High Yield Items

High Yield Items
Dr. Alisa Chebotarova, MD, PhD

4

DNA Replication and Repair

Purines and Pyrimidines
• De novo Synthesis of Purine Nucleotides: Purine Salvage Pathways:

Purine Nucleotide Degradation

De novo synthesis of Pyrimidine Nucleotides

Genetics expression and transcription

Carbohydryates

Amino Acids

Urea Cycle

Biochemistry and Genetics
Block 1 Review
Course:
Prepared by:
Dr. I.Vyshnytska, MSc, PhD
Dr. A.Chebotarova, MD, PhD
Water, Acids, Bases, and Buffers
HA ↔ H+ + A−
Week Acid conjugate base
• weak acid is 50% dissociated at a pH equal to its pKa - half is in acid form, half is in base form
• A buffer is usually effective at a pH = pKa ±1
• As the pH of a buffered solution changes from the pKa to 1 pH unit below the pKa , the ratio of [A-] to HA changes
from 1:1 to 1:10. If more hydrogen ions were added, the pH would fall rapidly because relatively little conjugate
base remains.
• Bicarbonate Buffer 20/1 when plasma pH=7,4
lungs
kidney
• Phosphate buffer. H2PO4− HPO42− + H+

Water, Acids, Bases, and Buffers
N pH: 7.35-7.45
N PCO2: 35-45 mmHg
N HCO3-: 22-26 mmol/L
= ↑ pH >7.44 = ↓ pH <7.36

Metabolic alkalosis ✮
• Metabolic alkalosis is characterized by an increased plasma pH due to a loss of H+
or as a direct result of a gain in HCO3−.
• In both instances there is a relative increase in HCO3−, raising the [HCO3−] / PCO2
ratio.
• In the acute stage, (clinically, may not be observed)
– pH is increased (greater than 7.45)
– [HCO3-] is increased (greater than 25mmol/L) (primary abnormality)
– PCO2 is almost normal

Metabolic alkalosis
Causes of metabolic alkalosis:
• Vomiting
• Pyloric stenosis resulting in vomiting
• Loss of acidic contents of the stomach, results in relative HCO3- excess.
• Nasogastric suction.
• Excessive consumption of antacids
• Renal loss of H+ (Cushing’s disease, bilateral adrenal hyperplasia)

Compensated metabolic alkalosis
• Increase in the pH (alkalosis) inhibits the respiratory center, there is a decrease
in the rate of respiration (hypoventilation) → Decreased washout of CO2 (CO2
retention) → ↑PCO2 ✮
• Renal system compensates, if it is functioning normally, by excreting more HCO3-
(alkaline urine)
• In the compensated state, ✮ (clinically more commonly observed)

üpH is higher than normal (closer to normal pH, when compared to acute stage)
ü[HCO3-] is increased (primary abnormality)
üPCO2 is increased (compensatory mechanism)

Compensated metabolic alkalosis

Respiratory acidosis ✮
• Respiratory acidosis is characterized by an increased arterial PCO2 (hypercapnia),
which decreases the [HCO3−] / PCO2 ratio. The underlying problem is due to CO2
retention, as a result of hypoventilation.
• the CO2 is NOT washed out resulting in elevation of PCO2 (primary abnormality)
• CO2 is an ACID, and its accumulation causes respiratory acidosis
• In the acute stage:
üpH is decreased (lower than 7.35)
üPCO2 is elevated (primary disturbance)
üHCO3- is almost normal

Respiratory acidosis
Clinical causes:
• Drugs that inhibit the respiratory center (opioids)
• Diseases/ injury of the phrenic nerve (supplies diaphragm)
• Lung diseases like chronic obstructive pulmonary disease, fibrosis of the lung,
respiratory distress syndrome in premature infants
• Obstruction to the respiratory tract – due to foreign body in trachea

Compensated Respiratory acidosis
• During compensation, the renal system comes to the rescue
• Kidneys excrete more H+, and generate more HCO3-, and thus the [HCO3-] levels
increase
• The excretion of phosphate and ammonia in urine increase
• In the compensated phase ✮

üpH is lower than normal (<7.35) - closer to normal pH, when compared to
acute stage
üPCO2: Elevated (as the primary defect is still not corrected – respiratory system
is still not functioning optimally)
ü[HCO3-]: Elevated (due to renal compensation)
Compensated Respiratory acidosis

Respiratory alkalosis ✮
• The primary abnormality in respiratory alkalosis is a decrease in PCO2
(hypocapnia), most commonly due to hyperventilation.
• This causes an increase in the [HCO3−] / PCO2 ratio with a consequent rise in the
plasma pH.
• Respiratory alkalosis is characterized by an increase in rate of respiration →
increased washout of CO2 → ↓↓PCO2 (primary disturbance)
• In the acute stage:

– pH is increased (<7.45)
– PCO2 is ↓↓ (<35mmHg)
– [HCO3-] is almost normal
Respiratory alkalosis
Causes of hyperventilation
• Anxiety
• Fever
• Hysteria
• Hypoxia (high altitude)
• Mechanical ventilation

Compensated Respiratory alkalosis
• During the chronic stage, the renal system tries to bring the pH back towards
normal
• The kidneys do NOT secrete H+ into urine
• There is increased excretion of HCO3- in urine and pH of urine becomes alkaline,
and serum HCO3- falls ✮
• In the compensated phase ✮:
– pH is higher than normal - closer to normal pH, when compared to acute
stage
– PCO2: Decreased (as the primary defect is still not corrected – respiratory
system is still hyperventilating)
– [HCO3-]:Decreased (due to renal compensation)
Compensated Respiratory alkalosis

Amino acids
• Essential: Arg, His, Ile, Leu, Thr, Lys, Met, Phe, Trp, Val
• Ketogenic: Leu, Lys

Associated Disease
AA name Classification Functions
/Comments
Alanine Nonpolar, N/E, Glucogenic most important glycogenic amino acids, in the
structure of transmembrane proteins
Valine Nonpolar, Essential, Glucogenic Branched-chain amino acid, in the structure of sickle-cell disease, MSUD
transmembrane proteins
Leucine Nonpolar, Essential, Ketogenic Branched-chain amino acid, in the structure of ferritin and other buffer
transmembrane proteins and transport proteins
Proline Nonpolar, N/E, Glucogenic helix breaker, present in β-turns
Isoleucine Nonpolar, Essential, Branched-chain amino acid
Gluco/ketogenic,
Glycine Nonpolar, N/E, Glucogenic inhibitory neurotransmitter, the smallest AA, inhibited by strichnine
formation of collagen, heme, purines, β-turns
Asparagine Polar, neutral, N/E, Glucogenic excitatory neurotransmitter, synthesis of
glycoproteins
Glutamine Polar, neutral, N/E, Glucogenic synthesis purines and pyrimidines, carrier of crosses the blood-brain
ammonia in plasma, barrier
Threonine Polar, neutral, Essential, protein-linking,
Gluco/ketogenic,
Serine Polar, neutral, N/E, Glucogenic protein-linking, In active site of proteases
Associated Disease
AA name Classification Functions
/Comments
Phenylalanine Aromatic, Essential, Gluco/ketogenic, can be converted into L-tyrosine PKU
Tyrosine Aromatic, N/E, Gluco/ketogenic, insulin and growth factor receptors, precursor tyrosinosis, tyrosinemia,
of dopamine, norepinephrine and adrenaline, albinism, Alkaptonuria
thyroxine, melanin
Tryptophan Aromatic, Essential, Gluco/ketogenic, precursor for serotonin, melatonin and niacin Hartnup disease
Aspartate Polar, Negative, N/E, Glucogenic urea cycle and in gluconeogenesis, purines
and pyrimidines synthesis
Glutamate Polar, Negative, N/E, Glucogenic excitatory neurotransmitter, post-translational
modification of blood coagulation proteins
Lysine Polar, Positive, Essential, Ketogenic synthesis of collagen and elastin; synthesis of
Histones
Arginine Polar, Positive, Essential*, Glucogenic precursor of nitric oxide, urea and ornithine,
synthesis of Histones
Histidine Polar, Positive, Essential, Glucogenic, Buffer action of Hemoglobin, allergenic
histamine, Neurotransmitter
Cysteine Sulfur-containing, N/E, Glucogenic forms disulfide bond Cystinuria
Methionine Sulfur-containing, Essential, methyl donor in transmethylation reactions, Homocystinuria
Glucogenic the 1st AA in translation
Cystinuria ✮
• One transporter is responsible for absorption by
intestine (active transport) of Cysteine, Ornithine, Lysine,
and Arginine (COLA) and is responsible for the
reabsorption of these amino acids by the kidney.
• In the inherited disease cystinuria, this transporter is

defective and all 4 amino acids occur in urine, intestinal
absorption is also impaired
• Clinically can cause kidney stones and block the urinary

tract – stones can damage kidneys and nearby organs –
death possible – oral hydration is important
• Cysteine molecules are not reabsorbed by the kidney
can be oxidized in urine to form cystine
• These form hexagonal white crystals, which grow into
pink or yellow stones
Hartnup disease ✮
• Disease affecting (re)absorption of neutral amino
acids, incl., tryptophan (a precursor of serotonin,
melatonin and niacin-vit B3)
• Infant form – photosensitivity, intermittent ataxia
and tremor
• Later, dermatitis, dementia and diarrhea (Pellagra-
like dermatosis) – provoked by sunlight, fever,
drugs and stress and preceded by poor nutrition

Secondary Structure - Supersecondary
Structures (Motifs)
• Combinations of secondary structures. They are intermediate between secondary
and tertiary structures smaller than a protein subunit.
• Connected by loop regions at the surface of the protein

• Eg. Zn finger domains allow hydrogen bonding interaction
between the specific response elements and the DNA sequences.

Protein folding, Protein structure-function
relationships
• Defect of primary structure - AA sequence of the polypeptide chain:
• Marfan syndrome (mutation of gene for glycoprotein fibrillin-1) – Arachnodactyly,
hypermobile joints, dissecting aortic aneurysms and valvular disease, Pectus carinatum,
dislocation of the lens (upward and outward and temporally)

Cystic fibrosis ✮
• Most common mutation leads to formation

a misfolded protein protein retained in
RER and not transported to cell membrane,
causing:
• ↓ Cl− (and H2O) secretion in airways and
GI;
• ↑intracellular Cl− results in compensatory
• ↑ Na+ reabsorption via epithelial Na+
channels, ↑ H2O reabsorption, abnormally
thick mucus secreted into lungs and GI
tract.
• Other functional defects in CFTR protein
reaching the cell membrane may also
contribute to the pathogenesis of cystic
fibrosis.

Protein folding, Protein structure-function
relationships
• Defects of secondary structure:
• Prion Disease - Mutated (PrPsc, PrPr), higher proportion of β-sheet structure in place of the
normal α-helix structure, Aggregations of amyloid fibers
Transmissible spongiform encephalopathies (TSEs):
• Creutzfeldt-Jakob Disease, Classic (CJD)
• Kuru
• Amyloidosis - β-pleated sheet- Amyloids:
• Alzheimer Disease – neurotoxic beta-amyloid (Ab), tau protein

Protein Misfolding
Misfolded Proteins ✮: (Ubiquitin-proteasome Proteolytic Pathway)
• Covalent attachment of ubiquitin chains tags proteins for degradation into a
proteasome
• The mutations in any gene for Ubiquitination processes lead to an accumulation

of misfolded proteins.

Protein maturation, targeting
• Trimming (Proteolysis) - Portions of the protein chain must be removed by
specialized proteases, (proenzyme are converted to active form by cleavage of a
propeptide from an N- or C-terminus)
• Phosphorylation – on serine, threonine, or, less frequently, tyrosine residues
catalyzed by protein kinases
• Glycosylation – synthesis of glycoproteins, used to target proteins to the matrix
of lysosomes
• Hydroxylation - vitamin C-dependent

Fibrous proteins
• Collagen:
• Gly (glycine)-X-Y:
• Glycine
• Proline
• Lysine
• Triple helical structure:
• three polypeptide (pro-a) chains
• Stabilized by H-bonds between individual polypeptide chains
• Elastin:
• Stretchy protein with recoiling
• monomer = tropoelastin
• desmosine linkages (covalent linkage between 3 allysine and 1 lysine)
• degraded by Elastase
• α1- antitrypsin deficiency - deficiency of Elastase inhibitor, excessive destruction of
Elastin in lung
Fibrous proteins

RER
• Collagen synthesis:
RER
prolyl hydroxylase
lysyl hydroxylase
covalent cross-linking Ehlers-Danlos

syndrome
Dr. Alisa Chebotarova, MD, PhD Menkes disease

Extracellular cleavage of procollagen molecules ✮
• N- and C- procollagen peptidases
remove the terminal propeptides -
Extracellular space:
proteolytic processing
• Problems with cleavage - Ehlers-
Danlos syndrome procollagen
S
S
OH
S
S
S
S S
S Sugar OH Sugar
OH
tropocollagen
Sugar OH Sugar

Osteogenesis imperfecta ✮
• A heterogeneous group of inherited disorders-
Also k/a brittle bone syndrome
• defect in bone matrix formation: production of

a chain of type I collagen (Problems forming
triple helix).
• It presents in early infancy with fractures secondary

to minor trauma, and may be suspected if prenatal
ultrasound detects bowing or fractures of long
bones.

Ehlers Danlos Syndrome ✮
• Heterogeneous group of generalized connective tissue disorders.

• mostly affects Type III, V collagen (blood vessels)
Pathogenesis and genetics:
• Deficiency of collagen processing enzyme (Lysyl hydroxylase, Lysyl oxidase,
Procollagen peptidase) - In most cases, the step of collagen synthesis which is
disturbed in Ehlers-Danlos is the Proteolytic processing and formation of
cross-links✮
• Joint hypermobility,
• hyperextensible skin,
• tissue fragility exemplified by easy bruising,
• atrophic scars following superficial injury,
• mitral valve prolaps
The Alport syndrome ✮
• hereditary nephritis - a number of genetic disorders
• Mostly X-linked dominant
• Mutations in several genes encoding type IV collagen fibers
Clinical presentation:
• hematuria (loss of collagen from basement membrane),
• ocular lesions and hearing loss (loss of collagen from cochlea),
• end- stage renal disease.
• Electron microscopy reveals characteristic abnormalities of the structure of the basement
membrane and lamina densa.

Menkes disease ✮
• impaired Copper absorption and transport due to defective Menkes protein

• ↓ activity of lysyl oxidase
• defective collagen
• depigmented brittle hair,
• cerebral degeneration,
• osteoporosis,
• anemia,
• low body T,
• hypotonia,
• seizures,
• usually fatal in childhood
Fibrillin
• Glycoprotein. Major component of microfibrils. Microfibrils serve as scaffolding
for the deposition of tropoelastin, an integral component of elastic fibers.
• Sheath that surrounds elastin core
• Abundant in the aorta
• Deficient fibrillin: Marfan syndrome
• Patients have a slender, elongated habitus with abnormally long legs, arms, and
fingers (arachnodactyly)
• Pectus carinatum or pectus excavatum (deeply depressed sternum)
• Spinal deformities, such as severe kyphoscoliosis, may be present
• Hypermobile joints
• Predisposition to dissecting aortic aneurysms
• Dislocation of the lens (typically upward and outward and temporally)
Wound healing
• Delayed wound healing is most commonly due to infection.
• Other causes include:
• Vitamin C deficiency (coenzyme for hydroxylation of proline and lysine
procollagen)
• Copper deficiency (cofactor for lysyl oxidase, which cross-links
extracellular collagen)
• Zinc deficiency (cofactor for collagenase)

a1- antitrypsin deficiency ✮
• Inherited mutation of a gene for α1-

antitrypsin
• Mutated α1-AT protein aggregates in
hepatocellular ER – may be associated with
liver damage
• Can not come to lungs and protect them
from degradation by elastase -
Emphysema

Enzymes
Apoenzyme + cofactor = Holoenzyme
• Cofactors
• Metal ions (for metalloenzymes)
• Coenzymes
• Cosubstrates (altered in reaction)
• Prosthetic groups (tightly bound)
• Active Sites
• binding pocket or cleft
• substrate specificity (amino acid side chains)
• Activation Energy
• Energy barrier • Enzymes do not alter:
• Transition state • Equilibrium of the reaction
• Decreased by enzymes • Overall free energy change of
Dr. Alisa Chebotarova, MD, PhD reaction - D G0’
Enzymes
• Factors Affecting Enzyme Activity
1. Substrate concentration
• Michaelis-Menten
• Vm = maximal velocity
• Km = Michaelis constant (particular to enzyme) = [S] at ½ Vmax
• High Km: Low affinity
• Low Km: High affinity
• When [S]>>Km; Vo independent of [S]
• When [S]<<Km; Vo proportional to [S]
2. Temperature
3. pH
4. Enzyme concentration
Enzymes
• Lineweaver-Burk Plots
• Obtain both Km and Vmax
• x intercept = -1/Km
• y intercept = 1/Vmax
1. Competitive inhibitors (inhibitors are structural analogues of S):

• Vmax unchanged
• Km increases
2. Non-Competitive inhibitors:
• Vmax decreases
• Km unchanged
3. Uncompetitive inhibitors:
• Vmax decreases
• Km decreases

Clinical Biochemistry: Drugs -Competitive
Inhibitors
• Sulfonomides and Trimethoprim ✭
• Bacteriostatic drugs
• Interfering with the folate metabolism of the
bacteria.
• Sulfonamides: Competitive inhibitor of Folate
Synthase ✭
• Trimethoprim: Competitive inhibitor of
Dihydrofolate Reductase ✭

Drugs-Competitive Inhibitors
• Methanol is an alcohol that forms in organism a toxic substances Formaldehyde
and Formic acid
• The first reaction is catalyzed by the enzyme alcohol dehydrogenase
Alcohol dehydrogenase Acetaldehyde dehydrogenase
Methanol Formaldehyde Formic acid
• the formic acid produced from methanol metabolism is primarily responsible for the
acidosis and visual disturbances that occur
Ethanol or fomepizole: These antidotes work via competitive inhibition; ethanol and
fomepizole are metabolized by ADH, just as methanol is, but the enzyme has a higher affinity
for both antidotes than it does for methanol.

Non-Competitive Inhibitors
• Cyanide is an example of a non-competitive inhibitor. Cyanide binds to the final

enzyme in the electron transport chain, and prevents this enzyme from catalysing
the reaction from oxygen to water. This prevents the flow of electrons down the
electron transport chain and no ATP can be generated, which results in death.
• Cyanide binds to the cytochrome c oxidase heme a3-Cu-binuclear center to
inhibit both cellular oxygen utilization and ATP production (Way, 1984).
• It can be fatal, depends on the Cyanide concentration.

Allosteric Regulation ✭
• Positive effectors: Increase catalytic activity of
the enzyme
• Positive effectors decrease the apparent Km.
• Negative effectors: Reduce or inhibit catalytic

activity of the enzyme
• Negative effectors increase the apparent Km.

Biological reactions: Energy
• ∆G = ∆H - T ∆S = Gproduct – Greactant
• If ΔG is negative - Net Loss of Free Energy, Spontaneous, Exergonic reaction

• If ΔG is positive - Net Gain of Free Energy, Not Spontaneous, Endergonic reaction
• At equilibrium the ratio of products to reactants is always the same, regardless of
the actual concentration (ΔG = 0)
• the total free energy change is equal to the sum of the free energy changes of
each step

ROS: Hydroxyl radical OH•
• Hydroxyl radical OH. is the most detrimental ROS!
• it is also formed from water during ionizing radiation (DNA damage).
• A crushing injury leads to hydroxyl radicals

DNA structure
• Purines (A, G) – 2 rings PURe As Gold
NA are always written and read from the 5'-end of the chain
• Pyrimidines (C, T, U) -1 ring CUT the PY(pie) to the 3'-end
• C with G = 3 H+ bonds (more stable than A-T base-pairs) - H bonds + the hydrophobic interactions between the
stacked bases = stable double helix
• GAG-amino acids necessary for purine synthesis (Glycine, Aspartate, Glutamine)
• Ribose has a hydroxyl group at the 2’ position

• Deoxyribose has a hydrogen at the 2’ position
• Heterochromatin - condensed, transcriptionally inactive, darker. Euchromatin - less condensed, lighter, active
• DNA (negatively charged)-histones (positively charged): H1 (outside), H2A, H2B, H3 and H4, rich in the lysine and
arginine (+)

RNA structure
• U, not T
• rRNA -component of the ribosomes
• tRNA - pseudouridine and ribothymidine, has anticodon (complementary and antiparallel to

codon), trinucleotide sequence 5’-CCA-3’ added to their 3ʹ-ends for binding amino acid
• hnRNA – precursor for mature mRNA, contains untranslated regions at its 5’ - and 3’ –ends, introns, exons
• mRNA - contains untranslated regions at its 5’ - and 3’ –ends, exons (polycistronic - prokaryotes , monocistronic -
eukaryotes )
• snRNPs - participate in splicing

• miRNAs- mRNA degradation (targeting the 3ʹuntranslated region)
• Ribozymes - RNAs with catalytic activity.

Human genome
• The mitochondrial genome - small circular double stranded molecule, 37 genes,

exclusively maternal inheritance, lack introns and histones, its own genetic code
• The Human Nuclear Genome:

• Single-Copy DNA Sequence - coding portion of genes
• Repetitive DNA Sequences - satellite DNAs (Alpha satellite - centromere ; Minisatellite -
Telomeric DNA; Microsatellite DNA – TG Lynch syndrome)
• Interspersed throughout the genome – Alu: recombination

Protein-coding gene
The Human Nuclear Genome
Repetitive DNA Sequences → Satellite DNAs: as DNA markers
Variable number tandem repeats may be used in DNA fingerprinting analysis -

often used in paternity and forensic identity
• VNTR patterns are extremely specific and

can be accurately compared. A child will
share one band with the biological mother
and one with the biological father.
• A persons’ VNTRs will never have
sequences that their parents do not have.
DNA Replication

Replication in Prokaryotes
Strand Separation:
3. Solving the problem of supercoils:

• DNA topoisomerases ✮
• remove supercoils in the helix
• 2 types:
• Type I topoisomerase: nicks one strand;
• Type II (DNA gyrase ✮) topoisomerase: nicks both strands - found in E. coli, Able
to introduce negative supercoils into relaxed circular DNA using energy from the
hydrolysis of ATP

DNA topoisomerase inhibitors✮
ØBacterial: Antibacterial
• Topoisomerase II (DNA gyrase) inhibitors:
Clinical Biochemistry
FLUOROQUINOLONES (nalidixic acid, ciprofloxacin)
ØEukaryotic: Anti cancer

• Topoisomerase I inhibitors: irinotecan, topotecan
• Topoisomerase II inhibitors: amsacrine, etoposide,
and doxorubicin.

II. Primer Synthesis:
• Both maternal strands of a DNA molecule are used as templates for the
synthesis of daughter strands
• Enzymes, involved to the synthesis of a daughter strand would not be able to
start synthesis without a primer – a short stretch of RNA
• Primase - An enzyme synthesizing RNA primer (DNA-dependent RNA polymerase)

1. Leading strand: The strand that is being copied in the direction of the advancing replication
fork is called the leading strand and is synthesized continuously ✮.
2. Lagging strand: The strand that is being copied in the direction away from the replication fork
is synthesized discontinuously, with small fragments of DNA being copied near the replication
fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually
joined (ligated) to become a single, continuous strand. The DNA synthesis of a new strand
lagging occurs in the 3‘→ 5‘ direction (while every Okazaki fragment growths in 5‘→ 3‘
direction).

DNA synthesis:
• DNA Polymerase III ✮
• has proofreading activity - 3’ à 5’ exonuclease activity ✮
• DNA polymerase III recognizes its mistake
• Makes 1 step back
• removes a nucleotide non-corresponding to the maternal strand in 3’ to 5’ direction (opposite to the
direction of synthesis)
• And keeps moving in the 5’ to 3’ direction again adding correct nucleotides
3’ 5’
dA dC dT dG dC dA dT dC Maternal strand
5’ 3’
U G A dC dG dT Pol III dC Daughter strand
RNA primer
dA
dC
Primer removal:
• DNA Polymerase I ✮
• 5’ to 3’ exonuclease activity ✮
• Able to remove the RNA primer
• 5’ to 3’ polymerase activity ✮
• Replaces the removed RNA primer with deoxyribonucleotides
• 3’ to 5’ exonuclease activity ✮
• “proofreads” the newly synthesized DNA chain

Eukaryotic DNA Replication
Eukaryotic polymerases:
•DNA polymerase a: responsible for the initiation of DNA replication at origins of
replication (on both the leading and lagging strands: RNA primer and DNA
extension)
•DNA polymerase e (epsilon): leading strand synthesis, has proofreading activity
•DNA polymerase d: Okazaki fragments of the lagging strand synthesis
•DNA polymerase g: mitochondrial DNA synthesis
•DNA polymerase b: DNA repair

Reverse transcriptase
• A reverse transcriptase (RT) ✮ is an enzyme used to generate complementary
DNA (cDNA) from an RNA template. It synthesizes a TTAGGG sequence repeats.
• Is used by retroviruses to replicate their genomes, by retrotransposon mobile
genetic elements to proliferate within the host genome. And some non-
retroviruses such as the hepatitis B virus also use RT.
• Retroviral RT has three sequential biochemical activities:
üRNA-dependent synthesis of DNA,
üribonuclease H (hydrolysis of the RNA strand of RNA/DNA hybrid),
üDNA-dependent DNA polymerase activity.
• Collectively, these activities enable the enzyme to convert single-stranded RNA
into double-stranded cDNA.

Reverse transcriptase
• In contrast to DNA polymerase, reverse transcriptase does not have 3’→ 5’
proofreading activity
• prone to errors and high rate of mutations
• Example: human immunodeficiency virus

• AZT resistance involves mutations in the viral enzyme reverse transcriptase
• One chemotherapeutic treatment of HIV is the use of AZT (3ʹ-azido-2ʹ,3ʹ-

dideoxythymidine) or structurally related compounds.
• Once AZT enters cells, it can be converted to the triphosphate derivative and used
as a substrate for the viral reverse transcriptase in synthesizing DNA from its RNA
genome. The replacement of an azide instead of a normal hydroxyl group at the 3ʹ
position of the deoxyribose prevents further replication by effectively causing
chain termination, because a reverse transcriptase lacks proofreading activity.
• Mechanism
Dr. Alisa Chebotarova, MD, PhD of AZT shortly: It inhibits viral reverse transcriptase
Signal Transduction – G proteins
cAMP is the second messenger for AC
system (a system incudes Protein
kinases A)
IP3, DAG and Ca2+ are the second

messengers for phosphatidyl inositol
diphosphate system (a system incudes
Protein kinases C)

Signal Transduction – G proteins
RAS
* Protein
kinases A,C,G are the serine, threonine kinases (phosphorylate Ser or Thr
amino acid residue on the surface of proteins)
ADP- Ribosylation by Bacterial Toxins
• Certain bacterial exotoxins are enzymes which attach the adenosine diphosphate
(ADP)-ribose residue of NAD to G-protein α subunits, an activity known as ADP-
ribosylation.
• Vibrio cholerae exotoxin ADP-ribosylates Gs α-subunit, leading to an increase in

cAMP and subsequently chloride secretion from intestinal mucosal cells, and causing
the diarrhea of cholera.
• Certain strains of Escherichia coli release toxins (heat-labile enterotoxin or LT) similar
to cholera toxin, producing traveler’s diarrhea.

Signal Transduction: cGMP
• Such drugs as Nitroglycerin, Nitroprusside act as a vasodilator, being precursors
of NO molecules, and they are used for angina pectoris treatment.
• Nitric oxide (NO) is synthesized by vascular endothelium, diffuses into the
surrounding vascular smooth muscle cytosol, where it directly binds the
guanylate cyclase, activating the enzyme.

Excision of DNA damage: Base Excision Repair (BER)
• Single bases in DNA can be chemically damaged by a
variety of mechanisms, the most common one is
Uracil formed from oxidative deamination of
Cytosine.
• Enzymes glycosylases in humans recognize and

remove the damaged base. This results in an
apurinic or apyrimidinic (AP) site in DNA.

Hereditary Nonpolyposis Colon Cancer
(HNPCC)-Lynch syndrome✮
• HNPCC results from a mutation in one of the genes (usually hMLH1 or hMSH2)
encoding enzymes that carry out DNA mismatch repair.
• Autosomal dominant ∼ 80% progress to CRC (Colorectal Cancer).
• One prominent type of error that accompanies DNA replication is microsatellite
instability.
• In a patient with HNPCC, cells from the resected tumor show microsatellite instability,
whereas normal cells from the individual (which still retain mismatch repair) do not show
microsatellite instability. Along with information from a family pedigree and histologic
analysis, microsatellite instability may be used as a diagnostic tool.

Double Strand Break Repair:
Homologous recombination
Cleavage of the two crossovers creates two complete recombinant products
• For double-strand break in DNA the Homologous recombination is the most

effective repair.

Diseases associated with defective DNA repair system
• Xeroderma Pigmentosum (Skin condition, easy sunburns) - Defect in the
nucleotide excision repair (T-T dimers repair)
• Hereditary Nonpolyposis Colon Cancer (HNPCC)-Lynch syndrome -
Defect in the mismatch repair
• Ataxia Telangiectasia - Defect in the double-strand DNA breaks repair via non-
homologous end joining (NHEJ)
• Breast and ovarian cancers due to inherited mutations in BRCA1 and BRCA2
genes - Defect in homologous recombination

Block 2 Review
Course:
Prepared by:
PDH
Wernicke-Korsakoff Syndrome - B1 (alcoholics)

1. Wernicke encephalopathy
Acute phase: confusion, ataxia, abnormal eye movements
2. Korsakoff's psychosis
Chronic phase: amnesia (anterograde, retrograde),
confabulation
Beriberi – B1
1. Wet type
cardiovascular presentations (Congestive Heart Failure)
2. Dry type
peripheral neuropathy
Pellagra – B3
Diarrhea
Dermatitis
Dementia
Transport of electrons into the Mitochondria
• The inner mitochondrial membrane is impermeable to NADH
• Shuttles are required for the oxidation of cytosolic NADH by the ETC

Glucagon destroy fructose 2,6-bisphosphate
to switch off glycolysis

Pyruvate kinase deficiency
• However, the effects of the anemia are frequently moderated by the twofold to
threefold elevation in 2,3-bisphosphoglycerate (2,3-BPG) concentration that
results from the blockage of the conversion of phosphoenolpyruvate to pyruvate.
• Because 2,3-BPG binding to hemoglobin decreases the affinity of hemoglobin for
oxygen, the red blood cells that remain in circulation are highly efficient in
releasing their bound oxygen to the tissues
• Affects mainly RBCs: Glycolysis is only the source of energy in RBCs!
1. Anemia (normocytic normochromic) due to lysis of RBCs;
2. No Heinz Bodies
3. High concentration of 2,3BFG decreases affinity of Hb to O2 contracted, shrunken, spiculated red cells
(echinocytes)
Erythrocytes with a lowered level
of reduced glutathione are more
susceptible to hemolysis and are
easily destroyed especially if they
are stressed with oxidant drugs
(for example, antimalarial drugs,
Primaquine).
•Heinz bodies—denatured globin

chains precipitate within RBCs due
to oxidative stress.
•Glutathione also helps maintain the reduced states of sulfhydryl groups in

proteins, including hemoglobin. Oxidation of those sulfhydryl groups leads to the
formation of denatured proteins (including denatured Hb) that form insoluble
masses (called Heinz bodies) that attach to the red cell membranes and damage
them – hemolytic anemia!
Bite cells: result from phagocytic removal of
Heinz bodies by macrophages
I-CELL DISEASE: Defect in protein trafficking
• I-cell disease is caused by a deficiency of the enzyme UDP-

N-acetylglucosamine-1-phosphotransferase.
• Deficiency of this enzyme leads to failure of mannose

phosphorylation (lack of recognition marker). In this case,
unmodified lysosomal enzymes cannot be incorporated
into the lysosome for normal use.
• Secretion of active lysosomal enzymes into blood and

extracellular fluid

Mucopolysaccharidoses ✮

TCA
Insulin, Ca2+
CoA-SH
NAD+
FAD
TPP Acetyl-CoA
Lipoic acid Citrate, ATP, NADH
ADP
Fluoroacetate
ADP, Ca+2
ATP, NADH
Arsenate
Ca2+ Succinyl CoA, NADH

Dr. Alisa Chebotarova, MD, PhD ATP
ETC, Oxidative Phosphorylation
4H+ 4H+ 2H+ 2,4-Dinitrophenol,
I
e- Aspirin, V
IV thermogenin
2e- 2e-
II III
Cyt b
Fe-S Cyt b
e- Cyt a3 Fo
FMN UQ Fe-S
Fe-S
Cyt c
e-
Cyt c1 Cyt a
2e- FAD Cu e-
Doxorubicin 2e- e- ½O H2O
e-
FumarateAntimycin A F1
Barbiturates NADH NAD+ Succinate
Cyanide, CO,
Rotenone Malonate
ADP + Pi ATP
Inhibitors of the ETC and Oxidative Phosphorylation: ↓ ATP synthesis with ↓ O2 consumption, ↑
NADH and FADH2
Uncouplers: ↓ ATP synthesis with ↑ O2 consumption, ↓ NADH Oligomycin
↑ permeability
• Chebotarova,
Dr. Alisa MD, PhDof membrane, causing a ↓ proton gradient and
Carbohydrates – chemistry, classification
Carbohydrates - mono:
A. Aldoses/ketoses B. D/L-sugars (Enantiomers): C. Epimers:
• The same sugar - mirror-images • Different sugars
• D: OH on the right • differ in configuration
• L: OH on the left about one asymmetric
carbon
D. Anomers:
• Ring structures
• OH group is below the ring a-anomers
• OH group is above the ring b-anomers
Carbohydrates - di:
1. Lactose: b galactose + glucose
2. Sucrose: a glucose + b fructose
3. Maltose: 2 D-glucose molecules
Digestion of carbohydrates
A. Salivary and Pancreatic α-Amylase
1. Salivary α-amylase
• endoglucosidase - hydrolyzes internal α -1,4-bonds
2. Pancreatic α-amylase
• continues to hydrolyze the starches and glycogen - α -1,4-bonds
• Used for diagnosis of pancreatitis
B. Disaccharidases of the Intestinal Brush Border Membrane
• Glucoamylase
• Sucrase–isomaltase complex
• 𝛃-Glycosidase complex (Lactase, Glucosyl–ceramidase) - Splits 𝛃 -glycosidic bonds
• Lactase deficiency

Transport of carbohydrates
2
UT
GL
GL
glucose UT
Na+ SGLT 1
galactose T2
GLU
glu Blood glucose
gal GLUT2 (absorbed from gut
fru and synthesized) GLU
T1
fructose GLUT5 GLU
4
UT
GLU T3
GL
T4

Properties of the GLUT 1 to GLUT 5 Isoforms
TRANSPORTER TISSUE DISTRIBUTION FEATURES
GLUT 1 Human erythrocyte a high-affinity glucose transport system
Blood–brain barrier Allow to import glucose even when
Blood–retinal barrier plasma levels are very low
Blood–placental barrier
Blood–testis barrier
GLUT 2 Liver A high-capacity, low-affinity transporter
Pancreatic b-cell May be used as the glucose sensor in
Serosal surface of intestinal mucosa cells – transport of glucose, the pancreas – regulation of insulin
fructose and galactose release
Renal tubular cells (the same as above)
GLUT 3 Brain (neurons), placenta a high-affinity system
GLUT 4 Adipose tissue a high-affinity system
Skeletal muscle Insulin-dependent
Heart muscle
GLUT 5 Intestinal epithelium fructose transporter
Dr. Alisa Chebotarova, MD,Spermatozoa
PhD
INSULIN
+
Glucokinase Hexokinase - G6P Anaerobic Glycolysis (1 mol of glucose to
h- ATP + Pi 2 mol of lactate): Total = 2 ATP in
+ glycolysis
- ATP Aerobic Glycolysis (1mol of glucose to 2
ATP mol of pyruvate -> TCA):
PFK2 F-2,6-BP
+
Phosphofructokinase-1(PFK-1)
- citrate Total = 32 ATP (30 if NADH is shuttled by
AMP +
- fructose-6-phosphate
glycerol 3-P shuttle)
GLUCAGON
ro b ic NADH
Glycolysis
A n a e
+ATP RBC 2,3bisphosphoglycerate

ATP 3-Phosphoglycerate
Fru-1,6-BisP -
+
Pyruvate Kinase
NADH +ATP PYRUVATE KINASE DEFICIENCY
1. Hemolytic anemia (RBCs have only this
glycerol 3-phosphate Lactate Dehydrogenase pathway for the ATP production)
and 2. Increased 2,3Bisphosphoglycerate
(causes ↓ O2 affinity to Hb, normally
malate–aspartate shuttle
Aerobic helps to release O2 from β subunit of
TCA HbA)
3. No Heinz Bodies (in difference to G6PD
ETC deficiency!!!)
Glycogen
GLYCOGEN SYNTHESIS / GLYCOGENESIS GLYCOGEN BREAKDOWN/GLYCOGENOLYSIS
1. Conversion of Glucose to Glucose 6-phosphate. 1. Release of Glucose 1-phosphate from glycogen.
Catalyzed by Glucokinase in the liver or Catalyzed by Glycogen Phosphorylase
Hexokinases in other tissues 2. Removal of branches. Catalyzed by Debranching
enzyme:
2. Conversion of Glucose 6-phosphate to Glucose 1-
As a transferase, the debrancher first removes a unit
phosphate. Catalyzed by phosphoglucomutase containing three glucose residues and adds it to the
3. Formation of uridine-diphosphate-glucose. end of a longer chain by an α-1,4-glycosidic bond
Catalyzed by UDP-Glc pyrophosphorylase The one glucosyl residue remaining at the α-1,6-branch
is hydrolyzed by the amylo-1,6-glucosidase activity of
4. Elongation of chain. Catalyzed by glycogen the debrancher, resulting in the release of free glucose
synthase - Makes a linear a-1,4-linked 3. Conversion of Glucose-1-P to Glucose-6-P. Catalyzed
polyglucose chain by phosphoglucomutase
5. Introduction of branches. Catalyzed by Branching 4. Conversion of Glucose-6-P to Glucose. Catalyzed by
enzyme (Glycosyl α1,4:α1,6 Transferase). glucose-6- phosphatase
Hydrolyzes an α1,4 bond, Transfers the In liver and kidney
oligoglucose unit and attaches it with an α1,6 In muscles, enzyme glucose-6-phosphatase is
bond to create a branch absent
Glycogen
• Regulation of Glycogen Metabolism:
• Covalent modification of major enzymes of
glycogen synthesis (glycogen synthase) and
glycogen degradation (glycogen phosphorylase):
• Glycogen synthase is active when

dephosphorylated (not covalently modified) -
glycogen synthase a
• When glycogen synthase is covalently modified
(phosphorylated), it is inactive in the form of glycogen
synthase b
• Glycogen phosphorylase is active when

phosphorylated (covalently modified) - glycogen
phosphorylase a
• When the covalent modification (phosphorylation) is
removed and the enzyme is inactive, it is referred to as
glycogenMD,
Dr. Alisa Chebotarova, phosphorylase
PhD b
Regulation of Glycogen Metabolism

