Haemopoietic

BIOCHEMISTRY
SYLLABUS
Haemoglobin: (P. 707)
Structure, functions, Structure-function relation, comparison with myoglobin (P. 708), Role of 2,3 - BPG (P. 708)
Heme Biosynthesis (P. 709): Building materials, reaction at commencement/Termination, general overview of
pathway. Key intermediates
Biosynthesis: Chromosomes globin genes, Chronology of gene expression, Significance of fetal Hb (P. 709)
Haemoglobinopathy (P. 710): Definition, genetic basis.
Abnormal haemoglobin: HbS (P. 710) as an example-genetic basis, Molecular disease concept, laboratory
identification of HbS.
Thalassaemias (P. 711): molecular basis of -thalassaemia, -thalassaemia.
Porphyrias (P. 712): Definition, Acquired-one example, congenital- one example.
Iron Metabolism: (P. 714)
Functions, absorption (P. 715), transport (P. 716), storage (P. 716), release utilization, requirement for
haemopoiesis, elimination, menstrual losses, Fe-cost per pregnancy.
Iron deficiency anaemia: Biochemical indicators, laboratory diagnosis.
Vitamin B12 Metabolism: (P. 717)
General metabolism, biochemical functions, biochemical impairments in deficiency (P. 718) III
Folic acid Metabolism: (P. 719)
General metabolism, biochemical functions as 1-C carrier, biochemical impairments in deficiency (P. 719)
Blood pH Regulation: (P. 720)
Blood hydrogen ion concentration, buffers-[H+] (P. 720) buffering clinical relevance of [H+] measurement,
respiratory acidosis/alkalosis, clinical intervention in respiratory acidosis (P. 721)/alkalosis (P. 722), arterial
blood gas analysis (P. 721), metabolic acidosis (P. 722)/alkalosis (P. 722)
Glycolysis: (P. 723)
Importance, definition, overview of pathway (P. 724), committed step, regulated steps, irreversible steps,
mechanisms of regulation (P. 724), hormonal regulation in fed/fasted condition, net energy yield,
intermediates at metabolic junction, glycolysis in RBC, hepatocyte, in adipocyte, entry of fructose galactose
into glycolysis
HMP Shunt: (P. 725)
Importance, NADPH generating steps, ribose generating step, transketolase, interconnection with glycolytic
pathway, G6PD deficiency (P. 727), haemolysis.
Glutathione (redox) System: (P. 728)
Composition, functions (P. 728), biochemical mode of action, glutathione reductase, GSH-Peroxidase, selenium
Plasma Proteins: (P. 728)
Introduction, classification (P. 728), diagnostic importance

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 Biochemistry

III

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BIOCHEMISTRY
HEMOGLOBIN
Past Questions:
1. Where and how heme is synthesized and show
how it is regulated in human body? Give an
example of porphyria leading to neuro-psychosis
and photophobia. (1 +2+2 +2 =7)[09 July]
2. Which is the rate-limiting step of heme
biosynthesis? Indicate the enzyme catalyzing the
reaction and its sub-cellular site. (1+3=4)[02 Dec]
3. Define porphyria. Give two examples of
porphyria indicating the defective enzyme.
(1+2+2= 5) [04 June]
4. What is prophyria? Write clinical sign and
symptoms of erythropoiticporphyrias.
(1+4=5) [08 July]
5 What is porphyria? Give two examples causing
neurological disturbance and show the deficient
enzymes. (1+4 = 5) [10 July]
6. Define prophyria. Write their types and mention
the clinical signs and symptoms of acute
intermittent porphyria. (5)[05 June]
7. Define porphyria. Explain Cutaneatarda and AIP.
(2+2+2=6) [11 July]
8. What is prophyria and give two examples.
Indicate how does 2,3-BPG, H+ and CO2 influence
on hemoglobin on O2 binding (explain with
graph) (1+2=3) [03 Dec]
9. List two different haemoglobinopathies and their
causes. (2+2=4)[08 July]
10.Discuss Hb-S and Hb-M. Show how they differ
functionally from normal Haemoglobin.
(1+1+2=4) [09 July]
11.What is the genetic basis and mechanism of
formation of Hbs? How it differs functionally
from normal Hb? How is Hbs identified in the
lab?
FAST TRACK BASIC
Haemopoietic
 Hemoglobin is a tetrameric conjugated heme
protein of erythrocytes, which transports O2 to
the tissue and returns CO2 and protons to lungs.
 It is an example of chromoprotein, metalloprotein,
carrier protein and allosteric protein.
Structure
i. Heme (prosthetic group)
- Consists of protoporphyrin IX and ferrous iron
(Fe++)
- Protoporphyrin IX is an asymmetrical porphyrin
- Porphyrins are cyclic compounds formed by
the union of four pyrrole rings through
methenyl bridges (= CH –).
- Ferrous iron (Fe++) resides at centre of planar
tetrapyrrole
- Fe++ form six co-ordinate bonds
- Four bonds are linked with Nitrogen of pyrrole
rings III
th
- 5 bond linked with nitrogen of proximal
Histidine (F8) of the globin
- 6th bond linked with O2 through distal Histidine
(E7) of the same globin.
ii. Globin (apoprotein)
- It is a tetrameric structure comprised of four
polypeptide chains.
- Usually comprised of 2- alpha and 2 - (, , , )
Major adult Hb (HbA): 22: 97%
Minor adult Hb (HbA2): 22: 2%
Fetal Hb (HbF): 22: 1%
- Each polypeptide chain contains one heme
molecule; therefore, a single haemoblogin
molecule contains four heme molecules.
-  and  subunits are connected by Vander
waal's force, hydrogen bonds and electrostatic
forces
SCIENCE MBBS -707-
 Biochemistry

Function - Rapaport Leubering cycle is a shunt pathway of

 Transports O2 to the tissue glycolysis.
 Returns CO2 and H+ to the lungs - This cycle is concerned with synthesis of 2, 3-
BPG in the RBC.
 Buffering action
 Function as anti-oxidant and regulator of iron - 1, 3- BPG produced in glycolysis is converted to
metabolism in the the substantia nigra, 2, 3- BPG by the enzyme 2, 3-
macrophages, alveolar cells and mesangial cells in bisphosphoglycerate mutase.
the kidney. - 2, 3- BPG is hydrolyzed to 3- Phosphoglycerate
Difference between hemoglobin and by the enzyme 2, 3- Bisphosphoglycerate
myoglobin phosphatase.
Action
Hemoglobin Myoglobin
- In normal condition, only 25% of oxygen is
i. Found in RBC i. Found in skeletal
delivered to tissue i.e. one out of four oxygen
muscles and heart
muscles molecules bound to a hemoglobin molecule.
- During hypoxic condition, RBC produces 2, 3-
ii. Has tetrameric ii. Has monomeric
BPG.
structure structure
- Normally in 100% oxygenated hemoglobin a
iii. It contains total 30 iii. It contains total 8 small cavity (about 5 A0) is present due to
helices ( - 7 helices helices spatial arrangement of globin chains.
and  - 8 helices) - When a single oxygen molecule is removed,
iv. Exterior of structure iv. Exterior contains only cavity enlarges (about 11 A0). Now, 2, 3- BPG
may contain non - polar amino acids can accommodate into the cavity (i.e. 2, 3- BPG
polar amino acids easily bind to deoxygenated hemoglobin).
III v. Molecular weight: 65 v. M.W: 17 KD - Ionic bond i.e. salt bridge is formed between
KD positively charged amino acids (Histidine and
Lysine) of β globin chain with negatively
vi. Transport gases and vi. Acts as reservoir of O2
charged phosphate group of 2, 3- BPG.
acts as buffer
- The binding of 2, 3- BPG to deoxyhemoglobin
vii. Oxygen dissociation vii. Oxygen dissociation allosterically promotes the release of
curve: sigmoid curve: hyperbolic remaining three oxygen molecules still bound
viii.One 2,3 -BPG bound in viii.No space for 2,3 - to hemoglobin.
R - form of Hb. BPG. - Thus, 2, 3- BPG enhances the ability of RBC to
ix. Allosteric property due ix. No such property deliver oxygen more efficiently to tissues.
to quaternary structure Clinical significance
x. Co-operative binding x. No such binding i. In Hypoxia
present - The concentration of 2, 3- BPG in RBC is
2, 3 - Bisphospho Glycerate (2,3-BPG) elevated.
Synthesis - So, in presence of 2, 3- BPG, hemoglobin
2,3- bisphospho glycerate mutase will deliver oxygen more efficiently to
1, 3 - bisphosphoglycerate tissues.
1,3 - ADP 2, 3 - ii. In Anemia
bisphospho bisphospho
glycerate ATP glycerate - 2, 3- BPG levels are increased in severe
kinase
2,3- bisphospho glycerate anemia in order to cope up with the oxygen
3 - phosphoglycerate phosphatase demand of the body.

