Abs
 Autoantibodies: Anti-thyroid microsomal Ab.
07/22/2024 Dr Ambayehu 901
Fc receptor
Fc receptor: protein found on the surface of

certain cells (including natural killer cells,
macrophages, neutrophils, and mast cells).
Fc receptors bind to antibodies that are attached
to infected cells or invading pathogens.
Their activity stimulates phagocytic or cytotoxic
cells to destroy microbes, or infected cells by
antibody-mediated phagocytosis or antibody-
dependent cell-mediated cytotoxicity.
07/22/2024 Dr Ambayehu 902

Immunoglobulin A (IgA)
10- 15% of the total Ig, average concentration: 150 to 350 mg/dl in serum.
The daily synthesis of IgA exceeds that of any other Ig.
Every day, the human body secretes about 5 to 15 g of secretory IgA into mucous secretions.
IgA exists in two forms . Vascular IgA present in serum and the Secretary IgA found in all
kind of external secretions of the body such as breast milk, saliva, tears, and mucus of the
bronchial, genitourinary, and digestive tracts.
Vascular IgA: In serum, IgA exists primarily as a monomer, but dimers, present.
Polymerization is facilitated by J-chain polypeptide identical to that found in pentameric IgM.
Secretary IgA: Most of the IgA found in external secretions, called secretory IgA, is dimeric.
Apart from the regular light and heavy chains and a J-chain polypeptide, secretary IgA also
contains another polypeptide called secretory component .
IgA is the most predominant antibody in the colostrum, the initial secretion from the mother’
breast after a baby is born.
Does not activate complement (unless aggregated)
Binds to Fc receptors on some cells
07/22/2024 Dr Ambayehu 903

Immunoglobulin M (IgM)
5 -10% of total serum Ig, average serum concentration :1.5 mg/ml.

Is the first class of antibodies generated during a primary immune response.
Monomeric IgM (MW 180,000) as membrane-bound antibody on B cells.
Circulate in plasma as a pentamer , 5 monomer units are held together by
disulfide bonds through their C-terminal heavy chain domains.
The 5 monomer subunits are arranged with their Fc regions in the center of the
pentamer and ten antigen-binding sites on the periphery of the molecule.
J-Chain: Each IgM pentamer carries an additional polypeptide called the J
(joining) chain, which joins the C-terminal domains of 2 polypeptides by
disulfide bonds.
Rest of the polypeptides are joined to each other by inter-chain disulfide
linkages.
The J chain is a small asp and glu rich polypeptide that facilitates
polymerization of the monomers to form pentameric IgM.
It appears to be added just before secretion of the pentamer.
J-chain is not absolutely essential for the polymerization of IgM monomers.
07/22/2024 Dr Ambayehu 904

Immunoglobulin M (IgM)…
Relative Efficiency of IgM: Because of its pentameric structure, IgM carries 10

antigen-binding sites and hence can bind 10 small hapten molecules. It is 100 to
1000 times more efficient for agglutination reactions of RBCs as well as viral
particles. IgM is also more efficient than IgG at activating the complement
because activation requires two Fc regions in close proximity that is easily found
on IgM pentamers.
Because of its large size, IgM does not diffuse well and therefore is found in
very low concentrations in the intercellular tissue fluids. IgM antibodies stick to
the mucosal surfaces of the secretary cells through their J-chains. Although IgA
is the major isotype found in these secretions, IgM plays an important accessory
role as a secretory Ig.
1st Ig produced in a primary response to an antigen and serve as first line of
defense.
07/22/2024 Dr Ambayehu 905

07/22/2024 Dr Ambayehu 906
Immunoglobulin E (IgE)
Primarily involved in the allergic reactions and hypersensitivity.
Present in serum at extremely low concentrations (~30 µg/dl).
Able to trigger hypersensitivity reactions characterized by symptoms of hay
fever, asthma, hives, and anaphylactic shock.
Structure : regular hetero-tetrapeptide composed of κ or λ light chains and ε
type of heavy polypeptide chains. The heavy chains are larger in size than IgG
and contain 4 constant domains compared to three in the IgG.
Upon binding with an allergen, IgE mediate allergic response by attaching
with the basophils in blood and mast cells in tissues through their Fc receptors.
IgE are produced against the parasites and a local (e.g. intestinal) mast-cell
degranulation may release mediators that attract the macrophages and
phagocytic cells necessary for antiparasitic defense. IgE are known to provide
the major defense against helminthic infections.
IgE also appears to participate in the inflammatory processes.
07/22/2024 Dr Ambayehu 907

Immunoglobulin D (IgD)
The serum concentration of IgD is very low (30 µg/ml).