Glycogen Storage Diseases (GSD)
• cmc

Glycogen Storage Disease Type I
(glucose-6-phosphatase deficiency, Von Gierke disease)
• Enzyme deficiency: Glucose-6-phosphatase. ✮
• The enzyme is absent in liver cells and also in kidney (“Hepatonephromegaly glykogenica”)
• Children with GSD I are unable to release glucose from liver glycogen
• Glycogen Structure – normal ✮
• Clinically:
• If untreated this results in prolonged periods when their blood sugar level is too low - Severe
fasting hypoglycemia ✮
• They present in early childhood with sweating, irritability, poor growth and muscle weakness
• Liver enlargement - hepatomegaly ✮ occurs due to excessive accumulation of glycogen that
cannot be broken down normally.
• ↑ glycogen with normal structure in liver and kidneys✮
• ↑ blood lactate ✮
• Hyperlipidemia, hyperuricemia, short stature, doll-like facies, protruding abdomen emaciated
extremities ✮
Glycogen Storage Disease Type II
(alpha glucosidase (acid maltase) deficiency, Pompe's Disease)
• Enzyme deficiency: Lysosomal acid α-1,4-glucosidase, GAA (Acid Maltase)✮
• Enzyme is present in lysosome and catalyzes break down of oligosaccharides.
• Glycogen Structure – Glycogen-like material ✮
Clinically:
• Glycogen-like material in inclusion bodies ✮.
• Generalized involvement of organs seen including heart, liver, smooth and striated muscles.
Nearly all tissues contain excessive amount of normal glycogen.
• Cardiomegaly, Cardiomyopathy ✮
• Muscle weakness ✮
• GSD II usually presents within the first months of life, death by 2 years ✮

Glycogen Storage Disease Type III
(debranching enzyme deficiency, Cori disease)
• Enzyme deficiency: Debranching Enzyme (α-1,6-glucosidase) ✮
• Milder form with normal blood lactate levels
• Glycogen Structure – abnormal ✮
• Clinically:
• Children with GSD III are often first diagnosed with a swollen abdomen due to a very
large liver with short outer branches, Single glucose residue at outer branch ✮
• “Limit Dextrin” type of Glycogen (Glycogen structure with short outer branches of at
most 4 glucose residues) deposit in the cytosol of liver &muscle cells
• Mild hypoglycemia ✮ but this is not as common as in GSD I.
• Treatment consists of a high protein diet and prevention of prolonged periods of fasting
Glycogen Storage Disease Type IV
(branching enzyme deficiency, Anderson disease)
• Enzyme deficiency: Branching Enzyme ✮
• GSD IV is a very severe but rare disorder that leads
to cirrhosis of the liver and heart involvement
• Glycogen Structure – abnormal ✮
Clinically:
• Large accumulations of glycogen seen with linear unbranched structure. Linear
glycogen easy precipitates.
• Main organs affected are: Liver (mainly affected), others are Heart, Muscle and kidney.
Infantile hypotonia, cirrhosis ✮
• Most children with this condition have died before two years of age. ✮
No treatment apart from liver transplantation has been found to prevent progression of
the disease.
Glycogen Storage Disease Type V
(muscle glycogen phosphorylase deficiency, McArdle disease)
• Enzyme deficiency: Muscle Phosphorylase ✮
• Glycogen Structure – normal ✮
• Clinically:
• ↑ glycogen in muscle, but cannot break it down - Muscle cramps on exercise,
pain, weakness and stiffness of muscles ✮
• No lactate is formed.
• Muscles recover on rest, due to utilization of Fatty acids for energy.
• The consequence is exercise-induced myalgia and fatigue
• Myoglobinuria ✮ The disorder affects all skeletal muscle, which results in
significant disability.
Substrates for gluconeogenesis:
Lactate Cori cycle
Glucose 6-phosphatase Glucogenic amino acids (particularly
INSULIN alanine) glucose/alanine cycle
AMP Glycerol
+ Fatty acids with an odd number of
carbon atoms
PFK2 F 2,6-BP Fructose 1,6 Bisphosphatase Not Substrates for gluconeogenesis:
- Acetyl CoA, Ethanol
Citrate Fatty acids
GLUCAGON
ATP
Glycerol 3P Leucine, Lysine
GLUCAGON
PEPcarboxykinase
Alanine
Gluconeogenesis
Alcohol metabolism in the liver leads
- GTP
CORTISOL to unregulated and rapid production
Lactate of NADH
1.Pyruvate → lactate (lactic acidosis)
Malate Shuttle 2.Oxaloacetate → malate (prevents
gluconeogenesis → fasting
- ATP
hypoglycemia)
3.DHAP → glycerol-3-P (combines
Pyruvate Carboxylase with fatty acids to make triglycerides
ADP Biotin
Acetyl-CoA hepatosteatosis)
Fructose metabolism
Essential Fructosuria - Deficiency of fructokinase
Fructosuria
Fructose accumulates, excreted in the urine
Depends on the time and amount of fructose and sucrose intake
Benign (Asymptomatic, May go undiagnosed)
Due to normal activity of hexokinase which converts fructose to
fructose 6-P, and directs it to glycolysis
Hereditary Fructose Intolerance -Deficiency of Aldolase B
Autosomal recessive
Phathobiochemistry:
Fructose 1-P accumulates (toxic)
Depletion of inorganic phosphate, Depletion of ATP
Gluconeogeneis is inhibited, Protein synthesis is inhibited
Kinetics of fructose metabolism: Accumulation of AMP, Catabolism to Uric Acid
• Rate of fructose metabolism is higher than glucose metabolism • Onset: 4-6 months, after weaning
• ↑ dietary fructose → sequestering of inorganic phosphate →
• Severe hypoglycemia and vomiting following fructose intake
↓ ATP
• ↑ dietary fructose → ↑ lipogenesis in liver Bleeding tendency, Hyperuricemia, Lactic acidosis,
• Fructokinase has a higher Vmax than aldolase B hepatomegaly, jaundice, Renal proximal tubule defect (Fanconi
Fructose 1-PMD,
• Chebotarova,
Dr. Alisa abundant
PhD in the liver, tendency to accumulate syndrome) – metabolic acidosis
Fructose metabolism
Fructose
Fructokinase
Fructose 1 - phosphate
Aldolase B
Glyceraldehyde Dihydroxyacetone
phosphate
ATP
Glyceraldehyde 3-phosphate
Intermediates of glycolysis
Inherited disorders of fructose metabolism
Fructose
Fructokinase Essential fructosuria
Fructose 1– phosphate
Essential fructosuria (Benign fructosuria) (AR)

• Deficiency of the enzyme fructokinase in liver
• On ingesting sucrose, fructose is not metabolized in liver
and is excreted in urine in these children
• No toxic metabolites of fructose accumulate in liver –
therefore persons are asymptomatic
• Clinitest test with urine reveals the presence of a reducing
sugar, that is not glucose or galactose

Inherited disorders of fructosemetabolism
Fructose 1- phosphate
Hereditary fructose intolerance
Aldolase B
Glyceraldehyde Dihydroxyacetone
phosphate

•Hereditary fructose intolerance (HFI)
• Deficiency of aldolase B in the liver

• Ingestion of sucrose or fructose, results in trapping of
fructose1-phosphate in hepatocytes
• Trapping of Pi, results in ATP deficiency, and inhibits
gluconeogenesis. AMP rises. In the absence of Pi, AMP is degraded →
hyperuricemia
• Trapping of Pi, also inhibits glycogenolysis

• A combined effect of inhibition of gluconeogenesis and
glycogenolysis, results in hypoglycemia (drowsy and
apathetic)
• Remember, the hypoglycemia occurs following consumption
of sucrose or fructose, and NOT in the fasting state!!!
• Urine analysis detects the presence of a reducing sugar
(fructose) that is not glucose
The symptoms of hereditary fructose intolerance:
• After ingesting fructose, individuals with hereditary

fructose intolerance may experience nausea,
bloating, abdominal pain, diarrhea, vomiting, and
low blood sugar (hypoglycemia).
• Affected infants may fail to grow and gain weight at
the expected rate (failure to thrive).
• No treatment can cure hereditary fructose
intolerance. Instead, a person should avoid
consuming fructose.

Sorbitol Pathway
In cases of hyperglycemia, glucose may be

converted to sorbitol by aldose reductase (has
a high Km value, only occurs in cases of
increased blood glucose)
Osmotically active – leads to water

accumulation
Cataract formation, damage to neurons, defects
in retina and vessels

Galactose Metabolism
Galactosemia
autosomal recessive
Deficiency of galactokinase
or galactose 1-P uridyltransferase (GALT)
• Begins around day 3 in a newborn
• Jaundice, hyperbilirubinemia do not resolve if the
galactokinase infant is treated with phototherapy.
• Cirrhosis.
GALT Classical Galactosemia • Vomiting and diarrhea after milk ingestion
• Severe bacterial infections
• Failure to thrive, lethargy, hypotonia, and mental
retardation
• Cataracts (galactitol synthesis by aldose reductase)
Significance of the polyol pathway
In uncontrolled diabetes mellitus, when

the blood glucose is elevated, glucose enters
the lens of the eye and is converted to sorbitol
by the polyol pathway
Sorbitol is osmotically active and it
increases the water content of the lens,
resulting in cataract (decreased transparency
of the lens).
Sorbitol formation in the tissues is partly implicated in the pathogenesis of
the microvascular complications of diabetes mellitus (neuropathy, nephropathy,
retinopathy) – glucose uptake in these tissues is independent of insulin.
Sorbitol is the sugar alcohol formed from glucose by the action of aldose
reductase. It catalases also reduction of Galactose to galactitol.
Defects in Carbohydrates Metabolism
Von Gierke’s Hereditary Fructose Intolerance Classical Galactosemia
Def. in Glucose-6-phosphatase Def. in Aldolase B Def. in GALT

Accumulate Glucose-6-P04 Accumulate Fructose-1-PO4 Accumulate Galactose-1-PO4
Organomegaly, severe fasting Usually presents after 6 Cataracts (galacticol), mental
hypoglycemia months of age – when child retardation, liver failure
Usually presents in school age switches to formula or Presents in first months of life –
children –lethargy and poor consumes fruit on a farm child is entirely breast fed
performance
All may present with hypoglycemia(+/-), lactic acidosis, and hyperuricemia
Dr. Alisa Chebotarova, MD, PhD Prepared by Dr. Inna Shypilova

Pentose Phosphate Pathway
G6PD DEFICIENCY
X-linked recessive (Africa and Asia,
and parts of the Mediterranean)
Hemolytic anemia
Insulin
Caused by the inability of RBCs
G6PD
NADPH to detoxify ROS by glutathione.
Heinz bodies(Hb precipitation)
Bite cells
Acute episodic hemolysis after
Antibiotics, Antimalarials,
Transketolase
Vit B1 Antipyretics, Fava beans,
InfectionsThe Glutathione System
• From Glucose 6-phosphate to ribulose 5-phosphate-

Irreversible reactions, produce NADP
• From ribulose 5-phosphate to fructose 6 phosphate
by Transketolase (Thiamin requiring enzyme!)
GAG, proteoglycans, Glycoproteins
Long, unbranched hetero-polysaccharide chains consisting of repeating disaccharide units. Highly negatively
charged; all GAGs are released into the extracellular matrix (EXCEPT for heparin)
Acidic sugars: Glucuronic, Iduronic
Amino sugars: Glucosamine/Galactosamine are acetylated and sulfated
Linkage between GAG and core protein: Carbs are linked to protein by serine via O-glycosylation; Xylose-Galactose-
Galactose-[Acidic Sugar-Amino Sugar]n
Proteoglycans Glycoproteins
Always negatively charged, usually sulfated Primarily proteins (some sugar)
Long straight chains May be negative; NOT sulfated
Glucuronic/Iduronic Acid, Xylose Short chains, highly branched
Support cellular and fibrous components; cell-cell Mannose, Sialic Acid, Fucose
signaling, adhesion Cell-surface recognition, Mucins, globular proteins
MUCIN
GAG, proteoglycans, Glycoproteins
Hurler syndrome (MPS IH) a-L-Iduronidase deficiency Hunter syndrome (MPS II)
(Iduronate sulfatase deficiency)
• Autosomal recessive, Affects degradation of heparan and dermatan • X-linked, accumulation of
sulfates
heparan and dermatan sulfates
• Corneal clouding,
• coarse facial features, • Mild Hurler + aggressive
• dwarfing, behavior
• mental retardation, • No corneal clouding
• upper airway obstruction • Macrocephaly,
• hepatosplenomegaly hepatosplenomegaly
• Deposition in coronary arteries leads to ischemia and early death • Mild to severe mental
• Treatable with enzyme replacement or with bone-marrow graft (before retardation
18/12)
I-cell Disease (deficiency of the UDP-N-acetylglucosamine:N-acetylglucosaminyl-1-phosphotransferase)

• failure of the Golgi to phosphorylate mannose residues (mannose-6-phosphate) on glycoproteins
• proteins are secreted extracellularly rather than delivered to lysosomes
• coarse facial features, gingival hyperplasia, clouded corneas, restricted joint movements, claw hand deformities,
kyphoscoliosis,
Dr. Alisa Chebotarova, MD,and
PhD high plasma levels of lysosomal enzymes
Fatty Acids
• Fatty acids are usually straight aliphatic chains with a methyl group at one end and a carboxyl group at the other
end
• Naturally occurring fatty acids are usually straight chain (unbranched) with an even number of Carbon atoms:
A. CLASSIFICATION:
ü Short chain (< 8C),
ü Medium chain (8-14C)
ü long chain (>16C)
vDouble bounds (cis-):

Each cis- double bond introduces a kink of about 120o into the molecule, lowering melting point:
• Saturated fatty acids – no double bonds
• Unsaturated fatty acids
• Monounsaturated - with 1 carbon-carbon double bond
• Polyunsaturated – with 2 or more carbon-carbon double bonds
• C18:2 (ω-6) and C18:3 (ω-3) are two essential fatty acids
• Linoleic acid (precursor for arachidonic acid/prostaglandins etc.) 18:2Δ9,12 ✮
• Linolenic acid (precursor for other ω-3 acids essential for growth and development) 18:3Δ9,12,15
Digestion Of Dietary Triacyclglycerols
A. Digestion in mouth and stomach
Lingual lipase:
• Ideal substrate- short chain FA, TGs - Milk fat
Gastric lipase
• Acts on triglycerides with short and medium chain fatty acids
C. Digestion in duodenum and small intestine
Emulsification of lipids - Bile salts
Pancreatic lipase (Requires the co-lipase)
Gastrointestinal hormones that aid fat digestion:
Cholecystokinin - Stimulates the pancreas to release digestive enzymes
Secretin - Stimulates the exocrine pancreas and gall bladder to release bicarbonate
Absorption Of Dietary Lipids
1. Large products form mixed micelles with bile salts (effectively recycled) and other dietary
lipids such as fat soluble vitamins, travel to microvilli on intestinal epithelial cells, and are
absorbed
2. Short and Medium Chain Fatty acids - Taken up by the enterocytes without the aid of mixed
micelles. Travel to liver in portal blood, carried by serum albumin
3. Resynthesis of triacylglycerols in intestinal epithelial cells
4. Packaging into nascent chylomicrons (Apo-B48)
5. Secretion into lymph and blood
6. Formation of mature chylomicrons (+Apo CII and ApoE from HDL)

de novo synthesis of fatty acids
• Precursors - Cytosolic Acetyl CoA (acetyl portion of acetyl CoA enters the cytosol as part of citrate and in the
cytosol ATP-citrate lyase forms Acetyl CoA), NADPH (the pentose phosphate pathway, malic enzyme).
1). Formation of Malonyl CoA from Acetyl CoA, by Acetyl CoA carboxylase (CO2 + Hydrolysis of ATP + biotin).
• Allosteric regulation: activation - polymerization by citrate, inactivation – depolymerization by long-chain fatty
acyl CoA (Palmitoyl CoA)
• Long-term regulation: activation - high-calorie diet increases the rate of transcription, inactivation - Low energy
levels, low-calorie diet
• Hormones: activation – Insulin- dephosphorylation, inactivation- Glucagon and Epinephrine – phosphorylation
(AMPK)
2). Fatty Acid Synthase- 7 enzyme activities and ACP (pantothenic group) Requires NADPH (from HMP shunt)
3). 7 Rounds of Synthesis Results in the Production of Palmitate
• Synthesis of long chain FAs by Elongases (Brain has elongases to synthesize very long chain fatty acids)
• Synthesis of unsaturated FA: Desaturases (Human have 9,6,5 and 4, but never beyond Δ9 )
Formation of TAGs
• Synthesis of Triacylglycerols in ADIPOSE TISSUE occurs only in the presence of Insulin and GLUT4 transporters-
glucose dependent
• HEPATOCYTES have the enzyme Glycerol kinase that allows TAG synthesis to be independent of Glucose entry

Lipolysis (fatty acid breakdown)
1. Breakdown of Triacylglycerols in Adipose by hormone
sensitive lipase (HSL) (releasing free FA)
• Activation: Low energy, Glucagon, Epinephrine,
Norepinephrine -phosphorylation (AMPK), Cortisol,
Growth hormones, Thyroid hormones - increase HSL
expression
• Inactivation: High energy state, Insulin –
dephosphorylation
2. Fatty acids are activated by fatty acyl CoA synthetase

(+ATP) and form Fatty Acyl CoA

β Oxidation of Fatty Acids
• LCFA translocation- Carnitine shuttle:
• 1. carnitine acyltransferase I (CAT)- transfers the fatty acyl group to carnitine (outer mitochondrial membrane) –
inhibited by Malonyl CoA
• 2. Fatty acylcarnitine is shuttled across the inner membrane.
• 3. Carnitine acyltransferase-2 transfers the fatty acyl group back to a CoA (mitochondrial matrix).
1. β Oxidation
The first step is the removal of two hydrogen atoms from the 2(α)
and 3 (β) carbon atoms, catalyzed by acyl-CoA dehydrogenase -
Chain length specific dehydrogenases
• Many Acetyl-CoA molecules are released -> TCA
• FADH2 and NADH are produced -> ETC
2. Oxidation of Odd Carbon Chain Fatty Acids: in final formation of
propionyl-CoA, than it is converted to succinyl-CoA (2 enzymes,
CAT Deficiency- CPTI (CAT-I), CAT-II deficiencies: biotin, vitamin B12)
Patients report feeling weak, reduced glucose 3. Very Long (Chain) Fatty Acids (VLCFA) Oxidized in Peroxisome
levels, diminished ketone levels, muscle biopsy 4. Peroxisomal a-Oxidation of Branched Chain Fatty Acids:
shows elevated muscle triglycerides detected as PHYTANIC ACID, Degraded by a-oxidation in peroxisome,
lipid
Dr. droplets
Alisa in the
Chebotarova, cytoplasm,
MD, PhD myoglobinuria mitochondria
β Oxidation of Fatty Acids
• Jamaican Vomiting Sickness - unripe fruit of the Ackee-Ackee Tree contains Hypoglycin A - Inhibitor
of Acyl CoA dehydrogenase. Vomiting (Within 2-6 hrs.), Severe Hypoglycemia, Convulsions, coma
and death.
• Deficiency of Medium-chain Acyl-CoA Dehydrogenase (MCAD) - fatigue, vomiting, increased
respiration, low glucose levels , increased plasma free fatty acids levels, low ketone levels, elevated
levels of organic acid metabolites of medium (C6-12) chain organic acids in urine, partially oxidized
medium chain fatty acids in blood, accumulation of monocarboxylic fatty acids and dicarboxylic
organic acids, urinary excretion of C8-C10 acyl-carnitine compounds, cerebral edema, coma leading
to death
• Zellweger Syndrome- Defect in PTS-receptors (Peroxisomal), Vision disturbances, prenatal growth
failure, lack of muscle tone, unusual facial characteristics, mental retardation, seizures and an
inability to suck and/or swallow
• Refsum disease- phytanic acid oxidase deficiency, over-accumulation of phytanic acid in cells and
tissues, neurologic damage, cerebellar degeneration, and peripheral neuropathy, ataxia, scaly skin
(ichthyosis), difficulty hearing, and eye problems

β Oxidation/FA synthesis
Synthesis Breakdown
Main goal Fatty acids formation Energy generation

Site and tissue Cytosol/Liver, Adipose tissue, Lactating mammary Mitochondria/Liver, Adipose tissue
glands Muscle
Precursor and transporter Acetyl-CoA and Citrate shuttle Acyl-CoA and Carnitine shuttle
Rate-determining enzyme Acetyl-CoA carboxylase Carnitine palmitoyltransferase I
Required substances ATP, Biotin (cofactor), CO2, NADPH, CoA-SH NAD+, FAD, H2O
Upregulation Insulin, ↑ glucose, ATP, and citrate Glucagon (indirectly)
Downregulation Glucagon, ↓ glucose, epinephrine, AMP, Malonyl-CoA, Insulin (indirectly)

and catecholamines, Acyl-CoA and palmitoyl-CoA
End products Palmitic acid, NADP Acetyl-CoA, Ketone bodies
Prepared
Dr. by Dr. Inna
Alisa Chebotarova, MD,Shypilova
PhD NADH+, FADH2
Ketone metabolism
Types of ketone bodies:
1. Acetoacetate → first ketone body produced

3. Acetone → volatile – formed spontaneously from acetoacetate – “fruity breath” (exhaled)
2. B-hydroxybutyrate → levels will increase as the level of NADH increase. Conversion of
acetoacetate to beta-hydroxybutyrate is used to regenerate NAD via dehydrogenase.
• Synthesis occurs in the mitochondria in the liver, when OAA is consumed for gluconeogenesis
and acetyl-CoA can not enter TCA, fast state.
• Most commonly formed in uncontrolled diabetes, starvation, defect in pyruvate carboxylase
• Ketone bodies are released to be utilized by peripheral tissues
• Thiophorase – liver does not have this enzyme and therefore cannot utilize ketone bodies;
• First two weeks – consumed mostly by skeletal muscle; second two weeks – brain and other
tissues will begin to utilize it
• Benefit – providing acetyl CoA, generating NADH in the peripheral tissues (decreasing
congestion in the liver)
Ketone metabolism
•Major roles of insulin in fat metabolism

–Activates fatty acid synthesis in hepatocytes
–Activates TAGs synthesis in Adipose Tissue
–Inhibits lipolysis in Adipose Tissue
•Diabetes Mellitus – Absolute/Relative Insulin Deficiency

–Inhibition of fatty acid synthesis in hepatocytes
–Inhibition of TAGs synthesis in Adipose Tissue
–Inactivation Lipoprotein Lipase:
•Plasma circulating triglycerides are not
hydrolyzed à hypertriglyceridemia
–Activation lipolysis in Adipose Tissue
•Fatty acids released
•Increased fatty acid oxidation –Ketogenesis -
Ketoacidosis
Phospho- and Sphingolipids

Phospho- and Sphingolipids

AOS for H2O2: Catalase or Glutathione
peroxidase
• Hydrogen peroxide is scavenged by catalase or by glutathione peroxidase [fast
reactions].
• Catalase (CAT) is found mainly in peroxisomes.
• It contains heme and can use two hydrogen peroxides as substrates at the same time
(most efficient, fast).
• The catalase enzyme causes the hydrogen peroxide to break down. This reaction forms
water and oxygen. The bubbles are oxygen gas.

GSH is the
main
cytosolic ROS
scavenger

• Phagocytosis by white blood cells use with their oxygen- dependent system important
enzymes for body defense:
• *NADPH oxidase which uses NADPH and molecular oxygen and forms superoxide
• *Myeloperoxidase which uses hydrogen peroxide and chloride ions and forms
hypochloric acid
• *Inducible Nitric oxide synthase which uses NADPH and Arginine and forms nitric
oxide and citrulline
Eicosanoids
Prostacyclin: Produced by the endothelium, Promotes vasodilation; inhibit platelet aggregation
Thromboxane: Produced by platelets, Promote vasoconstriction; promote platelet aggregation
Leukotrienes: LT-B4→ Chemotaxis and adhesion of white blood cells;
LT-C4, LT-D4, LT-E4 → Slow reacting substances of anaphylaxis (SRSA), Take over for histamine
(immediate responder)
• Pharmaceutical actions:
• Phospholipase A2: Inhibited by steroids – used as an anti-inflammatory
• Cox 1 and Cox 2: Inhibited by Nonselective COX inhibitors
• Reversible → ibuprofen; Irreversible → aspirin (contraindicated in patients with bronchial
asthma)
• Selective Cox-2: celecoxib, rofecoxib
• Thromboxane Synthesis is inhibited by low dose aspirin→ cardioprotective effect of aspirin
Dr. Alisa Chebotarova, MD, PhD Prepared by Dr. Inna Shypilova
Eicosanoids
Derivative Tissues Actions
TXA2
Platelets Platelet aggregation, vasoconstriction, clotting of blood
Thromboxane A2
PGI2
Vascular endothelium Inhibits platelet aggregation, vasodilation
Prostacyclin
PGF2α
Vasoconstriction, smooth muscle contraction, uterine contraction,
Prostaglandin 2α Most tissues
medical termination of pregnancy
PGE2 Vasodilation, smooth muscle relaxation, induces labor,

Most tissues esp. kidneys
Prostaglandin E2 bronchodilator
Rate Limiting Enzymes
Pathway RLE Positive effectors Negative effectors
Glycolysis Phosphofructokinase-1 (PFK-1) Fructose-2,6-biphosphate, AMP Citrate; ATP
HMP pathway Glucose-6-phosphate NADP+ NADPH
dehydrogenase
Gluconeogenesis Fructose-1,6-biphosphatase Citrate Fructose-2,6-biphosphate; AMP
Citric acid cycle Isocitrate dehydrogenase ADP; Ca2+ NADH + H+ ; ATP
Glycogenesis Glycogen synthase Dephosphorylation via insulin Phosphorylation by glucagon and
signalling; Glucose-6-P epinephrine signaling
Glycogenolysis Glycogen phosphorylase Phosphorylation by glucagon Dephosphorylation
and epinephrine signalling; upon insulin signalling; ATP;
AMP Glucose-6-phosphate
Fatty acid synthesis Acetyl-CoA carboxylase Citrate; Dephosphorylation by Acyl-CoA, palmitoyl-CoA
insulin signalling Phosphorylation by glucagon,
epinephrine and
norepinephrine, by AMPK
β-oxidation Carnitine acyltransferase I Induction of expression Malonyl-CoA
Dr. Alisa Chebotarova, MD, PhD by LCFA and thyroid hormones Prepared by Dr. Inna Shypilova
Transcription
• Only one RNA polymerase synthesizes all of the RNA (DNA-dependent RNA polymerase)
• RNA polymerase moves along the template strand in the 3ʹ to 5ʹ direction
• Synthesizes RNA in the 5’ → 3’ direction
• Does not require a primer
• Has no exonuclease or endonuclease activity (No ability to repair mistakes)
RNA polymerase RNA polymerase

Transcription
• The coding (antitemplate) strand:
• 5ʹ→3ʹ
• not used during transcription
• identical in sequence to the RNA molecule, has T
• Template DNA:
• 3ʹ→5ʹ
• mRNA product:
• synthesized in the 5ʹ to 3ʹ direction
• complementary and antiparallel to the template strand and identical to coding (T ->U)

Prokaryotic Eukaryotic
Gene regions polycistronic Always monocistronic, Genes have exons and

introns, Large spacer (noncoding) DNA between
genes
RNA polymerase Core enzyme: α2ββ (inhibited by rifampin( RNA pol I: rRNA (nucleolus)
Sigma factor RNA pol II: mRNA; snRNA (inhibited by amanitin)
RNA pol III: tRNA, 5S RNA
Initiation of transcription Promoter (–10) TATAAT and (–35) sequence Promoter (–25) TATA and (–70) CAAT
Sigma initiation subunit required to recognize Transcription factors (TFIID) bind promoter
promoter
mRNA synthesis (elongation) Template read 3ʹ to 5ʹ; mRNA synthesized 5ʹ to 3ʹ; Template read 3ʹ to 5ʹ; mRNA synthesized 5ʹ to 3ʹ;
gene sequence specified from coding strand 5ʹ to gene sequence specified from coding strand 5ʹ to
3ʹ; transcription begins at +1 base 3ʹ; transcription begins at +1 base
Termination of transcription Stem and loop + UUUUU. Stem and loop + rho Not well characterized
factor
Relationship of RNA transcript RNA is antiparallel and complementary to DNA RNA is antiparallel and complementary to DNA
to DNA template strand; RNA is identical (except U template strand; RNA is identical (except U
substitutes for T) to DNA coding strand substitutes for T) to DNA coding strand
Posttranscriptional processing none In nucleus: 5ʹ cap (7-MeG) 3ʹ tail (poly-A sequence)
of hnRNA (pre-mRNA Splicing (GU at the 5’ exon-intron boundary –
Splice Donor; AG at the 3’ exon-intron boundary –
Splice Acceptor). Alternative splicing yields variants
of protein product
Protein synthesis
Prokaryotic Eukaryotic
Preparation (Charging) aminoacyl-tRNA synthetases bind amino acid to tRNA (to adenine in the 3’ OH end CCA), requires 2 ATP,
proofreading or editing activity
Initiation mRNA Shine - Dalgarno + 16S rRNA small subunit = mRNA 5’cap + small subunit = locates the start
locates the start codon AUG – encodes fMET -> large codon AUG – encodes MET -> large ribosomal
ribosomal subunit joins the complex -> A,P,E sites subunit joins the complex -> A,P,E sites
Tetracycline, streptomycin inhibit 30S subunit
Elongation A charged tRNA binds in the A site -> Peptidyl The same + Diphtheria toxin ADP- ribosylation
transferase (large subunit) makes peptide bondà of eEF-2
Translocation. Requires 2 GTP
Cloramphenicol, erythromycin, inhibit by binding to 50S
subunit
Termination A site reaches stop codons: UAG, UAA, UGA

Replication Inhibitors
Prokaryotic (Antibiotic) Eukaryotic (Chemotherapeutic)
Nalidixic acid Gyrase Doxorubicin Type II Topoisomerase
Ciprofloxacin Gyrase Topotecan Type I Topoisomerase
Irenotecan Type I Topoisomerase
Transcription Inhibitors
Rifampicin Beta subunit of RNA polymerase alpha- Amanitin RNA polymerase II

(toxin)
Actinomycin D Intercalates DNA interferes with RNA

polymerase
Translation Inhibitors
Streptomycin Initiation - 30S Ribosome subunit Diphtheria toxin post-translational modification of eEF2
(Elongation Factor 2) by ADP- ribosylation
Tetracycline Inhibits binding of aminoacyl-tRNA,

30S Ribosome subunit
Erythromycin Translocation - 50S Ribosome subunit
Cloramphenicol 50S Ribosome subunit

Regulation of Gene Expression
Prokaryotic Operons
• Lac Operon – turned on with high lactose, low glucose
— Repressor: encoded by LacI, consitutive, binds Operator blocks transcription
— Inducer: Allolactose, sythensized from Lactose by β-galactosidase, ligand for repressor, induces
repressor dissociation from the DNA (operator)
— CAP: Transcriptional activator, binds CAP sequence when bound by cAMP ligand
— cAMP: synthesized by adenylyl cyclase, inhibited by glucose


Regulation of Eukaryotic Gene Expression
1. Chromatin remodeling (Acetylation of histones (lysine residues) = increased transcription,
methylation of DNA = repressed transcription: Fragile X syndrome, Genetic Imprinting in Prader-
Willi Syndrome, Rett syndrome)
2. Promoters (TATA box, CAAT box, GC box): mutation = dramatic decrease in gene transcription
(Reporter gene assays)
3. Enhancers and Silencers (DNA cys-elements): upstream, downstream, Consist of response
elements, Cis-acting elements
4. Transcription factors (proteins trans-elements): general (TFI, TF II, TFIII) – bind promoter;
special (Zinc fingers) – bind enhancer or silence
5. Gene Amplification - increasing the number of genes available for transcription
6. Gene Rearrangement (Ig) - extensive DNA sequence-coding changes
7. RNA splicing
8. RNA Editing (ApoB) - C-to-U editing
9. RNA interference (miRNA)
10. Translation of mRNA (in cytoplasm-P bodies)
Molecular Biology Techniques
1. Recombinant DNA and DNA Cloning:
• Obtaining large quantities of specific DNA fragments
• Target DNA is obtained from genomic DNA by digestion with restriction enzymes
• Restriction endonucleases produce DNA fragments with complementary (“sticky”) ends
• bacterial enzymes
• Natural defense mechanism in bacteria to protect against invading bacteriophages
• Methylase - transfers methyl groups to bases within the specific sequences of its own bacterial genome to protect it from digestion
by the restriction endonucleases
• highly specific for short nucleotide sequences (palindromes - both strands of DNA have the same sequence when read in a 5’ -
> 3’ direction)
• cleave both DNA strands within this region
• DNA fragments from different sources produced by cleavage with the same restriction enzyme and
containing complementary sticky ends anneal with each other
• Target DNA to be cloned is inserted into a cloning vector, forming recombinant DNA
• Cloning vectors are able to recombine with restriction fragments and replicate within a host cell to amplify the
target DNA.
• Vectors may be plasmids that grow within living host cells, phage vectors that destroy the host bacterium after replicating, or larger
artificial chromosomes from bacteria and yeast
• must possess Autonomous replication in a host cell, have antibiotic-resistance gene, restriction site
• A bacterial host culture containing an assortment of recombinant plasmids can be used as a library for
Dr. finding different
Alisa Chebotarova, target DNAs
MD, PhD
2. DNA libraries
• collections of restriction fragments cloned within suitable host cells
• A genomic DNA library is obtained from restriction enzyme digestion of the entire
genome of an organism and theoretically contains all of the nuclear DNA sequences.
3. Detection of Specific Nucleic Acid Sequences with Probes
• A specific DNA fragment in a DNA library that is immobilized by blotting on a
nitrocellulose filter can be identified by incubation with probes (synthetic single-strand
oligonucleotides, labeled with a radioactive isotope)
• In a mixture of denatured single-strand DNA, RNA fragments, or proteins, a probe can
hybridize only with fragments containing a complementary sequence:
• Samples are separated by electrophoresis -> gel is placed on nitrocellulose paper -> nucleic acids or
proteins transfer to the paper (blotting) -> bind -> nitrocellulose blot is treated with a radioactive
probe that binds to a specific target molecule of interest -> autoradiography
• Southern blotting - detects DNA with labeled DNA probes
• Northern blotting - detects RNA with labeled DNA probes (specific for a tissue)
• Western blotting - detects proteins by using labeled antibodies
4. Microarrays
• the screening for the expression of many genes at one time by arranging
fragments of known genes in known positions on a small grid and hybridizing a
fluorescently labeled RNA probe obtained from the cell type under study
5. Polymerase Chain Reaction
• in vitro enzymatic method for amplifying a target DNA sequence located between
two short flanking sites whose sequences are known
• Based testing for HIV is most useful in the first few months of contraction
• Alternating cycles of replication and denaturation - heating-cooling cycle:
• Single-strand oligonucleotide primers complementary to the two 3’ flanking sites
• A heat-stable DNA polymerase that remains functional throughout the heating phases
• four deoxyribonucleoside triphosphates (dNTPs)
Polymerase Chain Reaction – Steps ✮
1. Add excess amounts of DNA primers complementary and antiparallel to
both 3' flanks of the target sequence. This selects the region to be
amplified.
2. Add a heat-stable DNA polymerase (Taq DNA polymerase) and deoxy
ribonucleotides (dNTPs) for DNA synthesis.
3. Heat the sample to 95° C to melt the DNA (convert dsDNA to ssDNA)-
Takes 20-60 seconds
4. Cool the sample to 50°-55° C to anneal primers to the complementary
DNA at the 3' flanking sequence. -Takes 20-90 seconds
5. Heat the sample to 70-72° C to increase the activity of the Taq DNA
polymerase-Takes 10-60 seconds (to increase rate of reaction)
6. Primer elongation occurs, and new complementary strands are
synthesized.
6. Restriction Fragment Length Polymorphisms
• After target DNA has been obtained by PCR or cloning, it can be analyzed by
developing a physical map of its sequence
• Target DNA can be analyzed by developing a physical map of restriction sites
contained in its sequence
• When mutations occur in a restriction site, the lengths of fragments obtained in a
restriction digest vary from normal sizes because the corresponding restriction
enzyme can no longer cut the DNA
• A different length of a known restriction fragment found in a normal population -
a restriction fragment length polymorphism (RFLP).

7. DNA sequencing
• use of dideoxynucleotides to interrupt DNA synthesis using the unknown DNA as
the template
The sequence of the newly synthesized DNA strand in this example is:
5’-CTTGGAACTGTA-3'

A. Southern Blotting ✮
• DNA Blot
• Useful in identifying a specific restriction
fragment out of the millions present in a
restriction digest of an individual’s genomic
DNA
• DNA (or RNA) probe
• All tissues are the same in the same individual
• Detect:
• Size of DNA fragments
• Amount of each DNA fragment (intensity of the
band)
• The Southern blotting is a procedure for

analyzing specific DNA sequences in a
complex mixture of different sequences,
such as genomic DNA and mutant
homozygotes and heterozygotes.
B. Northern Blotting ✮
• RNA Target
• DNA or RNA probe
• Differences between tissues!
• Transcript abundance due to different transcription
factors
• Alternative splicing
• RNA editing
• Detect:
• Size of RNA transcripts
• Amount of each RNA transcript (intensity of the band)

C. Western Blotting ✮
• aka. Immunoblot. Western blotting is used to detect a target polypeptide or
protein from within a mixed sample.
• Protein Target
• Labeled antibody probe is used to bind to relevant protein
• Different in different tissues
• Transcriptional regulation
• Translational regulation
proteins separated
• Detect: by gel
electrophoresis
• Size of proteins
• Amount of protein (intensity of the band)
D. Southwestern Blotting ✮
• Detects DNA-binding proteins with labeled dsDNA probes.