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 Haemopoietic

iii. Fetal hemoglobin (HbF) Regulation of Heme Synthesis [09]

- 2, 3- BPG cannot bind with fetal hemoglobin  ALA synthase is rate - limiting enzyme.
due to absence of β globin chain.
 Heme inhibits the synthesis of ALA synthase by
- Therefore, HbF has higher affinity for acting as a co - repressor.
oxygen compared to adult hemoglobin.
 ALA synthase is also allosterically inhibited by
Heme synthesis hematin
 Building materials  Barbiturates induce heme synthesis
- Glycine, succinyl CoA, Vitamin B6 (PLP), Iron  Ferrochelatase and ALA dehydratase are inhibited
 Site [09] by lead.
- Can be synthesized in almost all cells  Isoniazid decreases availability of PLP hence
 85% in normoblasts (not in matured decreases heme synthesis
erythrocytes) Globin Synthesis
 15% in hepatocytes
 Synthesis of globins occur in ribosome of
- Synthesis is partly cytoplastic and partly
erythroblast
mitochondrial.
 Genes for globin chains are found in:
 General overview of pathway [09,02]
Succinyl CoA + Glycine
Chromosome 16: For  - chain, (zeta) chain
ALA synthase (+) PLP
Chromosome 11: For, ,  chain, ε chain
CoA - SH
- Amino -  - ketoadipic acid
Fetal Hemoglobin (HBF)
ALA synthase (+) PLP Mitochondria  Consists of heme and globin chains (2  and 2 )
CO2
 Each  chain contains 141 amino acids and -
2 ×  - amino - levulinic acid (ALA)
Cytoplasm contains 146 amino acids
ALA dehydratase III
2H2O Physiochemical properties
Porphobilinogen (PBG) - monopyrrole - Increased solubility of deoxy HbF
PBG - deaminase/HMB synthase/
Uroporphyrin I synthases
- Slow electrophoretic mobility for HbF
Hydroxy methyl Bilane (HMB) - Increased resistance of HbF to alkali
denaturation
Uroporphyrinogen - I - HbF has decreased interaction with 2, 3 - BPG
Uroporphyrinogen III synthase
4NH3 Note:
Uroporphyrinogen - III - HbF level remain elevated in children with anemia
Uroporphyrinogendecarbexylase and  - thalassemia
4CO2
Coproporphyrinogen - III - HbF level increases in high altitude
O2 NADP Oxygen Affinity
Coproporphyrinogen Oxidase
CO2 NADPH2  The ODC of fetus and new born is shifted to left.
Proto - porphyrinogen - III (PPG)
Mitochondria  This increase in O2 affinity is physiologically
advantageous in facilitating transplacental O2
4H Protoporphyrinogen oxidase
transport.
Protoporphyrin - III  The major reason for increased affinity is
++
Fe + heme synthase/ferrochelatase diminished binding of 2, 3 - BPG to HbF which is
due to the inability of  - chains to bind 2, 3 - BPG.
Heme

FAST TRACK BASIC SCIENCE MBBS -709-

 Biochemistry

Hemoglobinopathies [08] Sickle Cell Hemoglobin [09, 08]

 Disorders of hemoglobin caused by altered amino  Sickle cell anemia or sickle cell hemoglobin is a
acid sequence of globin chain or deficient globin hereditary blood disorder, characterized by red
chain biosynthesis or both or abnormality due to blood cells that assume an abnormal rigidity and
sickle shape.
toxins
 It is one of the most common
Classification of hemoglobinopathies hemoglobinopathies.
I. Structural hemoglobinopathies  Sickle cell disease: It is homozygous sickle cell
hemoglobin caused by inheritance of two mutant
- Abnormal hemoglobin polymerization - HbS genes.
Hemoglobin sickling  Sickle cell trait: It is heterozygous sickle cell
- Altered O2 affinity hemoglobin caused by inheritance of one mutant
gene and other normal gene. People with sickle
 High affinity: Polycythemia cell trait can lead a normal life.
 Low affinity: Cyanosis, pseudo - anemia Genetic cause
- Sickle cell anemia is caused by a point mutation
- Hemoglobins that oxidize readily
in the β globin gene on chromosome 11.
 Unstable hemoglobins: Hemolytic anemia - This mutation causes hydrophilic glutamic acid
to be replaced with the hydrophobic valine at
 M hemoglobin: Met-hemoglobinemia,
6th position of β globin chain.
cyanosis
Cause of sickling
II Thalassemia - defective biosynthesis of globin Substitution of hydrophilic glutamate with
chains hydrophobic valine
-  - thalassemia 
III Generation of ‘sticky patch’ on the surface of β
-  - thalassemia
globin chain
- , ,  thalassemia 
III. Thalassemic hemoglobin variants At low oxygen, polymerization of HbS

- HbE
Long stiff fibrous precipitates
- Hb constant spring 
- Hb lepore Distortion of erythrocyte into a sickle or crescent
shape
IV. Hereditary persistence of fetal hemoglobin
Lab identification
- Persistence of high levels of HbF into adult life - Electrophoresis
v. Acquired hemoglobinopathies  At alkaline pH, Hb-S shows a slower moving
band than Hb-A
- Methemoglobin due to toxic exposures
 At acidic pH, Hb-S moves faster than Hb-A
- Sulfhemoglobin due to toxic exposures
- Sickling test
- Carboxyhemoglobin  A blood smear is prepared
- HbH in erythroleukemia  Reducing agent such as sodium dithionate is
added
- Elevated HbF in states of erythroid stress and
 Blood smear under microscope show sickled
bone marrow dysplasia RBC

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 Haemopoietic

Note: Thalassemia syndrome [KU 10]

- HbS loses its oxygen binding capacity.  Thalassemia syndrome is heterogeneous group of
- Specifically, person with one of the two alleles of disorders caused by inherited mutations that
sickle cell hemoglobin shows less severe symptom decrease the synthesis of adult hemoglobin HbA
when infected with Malaria. (2 2)
Important hemoglobinopathies Classification
Hemoglobin Point Amino acid Codon and 1.  - thalassemia
mutation substitution base a. Hydrops fetalis
position substitution - Most severe form of  - thalassemia
HbS Beta 6 Glu  Val GAG  GUG - Absence of all 4  chains
- High chances of intrauterine death or
HbC Beta 6 Glu  Lys GAG  AAG
shortly after birth
HbE Beta 26 Glu  Lys GAG  AAG
b. Hemoglobin H diseases (HbH)
HbD Beta 121 Glu  Gln GAG  CAG - Absence of 3 out of 4  chains
HbM Proximal Glu  Tyr CAC  UAC - Common in Asians
or distal
- Unpaired  - chains accumulate and are soluble
histidine in
enough to form 4 tetramer called HbH.
α or β
chains c.  Thalassemia Triat
- Deletion of 2  - globin genes either from
Methemoglobin same chromosome or one from each of two
 Methemoglobin is a form of hemoglobin in which chromosomes
the iron in the heme group is oxidized from d. Silent carrier state
ferrous (Fe++) to ferric (Fe+++) state. III
- Single  - globin gene deleted
 Methemoglobin cannot bind oxygen.
- Completely asymptomatic
 It markedly decreased capacity of oxygen binding
and transport. 2. β - Thalassemia:
 Increase of Methemoglobin i.e. more than 5 g/dl Due to point mutation that leads to defect in
in blood is manifested as cyanosis. transcription, splicing or translation of β globin mRNA.
 According to the cause, Methemoglobin can be a. Thalassemia major
divided as: - Homogeneous
i. Congenital Methemoglobin - With two -thalassemia alleles (+/+, +/0,
- It is due to Cytochrome B5 reductase enzyme 0/0)
deficiency. - Most severe form of  thalassemia
- It is characterized by cyanosis from birth. - Severe anaemia, profound microcytosis,
ii. Acquired Methemoglobin massive splenomegaly
- It may develop by intake of water containing - Elevated level of HbF, HbA2 or both
nitrates or due to absorption of aniline dye.
b. Thalassemia intermedia
- Drugs producing Methemoglobin are: - It is heterogeneous variety with moderate
Acetaminophen, Phenacetin, Sulphanilamide, severity
Amyl nitrate and Sodium nitroprusside
- Milder variant of + or 0
- Glucose-6-phosphate dehydrogenase deficiency: - Moderate anaemia which can be
The condition can be manifested in such aggravated by infection, development of
person even with small dose of drugs. splenomegaly and hypersplenism.
FAST TRACK BASIC SCIENCE MBBS -711-
 Biochemistry

c. Thalassemia Minor Types

- Heterogeneous variety which is mostly 1. Congenital: Based on site where enzyme is
asymptomatic deficient congenital porphyria are further
- With one thalassemia gene and one normal classified as:
gene
a. Erythropoietic
- +  or 0 
- Usually present as profound microcytosis  Congenital erythropoietic porphyria
and hypochromia but only minimal or mild  Erythropoietic protoporphyria
anemia b. Hepatic porphyria
 Acute intermittent porphyria
Note:
 Hereditary coproporphyria
i. 0 - thalassemia
- Total absence of globin chain in homozygous  Variegate porphyria
state result from base substitutions  Porphyria cutanea tarda
ii. + - thalassemia 2. Acquired
- Reduced  - globin synthesis in homozygous - Secondary to disease: Cirrhosis, hepatitis,
state chronic renal failure
- Produced from defects in post - transcriptional - Drugs: Barbiturates, griseofulvin, steroids
processing of mRNA - Poisons: Lead, Hexachlorobenzene, aluminium,
Laboratory findings mercury, arsenic
1. Total haemoglobin: ed Enzyme deficiencies in porphyrias [04, 10]
2. Red blood cells: Glycine + succinyl CoA
- Microcytic, hypochromic ALA synthase
- Anisocytosis, poikilocytosis
III - Presence of target cells, tear drop cells and  - Aminolevulinic acid (-ALA)
normoblast ALA dehydratase