Constitutes about 0.2% of the total Ig in serum.
IgD, together with IgM, is the major membrane bound immunoglobulin expressed
by mature B cells.
IgD was first discovered from a multiple myeloma (a B-cell tumor that produces a
single kind of antibody) patient when it was observed that the myeloma protein
did not react with any anti-isotype antisera known at that time i.e. IgA, IgM, and
IgG. Antibodies were then raised against the myeloma antibodies and further
experiments revealed a new class of immunoglobulins called IgD.
The biological function of IgD immunoglobulins is yet to be identified but various
studies have claimed their activity against antigens like penicillin, diphtheria
toxoid, insulin and milk proteins along with thyroid and nuclear antigens.
They have also been implicated in cell differentiation.
No biological effector function has been identified for IgD.
07/22/2024 Dr Ambayehu 908

Summary of different classes of Immunoglobulin
07/22/2024 Dr Ambayehu 909

Biological properties of Immunoglobulins
Recognition and binding properties are inherent in the V regions, the biological
properties, also known as ‘Effector functions’ reside in the C-terminal (Fc) fragment.
There are 5 types of effector functions
1. Opsonization
 The secreted Ig, when bound to a target antigen like a pathogenic bacteria, facilitate
the phagocytosis of the bacteria. This coating of the microorganism or other foreign
bodies with the antibodies is known as ‘Opsonization’.
 Once inside the phagocytic cell, the pathogen is digested and neutralized by the
enzymes and various antibacterial peptides.
 The IgG, IgA and IgM class of antibodies can initiate the opsonization process.
2. Complement activation
 Complement system is a group of catalytic serum glycoproteins that are activated in
sequential manner and result in the perforation of cell membranes of the invading
micro-organisms.
 The collaboration between antibody and the complement system is important for the
inactivation and removal of antigens and the killing of pathogens.
 Activation of the complement proteins and their binding to the foreign
substances/cells is also known as Complement fixation.
 Most of the IgG subclasses can activate the complement system.
07/22/2024 Dr Ambayehu 910
Biological properties of Immunoglobulins...
3. Cell Mediated Cytotoxicity

