Biochemistry

BIOCHEMISTRY
A COMPLETE COURSE
PART II
DR. C. SHANMUGAPRIYA
PROFESSOR OF BIOCHEMISTRY
Dr. C. Shanmugapriya,
MBBS., M.D., (Biochemistry)
saipriyain@gmail.com
She is working as the Head, Research & Development (May 2018
till date) of KRIYA Medical Technologies Pvt. Ltd. She has been instru-
mental in products validation and in new products development includ-
ing a unique molecular transport media for SARS CoV2, molecular diag-
nostic kit for Omicron detection and for simultaneous detection of SARS
CoV2, Influenza A & B, RSV and is currently undergoing training in novel
Molecular Biology Techniques at KRIYA.
She has done her MBBS at Madurai Medical College and MD Bio-
chemistry at Madras Medical College. She was working as the Professor
and Head of the Department of Biochemustry, Government Thoothukudi
Medical College and Hospital, Thoothukudi. She has been teaching Bio-
chemistry and Physiology for medical and dental post-graduation aspir-
ants since 2006 and she has coached thousands of students for the past
16 years, who continue to come up with flying colours. She is known for
her conceptual teaching. She is the co-author of the book “A guide for
preparation of ALL INDIA NEET EXAM” Volume I 2016-2017.
She was representing India in the International Federation of Clini-
cal Chemistry – Task Force for Young Scientists for three consecutive
terms 2012 to 2015, 2015 to 2018, and 2019 to 2021. She has received
the “Young scientist award” twice, in the year 2010 for the work on Type
2 diabetes and in the year 2008 for the work on coronary atheroscle-
rosis. Her work on Type 2 diabetes was also chosen as the best scien-
tific paper in the National Diabetology Conference in the year 2010 at
Chennai and she received the Best oral paper presentation for her
work on “TCF7L2 Variant rs7903146 affects the Risk of Type 2 Diabetes
by Modulating Incretin secretion / action” at Asia Pacific Federation of
Clinical Biochemists 2013 held at Bali.
She is an educator in Prepladder, an online teaching platform for
medical postgraduation aspirants since March 2023 and she will be
teaching Biochemistry and Applied Biochemistry in the platform.
She is an educator in Unacademy in the NEET PG category where she
teaches Biochemistry and Physiology to NEET PG aspirants.
https://unacademy.com/@CShanmugapriya
She has a YouTube channel “Biochemistry Dr.C. Shanmugapriya” in which
she uploads conceptual videos on Biochemistry and Clinical Biochem-
istry.
https://www.youtube.com/channel/UCTge8U1HBZf7jUaBATr1iCw
For more details and more testimonials, check her page:
https://www.facebook.com/biochemneetpgdiscussionforum/
Contents
LIPID METABOLISM PART II 05 Translation inhibitors 61
Cholesterol synthesis 06 RT PCR or reverse transcription
Fatty acid oxidation 08 PCR 66
Lac operon 70
Fate of a fatty acid in cell 10
Crispr cas system 76
Lipoprotein metabolism 15
AMINOACID AND PROTEIN
Chylomicron metabolism 16 CHEMISTRY 78
Vdld metabolism 16 Classification of aminoacids 79
HDL Metabolism 17 Protein structure 81
Hyperlipoproteinemia 20 AMINOACID METABOLISM 84

Aminoacid breakdown 85
GENETICS 24
Urea Cycle and Disorders 88
Nucleotide structure and linkages
25 Individual Aminoacid metabolism
94
Human Genomic Sequences 36
VITAMINS 111
Replication 38
Vitamin A 112
Errors of replication and DNA
repair mechanisms 45 Vitamin E 114
RNA, Transcription and post Vitamin K 115