3. Reticulocytes: ed
Porphobilinogen (PBG)
4. Red cell indices: PBG deaminase: Acute
- MCV, MCH, MCHC Markedly ed intermittent porphyria

5. WBC: ed with myelocytes and Hydroxymethylbilane (HMB)

metamyelocytes (Uroporphyrinogen-I)
6. Platelet: Normal but decreased in UPG-III co-synthase: Congenital
erythropoietic porphyria
splenomegaly
7. Serum Bilirubin: ed Uroporphyrinogen-III (UPG-III)
8. Hb electrophoresis: HbF, HbA2 UPG-decarboxylase: Porphyria
cutanea tarda
9. Bone marrow aspirate: Normoblastic erythroid
hyperplasia with predominance of Coproporphyrinogen (CPG)
intermediate and late normoblasts CPG-oxidase: Hereditary coproporphyria

Porphyria [03, 04, 05, 08, 10] Protoporphyrinogen (PPG)

 A group of inborn errors of metabolism associated PPG – oxidase: Porphyria variegata
with the biosynthesis of heme.
Protoporphyrin (PP)
 Characterized by increased production and Heme synthase (Ferrochelatase):
excretion of porphyrins or their precursors (ALA + Erythropoietic protoporphyria

PBG)
Heme

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 Haemopoietic

Acute Intermittent porphyria [11] Congenital erythropoietic porphyria

- Hepatic porphyria
- Inherited as an autosomal dominant trait
- PBG diaminase or UPG - I synthase deficiency
- This leads to secondary increase in activity of
ALA synthase
- Level of ALA and PBG are elevated in liver,
blood and urine.
Clinical Features [05]
- Patients with intermittent abdominal pain with
nausea, vomiting and constipation
- Cardio vascular symptom: Tachycardia,
hypertension
- Neurological symptom: Anxiety, insomnia,
seizures, axonal degeneration, peripheral
neuropathy
- Urine: Freshly colourless but on standing
colour changes to red due to oxidation of PBG
to Porphobillin.
- Some drugs like Barbiturates, Griseofulvin and
Steroids can precipitate an attack.
These drugs induce Cyt - P 450

Heme consumed in synthesis of Cyt - P 450

Low hepatic concentration of heme induce
ALAS-I
 
Synthesis of harmful heme precursors
 
Precipitation of disease
Diagnosis
- Increased level of PBG and ALA in plasma and
urine
- Low activity of PBG - deaminase in hepatic cells
Treatment
- Analgesics for abdominal pain.
- Low dose of Benzodiazepam for sedation (DOC)
-Inherited as an autosomal recessive trait
-Uroporphyrinogen - III synthase deficiency
- Type I isomer is formed in larger amounts
(normally type III isomer formed considerably)
Type I isomer

Porphyrins Type I

Ineffective feedback inhibition

Further increase of type I porphyrins
Clinical features
- Increased type - I porphyrins level in blood lead
to photosensitivity
Presence of porphyrins in capillaries

Photosensitisation

Formation of reactive oxygen species (free
radicals)
 III
Rupture the lysosome and release proteolytic
enzyme

Skin damage vesicle formation, itching
Repeated attack of dermatitis and scarring
Monkey face
- Repeated ulceration and scarring may cause
mutilation of nose, ear and cartilage.
- Porphyrins in urine makes the urine dark red in
color (Port wine appearance)
- Erythrodontia: When UV light is reflected on to
teeth a red fluorescence is seen
Diagnosis

- Intravenous glucose - Type I porphyrins [UPG I] in plasma and urine

- Intravenous heme - CPG I in feces

FAST TRACK BASIC SCIENCE MBBS -713-

 Biochemistry
Treatment
- Primary goal is to avoid exposure and skin
trauma
- Barrier sun creams containing Zinc or Titanium
oxide are most effective products
Erythropoietic protoporphyria [08]
- Inherited as an autosomal dominant trait
- Ferrochelatase deficiency
- Most common erythropoietic porphyria
- Protoporphyrin - IX accumulated in tissues and
excreted in bile (faeces)
- Increased free protoporphyrin in RBC
Clinical features
- Photosensitivity
- Can cause liver damage
Diagnosis
- Decreased ferrochelatase activity in cultured
lymphocytes
- RBCs exhibit red fluorescence at 620 mm
Treatment
-  - Carotene, heme transfusion
III Porphyria cutanea tarda [11]
- It is a genetic photosensitive skin disease in
adult life in which uroporphyrins are present in
the urine due to deficiency of
uroporphyrinogen decarboxylase (UROD).
- It is one of the hepatic porphyrias.
- The disease becomes active when acquired
factors, such as iron, alcohol, Hepatitis C Virus
(HCV), HIV, estrogens (used, for example, in
oral contraceptives and prostate cancer
treatment) and possibly smoking, combine to
cause a deficiency of UROD in the liver.
Symptoms
- The hallmarks of porphyrin cutanea tarda (PCT)
are blisters, which become ulcerated in areas
of the skin exposed to sunlight, especially on
the face, ears and dorsum of the hands.
- The affected areas of the skin also tend to be
fragile and show hyperpigmentation and
hypertrichosis.
-714- FAST TRACK BASIC
Diagnosis
- The preferred screening test for PCT is a
measurement of porphyrins in plasma.
- The presence of porphyrins in urine
(predominately uroporphyrin and 7-
carboxylate porphyrin) and feces
(predominately isocoproporphyrin) help to
confirm the diagnosis.
Treatment
- PCT is the most treatable of the porphyrias.
- Factors that tend to activate the disease should
be removed
- The most widely recommended treatment is a
schedule of repeated phlebotomies (removal
of blood), with the aim of reducing iron in the
liver.
- Another treatment approach is a regimen of
low doses of either chloroquine (125mg twice
weekly) or hydroxychloroquine (100mg twice
weekly).
IRON METABOLISM
Past Questions:
1. List the functions of iron. Sketch a diagram
showing the absorption and transport of iron and
the regulation of its level in plasma.
[3+2+3=8] [04 Nov]
2. Sketch the diagram showing internalization and
storage of iron and its control. [6][03 June]
3. Write sources, absorption and transport of iron
in the body and mechanism of control of serum
iron, also mention when it increases and
decreases [10][05 Dec]
4. Sketch diagrammatically the mode of absorption
of iron in the intestine and its control. [4][06 Dec]
5. Write the source, absorption, transport and
storage of iron in the body [3][05 June]
6. List the functions of iron. Sketch with diagram
the absorption and transport mechanism of iron
and the regulation of its level in plasma.
[3+2+3=8][04 Nov]
SCIENCE MBBS