Binding of the antibodies to pathogenic cells or to the modified cells (like cancer or
virus infected cells) also attracts a number of T cells particularly the Natural Killer
(NK) cells and T Cytotoxic (TC) cells to the target.
The antibody acts as a newly acquired receptor enabling the attacking cell to
recognize and kill the target cell.
This process is called antibody-dependent cell-mediated cytotoxicity (ADCC).
4. Allergy and hypersensitivity
Allergy and hypersensitivity are the properties of IgE class of antibodies.
IgE antibodies bind to the antigens (allergens) and result in formation of a network
by cross-linking.
The blood basophils and tissue mast cells have receptors for Fc end of the IgE
immunoglobulins.
Once the basophils or mast cells get attached to the IgE-linked allergen, their
cytosolic granules, containing a number of bio-active molecules, are translocated to
the plasma membrane.
The release of these bio-active molecules, like histamine, gives rise to allergic
manifestations.
The local release of the bio-active mediators from the mast-cells also induces the
build up of macrophages and other cells at the site of the antigen (parasite) and
result in its neutralization.
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Biological properties of Immunoglobulins...
5. Transcytosis
 The secretion of Ig in breast milk, their transport across placenta to the fetus or
their delivery to the mucosal surfaces of the respiratory, gastrointestinal, and
urogenital tracts is called as transcytosis.
 All these processes require the passage of the proteins across the epithelial
membranes.
 Tranport across membranes is also a property of the constant region of Ig
molecule.
 In humans and mice, IgA is the major antibody species that is easily transported,
although IgM can also be transported to mucosal surfaces.
 Most of the subclasses of IgG are transported across the placenta from mother to
the fetus.
 In humans, the transfer of IgG to the fetus takes place during the 3rd trimester of
gestation.
 The wide spectrum of maternal antibodies transferred to the fetus confers
protection to the developing fetus against pathogens.
 The transfer of maternal IgGs to the fetus is a form of passive immunization.
 Passive immunity can be defined as the acquisition of preformed protective
immunity (antibodies) without activations of one’s (fetus’s) immune system after
exposure to antigen. This becomes more important due to the fact that immune
system of the fetus, or even the new born, is too immature to respond to the attack
07/22/2024 of various pathogenic insults on its own.
Dr Ambayehu 912
Monoclonal antibodies
Every foreign antigen / substance / organism carries a battery of
epitopes on its surface that can individually induce an immune
response.
As a result, the normal serum contains a spectrum of antibodies,
each specific for a different epitope on the same or different
antigen.
Such a mixture of antibodies, mostly obtained by immunization
of animal with a foreign antigen, is called as polyclonal or
heteroclonal.
The polyclonal antisera, although used extensively in research
and medicine, are found lacking in their specificity.
If a single antigenically committed lymphocyte is isolated, it does
07/22/2024 not multiply for a long time in cultures.
Dr Ambayehu 913
Monoclonal antibodies…
In 1975, Georges Köhler and Cesar Milstein devised a method for
preparing antibody that is obtained from a clone of single
lymphocyte. These mono-specific antibodies were called as
monoclonal antibodies (MoAb).
The technique involved fusing a normal activated, antibody-
producing B cell with a myeloma cell (a cancerous plasma cell).
The fused cell, called a hybridoma, possessed the characteristics of
the B-cell i.e. specificity for a single epitope, as well as those of the
myeloma cell i.e. the immortal growth.
The clones of hybridoma cells, which secrete large quantities of
monoclonal antibody, can be cultured indefinitely.
The antibodies thus generated are highly specific for a single epitope
and are of the same class/subclass.
07/22/2024 Dr Ambayehu 914
Uses of Monoclonal antibodies
Diagnostic uses:
• For the detection of bacteria, viruses and parasites in biological
fluids as well as for the detection of antibodies produced against
these microorganisms in an individual.
• In hematology and blood banks where they are being used for
blood grouping and separation of specific cell populations.
• In pathology laboratories, fluorescence labeled MoAb are used
for identification of cells and tissues by a technique known as
‘Immunofluorescence labelling’.
• All the Immunoassays used in biochemistry laboratories viz.
Radio-immunoassay (RIA), Enzyme-immunoassay (EIA) and
chemiluminiscence-immunoassay (CIA), for the measurement
of hormones, drugs, vitamins and other peptides employ
monoclonal antibodies etc…
Therapeutic uses :
• To treat a number of clinical diseases like Rheumatoid arthritis.
• Radiolabelled antibodies specific for a number of tumors have
been approved as drugs by Food and Drug Administration.
07/22/2024 Dr Ambayehu 915

Abzymes
The antibodies that can catalyse reactions just like enzymes.

These monoclonal antibodies with enzyme activities are called
‘Abzymes’, since they act as antibodies as well as enzymes.
A new field of research has emerged known as ‘catalytic
antibody research’. The objective of research in this field is
directed at generation of a battery of abzymes that cut peptide
bonds at specific amino acid residues, just as restriction enzymes
cut DNA at specific sites.
Such abzymes would be invaluable tools in the structural and
functional analysis of proteins.
It might also be possible to generate abzymes that can dissolve
blood clots and help in the treatment of coronary artery disease or
stroke.
The abzymes could also be generated that would cleave viral
glycoproteins at specific sites, thus blocking viral infection.
07/22/2024 Dr Ambayehu 916