transcriptional modifications 49 Vitamin D 118
Transcription 52 Water soluble vitamins 122
Genetic cod 56 ALL ABOUT A ABG
Translation 58 INTERPRETATION IN AN HOUR
132
Steps of translation 58
LIPID
METABOLISM
PART II
@dr.c.shanmugapriya Dr. C. Shanmugapriya BIOCHEMISTRY
CHOLESTEROL
SYNTHESIS
INTRODUCTION
>> Cholesterol is a steroid and hence it gets >> Phase II: Mevalonate undergoes three
synthesized partly in cytoplasm and partly in kinase reactions followed by decarboxylation
smooth endoplasmic reticulum. to form Isoprenoid units
>> Acetyl CoA is the precursor of cholester- >> Phase III: 6 Isoprenoid units condense to
ol. form squalene
BIOCHEMISTRY I A COMPLETE COURSE I Part 02
>> Cholesterol synthesis occurs in 5 phases. >> Phase IV: Squalene reaches Smooth
>> Phase I: Acetyl CoA is converted to endoplasmic reticulum. Squalene epoxidase
mevalonate. converts squalene to form lanosterol
• Two molecules of acetyl CoA are linked >> Phase V: Lanosterol becomes Zymoster-
by thiolase to form acetocetyl CoA ol, Desmosterol and finally cholesterol
• Acetoacetyl CoA reacts with a third mol- Byproducts of cholesterol synthesis include
ecule of acetyl CoA in the presence of Dolichol, Ubiquinone and Heme A
HMG CoA synthase to form HMG CoA. HMG CoA reductase is the rate limiting en-
HMG CoA reductase converts HMG CoA zyme of cholesterol synthesis. It is inhibited
to mevalonate by statins and it exhibits diurnal variation. It is
active in the night
PHASE I
Acetyl CoA Acetyl CoA
Thiolase
Acetoacetyl
CoA
Acetyl CoA HMG CoA synthase
HMG CoA MEVALONATE
HMG CoA
reductase
6 >>>
PHASE II
MEVALONATE Isoprenoid
Kinase Decarboxylation
PHASE III
6
Squalene
ISOPRENOID
PHASE IV
Squalene Lanosterol Zymosterol Desmosterol
Cholesterol
REGULATION OF HMG COA REDUCTASE
• Allosteric Regulation • It is activated by dephosphorylation

• Inhibited by all its products • Induction or Repression
• Mevalonate, Cholesterol and Bile acid • Insulin induces in adipose tissue
• Covalent Modification
7
MCQS
1. Cholesterol synthesis 2. Statins act by inhibiting: 3. All the following are
takes place in byproducts of cholesterol
A. HMG CoA reductase synthesis except:
A. Cytoplasm B. HMG CoA Lyase
B. Nucleus C. Acetyl CoA carboxylase A. Dolichol
C. Endoplasmic reticulum D. 7 alpha hydroxylase B. Ubiquitin
1.D; 2.A; 3.B.

D. a&c C. Heme A
D. Cholesterol
FATTY ACID OXIDATION

INTRODUCTION
• Very Long chain fatty acids get oxidised in peroxisome). In beta oxidation, the
in peroxisome beta carbon atom gets oxidised to form a
• Very short chain fatty acids get oxidised carboxyl group, hence the alpha carbon
in mitochondria atom and the carboxyl group come out
• Short chain, medium chain and long chain as acetyl CoA. Fatty acids with n number
fatty acids get oxidised in both mitochon- of carbon atoms get oxidised to form n/2
dria and peroxisome acetyl CoA
• Fatty acids of all chain lengths get oxi- • The difference between mitochondrial
dised by beta oxidation in mitochondria oxidation and peroxisomal oxidation is
or in peroxidome. (exception – branched that the former is coupled to ATP produc-
chain fatty acids undergo alpha oxidation tion and the latter is coupled to hydrogen
peroxide generation
8 >>>
DIFFERENCE BETWEEN MITOCHONDRIAL AND

PEROXISOMAL OXIDATION
S.No PROPERTY MITOCHONDRIA PEROXISOME
1 Type of fatty acids
2 Type of oxidation
3 Products
4 ATP
S.No PROPERTY MITOCHONDRIA PEROXISOME
1 Type of fatty acids Very short, short, Short, medium, long and very
medium and long long chain fatty acid
chain fatty acid
2 Type of oxidation β oxidation β oxidation
3 Products n/2 acetyl CoA n/2 acetyl CoA
4 ATP + No ATP, H2O2 peroxidation
9
FATE OF A FATTY
ACID IN A CELL
FATTY ACID
ATP → AMP Acyl CoA
synthetase
Acyl CoA
TAG CPT1
Cholesterol β OXIDATION
ester Acyl CoA
HIGH LOW Acetyl CoA

ENERGY ENERGY
• As soon as fatty acid gets into any cell, • If the energy status is low, fatty acid has
irrespective of the final fate of fatty acid, to be oxidised, for which the fatty acid
it is converted to acyl coA by acyl CoA enters into mitochondria, with the help
synthetase, using two high energy phos- of a carrier – carnitine and the enzyme is
phates Carnitine acyl transferase I
• Further fate of acyl CoA is dependent on • Carnitine acyl transferase I is the rate lim-
the energy status iting enzyme of fatty acid oxidation
• If the energy status is high, fatty acid is
stored as triacyl glycerol and cholesterol
ester
PHASES OF FATTY ACID OXIDATION

• Phase I: n C fatty acids → n/2 acetyl
CoA
• Phase II: Acetyl CoA gets into TCA
cycle
10 >>>
STEPS OF EVERY CYCLE OF BETA OXIDATION
Acyl CoA
ACYL COA
FADH2 DEHYDROGENASE
UNSATURATED ACYL CoA
H2O HYDRATASE
β HYDROXY ACYL CoA