Introduction
 Essential trace element of the body
 Total iron content of body: 3 to 4 gm
Iron containing compounds
i. Heme containing proteins
- Hemoglobin, myoglobin, cytochrome, catalase,
lactoperoxidase, tryptophan pyrrolase e.t.c.
ii. Iron - sulfur complexes
- Complex III Fe - s, Succinate dehydrogenase,
Xanthine oxidase
iii. Nonheme Iron - containing protein
- Ferritin, transferrin, Hemosiderin, Aconitase,
Phe - hydrolase
Daily requirement (ICMR - 1990)
- For adults: 20 mg/day
- Children between 13 - 15 yrs: 20 - 30 mg /day
- Pregnant women: 40 mg/day
- Lactating women: 32 mg/day
Sources [05]
Exogenous:
- Rich sources: Non-veg sources-meat
- Good sources: Leafy vegetables, pulses
- Poor source: Milk
Note: Iron found in plant sources is usually bound
with proteins or organic acids (Phytates or oxalates)
make it unavailable for absorption.
Endogenous
- Destruction of old RBCs and other heme
proteins
Note: Per day 200 billion RBCs are destroyed (20 ml
blood = 6 gm of Hb = can give 25 mg iron).
Absorption of Iron [06, 05, 04]
- Site: duodenum and jejunum
- Per day absorption: 1 - 1.5 mg
- Absorption of heme iron: 30%
- Absorption of non - heme iron: 1 - 5%
- Bioavailability of the Fe is more important
factor than Fe content of the diet
FAST TRACK BASIC
Haemopoietic
Factors affecting Iron absorption
- Gastric acid (HCl), ascorbic acid, Cysteine etc.
convert Fe3+ to Fe2+, thus help in absorption of
iron.
- Phytic acid (in cereals), oxalic acid (in leafy
vegetable) and tannic acid (in tea) inhibit
absorption of Fe by forming insoluble iron
complex.
- Calcium, copper, lead and phosphates will
inhibit iron absorption.
- Resection of Duodenum (Partial or total
gastrectomy) reduces surface area for
absorption.
- Achlorhydria, celiac spruce also reduces Fe
absorption.
- Presence of ferric reductase enzyme on the
brush border of duodenal enterocyte converts
ferric to ferrous.
Mechanism of Iron Absorption:
III
1. Internalization:
- Iron can only be absorbed in Fe++ form
- Food iron may be Heme iron (Fe++ state) or
non-heme iron (Fe++state).
- But non-heme iron, as such, can’t be
absorbed. It needs to be reduced to Fe++
state and reduction is carried out by
ferroreductase.
- Once non-heme iron gets reduced to Fe++, it
is transported into enterocytes via a
transporter called Divalent Metal
Transporter-1 (DMT-1)
SCIENCE MBBS -715-
 Biochemistry
2. Mucosal Block
- Iron metabolism is unique because
homeostasis is maintained by regulation at
level of absorption (mostly regulation is
done at level of excretion in other cases)
- Duodenal cell synthesize a protein called
apo-ferritin.
- Once iron is internalized by cell, apo-ferritin
traps the iron and forms ferritin (Mucosal).
- Only the unbound form or free iron can go
to circulation. Bound form (i.e. ferritin) gets
trapped and is lost in faeces by shedding of
cell.
- Thus amount of iron absorbed depends
upon the amount of apoferitin synthesized
by duodenal cells. (Inversely proportional)
- During iron-deficiency or ed iron
requirement, synthesis of Apoferritin is
inhibited. So, more amount of iron is
absorbed.
- When body has enough iron stores,
synthesis of apoferritin is increased. So,
most of iron gets trapped in mucosal cell
III and only few are absorbed.
- This mechanism is called mucosal block or
ferritin curtain
3. Transport of iron from enterocyte to plasma
- The iron destined for circulation is
transported from cytoplasm across
basolateral membrane by ferroportin.
- The process is coupled with oxidation of
Fe2+ to Fe3+, which is carried out by iron
oxidases like hephaestin and ceruloplasmin.
Note:
- Hepcidin is a circulating peptide synthesized by
liver in response to increase in intrahepatic iron
level.
- Hepcidin inhibits iron transfer from enterocyte to
plasma by binding to ferroportin and causing it to
be endocytosed and degraded
- When iron stores are reduced, synthesis of
hepcidin is inhibited.
-716- FAST TRACK BASIC
Transport of Iron:
- Free iron is highly toxic, so after absorption
when iron reaches to plasma, it is trapped by
transferrin (an iron binding glycoprotein
synthesized by liver).
- Transferrin carries iron to site of utilization and
storage of iron.
- A single molecule of transferrin carries two
molecules of Fe+++.
- Target cells (erythroid cells, liver cells, spleen
cells) express transferrin receptors (TGfR-1 and
TGfR-2)
- Transferrin (along with iron) binds to these
receptor and iron is internalized by these cells
by receptor mediated endocytosis.
Note:
- Total iron binding capacity (TIBC) refers to level of
free transferrin in serum.
- TIBC increases when
i. Low serum iron
ii. High serum transferrin
Storage of Iron:
- Iron is stored in liver, spleen, bone marrow,
skeletal muscles
- Free iron is highly toxic. So, it is stored by
binding to either ferritin or hemosiderin.
- In liver, most ferritin is stored within the
parenchymal cells and in other tissue such as
spleen and bone marrow; it is mainly stored in
macrophages.
Note:
- Plasma ferritin is an index of iron stores
- Hemosiderin is formed by partial deproteinization
of ferritin by lysosomes
- Iron in hemosiderin is chemically reactive and
turns blue-black color when exposed to potassium
ferrocyanide (basis for Prussian blue stain).
- With normal iron stores, only trace amount of
hemosiderin are found but in iron loaded cells,
most iron is stored in hemosiderin.
SCIENCE MBBS
 Haemopoietic

VITAMIN B12 Absorption of vitamin B12

Past Questions: - Site: Ileum

1. Physiological functions and deficiency symptoms - Absorption of vitamin B12 requires two binding
of vitamin B12 (3)[07 Dec, 04 Nov] proteins

2. Mention co-enzyme forms of B1, B2, B6 and folic a. First is the intrinsic factor (IF) of castle
acid and give one example of enzyme in which - Secreted by the gastric parietal cells
they act as co-enzyme. (4+4=8) [04 June] - One molecule of IF can combine with 2
3. List the functions of vitamin B12. Mention the molecules of B12
deficiency symptoms. (3+2=5) [04 Nov] - IF-B12 complex is attached with specific
4. Importance, functions and deficiency receptors on mucosal cells.
manifestations of vitamin B12 and B6. (5)[06 June] - Whole IF - B12 complex is internalized
 Extrinsic factor (EF) of castle and antipernicious b. Second factor is cobalophilin
anemia factor
- Secreted in the saliva
Chemistry
- Gastric pepsin release vitamin from proteins of
- Water soluble, heat stable and red in colour food
- Contain one cobalt atom.
- Then B12 binds with cobalophilin
- Four pyrrole ring co-ordinated with a cobalt
- In duodenum cobalophilin is hydrolysed by
atom is called a corrin ring.
trypsin of pancreatic juice
- The fifth valency of the cobalt is covalenty
- Then B12 is released, which binds to intrinsic
linked to a substituted benzimidazole ring so
factor
called cobalamin.
- The sixth valency of the cobalt is satisfied by Transport and storage III
any of the following groups: cyanide, hydroxyl, - In the blood, methyl B12 form is predominant.
adenosyl or methyl. - Transcobalamin, a glycoprotein, is the specific
a. Cyanocobalamin carrier.
- When cyanide is added at the R position. - It is stored in liver cells as Ado - B12 form, in
- Oral preparations are in this form. combination with transcorrin.
b. Hydroxycobalamin Note: Generally, B complex vitamins are not stored in
- When hydroxyl group is attached at the R the body, B12 is an exception
position.
- Injectable preparations are in this form. Biochemical function of B12
c. Adenosylcobalamin (Ado - B12) i. Synthesis of methionine from homocysteine
- When taken up by the cells, these groups are Homocysteine
removed and deoxyadenosylcobalamin or Ado-
B12 is formed. Methylcobalamine
Homocysteinemethyltransferase
- Major storage, seen in liver
Cobalamine
d. Methyl cobalamin
- When methyl group replaces adenosyl group
Methionine
- Major form seen in blood circulation

FAST TRACK BASIC SCIENCE MBBS -717-

 Biochemistry

ii. Isomerization of methylmalonyl CoA to - Methylmalonyl CoA acts as a competitive

succinyl CoA inhibitor of malonyl CoA in fatty acids
Propionyl CoA + CO2 synthesis. Hence, biosynthesis of fatty acids,
required for myelin formation is impaired.
Propionyl CoA carboxylase
- Methylmalonyl CoA can substitute malonyl
D-methylmalonyl-CoA CoA in fatty acid synthesis, resulting in a
Recemase new type of branched chain fatty acids.
These fatty acids will disrupt the normal
L-methylmalonyl-CoA
membrane structure.
Adenosyl Methylmalonyl CoA mutase b. In vitamin B12 deficiency, conversion of
cobalamine homocysteine into methionine is arrested. Due
to unavailability of active methionine,
Succinyl CoA
methylation of phosphatidyl ethanolamine to
- Vitamin B12 is necessary for the activity of phosphatidyl choline is not adequate. This
enzyme methylmalonyl CoA mutase i.e. leads to deficient formation of myelin sheaths
vitamin B12 acts as co-enzyme. of nerve, demyelination and neurological
lesions.
Deficiency manifestation:
iii. The elevated serum homocysteine increases
i. Folate trap:
the risk of atherosclerotic cardiovascular
- N5-Methyl tetrahydrofolate is inactive form, disease
while tetrahydrofolate is active form of folic iv. Megaloblastic anemia
acid. v. Achlorhydria
- Cobalamin converts N5-Methyl tetrahydrofolate
Assessment of Vitamin B12
into tetrahydrofolate and get changed itself into
i. Schilling test:
III methylcobalamin.
- Radioactive labeled (Cobalt-60) vitamin B12,
5 one microgram is given orally.
N -Methyl Methyl transferase Tetrahydrofolate
tetrahydrofolate - Simultaneously, an intramuscular injection of
unlabeled vitamin B12 is given in order to
Cobalamine saturate tissue with normal vitamin B12.
Methyl cobalamine
- Hence, the entire absorbed radioactivity will
pass to the urine.
Methionine Homocysteine
Homocysteine methyl - The patient’s urine is then collected over next
transferase 24 hours.
- Therefore, in absence of cobalamin, N5-Methyl - In a normal person, much of the ingested
tetrahydrofolate cannot be converted into its vitamin B12 will be excreted in the urine.
active form i.e. tetrahydrofolate and remains - In patients with pernicious anemia or with
trapped in its inactive form. This is called folate deficiency due to impaired absorption, less than
5% of the radioactivity is detected in urine.
trap.
- If abnormality is found, the test is repeated,
ii. Demyelination of nervous system:
with radioactive vitamin B12 plus intrinsic factor
a. In vitamin B12 deficiency, methylmalonyl CoA is given orally, and urine is collected for 24 hours.
not converted to succinyl CoA. Therefore, the - In pernicious anemia, there is lack of intrinsic
accumulation of methylmalonyl CoA takes factor so that the first test is abnormal, but the
place. second test is normal.