Disorders of Immunoglobulin synthesis
Immunodeficiency
Immunodeficiency disorders reflect the inadequacy of the immune
system to fight tumors or invading organisms.
Associated with a deficient force to fight against the foreign
substances or organisms like bacteria or viruses.
Immunodeficiency causes persistent or recurrent infections, severe
infections by organisms that are normally mild, incomplete
recovery from illness or poor response to treatment, and an
increased incidence of cancer and other tumors.
Immunodeficiency disorders may affect any part of the immune
system. Most commonly, this involves decreased functioning of T
or B lymphocytes (or both), or deficient antibody production
07/22/2024 Dr Ambayehu 917

Causes of immunodeficiency
a)Genetic or congenital (inherited) defects: Defect in genes responsible for Ig
synthesis can cause congenital immunodeficiency disorders of antibody
production (B lymphocyte abnormalities).
b)Acquired immunodeficiency: HIV infection and AIDS can result in impaired
immuno-surveillance leading to increased susceptibility to infections.
c) Immunosuppression: A number of drugs can suppress the immune system e.g.
corticosteroids or immunosuppressant medications.
Other causes of immunodeficiency:
malnutrition, particularly with lack of protein
Splenectomy, as it poses the patient to higher risk of infection from certain
encapsulated bacteria that the spleen would normally help fight.
Diabetes
Increasing age also reduces the effectiveness of the immune system to some
degree
Some immunodeficiency disorders are mild and result in occasional illness.
Others are severe and may be fatal.
Immunosuppression that results from medications is often reversible once the
medication is stopped.
07/22/2024 Dr Ambayehu 918
Excessive production of Immunoglobulins
Malignant proliferation of the lymphocytes is known as lymphoma, which

could arise from T-cells or B-cells.
Multiple myeloma is cancerous disorder where a clone of plasma cells in an
individual has escaped normal controls of cell division and hence proliferates
endlessly without any activation by antigen.
The myeloma cell continues to secrete molecularly homogeneous antibodies.
This antibody is indistinguishable from normal antibody molecules but is
called myeloma protein to denote its source.
In a patient afflicted with multiple myeloma, myeloma protein can account
for 95% of the serum immunoglobulins.
In most patients, the myeloma cells also secrete excessive amounts of light
chains. These excess light chains excreted in the urine of multiple myeloma
patients are called as Bence-Jones proteins, after the name of their
discoverer.
07/22/2024 Dr Ambayehu 919
A simple urine test for B-J protein is diagnostic of multiple myeloma.
Fig. Serum proteins from a patient with multiple myeloma
07/22/2024 Dr Ambayehu 920

Blood clotting & related proteins
Control of bleeding from cut skin or blood vessel : ‘hemostasis’.
Hemostasis: Constriction of blood vessels, platelet aggregation & formation of a
protein meshwork to ‘plug’ the injury site.
Plasma contains a series of proteins that participate in the process of blood clotting
(coagulation).
The formation of fibrin clot at the site of injury is very complex and involves the
sequential activation of the mediator proteins.
Each activation step multiplies the signal through a cascade of reactions which leads
to very rapid coagulation.
Three phases of Hemostasis
1. Formation of a loose plug formed by the aggregation of platelets at the site of
injury. The platelets bind to collagen fibers and release ADP and thromboxane
A2, which attract and activate more platelets to the site. The binding is mediated
by vonWillebrand factor and fibronectin. The platelets change shape and form an
aggregate to form the plug that stops bleeding.
2. Fibrinogen is converted to fibrin, which forms a meshwork around platelets that
traps platelets and form a strong and stable plug.
3. Hemostatic plug or clot is partially or completely dissolved by the proteolytic
action of plasmin.
07/22/2024 Dr Ambayehu 921
Fig. Primary contributors to hemostasis
07/22/2024 Dr Ambayehu 922