• Useful in detecting expression of transcription factors that interact with
regulatory sequences controlling specific genes

1. A pharmaceutical firm is interested in the bacterial production of thymidylate
synthase in large quantities for drug-targeting studies. An important step in the
overall cloning strategy involves the ligation of synthase cDNA into a plasmid vector
containing a replication origin, an antibiotic resistance gene, and a promoter
sequence. Which additional nucleotide sequence should be included in this vector
to ensure optimal production of the thymidylate synthase?
A. Operator sequence
B. PolyA sequence
C. Shine-Dalgarno sequence
D. Attenuator sequence
E. 3ʹ-splice acceptor sequence
2. Restriction fragment length polymorphisms may be produced by mutations in
the sites for restriction endonucleases. For instance, a single base change in the
site for the nuclear SalI produces the sequence GTGGAC, which can no longer be
recognized by the enzyme. What was the original sequence recognized by SalI?
A. GTAGAC
B. GCGGAC
C. CTGGAC
D. GTCGAC
E. GTGTAC

DNA primers anneal to specific complementary
sequences on the strands that will be amplified
• PCR primers are synthetic DNA oligonucleotides of approximately 15–30 bases. PCR primers
are designed to bind (via sequence complementarity) to sequences that flank the region of
interest in the template DNA. During PCR, DNA polymerase extends the primers from their
3ʹ ends. To amplify the sequence of DNA shown below by polymerase chain reaction, which
olygonucleotides would be the most appropriate DNA primer to use?
a. 5’-ATAGTACCG; 5’-GACTAGCAC
b. 5’-ATAGTACCG; 5’-GTGCTAGTC
c. 5’-CGGTACTAT; 5’-CACGATCAG
d. 5ʹ-CGGTACTAT; 5’-CTGATCGTG
e. 5’-CGGTACTAT; 5’-GTGCTAGTA
Block 3 Review
Chylomicrons Metabolism
Apo C-II on chylomicron activates lipoprotein
lipase and hydrolysis of core TAG begins.
• Lipoprotein Lipase (LPL) - An extracellular enzyme –

anchored by heparin sulfate (a GAG) on vascular
endothelial surface.
• On most tissues but at higher levels in skeletal and
cardiac muscle and adipose.
• Can remove fatty acids from glycerol yielding a
monoacylglycerol; can cleave a terminal FA from a
phosphoglyceride
• LPL synthesis induced by insulin

Type I hyperlipoproteinemia - Lipoprotein
lipase deficiency
Hyperchylomicronemia or familial fat-induced hyperlipoproteinemia
• presence of chylomicrons in fasting blood (milky appearance of fasting
plasma)
• ↑TAG: 1000 mg/dl (borderline 150-199 mg/dl)
• normal to high total cholesterol
• Familial form rare autosomal recessive - no link with coronary artery disease

Type I hyperlipoproteinemia - Lipoprotein
lipase deficiency
Clinically:
• present in childhood with recurrent episodes of severe
abdominal pain due to acute pancreatitis
• Opalescent retinal blood vessels (lipemia retinalis) on
fundoscopic examination
• Eruptive xanthoma (small yellowish white papules, lipids
accumulate in large foam cells within the skin) on buttocks,
knees & elbows
• Hepatosplenomegaly due to chylomicrons in liver & spleen
reticuloendothelial cells
• Fatty liver/hepatic steatosis: due to an imbalance between
hepatic TAG synthesis & secretion of VLDL
Type IIA hyperlipoproteinemia – LDL
receptor defect
Familial hypercholesterolemia or hyperbetalipoproteinemia (defective LDL
receptor, familial defective ApoB-100)
Autosomal dominance inheritance
• raised levels of LDL
• total cholesterol in homozygous: 600 – 1200 mg/dl, heterozygous: 270-550
mg/dl (High >240)
• TAG normal

Type IIA hyperlipoproteinemia – Apo B100 or
LDL receptor defect ✮
Clinically:
• Tuberous xanthoma on elbows, hands, knees, wrists, heels or buttocks
Corneal arcus
• Planar xanthomas on back of legs
• Xanthelasmas (lipid deposition in eyelids)
• Corneal arcus (white ring around cornea)
• Tendinous xanthomas on hands and Achilles tendon
Accelerating premature atherosclerosis in homozygous familial hypercholesterolemia

can result in death in childhood, rarely survive beyond 2nd decade
• Atherosclerosis often develops in aortic root
HDL Metabolism
When cholesterol is taken up by HDL, it is immediately esterified by the plasma

enzyme Lecithin:Cholesterol AcylTransferase (LCAT) ✮
• The enzyme is synthesized by the liver
• binds to nascent HDL, and is activated by apo A-I
• Esterification maintains the cholesterol concentration gradient, allowing continued efflux
of cholesterol to HDL
* Lecithin is the phosphatidylcholine

Reverse Transport of Cholesterol by HDL
• Reverse cholesterol transport involves efflux of cholesterol from peripheral cells
to HDL, esterification of cholesterol by LCAT , binding of the cholesteryl ester–
rich HDL (HDL2) to liver and steroidogenic cells, the selective transfer of the
cholesteryl esters into these cells, and the release of lipid-depleted HDL (HDL3)

PC - phosphatidylcholine; lyso-PC = lysophosphatidylcholine. LCAT = Lecithin cholesterol transferase.
CETP = cholesteryl ester transfer protein. ABCA1 = transport protein.
For convenience the size of VLDLs are shown smaller than HDL, whereas VLDLs are larger than HDL.
Cholesteryl Ester Transfer Protein (CETP)
• During lipolysis, there is some exchange of TAG and

CE between VLDL and HDL mediated by Cholesteryl
Ester Transfer Protein (CETP) ✮
• Triacylglycerols from the VLDL are transferred to HDL
• CE are transported from mature HDL to VLDL
• As the result, VLDLs are processed to IDL, and next
to LDL, which are removed from the circulation by
the LDL receptor pathway (hepatic and tissue
uptake of C&CE).

Transport of Ammonia
Glutamine:
• synthesized from glutamate by the addition of ammonia by Glutamine synthetase in most
tissues
• In the brain and other tissues (if concentration of NH3 is high):
Glutamate + ammonia + ATP → Glutamine + ADP + Pi
• In the liver, glutamine synthetase is located in cells surrounding the portal vein.
• Its major role is to convert any ammonia that has escaped from urea production into glutamine => free
ammonia does not leave the liver and enters the circulation

Carbamoyl Phosphate Synthetase I (CPS I )
• Carbamoyl phosphate synthetase I

• involved in urea synthesis
• uses free ammonia as the source of nitrogen
• occurs in liver mitochondria
• activated by N-acetylglutamate

Carbamoyl Phosphate Synthetase deficiency
• Autosomal recessive – both genders are affected
• Carbamoyl phosphate is NOT accumulated
• Symptoms:
• severe Hyperammonemia
• ↑ blood glutamine – major transport form of ammonia in blood
• within 24h - 72h after birth infant becomes lethargic, needs stimulation to
feed; vomiting, increasing lethargy, ↓BUN, hypothermia and
hyperventilation; without measurement of serum ammonia levels and
appropriate intervention infant will die.
• No orotate in blood!!!
N-acetylglutamate synthetase Deficiency
• Deficient enzyme: N-acetylglutamate synthetase
• Symptoms:
• severe hyperammonemia, recurrent diarrhea, ataxia, hyperornithinemia.

Ornithine transcarbamylase deficiency ✮
• Most common urea cycle disorder
• X-linked recessive - affects males predominantly ✮
• Often evident in the first few days of life, but may present later
Clinically:
• symptoms of hyperammonemia (Cerebral edema, Lethargy, convulsions, coma,
death)
• ↑NH4
• ↓BUN
• ↑ blood glutamine – major transport form of ammonia in blood
• Orotic aciduria ✮ - ↑ blood orotic acid level
• due to mitochondrial carbamoylphosphate is accumulated, entering cytosol and being
incorporated into pyrimidine nucleotides which leads to excess production of orotic acid
Synthesis of THF (tetrahydrofolate)
Sulfonamides compete with PABA
• Active form: tetrahydrofolate à Produced by the action of dihydrofolate reductase

Tetrahydrofolate Interconversions and the 1C Pathway
MTHFR

Folate trap hypothesis
In vitamin B12 deficiency, the utilization of the N5-methyl form of

tetrahydrofolate in the B12-dependent methylation of homocysteine to
methionine is impaired. Because the methylated form cannot be
converted directly to other forms of tetrahydrofolate, folate is trapped in
the N5-methyl form, which accumulates.
The levels of the other forms decrease. Thus, cobalamin deficiency
is hypothesized to lead to a deficiency of the tetrahydrofolate forms
needed in purine and TMP synthesis (DNA synthesis), resulting in the
symptoms of megaloblastic anemia.

Comparison of Folate and B12 deficiency
Folate Vitamin B12
• Megaloblastic, hyperchromic • Megaloblastic. hyperchromic
anemia (blood color index > 1.1) anemia (blood color index > 1.1)
• High level of homocysteine in blood • High level of homocysteine in blood
(hyperhomocysteinemia/ (hyperhomocysteinemia/ homocystinuria),
homocystinuria), increased risk for increased risk for cardiovascular disease
cardiovascular disease • Progressive peripheral
• FIGlu accumulation neuropathy
• Methylmalonyl aciduria
• Deficiency is important in • Deficiency associated with:
pregnancy, can occur in pernicious anemia, gastric
alcoholics and malnourished resection, chronic pancreatitis,
individuals. vegan, malnutrition
• Develops in 3-4 months • Develops in years
Propionyl-CoA path

Degradation of Branched Chain Amino Acids
• Isoleucine, Leucine and Valine have a similar route of catabolism
• In contrast to other amino acids, they are metabolized primarily by the peripheral tissues
(particularly muscle), rather than by the liver
1) Branched-chain a-amino acid aminotransferase removes the amino groups to

form branched chain a-keto acids
2) Carboxyl groups removed by a single enzyme complex – branched chain a- keto
acid dehydrogenase complex ✮
• uses thiamine pyrophosphate (TPP), lipoic acid, FAD, NAD+ and coenzyme A
✮ as coenzymes

Maple Syrup Urine Disease (MSUD) ✮
• Recessive disorder – partial or complete loss of branched chain a- ketoacid
dehydrogenase ✮
• Enzyme decarboxylates branched chain a- ketoacids from Leu, Ile and Val
• These amino acids and corresponding a- ketoacids accumulate in the body and have a toxic
effect on the brain ✮
Clinical presentations:
• Feeding problems, vomiting, dehydration, severe metabolic acidosis because of
elevated levels of branched chain AA and their a-ketoacids in blood
• Urine contains large amounts of branched chain amino acids and keto analogs –
smells sweet like maple syrup ✮
• Neurologic problems are common
Biosynthetic reactions involving amino acids
and tetrahydrobiopterin

Dopamine, Norepinephrine, and Epinephrine
SYNTHESIS OF THE CATECHOLAMINE NEUROTRANSMITTERS:
• synthesized from Tyrosine:
• Tyrosine is hydroxylated by Tyrosine hydroxylase ✮(BH4-requiring enzyme) to form DOPA - the
rate-limiting step of the pathway
• DOPA is decarboxylated by DOPA decarboxylase (pyridoxal phosphate-requiring enzyme) to
form dopamine
• dopamine is hydroxylated by dopamine β–hydroxylase to norepinephrine in a reaction that
requires Vitamin C and copper
• Norepinephrine is converted to Epinephrine by an N-methylation reaction using S-
adenosylmethionine (SAM) as the methyl donor
PARKINSON DISEASE, a neurodegenerative movement

disorder, is due to insufficient dopamine production as a result
of the idiopathic loss of dopamine-producing cells in the brain.
Administration of L -DOPA (levodopa) is the most common
treatment
INACTIVATION AND DEGRADATION OF CATECHOLAMINE NEUROTRANSMITTERS:
• The action of catecholamines is terminated through reuptake into the
presynaptic terminal and diffusion away from the synapse
• Degradative enzymes are present in the presynaptic terminal and in adjacent cells
• MAO (monoamine oxidase) ✮ inactivates catecholamines that are not protected in
storage vesicles (norepinephrine, dopamine, or serotonin)
• COMT (catechol-O-methyltransferase)
• The end products of these reactions are excreted in the urine:
• vanillylmandelic acid (VMA) from epinephrine and norepinephrine - is increased with
pheochromocytomas, tumors of the adrenal characterized by excessive production of
catecholamines
• homovanillic acid from dopamine

In addition to the catecholamines, serotonin is also inactivated by

monoamine oxidase
may irreversibly or reversibly inactivate the

MAO INHIBITORS:
enzyme, permitting neurotransmitter molecules to
escape degradation and, therefore, accumulate within the
presynaptic neuron and to leak into the synaptic space -
reduce the severity of depression (by maintaining higher
levels of serotonin)

Inborn errors of phenylalanine metabolism
• Hyperphenylalaninemia – an error of phenylalanine metabolism
• Most forms of hyperphenylalaninemia are caused by mutations in the PAH gene

(results in Phenylketonuria - PKU), or due to defects in the BH4 (resulting in non-
PKU hyperphenylalaninemia)
• Phenylalanine Hydroxylase Deficiency (Classic Phenylketonuria) ✮
• most common clinically encountered defect
• In the United States, PKU occurs in 1 in 10,000 to 15,000 newborns (The incidence of PKU in
Caucasian populations)

Phenylalanine Hydroxylase Deficiency (Classic
Phenylketonuria)
Clinical presentations: ✮
• Hypopigmentation – fair hair, light skin color, and blue
eyes – due to:
• defect in tyrosine (and as the result melanin) synthesis from
phenylalanine. The hydroxylation of tyrosine is the first stage
in the pigment melanin formation
• Tyrosinase (the enzyme of the first step in the formation of the
pigment melanin) is competitively inhibited by the high levels of
phenylalanine
• CNS symptoms:
• Mental retardation by the age of 1 year, failure to walk or talk,
seizures, hyperactivity, tremor, microcephaly, and failure to
grow
• Eczema
• Characteristic musty (“mousey”) odor due to
accumulation of phenylacetate, and phenylpyruvate
PKU because of BH2 reductase deficiency ✮
• Dihydropteridine reductase (DHPR) deficiency is an autosomal recessive disorder
in the regeneration pathway of tetrahydrobiopterin (BH4) ✮
• Hyperphenylalaninemia in newborn screening
• Simple dietary restriction doesn’t reverse the CNS effects due to neurotransmitter
deficiency (serotonin, DOPA deficiency)
• microcephaly,
• hypotonia,
• mental retardation,
• convulsions

BH2 reductase deficiency
Treatment:
• low phenylalanine diet
• L-dopa
• 5-hydroxytryptophan supplementation
• BH4 supplementation

Albinism ✮
• A group of conditions in which a defect in tyrosine metabolism leads to a
deficiency of melanin
• deficiency of Tyrosinase ✮
• copper-requiring tyrosinase (Tyrosine hydroxylase)
• Partial or full absence of pigment in skin, hair and eyes
(red pupils)
• May have vision defects and photophobia
• Get easily burned by sun – no tanning

Tyrosine yields Fumarate in hepatocytes
• Tyrosine is converted to Fumarate and

acetoacetate
• Tyrosine → Homogentisic Acid✮ →(enzyme -
Homogentisate oxidase ✮) → Maleylacetoacetate →
Fumarylacetoacetate → Fumarate
These AA enter TCA cycle or used

for glucose synthesis, or ketone
synthesis
Alkaptonuria ✮
• Rare deficiency of homogentisic acid oxidase ✮ – not

considered life-threatening

Alkaptonuria ✮
• Homogentisic aciduria – patients urine turns black
after exposure to air
• Ear wax exposed to air turns red or black, dark spots on
the sclera and cornea
• Ochronosis - pigmentation of cartilage (Alkapton -
black ochronotic pigment)
• Ochronosis of cartilage leading to osteoarthritis –
precocious lower back pain – early joint replacements
(hip and shoulder). Large joint arthritis
• Sufferers may be symptomless until aged 40+ -
arthritis, but disease may be recognized earlier by
black-soiled diapers
Treatment – dietary restriction of Tyr and Phe
β
Нomocystinuria
PLP
Сystathioninuria
Homocystinuria ✮
•Autosomal recessive defects in metabolism of
homocysteine
•High plasma and urine levels of homocysteine and
methionine and low levels of cysteine
• The most common defect is deficiency of

cystathionine b-synthase ✮
• vitamin B6 (Pyridoxine) is a coenzyme
• Supplements of Pyridoxine is a treatment

Homocystinuria ✮
• homocysteinemia
• homocysteine (and homocystine) in urine,
• intellectual disability,
• osteoporosis develops during childhood,
• tall stature,
• kyphosis (reminiscent of Marfan syndrome patients habitus),
• lens subluxation (downward and inward),
• deep vein thrombosis, and early atherosclerosis
(stroke and MI before 20 y.o.). Homocysteine ↑ lipid oxidation and increased platelets
aggregation leading to fibrosis and calcification of
Dr. Alisa Chebotarova, MD, PhD atherosclerotic plaques
Cystinuria
• One transporter is responsible for absorption by
intestine (active transport) of Cysteine, Ornithine,
Lysine, and Arginine (COLA) and is responsible for
the reabsorption of these amino acids by the kidney.
• In the inherited disease cystinuria, this transporter is

defective and all 4 amino acids occur in urine,
intestinal absorption is also impaired
• Clinically can cause kidney stones and block the

urinary tract.
• Treatment: Restriction of dietary methionine (as a

Dr. precursor
Alisa Chebotarova,of
MD,Cysteine)
PhD
De Novo Synthesis of Purine Nucleotides
• Before purine synthesis can take place, 5-phosphoribosyl-1-pyrophosphate (PRPP) ✮.
• Occurs in many tissues (most actively in the cytosol of liver cells)
• The reaction is catalyzed by PRPP synthetase ✮ Requires 1 ATP as donor of phosphates
• Overactivity of PRPP synthetase → overproduction of purines → over degradation of

purines → gout symptoms
Lesch-Nyhan Syndrome
• X-linked recessive disease due to mutation in HGPRT
• HPRT is absent or deficient
Pathogenesis:
↓ HGPRT → inhibition of salvage pathway → 100 %
purines are passed into the degradation (excretory)
pathway → excess of PRPP → activation of PRPP
amidotransferase → overproduction of purines
throughout the body → severe hyperuricemia
• de novo synthesis pathway for purines must
compensate, which utilizes folate → macrocytic anemia

Lesch-Nyhan Syndrome ✮
Presentation:
• males
• The appearance of orange crystals of sodium urate in the
diapers of an affected infant (the earliest sign).
• Hyperuricemia - gout
• Intellectual disability
• Self-mutilation (hands and lips)
• Spastic cerebral palsy
• Kidney failure and death often in the first decade
• Hyper segmented neutrophils (typically seen in
megaloblastic anemia)
• Hypotonia

Inhibition of Xanthine oxidase can prevent the
occurrence of gout symptoms

CPS I vs. CPS II
• Carbamoyl phosphate synthetase I

• involved in urea synthesis
• uses free ammonia as the source of nitrogen
• occurs in liver mitochondria
• activated by N-acetylglutamate
• not affected by UDP or UTP
• Carbamoyl phosphate synthetase II

• involved in pyrimidine synthesis
• uses glutamine as the source of nitrogen
• occurs in the cytosol of all nucleated cells
• is inhibited by UDP and UTP
• not activated by N-acetylglutamate
Orotic Acid accumulation

Orotic aciduria
• Orotic aciduria is an autosomal recessive disorder caused by a defect in UMP synthase. This
enzyme contains two activities, orotate phosphoribosyltransferase and orotidine decarboxylase.
Pathogenesis and presentation:
• ↓ pyrimidines → ↓ nucleic acid synthesis → ↓ hematopoiesis → megaloblastic anemia.
• Growth retardation
• Orotic acid accumulates in the blood and spills into the urine → orotic acid crystals, white
precipitate in urine.
• Leukopenia, and severe anemia caused by hypochromic erythrocytes and megaloblastic bone
marrow (cannot be cured by folate or B12)
• No hyperammoniemia (vs. ornithine transcarbamoylase deficiency)
Treatment:
• uridine and/or cytidine nucleosides (feedback allosteric inhibition of CPS II)
• UMP and CMP production via the action of nucleoside kinases
• these then inhibit CPS II – less orotic acid produced

Two Orotic Acidurias
• Hyperammonemia (no megaloblastic anemia)
– Pathway: urea cycle
– Enzyme deficient: Ornithine transcarbamylase (OTC)
- The rise of uracil in urine can indicate excess pyrimidine biosynthesis due to divergence
of unutilized Carbamoyl-P towards the pathway of pyrimidine biosynthesis
• Megaloblastic anemia (no hyperammonemia)

– Pathway: pyrimidine synthesis
– Enzyme deficient: UMP synthase
*Folate and B12 deficiency (megaloblastic anemia but no orotic aciduria)

Conversion of Ribonucleotides to
Deoxyribonucleotides
Ribonucleotide Reductase
Ribonucleotide reductase is required for the formation of the deoxyribonucleotides for
DNA synthesis.
• All 4 nucleotide substrates must be diphosphates.
• dADP and dATP strongly inhibit ribonucleotide reductase.
• Hydroxyurea, an anticancer drug, blocks DNA synthesis indirectly by inhibiting
ribonucleotide reductase

Drugs – inhibitors of the nucleotides
synthesis (S phase inhibitors)
Purine synthesis:
• 6-mercaptopurine (6-MP) and its prodrug azathioprine: inhibit de novo purine
synthesis
• Mycophenolate and ribavirin: inhibit inosine monophosphate dehydrogenase
Pyrimidine synthesis:
• Leflunomide: inhibits dihydroorotate dehydrogenase
• 5-fluorouracil (5-FU) and its prodrug capecitabine: form 5-F-dUMP, which inhibits
thymidylate synthase (inhibits dTMP production)
Purine and pyrimidine synthesis:

• Hydroxyurea: inhibits ribonucleotide reductase
• Methotrexate (MTX), Trimethoprim (TMP), and Pyrimethamine: inhibit dihydrofolate
reductase (dTMP synthesis) in humans, bacteria, and protozoa, respectively.
Erythrocytes have unique and relatively simple metabolism:
▪ Main source of energy is glucose. Source of ATP is anaerobic glycolysis.
▪ The formation of 2,3-bisphosphoglycerate (2,3-BPG or 2,3-DPG) from 1,3-
bisphosphoglycerate is very important for regulation of affinity of Hb to O2. 2,3-
BPG decrease affinity of Hb to O2.
▪ 5 -10% of glucose are metabolized by means of pentose phosphate pathway.
NADPH is necessary for reduction of glutathione. Deficiency of Glucose-6-
phosphate dehydrogenase (G6PDH) is the cause of a hemolytic anemia.
Glutathione (reduced) performs specialized functions in erythrocytes:

(i) It maintains RBC membrane structure and integrity.
(ii) It protects hemoglobin from getting oxidized by agents such as H2O2 and other R∙.
Oxygen Dissociation Curve
Hemoglobin is less
saturated at a given
partial pressure of
oxygen
Carbon mono oxide (CO) poisoning
• Carbon monoxide (CO):
• a colorless, odorless, nonirritant gas that is generated as a byproduct of incomplete
hydrocarbon combustion.
• can result in carbon monoxide poisoning in poorly ventilated spaces
• has 240 times more affinity for hemoglobin than does oxygen.
• Inhaled CO rapidly diffuses across the alveolar membrane and binds tightly with
heme-bound iron in hemoglobin and other hemeproteins.
• binding to hemoglobin results in formation of carboxyhemoglobin.
• decreases the oxygen content of the blood by occupying oxygen binding sites. ✮
• inhibits the release of oxygen from hemoglobin in tissues by altering hemoglobin
conformation ✮.
• inhibits mitochondrial respiration and ATP production cease leading to rapid cell
death (inhibits cytochrome c oxidase).

Sickle cell anemia
• Because of a single amino acid substitution at position 6 in the two β chains, HbS
has one less negative charge✮
• Valine (Nonpolar, aliphatic amino acid) instead of a Glutamate (Negative amino acid)
• Point missense mutations (nonconservative substitution mutations)
• It causes a hydrophobic interactions among hemoglobin molecules after

deoxygenation

Molecular and cellular events leading to sickle cell crisis.
Rigid erythrocytes occlude blood flow in the capillaries. Microinfarcts produce tissue anoxia, resulting in severe pain.
Peripheral blood smear

with the sickle cells
HbS patient’s hemoglobin would

most likely aggregate upon the
Oxygen unloading
(after deoxygenation)
Sickle cell anemia
Clinically - Homozygotes : ✮
• Anemia, Failure to thrive, Splenomegaly

• Hemolysis
• Repeated crises brought on by physical exertion
(extreme abdominal pain):
• Sickled cells are not flexible enough to pass through
microvasculature - sickle shape of these RBCs can
obstruct blood vessels
• By impeding blood flow through these vascular beds, the
sickled cells cause microinfarcts in tissues and painful
vasoocclusive crises ✮
Sickle cell
The presence of the anemia
HbS hemoglobin
in the
erythrocytes of sickle cell patients leads to
reduced overall oxygen-carrying capacity. This
results in a compensatory increase in the
expression of fetal hemoglobin (HbF) in an
attempt to increase oxygen capacity.
Treatment:
• Hydroxyurea✮- increases levels of HbF.
• Increasing the level of fetal hemoglobin (HbF),
which binds oxygen with a higher affinity then adult
hemoglobin, inhibits the polymerization of sickle
hemoglobin (HbS).

Methemoglobinemia
Methemoglobinemia: blood disorder in which an abnormal amount of
methemoglobin is produced: ↓ O2 saturation, ↓ O2 content → tissue hypoxia
Classical presentation includes cyanosis with chocolate-colored blood,
as a result of the dark-colored methemoglobin and because methemoglobin is unable to
carry oxygen, a state of functional anemia is induced.
NB!!! Methemoglobin formation shifts dissociation O2 curve to the left

Hemoglobin in adults
1. HbA (α2β2)–90-95%.
2. HbA2 (α2δ2)–2.5%.
3. HbF – 0.5%
4. HbA1c (glycated): in normal individuals <5.7%;

in diabetes mellitus ≥6.5%
The secondary test for DM is a glycated hemoglobin test, or A1C test. This test measures the average
amount of glucose in a person’s bloodstream over the past 90 days as a percentage. HbA1c is glycosylated
HbA and is produced slowly whenever the glucose in blood is elevated. It persists until the RBC is destroyed
and the Hb degraded and so is useful as a long-term indicator of glucose level.
Hemoglobin H disease
• The α-thalassemias result when there is reduced expression at one or both of
the α-globin genes. In the α-thalassemias, normal amounts of β-globins are
made.
• The β-globin proteins are capable of forming homotetramers (β4) and these
tetramers are called hemoglobin H, HbH. An excess of HbH in red blood cells
leads to the formation of inclusion bodies commonly seen in patients with α-
thalassemia.
• In addition, the HbH tetramers have a markedly reduced oxygen-carrying
capacity. The α-globin homotetramers are extremely insoluble which leads to
premature red cell destruction in the bone marrow and spleen. Clinically this is
referred to as hemoglobin H disease.

Reaction 1:
Heme synthesis starts in

mitochondria with succinyl CoA
and glycine. Glycine is
decarboxylated , hemin
Low levels of intracellular iron
PLP
δ-Aminolevulinate synthase
needs pyridoxal phosphate (PLP)
as coenzyme.
• PLP is formed from vitamin B6.
[Isoniazid, a drug used for
tuberculosis treatment, depletes PLP,
as well as oral contraceptives]
• Deficiency of vitamin B6: heme
synthesis cannot start efficiently
!

Inhibition of heme synthesis
Lead can lead to anemia
poisoning
l Lead inhibition (poisoning):

• this heavy metal can inhibits ALA
dehydratase and ferrochelatase.
• ALA and protoporphyrin IX
accumulate in urine
COMPARISON OF MICROCYTIC ANEMIAS
, Hypochromic , Hypochromic , Hypochromic

↑ Fe in mitochondria
And oral contraceptives

Heme synthesis in erythroid cells (Nuclear Red Cells)
l heme is mainly needed for Hb

l is connected to protein synthesis of the globin chains
l Accumulation of heme will lead to more globin chain
synthesis for hemoglobin (ALAS2 is not inhibited by
hemin)
l Synthesis of heme in erythroid cells is under control of
erythropoietin and the availability of intracellular iron
l Blood loss stimulates bone marrow to synthesize heme
l Erythroid cells contain the isoform ALAS2 which is

expressed only in fetal liver and adult bone marrow
Porphyria:
• Autosomal dominant: Acute Intermittent

Porphyria (AIP) is mainly seen in adults of 40-50
years old.
• Autosomal recessive: Congenital Erythropoietic
Porphyria (CEP) is seen in early childhood.
• Autosomal dominant: Porphyria Cutanea Tarda

(PCT) is found in adults and can be hereditary and
acquired.

!
!
!
!

Acute intermittent porphyria (AIP) symptoms:
l very severe abdominal pain, abdominal colic
l highly agitated state, tachycardia, respiratory
problems, nausea
l weakness of lower extremities
lALA and porphobilinogen accumulate and at very
high levels can act as neurotransmitters. And they are
neurotoxins!
l δ-Aminolevulanic acid and porphobilinogen are
excreted and measured in urine
Congenital Erythropoietic Porphyria (CEP):
Uroporphyrin I and coproporphyrin I in tissues and
blood and urine (red)
Heme synthesis
Uroporphyrinogen I Uroporphyrin I
HMB
Coproporphyrinogen I Coproporphyrin I
Deficiency of Uroporphyrinogen III synthase

Genetic defect autosomal recessive trait
Porphyria Cutanea Tarda
Erosions and bullous

lesions in sun-exposed
areas of the patients are
produced by minor
trauma because of
increased skin fragility.
These lesions heal with

scarring, pigment
changes, and formation
of small white papules
Heme degradation
• RBCs have a lifespan of
about 120 days
• Senile RBCs are sequestered
by RES (Spleen, Kupffer
cells of liver, macrophages) Hemoglobin
• RBCs contain hemoglobin
that is broken down to heme
and globin (amino acids
enter amino acid pool) Heme Globin
• Heme containing proteins like

myoglobin and cytochromes Bilirubin Amino acids
also release heme on are reused
degradation, that also forms
bilirubin
Heme degradation
UDPglucuronyl
transferase
36
• Unconjugated (indirect) bilirubin is not water soluble
• Conjugated (direct) bilirubin is soluble→ present in blood (normal: 0.0 - 0.3
mg/dL) and can be excreted via urine
• Norm of total bilirubin in blood: 0.1 to 1.2 mg/dL

Formation of bilirubin in macrophages
1
3
Heme oxygenase
• Heme oxygenase
– oxidizes Fe2+(ferrous) to Fe3+(ferric)
– Porphyrin ring is cleaved A B
– CO is formed in the reaction

– Biliverdin is a green pigment
Biliverdin reductase
• Biliverdin reductase
– Bilirubin formed has a orange-yellow
color

Conjugation of bilirubin inliver
Defect in Gilbert and
Crigler Najjar syndrome
3
UDPglucuronyl UDPglucuronyl
transferase transferase
UDP
UDP-glucuronic acid
Bilirubin
Liver diglucuronide
• Bilirubin is converted to conjugated bilirubin (more water soluble) by
the addition of two molecules of glucuronic acid
• Enzyme: Microsomal UDP-glucuronyl transferase
(phenobarbital activates this enzyme) !!!
• The donor of glucuronic acid is UDP-glucuronicacid
Prehepatic (Hemolytic) jaundice
• Massive hemolysis may produce
bilirubin faster than it can be
conjugated – unconjugated and
total bilirubin levels increase in
blood causing jaundice
-Toxins
- ABO/Rh incompatibility
- Enzymatic defects – Pyruvate
kinase deficiency, G6PD
deficiency
- Sickle cell anemia or malaria

Laboratory findings in Hemolytic jaundice
Serum Serum Serum Urine Urine

total conjugated unconjugated bilirubin urobilinogen
bilirubin bilirubin bilirubin
Prehepatic N Absent
jaundice ↑↑ ↑↑ (acholuric ↑↑
jaundice)
Аcholuric jaundice. Jaundice without bile pigments in the urine. There

are excessive amounts of unconjugated bilirubin in the blood.
Why is bilirubin not found in urine in prehepatic jaundice?:

unconjugated bilirubin is not filtered in glomeruli.
Changes in bilirubin metabolism in hepaticjaundice
increase in both direct and indirect bilirubin in blood
Decreased
Decreased excretion of
excretion of stercobilin in
urobilin in urine feces
Loss of
Formation of bilirubin bilirubin in
urine
Normal
Increased unconjugated Decreased formation of

bilirubin in circulation urobilinogen in intestine
Increased conjugated
(albumin-bound)
bilirubin in circulation
due to regurgitation
Decreased uptake and Decreased excretion of conjugated

conjugation of bilirubin by bilirubin into bile and intestine
liver & decreased secretion
Dr. Alisa Chebotarova, MD,of
PhDconjugated bilirubin
Laboratory findings in hepaticjaundice

Hepatic Present
jaundice ↑↑ ↑ ↑ N or ↓
• Serum conjugated bilirubin is elevated as the capacity to excrete

conjugated bilirubin into biliary canaliculus is reduced. Conjugated
bilirubin regurgitates into plasma (as it cannot be excreted into bile)
• Serum unconjugated bilirubin is elevated as hepatocellular damage
results in lower uptake of unconjugated bilirubin into liver. Also, liver
damage can reduce the conjugating capacity of the liver.
• Urobilinogen in urine is normally very low and the stercobilin if
feces is decreased. Since conjugated bilirubin is soluble it is excreted
in the urine (making it dark).
• Additional lab findings are elevated levels of ALT and AST, LDH4
and LDH5
Laboratory findings in obstructive jaundice

Posthepatic N Present
jaundice ↑↑ ↑↑ ↓/ Absent
• Serum conjugated bilirubin is elevated as the excretion of

conjugated bilirubin into biliary canaliculus is reduced due to the
biliary obstruction. Conjugated bilirubin regurgitates into plasma
(as it cannot be excreted into bile)
• Urobilinogen in urine is absent to low (depending on the extent of
obstruction) and decrease of stercobilin in feces.
• Elevation of serum enzyme ALP (Alkaline phosphatase) is most
diagnostic in obstructive jaundice
Neonatal jaundice
• Unconjugated bilirubin crosses the blood
brain barrier (as it is lipid soluble) and
deposits in the basal ganglia of brain,
resulting in ‘kernicterus’
• Hypoalbuminemia, low pH, which weakens

the albumin-bilirubin bond, and drugs
(salicylates, sulfonamides) that
compete with bilirubin for albumin
binding, increase the amount of
unconjugated unbound bilirubin (thus
increase the risk of kernicterus)

Inherited unconjugated hyperbilirubinemia
• Crigler-Najjar syndrome type I – symptomatic:

- a rare autosomal recessive disorder
- Absent UDP-glucuronosyltransferase
- increased concentration of indirect-reacting
bilirubin
- premature death and early clinical findings.
Findings: jaundice, kernicterus (bilirubin deposition
in brain)

Inherited unconjugated hyperbilirubinemia
• Gilbert’s syndrome
– a rare autosomal recessive disorder, present in 3-7%
of population
– Characterized by occurrence of mild jaundice (2-5
mg/dL) usually following an infection or stress or
starvation
– UDP-glucuronyl transferase activity is about 50% of
normal (Defective conjugation of bilirubin with
glucuronic acid)
– Characterized by a mild increase in unconjugated
bilirubin
– Other hepatic enzymes are normal!
Wilson disease. Biochemical basis:
Autosomal recessive mutations in hepatocyte
copper-transporting ATPase. In norm, ATP7B helps in
copper incorporation into apo-ceruloplasmin, the transport
of copper from the liver to other parts of the body and
required for biliary copper excretion. Biliary copper
excretion provides a mechanism that normally prevents
systemic copper overload.
Defect leads to Excessive deposition of copper in
liver, brain and other tissues. Impaired incorporation of
copper into ceruloplasmin, low serum ceruloplasmin
leads to hepatic copper overload.
Excess copper - promotes free radical formation that
results in oxidation of lipids and proteins.
Copper excess is seen in Wilson disease
Main cause: Defective Accumulation of toxic levels in
incorporation of copper into liver
Ceruloplasmin, defective biliary
excretion of copper ↓
↓
Excess copper spills into the Liver damage – hepatitis going on
plasma to cirrhosis (most common
↓ presenting symptom)
RBC damage – hemolysis
Eye – KF rings (deposition of copper
in the cornea)
Brain – neurological damage causing Kayser Fleischer
neuropsychiatric symptoms (basal rings
ganglia)
↓
Lab Diagnosis: Decreased serum
ceruloplasmin.
Increased urinary excretion of copper
Increased hepatic copper content
Wilson disease: Low levels of Ceruloplasmin
Clinical features:
Hepatolenticular Degeneration
Central nervous system –
psychiatric problems with movement
abnormalities
Clumsiness, tremors, difficulty
walking, speech problems, impaired
thinking ability, depression, anxiety,
and mood swings
Kayser-Fleischer rings –
deposits of copper in the descemet
membrane in the limbus of cornearing
(yellow or green ring).
α2 -Haptoglobin (Hp)
It (90 kDa) is a α2 globulin. It is composed of two kinds of
polypeptide chains, two α-chains (possibly three) and only one
form of β-chain.
It can bind with the free hemoglobin (extra-corpuscular Hb)
in a tight noncovalent complex.
It binds free Hb by its α-chain and minimises urinary loss
of Hb. Abnormal Hb such as Bart’s-Hb (4γ) and Hb-H (4β) have
no α-chains and hence cannot be bound.
Hp-Hb (155 kDa) cannot pass through glomeruli of kidney
while free Hb (65kDa) can and Hp prevents the loss of free Hb
into urine, Hp-Hb complex is destroyed by RES cells.

Genetics

Dominant negative mutation
• Dominant negative mutation – A mutation who se gene product (i) adversely

affects the normal, wild-type gene product (ii) within the same cell. This usually
occurs if the product can still interact with the same elements as the wild-type
product, but block some aspect of its function.
• Exerts a dominant effect. A heterozygote produces a nonfunctional altered
protein that also prevents the normal gene product from functioning.
• Examples:
• A single mutation in p53 tumor suppressor gene results in a protein that is able
to bind DNA and block the nonmutated p53 from binding to the promoter.