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 Haemopoietic

ii. FIGLU excretion test: Deficiency manifestations

- Histidine is normally metabolized to Formimino i. Reduced DNA synthesis
glutamic acid (FIGLU) from which formimino In folate deficiency
group is removed by THFA. 
- In vitamin B12 deficiency, THFA is not activated, THFA reduced and thymidylate synthase enzyme
FIGLU is excreted in urine. inhibited
iii. Serum vitamin B12: By radio-immuno-assay or 
by ELISA. dUMP not converted to dTMP
IV. Methyl malonic acid: It is seen in urine. 
dTTP not available
FOLIC ACID 
Past Question: Reduced DNA synthesis
1. What are the co-enzyme forms of folic acid? Give ii. Megaloblastic anemia
two biochemical functions of the vitamin. - During erythropoiesis, DNA systhesis is delayed
(4)[02 Dec] but protein synthesis is continued.
 Abundant in vegetables - This dissociation between the maturity of
nucleus and cytoplasm leads to abnormal RBC.
Chemistry
- Abnormal RBC is rapidly destroyed in spleen.
- Pteridine group linked with para-amino benzoic - Reduced generation and increased destruction
acid is called pteroic acid of RBC result in Anaemia
- It is attached to glutamic acid to form pteroyl - Leukopenia and thrombocytopenia are also
glutamic acid or folic acid manifested
Absorption of folic acid iii. Homocysteinemia
- Site: Upper part of jejunum - Folic acid deficiency cause increased III
homocysteine level in blood
- Transported in blood: by  - globulins
- Increased risk of coronary artery disease
- Taken up by the liver where the co – enzymes
iv. Birth defects
are produced - Folic acid deficiency in pregnancy may lead to:
Biochemical function homocysteinemia and neutral tube defect
- Folic acid first reduced to 7, 8 - dihydrofolic v. Cancer
acid and further reduced to 5, 6, 7, 8 - tetra - Folic acid deficiency leads to etiology of
bronchial carcinoma and cervical carcinoma
hydrofolic acid
Assessment of folic Acid deficiency
- THFA is the carrier of one - carbon groups, they i. FIGLU excretion test
are: - Histidine is normally metabolized to formimino
i. Formyl glutamic acid (FIGLU) from which formimino
ii. Formimino group is removed by THFA. Therefore, in folate
deficiency, FLGLU is excreted in urine.
iii. Methenyl ii. AICAR excretion
iv. Hydroxymethyl - In Purine nucleotide synthesis, the last step is the
v. Methyl addition of C2 with the help of N10 – formyl THFA.
- In folic acid deficiency, this step is blocked so
- Methyl group in N5 - methyl THFA is required
the precursor i.e. amino imidazole
for synthesis of active methionine which takes
carboxamide riboxyl-5-phosphate (ALCAR)
part in transmethylation reactions. accumulates and is excreted in urine.

FAST TRACK BASIC SCIENCE MBBS -719-

 Biochemistry

BLOOD PH REGULATION a. Bicarbonate buffer system

Past Questions: - Strong acid replaced by weak acid  least pH
change
1. Define respiratory acidosis and alkalosis and
sketch an explanatory diagram showing HCl + NaHCO3 H2CO3 + NaCl
compensatory mechanism (respiratory alkalosis - Strong base replaced by weak base  least pH
and acidosis) (1+1+5=7) [11 July] change
NaOH + H2CO3 NaHCO3 + H2O
2. Sketch the diagram showing mechanism of
respiratory acidosis and its compensation. Henderson - Hasselbach equation

(2) [04 Nov] HCO3–

3. Sketch explanatory diagram of respiratory
acidosis and alkalosis and its compensation
(3+3=6)[05 June]
4. List the causes and explain the process of
compensation of respiratory acidosis using
diagram. (2+2=4) [06 Dec]
5. List important buffers of plasma. How the blood
pH is regulated during metabolic acidosis (explain
with diagram) (4+6=10) [07 July]
6. List important buffer systems present in blood.
List importance of bicarbonate buffer. (3)[07 Dec]
III  Normal pH of arterial blood: 7.4 (7.38-7.42)
 Normal pH of venous blood and interstitial fluid:
7.35
 Intracellular pH: 6 to 7.4 (av. 7.0)
 Threshold of pH of blood: Lower limit - 6.8, upper
limit - 8.0
pH = pK (6.1) + log CO
2
 HCO3– alkalosis (maintained by renal system)
 CO2 acidosis (maintained by respiratory
system)
- Buffering power is reasonably effective up to
ratio of 1:8 or 8:1 (normal 20:1)
 When acid added, HCO3– converted to
H2CO3 pH decrease
 When base added, H2CO3 converted to
HCO3– pH increase
 Buffering power is greater when: pH = pK
(both Concentration HCO3– ~ concn H2CO3)
 Buffering action of buffer is directly
proportional to concentration of buffer
systems
 Drawbacks of bicarbonate buffer: pK= 6.1,
act on buffering curve where buffering
power is poor.

 Defense against change in pH b. Phosphate buffer system

- Strong acid replaced by weak acid: Least
i Buffer system: Acts within fraction of second
change in pH
ii Respiratory system: Within 1 to 12 minutes
HCl + Na2HPO4 NaH2PO4 + NaCl
iii Excretory (renal) system: Hour to several days
- Strong base replaced by weak base: Least
Buffer system [07] change in pH
 Buffers are solutions that can resist change in pH, NaOH + NaH2PO4 Na2HPO4 + H2O
when acid or alkali is added. Henderson - Hasselbach equation
a. Mixture of weak acid and their salt of strong HPO4– –
base e.g. H2CO3/NaHCO3 (bicarbonate buffer) pH = pK (6.8) + log H PO –
2 4

b. Mixture of weak base and their salt of strong - However, pK near to maximum buffering
acid e.g. Na2HPO4/NaH2PO4 (phosphate buffer) action, it is less effective than bicarbonate as it
is 1/12th than bicarbonate concn in e.c.f.

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 Haemopoietic

- On the other hand, very important in tubular Effect of H+ concn on alveolar ventilation: (Medulla
fluid in kidney because oblongata)
i. [PO4]3- usually concentrated in tubules, thus - If pH fall to 7.0,ed alveolar ventilation four to
 buffering power five time
ii. Tubular fluid acidic, so bring close to - If pH rise to 7.6, ed alveolar ventilation
buffering (maximum) action of system fraction of normal level
- Also important intracelllularly, as 1
concentration is greater inside than outside. pH  alv. ventilation

c. Protein buffer system Classification of Acid base disturbance

- CO2 diffuses out of cell membrane quickly but I. Acidosis (fall in pH)
H+ and HCO3– takes hour to diffuse through it a. Respiratory acidosis: Primary excess of
except RBC. carbonic acid
- The rate of diffusion of these three substances b. Metabolic acidosis: Primary deficit of
corresponds to proportionate change in the bicarbonate
e.c.f. and i.c.f. (intracellular). II. Alkalosis (rise in pH)
 Major buffering action as of highest a. Respiratory alkalosis: Primary deficit of
concentration carbonic acid
 Main amino acid - Histidine imidazole group b. Metabolic alkalosis: Primary excess of
pK = 6.1 (as bicarbonate buffer) bicarbonate
Note: Respiratory acidosis [11, 06, 05, 04]
- 52% buffer activity in tissue cells - Respiratory acidosis occurs when there is
accumulation of CO2 due to reduced effective
- 6% buffer activity in RBC
- 42% buffer activity in extracellular buffer
alveolar ventilation. III
- Ratio of bicarbonate to carbonic acid is less
- Among these, than 20
40%  bicarbonate buffer Cause
1%  protein buffer i. Airway obstruction: COPD, bronchospasm,
1%  phosphate buffer asthma, pneumothorax
ii. Depression of respiratory centre: Anaesthesia,
Respiratory regulation of acid - base balance
sedative, cerebral trauma, tumors, narcotics
- Mainly act on the concn of CO2
iii. Neuromuscular disease: Poliomyelitis, tetanus,
- Average of 1.2 mmol/L by dissolved CO2 in e.c.f. motor neuron disease
- If metabolic rate ed: CO2 in e.c.f. iv. Pulmonary disease: Pulmonary fibrosis,
Pulmonary ventilation ed: CO2 in e.c.f. Pneumonia
Effect of increasing or decreasing alveolar v. CNS trauma, tumor
ventilation on pH of e.c.f vi. Ascites, peritonitis
1 vii. Sleep apnea
CO2 alv. ventilation
Blood gas analysis
- In alveolar ventilation 2 times:  pH by 0.23 pH PCO2 HCO3–
i.e. 7.63
Acute resp. acidosis   Normal or 
- In alveolar ventilation 1/4 times: pH by 0.45
i.e. 6.95 Chronic acidosis   

FAST TRACK BASIC SCIENCE MBBS -721-

 Biochemistry

Compensatory mechanism Compensatory mechanism

- Excess carbonic acid is buffered by hemoglobin - Reduction in plasma bicarbonate
and protein buffer system. - Decrease in renal re-absorption of bicarbonate
and decreased urinary excretion of H+
- Kidney responds by conserving HCO3- and
Clinical features
excreting H+ as NH4+.
- Hyperventilation, muscle cramps and paraesthesia
Clinical features - Perioral and digital tingling
- Decreased respiratory rate, hypotension and - Trousseau's sign: Positive
coma - Chvostek's sign: positive
- Dominated by the cause of hypoventilation (eg. - Tetany or seizures
paralysis, chest wall injury, chronic obstructive Metabolic acidosis [07]
lungs disease) - Metabolic acidosis occurs due to a primary
deficit in the bicarbonate resulting from
- CO2 accumulation leads to drowsiness,
 Accumulation of acid
peripheral vasodilation, tachycardia and
 Depletion of bicarbonate
tremors.
- Ratio of bicarbonate to carbonic acid less than 20
Respiratory Alkalosis [11, 06, 05, 04] Cause
- Respiratory alkalosis develops when there is a i. High anion gap
period of sustained hyperventilation resulting - Diabetic ketosis
in a reduction of pCO2 and increase in plasma - Lactic acidosis
pH. - Renal failure
- Ratio of bicarbonate to carbonic acid is more ii. Normal anion gap
than 20 - Renal tubular acidosis
III - CA inhibitors
Cause
- Diarrhoea
i. Hypoxia
ii. Increased intracranial pressure - Addison’s disease
iii. Stimulation of respiratory centre by drugs like Compensatory mechanism
salicylates - Stimulation of respiratory compensatory
iv. High altitude mechanism and produces hyperventilation
v. Excessive artificial ventilation - Renal compensation Na+- H+ exchange,
vi. Psychoneurosis NH4+excretion and HCO3- reabsorption
vii. Hysteria Clinical features
viii.Septicaemia - Marked increase in respiratory rate and depth
ix. Febrile conditions of respiration i.e., Kussmaul respiration
x. Meningitis - Depressed myocardial contractility
xi. Congestive cardiac failure - CNS depression: Headache, lethargy, coma
Blood Gas Analysis - Acetone breath in diabetic ketoacidosis (DKA)
pH PCO2 HCO3 Metabolic Alkalosis
- Metabolic alkalosis occurs due to a primary
Acute resp. alkalosis    excess of bicarbonate resulting from
Chronic resp.   Normal or   Excess base addition
alkalosis  Defective base excretion
 Acid loss
-722- FAST TRACK BASIC SCIENCE MBBS
 Haemopoietic