Phases of the formation of a haemostatic plug
07/22/2024 Dr Ambayehu 923

Pathways for the formation of fibrin clot
1) Intrinsic Pathway
Blood clotting through intrinsic pathway begins when negatively charged
surface of the exposed sub-endothelial tissue at the site of injury triggers the
activation of prekallikrein, HMW kininogen, factors XII and factor XI.
This phase is known as the ‘contact phase’.
Kallikrein cleaves factor XII to convert it to active XIIa.
Active factor XIIa acts on prekallikrein and factor XI to activate both of them
so that the contact phase reactants are accumulated at the site of injury.
The cascade of activation then leads to the assembly of factors VIIIa, IXa,
calcium and X called as ‘the tenase complex’.
Factor X is converted to Xa (a serine protease). The activated Xa then acts on
prothrombin to convert it to thrombin.
A number of factors (II, VII, IX, X) in the intrinsic pathway are zymogens
containing γ-carboxyglutamate (Gla) residue. The negatively charged Gla
residues bind calcium (Ca++) ions.
Factor VIII is activated by thrombin and is a cofactor that serves as receptor
for factors IXa and Xa on the platelets.
07/22/2024 Dr Ambayehu 924

2) Extrinsic Pathway
Extrinsic pathway starts when the tissue factor is exposed on
the surface of endothelial cells and monocytes at the site of
tissue injury.
The tissue factor activates factor VII to VIIa. Further, tissue
factor also acts as a cofactor for enzymatic activation of
factor X by factor VIIa.
The 53 kD factor VII is also a proenzyme containing Gla
residue, and is synthesized in liver.
The tissue factor and factor VIIa, known as the tissue factor
complex, also activate factor IX of the intrinsic pathway,
thus providing a point of a ‘cross talk’ between the two
pathways. This cross talk is very important for the initiation
of blood clotting in vivo.
07/22/2024 Dr Ambayehu 925

Fig. Coagulation cascade
07/22/2024 Dr Ambayehu 926

Nomenclature of various clotting factors proteins
Factor Name Mol.Wt. Factor Name Mol.Wt.

(kD) (kD)
I Fibrinogen 340 VIII Antihemophilic factor A 1200
II Prothrombin 69 IX Antihemophilic factor B 62

(Plasma thromboplastin
component)
III Tissue Factor - X Stuart factor 59
IV Ca++ - XI Plasma Thromboplastin 200

antecedent
V Pro-accelerin 200 XII Hageman factor 80
VII Proconvertin 45.5 XIII Fibrin stabilizing factor 320
07/22/2024 Dr Ambayehu 927

Prothrombin
69 kD, Single polypeptide and α2-globulin
At the time of tissue injury, prothrombin is activated to
thrombin by activated factor Xa by the cleavage of an N-
terminal peptide.
Thrombin is a serine protease with MW of 34 kD.
Thrombin gets attached, through negative charges on the
surface of platelets.
Calcium helps in its interaction with the platelets.
When active, thrombin acts on circulating fibrinogen molecules
around the platelets and cleaves ‘A’ and ‘B’ peptides from Aα
and Bβ polypeptides, thus exposing the binding regions of
central ‘E’ domain which help in making fibrin dimers.
07/22/2024 Dr Ambayehu 928

Fibrinogen
Fibrinogen and fibrin play important, overlapping roles in blood clotting,

fibrinolysis, cellular and matrix interactions, inflammation, wound healing,
and neoplasia.
Fibrinogen is a dimeric molecule that is composed of two sets of three
polypeptide chains termed ‘Aα’, ‘Bβ’, and ‘γ’.
The molecules are elongated 45-nm structures consisting of two outer D
domains, each connected to a central E domain by a coiled-coil segment.
The polypeptides are joined by disulfide bridging within the N-terminal E
domain. These domains contain constitutive binding sites that participate in
fibrinogen conversion to fibrin, fibrin assembly, crosslinking, and platelet
interactions
07/22/2024 Dr Ambayehu 929