Mosaicism
• Mosaic, or mosaicism, involves the presence of two or more

populations of cells with different genotypes in one individual who
has developed from a single fertilized egg
• All cells in a body replicate to create daughter cells, if one cell develops a
mutation during mitosis it gives rise to a new population of cells with different
genotype (somatic mosaicism)
• Presence of genetically distinct cell lines in the same individual.

Mosaicism
• Somatic mosaicism - mutation arises from mitotic errors after fertilization
and propagates through multiple tissues or organs.
• Symptomatic
• Somatic cells are affected
• It is not inherited
• Germline mosaicism - mutation only in egg or sperm cells – any gametes

developed from the mutant cell will have the same mutation
• Can be passed to offspring
• Pure germline mosaicism difficult to detect
• Not present in blood/tissue samples used for analysis
• Offspring disease may appear sporadic
Autosomal Dominant Diseases
• Familial hypercholesterolemia (LDL receptor deficiency)
• Marfan syndrome
• Achondroplasia
• Osteogenesis Imperfecta

Autosomal Dominant Inheritance - The
recurrence risk:
1. Autosomal dominant alleles are
relatively rare in populations, so the
typical mating pattern is a heterozygous
affected individual (Aa) mating with a
homozygous normal individual (aa)
• 50% are Aa, means that a half of the children
will be affected with the disease.

Autosomal Recessive (AR) Inheritance
• Cystic Fibrosis
• Phenylketonuria (PKU)
• Albinism
• Mucopolysaccharidoses (except Hunter
syndrome)
• All Sphingolipidoses (except Fabry disease)
• Classical galactosemia

Adenosine deaminase (ADA)
• Adenosine must be obligatory converted first to inosine by Adenosine deaminase

(ADA) to be father degraded.
• Adenosine Deaminase (ADA) Deficiency, an autosomal recessive disorder, causes
a type of severe combined immunodeficiency (SCID). High levels of dATP
accumulate in red cells of ADA patients and inhibit ribonucleotide reductase,
thereby inhibiting the production of other essential deoxynucleotide precursors
for DNA synthesis. Although it is believed that the impaired DNA synthesis
contributes to dysfunction of T cells and B cells, it is not known why the main
effects are limited to these cell types.

Adenosine Deaminase Deficiency ✮
• Pathogenesis:
• ADA is deficient → adenosine is
accumulated → can be converted to
dATP by adenosine kinase → dADP and
dATP are accumulated in blood cells of
ADA patients and inhibit
ribonucleotide reductase → inhibiting
the production of other essential
deoxynucleotide precursors for DNA
synthesis → rapidly dividing cells are
most affected (B, T cells)

X-Linked Recessive Diseases
• Ornithine transcarbamoylase deficiency
• Fabry disease
• Hunter syndrome
• Hemophilia A and B
• Lesch-Nyhan syndrome
• Red-green color blindness
• SCID (severe combined
immunodeficiency)

X-linked Recessive Inheritance
• The recurrence risk:
2. Normal male XAY + carrier female XAXa :
• ½ of the sons will be affected XaY
• ½ of the daughters will be carriers XAXa.

Mitochondrial Inheritance
1. Both males and females are
affected.
2. Transmission of the disease is
only from a female.
3. All offspring of an affected
female are affected.
4. None of the offspring of an
affected male is affected.
5. Diseases are typically
neuropathies and/or myopathies

Mitochondrial myopathies
• Mitochondrial myopathies - rare disorders; often
present with myopathy, lactic acidosis, and CNS
disease
• USMLE: MELAS syndrome
• Mitochondrial Encephalomyopathy
• Lactic Acidosis
• Stroke-like episodes
• Secondary to failure in oxidative phosphorylation
• Muscle biopsy often shows “ragged red fibers” (due
to accumulation of diseased mitochondria)
• Blotchy red muscle fibers on Gomori trichrome stain
are characteristic of the mitochondrial myopathies
(a histological stain used on muscle tissue)
Autosomal dominant trait
Examples of pedigree
X-linked recessive trait

Autosomal recessive trait

Structural Chromosomal Abnormalities
A. Translocations:
1. Reciprocal ✮
• occur when genetic material is exchanged between nonhomologous
chromosomes
• involve breakage of at least two chromosomes with exchange of the fragments
• The resulting chromosomes are called derivative chromosomes.
• If this happens during gametogenesis, the offspring will carry the reciprocal
translocation in all his or her cells and will be called a translocation carrier.
• The chromosome number remains at 46.
• The karyotype would be 46,XY,t(2p;8p) or 46,XX,t(2p;8p).
• Chronic Myelogenous Leukemia - rearrangements in somatic cells can lead to the formation of
cancers (Philadelphia chromosome): 46,XY,t(9p;22p) or 46,XX,t(9p;22p).
Structural Chromosomal Abnormalities
A. Translocations:
2. Robertsonian ✮
• much more common than reciprocal translocations
• occur only in the acrocentric chromosomes (13, 14, 15, 21, and 22)
• involve the loss of the short arms of two of the chromosomes and
subsequent fusion of the long arms.
The karyotype of this (male)

translocation carrier is:
- more often 45,XY,–14,–21,+t(14q;21q)
or 46,XY, ,–14,+rob (14q;21q)
Numerical chromosome abnormalities
The result of nondisjunction of one

homologous pair during the first
meiotic division:
All other homologs segregate (disjoin)
normally in the cell.
Two of the gametes are diploid for one
chromosome.
• When fertilization occurs, the
conception will be a trisomy of
non-segregated chromosome

Gene Mutations - Splice site mutations
• Substitution mutations (point mutation) of splice donors (GU) or acceptors (AG)
sites
• A genetic alteration in the DNA sequence that occurs at the boundary of an exon
and an intron (splice site).
• Splice site mutations affect the accuracy of intron removal from hnRNA during
posttranscriptional processing. If a splice site is lost through mutation it can lead to addition
of amino acids to translated protein (synthesis of abnormally longer protein).

Gene Mutations - Insertions and Deletions
2. An insertion changes the number of DNA bases in a gene by adding

of one or more nucleotides. As a result, the protein made by the gene
may not function properly.
3. A deletion changes the number of DNA bases by removing of one or

more nucleotides. The deleted DNA may alter the function of the
resulting protein(s).

• If insertion or deletion of multiple of 3 nucleotides (3, 6, 9) – non-

frameshift mutation (or in-frame mutation ✮)
• Protein has almost the same structure – all amino acids are at the
same place, simply the amino acids are added or removed
• Effect on Protein: may be nonfunctional

• An insertion or deletion of not a multiple of 3 nucleotides shifts reading frame
• That leads to the Frameshift mutation ✮.

• A reading frame consists of groups of 3 bases that each code for one amino acid.
• A frameshift mutation shifts the grouping of these bases and changes the code
for amino acids. Effect on Protein: usually nonfunctional; often shorter than
normal (truncated)

stop codons in RNA: UAA, UAG and UGA
Fragile X syndrome CGG
• Fragile X syndrome is an X-linked dominant disorder representing the most

common form of inherited mental retardation.
• Fragile chromosomal sites are only detectable in vitro when cells are exposed to
chemical agents that disrupt the DNA replication process.
• The CGG repeat resides in the 5ʹ-untranslated region (UTR) of the FMR1 gene.

Fragile X syndrome
• Clinical presentation:
• Males: 100% penetrance

• Mental retardation, autism,
• Long face
• Large everted ears
• Prominent jaw
• Mitral valve prolapse,
• Hypermobile joints
• Macroorchidism (usually postpubertal)
• Females: 60% penetrance
• Mental retardation

Turner Syndrome
Sex chromosome aneuploidy: Monosomy
• karyotype: 45,X
• is a genetic condition in which a female is partially or
completely missing an X chromosome (10% are mosaics)
• Short stature
• Edema of wrists and ankles in newborn
• Cystic hygroma in utero resulting in excess nuchal skin
and "webbed” neck
• Primary amenorrhea
• Coarctation of the aorta or other congenital heart
defect in some cases
Dr. Infertility
• Alisa Chebotarova, MD, PhD
Down Syndrome
• Autosomal aneuploidy
a) Trisomy 21 - Results from meiotic nondisjunction of the chromosome 21 pair

• High percentage of cases of trisomy 21 are due to abnormal gamete with an origin during
maternal meiosis I, related to advanced maternal age
b) Translocation - Robertsonian Translocation affecting chromosome 14 and
chromosome 21
• When a translocation carrier produces gametes, the translocation chromosome can
segregate with the normal 14 or with the normal 21.

Patau syndrome
Autosomal aneuploidy
• karyotype:
Trisomy 13 [47,XY,+13 or 47,XX,+13]
• Polydactyly (extra fingers and toes)
• Cleft lip, palate
• Microphthalmia (small eyes)
• Microcephaly, mental retardation
• Cardiac and renal defects
• rocker-bottom feet
• weak muscle tone (hypotonia).
• Very poor prognosis (Affected children typically die within the first
week of life, with only 5% surviving the first six months)
Patau syndrome

Edwards syndrome
• Autosomal aneuploidy
• the second most common autosomal trisomy
• karyotype:
Trisomy 18 [47,XY,+18 or 47,XX,+18]
• results from nondisjunction, usually in formation of the eggs or sperm, where
one gamete ends up with an extra chromosome 18; is associated with advanced
maternal age.

Edwards syndrome ✮
• slow growth before birth (intrauterine growth
retardation)
• a low birth weight
• a small, abnormally shaped head (prominent occiput)
• a small jaw and mouth (micrognathia)
• Clenched fist with overlapping fingers
• Inward turning, "rocker-bottom" feet
• Congenital heart defects
• Low-set ears,
• Mental retardation
•Dr. Very poor prognosis (Death usually in 1st year of life )
Alisa Chebotarova, MD, PhD
DiGeorge/ velocardiofacial syndrome
Structural Abnormality - a microdeletion in long arm of
chromosome 22
• The acronym CATCH-22
• C = cardiac defects,
• A = abnormal facies (hypertelorism) - abnormally
increased distance between two organs or bodily
parts, usually referring to an increased distance
between the orbits (eyes), or orbital hypertelorism
• T = thymic hypoplasia,
• C = cleft palate, cleft lip
• H = hypocalcemia because of hypoparathyroidism,
• 22 = Partial deletion on chromosome 22
• A child may exhibit a positive Chvostek sign
• ipsilateral twitching of the facial muscles occurs, whereas
there is no movement when the sign is negative. A positive
Chvostek sign may indicate hypocalcemia.
Chromosome 15
Prader-Willi Syndrome
Angelman Syndrome Imprinting defects

Imprinting
• Imprinting refers to the fact that a small
number of genes are transcriptionally
active only when transmitted by one of the
two sexes. The homologous locus in the
other parent is rendered transcriptionally
inactive. Thus, for imprinted loci, it is
normal to have only the maternal (for
some loci) active, or only the paternal (for
other loci) active. PW genes
• Involves methylation and possibly other
mechanisms to imprint or inactivate the
appropriate loci
• Occurs in specific loci on several
chromosomes only
Dr. Alisa Chebotarova, MD, PhD Loci normally imprinted on chromosome 15
Imprinting
• Normal function of Prader-Willi genes cluster “PWS/AS region”

in the paternal chromosome 15 is to produce
a long noncodind RNA, that silences some
genes which must not be expressed in a
healthy person
• Only one, paternal allele is active; the other –
maternal, is inactivated by methylation
PW genes
UBE3A gene
Dr. Alisa Chebotarova, MD, PhD Loci normally imprinted on chromosome 15
Prader-Willi Syndrome (PWS)
lack of the paternal contribution of the chromosome
15q11-q13 region leads to PWS
• the loss of function of PW genes in a particular

region of the paternal chromosome 15.
• A lack of paternal chromosome 15 because of a
deletion in normal chr. 15 (q 11-13 region). leads to
inability to express those genes and development
of disease
• Paternal allele is lost by deletion mutation and the
maternal allele is imprinted and is not expressed
*Maternal uniparental disomy of chromosome 15 has also been reported

for PWS patients.
Dr. Alisa Chebotarova, MD, PhD Deletion causing Prader-Willi syndrome
Prader-Willi Syndrome
In infancy:
• weak muscle tone (hypotonia),
• feeding difficulties,
• poor growth
• delayed development
• hyperphagia, obesity
• mental retardation

Good luck!

BLOCK 4 Review

Course: Biochemistry & Genetics
Prepared by: Dr. M. Vysochyn, MD, PhD
NUTRITION (I)
At the end of this lecture students will be able to:
Describe water-soluble vitamins;
Describe lipid-soluble vitamins;
MICRONUTRIENTS
The micronutrients are all those nutrients required in trace
(micro) amounts to maintain normal body function, but that are
not used as sources of energy.
They are divided into the organic compounds (vitamins), and

inorganic compounds (minerals).
VITAMINS
The name comes from the original idea that they were all amines,
hence vital amine, or vitamin. They are classified as either water or
lipid soluble.
The water soluble are: The lipid soluble are:
B1 - thiamin
B2 - riboflavin A - retinoids
B3 - niacin D - calciferols
B5 - pantothenate E - tocopherols
B6 - pyridoxine K - quinones
B12 - cobalamin Pro-A - carotenoids
B9 Folate - folate
H - biotin
C - ascorbate All groups of similar compounds.
VITAMINS
VITAMINS
Most water-soluble vitamins are not stored, but when in excess are
excreted in the urine.
The exceptions are vitamin B9 and B12, both of which are stored by
the liver.
All are present in low concentrations in most foods, thus circulating
levels are never high enough to induce any toxicity, and all are
considered non-toxic.
Lipid-soluble vitamins cannot be excreted in urine and all are stored to

at least some extent.
Two lipid-soluble vitamins, A and D, can induce toxic reactions after
either acute megadosage or chronic high dosage, especially vitamin A.
Synthetic vitamin K can also be toxic.
WATER SOLUBLE VITAMINS
VITAMIN B1 (THIAMINE)
THIAMINE THIAMINE PYROPHOSPHATE
PP
Functions as thiamine pyrophosphate, and is required by:

• pyruvate dehydrogenase in the conversion of pyruvate to acetylCoA,
• α-ketoglutarate dehydrogenase to convert α-ketoglutarate to
succinylCoA,
• transketolase in the pentose phosphate pathway which translocates 2-
carbon units.
• branched-chain ketoacid dehydrogenase
valine, isoleucine to Propionyl-CoA
Leucine to Acetyl-CoA
VITAMIN B1
transketolase in the pentose phosphate pathway which translocates

2-carbon units.
pyruvate dehydrogenase in the conversion of pyruvate to acetylCoA,
α-ketoglutarate dehydrogenase to convert α-ketoglutarate to succinylCoA,
VITAMIN B1
Thiamine deficiency, also known as beriberi, is a condition that
occurs due to not enough thiamine (vitamin B1). There are two
main types: wet beriberi, and dry beriberi.
Dry beriberi causes wasting and partial paralysis resulting from damaged
peripheral nerves. It is also referred to as endemic neuritis.
It is characterized by:
• Difficulty in walking
• Tingling or loss of sensation
(numbness) in hands and feet
• Loss of tendon reflexes
• Loss of muscle function or paralysis
of the lower legs
• Mental confusion/speech
difficulties
• Pain, Vomiting
• Involuntary eye movements
(nystagmus)
Key steps in the induction of
beriberi.
ATP = adenosine
triphosphate; CNS = central
nervous system; CoA =
coenzyme A; eNOS =
endothelial nitric oxide
synthase; KGDH = alpha-
ketoglutarate dehydrogenase;
NAD = nicotinamide adenine
dinucleotide; NADPH =
nicotinamide adenine
dinucleotide phosphate; PDH
= pyruvate dehydrogenase;
TCA = tricarboxylic acid cycle;
TK = transketolase.
Brain disease
VITAMIN B1
Wernicke's encephalopathy (WE), Korsakoff's syndrome (alcohol
amnestic disorder), Wernicke–Korsakoff syndrome are forms of dry
beriberi.
Alcoholics may have thiamine
deficiency because of the following:
Inadequate nutritional intake.
Decreased uptake of thiamine from
the GI tract.
Liver thiamine stores are reduced due
to hepatic steatosis or fibrosis.
Impaired thiamine utilization.
Ethanol per se inhibits thiamine
transport in the gastrointestinal
system and blocks phosphorylation
of thiamine to its cofactor form
(ThDP).
VITAMIN B1
Wet beriberi affects the heart and circulatory system. It is
sometimes fatal, as it causes a combination of heart failure
and weakening of the capillary walls, which causes the
peripheral tissues to become edematous.
Wet beriberi is characterized by:
• Increased heart rate
• Vasodilation leading to
decreased systemic vascular
resistance, and high output
cardiac failure
• Elevated jugular venous
pressure
• Dyspnea on exertion
• Paroxysmal nocturnal dyspnea
• Peripheral edema (swelling of
lower legs)
VITAMIN B1
Wernicke - Korsakoff Syndrome
Most common manifestation of deficiency in developed countries
(commonly observed in chronic alcoholics)
• Impaired intestinal absorption of thiamine
• Poor diets, carbohydrates (ethanol) increase thiamine demand,
magnesium deficiency
Wernicke's encephalopathy
• severe acute deficiency
• neurological damage
• horizontal nystagmus, ophthalmoplegia, ataxia, confusion
Korsakoff's psychosis
• Chronic neurologic sequel after Wernicke's encephalopathy
• amnesia (anterograde, retrograde)
• confabulation
VITAMIN B1
Beri beri is a disease of human
origin. It arose in populations
whose staple food was rice.
When rice was removed from
the husk, they became deficient
because most thiamine is in the
husk. Parboiling rice prior to
dehusking transfers some
thiamine to the rice kernel and
avoids beri beri.
Good sources of thiamine are
meats, grains, eggs, nuts, liver,
yeast and milk. The RDA is
0.9mg/day.
VITAMIN B2 (RIBOFLAVIN)
Riboflavin functions as a coenzyme, meaning that it is
required for enzymes (proteins) to perform normal
physiological actions.
Specifically, the active forms of riboflavin flavin
mononucleotide (FMN) and flavin adenine
dinucleotide (FAD) function as cofactors for a
variety of flavoproteine enzyme reactions
(for redox reactions).
FMN FAD
VITAMIN B2
This functions as part of flavin adenine
dinucleotide (FAD), a key hydrogen
acceptor in the TCA cycle, in the
conversion of succinate to fumarate
via succinate dehydrogenase.
It forms part of complex I in the ETC as

FMN, required for the uptake of
hydrogens from NADH2.
VITAMIN B2
It is also important in β-oxidation

when acyl-CoA is converted to
dehydroacyl-CoA via acyl-CoA
dehydrogenase.
Symptoms of deficiency: VITAMIN B2
• Skin cracking (including cracked corners of the mouth)
• Blurred vision and itching, watering, sore, or bloodshot eyes
• Eyes becoming light-sensitive and easily fatigued
• Weakness,
• Throat swelling/soreness
• Swollen tongue
• Dermatitis (seborrheic dermatitis)
• Anemia (normocytic normochromic)
•Corneal Vascularization
The 2 C’s: Cheilosis (crackling around

the corners of the mouth) and
Corneal Vascularization
Good sources of riboflavin are meats,
eggs, grains, nuts, peas and peanuts.
The RDA is 1.3mg/day.
VITAMIN B3 (NIACIN, NICOTINAMIDE)
Niacin can be synthesised
from tryptophan, but the rate
is very slow and normally not
adequate for the needs of the
body.
VITAMIN B3
It functions as part of nicotinamide adenine dinucleotide
(NAD), a key hydrogen acceptor in:
• glycolysis (glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate
and pyruvate to acetyl-CoA),
• TCA cycle (isocitrate to α-ketoglutarate, α-ketoglutarate to succinyl-
CoA and malate to oxaloacetate),
• β-oxidation (hydroxyacyl-CoA dehydrogenase reaction).
It also forms part of NADP in the pentose phosphate pathway.

VITAMIN B3
Severe deficiency, called pellagra, can cause symptoms related to
the skin, digestive system, and nervous system.
Symptoms of deficiency:
• thick, scaly pigmented rash on skin
exposed to sunlight
• swollen mouth and bright red tongue
• vomiting and diarrhea
• headache
• apathy
• fatigue
• depression
• disorientation
• memory loss
If not treated, pellagra can lead to death.
VITAMIN B3
Carcinoid tumors of the lung comprise roughly 5% of all lung tumors.
Serotonin and kallikrein are vasoactive substances produced by tumor
carcinoid neuroendocrine cells. Carcinoid tumors can rarely present
with carcinoid syndrome, which is caused by the release of vasoactive
amines from neoplastic cells.
Symptoms of carcinoid syndrome include:

• Flushing of the skin
• Secretory (watery, voluminous) diarrhea
• Abdominal cramps with nausea and
vomiting
• Wheezing, caused by bronchoconstriction
and/or bronchospasm
• Tricuspid insufficiency or pulmonic valve
stenosis (TIPS).
Niacin (B3) may be deficient in
carcinoid syndrome as a result
of increased tryptophan
metabolism into serotonin.
Tryptophan is a common
precursor to both serotonin and
niacin.
VITAMIN B3
Pellagra-like dermatitis also results from the treatment of
tuberculosis by isoniazid. This is due to a niacin deficiency
caused by depletion of pyridoxal phosphate (vitamin B6).
Hartnup syndrome (rare, autosomal recessive)
impaired synthesis of niacin from tryptophan caused by defective
transport of tryptophan (and other neutral amino acids) in the intestine
and kidney. Pellagra like symptoms.
High intakes may result in:
• prostaglandin induced facial flushing.
• liver damage in a few patients (liver function
should be monitored).
"Niacin flush," characterized by redness of the face
and neck after oral niacin administration, is not fully
understood, but is thought to involve both early and
late acting mediators similar to a hypersensitivity
reaction.
VITAMIN B3
Secondary pellagra occurs when your body can’t absorb niacin.
Things that can prevent your body from absorbing niacin
include:
• Gastrointestinal diseases, such as Crohn’s disease and ulcerative colitis
• Certain medications, including anti-convulsants and immunosuppressive
drugs
• Alcoholism, eating disorders
• Cirrhosis of the liver
• Carcinoid tumors
Lots of vitamin B3
can be found in
meats, fish, grains,
nuts and legumes.
The RDA is 16mg/day.
VITAMIN B5 (PANTOTHENATE)
Pantothenate functions as the central part of coenzyme A and
acts as the activator of fatty acids. It also allows for the
metabolism of acetyl-CoA, succinyl-CoA and malonyl-CoA.
Energy metabolism, cholesterol synthesis and ketone
formation all depend on coenzyme A as a key component
of metabolic intermediates.
VITAMIN B5 (PANTOTHENATE)
There are no specific deficiency symptoms as there has been
little historical record of deficiency.
Symptoms of deficiency:
• Fatigue
• Insomnia
• Depression
• Irritability
• Vomiting
• Stomach pains
• Burning feet
• Respiratory infections.
Good sources of vitamin B5 are kidney, liver, yeast, grains and green
vegetables. The RDA is 7mg/day.
VITAMIN B6 (PYRIDOXINE)
Pyridoxine is the major dietary input of vitamin B6, but it
functions as pyridoxal phosphate (PLP) ßAztive form.
PYRIDOXINE
PYRIDOXAL PHOSPHATE
Metabolically active form of vitamin B6, is involved in many aspects

of macronutrient metabolism, neurotransmitter synthesis, histamine
synthesis, hemoglobin synthesis and function, and gene expression.
PLP generally serves as a coenzyme (cofactor) for many reactions
including decarboxylation, transamination, racemization,
elimination, replacement, and beta-group interconversion.
The liver is the site for vitamin B6 metabolism.
Coenzyme for a large number of enzymes, particularly those that

catalyze reactions involving amino acids.
• Transamination (AST, ALT) à in liver (urea cycle)
• Deamination Serine à pyruvate + NH3
• Decarboxylation Histidine à histamine +CO2
• Condensation Glycine + succinyl CoA (TCA) à aminolevulenic acid
(Heme Synthesis)
• Required for the synthesis of niacin (B3) from tryptophan
• Required for the synthesis of neurotransmitters serotonin,
norepinephrine, epinephrine and γ-aminobutirate
• It is bound to glycogen phosphorylase (breakdown of glycogen)
(70-80%)
• Functions in glycogenolysis
A vitamin B6 deficiency cause symptoms including:
• Changes in mood, such as irritability, anxiety and depression.
• Confusion.
• Muscle pains.
• Low energy, or fatigue.
• Worsening of PMS symptoms.
• Worsening symptoms of anemia.
Microcytic (Sideroblastic) Anemia: depositing
iron (with mitochondria) around the red blood
cells
Decreased hemoglobin synthesis.
Erythrocytes smaller than normal, lack enough
hemoglobin to carry adequate oxygenà
Microcytic Hypochromic anemia.
Sideroblastic Anemia
Ringed Sideroblasts (abnormal erythroblasts)
Caused by defect in Heme synthesis
Iron accumulates in RBC mitochondria forming a
ring surrounding nucleus.
Good sources of vitamin B6 are fish, green vegetables, grains,
bananas, tomatoes and yoghurt. The RDA is 1.4mg/day.
VITAMIN B12 (COBALAMIN)
Has a complex ring structure (corrin ring), similar to a porphyrin
ring, to which is added a cobalt ion at its center.
• Synthesized exclusively by microorganisms
• No plant source
• Conserved in animals in the liver
Good sources are meat, fish, eggs, milk, liver, cheese - all animal
products. Some seaweeds and algae also contain some of it. The RDA is
1.5μg/day.
ABSORPTION AND TRANSPORT
OF VITAMIN B12
• Dietary B12 binds to R-binders
(haptocorrins) in the stomach and
travels to the intestine, where the R-
binders are destroyed by pancreatic
proteases
• The freed B12 then binds to intrinsic
factor (IF)- a glycoprotein, secreted by
the parietal cells of stomach
• B12–IF is absorbed by mucosal cells in
the ileum, enters the blood, and is
carried by proteins named
transcobalamins
• 50% to Liver
• 50% to Other tissues
ABSORPTION AND TRANSPORT
OF VITAMIN B12
• transcobalamin I – to the liver for
storage à 50%
• transcobalamin II – to other
tissues à 50%
• free cobalamin is released into
the cytosol of cells as
hydroxocobalamin
Converted into the active forms:
In cytosol to methylcobalamin
In mitochondria to 5’-
deoxyadenosylcobalamin
VITAMIN B12
Deoxyadenosylcobalamin: Coenzyme for Methylmalonyl-CoA
mutase (conversion of methylmalonyl-CoA to succinyl-CoA).
Odd-carbon fatty acids, Val, Met, Ile, Thr
Methionine
Threonine
Valine
Isoleucine
Thymidine
degradation
B12 deficiency: abnormal fatty acids accumulate and

become incorporated into cell membranes, including
cells of the nervous system.
May account for some of the neurologic manifestations of B12 deficiency.
VITAMIN B12
Methylcobalamin: Coenzyme for homocysteine methyltransferase in
the combined conversion of homocysteine to methionine.
B12 deficiency: N5-methyl-
tetrahydrofolate
accumulates while the level
of the other forms of THF
decrease (folate trap).
Leads to a decrease in the
forms of tetrahydrofolate
needed for purine and
thymine synthesis.
megaloblastic anemia
VITAMIN B12
Vitamin B12 deficiency symptoms:
• Weakness, tiredness, or lightheadedness
• Heart palpitations and shortness of breath
• Pale skin, smooth tongue
• Constipation, diarrhea, loss of appetite, or gas
• Vision loss – blind spots in the central portions of the visual fields
• Nerve problems – numbness or tingling, muscle weakness, and problems
walking alterations in gustatory (taste) and olfactory (smell) function
• Mental problems – depression, memory loss, or behavioral changes
The classical clinical presentation of the neurologic dysfunction associated
with a B12 deficiency includes:
• symmetric numbness and tingling of the hands and feet,
• diminishing vibratory and position sense,
• progression to a spastic gait disturbance.
The patient may become somnolent or may become extremely irritable
(“megaloblastic madness”).
VITAMIN B12
This is believed to be caused by hypomethylation within the nervous system
brought about by an inability to recycle homocysteine to methionine and
from there to S-adenosylmethionine (SAM). The nervous system lacks the
betaine pathway of methionine regeneration and is dependent on the B12
system.
VITAMIN B12
Megaloblastic Anemia: MCV > 100
Commonly due to failure to absorb the vitamin in the intestine
Rarely due to an absence of the vitamin in the diet
Symptoms:
Megaloblastic Anemia (Pernicious Anemia - if it is
due to a lack of Intrinsic Factor (deficient
absorption)). Pernicious anemia is an autoimmune
destruction of gastric parietal cells. Laboratory
testing will show anti-parietal or anti-intrinsic
factor antibodies.
Homocystinuria and methylmalonic aciduria
Elevated Methylmalonyl CoA in serum can be used
for differential diagnosis with Folic Acid deficiency
Neurologic deterioration - caused by progressive
demyelination of nervous tissue. Paresthesias.
VITAMIN B12
Diphyllobothrium latum is a fish tapeworm which patients
may get from eating raw or undercooked freshwater fish.
The larvae mature within
the gastrointestinal tract and
absorbs vitamin B12.
Diphyllobothrium latum is a
potential source of vitamin
B12 deficiency.
Schilling Test
Stage I consists of two doses of
vitamin B12 (cobalamin). The
first dose is radioactive and
taken by mouth (radioactive
cobalt). The second dose is not
radioactive and is given as a
shot 2 – 6 hours later.
The intramuscular injection of vitamin B12 is given to saturate the
liver and serum protein-binding sites, which allows radioactive
vitamin B12 to be excreted in the urine. 24-hour urine analysis will
then be done, to measure the body’s ability to absorb vitamin B12
normally.
Schilling Test
Stage II consists of radioactive B12 along with intrinsic factor. Stage II
of the test can determine if the vitamin B12 deficiency is caused by
damages of the stomach that prevent it producing intrinsic factor.
Stage III requires you to take antibiotics for 2 weeks, stage II is then
repeated. This stage can determine whether the low vitamin B12
levels are the result of abnormal bacterial growth.
Stage IV - pancreatic
enzymes for three days,
followed by a radioactive
dose of vitamin B12. This
shows if deficiency of
vitamin B12 is caused by
pancreas problems.
FOLIC ACID (B9, FOLATE)
Consists of the base pteridine attached to a molecule of para-
aminobenzoic acid (PABA) and a glutamic acid.
Plays a key role in one-carbon
metabolism of various compounds.
Carrier of one-carbon units: THF (FH4)
receives one carbon fragments from
donors like serine, glycine, and histidine N5-METHYL-TETRAHYDROFOLATE
and transfers them to intermediates in
the synthesis of amino acids
(methionine), purines, and thymidine.
Circulated in plasma primarily in the
form of N5-methyl-tetrahydrofolate.
Good sources of folate are green

vegetables and liver. The RDA is
200μg/day.
TETRAHDYROFOLATE INTERCONVERSIONS AND THE
1C PATHWAY
FOLIC ACID
Common signs a folate deficiency:
• Poor immune function; frequently getting sick
• Chronic low energy (including chronic fatigue syndrome)
• Poor digestion; issues like constipation, bloating and IBS
• Developmental problems during pregnancy and infancy, including
stunted growth
• Anemia, Pale skin
• Canker sores in the mouth and a tender, swollen tongue
• Changes in mood, including irritability
• Premature hair graying
Can be caused by:
Increased demand (pregnancy and lactation)
Poor absorption (pathology of the small intestine, alcoholism)
Treatment with drugs
• that are dihydrofolate reductase inhibitors (Methotrexate)
• sulfanilamide - inhibits the synthesis of folic acid in microorganisms
(synthesis of nucleotides for replication of DNA and RNA) as antimicrobial
agents.
FOLIC ACID
Megaloblastic anemia
Macrocytic (Large Cells).
Caused by diminished synthesis of purines
and thymidine leading to an inability of cells
to make DNA and to divide.
Cell growth without division.
Hyperhomocysteinemia leads to
hypercoagulability and is therefore
associated with thromboembolic
events. The underlying mechanisms
include decreased endothelial
antithrombotic activity and activation
of procoagulatory factors (e.g., factor
V, factor VII).
FOLIC ACID
Neural tube defects (Spina Bifida and
Anencephaly). There are three types
of spina bifida: occulta, meningocele,
and myelomeningocele.
Occulta is the mildest form,
myelomeningocele results in the most
complications.
This is due to a portion of the spinal cord and
corresponding meninges herniating through the
skin defect and being exposed to the amniotic fluid.
Upon delivery, there is an increased risk of
central nervous system infection if the neonate is
not immediately treated with antibiotics.
Surgery to repair the defect is performed within 72
hours to minimize further damage
and neurological deficits.
SULFONAMIDES - structural analogs of PABA
Competitively inhibit folic acid synthesis of
microorganisms and inhibit nucleotide
synthesis for DNA and RNA production.
Used as antimicrobials.
METHOTREXATE - folic acid analog inhibits dihydrofolate
reductase
• Inhibit nucleotide synthesis for DNA and RNA synthesis.
• used in the treatment of acute leukemia in children.
VITAMIN H (BIOTIN, B7, coenzyme R)
Sulfur containing imidazole derivative widely distributed in natural foods.
In most people intestinal flora can supply much of the biotin needs, but
in some people this is inadequate and can lead to deficiency.
In fatty acid synthesis: acetyl-

CoA carboxylase
Acetyl-CoA → malonyl-CoA
In gluconeogenesis: pyruvate carboxylase
Pyruvate → oxaloacetate
In fatty acid reduction: propionyl-
CoA carboxylase
Propionyl-CoA → methylmalonyl-CoA
Good sources of biotin are egg yolk, yeast, liver and kidney.
Raw egg white contains a protein, avidin, which chelates (binds) vitamin H
and makes it unabsorbable.
Anyone who eats significant quantities of raw eggs (quite common amongst
extreme body builders) can become biotin deficient. The RDA is 300μg/day.
VITAMIN H
Biotin deficiency symptoms may develop:
•red rashes on the skin, especially the
face.
•dry or scaly skin, dry eyes.
•brittle hair, hair loss.
•fatigue, insomnia or difficulty sleeping
•loss of appetite, nausea.
•depression.
•burning or prickling sensation in the
hands and feet.
•muscle pain, difficulty walking.
•changes in the intestinal tract (frequent
upset stomach).
•cracking in the corners of the mouth.
•seizures.
VITAMIN C (ASCORBATE)
Most mammals can synthesise vitamin C, but humans, primates,
guinea pigs and fruit-eating bats cannot.
Good sources of vitamin C are citrus and soft fruits, tomatoes and green
vegetables. The RDA is 40mg/day.
Collagen synthesis: It participates in collagen hydroxylation. Add

hydroxyl groups to proline or lysine in collagen molecule prolyl
hydroxylase and lysyl hydroxylase – collagen to assume its triple helix
structure. Development & maintenance of scar tissue, blood vessels,
and cartilage.
Degradation of tyrosine: Step catalyzed by homogentisate dioxygenase
(ferrous iron-containing enzyme).
Synthesis of norepinephrine and dopamine from tyrosine. Dopamine
beta hydroxylase participates in the biosynthesis of norepinephrine
from dopamine.
Bile acid formation: At the initial 7a-hydroxylase step
Absorption of iron: Significantly enhanced by the presence of vitamin C
May act as a general water-soluble anti-oxidant.
Symptoms and signs of severe scurvy may include:
Many symptoms may be explained by defective
collagen synthesis
•swollen, spongy and purplish gums that are prone
to bleeding, loose teeth
•bleeding into the skin (severe and easy bruising),
scaly, dry and brownish skin (perifollicular
hemmorrhages)
•very dry hair that curls and breaks off close to the
skin
•slow-healing wounds
•bleeding into the joints and muscles, which causes
areas of swelling over the bones of the arms and
legs
•premature stopping of bone growth (in babies and
children).
Synthesis of
norepinephrine
and dopamine
Symptoms and signs of excess (overdose) may include:

• Nausea
• Diarrhea, bloating
• Fatigue
• Increased risk of iron toxicity in transfusion patients and hereditary
hemochromatosis
• Nephrolithiasis (Vitamin C increases oxalate (kidney stones) formation)
NUTRITION (II)
LIPID SOLUBLE VITAMINS
VITAMIN A (RETINOL, RETINAL, RETINOIC ACID)
Generic term referring to
all compounds that exhibit
the biological activity of
vitamin A. Main functions
are carried out by retinol
and its 2 derivatives retinal
and retinoic acid.
Retinoids = term used to

describe both the natural
forms and the synthetic
analogs of retinol
VITAMIN A
Vitamin A occurs in vegetables as a provitamin:
•b-carotene a yellow pigmented molecule
•α-carotene is also a source
Cleaved in the intestinal mucosa to
give 2 molecules of retinal.
Not efficiently converted to vitamin

A (depends on amount in diet and
possibly food source). β-carotene
vitamin A activity is only one-sixth
of retinol (by weight).
Excess carotenoids are used in the body as anti-oxidants

VITAMIN A
Dietary Vitamin A, from animal sources is available in the form of Retinyl
esters. The absorption of retinol requires the presence of bile salts.
In the intestinal cells, retinol is esterified back and secreted with
chylomicrons.
b -Carotene is cleaved in the intestinal mucosa, esterified and secreted in
chylomicrons together with esters formed from dietary retinol.
The liver contains approximately 90% of the vitamin A reserves and secretes
vitamin A in the form of retinol, which is bound to retinol- binding protein.
The retinol-binding protein complex interacts with a second protein,
Transthyretin.
This trimolecular complex functions to prevent vitamin A from being filtered
by the kidney glomerulus, to protect the body against the toxicity of retinol
and to allow retinol to be taken up by specific cell-surface receptors that
recognize retinol-binding protein.
VITAMIN A
VITAMIN A
Visual Cycle - Retinal
Growth and Differentiation of Cells - Retinal, Retinoic Acid
Reproduction (promotes spermatogenesis, blocks resorption) - Retinol, Retinal
VITAMIN A DEFICIENCY
Night Blindness (nyctalopia)
• One of the earliest signs of Vitamin A deficiency.
• Visual threshold is increased making it difficult to see in dim light.
• Prolonged deficiency leads to an irreversible loss of visual cells.
Xerophthalmia
• Pathologic dryness of the conjunctiva and cornea.
• If untreated, may result in corneal ulceration and ultimately blindness
because of the formation of opaque scar tissue
• Most frequently seen in children of developing tropical countries
Dry, scaly skin (xerosis cutis)
Alopecia
Immune suppression
Xerosis (Dry Eyes) - The sclera loses Bitot's Spots - Patches of little
its shine and begins to wrinkle. gray bubbles on the sclera.
Corneal Ulceration - Dullness Keratomalacia - Soft or bulging

or damage to the cornea. cornea. Opacities (keratin deposits).
VITAMIN A TOXICITY (HYPERVITAMINOSIS A)
Due to excessive intake of vitamin A
• Intake of > 7.5 mg of retinol per day must be avoided
• Polar bear liver
Signs & Symptoms:
• Arthralgias,
• Skin changes (e.g., scaliness),
• Alopecia,
• Cerebral edema,
• Pseudotumor cerebri,
• Osteoporosis,
• Hepatic abnormalities.
May cause congenital malformations in the developing fetus of a
pregnant woman.
VITAMIN A
b-carotene
Consumption of foods rich in b-carotene is associated with:
• Decreased incidence of heart disease
• Decreased incidence of lung and skin cancer
• Reduced risk of cataracts and macular degeneration
May be due to its anti-oxidant properties or its enhancement of the
immune system.
Health benefits are independent of its role as a precursor of Vitamin A
Not toxic even at high doses for extended periods (unlike other forms
of Vitamin A which have toxicity)
Good sources are oily fish, fish oils, milk, butter, carotenes (red foods
and vegitables) and liver. The RDA is 700μg/day.
VITAMIN D (D2, D3)
Dietary vitamin D occurs in several forms. Some PLANT
active vitamin D3 is in food, but most is in the SOURCES
form of cholecalciferol (D3, animals) and
ergosterol (D2, fungi).
D2 and D3 only differ in 1 double bond in the
side chain.
Group of sterols that have a hormone-like
function ANIMAL
Functions: SOURCES
↑ intestinal absorption of calcium and
phosphate, Mg2+ in small intestine
↑ bone mineralization.
Good sources of vitamin D are milk,
margarine, eggs, oily fish, fish oils and liver.
The RDA is 10μg/day.
Vitamin D
• Regulation of Vitamin D Synthesis
• depends on the “status” of Ca2+ in the body
• When Ca2+ is sufficient (adequate dietary intake and N or
increased plasma Ca2+) - the inactive metabolite is
preferentially synthesized
• When Ca2+ is insufficient (low dietary intake, decreased
plasma Ca2+ ) - the active metabolite is preferentially
synthesized to ensure that additional Ca2+ will be absorbed
from the gastrointestinal tract.
• 1α -Hydroxylase activity is increased by:
• decreased plasma Ca2+ concentration,
• increased circulating levels of PTH, and
• decreased plasma phosphate concentration.
VITAMIN D DEFICIENCY
Infantile Rickets
• Rickets can present in young children that
are exclusively breastfed. They classically
present with bowed legs, rachitic rosary, and
a soft skull with frontal bossing.
• Serum laboratory values will show
low calcium and phosphate along with
elevated alkaline phosphatase and PTH.
Osteomalacia in adults
• is caused by vitamin D deficiency, can lead to
defective mineralization of osteoid (soft
bone).
• Laboratory test usually shows decreased
serum calcium level and
increased parathyroid hormone level.
VITAMIN D TOXICITY
Vitamin D is the most toxic of all vitamins.
Symptoms include:
•fatigue, loss of appetite, weight loss
•excessive thirst, excessive urination, dehydration, constipation
•irritability, nervousness, ringing in the ear (tinnitus)
•muscle weakness
•nausea, vomiting
•confusion, disorientation
•high blood pressure, heart arrhythmias
Long-term complications of untreated hypervitaminosis D include:
•kidney stones
•kidney damage
•kidney failure
•excess bone loss
•calcification (hardening) or arteries and soft tissues
In addition, increased blood calcium can cause abnormal heart rhythms.
VITAMIN E (VARIOUS TOCOPHEROLS)
Occurs in the diet as mixture of several
closely related compounds called,
tocopherols and tocotrienol. All are
isoprenoids.
α-tocopherol has the widest natural
distribution and the greatest biological
activity (scavenger of reactive oxygen
species).
Vitamin E is not toxic even if 50 times the

RDA is consumed.
Good sources are plant oils, grains, nuts
and green vegetables.