Causes GLYCOLYSIS
- Severe vomiting Past Questions:
- Cushing syndrome 1. Show the site and pathways of glycolysis and
explain its control. (5)[10 Jan]
- Milk alkali syndrome
2. Sketch the steps involved in glycolysis (mention
- Diuretic therapy (potassium loss) enzymes involved). How is the pathway
Compensatory mechanism regulated? (4+3=7) [04 June]
3. Sketch the steps involved in glycolysis (mention
- Renal compensation: H+-K+ exchange the enzymes involved). How is the pathway
Clinical features regulated? (4+3=7) [04 June]

- Hypoventilation Definition
 Glycolysis is the pathway in which glucose is
- Increased neuromuscular activity splitted into pyruvate or lactate under aerobic
- Hypokalemia and anaerobic conditions respectively, along
with production of a small quantity of energy.
- Hypertension
Site:
- CNS symptoms: Confusion, seizures, muscle - In cytoplasm of every cell
cramps, tetany paresthesias. Importance
- Only pathway taking place in all cells of the body
Expected renal & respiratory compensations
- Only source of energy in erythrocytes
Acid base - In strenuous exercise, anaerobic glycolysis
Compensations forms the major sources of energy of muscles
disturbances
- Preliminary step before complete oxidation
Metabolic acidosis - Expect pCO2 to be - Provides carbon skeletons for synthesis of non-
essential amino acids as well as glycerol part of fat.
III
reduce3d by 1 mm Hg for
every 1 mmol/L drop in Glucose transporter
bicarbonate. Transporter Present in Properties
GluT1 RBC, brain, Glucose uptake in
Metabolic alkalosis - Expect pCO2 to be kidney, colon, most of cells
increased by 0.6 mm Hg retina, placenta
for every 1 mmol/L rise in GluT2 Serosal surface of Low affinity; glucose
intestinal cells, uptake in liver;
bicarbonate
liver, beta cells of glucose sensor in beta
Chronic respiratory - Expect 1 mmol/L increase pancreas cells
GluT3 Neurons, brain High affinity; glucose
acidosis in bicarbonate per 10 mm
into brain cells
Hg rise in PCO2 GluT4 Skeletal, heart Insulin mediated
muscle, adipose glucose uptake
Acute respiratory - Expect 2 mmol/L
tissue
alkalosis de3crrease in bicarbonate GluT5 Small intestine, Fructose transporter;
per 10 mm Hg fall in pCO2 testis, sperms, poor ability to
kidney transport glucose
Chronic respiratory - Expect 4 mmol/L decrease GluT7 Liver endoplasmic Glucose from ER to
alkalosis in bicarbonate per 10 mm reticulum cytoplasm
Hg fall in PCO2 SGluT Intestine, kidney Co-transport; from
lumen into cell

FAST TRACK BASIC SCIENCE MBBS -723-

 Biochemistry

Overview of pathway [10, 04] Energy yield in aerobic conditions

No. of ATP per
Glucose Step Enzyme
ATP glucosenal
*Hexokinase
ADP 1 Hexokinase minus 1

Glucose - 6 - phosphate 3 Phosphofructokinase minus 1

*Phosphohexoseisomerase 5 Glyceraldehyde - 3 – phosphate 2.5 × 2 = 5
dehydrogenase
Fructose - 6 - phosphate

Phosphofructo kinase
ATP 6 1, 3 - bisphosphoglycerate kinase 1×2=2
ADP
9 Pyruvate kinase 1×2=2
Fructose - 1, 6 - disphasphate
Net energy yield = 9 - 2 =7 ATP
Aldoase
Energy yield in anaerobic conditions
Glyceraldehyde - 3 - phosphate + DHAP
No. of ATP per
Glyceraldehyde - 3 - phosphate - Step Enzyme
deydrogenase glucose mol

1 Hexokinase minus 1
1, 3 - bisphosphoglycerate
3 Phosphofructokinase minus 1
#1, 3 - bisphosphoglycerate kinase
6 1 - 3 bisphosphoglycerate kinase 1 × 2 = 2
3 - phosphoglycerate 9 Pyruvate kinase 1×2=2
III
Phosphoglycerylmutase
 Net energy yield = 4 - 2 = 2 ATP

2 - Phosphoglycerate Note:

Enolase - Complete oxidation of glucose through glycolysis

plus citric acid cycle will yield a net 32 ATPs.
Phosphoenol pyruvate
Mechanism of Regulation [04]
ADP
#*Pyruvate kinase
ATP The regulatory enzymes of glycolysis are
i. Hexokinase
Pyruvate
ii. Phosphofructokinase
+
NADH + H
#*Lactate dehydogenase
+
iii. Pyruvate kinase
NAD
1. Hexokinase:
Lactate
- Hexokinase is inhibited by glucose-6-
Note: phosphate i.e., feedback inhibition.
- "*" denotes key enzymes - This enzyme prevents the accumulation of
- "#" denotes substrate level phosphorylation glucose-6- phosphate due to product
- Both "#"and "*" denotes irrevessible steps inhibition.

-724- FAST TRACK BASIC SCIENCE MBBS

 Haemopoietic

- Glucokinase acts when the glucose level is

higher than 100 mg/dl.
HEXOSE MONOPHOSPHATE PATHWAY
Past Questions:
- Glucokinase specifically phosphorylates
1. List the steps of HMP pathway and list its
glucose.
importance. (4+1=5) [08 July]
- Glucokinase is induced by insulin. 2. List the significance of HMP shunt pathway
2. Phosphofructokinase: (when, where and why) (3)[03 Dec]
- It is the most important regulatory enzyme in 3. Sketch the diagram of HMP shunt pathways.
glycolysis. Show sites of formation of NADPH. List the
importance of NADPH. (3+1+1=5) [10 July]
- It is allosterically inhibited by ATP, citrate and
4. List source and sketch the diagram showing steps
H+ ions, whereas fructose-2, 3-bisphosphate
of synthesis of NADPH and also list its important
and AMP are the allosteric activators.
functions. (4+4=8) [06 Dec]
3. Fructose-2, 6-biophosphate: 5. Explain the significance of HMP shunt.
- It most important regulatory factor for (4, 5)[02 Dec, 05 Dec]
controlling PFK and ultimately glycolysis in the  HMP shunts pathway or pentose phosphate
liver. pathway or phospho-gluconate oxidative
- Fructose-2, 6-biophosphate is formed from pathway.
fructose-6-phosphate by action of an enzyme  An alternative pathway to glycolysis and TCA cycle
called Phosphofructokinase-2. for the oxidation of glucose
- Then, fructose-2, 6-bisphosphate is hydrolyzed  Anabolic in nature since biosynthesis of NADPH
to fructose-6-phosphate by action of an and pentose occurs
enzyme called fructose-2, 6-bisphosphatase. Site
- The activities of both the enzymes are - Cytoplasm of RBCs, adrenal cortical cells, liver III
reciprocally regulated. cells, testicular cells, mammary cells, lens of
- When glucose supply increases, PFK-2 is eye, adipose tissue
activated so F-2, 6-BP concentration increases, Importance [02, 03, 05, 08, 10]
this in turn activates PFK. Thus, glycolysis is Related with biosynthesis of
favoured. - Reduced NADPH
4. Pyruvate kinase: - Pentose phosphate
- Pyruvate kinase is inhibited by ATP and i. Pentose: Synthesis of nucleic acids (DNA and RNA)
activated by F-1, 6-BP and many nucleotides such as ATP, NAD+, FAD & CoA
5. Insulin activates all three regulatory enzymes. ii. NADPH
- Free radical scavenging
6. Glucagon and glucocorticoids inhibit glycolysis.
- Erythrocyte membrane integrity
Note:
- Prevention of accumulation of met -
- Fluoride irreversibly inhibits enolase enzyme. So, hemoglobin
during blood sugar estimatio fluoride is added in - Detoxification of drugs
order to arrest glucose metabolism by the blood - Lens of eye (for transparency)
cells. Hence, the correct blood glucose level is - Bactericidal activity of macrophage
measured.
- Biosynthesis of fatty acids, cholesterol and
steroids

FAST TRACK BASIC SCIENCE MBBS -725-

 Biochemistry

Overview of pathway [10, 08, 06]