Formation of fibrin clot
At the site of injury, platelet associated thrombin cleaves 2 peptides
(‘A’ and ‘B’ ) from fibrinogen converting it to fibrin.
The cleavage of fibrinopeptide A from Aα alpha chains exposes two
EA polymerization sites, and subsequent fibrinopeptide B cleavage
exposes two EB polymerization sites that can also interact with
platelets, fibroblasts, and endothelial cells.
The fibrin molecules interact with each other through D to EA
associations to form fibrin dimers .
The dimers then get aligned side-to-side to make fibrils that
converge in parallel arrays to make bilateral network branches and
multistranded thick fiber cables.
Once thin temporary fibrin clot has been formed, thrombin-
activated factor XIIIa comes into play.
This factor is a highly specific transglutaminase that covalently
cross-links fibrin molecules by forming peptide bonds between the
amide groups of glutamine and the € -amino groups of lysine
residues
07/22/2024 Dr Ambayehu 930
Fig. Formation of fibrin clot
07/22/2024 Dr Ambayehu 931

Fibrin Clots Are Dissolved by Plasmin
Plasmin, the serine protease mainly responsible for degrading fibrin
and fibrinogen, circulates in the form of its inactive zymogen,
plasminogen (90 kDa), and any small amounts of plasmin that are
formed in the fluid phase under physiologic conditions are rapidly
inactivated by the fast-acting plasmin inhibitor, α2 -antiplasmin.
Plasminogen binds to fibrin and thus becomes incorporated in clots
as they are produced; since plasmin that is formed when bound to
fibrin is protected from α2 -antiplasmin, it remains active. Activators
of plasminogen of various types are found in most body tissues, and
all cleave the same Arg-Val bond in plasminogen to produce the two-
chain serine protease, plasmin.
Tissue plasminogen activator (t-PA) is a serine protease that is
released into the circulation from vascular endothelium under
conditions of injury or stress and is catalytically inactive unless
bound to fibrin.
07/22/2024 Dr Ambayehu 932

Fig. Plasmin and Fibrinolysis
07/22/2024 Dr Ambayehu 933

Recombinant t-PA & Streptokinase Are Used as Clot Busters
Alteplase, t-PA produced by recombinant DNA technology, is
used therapeutically as a fibrinolytic agent, as is streptokinase.
However, the latter is less selective than t-PA, activating
plasminogen in the fluid phase (where it can degrade circulating
fibrinogen) as well as plasminogen that is bound to a fibrin clot.
The amount of plasmin produced by therapeutic doses of
streptokinase may exceed the capacity of the circulating α 2 -
antiplasmin, causing fibrinogen as well as fibrin to be degraded
and resulting in the bleeding often encountered during
fibrinolytic therapy
Streptokinase has also been widely used in the treatment of
coronary thrombosis, but has the disadvantage of being antigenic.
07/22/2024 Dr Ambayehu 934

Disorders of blood coagulation
1) Hemophilias A and B:
Two most common diseases among bleeding disorders.
Hemophilia A : X-linked and runs in males in the family, females
being carriers, unless they are homozygous for the defect.
Seen in a number of members of the royal families of Europe.
Patients are deficient in factor VIII.
Hemophilia B : also known as Christmas disease, named after the
first patient in which this was demonstrated. The disorder is due to
the deficiency of factor IX.
Management of haemophilias : Administration of cryoprecipitates
(rich in factor VIII and IX) from pooled human plasma, but now
recombinant factors are available and are used to control
07/22/2024 bleeding . Dr Ambayehu 935
Disorders of blood coagulation…
2) Von Willebrand disease:
Most common bleeding disorder
Characterized by deficiency of von Willebrand factor (vWF).
The factor is secreted from endothelial cells and helps the platelets to adhere to the site injury.
vWF : Also known as platelet adherence factor (PAF) and stabilizes factor VIII during the
formation of fibrin clot.
Inheritance: Autosomal dominant
3) Disseminated intravascular coagulation :
A complication of prolonged labour in pregnancy.
Placenta secretes some proteolytic substances into maternal blood which trigger thrombin
activity and result in intravascular clotting.
The clots are dissolved by plasmin but the excessive demand on the fibrinogen and thrombin
results in their depletion.
This acquired hypofibrinogenemia may lead to massive post-partum bleeding.
4) Trousseau’s triad:
Carcinoma of pancreas has long been associated with increased intravascular coagulation and
is manifested as thrombophlebitis.
The combination of carcinoma of pancreas, thrombophlebitis and coagulopathy is known as
Trousseau’s triad, named after the person who diagnosed the disease in himself.
Primarily caused by secretion of large amounts of trypsin by the tumor tissue that overwhelms
the anti-trypsin activity and causes activation of the clotting factors.
07/22/2024 Dr Ambayehu 936