VITAMIN E
Acts as an antioxidant (even in the gut – prevents vitamin A oxidation).
First line of defense against peroxidation of polyunsaturated fatty acids
contained in membrane phospholipids inhibits oxidative degradation of
lipids by free radicals.
Typically oxygen molecules with unpaired electrons created as a byproduct
of normal oxygen consumption. Damage intracellular molecules by
breaking critical intramolecular bonds, leading to extensive cell injury
A single circulating free radical in the cell can damage tens or even
hundreds of enzymes, membrane lipid or DNA molecules through a
destructive chain reaction of chemical damage that cascades through a cell
until it is ultimately quenched.
Important in preventing macular degeneration and cognitive decline with
age
Epidemiological studies suggest that people consuming 100 mg/day have
reduced risk of myocardial infarction.
Vitamin C maintains Vitamin E in it’s reduced form.
VITAMIN E
Vitamin E Deficiency Symptoms:
1. Low levels of vitamin E can cause many digestive system problems,
which will lead to poor absorption of nutrients from the digestive
tract. This leads to diseases in pancreas, liver, gall bladder etc.
2. Nervous system related problems in hands, legs, arms and feet.
3. Pain, tingling, loss of sensation, gastrointestinal diseases.
4. Dry hair or loss of hair.
5. Muscular weakness.
6. Slow tissue healing.
7. Leg cramps.
VITAMIN K (K1, K2, K3)
Phylloquinone (K1) – major form of
vitamin K found in plants.
Menaquinones (K2) – a series of
polyprenoid unsaturated forms of vitamin
K found in animal tissues and synthesized
by bacteria in the intestine.
Menadione – Vicasol (K3) – parent
compound of the vitamin K series. Does
not occur naturally. If administered, it is
alkylated in vivo to one of the
menaquinones.
Good dietary sources are green
vegetables and liver.
The RDA is 80μg/day

VITAMIN K
Requires normal fat absorption
Naturally occurring K derivatives are absorbed only in the presence of
bile salts
Distributed in the bloodstream via the lymph, in chylomicrons
Exception: Menadione (K3) – water-soluble and absorbed even in the
absence of bile salts and passes directly into the hepatic portal vein
Vitamins A & E compete for absorption with vitamin K. Excess of these
vitamins can decrease vitamin K absorption.
After a meal, vitamin K levels increase initially in liver, but decline
rapidly – little storage
VITAMIN K
Vitamin K is required to introduce Ca2+ binding sites on several calcium-
dependent proteins.
Required for conversion of glutamic acid residues to γ- carboxyglutamic acid
(Gla) residues in several precursor proteins.
Vitamin K is a coenzyme for enzymes involved in the γ-carboxylation of the
glutamate residues of the precursor proteins.
Targets of γ-carboxylation:
K is for Koagulation. Necessary for the activation of clotting factors II, VII, IX,
X, and proteins C and S.
The liver produces the vitamin-K dependent clotting factors (II, VII, IX, X).
Therefore, liver disease can mimic vitamin K malabsorption. Factors V & VII
measurements can distinguish between the liver dysfunction & vitamin K
deficiency.
Bone formation: osteocalcin (bone Gla protein), matrix Gla protein
The g-carboxyglutamate of osteocalcin is required to bind hydroxyapatite
crystals in the bone and thereby is required for bone mineralization
Coagulation in Platelets
• γ- Carboxyglutamate of prothrombin (factor II) is a good chelator of
positively charged calcium ions because of the 2 adjacent, negatively
charged carboxylate groups
• The prothrombin -calcium complex is then able to bind phospholipids
essential for blood clotting on the surface of platelets.
• The binding of prothrombin to phospholipid surfaces is crucial because
it brings it into close proximity of two clotting proteins that catalyze its
conversion intro thrombin.
BLOOD COAGULATION CASCADE
DEFICIENCY OF VITAMIN K
Unusual because adequate amounts are generally produced by intestinal

bacteria or obtained from the diet
Possible causes:
Decrease in the intestinal bacterial population (eg. Antibiotics-
Trimethoprim/sulfamethoxazole and ciprofloxacin are common causes),
combined with marginal malnourishment
Fat malabsorption:
• Associated with pancreatic dysfunction, biliary disease or atrophy of the
intestinal mucosa.
• Vitamins A & E compete for absorption with vitamin K. Excess of these
vitamins can decrease vitamin K absorption.
Breast-fed newborns (have sterile intestine) (little intestinal flora, breast milk
very low in vitamin K), especially in a home-birth where a postnatal injection
of vitamin K may not be given. Vitamin K does not cross the placenta.
Infants whose mothers have been treated with certain anticonvulsants during
pregnancy such as phenytoin (Dilantin).
VITAMIN K
Signs and symptoms vitamin K deficiency may include:
• Easy bruising
• Oozing from nose or gums
• Excessive bleeding from wounds, punctures, and injection or
surgical sites
• Heavy menstrual periods
• Bleeding from the gastrointestinal (GI) tract
• Blood in the urine and/or stool
• Increased prothrombin time (PT)
Hypoprothrombinemia
Deficiency in prothrombin (factor II)
Increased coagulation times, bleeding tendency - ↑ PT but normal
bleeding time.
NUTRITION (III)

MINERALS
Minerals: The chemical elements required for normal function of the body
(generally excluding C, H, N, O, S).
May be divided into 2 groups:
Macrominerals: Required in amounts greater than 100 mg/day. More than
0.05% body mass.
Microminerals: (trace/ ultratrace elements) Required in amounts less than
100 mg/day. Less than 0.05% body mass.
Ultratrace minerals < 0.0001% of body mass.
In general, require carrier proteins for absorption.
Transport and storage also require special proteins.
Excretion occurs in the feces (unabsorbed minerals and from bile) and urine
and sweat.
Excess intake produces toxic symptoms
Dietary Requirements are met by a varied intake of adequate amounts of
whole-grain cereals, legumes, leafy vegetables, meat, and dairy products
GENERAL FUNCTIONS OF MINERALS
Components of essential body compounds

Ca: bones and teeth, Cl: HCl in stomach, I: hormone thyroxine
Cofactors in biological reactions
Mg, Fe, Zn, Cu: metalloenzymes, metalloproteins (hemoglobin)
P: protein regulation
Facilitate absorption, digestion and transport
Na, Mg: carbohydrate, Ca: vitamin B12
Acid-Base balance
Na, K: alkali forming, Cl: acid forming
Water balance
Na, K, Cl: intracellular and extracellular osmotic pressures
Nerve Impulses, Muscle Contraction
Na, Ca: action potentials
ESSENTIAL MACROMINERALS
Calcium (Ca)
Total Calcium in human body: 1 - 1.5 Kg (bones - 99 %, extra cellular fluid - 1 %).
Absorption: Duodenum, against concentration gradient, requires carrier protein.
Sources: Milk, Egg, Fish, Vegetables – moderate. The RDA is 700mg/day.
Most important quantitative function is as calcium hydroxyl apatite:

3[Ca3(PO4)2].Ca(OH)2
This forms the structure of bone and teeth and the vast majority of body calcium is in this
form.
• Plasma-free calcium must be maintained within a very narrow range
– Hypercalcemia - depression of the nervous system- slower reflexes
– Hypocalcemia - excitability of the nervous system - is muscle tetany.
The interstitial fluid is almost entirely free calcium but in the plasma only about 50% is free,
50% is bound mainly to protein.
ECF phosphate can change
Phosphate circulates mainly as H2PO4-2 along with a small amount of H2PO4-(acidemia
increases the proportion of the latter).
Calcium (Ca) Functions
1. Bones & Teeth : 3. Nerve conduction

• Formation of bone & teeth. • From presynaptic to
• Bones are reservoir for Ca in postsynaptic region
the body. 4. Secretion of hormones
• Osteoblasts -» bone deposition • Insulin, PTH, Calcitonin, ADH
• Osteoclasts -» 5. Second messenger
demineralization. 6. Membrane integrity
2. Muscle contraction 7. Blood coagulation
• Ca interacts with Troponin C to • Factor IV
trigger muscle contraction 8. Prolongs heart systole
• Ca activates ATP-ase, 9. Activation of enzymes
↑interaction between actin • Calmodulin
and myosin
FORMS OF Ca2+ IN BLOOD
The total Ca2+ concentration in blood is normally
10 mg/dL
• 40% is bound to plasma proteins, mainly
albumin.
• 60% is not protein bound - ultrafilterable.
10% - complexed to anions (e.g., phosphate, sulfate,
•
citrate)
• 50% of the total - free , ionized Ca2+ the only
biologically active form
Hypocalcemia is a decrease in the plasma Ca2+
concentration.
Hypercalcemia is an increase in the plasma Ca2+
concentration.
Ø Alkalosis lowers free calcium by increasing
protein-binding - ↑ in pH - ↑ affinity of
albumin (↑ negative charge) to bind Ca2+ -
hypocalcemia
Ø Acidosis raises free calcium by decreasing
protein-binding.
CALCIUM HOMEOSTASIS
Ca2+ homeostasis
involves the coordinated
interaction of three
organ systems:
• bone
• Kidney
• Intestine
• and three hormones:
• PTH
• Calcitonin (decrease plasma Ca2+)
• vitamin D
• in a healthy person in Ca2+ balance, net excretion of Ca2+ by the kidney is
equal to net absorption of Ca2+ from the gastrointestinal tract.
PARATHYROID HORMONE
There are four parathyroid glands in humans, located in the neck under the thyroid gland.
The chief cells of the parathyroid glands synthesize and secrete PTH, a single-chain
polypeptide with 84 amino acids
Storage & release:
• synthesized on the ribosomes as prepro-PTH
• Storage in vesicles
• Cleavage of mature PTH into N and C terminal segments (circulation, liver)
• The molecule’s biologic activity resides entirely in the N terminal 34 amino acids.
• Pattern of secretion – diurnal variations with ↑ secretion at night
• T1/2 of PTH – 5-8 min, N-terminal fragment has much shorter T1/2, C-terminal
fragment – 6-12 h
Regulation of Parathyroid Hormone Secretion:
PTH is a peptide hormone released from the parathyroid glands in response to lowered
plasma free Ca2+ (the primary regulator of PTH)
In most cells, exocytosis depends on a rise in intracellular free calcium (insulin secretion,
ADH, Oxytocin secretion, etc.)
In the parathyroid gland, a fall in intracellular free calcium causes release.
↑ extracellular Ca2+ concentration - Ca2+ binds to the parathyroid cell membrane receptor
- ↑ phospholipase C - ↑ levels of IP3 /Ca2+ - ↓ PTH secretion.
PARATHYROID HORMONE
Regulation of Parathyroid Hormone Secretion:
• chronic (long-term) changes in plasma Ca2+
concentration alter transcription of the gene for
prepro-PTH synthesis and storage of PTH, and
growth the parathyroid glands.
• chronic hypocalcemia - causes secondary
hyperparathyroidism
• chronic hypercalcemia - causes decreased
synthesis and storage of PTH, increased
breakdown of stored PTH, and release of
inactive PTH fragments into the circulation.
The negative feedback relationship between plasma calcium and PTH secretion is highly
sigmoidal, with the steep portion of the curve representing the normal range of plasma free
calcium.
• ↓ magnesium - ↑ PTH
• ↑ Phosphate binds Ca2+ → ↓ blood [Ca++] → ↑ PTH secretion
• Vitamin D - ↓ rate of PTH transcription
PARATHYROID HORMONE
Actions of Parathyroid Hormone

• PTH has actions on bone, kidney, and
intestine, all of which are coordinated
to produce an increase in
2+
plasma Ca concentration
• The actions on bone and kidney
are direct and are mediated by
cAMP;
• the action on intestine is indirect,
via activation of vitamin D.
Activated protein kinases phosphorylate intracellular proteins - inhibition of Na+ -

phosphate cotransport - decreased phosphate reabsorption and phosphaturia
Parathyroid Hormone
• Actions of Parathyroid Hormone

• ↑ bone resorption of Ca2+ and PO43−.
• ↑ kidney reabsorption of Ca2+
in distal convoluted tubule.
• ↓ reabsorption of PO43−
in proximal convoluted tubule.
• ↑ 1,25-(OH)2D3 (calcitriol) production
by stimulating kidney 1α hydroxylase
in proximal convoluted tubule
PARATHYROID HORMONE

1. Bone
• PTH receptors are located on osteoblasts but not on osteoclasts.
• Initially and transiently, causes an increase in bone formation by a direct
action on osteoblasts
• In a second, long-lasting action on osteoclasts, PTH causes an increase in bone
resorption ( indirect and mediated by cytokines released from osteoblasts)
• Binding of PTH stimulates the osteoblast to release RANK-L. This in turn, increases
osteoclastic activity resulting in bone resorption and the release of calcium and
phosphate into the blood
• The overall effect of PTH on bone is to promote bone resorption, delivering
both Ca2+ and phosphate to ECF.
Parathyroid Hormone
2. Kidney
• inhibits phosphate reabsorption by
inhibiting Na+-phosphate cotransport
in the proximal convoluted tubule -
phosphaturia, urinary cAMP
• stimulates Ca2+ reabsorption Stimulation of
calcitriol formation
(Excreting phosphate in urine “allows” via activation of 1α-
hydroxylase
the plasma ionized Ca2+ concentration (proximal tubule)
to increase) ↓ proximal tubular

phosphate reabsorption (↓
3. Small intestine tubular Tm) – inhibition of
the Na-Pi cotransporter
• indirectly stimulates intestinal (cAMP mediated effect → ↑

urinary excretion of cAMP)
↑ Ca2+
reabsorption mainly
Ca2+ absorption via activation of in the distal
convoluted tubules
(↑ tubular Tm) (vit
vitamin D (stimulates renal 1α - D has synergetic
action)
hydroxylase, the enzyme that converts
25-hydroxycholecalciferol to the active
form, 1,25-dihydroxycholecalciferol)
Parathyroid Hormone-Related Peptide
• PTHrP is a paracrine factor secreted by many tissues (lung, mammary tissue,

placenta)
• It may have a role in fetal development.
• In postnatal life, its role is unclear.
• The majority of humoral hypercalcemias of malignancy are due to

overexpression of PTHrP.
• PTHrP has a strong structural homology to PTH and binds with equal affinity
to the PTH receptor.
CALCITONIN
• It is synthesized and secreted by the parafollicular or C (“C” for calcitonin) cells of the thyroid
gland.
• Preprocalcitonin – procalcitonin - calcitonin, is stored in secretory granules for subsequent
release.
• Regulation of secretion - major stimulus for calcitonin secretion is increased plasma
Ca2+ concentration
• Actions:
• lowers plasma calcium by decreasing the activity of osteoclasts, decreasing bone resorption.
• Calcitonin is useful in the treatment of Paget’s disease, severe hypercalcemia, and
osteoporosis.
• Calcitonin is not a major controller of Ca2+ in humans.
• !!! Removing the thyroid (with the C cells) or excess of calcitonin via a C cell tumor (medullary
carcinoma of the thyroid) has little impact on plasma calcium.
• No deficiency or excess disease has been described.
Vitamin D
Regulation of Vitamin D Synthesis
! depends on the “status” of Ca2+ in the body
• When Ca2+ is sufficient (adequate dietary intake and N or increased

plasma Ca2+) - the inactive metabolite is preferentially synthesized
• When Ca2+ is insufficient (low dietary intake, decreased plasma Ca2+ ) -

the active metabolite is preferentially synthesized to ensure that
additional Ca2+ will be absorbed from the gastrointestinal tract.
• 1α -Hydroxylase activity is increased by:
• decreased plasma Ca2+ concentration,
• increased circulating levels of PTH, and
• decreased plasma phosphate concentration.
Vitamin D
Actions of Vitamin D
•1. Intestine
• increases Ca2+ absorption
1. Ca2+ diffuses from the lumen
into the cell, down its
electrochemical gradient
2. It is bound inside the cell to
calbindin D-28K
3. subsequently is pumped
across the basolateral
membrane by a Ca2+ - ATPase
•increases PO4 absorption
• Na-Pi cotransporter →↑ Pi
entry into the mucosal cells
Vitamin D
Actions of Vitamin D
2. Kidney
• - parallel to its actions on the intestine - it stimulates both Ca2+ and phosphate
reabsorption.
3. Bone
• synergistically with PTH - stimulate osteoclast activity and bone resorption.
• mineralized “old” bone is resorbed to provide more Ca2+ and phosphate to ECF
so that “new” bone can be mineralized
• Indirect action
• ↑ calcification of newly formed bones (indirect antirachitic action)
primarily by intestinal and renal actions and by rising plasma [Ca2+ ] &
[HPO42-] ® osteoid calcification
Disorders In Calcium And Phosphate
Hyperparathyroidism
PRIMARY HYPERPARATHYRODISM
Increased PTH secretion.
Primary hyperparathyroidism is the main cause or hypercalcemia.
• In most cases caused by a single parathyroid adenoma (80%).
• The remainder generally are hyperplasia of the parathyroids.
Screening involves measuring plasma Ca++
• Plasma:
• ↑ PTH, ↑ Ca++ , ↓ PO4 (increased renal secreation)
• Patients most often do not have symptoms.
• Consequences include excessive bone turnover (↑ alkaline phosphatase).
• Typical consequence is osteitis fibrosa cystia (cystic bones spaces filled with brown
fibrous tissue).
• Increased scalloped areas of bone with replacement containing fibrous tissue.
Decreased bone mass
PRIMARY HYPERPARATHYRODISM
Renal function is compromised - reduced ability to concentrate the urine. Even
though PTH Is elevated, the filtered load of calcium is elevated and calcium
appears in the urine
• Calcium stones in kidney, polyuria - dehydration.
• Increased cAMP in urine (second messenger of PTH).
High calcium can lead to nephrogenic diabetes insipidus. This is why there is
massive volume deficit in hypercalcemia.
Symptoms of hypercalcemia, when they appear, focus on CNS depression. High

calcium makes it harder to depolarize neural tissue
• fatigue,
• lethargy,
• neuromuscular weakness,
• mental depression (psychiatric overtones)
• confusion, and constipation (groans)
• Ectopic hypersecretion Is PTHrP.
• Hypercalcemia decreases QT interval and in some cases causes cardiac
arrhythmias.
DISORDERS IN CALCIUM AND PHOSPHATE
Related causes of hypercalcemia
• Lithium shifts the sigmoid Ca/PTH curve to the right.
• Higher calcium levels are thus needed to suppress PTH. Similarly, the CaSR is mutated in
patients with familial hypocalciuric hypercalcemia, resulting in more PTH for any given
calcium concentration in the plasma.
• Sarcoidosis and other granulomatous disorders (10%) due to increased activity of 1-alpha
hydroxylase activity in granulomas.
• Thyrotoxicosis, milk-alkali syndrome
• Thiazide diuretics increase renal calcium absorption.
TREATMENT
• usually surgery - removing the adenoma or with hyperplasia removing most of the
parathyroid tissue
• Treat with high volume fluid replacement
• Bisphosphonates need 2–3 days to be fully effective
• Calcitonin rapidly inhibits osteoclastic activity
Hyperparathyrodism
Secondary Hyperparathyroidism of Chronic Renal Failure
Origin is the hyperphosphatemia of renal failure.

The ↑ PO4 induces hypocalcemia and the ↓ Ca++ drives a secondary
hyperparathyroidism.
As long as the PO4 remains elevated, the PTH cannot elevate the plasma Ca++
adequately.
• Plasma:
• ↑ PO4 , ↓ Ca++, ↑ PTH plus signs of renal failure.
Hyperparathyrodism
Secondary Hyperparathyroidism of Vitamin D Deficiency
Origin is a dietary deficiency in vitamin D and/or inadequate sunlight exposure,
which lowers plasma calcium.
• Secondary hyperparathyroidism due to the decrease in plasma calcium.
• The increased excretion of PO4 causes the ↓ plasma PO4
• Plasma:
• ↓ Ca++, ↑ PTH, ↓ PO4
• Low plasma 25-hydroxy vitamin D diagnostic for the disorder.
• The increased PTH increases bone resorption to help maintain plasma calcium.
Consequence:
• in children is rickets, and osteomalacia in adults.
Treatment is a vitamin D supplement to elevate circulating 25-hydroxy vitamin D.
Note: This will not work for chronic renal failure. Because of the 1a-hydroxylase
deficiency, 1,25-dihydroxy vitamin D replacement is more appropriate.
Hypoparathyroidism
PRIMARY HYPOPARATHYROIDISM
The problem is inadequate PTH secretion . The most common cause Is surgery on
the neck: Thyroidectomy, partial parathyroidectomy, cancer surgery.
Plasma:
• ↓ PTH, ↓ Ca ++, ↑ PO4
Phosphate increases because the phosphaturic effect of PTH is lost.

Most or the symptoms occur because of hypocalcemia-induced increased
neuromuscular excitability.
• Tetany (not unique to hypocalcemia) - also occurs with hypomagnesemia.
• Seizures
Classic sign is carpopedal spasm. The muscle contractions are painful.

• Trousseau sign: Elicited from an inflated pressure cuff to 20 mmHg above systolic
blood pressure. Produces carpal spasm.
• Chvostek sign : Tapping the facial nerve causes the facial muscles to contract
(specificity of the test is low).
Hypoparathyroidism
Carpopedal spasm Trousseau sign

Hypoparathyroidism
Primary Hypoparathyroidism
Hyperventilation causes tetany

Hyperventilation increases plasma pH due to Respiratory alkalosis. At this alkaline pH,
plasma proteins are more ionized, these protein anions bind with Calcium
This in turn leads to a decreased Serum ionic Calcium levels causing a clinical condition
known as Tetany.
Pseudohypoparathyroidism
This is a rare familial disorder characterized by target tissue resistance to parathyroid
hormone.
Exhibits same signs and symptoms as primary hypoparathyroidism except PTH elevated
It is usually accompanied by developmental defects: mental retardation, short and
stocky stature, one or more metacarpal or metatarsal bones missing (short 4th or 5th
finger).
Hypoparathyroidism
Secondary Hypoparathyroidism of Excess Vitamin D
• The excess vitamin D raises the plasma calcium, which induces the
secondary hypoparathyroidism
• Plasma:
• ↑ Ca ++, ↑ PO4 ↓ PTH,
• The elevated phosphate is due to the decreased phosphate excretion
by the kidney.
High plasma 25-hydroxy vitamin D is diagnostic for the disorder.
• One of the toxic effects of vitamin D is that it increases the activity of
osteoclasts and increases bone resorption.
As a result, there is a negative calcium balance and bone loss
Calcium (Ca)
Symptoms of hypercalcemia:
•Loss of appetite
•Nausea and vomiting
•Constipation and abdominal pain
•Increased thirst and frequent urination
•Fatigue, weakness, and muscle pain
•Confusion, disorientation, and difficulty thinking
•Headaches
•Depression
Symptoms of severe hypercalcemia may include:
•Kidney stones, a painful condition in which salt and minerals form solid
masses called “stones” in the kidneys or urinary tract
•Irregular heartbeat
•Heart attack
•Loss of consciousness
•Coma
Functions: Phosphorus (P)
Constituent of bones, teeth (hydroxyapatite)
Energy & component of ATP, nucleic acids
Phosphorylated metabolic intermediates
Absorption of nutrients
Regulation of protein activity
Acid-Base Balance (buffering)
Metabolism :
Regulated by Parathyroid Hormone, Vitamin D
vitD: ↑intestinal absorption;
PTH: ↑ kidney excretion – net loss of P
Serum levels regulated by kidney reabsorption
Deficiency has never been shown as it is so common in foods. The RDA is
550mg/day.
An important consideration is the dietary calcium:phosphorus ratio.
Should be 1.1-1.3:1.0.
Phosphorus (P)
Deficiency states (rare): hypophosphatemia
• Rickets in children, phosphate diabetes, Fanconi syndrom
• Osteomalacia in adults
Toxicity states: hyperphosphatemia, caused by:
• hypoparathyroidism (low PTH)
• renal failure (reduced excretion)
An important consideration is the

dietary calcium:phosphorus ratio.
Should be 1.1-1.3:1.0
Too high and calcification of soft tissues can result.
Too low and highly acid urine can damage kidneys.

Sodium (Na)
Functions:
• Principal cation in extracellular fluid
• Regulates plasma volume, acid-base balance, nerve and muscle
function, Na+/K+-ATPase, glucose absorption
Metabolism :
• Regulated by hormone aldosterone – stimulates reabsorption of Na in
kidneys – increases blood levels, decreases excretion in urine.
• Also by enzyme renin - secreted by kidney in response to low blood Na
converts angiotensinogen into angiotensin I angiotensin I is
converted into angiotensin II, stimulates aldosterone secretion from
adrenal gland.
Sources: NaCl - Table salt, added to prepared food, naturally in food.

Deficiency states:
Sodium (Na)
Hyponatremia: Unknown on normal diet.
• Usually caused by dilution by excess water, not sodium deficiency
• May occur secondary to injury or illness (↑ADH, 21-hydroxylase
deficiency).
Symptoms: nausea and vomiting, headache, fatigue, loss of appetite,
restlessness and irritability, muscle weakness, spasms, or cramps,
convulsions, confusion and decreased consciousness or coma.
Toxicity states:
Hypernatremia: usually caused by insufficient water (dehydration), rarely
from sodium intake, 17-hydroxylase deficiency, Cuchings disease
Symptoms: hypertension (in susceptible individuals), lethargy,
weakness, irritability, neuromuscular excitability, and edema
convulsions and coma in severe cases.
Potassium (K)
Functions:
• Principal cation in intracellular fluid (acts with Na to maintain water
balance).
• Regulates intracellular osmotic pressure, acid-base balance, nerve
and muscle function, Na+/K+-ATPase, glycogen synthesis.
Metabolism: regulated by aldosterone (stimulates excretion of K into the
urine). Lowers blood K levels.
Sources:
• Many foods, highest in some vegetables, fruits and nuts
• food additives: KNO2 KNO3 (preservative), KCl (salt substitute)
Potassium (K)
Deficiency states:
Hypokalemia: Occurs secondary to illness, injury, or diuretic therapy.
eg. Diarrhea in infants.
Symptoms: cardiac dysrhythmia, constipation, muscular weakness,
paralysis, fatigue, mental confusion.
Toxicity states:
Hyperkalemia: Caused by renal insufficiency, medications that block
excretion, Mineralocorticoid deficiency (Addison’s disease), 21-
hydroxylase deficiency.
Symptoms: generally asymptomatic, nausea, muscle weakness,

cardiac arrhythmia, cardiac arrest (KCl used in lethal injection).
Chloride (Cl)
Functions:
• Fluid and electrolyte balance (counter ion to Na and K)
• Gastric fluid, cerebrospinal fluid
• Primarily extracellular
Deficiency states:
Hypochloremia: rarely occurs in isolation
Secondary to vomiting, diuretic therapy, renal disease.
• few symptoms - dehydration, fluid loss
• in infants: anorexia, lethargy, weakness
Sources: Table salt, water supply (chlorination)
Functions: Magnesium (Mg)
• constituent of bones, teeth (60% of body magnesium).
• enzyme cofactor - enzymes that utilize or synthesize ATP (kinases,
polymerases, ATPases…)
• antagonizes calcium in nerve and muscle action potentials
Deficiency states:
Hypomagnesemia: secondary to malabsorption or diarrhea, alcoholism.
Symptoms: irritability and fatigue, nervousness, nystagmus,
convulsions (overstimulation of nerves), muscular contraction (spasms
or cramps).
Toxicity states:
Hypermagnesemia – often due to renal failure (depressed deep tendon
reflexes and respiration)
Sources: Most foods, Green leafy vegetables (containing chlorophyll).
Magnesium and calcium compete for a transporter mechanism in the GIT,
thus magnesium deficiency may result from excess dietary calcium.
Sulphur (S)
Sulphur functions in sulphydryl (cysteine/cystine) bridges in
proteins, as part of coenzyme A, thiamine and biotin, as well as in
methionine.
It is also important as part of collagen and glutathione structures.
Again it is very common in foods, especially protein rich foods like

beans, so deficiency has never been proven. The RDA is
800mg/day.
ESSENTIAL MICROMINERALS TRACE ELEMENTS
Functions: Copper (Cu)
Constituent of metalloenzymes:
•Lysyl oxidase (collagen synthesis)
•Cytochrome C oxidase (electron transport chain)
•Superoxide dismutase (oxidative stress)
•Dopamine β-hydroxylase (norepinephrine synthesis)
•tyrosinase (catecholamine biosynthesis)
•MAO (catecholamines degradation)
Role in iron absorption: Ceruloplasmin (oxidizes iron from Fe+2 to Fe+3 to
enable binding to transferrin)
Metabolism :
Absorbed through all of gastrointestinal trackt
Removed from plasma by liver
• Ceruloplasmin synthesis
• Stored in protein complex
• Excreted in bile
Major carrier in plasma is ceruloplasmin
Deficiency states: Rare
• Secondary to Zn Toxicity
• Secondary to Menke’s syndrome
• Infants on total parenteral nutrition Menke’s syndrome
X-linked, recessive disorder of Cu Metabolism
(Mutation in ATP7A gene)
Clinical Presentation:
Onset during infancy (primarily males).
Hypotonia, sagging facial features, osteoporosis,
seizures, mental retardation, developmental
delay, failure to thrive, anemia, arterial tortuosity
Unusual hair: kinky, brittle, colorless or steel
color. Often fatal within first decade.
Normal Function: ATP7A - widely distributed Cu2+ cation pump. P-type ATPase.
Transfers Cu to Golgi for Cu-containing metalloenzymes.
Mutations: Copper can be absorbed into the mucosal cell, but it cannot be
transported into the bloodstream.
Toxicity states: Rare. Secondary to Wilson’s disease
Wilson’s Disease (hepatolenticular degeneration)
Autosomal recessive disorder of Cu Metabolism. Inadequate hepatic copper
excretion and failure of copper to enter circulation as ceruloplasmin.
Accumulation of Cu in Tissues (especially in liver, brain, cornea, kidneys, and
joints).
Clinical Presentation: “Copper is Hella BAD.”
• ↓ Ceruloplasmin,
• Cirrhosis,
• Corneal deposits (Kayser-Fleischer rings),
• Copper accumulation,
• Carcinoma (hepatocellular)
• Hemolytic anemia
• Basal ganglia degeneration (parkinsonian symptoms)
• Asterixis
• Dementia,
• Dyskinesia,
• Dysarthria
Functions:
Iodine (I)
• Dietary iodine is efficiently absorbed and transported
to the thyroid gland
• In the thyroid gland: utilized for synthesis of T3 and T4. These
hormones function in regulating basal metabolic rate of adults and in
the growth and development of children.
Metabolism: Stored in thyroid as thyroglobulin
Sources: Iodized salt,
saltwater seafood, in
other foods levels vary
depending on location
that they were grown,
spinach leaves.
Iodine (I)
Iodine (I)
Deficiency states:
• Neonatal hypothyroidism (Cretinism) in
children - stunted physical and mental growth.
• Goiter (enlargement of the thyroid gland) and
hypothyroidism in adults
Toxicity states: Thyrotoxicosis (increased thyroid

hormones in the blood)
Symptoms associated with increased metabolic

rates: weight loss, increased appetite, anxiety,
intolerance to heat, hair loss, muscle aches,
weakness, fatigue, hyperactivity, irritability,
hypoglycemia
Functions: Iron (Fe)
• Constituent of Heme (hemoglobin, cytochromes, etc.)
• Cofactor in Heme and non- Heme containing proteins
Metabolism:
• Transported as transferrin
• Stored as ferritin or hemosiderin (binds excess Fe3+ to prevent
escape of free Fe3+ into the blood, where it is toxic)
It is lost from the body with bleeding, sloughed-off cells, sweat, urine, and
feces. ceruloplasmin (a Cu2+ protein) oxidizes Fe2+
to Fe3+ for transport and storage
Hemosiderin is the amorphous iron

deposition adjacent to ferritin spheres,
binds excess Fe3+ to prevent escape of
free Fe3+ into the blood, where it is
toxic.
Iron Deficiency: Anemia
Anemia: reduction in the number, size, & color of RBC’s
microcytic hypochromic anemia
Symptoms: Weakness, paleness, headaches, reduced immunity, inability to
concentrate, cold intolerance, tachycardia, conjunctival pallor, fatigue,
koilonychia, a smooth tongue, and low serum ferritin levels, and pica
(an eating disorder in which the patient will have a craving for items such
as ice chips and dirt.)
Causes:
Decreased iron intake, achlorhydria
Increased iron loss: GI bleeding, Excessive menstrual flow, blood donation
Increased iron needs: infancy, pregnancy, lactation, riboflavin deficiency
Unknown (idiopathic hypochromic anemia)
Colon cancer often causes iron deficiency anemia in elderly
patients because of chronic intestinal blood loss.
Iron Overload
Hemochromatosis
Primary - Hereditary Hemochromatosis
Secondary - Hemophilic children (require
repeated blood transfusion and excessive
parenteral iron intake).
Hemosiderosis is the deposition of
hemosiderin (iron);
And hemochromatosis is the disease caused
by this iron deposition.
Classic triad of micronodular Cirrhosis(liver
cell death), Diabetes mellitus(pancreatic cell
death), and skin pigmentation “bronze”
diabetes.
Zinc (Zn)
Storage in liver with a specific protein metallothionine
Functions:
• Cofactor of many enzymes (lactate dehydrogenase, alkaline
phosphatase, carbonic anhydrase, ALA dehydratase is Zn-containing
enzyme, sensitive to inhibition by lead, nterstitial collagenas).
• Formation of zinc fingers - type of DNA Binding domain. In transcription
factors like the Nuclear Receptor Family (receptors for Steroid Hormones,
Retinoic Acid, Thyroid Hormones, Vitamin D).
• Stabilizes insulin when stored in b-cells.
• Promotes synthesis of Retinol-binding protein.
• Gusten, Zn containing protein in saliva, is important for taste sensation
• Role in growth, reproduction and wound healing.
Sources: Widely distributed in foods, better absorbed from animal products.
Meat, poultry, fish, dairy, whole grain cereals.
Zinc (Zn)
Deficiency states:
• Secondary to acrodermatitis enteropathica or
parenteral nutrition without Zn
• Approximately 60% of the
total body zinc content is located within
muscle. Therefore, conditions associated with
muscle catabolism are associated with marked
release of zinc and subsequent increased
excretion in the urine. Zinc deficiency can also
develop whenever there are protracted losses
of gastrointestinal secretions.
• Dermatitis (In acrodermatitis enteropathica)
• Hypogonadism, growth failure, impaired wound
healing, impaired immune function, decreased
taste and smell acuity
Functions:
Selenium
• Constituent of glutathione peroxidase (breakdown of peroxides, synergistic
antioxidant with vitamin E).
• Type I iodothyronine-5-deiodinase (removes Iodine from T4 to yield T3)
Metabolism: two forms. Protein carrier in plasma, excreted in urine and feces.
• organic - selenomethionine, selenocysteine from foods
• inorganic – selenium from supplements
Deficiency states:
• Marginal deficiency when soil content is low
• Secondary to parenteral nutrition, protein energy malnutrition
• results in high levels of T4 (thyroxine)
Toxicity states- selenosis:
• Toxic levels in some soil
• Megadose supplementation
• Hair loss, dermatitis, and irritability
• Garlic, breath
Sources: Organ meats, meats, cereals and grains - varies with soil content
ESSENTIAL MICROMINERALS
ULTRATRACE ELEMENTS
Chromium (Cr)
Functions: Constituent of “glucose tolerance factor” that binds
to and potentiates insulin.
Deficiency states:
• Impaired glucose tolerance
• Secondary to parenteral nutrition
Toxicity states:
• from contaminated drinking water or pollutants (not diet)
• effects vary depending on the compound
Sources: Meat, liver, whole grains, nuts, cheese, brown sugar
Cobalt (Co)
Functions: Required only as a constituent of vitamin B12
Metabolism: As for vitamin B12
Deficiency states: Vitamin B12 deficiency
Toxicity states:
• Previously a beer additive. Excessive consumption - toxicity
• Goitrogenic
• Polycythemia (increased red blood cells)
Sources: Foods of animal origin: organ meats, meats, dairy
Molybdenum (Mo)
Functions: Constituent of oxidase enzymes
• xanthine oxidase
• aldehyde oxidase
• sulfite oxidase
Deficiency states: Secondary to parenteral nutrition (only one reported
case)
Manganese (Mn)
Functions:
• Cofactor of hydrolase, decarboxylase, and transferase enzymes
• Required for:
• Glycoprotein and proteoglycan synthesis
• Mitochodrial superoxide dismutase
Metabolism
• Absorption similar to Fe (major Mn binding protein is transferrin)
• Levels regulated by bile excretion
Deficiency states: unknown
Toxicity states:
• Inhalation poisoning - psychotic symptoms and parkinsonism
Sources:
• Whole grain cereals and green vegetables
Fluoride (F)
Functions: Increases hardness of bone and teeth
Deficiency states:
• Dental caries
• osteoporosis?
Toxicity states: Dental fluorosis
• white deposits in enamel
• brown discoloration, pitting
• NaF is mildly toxic
Sources:
• Drinking water (natural or supplemented)
• Fluoridated toothpastes and rinses
• Food supply (tea and fish are rich sources)
The RDA is 4mg/day.
NUTRITION (IV)
NUTRITION
Nutrition - science that interprets the interaction of nutrients and other
substances in food in relation to maintenance, growth, reproduction,
health and disease of an organism. It includes food intake, absorption,
assimilation, biosynthesis, catabolism, and excretion.
• Many diseases are to a greater or lesser extent related to poor or
imbalanced nutrition.
• Some diseases are truly nutrition diseases, ie. the cause is a problem
with a nutrient and the cure is treatment involving that nutrient. In
some cases it may be more than one nutrient.
• Some diseases cause nutritional problems.
• Other diseases have nutritional components, ie. the etiology can be
improved or worsened by nutritional changes.
Very important to understand:
Both Undernutrition and Overnutrition are equally important forms
Malnutrition.
NUTRITION
Early studies in nutrition arose from observations of
deficiencies that could be cured by addition of a single
nutrient. This was how many of the micro-nutrients were
identified as such (Scurvy, Goitre, Beri-beri).
Not all the cases were deficiency-related.