(1) G6PD (2) Gluconolactone
6 × glucose - 6 - P 6 × 6 phospho 6 × 6 -Phosphogluconate
hydrolase
+ gluconolactone +
6NADP 6NADPH 6 × NADP
(3) Dehydrogenase
5 × glucose - 6 - P

6 × NADPH
6 × 3 - Keto - 6 -
phophogluconate

(3) Dehydrogenase
5 - fructose - 6 - P

6 × CO2

6 × Ribulose - 5 - P

(4B) Epimerase (4A) Isomerase

4 × xylulose - 5 - P 2 × Ribose - 5 - P

2 × Xylulose - 5 - P 2 × Xylulose - 5 - P
(5) Transketolase

III
2 × glyceraldehyde - 3 - P 2 × Sedoheptulose-7 - P

(6) Transaldolase

2 × erythrose - 4 - P 2 × Fructose - 6 - P
(7)
Second Transketolase

2 × fructose - 6 - P
2 × glyceraldehyde - 3 - P
(8)

1 × fructose - 6- P

2 × fructose - 6- P

2 × fructose - 6 P

Interconnection with glycolytic pathway phosphate. These intermediates can be further

i. During non - oxidative phase, pentose phosphate catabolised through glycolysis and TCA cycle.
is converted to intermediates of glycolysis i.e. iii. Glucose can also be synthesised from these two
fructose - 6 - phosphate and glyceraldehyde - 3 - intermediates

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 Haemopoietic

Regulation of HMP shunt pathway - These free radicals are inactivated by enzyme
i. The first reaction catalyzed by GPD is the rate systems containing Superoxide dismutase
limiting step and it is inhibited by NADPH. (SOD), Peroxidase (POD) and Glutathione
reductase (GR).
ii. The oxidative phase: Controlled by level of
NADPH. - Glutathione reductase is regenerated with the
help of NADPH produced during HMP shunt
The non-oxidative phase: Controlled by
pathway.
requirement of pentoses.
III. Lens of Eye:
iii. Insulin induces GPD hence increase the overall
pathway. - Maximum concentration of NADPH is seen in
Physiological significance lens of eyes.
I. RBC membrane integrity - NADPH is required for preserving the
transparency of lens.
 IV. For lipid or steroid synthesis
O2 (superoxide)
- Oxidative phase of the pathway is seen in
Superoxide dismutase following organs, where NADPH generation is
required for lipid synthesis or steroid synthesis.
H2O2 (peroxide) a. Liver
+
2GSH (reduced) 2NADP b. Adipose tissue
Glutathione
c. Adrenal cortex
Glutathione
peroxidase reductase d. Mammary glands
e. Tester and ovaries
+
GS-SG (oxidized) 2NADPH + 2H f. RBCs
Clinical significance:
H2O
i. G-6-PD deficiency:
- Free radicals are continuously produced in RBC III
- In G-6-PD deficient cell, the level of NADPH is low.
and if they are not neutralized, they will
- Due to low concentration of NADPH,
destroy the membrane.
glutathione is not reduced as a result
- Glutathione neutralizes these free radicals to destruction of the free radicals cannot take
non-toxic form by reducing them and in the place.
process it itself undergoes oxidation.
- When, such person is exposed to certain drugs
- For functioning on next free radical, Glutathione or toxin like Primaquine, further production of
must return to its original reduced state. peroxides will take place.
- The reduction of Glutathione is carried out by - Hence, drug- induced hemolytic anemia is
Glutathione reductase, which is NADPH manifested.
dependent.
ii. Methaemoglobin:
- HMP shunt by ensuring availability of NADPH
- Normal blood has 1% of methaemoglobin.
helps to regenerate Glutathione to its active
- Methaemoglobin formed in the RBCs is readily
form and neutralize free radicals and thus
reduced back to ferrous state by
prevents lysis of RBC membrane.
methaemoglobin reductase enzyme system.
II. Free radical scavenging
- About 20% of reducing activity is due to
- Free radicals are continuously produced in all
NADPH dependent system.
cells.
- In G-6-PD deficient person, synthesis of NADPH
- These free radicals destroy DNA, proteins, fatty
is impaired.
acids and all biomolecules and in turn cells are
- Hence, methaemoglobin level rises in such
destroyed.
patient.

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 Biochemistry

iii. Thiamine deficiency: iv. Reduction of met -hemoglobin

- Transketolase enzyme use TPP as co-enzyme. v. Conjugation for detoxification
- So, in thiamine deficiency, HMP shunt pathway Note:
is inhibited, this results into impaired synthesis
of NADPH. Glutathione peroxidase is Selenium containing enzyme
- Thus, decreased NADPH manifests the
symptoms of Wernicke's Korsakoff's syndrome. PLASMA PROTEINS
Normal value
GLUTATHIONE (GSH)
- Normal concentration of the plasma proteins are
Past Questions:
Total proteins: 7.3 g/dl (6.4 to 8.3 g/dl)
1. What is glutathione? Outline its important
Serum albumin: 4.7 g/dl
functions. (4)[02 Dec]
2. What is the composition and biochemical Serum globulin: 2.3 g/dl
functions of glutathione. (1 +3 = 4)[10 Jan] Fibrinogen: 0.3 g/dl
3. Composition and functions of glutathione(3)[07 Dec]
- Molecular weight:
 Gamma - glutamyl cysteinyl glycine
 Tripeptide containing glutamate, cysteine and Albumin: 69,000 D
glycine Globulin: 1,50,000 D
Glutamate + cysteine  - glutamyl cysteine Fibrinogen: 4,00,000 D
 - glutamyl cysteine + glycine  Glutathione
- Specific gravity of the plasma proteins is 1.026.
Functions [10, 02]
Origin of plasma proteins
i. RBC membrane integrity
I. In embryo

O2 (superoxide) - In embryonic stage, the plasma proteins are
III Superoxide dismutase synthesized by the mesenchyme cells.
- The albumin is synthesized first.
H2O2 (peroxide)
2NADP
+ II. In adults
2GSH (reduced)
- In adults, the plasma proteins are synthesized
Glutathione Glutathione
peroxidase reductase mainly from reticuloendothelial cells of liver.

+
- The plasma proteins are synthesized also from
GS-SG (oxidized) 2NADPH + 2H
spleen, bone marrow, disintegrating blood cells
H2O and general tissue cells.
ii. Amino acid transport - Gamma globulin is synthesized from B
- Via Meister cycle lymphocytes.
iii. Co – enzyme role Types of plasma proteins
- Metabolic role of GSH is mainly in reduction I. Transport protein:
reactions.
- Albumin, Pre-albumin or transthyretin, Retinol
2GSH  GS - SG + H2
binding protein (RBP), Thyroxine binding
Thus, released hydrogen is used for reducing
other substrates as: globulin (TBG), Transcortin
i. Malonyl acetoacetatefumaryl acetoacetate II. Clotting factors:
ii. Cysteic acid  taurine - Prothrombin, Thrombin, Fibrinogen
iii. I2 + 2GSH  2HI + GS - SG

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III. Acute phase proteins (APP):
- Acute phase proteins are those proteins in blood
whose level increases (50 to 1000 times) in various
inflammatory and neoplastic conditions.
- Important acute phase proteins are:
a. C-reactive protein: C-reactive protein
increases in infection.
b. Ceruloplasmin:
 Ceruloplasmin increases in active hepatitis,
biliary cirrhosis, hemochromatosis,
obstructive biliary disease, pregnancy,
estrogen therapy, inflammatory condition,
collagen disorders and in malignancies
 It decreases in Wilson's disease,
malnutrition, Nephrosis and cirrhosis
c. Alpha-1 Anti-trypsin (AAT):
 It decreases in Nephrotic syndrome.
 Deficiency of Alpha-1 Anti-trypsin causes
emphysema.
Clinical features
Plasma protein Conditions when increase
Total proteins Hyperproteinemia:
1. Dehydration
2. Hemolysis
3. Acute infections like acute hepatitis and 3. Burns
acute nephritis
4. Respiratorydistress syndrome
5. Excess of glucocorticoids
6. Leukemia
7. Rheumataoid arthritis
8. Alcoholism
Albumin 1. Dehydration
2. Excess of glucocorticoids
3. Congestive cardiac failure
Globulin 1. Cirrhosis of liver
2. Chronic infections
3. Nephrosis
4. Rheumatoid arthritis
Fibrinogen 1. Acute infections
2. Rheumatoid arthritis
3. Glomerulonephritis
4. Trauma and Stroke
A/G ratio 1. Hypothyroidism
2. Excess of glucocorticoids
3. Hypogammaglobulinemia
4. Intake of high carbohydrate or protein diet
FAST TRACK BASIC
Haemopoietic
IV. Negative acute phase proteins:
- During an inflammatory response, some
proteins are decreased in blood; those are
called negative phase proteins.
- For example: Albumin, transthyretin, retinol
binding protein and transferring.
Functions of Plasma Protein
- Help in coagulation of blood
- Help in defense mechanism of body
- Help in transport mechanism
- Help in maintenance of osmotic pressure in blood
- Help in regulation of acid - base balance
- Help in viscosity of blood
- Help in erythrocyte sedimentation rate
- Help in suspension stability of red blood cells
- Help in production of trephine substances
- Help as reserve proteins
Conditions when decrease
Hypoproteinemia:
1. Diarrhea
2. Hemorrhage
4. Pregnancy III
5. Malnutrition
6. Prolonged starvation
7. Cirrhosis of liver
8. Chronic infections like chronic
hepatitis or chronic nephritis.
1. Malnutrition
2. Cirrhosis of liver
3. Burns
4. Hypothyroidism
5. Nephrosis
6. Excessive intake of water
1. Emphysema
2. Acute hemolytic anemia
3. Glomerulonephritis
4. Hypogammaglobulinemia
1. Liver dysfunction
2. Use of anabolic steroids
3. Use of Phenobarbital
1. Liver dysfunction
2. Nephrosis
SCIENCE MBBS -729-
 Biochemistry