Therapeutic blood components
Blood transfusion: administration of blood group cross matched
whole blood intravenously to the diseased patient.
All patients do not need whole blood.
A patient with low Hb might need only RBC
A burn patient benefits from plasma separated from cells
Nephritic syndrome patients require mainly albumin
Hemophilia patients might need only the clotting factors.
Separation of blood and plasma components for therapeutic
purposes is very common.
The different blood/plasma components are administered in
different conditions – thus transfusion medicine has become very
focused as per the requirements.
Fractionation of the components also means that from a single
unit of blood, a number of patients can be treated.
07/22/2024 Dr Ambayehu 937
Plasma components available for therapeutic use
Blood preparation Application

Fresh frozen plasma Liver cirrhosis, Blood clotting disorders and
Disseminated intravascular coagulation
Cryoprecipitate Rich in fibrinogen, factor VIII and fibronectin.
Used in Clotting disorders,
hypofibrinogenemia, hemophilia, von
Willebrand disease
Factor VIII Hemophilia A
concentrate
Albumin Hypovolemic shock, cerebral edema
Immune serum Immunodeficiency states, Passive immunization

globulin against tetanus & hepatitis
07/22/2024 Dr Ambayehu 938

Enzymes in plasma
Enzymes of different origin are present in plasma and are used for
diagnosis. Because of normal cell turn over they present in small
quantity , however, increased quantity is due cells injuries.
1. Acid phosphatase: Tumor marker in prostate carcinoma
2. Alanine aminotransferase(ALT): An indicator of hepatocellular
damage.
3. Alkaline phosphatase: Increase in cholestatic liver disease and is
marker of osteoblast activity in bone disease.
4. Amylase: an indicator of cell damage in acute pancreatitis.
5. Aspartate amino transferase (AST): An indicator of hepatocellular
damage or as a marker of muscle damage such as myocardial
infarction(MI).
6. Creatine Kinase(CK): A marker of muscle damage and acute MI.
7. γ-glutamyl transpeptidase: A sensitive marker of liver cell damage
8. Lactate dehydrogenase: A marker of muscle cell damage.
07/22/2024 Dr Ambayehu 939
Enzymes that have been shown to have diagnostic value are present in plasma
07/22/2024 Dr Ambayehu 940

The end!
07/22/2024 Dr Ambayehu 941

Chapter Fourteen
Blood group antigens and antibodies
07/22/2024 Dr Ambayehu 942

Blood grouping system classified in to:
1. ABO Blood Group
2. Rh Blood Group System
07/22/2024 Dr Ambayehu 943

ABO Blood Group
First blood groups discovered by Landsteiner(1900).
Most significant for transfusion practice.
ABO compatibility is essential before other pre-transfusion
test is performed.
ABO antigens are the only Ags for which reciprocal
antibodies consistently and predictably exist in serum of
normal individuals.
07/22/2024 Dr Ambayehu 944

ABO blood group
Blood Group RBC Ag Antibodies
A A Anti-B
B B Anti-A
AB A&B None
O None Anti-A & Anti-B
1. Group A: Express A antigen on RBC surface & Anti-B in serum.