Early 20th century Arctic explorers ignored advice from local Inuit
and ate polar bear liver (vitamin A poisoning).
FACTORS AFFECTING FOOD CHOICE
There are internal and external factors
affecting food selection.
Internal factors can be physiological -
hunger, satiety, appetite, aversion,
preference, emotion, personality.
External factors include culture, religion,
ethics, economics, controls, society,
education, media.
ASSESS THE ADEQUACY OF A DIET
An adequate diet should:
• Contain all nutrients and in appropriate amounts;
• Support health and allow for physical activity;
• Provide adequate reserves;
• Protect against disease.
All models of an adequate diet indicate that plant foods should
predominate, and provide about 73% of intake.
THE ADEQUACY OF A DIET
EU relies on the “plate” model with foods assigned to different
groups.
Cereals and starchy foods should be 33%, as should fruit and
vegetables. Meat and fish should be 12%, while dairy should be 15%.
The other 7% should be high energy foods rich in CHO or fat. The
different food groups have been defined as the following:
• Cereals/starches - bread, cereals, starchy
foods, potatoes.
• Fruit/vegetables - fruit and vegetables in
any form.
• Meat/fish - meat, fish, eggs, pulses.
• Dairy - milk, cheese, yoghurt, etc.
• High energy - all foods with high levels of
fat or CHO, especially concentrated foods.
One problem is how to deal with food combinations?
THE ADEQUACY OF A DIET
Another way to classify foods is into core,
secondary and peripheral foods.
Core foods usually occur several times a day
and/or form the basis of the meal, eg. rice,
potatoes.
Secondary foods enhance the core by filling
in any nutritional gaps, eg. fish stew, steak.
Peripheral foods are not essential but may
be a pleasant addition, eg. ice cream, tea,
coffee.
The bottom line is everyone's diet is different to some extent, so all
guides are just that, guides.
Each individual's nutrient requirements are different and may vary with
other circumstances, thus any assessment should be viewed in this light.
If no symptomology of deficiency, no dramatic mass change with time,
no clinical reason, then no intervention.
APPROPRIATE AMOUNTS OF NUTRIENTS
Highly variable between individuals, but are normally distributed about
the mean. Every nutrient is used by the body every day and needs
replacement.
The urgency of replacement depends on the amount and whether the
body can store the nutrient.
The requirement is defined as the amount to avoid clinical deficiency.
USA and EU this is the recommended daily allowance (RDA). In the UK
its the reference nutrient intake (RNI). Same number different name.
Too high or low an intake will cause malnutrition. This is the estimated
average requirement (EAR).
IRON EAR RNI/RDA
MALES 6.7MG 8.7MG
FEMALES 11.4MG 14.8MG
CHO
MALES 90G 150G
FEMALES 80G 135G
FOOD REACTIONS
There are several adverse reactions to food:
• Food intolerance (including allergic);
• Food poisoning;
• Food aversion.
FOOD INTOLERANCE covers a variety of responses:
• allergic,
• pharmacological
• enzymatic.
Allergic reactions can be IgE-mediated, and if so, lead to anaphylactic
shock via histamine release. Legislation requires the identification of
allergens on food labels:
• wheat, rye, barley, oats, spelt and various hybridised strains (because
of gluten);
• almonds, hazelnuts, walnuts, cashews, pecans, brazils, pistachios,
macadamias, Queenslands (because of nut proteins);
• crustaceans, eggs, fish, peanuts, soybeans, milk, celery, mustard,
sesame, SO2 and sulphites (because of various allergens).
FOOD REACTIONS
FOOD INTOLERANCE covers a variety of responses:
• allergic,
• pharmacological
• enzymatic.
Allergic reactions can be IgE-mediated, and if so, lead to anaphylactic
shock via histamine release.
Current legislation requires the identification of a wide range of
allergens on food labels:
• wheat, rye, barley, oats, spelt and various hybridised strains (because
of gluten);
• almonds, hazelnuts, walnuts, cashews, pecans, brazils, pistachios,
macadamias, Queenslands (because of nut proteins);
• crustaceans, eggs, fish, peanuts, soybeans, milk, celery, mustard,
sesame, SO2 and sulphites (because of various allergens).
FOOD REACTIONS
Non-IgE mediated reactions are generally reactions within the
GIT itself and are often also gluten-related, eg. coeliac disease.
Some foods contain pharmacologically active compounds, egs. some
cheeses, wines, bananas, yeasts, avocados, oranges, chocolate,
tomatoes, strawberries and fish. Most of these stimulate histamine
release directly.
Enzyme defects are not common, but do occur.
Lactose intolerance reflects lack of lactase and inability to catabolise
lactose.
Fructose intolerance reflects a deficiency in entry of fructose into
glycolysis.
Galactose intolerance reflects a deficiency in the isomerisation of
galactose to glucose and causes galactosemia.
Alcohol dehydrogenase deficiency leads to impaired ability to catabolise
alcohol, often characterised by hot flushes of the skin.
FOOD POISONING
This is defined as any disease of an infectious or toxic nature
associated with food or water.
Usually food poisoning is a result of poor sanitation or poor food
preparation practices.
FOOD AVERSION
This is primarily a conditioned response and not related to a
reaction induced by consumption of the food, more the thought of
its consumption, eg. vegetable consumption in children.
However, it is real as it interferes with the consumption of a
normal diet.
NUTRIENTS IN GROWTH AND DEVELOPMENT
Proper nutrition is critical for normal fetal development, especially in
the early developmental stages.
Folate deficiency causes failure of the neural tube to close and results
in neural tube defects.
Iron deficiency is seen in pregnancy, although supplementation often
reverses this.
Calcium deficiency in pregnancy can be found where culture demands
skin coverage.
Breast milk composition is tailored to the needs of the infant human,
and changes with the length of lactation and even during a single
session.
Human milk contains much higher levels of n6 and n3 polys, lactose,
whey protein, taurine, IgA, and lower levels of NaCl compared to cows
milk.
NUTRIENTS IN GROWTH AND DEVELOPMENT
However, the body composition of a fetus is not the same as
that of a child or an adult.
NUTRIENTS IN AGEING
Recent trends are to living longer.
Ageing results from 3 factors in combination:
• genetics,
• lifestyle,
• nutrition.
The last 2 are under an individuals' at least partial control.
Elderly people:
Diverse group at risk for nutritional problems resulting from
combination of environmental, social, economic factors; compounded
by numerous physiologic changes occurring at different rates as
individuals age.
Risk factor checklist «DETERMINE» identifies several warning signs for

individuals at risk for poor nutritional status.
NUTRIENTS IN AGEING
“DETERMINE”
DISEASE: change in eating (hard to eat, cook, shop)
• Confusion-Memory loss (remember what/how to eat)
• Depression cause changes in appetite, energy level, weight
EATING POORLY: eating too little/too much, drinking too much alcohol,
not eating healthy foods each day. Diminished sense of taste/smell can
decrease appetite and influence food choices (↓ability taste salt)
Reduced sense of smell difficult elderly detect spoiled food (advised to
read all dates stamped on food products)
TOOTH LOSS OR MOUTH PAIN: loss teeth or have problems with their
mouth, teeth, gums.
ECONOMIC HARDSHIP: little food money may not eat enough food or
may eat foods insufficient vitamins, minerals, calories.
Buy prepackaged/convenience foods (typically high sodium, potassium,
sugar)
NUTRIENTS IN AGEING
REDUCED SOCIAL CONTACT: cooking/eating alone are hard
• not feel like shopping/preparing food they need.
• loss of a spouse, retirement, social isolation can lead to loneliness,
depression, lack of motivation to eat
MULTIPLE MEDICATIONS/DRUGS: drugs/other medications can depress
appetite, alter nutrient absorption/excretion
• drugs can further alter sense of taste/smell, change secretion saliva,
irritate stomach, cause nausea.
• some drugs contribute directly to dietary deficiencies
(antacids: absorb folic acid/calcium; laxatives: absorb fat-soluble
vitamins; aspirin: which increases excretion of folic acid)
INVOLUNTARY WEIGHT LOSS/GAIN
NEED FOR ASSISTANCE WITH SELF-CARE
ELDERLY: patients older than 80yr
ETHNIC GROUPS AND RELIGION AND NUTRIENTS
Human migration has always been around, and most groups take their
traditional foods and food preparation procedures with them.
In many cases this has enriched the variety of foods for the host
country, but in some cases the differences have led to marginalisation
and discrimination, especially if the food practices are seen as strange
or contrary to the host country.
Probably the most common practice is vegetarianism for cultural,

moral or religious reasons.
Vegetarianism in itself is acceptable nutritionally as long as the diet is
balanced.
One group of vegetarians, vegans, have nothing of animal origin and as
such make themselves more liable to B12 deficiency long term.
ANTHROPOMETRY
This is the comparative measurement of various parameters of the
human body, and the analysis of the data in relation to population
norms.
It is not possible to get accurate data on living people as to the absolute
composition of their body. There are a variety of means of
anthropometric analysis, all of differing degrees of accuracy, depending
on assumptions made about the individual.
• Skinfold measurements,
• Waist:hip ratio,
• Bioimpedance analysis (BIA),
• Body mass index (BMI),
• Underwater weighing (UWW),
• Dual energy X-ray absorptiometry (DEXA),
• Peripheral quantitative computerized tomography (PQCT),
• Air displacement plethysmography (ADP).
There are also several models of body composition with
varying degrees of complexity.
Two component model.
This is the most simple model, and all of the body falls
into one of two component substances: fat mass, and fat-
free mass.
Three component model.
This divides the body into three component substances:
fat mass, water mass, and other fat-free mass.
Four component model.
This divides the body into four component substances:
fat mass, water mass, protein mass, and mineral mass.
Which model is used depends on the data required from the analysis.
The simpler the model, the easier it is to get data, but the less
representative it is of the 'real' world, and the converse.
SKINFOLD MEASUREMENTS
There are 4 sites commonly used.
It is a two component model, assuming only
fat and non-fat.
It assesses the thickness of the skin fat
layer.
It is highly subjective unless an experienced
person is carrying out the technique.
SKINFOLD MEASUREMENTS
TRICEPS SKINFOLD ABDOMINAL SUBSCAPULAR

BICEPS SKINFOLD SKINFOLD SKINFOLD
WAIST TO HIP RATIO (WHR)
The waist and hip circumferences are measured and the ratio
calculated.
This is a measure of the distribution
of body fat. A WHR of <0.91 is
normal for males and <0.81 normal
for females. A WHR of 1 or higher
signals increased health risk.
WAIST TO HIP RATIO (WHR)
Android obesity (male pattern or abdominal obesity) = high risk.
Gynoid obesity (female pattern or gluteal obesity) = low risk.
BIOIMPEDANCE ANALYSIS (BIA)
BIA is a measure of the opposition to the flow of an electric
current through the tissues. The measurement of the
bioimpedance of humans and animals has proved useful as a
non-invasive method for measuring blood flow (often referred
to as bioimpedance plethysmography) and body composition.
Advantages:
• fast,
• portable,
• easy to use.
Disadvantages: cheaper units tend to consistently overestimate

lean people and underestimate obese people.
BODY MASS INDEX (BMI)
Equations:
BMI = weight in kilograms/(height in meters)2, or
BMI = weight in pounds/(height in inches)2 x 705
Females Males
Severely underweight: <16 <17
Underweight: 16-19 17-20
Normal: 19-24 20-25
Overweight: 24-29 25-30
Mild obesity: 29-34 30-35
Very obesity: 34-39 35-40
Severe obesity: >39 >40
BODY MASS INDEX
However, these are generalisations. Some people
have denser/larger bones and thus are heavier than the average. Others
have different body compositions.
UNDERWATER WEIGHING (UWW)
The dry weight of the subject is first determined, they then sit on a
seat, expel all the air from their lungs, and are lowered into a tank
until all body parts are emerged. The person must remain
motionless underwater while the underwater weight is recorded.
Advantages:
• most widely used test
of body density.
Disadvantages:
• equipment required
expensive,
• discomfort of subject.
DUAL ENERGY X-RAY ABSORPTIOMETRY (DEXA)
DEXA is a means of measuring bone mineral density (BMD). Two X-
ray beams with differing energy levels are aimed at the patient's
bones. When soft tissue absorption is subtracted out, the BMD can
be determined from the absorption of each beam by the bones.
DEXA scans can also be used to measure total body composition and
fat content, but it is recommended to be used in conjunction with
another method (eg. under water weighing)
Advantages: low radiation

exposure, quick, no preparation
on the part of the subject.
Limitations: expensive, most
accurate for bone density only.
PERIPHERAL QUANTITATIVE
COMPUTERIZED TOMOGRAPHY (PQCT)
PQCT is used for measuring bone mineral density (BMD) in a
peripheral part of the body. It is useful for measuring bone strength
unlike most other common techniques for measuring BMD, a PQCT
scan is able to measure volumetric bone mineral density, plus other
measures such as the stress-strain index (SSI) and the geometry of
the bone. DEXA is only able to provide the bone mineral density.
Advantages: can generate 3 dimensional

images, can estimate actual strength of
bone.
Limitations: expensive, subject must
remain still for 15 minutes.
AIR DISPLACEMENT
PLETHYSMOGRAPHY (ADP)
The ADP system measures body composition by determining body
volume and body weight. Body weight is measured first, then body
volume is measured by first measuring the volume of the chamber
while empty. Then the volume of the subject chamber is measured
with the subject inside. Once body volume and weight are
determined, body density can be calculated.
Advantages:
• portable,
• subject can breath during
test,
• suited for special needs
subjects.
Disadvantages: very expensive.
ENERGY SOURCE AND USE
Energy is inherent in the chemical bonds of organic molecules.
This energy is liberated during metabolism, conserved as specific
chemical bonds and then used to drive the processes required by
the human body.
While most of this energy appears initially as internal heat, some
may be used to perform physical activities and any excess above
immediate requirements is stored as fuel molecules for future use.
The only chemical energy losses are in the form of excretion
products lost through the urine.
The balance is:

internal heat produced + energy stored + external work
energy + urine losses = energy of food absorbed.
CALORIC VALUE OF FOODS
Definition: Caloric value is defined as amount of heat- energy
obtained by burning 1.0 gm of the food stuff completely in the
presence of O2.
Unit of Energy: The unit of energy is calorie (kilo-calorie) - It is defined
as the amount of heat required to raise the temperature of 1.0 gm of
water by 1oC
Taking into consideration, the
variations in caloric value of individual
carbohydrate/fat/protein, their
average energy value when
metabolized may be represented as
follows in:
Mr. X consumes a daily average of
- 500g carbohydrates - 195g protein
- 70g fat - 20g alcohol
What is his daily energy intake? 2000+780+630+140=3550 kcal
CALORIC REQUIREMENTS
To maintain caloric balance in an adult, it is necessary to supply
enough foods to replace the calories expended per day.
These include the following:
üA supply of “basal” requirements (BMR)
üSupplies of calories to meet the extra requirement caused by
specific dynamic action (SDA), also called “calorigenic action” of
foods.
The amount of energy required for any individual varies directly with
the degree of activity and environmental conditions, but the rate of
energy production in an individual by its over-all cellular metabolism
is more or less constant under some standard conditions basal
conditions and is known as basal metabolism.
Basal/Resting metabolic rate (BMR/RMR) - energy expenditure
necessary to maintain basic physiologic functions while at rest in a
post-absorptive state. Measured under standardized conditions.
FACTORS THAT AFFECT BMR
METHODS FOR CALCULATING BMR
Rough Estimate: BMR = 24 x weight in kg (kcal/day)
Gender Specific Estimate:

BMR women = 24 x 0.9 x weight in kg (kcal/day)
BMR men = 24 x 1.0 x weight in kg (kcal/day)
Owen equations:
BMRwomen= 795 + (7.18 x weight in kg)
BMRmen= 879 + (10.2 x weight in kg)
Harris and Benedict Equations:

BMRwomen= 655 + (9.6 x W) + (1.8 x H) – (4.7 x A)
BMRmen= 66 + (13.7 x W) + (5 x H) – (6.8 x A)
Where W is weight in kg, H is height in cm, and A is age in yrs.

PATHOLOGICAL VARIATIONS IN BMR
1. Fever: Infections and febrile diseases elevate the BMR, usually in
proportion to the increase in temperature.
2. Diseases: They are characterized by increased activity of cells, also
increased heat production due to increased cellular activity.
• Leukaemias (21 to 80%)
• Polycythemia (10 to 40%)
• Some types of anaemias, cardiac failure, hypertension (25-80%).
3. Endocrine diseases: The most important factor which alters BMR is
the state of function of thyroid.
In hyperthyroidism: BMR is increased to +75% or more.
In hypothyroidism (myxoedema): BMR is reduced to 40% or more.
BMR increased - Cushing’s disease, Cushing’s syndrome, Acromegaly.
BMR is decreased - Addison’s disease (Hypofunction of Adrenal
cortex).
SPECIFIC DYNAMIC ACTION (SDA)
In an adult individual, whose BMR is 1600 C, is fed with just enough
food to provide 1600 C and is kept under basal conditions (except that
he is not under post absorptive state), it is found that his energy
output has increased beyond the basal output of 1600.
The increase varies with the type of food that has supplied the calories.
This stimulant action of foods on the metabolism is known as the
“specific dynamic action” (SDA) or calorigenic action of food.
Definition: SDA may be defined as “extra heat” production, over and
above the actual heat ought to be produced outside from a given
amount of food, when this food is metabolized inside the body.
To provide for this increase in metabolism, an extra provision 5 to 10%
of basal requirement (usually take 6%) has to be made.
Thus for a 70 kg man with BMR of 1600 C, another 80 to 160 C (96 C)
have to be added on this account.
Typical Activities with
Corresponding Activity Factors
What is Mr. X (127 kg, 213 cm) Daily Energy
Expenditure?
• BMR=24*1*127=3048 kcal/day
• Hourly metabolic rate =3048/24=127

• Physical Activity=75.5:
• Sleeping 9*1=9
Activity Hours • Walking 3*2.5=7.5
Sleeping 9 • Boxing 6*7=42
Walking 3 • Scuba Diving 2.5
Boxing 6 • Sax + Eating 3*1.5=4.5
Scuba Diving 1 • Swimming = 2*5 = 10
Swimming 2
• Daily Energy Expenditure = 75.5 x hourly
Saxophone 1 metabolic rate = 9588.5
Eating 2
NUTRITION-RELATED DISEASES
NON-SPECIFIC/MACRO- SPECIFIC NUTRIENT
NUTRIENT RELATED RELATED
Kwashiorkor - ↓prot+±en Beri beri - B1

Marasmus - ↓prot+↓en Pellagra - B3
Diabetes - ↓ insulin Ricketts - D
Atherosclerosis - ↑ fat Scurvy - C
Cancer - diet Anemia (M) - B12
Obesity - indulge/genes Anemia (H) - Fe
Bulimia - psychology Goitre - I
Anorexia - psychology
Bingeing - psychology
But specific diseases have been covered under vitamins and
minerals and will not be repeated.
BULIMIA NERVOSA
This is characterised by episodes of binge eating
during which energy intakes are very high, followed
by laxatives, diuretics and self-induced vomiting.
Bouts are reportedly triggered by 'loss of control'.
Body mass can be low or normal, with food intake
normal between bouts, although in some cases
excessive exercise and strict dieting are interspersed
between bouts.
The bingeing, vomiting and purging result in GIT,
teeth and salivary gland damage, as well as
electrolyte (K+, Na+, Ca2+, Mg2+, PO4-) imbalance
and dehydration. Kidney damage is also reported
from excessive diuretic use.
Dorsal hand calluses from induced vomiting (Russell
sign).
ANOREXIA NERVOSA
Related to poor body image. Characterised by
extremely low energy intake over a prolonged
period causing excessive mass loss. May be
excessive exercise and self-induced vomiting in
some cases. Physiological changes are typical of
starvation. GIT shows bloating, constipation,
poor motility. Muscles are wasted and bones
demineralise.
Thyroid hormones reduced so low BMR (BMI <
18.5 kg/m2), but increased cortisol causing
protein loss. Loss of adipose tissue causes failure
of reproductive capacity. CNS disturbance causes
depression, irritability, poor concentration and
memory loss. Heart muscle, BP, blood cells,
immunity and peripheral circulation all reduced.
Cardiac arrhythmias common.
BINGE EATING
While binge eating is similar to bulimia
in that eating is excessive, there are not
the concomitant purges to remove the
excess energy intake. Thus binge eating
is often accompanied by overweight
leading to obesity. The initial trigger is
still poor body image, but the reaction
and outcome is different.
OBESITY
Obese persons are more prone to
certain disease:
• Diabetes mellitus type II.
• Cardiovascular disorders
(нypertension, angina,
atherosclerosis, varicose veins
and thromboembolism)
• Liver diseases (fatty liver,
cholelithiasis, cholecystitis)
• Musculoskeletal system
(ackache, arthritis of hips and
knee joints, flat feet)
• Metabolic diseases
(hyperuricaemia).
• Gynaecological disorders
(amenorrhoea, oligomenorrhoea,
endometrial carcinoma)
OBESITY
TYPES OF OBESITY
Immediate cause of obesity is always a positive energy balance, but there are
many ways in which the balance may be tilted towards the positive side.
Thus, obesity is often divided into 2 types:

- Exogenous Obesity - overfeeding and gluttony with less physical activity.
- Endogenous Obesity - endogenous factors: endocrinal, metabolic, hypothalamic
lesion.
OBESITY
Pathologically, the types of
obesity are:
a. Hyperplastic type - life long
obesity, increase in adipose cell
number and increase in adipose
cell size. Fat distribution -
peripheral and central. Long-
term response to treatment is
not good.
b. Hypertrophic type - adults
after twenty years of age (adult
onset type). Hypertrophy of
adipose tissue cells (increase
size only). Fat distribution -
central. Long term response to
treatment is fairly good.
OBESITY
Obesity is most commonly due to overeating than the caloric requirement.
Obesity can be encountered with other diseases, viz. certain metabolic disorders,
and endocrine disorders.
Pathogenesis
- Age - more common in middle life.
- Sex - adult women are more prone to obesity
- Genetic factor - members of certain families
- Psychological factors - psychologically
imbalanced, anxieties, worries.
- Hypothalamic factor:
Two mechanisms regulate food intake:
• If certain lateral centres are bilaterally destroyed - aphagia results.
• When the medially controlled centres are bilaterally destroyed. The individual
eats more than requirements and obesity results.
- Epidemic encephalitis - hypothalamic lesions - obesity.
OBESITY
- Endocrine factors:
• Fröhlich’s syndrome - hypogonadism and obesity, has been considered the result of
hypopituitarism.
• Cushing’s syndrome (Adrenocortical hyperfunction) - fat mainly confined to the
head, neck and trunk (truncal obesity and buffalo hump) but spares the limb.
• Hypothyroidism - diminished BMR and energy expenditure may be associated with
gain in weight and obesity.
• Hypogonadism - adiposity characteristic of hypogonadism involves chiefly the
breast, abdomen, hips and thighs.
The endocrine disorders do not cause the obesity as such but may favour its
development by increasing food intake or decreasing energy expenditure or both.
METABOLIC CHANGES IN OBESITY
Various metabolic abnormalities observed in obesity are not permanent in nature.
They are induced with weight gain and are reversible with weight reduction.
1. Changes in Fat Metabolism

Increased level serum triglyceride (hypertriacylglycerolaemia).
Serum cholesterol level - obesity associated with type IV and type V
hyperlipoproteinaemias, hypertriglyceridaemia - slight to moderate
hypercholesterolaemia.
Mobilisation of FFA increase
Lipoprotein lipase activity increase - increased FFA assimilation
in adipose tissue and thus it can lead to increased fat deposition, in adipose tissue.
2. Changes in carbohydrate metabolism - obesity is associated with

hyperinsulinaemia. The β-cells are stimulated to produce more insulin.
Prolonged hyperinsulinism - exhaustion of β-cells - individuals who are genetically
susceptible to diabetes mellitus.
Insulin resistance is associated with obesity.
METABOLIC CHANGES IN OBESITY
Obesity has been found to be associated with fewer numbers of insulin
“receptors”, on adipose tissue, liver and muscle. A high blood insulin level
(hyperinsulinaemia) decreases the number of insulin receptors on target cell
membrane, probably through internalisation of the insulin-receptor complex into
the cell and thus decreases the insulin sensitivity of the target tissues, thus
contributing, to insulin resistance and impaired glucose utilisation by the cells.
3. Changes in acid-base status - massive obesity may be associated with alveolar

hypoventilation - PCO2 may be high ↑ - certain personality changes, fatiguability,
dyspnoea and somnolence, called as “obesity-hypoventilation syndrome”
(Pickwickian syndrome).
4. Energy metabolism in obesity - BMR usually normal in obese subjects. Their

energy expenditure per unit mass is the same as in normal people.
HORMONES OF ADIPOSE TISSUE AND ITS ROLE IN OBESITY
Two peptide hormones are produced by adipose tissue cells (adipocytes):
1. Leptin
2. Adiponectin
LEPTIN
Leptin - protein hormone
produced by adipose tissue cells
(adipocytes) abut acts through
brain receptors. Other sources
of leptins: brown adipose
tissue, placenta, ovaries,
mammary epithelial cells, bone
marrow pituitary and liver.
Leptin circulates at levels proportional to body fat.

Receptors for leptin - brain neurons involved in regulating energy intake and
expenditure. Control food intake (down appetite) and energy expenditure by acting
on receptors in mediobasal hypothalamus.
LEPTIN RESISTANCE AND OBESITY
Leptin resistance and obesity:

Although leptin is a circulating
signal that reduces appetite, in
general, obese people have an
unusually high circulating
concentration of leptin. These
obese people are considered
to be resistant to the effects of
leptin, in much the same way
that people with type 2
diabetes are resistant to the
effects of insulin.
ADIPONECTIN
Adiponectin - protein hormone produced and secreted by adipose tissue cells

(adipocytes), effects through brain.
Metabolic effects and functions of adiponectin:

– Increases glucose by glucose uptake and gluconeogenesis (glucose flux).
– Lipid catabolism—β-oxidation increases and triglyceride clearance.
– Protection from endothelial dysfunction (important
facet in atherosclerosis formation).
– Insulin sensitivity.
– Weight loss—Adiponectin exerts some of its weight reduction effects via the
brain.
– Control of energy metabolism.
Levels of the hormone are inversely correlated with body fat percentage in adults.
The hormone plays a role in the suppression of the metabolic derangements that
may result in type-2 diabetes, obesity, atherosclerosis,
nonalcoholic fatty liver disease (NAFLD).
HOSPITAL MALNUTRITION SYNDROME
This results from patients having increased nutrient requirements post
trauma or surgery, but decreased ability to consume adequate amounts of
food to meet these increased nutrient needs.
Most muscles are less active so protein is mobilised to provide glucose and
protein synthesis substrates to make immune system components.
Severity depends on the degree of

trauma, but may lead to as much
as 70g/day of protein loss.
Can cause immune inadequacy,
increased infection risk and poor
wound healing. Parenteral feeding
and/or very concentrated
liquidised foods ameliorate.
KWASHIORKOR
Caused by normal to high CHO intake, but low or poor quality protein. Thus
a severely distorted nutritional profile.
Usually related to the arrival of a subsequent child in a low income family.
Energy supply normal so adipose tissue usually normal at least in the early
stages. BMR also normal.
Protein synthesis decreased, leading to
reduced collagen albumin, immune
system, transport proteins.
Energy intake at least normal but
ability to transport synthesised fat
reduced, so stored in liver, hence
enlarged liver (hepatomegaly).
Reduced muscle mass causes reduced mobility,
and muscle weakness.
Enlarged liver and weak abdominal muscles leads
to adbominal protuberance. Limbs often wasted.
Reduced albumin and collagen causes excessive
fluid loss from the circulation and edema.
Poor protein synthesis causes reduced epithelial
cell turnover, skin lesions, reduced hair growth,
GIT atrophy.
Hair pigmentation reduced from reduced tyrosine.
Liver damage may occur from increased free
radical damage.
Reduced protein may also cause impaired brain
development.
Reduced immune system proteins leads to
increased susceptibility to infection.
MARASMUS
This is really just starvation of a child. All
components of the nutritional profile are
decreased, so no distorted pattern as in
kwashiorkor.
Very little adipose tissue since energy intake
decreased.
BMR reduced significantly.
Protein synthesis decreased and muscle mass
reduced to provide glucose for brain and RBCs,
thus generalised muscle wasting.
Epithelial tissues and GIT mucosa atrophied, as
are internal organs, and immune system.
Highly susceptible to infection.
MARASMIC-KWASHIORKOR
The extremes of both marasmus and
kwashiorkor are very similar and called
marasmic- kwashiorkor.
Must be remembered that both conditions

are progressive deteriorating situations.
Once they have both become severe, the
distinctions blur as overall body functions
start to fail.
Prognosis for marasmic- kwashiorkor is

extremely poor.
THE END
INTEGRATION
OF
METABOLISM
BY DR. M. VYSOCHYN, MD, PhD

Integration of Metabolism Well-Fed
State
MAJOR ORGAN
INTEGRATION OF METABOLISM
• All tissues of the body carry out metabolic processes for survival and
growth but not all of the major metabolic pathways are operational at
the same time, nor at the same level in all tissues.
• Changes in nutritional, hormonal, physiological, and pathological
status lead to alterations in metabolism in each tissue.
• In addition, and critical to an understanding of metabolic integration, is
that changes in metabolic function in one tissue can, and often do, have
potentially profound inpacts on the metabolic processes of other
tissues.
• In the context of overall tissue integration, it is most important to
demonstrate how the metabolic processes of the brain, liver, adipose
tissue, and skeletal muscle are interconnected
MAJOR ORGAN
INTEGRATION OF METABOLISM
• Within the context of organ integration of metabolism, the pathways that
are involved include glycogen synthesis (glycogenesis) and breakdown
(glycogenolysis), glycolysis, gluconeogenesis, TCA cycle, lipid synthesis
(lipogenesis) and breakdown (lipolysis), lipid oxidation, ketogenesis,
amino acid catabolism, urea cycle, protein synthesis, and proteolysis.
• With respect to metabolic integration, the best way to gain an
understanding of the processes involved is to examine how these major
metabolic pathways respond to everyday feeding and then fasting
between meals. Prolonged absence of food results in death within a very
short period of time. This fact explains why humans have evolved a
complex system of regulatory circuits that were evolutionarily designed
to stimulate our desire to seek and consume food.
ENERGY REQUIREMENTS AND RESERVES
• The 2 major hormones that control the use and storage of energy are
the pancreatic hormones insulin, and glucagon.
• These 2 hormones serve as counterregulatory hormones in that they
oppose the primary actions of each other.
• Insulin is released following food intake to prepare the organs to
absorb, utilize, and store the energy of the food.
• Conversely, glucagon is released during fasting in response to falling
levels of blood glucose.
METABOLIC STATES
• Fed / absorptive / post-prandial state
• Occurs during the ingestion of food and for several hours after
a meal
• high insulin / low glucagon state
• Fasting
- low insulin / high glucagon state
• Post-absorptive state – early period of fasting
- between 6 and 12 hours after the last meal
• Prolonged fasting / starvation
– more than 12 hours after the last meal
AFTER AN ORAL GLUCOSE LOAD
• The concentration of glucose
in the blood rises rapidly
after the ingestion of glucose
(or a high carbohydrate meal)
• The increase in blood
glucose concentration is
closely followed in time by an
increase in plasma insulin
concentration
• Peak glucose concentration
occurs within the first hour
and a return to basal levels
occurs within two hours.
A protein-rich meal leads to the release
of both insulin and glucagon
• Glucagon stimulates
gluconeogenesis and the release
of the newly formed glucose
from the liver to the blood
stream.
• Glucagon rise is due to the rise of
amino acids such as arginine that
promote its secretion from the
pancreatic a-cells
• The very moderate rise in insulin
stimulates uptake of the sugar
formed in the liver by muscle
and adipose tissue.
• Insulin is high enough the
promote the uptake of amino
acids in the tissues
WELL FED/ ABSORPTIVE PHASE
Elevated concentrations of glucose in blood stimulate release of insulin
from b cells of the pancreas
Glucokinase is most important in liver and islet cells of pancreas.
Both organs respond to fluctuations in plasma glucose levels.
1.Glucose enters beta cells via GLUT2 (low affinity)
2.Phosphorylated to glucose-6-phosphate by glucokinase (trapped in
cell)
3.Glycolysis/TCA Cycle/Oxidative Phosphorylation – increased ATP
levels
4.ATP-dependent Potassium channel closes
5.Membrane depolarization
6.Activation of a voltage-gated calcium channel
7.Ca2+ increases in the cell
8.Ca2+ stimulates fusion of insulin-containing exocytotic vesicles with
the plasma membrane
9.Insulin is secreted through the plasma membrane
INSULIN RELEASE IN RESPONSE TO ELEVATED
BLOOD GLUCOSE
Metabolic Actions of Insulin:
• Membrane effects: increase number of GLUT4 transporters
• Covalent modification: acts on carbohydrate and fat metabolism
• Induction-repression of enzyme synthesis
• Leads to an alteration in the total population of active sites
(enzyme molecules), rather than influencing the efficiency of
existing enzyme molecules
Time course of Insulin action:
The binding of Insulin provokes a wide range of actions.
1. Within seconds of binding of the Insulin to its receptor: The most
immediate response is an increase in glucose transport into
adipocytes and skeletal muscle cells
2. Minutes to hours: Insulin induced change in enzyme activity
3. Hours to days: Increase in amounts of enzymes by stimulation of
gene expression through transcription and translation
Well-fed state in liver (control of carbohydrate
metabolism)
V
Insulin changes the expression of many genes involved in
carbohydrate and fat metabolism
LIVER (CARBOHYDRATE METABOLISM)
The liver retains 60% of glucose presented by the portal system