SPECIAL POINTS FOR MCQs

1. Hemoglobin normal level (m) : 14-16 g/dl, (F) : 13-15 g/dl
2. Adult Hb (HbA) is 66,684 Dalton.
3. HbA has 2 and 2 chain, HbF has 2 and 2 gamma.
4. -chain coded by gene on chromosome 16. β, Ύ and δ chain coded by gene on chromosome 11.
5. -chain: 141 amino acids.β, γ ,  chain: 146 amino acids.
6. There are 36 histidine residues in Hb molecules.
7.  and B chain are connected by relatively weak non-covalent bonds (Van-der-waal’s force, H-
bond, Electrostatic forces)
8. 4 heme residues in each Hb molecule account for 4% of whole mass of Hb.
9. Iron atom in heme is in reduced ferrous state.
10. Each Hb can bind with 4 oxygen molecules.
11. Binding of oxygen to one heme residue increases the affinity of remaining heme residues for
oxygen. It is homotropic interaction.
12. Binding of 2, 3- BPG at site other than oxygen binding site lowers the affinity for oxygen. It is
heterotropic interaction.
13. Influence of pH and pCO2 to facilitate oxygenation of Hb in lungs and deoxygenation at tissues is
known as Bohr’s effect.
14. In adult p50 of normal Hb at 37°C is 26 mm Hg. In fetus and newborn p50 of normal Hb is 20 mm
Hg.
III
15. Red cell 2,3-BPG level is decreased in acidosis and increased in alkalosis.
16. HbA2 comprises about 2% of total Hb in adult and consists of 2 and 2 chain.
17. Affinity of CO to Hb is about 200 times that of oxygen.
18. When one molecule of CO binds to one monomer of the Hb molecule, it increases the affinity of
others to O2 so that O2 bound to these monomers are not released.
19. Heme consists of 4 pyrrole rings linked by Methenyl Bridge.
20. Heme can be synthesized by almost all tissues in the body. Heme is synthesized in normoblast but
not in matured erythrocytes.
21. Heme synthesis occurs partly in cytoplasm and partly in mitochondria.
22. ALA synthase is rate-limiting enzyme in Heme synthesis.
23. Amino acid required for heme synthesis is glycine.
24. Most common type of porphyria is Acute Intermittent Porphyria. (Autosomal dominant trait)
25. Urine is dark red in colour (port wine appearance) in congenital erythropoitic porphyria.
26. Heme is degraded primarily by microsomal enzyme system- Heme oxygenase system.
27. End products of heme catabolism are bile pigments.
28. Enzyme for conjugation of Bilirubin is UDP glucuronyl transferase.
29. ALA-dehydratase is inhibited by lead.
30. Bilirubin in serum is measured by van den Bergh reagent.
31. Methemoglobin: Iron is in ferric state.

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 Haemopoietic

32. HbS; glutamic acid in 6th position of β-chain of HbA is converted to valine
HbE; B-26: Glutamic acid replaced by lysine
HbD; B-121: Glutamic acid replaced by glutamine
33. Myoglobin can bind with only one oxygen.
34. Daily requirement of iron: 20 mg/day
35. Iron is absorbed from duodenum and jejunum.
36. Iron in diet is in ferric from but absorbed only in ferrous form. Fe+3 converted to Fe+2 by HCl of
gastric juice, Vitamin C etc.
37. Divalent metal cation transporter helps in absorption of iron from enterocyte.
38. Iron binding capacity of serum is 400 ng/dl.
39. Iron absorbed in enterocyte is release into blood via ferroportin channel.
40. Hepcidin binds with ferroportin channel causing endocytosis of channel.
41. Most reliable factor or parameter indicating iron deficiency is plasma transferrin level.
42. Bronze diabetes: when there is 25-30 gm of iron in body. It is triad of liver cirrhosis,
hemochromatosis, and pancreatitis with diabetes.
43. Folic acid is derived from pteridine, PABA and Glutamic acid.
44. Folic acid is absorbed by upper part of jejunum and transported in blood by -globulins.
45. Folic acid is mainly involved in one carbon metabolism.
46. N5-methyl THFA is converted to THFA by Vitamin B12. If B12 is deficient, this reaction cannot take
place leading to folate deficiency and accumulation of Methyl THF called folate trap.
47. Folic acid deficiency causes neural tube defect.
48. FIGLU test done to assess folate deficiency.
49. Vitamin B12 is absorbed from ileum (terminal part)
III
50 Vitamin B12 is cobalt containing vitamin.
51. Vitamin B12 consists of corrin ring.
52. Various form of Vitamin B12 are; Hydroxycobalamin, Methylcobalamin, Adenosylcobalamin and
Cyanocobalamin
53. One molecule of Intrinsic factor can combine with 2 molecules of B12.
54. Injectable form of vitamin B12: Hydroxycobalamin
55. Storage form of Vitamin B12: Adenosylcobalamin
56. Abundant form seen in blood: Methylcobalamin
57. Test for assessment of B12 deficiency: Schilling test
58. Phosphate buffer is the intracellular buffer.
59. In respiratory acidosis; patient will show reduced rate and depth of respiration
60. In metabolic acidosis; there will be hyperventilation.
61. Use of diuretics cause loss of potassium leading to metabolic alkalosis.
62. Normal bicarbonate (base): Carbonic acid (acid) ratio is 20:1.
63. Glycolysis occurs in cytosol. It takes place in all cells of the body.
64. A step 1, 3 and 9 of glycolysis is irreversible steps and enzymes required for these steps are key
enzymes of glycolysis.
65. Hexokinase acts on glucose, fructose and mannose and functions even when blood sugar level is
low.
66. Glucokinase acts only on glucose and functions only when glucose level is >100 mg/dl.
FAST TRACK BASIC SCIENCE MBBS -731-
 Biochemistry

67. RBC, skeletal muscle undergo anaerobic glycolysis.

68. Enolase is inhibited by fluoride ion.
69. Rapoport- Leubering cycle in RBC produces 2, 3-BPG which enhances oxygen dissociation.
70. NADPH is generated by Glucose-6-Phosphate dehydrogenase.
71. Transketolase activity is decreased by deficiency of thiamine pyrophosphate.
72. Excessive alcohol intake produces NADH, which converts pyruvate to lactate and hence cause
lactic acidosis.
73. Mannose derivatives are produced from N-acetyl glucosamine.
74. Gluose-6-phosphate dehydrogenase is the rate limiting enzyme in HMP-pathway.
75. G-6-PD is inhibited by NADPH.
76. ATP is neither generated nor utilized in HMP-Shunt pathway.
77. Normal blood albumin level is: 3.5-5.0 g/dl.
78. Hemopexin carries free Heme.
79. CRP, ceruloplasmin, Haptoglobinetc are acute phase reactant proteins
80. Albumin do not show polymorphism
81. Alpha-1 antitrypsin is a protease inhibitor the deficiency of which causes emphysema.
82. There are 36 histidine residues in Hb molecule which are important in buffering action.
83. In oxy - hemoglobin, the 6thvalency of Iron binds to oxygen atom and forms Hydrogen bond with
imidazole nitrogen of the distal histidine.
84. In deoxy - hemoglobin, a water molecule is present between the iron and distal histidine.
85. The binding of oxygen to one heme residue increases the affinity of remaining heme residues for
oxygen is called positive co-operativity. (Homotrophic reaction)
86. The deoxygenated hemoglobin carries three oxygen molecules i.e. 75% Oxygen saturation.
III
87. Chloride shift or Hamburger effect: When CO2 is taken up, the HCO3– concentration within the
cell decreases which diffuse out into the plasma. Simultaneously, chloride ions from plasma would
enter in the cell to establish electrical neutrality.
88. Oxy - Hb is more negatively charged than Deoxy - Hb.
89. The red cell 2, 3 - BPG level decreased in acidosis and increased in alkalosis.
90. Type III prophyrin is the most predominant in biological systems.
91. Coproporphyrinogen oxidase specifically acts only on type III series.
92. Oxidation and reduction of the Fe and Cu atoms of cytochromes are essential to their, biological
functions as carriers of electrons. By contrast, oxidation of the Fe++ of myoglobin or hemoglobin to
Fe+++ destroys their biological activity.
93. Co-operative binding of Hemoglobin permits hemoglobin to maximize both quantity of O2 loaded
at pO2 of the lungs and quantity of O2 released at pO2 of peripheral tissues.
94. Protons arise from rupture of salt bridge when O2 binds.
95. P50: A measure of O2 concentration is the partial pressure of O2 that half saturates given
hemoglobin.
96. BPG forms salt bridges with the terminal amino groups of both  chains via Val NA1 and with Lys
EF6 and His H21 hence stabilizes the de-oxygenated hemoglobin.
97. The carbon of CO2 is derived from COOH group of Gluconic acid
98. GPD deficiency offers resistance to plasmodium infection.

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