Genotypes AA or AO
2. Group B: Express B Ag on RBC surface & Anti-A in serum.
 Genotypes BB or BO
3. Group O - Have neither A nor B antigens on their RBC & both
anti-A & anti-B in serum.
 Genotype OO (“universal donors”)
4. Group AB: Express A and B Ag on RBC surface & no ABO
antibodies (“universal recipients”)
07/22/2024
Genotypes A1B or A2B Dr Ambayehu 945
ABO blood group
07/22/2024 Dr Ambayehu 946

Possibilities of ABO blood group donation
Blood Group Donate to Received from

A A & AB A &O
B B & AB B&O
AB To It self only (A B) All (A,B,AB,O)
O All (A,B,AB,O) From it self only (O)
Note:
Blood group ‘O’ is universal donor and
Blood group ‘AB’ is universal recipient
07/22/2024 Dr Ambayehu 947
Rh blood group
 Complex blood group with >50 described
antigens
 D Ag is more potent
 Rh Antibodies are Not naturally occurring
 Produced as a result of immune stimulation from
exposure to the antigen through transfusion
and/or pregnancy
Implicated in transfusion reactions
Because they are IgG, they can cross placenta
and cause HDN(hemolytic disease of new born)
07/22/2024 Dr Ambayehu 948

Hemolytic disease of the newborn (HDN)
 Also referred to as erythroblastosis fetalis
 Occurs when the mother is alloimmunized to antigen(s) found on the
RBC of the fetus
 Destruction of 2nd birth fetal RBCs by mother’s IgG antibodies
1. Rh Incompatibility Rh (-) mother & Rh (+) baby
2. ABO Incompatibility “O” mother & “A” or “B” baby
07/22/2024 Dr Ambayehu 949

1. HDN due to Rh incompatibility
 Set-up: Rh(-) mother + Rh(+) baby.

 Rh (-) person exposed to Rh(+) blood will develop reaction
after 2 – 4 weeks.
 Mother develops antibody against the Rh(+) blood coming
from the baby.
 First baby is not affected; but HDN occurs during subsequent
pregnancies.
 May be prevented by giving anti-Rh to Rh (-) mother in the
ante-natal (28 weeks) and immediate postnatal period (within
72 hours after delivery).
07/22/2024 Dr Ambayehu 950
HDN due to Rh incompatibility cont..
07/22/2024 Dr Ambayehu 951

HDN due to Rh incompatibility cont..
Rh(-) RBC Antibody
Rh(+) RBC
07/22/2024 Dr Ambayehu 952

2. ABO Hemolytic Disease
 Materno-fetal ABO incompatibility
 A and B got IgM in blood which cannot cross
placenta.
 O got IgG antibody; so, cross placenta and cause
hemolytic disease.
 Occurs most frequently in group A or B babies
born to group O mothers.
 ABO incompatibility often affects the first
pregnancy because of the presence of non-RBC
stimulated ABO antibodies.
 Red blood cell destruction by ABO antibodies is
more common than by anti-D.
 These cases can usually be treated with
phototherapy.
07/22/2024 Dr Ambayehu 953
The end!
07/22/2024 Dr Ambayehu 954

Biochemistry Final

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BIOCHEMISTRY

Dr. Ambayehu (MD)

Dr. Ambayehu (MD)

• The study of biochemistry is essential to understand basic functions of

Dr. Ambayehu (MD)

• Attraction between particles of the same substance

Dr. Ambayehu (MD)

A. Simple protein: Yields only amino acids and no other major

• Eg: Albumins, Globulins, Glutelins, albuminoids, histones, and

B. Conjugated Proteins: Yields amino acids and other organic

• E.g. Nucleoprotein: For storage & transmission of genetic information

• e.g. Casein: bring phosphorous to growing infant

• Metalloprotein: Metal storage proteins (e.g. ferritin) and Metal

• e.g. Fibronectin (adhesive glycoproteins) and proteoglycans for ECM.

• IgG: circulate in plasma.

• Many glycosylated membrane proteins on extracellular segment.

• Blood group antigens

• Flavoproteins - Containing flavin (vitamin B2 derivative) E.g.

• Hemeproteins are a group of specialized proteins that contain heme as

• The binding of CO2 stabilizes the T or deoxy form of hemoglobin,

• Defined as a group of genetic disorders caused by production of

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• Do not change the equilibrium points /Have no effect on Keq/ : only

• Apply to the thermodynamically allowable reactions/ do not change

• Can be viewed from two different perspectives:

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• The regulation of the reaction velocity of enzymes is essential if

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• Monosaccharides vary from trioses to heptoses.

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