Increased glucose metabolism brought about by:
1. Increased glycolysis
2. Increased glycogen synthesis; decreased glycogenolysis
3. Decreased gluconeogenesis
4. Increased activity of the hexose monophosphate shunt (HMP)
o Accounts for 5% to 10% of the glucose metabolized by the liver
o Due to increased availability of glucose 6-phosphate and active
utilization of NADPH in hepatic lipogenesis
Fed state: most of the enzymes regulated by covalent modification are
in the dephosphorylated state
Glucokinase -in liver is indused by insulin
Glucose is oxidized to CO2 and H2O to meet the immediate energy
needs of the liver.
Glycolysis in the liver is stimulated following a meal by an increase in
fructose 2,6-bisphosphate, an allosteric activator of
phosphofructokinase-1 by insulin (indirectly by activation of PFK2)
2. Gluconeogenesis is inhibited by fructose 2,6-bisphosphate, an
inhibitor of fructose 1,6-bisphosphatase
3. Excess glucose is stored in the liver as glycogen, which is used
during periods of fasting to - glycogen synthase activated by insulin.
Glycogen Phosphorylase inhibited by insulin
4. HMP is stimulated (G6PD is activated by insulin)
5. PDH (pyruvate dehydrogenase) is activated by insulin
LIVER (FAT METABOLISM)
1. Increased de novo fatty acid synthesis
Liver is the primary tissue for de novo synthesis of fatty acids:
Increased availability of substrates (acetyl CoA and NADPH from glucose
metabolism)
Activation of acetyl CoA carboxylase by insulin (dephosphorylation)
2. Increased triacylglycerol synthesis
Favored because substrates are available
Fatty acyl CoA –
• from de novo synthesis from acetyl CoA
• from hydrolysis of the TAG component of chylomicron
remnantremoved
• from the blood by hepatocytes
Glycerol 3-phosphate - from glycolytic metabolism of glucose
(glycerol kinase makes the liver insulin-independent for the metabolic
control of triacylglycerides biosynthesis)
Packaged into VLDL and secreted into blood: For use by extrahepatic
tissues, particularly adipose and muscle tissue
LIVER (AMINO ACID METABOLISM)
1. Increased protein synthesis
v Results in replacement of any proteins that may have been
degraded during the previous post-absorptive period
2. Increased amino acid degradation.
After a meal, most of the energy needs of the liver are met by the
oxidation of excess amino acids.
a. Deaminated, with the resulting carbon skeletons being
degraded by the liver to pyruvate, acetyl CoA, or TCA
cycle intermediates
b. Liver has limited capacity to degrade the branched-
chain amino acids leucine, isoleucine, and valine
ü Pass through the liver essentially unchanged and are
preferentially metabolized in muscle
Major metabolic pathways in liver
in the absorptive state
ADIPOSE TISSUE (CARBOHYDRATE METABOLISM)
1. Increased glucose transport
v Glucose transport into adipocytes is sensitive to the
concentration of insulin in the blood: GLUT4 transporters
insulin à influx of glucose into adipocytes
v Increased intracellular availability of glucose results in an
enhanced rate of glycolysis
v Serves a synthetic function by supplying glycerol phosphate
for TAG synthesis
3. Increased activity in the hexose monophosphate pathway
(HMP)
v Glucose metabolism via the HMP produces NADPH that is
essential for fat synthesis
ADIPOSE TISSUE (FAT METABOLISM)
1. Increased TAG synthesis
After consumption of a lipid-containing meal, hydrolysis of the TAG of
chylomicrons and VLDL (from liver) provides adipose tissue with fatty
acids
Dietary glucose provides DHAP (Dihydroxyacetone) and Glycerol 3-phosphate
2. Decreased TAG degradation

Insulin inhibits the hormone sensitive lipase
Most of the fatty acids added to the lipid stores of

adipocytes is provided by dietary fat (from chylomicrons)
and a lesser amount is supplied by VLDL from the liver
Mechanisms that affect triacylglycerol storage
in adipose tissue
v Insulin stimulates adipose cells to synthesize and secrete lipoprotein lipase (LPL)
which hydrolyzes the chylomicron and VLDL triacylglycerols
v Insulin causes the number of glucose transporters in adipose cell membranes
to increase
SKELETAL MUSCLE
Energy metabolism is unique
Able to respond to substantial changes in the
demand for ATP that accompany muscle
contraction
üAt rest: accounts for approximately 30%
of the oxygen consumption of the body
üDuring vigorous exercise: responsible for
up to 90% of the total oxygen
consumption
Major metabolic pathways in skeletal muscle in the
Absorptive state
A. Carbohydrate metabolism
v Increased glucose transport
v Increased glycogen synthesis
B. Fat metabolism
v Fatty acids are released from chylomicrons and VLDL by the action of
lipoprotein lipase
v Fatty acids are of secondary importance as a fuel for muscle during the well
fed state, in which glucose is the primary source of energy
C. Amino acid metabolism
v Increased protein synthesis
v Increased uptake of branched-chain amino acids 602
SKELETAL MUSCLE (Carbohydrate Metabolism)
1. Increased glucose transport
v Due to a transient increase in plasma glucose and insulin
after a carbohydrate-rich meal
v Glucose is phosphorylated to glucose 6-phophate and
metabolized to provide the energy needs of the cells
q Contrasts with the post-absorptive state in which ketone
bodies and fatty acids are the major fuels of resting
muscle
2. Increased glycogen synthesis
vFavored by :
qThe increased insulin to glucagon ratio
qthe availability of glucose 6-phosphate
SKELETAL MUSCLE (Fat Metabolism)
Fatty acids are released from chylomicrons and VLDL by the action of
lipoprotein lipase
q Fatty acids are of secondary importance as fuel for muscle in the
well-fed state in which glucose is the primary source of energy
q Cardiac muscle – FA as energy

SKELETAL MUSCLE (Amino Acid Metabolism)
1. Increased protein synthesis
v A spurt in amino acid uptake and protein
synthesis occurs after ingestion of a meal
containing protein
q Replaces protein degraded since the previous
meal
2. Increased uptake of branched-chain amino acids
v Muscle is the principal site for degradation of
branched-chain amino acids
q Used for protein synthesis and as sources of
energy.
BRAIN
vContributes only 2% of the adult weight

vAccounts for 20% of the basal oxygen consumption of
the body at rest
vUses energy at a constant rate
vFuel needs is prioritized by the body
qFuels must be able to cross the endothelial cells that
line the blood vessels in the brain (called the “blood-
brain barrier”)
v Uses glucose exclusively as fuel
v Contains no significant stores of glycogen and is therefore
completely dependent on the availability of blood glucose
v Brain has no significant stores of TAG
v Fatty acids do not efficiently cross the blood-brain barrier
607
Intertissue
relationships in the
absorptive state
INTEGRATION
OF
METABOLISM

Integration of Metabolism Starved
State
STARVATION
¯ Plasma levels of
No
glucose, amino acids ¯ Insulin secretion
food
and Triacylglycerols Glucagon release
(TAGs)
Possible causes:
qInability to obtain food
qDesire to lose weight rapidly
qInability to eat due to trauma, surgery, neoplasm, burn, etc
The decreased insulin to glucagon ratio, and the decreased

availability of circulating substrates, makes the period of nutrient
deprivation a catabolic period characterized by the degradation of
TAG, glycogen and protein.
612
STARVATION
MAJOR INSULIN COUNTERREGULATORY HORMONES
Epinephrine, secreted in
response to fasting, stress,
trauma, and vigorous exercise,
decreases the release of
insulin
Epinephrine release signals
energy utilization, which
indicates that less insulin needs
to be secreted, as insulin
stimulates energy storage
STARVATION
The exchange of substrates among liver, adipose tissue, muscle and

brain is guided by two priorities:
1. The need to maintain adequate plasma levels of glucose to
sustain energy metabolism of the brain and other glucose-
requiring tissues
2. The need to mobilize fatty acids from adipose tissue and
ketone bodies from liver to supply energy to all other
tissues, mainly the cardiac muscle
Humans are exquisitely equipped to provide nutrients from stored sources for all tissues and
organs during periods of fasting, even long periods
POSTABSORPTIVE STATE
• Glucagon and epinephrine levels rise during an overnight fast.
• These hormones exert their effects on skeletal muscle, adipose
tissue, and liver.
• In liver, glycogen degradation and the release of glucose into the
blood are stimulated.
• Hepatic gluconeogenesis is also stimulated by glucagon, but the
response is slower than that of glycogenolysis.
• The release of amino acids from skeletal muscle and fatty acids
from adipose tissue are both stimulated by the decrease in insulin
and by an increase in epinephrine.
• The amino acids and fatty acids are taken up by the liver, where
the amino acids provide the carbon skeletons and the oxidation of
fatty acids provides the ATP necessary for gluconeogenesis.
PROLONGED FAST (STARVATION)
• Levels of glucagon and epinephrine are markedly elevated during
starvation.
• Lipolysis is rapid, resulting in excess acetyl-CoA that is used for ketone
synthesis.
• Levels of both lipids and ketones are therefore increased in the blood.
• Muscle uses fatty acids as the major fuel, and the brain adapts to
using ketones for some of its energy.
• After several weeks of fasting, the brain derives approximately two
thirds of its energy from ketones and one third from glucose.
• The shift from glucose to ketones as the major fuel diminishes the
amount of protein that must be degraded to support gluconeogenesis.
• There is no “energy-storage form” for protein because each protein has
a specific function in the cell.
• Therefore, the shift from using glucose to ketones during starvation
spares protein, which is essential for these other functions.
• Red blood cells (and renal medullary cells) that have few, if any,
mitochondria continue to be dependent on glucose for their energy.
Fasting and starvation
Glycogen reserves
depleted after day 1.
The liver first uses glycogen degradation, then gluconeogenesis, to

maintain blood glucose levels to sustain energy metabolism of the
brain and other glucose-requiring tissues.
1. Increased glycogen degradation
Liver glycogen is nearly exhausted after 10-18 hours of fasting
Glycogen Phosphorylase is activated by Epinephrine and
Glucagon:
1. Both increase in cAMP and Protein kinase A,
2. Epinephrine also activates the PIP2 pathway to cause release
of Ca+2 and activation of the calmodulin kinases.
3. PKA and PKC phosphorylate and activate Phosphorylase
Kinase
4. Phosphorylase Kinase phosphorylates and activates Glycogen
Phosphorylase – glycogen degradation
623
2. Glycolysis is inhibited (glucagon
inhibits PFK-1 in hepatocytes by
an indirect mechanism involving
PFK-2-phosphorylation and
fructose 2,6-bisphosphate)
3. Gluconeogenesis is stimulated
vPyruvate carboxylase activated
by increased acetyl CoA from fatty
acid oxidation.
vPhosphoenolpyruvate
carboxykinase (PEPCK) is induced
by glucagon and cortisol.
vFructose-2,6-bisphosphatase
glucagon will lower F2,6-BP and
stimulate gluconeogenesis
Regulation of glucose metabolism by
fructose 2,6-bisphosphate
Gluconeogenic precursors in starvation
vLactate conversion to pyruvate

qRed blood cells
qSkeletal muscle (less
common during starvation)
vAlanine conversion to pyruvate
qSkeletal muscle
vGlycerol conversion to
dihydroxyacetone
qAdipose tissue via lipolysis
CORI CYCLE AND GLUCOSE-ALANINE CYCLE
During fasting, lactate from red blood

cells (and possibly exercising skeletal
muscle) is converted in the liver to
glucose that can be returned to the red
blood cell or muscle.
vThis is called the Cori cycle.
vThe alanine cycle is a slightly different
version of the Cori cycle, in which muscle
releases alanine, delivering both a
Gluconeogenic substrate (pyruvate) and
an amino group for urea synthesis.
LIVER (Fat Metabolism)
1. Decreased fatty acid synthesis
Acetyl-CoA carboxylase (ACC) is inactivated by glucagon by cAMP dependent
phosphorylation
2.Increased fatty acid (beta) oxidation
The oxidation of fatty acids derived from adipose tissue is the major source of
energy in hepatic tissue in the post-absorptive state (decreased FA synthesis ➝
↓ Malonyl CoA ➝ ↓ inhibition of CAT1 (carnitine acyltransferase I) = activation
of FA transport to mitochondria for the b-oxidation)
3. Decreased Cholesterol synthesis
HMG-CoA reductase (β-Hydroxy β-methylglutaryl-CoA) is inactivated by glucagon by
cAMP dependent phosphorylation
4. Increased synthesis of ketone bodies
Favored when the concentration of acetyl CoA, produced from the oxidation of fatty
acids, exceeds the oxidative capacity of the TCA cycle
q Starts during the first days of starvation
q Important because ketone bodies can be used for fuel by most tissues
including brain
ü Reduces the need for gluconeogenesis from amino acid carbon
skeletons, thus slowing the loss of essential protein
qLevel of blood ketones can reach 20-30 mg/dl during prolonged starvation (N
1-3 mg/dl)
Coordinated regulation of fatty acid synthesis and
breakdown in liver (covalent control)
Conditions that enhance ketone body formation
vThe production and export of ketone
bodies by the liver allows continued
oxidation of fatty acids with only minimal
oxidation of acetyl-CoA.
vWhen intermediates of the citric acid
cycle are being siphoned off for glucose
synthesis by gluconeogenesis, for
example, oxidation of cycle intermediates
slows and so does acetyl-CoA oxidation
vThe liver contains only a limited
amount of coenzyme A, and when most
of it is tied up in acetyl CoA, beta
oxidation slows for want of the free
coenzyme
vThe production and export of ketone
bodies frees coenzyme A, allowing
continued fatty acid oxidation
LIVER (Amino Acid Metabolism)
Amino acids are degraded to carbon skeletons
Carbon skeletons are used for gluconeogenesis
Main amino acids are alanine and glutamic acid
The amino groups are converted to urea
634
Fate of amino acid nitrogen in the fasting state
Amino acids are released
from muscle proteins
Some amino acids enter the
blood
Other amino acids are
partially oxidized and the
nitrogen stored in the form of
alanine and glutamine and
enter the blood
Alanine (the major
Gluconeogenic amino acid)
and other amino acids enter
the liver
Amino acid metabolism in liver during fasting
Hormonal regulation of hepatic

amino acid metabolism in the
Postabsorptive state:
q Glucagon mediated
activation of enzymes or
protein
q Induction of enzyme
synthesis mediated by
glucagon and glucocorticoids
qInduction of urea cycle

enzymes occurs during
fasting and after a high
protein meal
Summary: Liver in the Fasting state
Adipose Tissue (Carbohydrate Metabolism)
1. Does not work GLUT 4 transporters.

Glucose transport and its subsequent metabolism are depressed
2. decreased synthesis of triacylglycerol
Less glucose – less DHAP – less TAG synthesis (adipose tissue lacks
Glycerol kinase)
Adipose Tissue (Fat Metabolism)
1. Increased degradation of Triacylglycerols
q The low insulin to glucagon ratio leads to the activation of
hormone-sensitive lipase (phosphorylation)
q Enhanced by the elevated catecholamines
ü Epinephrine and norepinephrine (from the sympathetic nerve
endings in adipose tissue) also activate hormone-sensitive lipase
2. Increased release of fatty acids
Fatty acids from the degradation of TAG are released into the
blood
q Transported to other tissues (bound to albumin) for use as fuel
q Fatty acids are efficiently removed from the circulation by most
tissues
Epinephrine and glucagon mobilized stored Triacylglycerols
during starvation
Skeletal Muscle (Carbohydrate Metabolism)
1. Decreased glucose transport
into skeletal muscle cells leads to subsequent decrease in
glucose metabolism
2. Increased glycogen degradation
The muscle does not have receptors for Glucagon

Glycogenolysis in the muscle is stimulated by Epinephrine
Epinephrine and Cortisol:

As in acute stress or prolonged exercise
By binding to the β adrenergic receptors and activation by
protein kinase A – activation of glycogen phosphorylase
Glucagon and epinephrine both modulate glycogenolysis
in liver but only epinephrine regulates glycogen
degradation in muscle cells
Skeletal Muscle (Lipid Metabolism)
•In the fasting state, resting muscle uses fatty acids derived
from free fatty acids in the blood.
•Ketones may be used if the fasting state is prolonged.
Skeletal Muscle (Protein Metabolism)

v First few days of starvation:
q rapid breakdown of muscle protein, providing amino
acids for gluconeogenesis in the liver
ü Alanine and glutamine are quantitatively the most
important Gluconeogenic amino acids released from
muscle
v After about a week of starvation:
q rate of muscle proteolysis decreases due to a decrease in
the need for glucose as a fuel for brain
Brain
v First few days of starvation:
q Brain continues to use glucose as fuel exclusively
v Prolonged starvation (greater than a week):
q Plasma ketone bodies reach markedly elevated levels and are
used as fuel by the brain
ü Decreases the need for protein catabolism for
gluconeogenesis
Glucose homeostasis has five phases
vPhase I is the well fed state, in which glucose is provided by dietary
carbohydrate
vPhase II hepatic glycogenolysis maintains blood glucose once glucose from
phase I is exhausted
vPhase III hepatic gluconeogenesis from lactate, glycerol, and alanine
becomes increasingly important
vPhase IV in this phase ketone bodies accumulate high enough so the brain
starts to use them
vPhase V this is characterized by even less dependence in gluconeogenesis. In
this phase, the energy needs of almost every tissue are largely met by fatty acid or
ketone body oxidation
Course: Biochemistry
INTEGRATION
OF
METABOLISM

Metabolic Response to Stress
648
Injury to the body
vExample: from trauma, surgery, infection, or
burns
vTriggers a characteristic hypermetabolic state
in which the resting energy expenditure is
increased along with body temperature
vExtent and pattern of the metabolic changes
vary with the type as well as the severity of
injury or infection
649
Tissue Injury
vLeads to increased
levels of:
qInsulin
qGlucagon
qCatecholamines
(epinephrine)
qCytokines (like
interleukin-1 and
interleukin-6)
qGlucocorticosteroids
(Cortisol)
650
Insulin - does not display its usual
spectrum of action
Insulin resistance is promoted by high concentrations of
counterregulatory hormones (cortisol, and epinephrine)
1. Glucose utilization by the insulin-sensitive tissues is

minimized *reduced GLUT4 transporters
2. Stress induces insulin resistance in muscle, adipose

tissue and liver at a post-receptor level
651
High concentrations of epinephrine and glucagon
stimulate glycogenolysis and gluconeogenesis
vHigh levels of catecholamines (epinephrine and
norepinephrine) cause insulin resistance
vThis leads to moderate hyperglycemia caused
in part by a decreased peripheral uptake of
glucose
vThe catecholamines act through two major
types of receptors on the plasma membrane of
target cells, the a-adrenergic and the b-adrenergic
receptors and epinephrine presence signal
impending activity
vThe actions of epinephrine in the liver, the
adipocyte, the skeletal muscle, and the a and b
cells of the pancreas directly influences
metabolism
vEpinephrine is a counterregulatory hormone
that has metabolic effects directly toward
mobilization of fuels from their storage
sites for oxidation by cells to meet the increase
energy requirements of acute and chronic stress
Glucagon and epinephrine both modulate glycogenolysis in
liver but only epinephrine regulates glycogen degradation
in muscle cells
653
vGlucocorticoids
qFacilitate gluconeogenesis by
induction of glucose-6-phosphatase
and PEP carboxykinase
vCytokines
qStimulate lipolysis in adipose tissue
and contribute to muscle degradation
Cortisol signals stress, including low blood glucose
vA variety of stressors
(anxiety, fear, pain,
hemorrhage, infection, low
blood glucose,
starvation) stimulate
release of the corticosteroid
hormone, cortisol from the
adrenal
cortex
vCortisol acts on muscle, liver, and adipose tissue to supply the organism with fuel
to withstand stress
vCortisol is a relatively slow-acting hormone that alters metabolism by changing
the kinds and amount of certain enzymes synthesized in its target cells, rather
than regulating the activity of existing enzymes molecules
Metabolism During
Exercise
656
Skeletal Muscle:
vCan increase energy turnover 18-20 fold under exhaustive work loads.
vUses fatty acids and glucose as substrates for aerobic metabolism and
ATP production
vIs dependent upon glucose as a substrate for anaerobic energy
production.
During physical exertion/exercise:
vMechanisms very similar to those that are used during fasting operate
to maintain blood glucose levels
vThis low insulin / glucagon ratio:
qprompts the liver to break down glycogen and start
gluconeogenesis
vThere is decreased secretion of insulin and a marked increase in
glucagon secretion, i.e., low insulin/glucagon ratio
qProvides the glucose required to balance the glucose uptake by
the muscle
657
Activation glycogenolysis and glycolysis during exercise
vGlycolysis is activated by the stimulation of phosphofructokinase

vAMP activates glycogenolysis and glycolysis
vGlycogenolysis and glycolysis are activated together because both
PFK-1 and glycogen phosphorylase b are allosterically activated by
AMP
658
Production of blood glucose by the liver from various
precursors during rest and during prolonged exercise
vMild to moderate-
intensity exercise can be
performed for longer
periods of time than can
high-intensity exercise
vThis is because of the

aerobic oxidation of glucose
and fatty acids, which
generates more energy per
fuel molecule than
anaerobic metabolism, and
which also produces lactic
acid at slower pace than
anaerobic metabolism
Energy substrates in exercising muscle
vThe main types of "fuel" used by muscle for energy metabolism are glycogen,
glucose, and free fatty acids
vThe particular energy sources used by working muscle for aerobic metabolism
depend upon a number of factors including the intensity, type, and duration of
exercise, physical conditioning, and diet
vDuring high-intensity, isometric exercise, anaerobic glycolysis, and the creatine
kinase reaction, in which phosphocreatine is converted to ATP, are the primary
sources of energy
Energy substrates in exercising muscle
vWith submaximal exercise, the type of

substrate used by muscle is heavily dependent
upon the relative intensity of exercise.
vDuring low-intensity submaximal exercise, the
main sources of energy are blood glucose and
free fatty acids.
vWith high-intensity submaximal exercise, the
proportion of energy derived from glycogen
and glucose is increased, and glycogen
becomes the main source.
vFatigue is experienced when glucose and
glycogen stores are depleted (as when a
marathon runner hits the "wall").
Fuels used during exercise
Mild and moderate-intensity long term exercise
vLactate release decreases with duration of exercise
vBlood glucose as fuel (glycogenolysis and gluconeogenesis)
vFree fatty acids as a source of ATP
qThe longer the duration
of the exercise, the greater
the reliance of the muscle
on free fatty acids for the
generation of ATP
qDuring prolonged
exercise, the small
decrease of insulin, and
increases of
glucagon, epinephrine and
norepinephrine, cortisol all
activate adipocyte lipolysis
Diabetes Mellitus
v Classification:
Ø Type 1 - IDDM - autoimmune destruction of b cells.
Ø Type 2 - NIDDM – partial b-cell failure and insulin

resistance.
Ø Gestational Diabetes – Type 3 - appears in 2-5% of all
pregnancies.
qPregnancy is a relatively insulin-resistant state that is
somewhat
exacerbated in some females to the point at which
gestational diabetes develops.
qPregnant females with gestational diabetes are at increased
risk for giving birth to large-for-gestational-age infants, with
increased muscle mass and body fat
665
Type I Diabetes
vAlso known as insulin-dependent diabetes mellitus (IDDM) or
juvenile diabetes
vCharacterized by an deficiency of insulin caused by massive
autoimmune attack on the β cells of the pancreas
vUsually appears before the age of 35 and most often between 10
and 16 years of age
666
Metabolic events in Diabetes Mellitus type 1
vIn diabetes type I the insulin/glucagon ratio cannot increase
v The liver is always gluconeogenic and ketogenic
v The liver contributes to in the well fed state
v Accelerated gluconeogenesis, fueled by uncontrolled proteolysis in
the skeletal muscle, maintains the hyperglycemia even in the starved
state
v Uncontrolled lipolysis in adipose tissue increases plasma fatty acid
levels and ketone body production by the liver
v Fatty acid oxidation and ketogenesis cannot completely dispose of
fatty acids taken up by liver and the excess is esterified and
directed into VLDL
vHypertriacylglycerolemia results since VLDL and chylomicrons
cannot be cleared from the blood by lipoprotein lipase, which
expression is dependent on insulin
1. Liver Metabolism in Type 1 Diabetes
• The liver interprets the low insulin/glucagon ratio as a signal of low blood
sugar, leading to stimulation of gluconeogenesis. Thus the hepatic output
of glucose is increased despite ample or excessive glucose in the blood.
Amino acids mobilized from muscle are used as the carbon skeletons, as
described for fasting metabolism. Excessive amounts of acetyl-CoA
produced by mobilization of FFAs are shunted away from the already
saturated citric acid cycle and into production of ketone bodies.
Significantly, the rate of ketone production in diabetes is much greater
than in starvation, making this a life-threatening condition.
2. Adipose Tissue Metabolism in Type 1 Diabetes
• The absence of insulin leads to uncontrolled mobilization of FFAs, which
serves as the source of excess ketone body production by the liver.
Lipoprotein lipase, which is increased by insulin, is decreased in its
absence, leading to an elevation in chylomicrons and VLDL levels. Because
glucose uptake in adipose cells is insulin dependent, the defective
transport further contributes to an abnormally elevated blood glucose
level. 669
3. Muscle Metabolism in Type 1 Diabetes

• The lack of insulin prevents glucose uptake by muscle tissue,
further contributing to abnormally high blood glucose
concentrations. Protein synthesis is decreased and degradation
increased in the diabetic state, as would be seen during fasting,
to mobilize carbon skeletons for use in gluconeogenesis, even
though it is not needed. Muscle amino acids are also consumed
in the citric acid cycle to make up for the loss of glucose, which
cannot be transported into the cell.
4. Brain Metabolism in Type 1 Diabetes
• In the untreated diabetic, blood glucose remains the sole source
of fuel because it is in plentiful supply. Therefore ketones are not
used by the brain as they are during starvation.
vUsually recognized by the abrupt appearance of :
• Polyuria (frequent urination)
• Polydipsia (excessive thirst)
• Polyphagia (excess hunger)
• Often triggered by stress or illness
vOther symptoms: fatigue, weight loss, weakness
vKetoacidosis may be the initial presentation
672
Type II Diabetes
vAlso known as non-insulin-dependent diabetes

mellitus (NIDDM) or adult-onset diabetes
vDue to a combination of 2 factors:

• dysfunctional β cells and insulin resistance
vUsually seen after age 35
673
Type II Diabetes caloric surplus
v Due to a combination of 2 factors:
1. Insulin resistance hyperinsulinemia
1 Tissues fail to respond
normally to insulin
q Liver: uncontrolled ñSREBP-1c
hepatic glucose
production
ñadiposity
q Muscle and adipose:
decreased glucose uptake
2 Mainly due to defects in signal ectopic lipid deposition
transduction
2. Dysfunctional b cells (in advanced insulin resistance
stages)
b-cell lipotoxicity
Fail to secrete enough insulin to
correct the prevailing hyperglycemia
Hyperglycemia 67
4
Hyperinsulinemia precedes diabetes
675
Metabolic components of diabetes type 2
Excess amount of FFA and TG
Intracellular ectopic accumulation
Pancreas (b cells) Liver Muscle
1- chronic insulin secretion 1. Gluconeogenesis 1. GLUT-4 translocation

(hyperisulinemia) impairment
2. Lipogenesis
2.- apoptosis
well fed state starvation diabetes type 1 diabetes type 2
Insulin high low Not present high

Glucagon low high high low
Epinephrine Not present Present Present Not present
Cortisol Not present Present Present Not present
Gluconeogenesis Inhibited Activated Activated Activated
Glycolysis Activated Inhibited Inhibited Inhibited
Glycogen breakdown Inhibited Activated Activated Activated
Glycogen synthesis Activated Inhibited Inhibited Inhibited
Lipogenesis Activated Inhibited Inhibited Activated
b-oxidation Inhibited Active highly activated Normal
Ketogenesis Inhibited Active highly activated Normal
Hyperglycemia Transient Not present Present Present
Hypertriacylglycerolemia Transient Not present Present Present
Metabolic acidosis Not present Present Present Not present
Population Genetics
Course:
Biochemistry and Medical Genetics
Prepared by: Dr. Alisa Chebotarova, MD, PhD
Modified by: Dr. I. Vyshnytska, MSc, PhD
INTENDED LEARNING OBJECTIVES
By the end of the lecture students must be able to:
1. Give a definition to Population genetics
2. Be able to calculate allele and genotype frequencies for a population
3. Understand how to apply the Hardy-Weinberg equation to test whether populations appear
to be in Hardy-Weinberg equilibrium
4. Be able to explain why HWE is a null hypothesis for evolutionary change
5. Be able to use Hardy-Weinberg equation to predict genotype frequencies for populations
assumed to be in equilibrium
6. Be able to use Hardy-Weinberg equation to predict genotype frequency in an Autosomal
Recessive, Autosomal Dominant, X-Linked disorders
7. Define Stratified Mating, Assortative Matings, Consanguineous Mating
8. Explain Heterozygote advantage, Genetic Drift, Gene Flow
Introduction
• Population genetics is the quantitative study of the distribution of genetic
variation in populations and of how the frequencies of genes and genotypes are
maintained or change.
• Population genetics is concerned both with genetic factors, such as mutation and
reproduction, and with environmental and societal factors, such as selection and
migration, which together determine the frequency and distribution of alleles
and genotypes in families and communities.
• A mathematical description of the behavior of genes in populations is an
important element of many disciplines, including anthropology, evolutionary
biology, and human genetics.

Population Genetics
Population genetics is the study of genetic variation in populations. Basic concepts
of population genetics allow us to understand how and why the prevalence of
various genetic diseases differs among populations.
Population genetics is concerned with:

• Gene and genotype frequencies in populations
• The factors that tend to keep them constant
• The factors that tend to change them
It is largely concerned with the study of polymorphisms and their distribution in

populations.
It directly impacts counseling, forensic medicine, and genetic screening.
Population Genetics
A population is:
Ecologically: A group of individuals of the same species living within a restricted

geographical area that allows any two individuals to interbreed.
Genetically: A group of individuals who share a common gene pool and have the
potential to interbreed.

Population Genetics
• With regards to genetic disorders, knowledge about the different disease genes
that are common in different populations and about their role in health and
disease can be a valuable asset in diagnosis, treatment, genetic counseling, as
well as in health care planning.
• The human population (8 billion people as of October 2022):
• The only living species in the human family, Homo sapiens, has become a highly diversified
global array of populations.
• Humanity cannot be classified into discrete geographic categories with absolute
boundaries. Partly as a result of gene flow, the hereditary characteristics of human
populations are in a state of perpetual flux.
• Certainly, groups of people living in separated geographic regions differ statistically in
certain genetic traits, but these genetic differences are a property of local human
populations – POLYMORPHISM.
Population frequency
• Allele (gene) frequency: proportion of chromosome that contain a specific type
of allele in a population
• Normal allele (p); (to remember: most popular allele = p)
• Mutant allele (q);
• Genotype frequency: proportion of each genotype in a population:

• Homozygous normal (p2);
• Heterozygous (2pq);
• Homozygous affected (q2);

Genotype Frequencies
Allele 1
For a given locus, the genotype
frequency measures the proportion
of each genotype in a population.
Allele 2
Genotype 1 Genotype 3
(Homozygous 1-1) Genotype 2 (Homozygous 2-2)
(Heterozygous 1-2)
# of individuals with deﬁned genotype in the populajon

Genotype frequency =
total # of individuals in the populajon

Genotype Frequencies
• For example, suppose that a population of 100 individuals has been assayed for
an autosomal restriction fragment length polymorphism.
• If the RFLP has two possible alleles, labeled 1 and 2, there are three possible
genotypes: 1-1, 1-2, and 2-2.
• Visualization of a Southern blot allows us to determine the genotype of each
individual in our population, and we find that the genotypes are distributed as
follows:
# of individuals with deﬁned genotype in the popula;on

= total # of individuals in the popula;on

Gene Frequencies
• By definition, the total number of autosomal alleles in a population is twice the
number of individuals in the population (excepting individuals with trisomies,
duplications, or translocations)
# of copies of an allele in the populajon

Allele (Gene) frequency =
total # of alleles in the populajon
• The allele frequency measures the proportion of chromosomes that contain a

specific allele.
• Allele frequencies can be represented as a decimal, a percentage, or a fraction.

Gene Frequencies
• Consider a population of 1000 individuals all typed for the simplest test at the
MN locus.
• In its most simplistic form this locus can be reduced to a codominant system with
two alleles M and N
• Every individual in the population will be either MM (having two M alleles), MN
(heterozygous), or NN (having two N alleles).

Calculating Gene Frequencies
Suppose the codominant trait typing results were as follows: 300 MM individuals,
600MN individuals, and 100 NN individuals.
Calculate M gene (allele) frequency:

1000 individuals each have two alleles at the MN locus = 2000 total alleles
• Each MM individual has 2 M alleles:
• 300 x 2 = 600 M alleles
• Each MN individual has 1 M allele:
• 600 x 1 = 600 M alleles
There is a total of 1200 M alleles in a population of 2000 alleles.
The gene (allele) frequency of the M allele is:
1200/2000 = 0.6
Hardy-Weinberg Law
• The Hardy-Weinberg Law states: In a large, random-mating population that is not

affected by the evolutionary processes of mutation, migration, or selection,
both the allele frequencies and the genotype frequencies are constant from
generation to generation.
• Helps predict the distribution of phenotypes & genotypes in large populations

Hardy-Weinberg Law
• To be in the Hardy-Weinberg Equilibrium,

the population must be:
• Large
• Completely random mating
• No mutations
• No migration in/out of population
• No natural selection

Hardy-Weinberg Equilibrium
• For many human autosomal recessive traits the heterozygote cannot be
distinguished phenotypically from the normal homozygote.
• Can we predict the heterozygote (carrier) genotype frequency without doing

genetic testing on large numbers of people in a population?
• The Hardy-Weinberg law allows us to make these estimates

• Is used to calculate gene or genotype frequencies if they can't be counted directly
• The Hardy - Weinberg equation, allows one to estimate genotype frequencies if

one knows allele frequencies, and vice versa.
Conditions:
If there are 2 alleles in a population, A and a, where A is the dominant and a is recessive:
• p is the frequency of allele A (conventionally the most common, normal allele)
• q is the frequency of allele a (conventionally a minor, disease-producing allele)
• alleles combine into genotypes randomly
Expressions:
The AA genotype frequency is p2 (genotype frequency of homozygotes)
The aa genotype frequency is q2 (genotype frequency of homozygotes)
The Aa genotype frequency is 2pq (genotype frequency of heterozygous)
• Since the A allele could be inherited from the mother and the a allele from the father, or vice
versa).

The first important property of the Hardy-Weinberg law:

• Since there are only two alleles in the population:
• p + q = 1 (100%) [frequencies of the 2 alleles add to 1]
• And 3 genotypes in the population (AA, Aa, aa):
• p 2 + 2pq + q2 = 1 (frequencies of the 3 genotypes add to 1)

The first important property of the Hardy-Weinberg law:
• In most cases where this equation is used, a simplification is possible.

• Generally p, the normal allele frequency in the population, is very
close to 1 (e.g., most of the alleles of this gene are normal-100%).
• In this case, we may assume that p ~ 1 , and the equation simplifies
to:

• A second crucial implication of the Hardy-Weinberg law is that the

proportions of the genotypes do not change from generation to
generation.
• Such a population is said to be at Hardy-Weinberg Equilibrium.

• This occurs in populations only when the assumptions of the Hardy-
Weinberg law are met.

Usage of the Hardy Weinberg law for Autosomal Recessive
disorders
• In recessive disorders, the frequency of the disease (incidence) corresponds to

the frequency of the homozygous state for the disease allele, which is q2
according to the Hardy-Weinberg relationship.
• The allele frequency is thus:
• Since (p + q) =1, p can also be determined as can the heterozygote or carrier

frequency, 2pq

disorders
Example 1 – carrier genotype frequency:
If the frequency of an AR disorder (aa genotype) is approximately 1/4500 in
Ireland. What is the carrier of this disorder (Aa heterozygote) frequency?
Because all affected individuals are homozygous (aa) for a mutant allele:
• Let q = frequency of mutant allele (a)
• and p = frequency of normal allele (A)
1. q2 (aa) = 1/4500 given ®

2. q = 1/4500 = 0.00022 = 0.015 (~1.5 %)
3. p = 1 (the normal allele frequency in the population, is very close to 1, according to the
first important property of the HWL)
4. 2pq (Aa) = 2 x 1 x 0.015 = 0.03 (~ 3 %)
The percentage of heterozygote carriers in this population therefore is about 3%
disorders
Example 2 – carrier genotype frequency:
PKU incidence in China: 1 in 100,000
What is a carrier frequency ?
• p = frequency of normal allele (A)
• q = frequency of mutant allele (a)
1. q2 (aa) = 1/100000 ®
2. q = 1/100000 = 1/316
3. p = 1 (the normal allele frequency in the population, is very close to 1)
4. 2pq (Aa) = 2 x 1 x 1/316 = 2/316 = 1/158 or 0.0063 = 0.63%

disorders
Example 3 – allele frequency and genotype frequency:
Tay-Sachs disease is an autosomal recessive disorder for which the carrier
frequency in individuals of Ashkenazi Jewish descent is about 1 in 30.
How frequent is Tay-Sachs disease in this population?
• p = frequency of normal allele (A)
• q = frequency of mutant allele (a)
1. p = 1 (the normal allele frequency in the population, is very close to 1 )

2. Aa = 2pq = 1/30 given
3. q = (1/30)/2 = 1/60 – this is mutant allele frequency
4. Answer: aa = q2 = (1/60)2 = 1/3600 – this is disease frequency

Usage of the Hardy Weinberg law for
Autosomal Recessive disorders
4a. A 20-year-old female from one of European country is aware that she has an
AR genetic disease that has required her lifelong adherence to a diet low in
natural protein with supplements of tyrosine and restricted amounts of
phenylalanine. She asks her geneticist about the chances that she would marry a
man with the disease-producing allele.
• The geneticist tells her that the known prevalence of PKU (a disease frequency, aa genotype)
in the in European populations is 1/10,000 live births. Her greatest risk comes from marrying a
carrier for two reasons. First, the frequency of carriers for this condition (Aa) is much higher
than the frequency of affected homozygotes, and second, an affected person would be
identifiable clinically. The geneticist used the Hardy-Weinberg equation to estimate the carrier
frequency from the known prevalence of the disease in the following way:
• genotype frequency (disease prevalence) = q2 = 1/10,000 live births
• q = square root of 1/10,000, which is 1/100
•Dr. Carrier frequency = 2q = 2/100, or 1/50 is the carrier frequency
4b. The woman now asks a second question: “Knowing that I have a 1/50 chance
of marrying a carrier of this allele, what is the probability that I will have a child
with PKU?”
• The probability that she will marry a heterozygous carrier = 1/50
• If he is a carrier, the probability that he will pass his PKU allele (a) versus the
normal allele (A) to the child = 1/2.
• She is homozygote (aa), so she will definitely pass affected allele = 1 (100%)
• These probabilities would be multiplied to give:
1 × 1/50 × 1/2 = 1/100, the probability that she will have a child with PKU.


Hardy-Weinberg Equilibrium for Dominant
Diseases
• Frequency of normal allele “a” = p
• Frequency of mutant allele “A” = q
• Most of the affected individuals will be heterozygous (Aa).
• p2 = frequency of healthy (normal) homozygote (aa)
• 2pq = frequency of affected heterozygote (Aa)
• q2 = frequency of affected homozygote (AA)

Hardy-Weinberg Equilibrium for Dominant
Diseases
Example 5:
• 1/500 people in the United States have a form of LDL-receptor deficiency (semi-dominant type,
means most of affected people are heterozygotes Aa) and are at increased risk for
cardiovascular disease and myocardial infarction. What is a frequency of affected homozygotes
in the United States?
1. 2pq (Aa) = 1/500 given
2. q = (1/500)/2 = 1/1000
3. q2 (AA) = (1/1000)2 = 1/1000000 = 1/106
• or one in a million live births are homozygous for the condition.
• These individuals have greatly elevated LDL-cholesterol levels, a much-higher risk for cardio-
vascular disease than heterozygotes, and are more likely to present with characteristic
xanthomas, xanthelasma, and corneal arcus.

X-linked Recessive Inheritance
1. Since a recessive disease-causing mutation occurs
on the X chromosome, a male will be affected with
the disease.
2. Because males require only one copy of the
mutation to express the disease and females require
two copies, X-linked recessive diseases are seen
much more commonly in males than in females.
3. All the daughters of an affected male are
heterozygous carriers.
4. A heterozygous (carrier) female transmits the
disease causing mutation to 50% of her sons (which
are affected) and 50% of her daughters (which are
carriers).
The Hardy-Weinberg Law in X-Linked
Recessive Disease
• Frequency of normal allele “XA” = p
• Frequency of mutant allele “Xa” = q
• Female genotypes:
• p2 = frequency of healthy homozygote (XAXA)
• 2pq = frequency of carrier heterozygote (XAXa) Females mainly are carriers
• q2 = frequency of affected homozygote (XaXa) ~ 0
• Only 2 possible male genotypes:
• p = XAY – healthy
• q = XaY – affected
• For males the genotype frequencies is q (disease frequency – XaY genotype) and gene
frequencies in the population is q (affected allele frequencies – only one X chromosome is
enough for disease) are equal !!!!
Recessive Disease
• The frequencies of the normal and mutant alleles in the population can be
determined directly from the incidence of the corresponding phenotypes in
males by simply counting the genotypes.
• 1/10,000 males has hemophilia A also gives the allele frequency for the disease-
producing allele: 1/10,000 in the population
• Because females have two X chromosomes, their genotypes are distributed

binomially, exactly like autosomal genotypes
• As most X-linked alleles are recessive, the normal homozygotes and
heterozygotes are not distinguishable in phenotype in females

Recessive Disease
Example 6:
• 8% Caucasian males have color blindness (which is X-linked recessive disorder):
• q = 8% = 0.08 given*
• p = 92% = 0.92
• Counts In females:
• Normal non-carrier = XAXA = p2 = 0.922 = 0.8464
• Normal carrier = XAXa = 2pq = 2 x 0.08 x 0.92 = 0.1472
*X-linked recessive disorders, males receive either the normal allele or the mutant
allele in proportion to their frequency in the population.
Factors That Disturb
Hardy - Weinberg Equilibrium
Natural Selection
• Natural selection is one of the basic mechanisms of evolution, along with mutation, migration,
and genetic drift.
• Natural selection is the differential survival and reproduction of individuals due to differences
in phenotype (alleles frequency change).
Natural selection acts on the phenotype, the
characteristics of the organism which actually interact with
the environment, and the genetic (heritable) basis of any
phenotype that gives that phenotype a reproductive
advantage may become more common in a population.
Over time, this process can result in populations that
specialise for particular ecological niches (microevolution)
and may eventually result in speciation (the emergence of
new species, macroevolution).
• Hardy - Weinberg law cannot be applied with respect to long time period. HWL is a null
hypothesis for the evolutionary changes.
Natural Selection
• Natural variation occurs among the individuals of any population of
organisms. Some differences may improve an individual's chances of
surviving and reproducing such that its lifetime reproductive rate is
increased, which means that it leaves more offspring. If the traits that
give these individuals a reproductive advantage are also heritable, that
is, passed from parent to offspring, then there will be differential
reproduction, that is, a slightly higher proportion of fast rabbits or
efficient algae in the next generation.
• The concept of fitness is central to natural selection. In broad terms,
individuals that are more "fit" have better potential for survival. In
other words, natural selection is a key process in the evolution of a
Dr. population.
Factors That Disturb Hardy - Weinberg
Equilibrium
• Although human populations are typically in Hardy-Weinberg equilibrium for
most loci, deviations from equilibrium can be produced by new mutations, the
introduction of a new mutation into a population from outside (founder effect),
nonrandom mating (for example, consanguinity), the action of natural selection,
genetic drift, and gene flow.
In general:
• Violating the assumption of random mating can cause large deviations from the
frequency of disease based on allele frequencies.
• Changes in allele frequency due to mutation, selection, or migration cause more

minor and subtle deviations.
Factors That Disturb Hardy-Weinberg
Equilibrium
• In random mating, for any locus, an individual of a given genotype (AA) has a
purely random probability of mating with an individual of any other genotype (Aa
or aa).
• But simple observation shows that a number of factors enter into one's choice of
mate.
• 3 ways the random mating assumption breaks down:

• Stratified mating
• Assortative mating
• Consanguineous mating

1. Stratified Mating
• Stratified population is one divided into sub-groups
• e.g. in USA, the population can be sub-divided into Caucasians, African Americans,
Hispanics, Native Indians, Asian, Mediterranean origin, etc.
• It is apparent that there are more mating within the subgroups than Inter-group
mating
• Result: the presence of a systematic difference in allele frequencies between

subpopulations in a population - Increased proportions of homozygotes for
certain genetic traits (alleles) common within the sub-group
• incidences of thalassemia in East Indian and Mediterranean populations

• incidences of CF and PKU in Caucasian population.

2. Assortative Mating
• It is the choice of a mate because the mate
possesses some particular trait. Individuals with
similar phenotypes mate with one another more
frequently than would be expected under a
random mating pattern.
• Positive assortative mating:

• when mates with similar characteristics are
chosen (native language, intelligence, stature,
skin color, musical talent or athletic ability)
• Negative assortative mating:

• when mates with dissimilar characteristics are
preferred, less common.
2. Assortative Mating
• Clinically significant if characteristics selected are

“genetic”
• People with genetic deafness, vision deficiencies or
dwarfism selecting similar mates
• Result: Increased proportions of homozygotes

and decreased heterozygotes for the alleles (and
diseases) which are common in both partners.

3. Consanguineous Mating
• It may lead to an increase in the frequency of AR disease.
• Differentiate from stratified matings

• In stratified populations, each sub-group is expected to have a higher
frequency of a few alleles relative to the general population. If those alleles
are disease causing there will be an increased frequency of the disease
• In consanguineous matings rare alleles are much more likely to become

homozygous and so rare disorders are much more likely to occur

Exceptions to Constant Allele Frequency
• Heterozygote advantage
• Genetic Drift
• Gene Flow

1. Heterozygote advantage
• Heterozygotes for some diseases have increased fitness over both types of
homozygous genotypes
• Sickle cell anemia (Heterozygotes have advantage to malaria)

• Tay Sachs (Heterozygotes have protection against Mycobacteria tuberculosis)
• Cystic fibrosis (The carrier of the CFTR mutation will not have Cystic Fibrosis, but will have
only half as many chloride channels as a non-carrier. This limits the amount of fluid lost to
diarrhea in the event a carrier gets infected with a disease like Typhoid or Cholera)

• Natural selection can act upon the genetic variation to increase frequencies of
genes that promote survival and decrease frequencies of genes that decrease
survival.
SICKLE CELL DISEASE
Affects 1 in 600 African-Americans and 1 in 50 individuals in
some parts of Africa
RBC of normal people (homozygotes for normal alleles) are
conducive to malarial infection/survival - more malaria induced
deaths.
Heterozygotes with moderate sickle cell disease are resistant
to malaria (the malarial parasite does not survive well in the
RBCs of sickle cell heterozygotes). These individuals are thus
protected from the lethal effects of malaria
• Greater survival compared to homozygotes and normal.

Other Examples of Heterozygote Advantage
1. Hemoglobin C
2. Thalassemia
3. G6PD deficiency
• All linked with advantage due to the RBC's resistance against malaria and
similar infections

1. Heterozygote advantage:
An African American couple has produced two children with sickle cell disease. They have
asked why this disease seems to be more common in the African American population than in
other U.S. populations. Which of the following factors provides the best explanation?
A. Consanguinity
B. Genetic drift
C. Increased gene flow in this population
D. Increased mutation rate in this population
E. Natural selection
The frequency of sickle cell disease is elevated in many African populations because
heterozygous carriers of the sickle cell mutation are resistant to malarial infection but do not
develop sickle cell disease, which is autosomal recessive. Thus, there is a selective advantage
for the mutation in heterozygous carriers, elevating its frequency in the population.
2. Genetic Drift:
• is the change in the frequency of an existing gene variant (allele) in a small
population due to random sampling of organisms during change of generations
• The alleles in the offspring are a sample of those in the parents, and chance has
a role in determining whether a given individual survives and reproduces
• fluctuation in allele frequency due to chance operating on the small gene pool
contained within a small population
• In a small population, allele frequency may fluctuate by random chance (for
reasons unrelated to carrying the mutant allele i.e., changed fertility) from
generation to generation.

2. Genetic Drift:
May cause extreme reduction in population by two mechanisms:
• Bottleneck effect - Disaster that kills (a tidal wave, earthquake etc.)
• Founder effect - The loss of genetic variation when a new colony is established by a very small
number of individuals from a larger population
In each generation, some individuals may, just

by chance, leave behind a few more descendants (and
genes, of course!) than other individuals.
The genes of the next generation will be the
genes of the "lucky" individuals, not necessarily the
healthier or "better" individuals.
It happens to ALL populations.
3. Gene Flow:
• is the transfer of genetic variation from one population to another
• slow diffusion of genes across a barrier, involving large populations and resulting
in a gradual change in gene frequencies
Gene flow — also called migration — is any movement of
genes from one population to another. Gene flow includes
lots of different kinds of events, such as pollen being blown to
a new destination or people moving to new cities or
countries.
If genes are carried to a population where those genes
previously did not exist, gene flow can be a very important
source of genetic variation

Genetics of Cancer
Course:
Biochemistry and Medical Genetics
Prepared by: Dr. Alisa Chebotarova, MD, PhD
Modified by: Dr. I. Vyshnytska, MSc, PhD
INTENDED LEARNING OBJECTIVES
By the end of the lecture students must be able to:
1. Outline the current state of knowledge about the biochemical and molecular basis of cancer
2. Describe the Main Forms of Cancer
3. Explain the main difference between Benign and Malignant Tumors
4. List hallmarks of Cancer
5. List Cancer-causing agents
6. Describe Proto-oncogenes and Oncogenes
7. Explain Mechanisms of activation of Oncogenes
8. Describe Tumor Suppressors – definition, functions, classification (Gatekeepers, Caretakers)
9. List Mutations in Tumor suppressors
10. Explain Loss of Heterozygosity

High-Yield Terms
• Carcinogen ✮ : any substance, such as a chemical, or any type of radiation that is
directly involved in causing cancer
• Neoplasm: any abnormal mass of tissue, medically synonymous with the term tumor
• Malignancy: medically describes any condition that becomes progressively more severe,
with respect to cancers the term characterizes invasiveness and the tendency to
metastasize
• Metastasis: refers to the spread of cancer from a site of origin to a new organ or
another part of the body
• Proto-oncogene ✮ : any normal gene that can become an oncogene due to gene
mutation or abnormal expression
• Oncogene ✮ : any gene that has the potential to cause cancer, in tumor cells, these
genes are often mutated or expressed at high levels
• Tumor suppressor ✮ : any gene that prevents the unregulated growth of cells, loss of
function of this class of gene can result in cancer, also called antioncogene
Cancer genetics
Transformation of a cell is due to the action of one or more “oncogenes”

(gain of function) and/or due to the loss of one or more “tumor suppressor
genes” (loss of function).
Basics of Cancer Biology ✮
• Tumors or Neoplasms
• a disease process characterized by uncontrolled cellular proliferation leading to a
mass formation
• Benign tumor
• does not metastasize
• grows in a confined local area
• Malignant tumor
• Uncontrolled growth
• capable of invading neighboring tissues or spreading (metastasizing)
• Cancer: a general term for a malignant tumor

Benign vs. Malignant ✮
Benign Malignant
• Slow growing • Fast growing
• Capsulated • Non capsulated
• Non-invasive • Invasive and Infiltrate
• Do not metastasize • Metastasize
• Well differentiated • Poorly differentiated
• Suffix ‘oma’ eg., Fibroma • Suffix “Carcinoma” or
“Sarcoma”

Benign to Malignant
• Cancer arises from a loss of normal growth control. In normal tissues,
the rates of new cell growth and old cell death are kept in balance.
• In cancer, this balance is disrupted.
• This disruption can result from uncontrolled cell growth or loss of a
cell's ability to undergo cell suicide by a process called "apoptosis"
• In normal tissues, apoptosis, or "cell suicide," is the mechanism by
which old or damaged cells normally self-destruct.

Three Main Forms of Cancer
• Carcinomas
– originate in epithelial tissues
– Account for 90% of all cancers
• Sarcomas
– originating in mesenchymal tissue (bone, cartilage,
fat, connective tissue, muscle, nervous tissue)
• Leukemias and lymphomas
– cancer cells reside and proliferate mainly in the
bloodstream, lymphatic system and bone marrow

Biochemical Characteristics of Cancer Cells:
1. Increased synthesis of DNA and RNA.
2. Increased rate of glycolysis both aerobic and anaerobic. ✮
3. Alterations of permeability and surface charge.
4. Changes in composition of glycoproteins and glycosphingolipids on cell surfaces (alterations
of the oligosaccharide chains).
5. Increased activity of ribonucleotide reductase and decreased catabolism of pyrimidines ✮
6. Secretion of certain proteases and protein kinases.
7. Alterations of isoenzyme patterns often to a fetal pattern and synthesis of fetal proteins, ✮
e.g. carcinoembryonic antigen (CEA), α-fetoprotein (AFP), etc.
8. Appearance of new antigens and loss of certain antigens.
9. Inappropriate synthesis of certain hormones and growth factors.
10. Cancer cells continue to express telomerase activity which prevents telomere shortening.
*Hallmarks of Cancer: The Next Generation, 2011; https://www.cell.com/fulltext/S0092-
8674(11)00127-9
Cancer Causing Agents
Carcinogens
• Carcinogens are cancer-causing agents
• Ionizing Radiation
• Chemical Carcinogens
– Asbestos and lung cancer
– Alcohol and liver cancer
– Smoking and lung cancer
• Biological agents
– Aflatoxin B1 and liver cancer
– Hepatitis B (HBV) and hepatocellular cancer
– Human Papilloma Virus (HPV) and cervical cancer
– Human immunodeficiency virus (HIV) and Kaposi
sarcoma
– Helicobacter pylori and stomach cancer
– Schistosomiasis and bladder cancer
Radiation and Cancer
• Ionizing and ultraviolet radiation
• DNA mutations that lead to cancer
• Risk is age dependent
- under 10 and elderly
• Significant risk for individuals with inborn
defects of DNA repair

Radiation and Cancer
1. Exposure to ultraviolet radiation in the form
of sunlight is related to the frequency of skin
cancers, such as squamous cell and basal cell
carcinomas and melanomas.
The process is thought to act by inducing
dimer formation between neighboring thymine
pairs in DNA. In most cases, such dimers are
successfully repaired by enzymatically
mediated mechanisms (excision
endonuclease).
• xeroderma pigmentosum, an AR disorder
characterized by failure of DNA excision repair
mechanisms.
2. Ionizing radiation is a classic cause of
cancer, exemplified by the increased incidence
of cancers in those exposed to radiation.
Chemical Carcinogens
• When chemical carcinogens are internalized by cells, they are often metabolized,
and the resulting metabolic products are either excreted or retained by the cell.
• Inside the cell, carcinogens or their metabolic products can either directly or
indirectly affect the regulation and expression of genes involved in cell-cycle
control, DNA repair, cell differentiation or apoptosis.
• For example, carcinogenic ions or compounds of nickel, arsenic and cadmium can induce
structural and numerical chromosome aberrations.
• Together genotoxic and non-genotoxic mechanisms can alter signal-transduction

pathways that finally result in hypermutability, genomic instability, loss of
proliferation control, and resistance to apoptosis — some of the characteristic
features of cancer cells.
Oncogenes
Oncogenesis ✮
• Oncogenes are abnormal (mutated) versions of “Proto-oncogenes” that are part
of normal cellular function. ✮
• The process of activation of proto-oncogenes to oncogenes can include retroviral
transduction or retroviral integration, point mutations, insertion mutations, gene
amplification, chromosomal translocation, and/or protein-protein interactions.
• Retroviruses: RNA viruses that synthesize DNA as the replicative form and cause insertion
of DNA into the host DNA. This DNA carries a mutant form of the genes.
• Modification of the structure and/or level of expression of a protooncogene may
convert it into an oncogene.
• Oncogene formation is usually an autosomal dominant mutation (gain of
function mutation)
Oncogenes
Oncogenes arise from the mutation of proto-
oncogenes.
They resemble proto-oncogenes in that they code
for the production of proteins involved in growth
control.
However, oncogenes code for an altered version (or
excessive quantities) of these growth-control
proteins, thereby disrupting a cell's growth-
signaling pathway
By producing abnormal versions or quantities of
cellular growth-control proteins, oncogenes cause
a cell's growth-signaling pathway to become
hyperactive.
GF signaling in norm
Dr. Alisa Chebotarova, MD, PhD *MAP - the mitogen-activated protein kinase
GROWTH FACTORS
Action at Molecular Level
1. Most of the growth factors have high affinity protein receptors on the
membranes of target cells.
2. Genes for receptors of EGF and IGF have been extensively studied. Structurally
they are found to have short membrane spanning segments and external and
cytoplasmic domains of varying lengths.
3. Most of the receptors, e.g. of PDGF, EGF, etc. have been found to exhibit protein
tyrosine kinase activity which is located in cytoplasmic domain.
4. The kinase activity brings about autophosphorylation of the receptor protein
and also phosphorylation of other proteins of target cells.
5. Growth factors interact with the specific membrane receptor and transmits the
message across the plasma membrane to the interior of the cells by
transmembrane signal transduction, which finally affect one or more processes
involved in mitosis of the cells.
Growth Factor signaling: key players
• Growth Factors: ligands that stimulate cell proliferation
• Growth Factor Receptors: a mutated receptor may remain constitutively active in

the absence of a ligand: c-erbB2, ret
• Plasma membrane G proteins: ras
• Nonreceptor Protein Kinases: Abl
• Transcription factors
• Myc → Altered gene expression.
• Cell cycle or cell-death regulators

• Cdk4, cyclin D, Bcl-2
Growth Factors (Mitogens)
• Are usually polypeptides.

• Play a major role in regulating differentiation of stem cells to form various types
of mature cells.
• Products of several oncogenes are either growth factors or parts of receptors for
growth factors.
• Chronic exposure of target cells to increased amounts of a growth factor or to
decreased amounts of a growth inhibitory factor may alter the balance of cellular
growth (growth autonomy).

Growth Factors
• Growth factors may act in the following 3 ways:

• Endocrine action: Similar to hormone action. May be synthesized in one place
and then carried by bloodstream to target cells where they exert their effects.
• Autocrine action: Synthesized and act on the same cells.
• Paracrine action: Synthesized in certain cells and secreted from them to affect the
neighbouring cells.

Growth Factors

Growth Factors: A modified receptor
In contrast, receptors
Under normal circumstances
encoded by oncogenes do
membrane-bound receptors require the
not require the regulatory
binding of their ligand to be in an
activated state. step of ligand binding to be
active (constitutively active
Dr. Alisa Chebotarova, MD, PhD receptores).
RET proto-oncogene ✮
• A point mutation in RET gene makes it active oncogene
• Encodes for a Receptor tyrosine kinase (RTK)
• RET autophosphorylation and intracellular signaling: phosphorylated tyrosines

become docking sites for intracellular signaling proteins
• Abnormal gene product: Mutations in codons in the cysteine-rich extracellular

domain results in Ligand-independent RET
• Constitutive activation (i.e., gain of function) of tyrosine kinase

RET and Multiple Endocrine Neoplasia type 2
• Autosomal Dominant
• Associated with RET gene mutations (10q11.2)
• Receptor tyrosine kinase
• MEN 2A:
• Medullary thyroid carcinoma (secretes calcitonin)
• Pheochromocytoma
• Parathyroid hyperplasia
• MEN 2B:
• Medullary thyroid carcinoma (secretes calcitonin)
• Pheochromocytoma
• Oral/intestinal ganglioneuromatosis (mucosal neuromas).
• Associated with marfanoid habitus.
RAS proto-oncogene ✮
• A point mutation in RAS gene makes it active
oncogene
• One of the first oncogenes discovered was a

mutant RAS gene that encoded a large family
of small GTP binding (G) proteins.
• Point mutations in the RAS gene result in the
ras protein that is locked in its active, GTP-
bound form.
• Transfection of normal cells with RAS (and
other oncogenes) causes these cells to grow
in uncontrolled fashion (transformation).
• RAS has been found to be associated with a
large number of cancers (colon, lung,
pancreatic)
HER2 / neu ✮
• HER2 is a protein receptor that in humans is
encoded by c-erbB2 gene, a known proto-
oncogene located at the long arm of human
chromosome 17
• Amplification of the c-erbB2 leads to
addition the extra copies of chromosomal
regions containing proto - oncogenes.
• The up to 200-fold amplification of proto-
oncogene products like HER2/neu-human
epidermal growth factor receptor (in
breast cancer in older females, ovarian,
and gastric carcinomas) can be found.
HER2 gene status
identified by FISH.
• Chromosome 17
centromere (CEP17) gain is
frequently observed in
breast cancer by FISH. Red
signals represent HER2
gene, green signals
represent CEP17.
• A: HER2 gene negative
case;
• B: HER2 gene positive case,
HER2/CEP17 ratio of higher
than 2.0;
• C: HER2 gene positive case,
HER2 signals are clustered;
• D: HER2 gene with CEP17
gain.
Chromosomal Translocation
• A proto-oncogene may also be activated in some instances by a chromosomal
mutation, most usually, a translocation.
• Three important examples:
• Chronic myelogenous leukemia (CML):
• translocation between chromosomes 9 and 22.
• Burkitt lymphoma:
• translocation between chromosomes 8 and 14.
• B-cell lymphoma:
• Translocation involving chromosomes 18 and 14 leads to over-expression of the anti-
apoptotic gene bcl-2.

Chronic myelogenous leukemia (CML) and
Philadelphia chromosome ✮
• A reciprocal translocation of the long arms of
chromosomes 9 and 22, termed the Philadelphia
chromosome.
• This translocation alters the activity of the abl proto-
oncogene (chr 9)
• The BCR locus was originally identified as a region of
frequent chromosomal breakage and thus, termed the
breakpoint cluster region, BCR (chr 22)
The chimeric BCR-ABL fusion gene on the Philadelphia

chromosome encodes a membrane-associated protein
tyrosine kinase that remains constitutively active
Chromosomal translocation and Burkitt
Lymphoma ✮
• Translocation between chromosome 8 and 14
occurs in Burkitt lymphoma.
• The gene for the transcription factor c-myc
(from 8) comes under the influence of strong
promotors of the heavy immunoglobulin chains
(14).
• rare form of cancer
• B-cell lymphoma
• predominantly affecting young children in
Central Africa
– associated with Epstein Barr virus infection
– pathogenic mechanism involves EBV-induced latent
membrane protein (LMP-1)
Chromosomal translocation and B-cell
Lymphoma ✮
• Translocation involving chromosome 14 and 18 in
B-cell lymphoma (Follicular and undifferentiated
lymphomas).
• Translocation places bcl-2 (from 18) near the
strong promoter for immunoglobulin heavy chain
gene (14)
• Thus leading to over-expression of the anti-
apoptotic gene BCL2.
• Damaged cells may then escape apoptosis

Tumor Suppressors
Tumor Suppressors
• Tumor suppressor genes are genes that regulate the growth of cells and when
these genes are functioning properly, they can prevent and inhibit the growth of
tumors.
• There are 3 main classes of tumor suppressor genes:
• These include genes that encode proteins that tell cells to stop growing and
dividing.
• Another class encodes proteins responsible for repairing DNA damage prior to
allowing cells to complete the cell cycle.
• The third class encodes proteins that are involved in regulating cell death
processes, called apoptosis.

Mutations in Tumor suppressors can cause
cancer ✮
• Tumor suppressor proteins arrest cell cycle at checkpoints and keep cell division
in control
• Mutations in tumor suppressor genes cause a loss of function of these proteins
• Both copies of tumor suppressor genes need to be mutated for development of
cancers

Oncogenes vs. Tumor suppressors

The two-hit model of cancer ✮
• Tumor-Suppressor Genes
• Both alleles of the tumor suppressor gene
need to be mutated
• 1st hit can be inherited
• 2nd hit can arise spontaneously
• By mutation
• Epigenetic origin
• gene silencing
• X inactivation
• imprinting

The two-hit model of cancer ✮
• Tumor-Suppressor Gene: Retinoblastoma gene, RB1
• In hereditary retinoblastoma, mutations in the RB1 gene appear to be inherited
in an autosomal dominant pattern (means that one copy of the altered gene in
each cell is sufficient to increase the risk of cancer).
• 1st hit: A person with hereditary retinoblastoma may inherit an altered copy of
the RB1 gene from one parent, or the altered gene may be the result of a new
mutation that occurs in an egg or sperm cell or just after fertilization.
• 2d hit: For retinoblastoma to develop, a mutation involving the other copy of the
RB1 gene must occur in retinal cells during the person's lifetime. This second
mutation usually occurs in childhood, typically leading to the development of
retinoblastoma in both eyes (bilateral).

Loss of Heterozygosity - LOH ✮
• When normal tumor suppressor genes are absent or nonfunctional
due to LOH, the cell may begin to divide abnormally and become
cancerous.
• If a patient 1) inherits or develops a mutation in a tumor suppressor
gene, 2) the complementary allele must be deleted/mutated before
cancer develops.
• In case of Retinoblastoma:
• One RB1 allele is mutant
• The remaining normal RB1 allele is lost in heterozygotes (LOH)

Loss of function mutations in proteins
that can lead to carcinogenesis
1. Proteins that regulate a particular phase of the cell cycle or monitor

checkpoints to arrest cell cycle (e.g., Rb, p53 block G1→S phase)
2. Proteins that are components of growth-inhibitory signaling pathways (APC
protein)
3. Proteins that promote apoptosis (Fas, Bax)
4. Proteins that participate in DNA damage repair (NER proteins implicated in
Xeroderma pigmentosum, BRCA1, BRCA2)

Checkpoints and Cell Cycle Regulation
•The processes that drive a cell through the cell cycle must be highly regulated so
as to ensure that the resultant daughter cells are viable and each contains the
complement of DNA found in the original parental cell.
•Many important genes involved in the regulation of cell cycle transit have been
identified and are referred to as cell division cycle genes (or cdc genes). Many of
these genes encode proteins that control progression through the phases of a cell
cycle at specific points called checkpoints or restriction points.

•The heart of this timing control is the responsibility of a family of

protein kinases that are called cyclin-dependent kinases, CDK.
•Different CDK operate at different points in the cell cycle.
•The kinase activity of these enzymes rises and falls as the cell progresses
through a cell cycle.
•Constitutively expressed but inactive when not bound to cyclin.

•The cyclical activity of each CDK is controlled by the proteins cyclins

• Regulatory proteins that control cell cycle events;
• phase specific;
• activate CDKs.
•Four different classes of cyclins have been defined on the basis of the stage of the
cell cycle in which they bind and activate CDKs:
• G1-cyclins,
• G1/S-cyclins,
• S-cyclins,
• M-cyclins.
• Cyclin-CDK complexes:
• Phosphorylate other proteins to coordinate cell cycle progression;
• must be activated and inactivated at appropriate times for cell cycle to progress.
• p21 is CDK inhibitory proteins (CIP): protein that binds to and inhibits cyclin-CDK
complexes
• and there are other CIP: p27, p57, p15, p16, p18, and p19 in mammalian cells.

Rb

Retinoblastoma protein
• The retinoblastoma protein (protein name abbreviated pRb; gene name
abbreviated RB or RB1) is a tumor suppressor protein that is dysfunctional in
several major cancers.
• This protein acts as a tumor suppressor, which means that it regulates cell growth
and keeps cells from dividing too fast or in an uncontrolled way.
• One function of pRb is to prevent excessive cell growth by inhibiting cell
cycle progression (G1→S) until a cell is ready to divide.
• The Rb protein is present in one of two forms:
• Active – hypophosphorylated
• Inactive – hyperphosphorylated
• The resting cell in G0 phase contain active – hypophosphorylated Rb protein

Role of Retinoblastoma protein
• Active: Early in G1/G0, Rb is in its hypophosphorylated active form, and it binds to
and inhibits the E2F family of transcription factors, preventing transcription of
cyclin E.
• The initiation of DNA replication (S phase) requires the activity of cyclin E/CDK2
complexes.
• Inactive: Growth factor signaling (or mitogenic signaling) leads to cyclin D

expression and activation of cyclin D–CDK4/6 complexes. These complexes
phosphorylate Rb, inactivating the protein and releasing E2F to induce target
genes such as cyclin E gene.
• Expression of cyclin E then stimulates DNA replication and progression through
the cell cycle.
Retinoblastoma ✮
• A rare type of eye cancer that forms in

immature cells of the retina and most often
manifest in early childhood prior to the age
of 5.
• Diagnosis of retinoblastoma after the age of 6 is
extremely rare due to the terminal differentiation of
retinal epithelial cells.
• Autosomal dominant
• Mutation in the Rb tumor suppressor gene

• microdeletions in chromosome 13 in
affected individuals, in the RB gene
Retinoblastoma ✮
• Two forms:
• sporadic-always unilateral - 60% of the total cases
• Requires two somatic “hits"
• Two mutations in same cell
• Often a single tumor
• Occurs at a later age
• familial-autosomal dominant - 40% of the total cases ( 80%

bilateral, 15% unilateral, 5% asymptomatic)
• One gene mutated in all cells at birth (germline
mutation)
• Second somatic mutation “hit"
• Frequent, multiple tumors
• Tumors at younger age
• high risk of other primary tumors (osteosarcoma)
Retinoblastoma ✮
• Tumors develop due to unchecked division of precursor cells in the immature
retina.
• In the non-hereditary form, typically only one eye is affected and there is no
family history of the disease. Affected individuals are born with two normal
copies of the RB1 gene. Then, usually in early childhood, both copies of the RB1
gene in certain retinal cells acquire mutations. People with non-hereditary
retinoblastoma are not at risk of passing these RB1 gene mutations to their
children.
• In the hereditary form of the disease, one copy of the Rb gene is mutated in
every cell and the cells become predisposed to become cancerous. When the
good second copy of the gene is damaged due to a somatic mutation, the cells
lose the control of G1-S checkpoint.
p53 is an important tumor suppressor protein and
is induced during DNA damage ✮
• The p53 tumor suppressor protein is encoded by the TP53 gene, located in
chromosome 17
• Expression of p53 is very low in normal cells. p53 is stimulated by cellular stress
like ionizing radiation, hypoxia, carcinogens, and oxidative stress.
• The p53 gene is the most common genetic alteration in cancer, and patients who
inherit a mutated copy of the p53 gene have an increased predisposition to soft
tissue neoplasms, leukemias, central nervous system tumors, and breast cancer.

The p53 tumor suppressor gene acts as the
‘Guardian of the Genome’ ✮
• The p53 has three major effects:
1. p53-mediated cell cycle arrest. It occurs late in
the G1 phase and is caused mainly by p53-
dependent transcription of the CDK inhibitory
proteins gene (p21). The p21 protein inhibits
cyclin–CDK complexes and prevents
phosphorylation of Rb, thereby arresting cells in
the G1 phase.
2. p53-induced senescence is a permanent cell
cycle arrest
3. p53-induced apoptosis of cells with irreversible
DNA damage
p53 Tumor Suppressor and Cancer
• More than 50% of all human cancers exhibit mutations in the TP53 gene.
• Examples of cancers in which p53 fails to activate include Li-Fraumeni syndrome
and Ataxia telangiectasia.
• Metabolic activation of benzo(a)pyrene, which is present in cigarette smoke and
charbroiled meat, produces a potent mutagen.
• Activated benzo(a)pyrene causes mutations in genes such as TP53 by G→T
transversions.
• Aflatoxin, a fungal metabolite that is present as a contaminant in moldy grain and
peanuts also induces G→T transversions in TP53 gene.
• Both benzo(a)pyrene and aflatoxin are the carcinogens because the mutations they cause
lead to cancer.

Li - Fraumeni Syndrome ✮
• Li-Fraumeni syndrome is a rare autosomal
dominant disorder that greatly increases the risk
of developing several types of cancer.
• Is a familial DNA repair syndrome caused by
mutation in the tumor-suppressor gene p53.
• The cancers most often associated with Li-
Fraumeni syndrome include breast cancer, a form
of bone cancer (osteosarcoma), and cancers of
soft tissues (such as muscle) called soft tissue
sarcomas.
• Other cancers commonly seen in this syndrome
include brain tumors, cancers of blood-forming tissues
(leukemias), and a cancer called adrenocortical
carcinoma that affects the outer layer of the adrenal
glands.
Li - Fraumeni Syndrome ✮
• A 23-year-oldwoman develops a tumor of the soft tissue of her arm (sarcoma)
and is being evaluated by an oncologist. The physician learns that her older
brother had leukemia when he was 12 years old, her grandmother died of a brain
tumor, and her aunt has breast cancer. With this clustering of tumors, the
oncologist suspects Li-Fraumeni syndrome and orders molecular studies on the
patient’s tumor for which of the following genes?
a. p53
b. RET
c. WT-1
d. Rb
e. TNF
BRCA1, BRCA2 and breast and ovarian cancer ✮
• BRCA1:
• Tumor-suppressor gene, BRCA-1, has been identified at the chromosomal locus
17q21.
• Functions of BRCA-1 gene product
• This gene encodes a zinc finger protein and the product therefore may function
as a transcription factor.
• The gene product also appears to be involved in Double Strand Break repair.
• BRCA1 gene mutations – autosomal dominant! - do not always result in breast or ovarian
cancer – Incomplete penetrance

About 5% to 10% of breast and 10% to 15% of
ovarian cancers are hereditary.
• BRCA1:
• Women who inherit a mutated allele of this gene from either parent have at
least a 60-80% lifetime chance of developing breast cancer and about a 33%
chance of developing ovarian cancer.
• Men who carry a mutant allele of the gene have an increased incidence of
prostate cancer and breast cancer.
• family history of breast cancer, often at a young age.
• BRCA2:
• This gene has been localized to chromosome 13q12; it is also associated with an
increased incidence of breast cancer in men and women.

APC gene and Familial Adenomatous
Polyposis ✮
• Familial Adenomatous Polyposis (FAP) is an
inherited disorder characterized by a highly elevated
risk of developing colorectal carcinoma. FAP is
associated with germline mutations in the
adenomatous polyposis coli (APC) gene.
• FAP adenomas appear as a result of loss of function
mutations to the APC gene which characterizes the
gene as a tumor suppressor
• Multiple colonic polyp development, within the first
decades of life, is characteristic of FAP. These polyps
become malignant carcinomas and adenomas later
in life.
• Colectomy (removal of colon) prevents development of malignancies
NF 1 gene ✮
• NF-1 is a tumor suppressor gene on the
chromosome 17
• NF1 gene encodes a GTP ase-activating protein
(GAP) that accelerates GTP hydrolysis of active GTP-
Ras to inactive GDP-Ras.
• Ras is a signal transducer protein that is active when
bound to GTP. Active Ras promotes cell proliferation.
• Loss of NF1 function (due to mutation) allows ras to
remain in its activated GTP-bound state for an
extended period.

Neurofibromatosis 1 (von Recklinghausen disease)✮
• Neurofibromatosis type 1 is an autosomal dominant disorder, though some

cases result from spontaneous new mutations (no prior family members with the
mutation).
• NF-1 exhibits variable expressivity, because the manifestations (location and
types of neoplasms) are not the same in all patients:
• changes in skin coloring (pigmentation): 6 or more café au lait spots
• 2 or more neurofibromas: the growth of tumors along nerves in the skin, brain, and other
parts of the body.
• Axillary or inguinal freckling
• Optic glioma

WT genes
• The WT1 gene provides instructions for making a protein that is necessary for the
development of the kidneys and gonads (ovaries in females and testes in males).
• chromosome 11p13
• The WT1 protein plays a role in cell growth and in apoptosis. To carry out these
functions, the WT1 protein regulates the activity of other genes by binding to
specific regions of DNA. On the basis of this action, the WT1 protein is a
transcription factor.

Wilms' tumor ✮
• aka. Nephroblastoma
• Most common renal malignancy of early childhood

(ages 2–4). Contains embryonic glomerular structures.
Presents with large, palpable, unilateral flank mass
and/or hematuria.
• “Loss of function” mutations of tumor suppressor

genes WT1 or WT2

Important oncogenes and associated tumors

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Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

• Phosphate buffer. H2PO4− HPO42− + H+

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

• In the compensated state, ✮ (clinically more commonly observed)

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

• In the compensated phase ✮

Dr. Alisa Chebotarova, MD, PhD

• In the acute stage:

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

• Ketogenic: Leu, Lys

Dr. Alisa Chebotarova, MD, PhD

• In the inherited disease cystinuria, this transporter is

• Clinically can cause kidney stones and block the urinary

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• Connected by loop regions at the surface of the protein

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

• Most common mutation leads to formation

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

• The mutations in any gene for Ubiquitination processes lead to an accumulation

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

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covalent cross-linking Ehlers-Danlos

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• defect in bone matrix formation: production of

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Dr. Alisa Chebotarova, MD, PhD

• impaired Copper absorption and transport due to defective Menkes protein

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• Inherited mutation of a gene for α1-

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• When [S]<<Km; Vo proportional to [S]

1. Competitive inhibitors (inhibitors are structural analogues of S):

Dr. Alisa Chebotarova, MD, PhD

Dr. Alisa Chebotarova, MD, PhD

Alcohol dehydrogenase Acetaldehyde dehydrogenase

Methanol Formaldehyde Formic acid

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• Negative effectors: Reduce or inhibit catalytic