β HYDROXY ACYL COA
NADH DEHYDROGENASE
KETOACYL CoA
ACETYL CoA
DETAILS OF PHASE I – EVERY CYCLE

ENZYMES INVOLVED ENERGETICS PRODUCT
• Acyl CoA 4 Acetyl CoA

Dehydrogenase
• Hydratase
• Beta Hydroxyacyl CoA
dehydrogenase
• Thiolase
REGULATION OF CPT1
STIMULATORS: • Glucagon INHIBITORS • Insulin
• ADP • Acyl CoA • ATP • Malonyl CoA
• NAD • NADH
• FAD • FADH2
FORMULA FOR CALCULATION OF ENERGETICS OF FATTY

ACID OXIDATION
• (n - 1) x 4 + (n) x 10 - 2
• 2 2
• Palmitic acid with 16 carbon atoms provides 106 ATPs
• Stearic acid with 18 carbon atoms provides 120 ATPs
11
METABOLIC CHANGES IN DKA

• Low Insulin in diabetes stimulates Hor- • Formed acetyl CoA will not be able to en-
mone Sensitive Lipase and hence periph- ter into citric acid cycle, as oxaloacetate
eral lipolysis in adipose tissue is unavailable
• Fatty acids and glycerol reach liver • Oxaloacetate is unavailable because, in
• Fatty acids are oxidised in the liver to diabetes, gluconeogenesis is stimulated
form acetyl CoA and oxaloacetate is used for gluconeo-
genesis
• Acetyl CoA accumulates and condenses
to form ketone bodies
KETONE BODY SYNTHESIS

1 Acetyl CoA Acetyl CoA β HYDROXYBUTYRATE
β HYDROXYBUTYRATE Thiolase
DEHYDROGENASE
Acetoacetyl Acetone
CoA 1
Acetyl CoA HMG CoA

synthase
HMG CoA Acetoacetate
HMG CoA
lyase
FACTS ABOUT KETONE BODY METABOLISM

• Ketone bodies are products of incom- • Ketone bodies are utilised by extrahepat-
plete fatty acid oxidation ic tissues
• They are synthesised in diabetes and • Liver can not utilise ketone bodies be-
starvation because of low availability of cause they lack thiophorase or succinyl
oxaloacetate CoA acetoacetate coA transferase
• Ketone bodies are synthesised in liver
mitochondria
12 >>>
CAUSES OF DEFECTS OF BETA OXIDATION

OF FATTY ACIDS
>> Congenital: >> Acquired:

• Genetic defects of any • Jamaican Vomiting
of the following en- sickness due to the
zymes involved in fatty intake of unripe ackee
acid oxidation fruit
» CPT1 • Mysterious deaths of
» CPTII children in Muzaffarpur,
» Acyl CoA dehydro- India
genase
» Hydratase
» Beta hydroxyacyl
CoA dehydrogenase
» Thiolase
BIOCHEMICAL FEATURES OF FATTY ACID

OXIDATION DEFECTS
Non ketotic hypoglycemia: • Acetyl CoA is a stimulator of Pyruvate
• Fatty acid oxidation defect causes decarboxylase (enzyme of gluconeogene-
ficiency of ATP. Gluconeogenesis is sis) Lack of acetyl CoA causes inhibition
dependent on fatty acid to provide the of gluconeogenesis
necessary energy. Lack of energy causes • As acetyl coA is not formed in fatty acid
hypoglycemia. oxidation defect, ketone bodies are not
formed
Hyperammonemia:
• Fatty acid oxidation defect causes stimulation of aminoacid oxidation
To be filled
up after
learning
about
regulation of
urea cycle !!
Dicarboxylic aciduria tion). Peroxisomes can not oxidise very

• When mitochondrial enzymes are defec- short chain fatty acids by beta oxidation.
tive, all fatty acids get oxidised in perox- Hence, they mediate omega oxidation,
isomes (not coupled to energy produc- which causes dicarboxylic acids to be
generated
13
MCQS
1. Fatty acid Oxidation 5. How many cycles of 9. Ketosis is observed in
occurs in: beta oxidation does Palmitic Diabetes because of:
acid go through :
A. Mitochondria A. Low availability of oxalo-
B. Peroxisome A. 7 acetate
C. Both B. 8 B. Excess oxaloacetate
D. None C. 9 C. Low energy
2. Very short chain fatty

D. 16 D. Low fatty acid oxidation
acid Oxidation occurs in: 6. CPT1 is activated by all, 10. Liver can not utilize
EXCEPT: ketone bodies because of
A. Mitochondria lack of:
B. Peroxisome A. Acyl CoA
C. Both B. Malonyl CoA A. Thiolase
D. None C. High ADP/ATP ratio B. Thioesterase
3. Very long chain fatty

D. Glucagon C. Thiophorase
7. Number of ATP gener-

D. Aconitase
11. Fatty acid oxidation

acids undergo
ated in the liver by complete
A. β Oxidation oxidation of Palmitate: defects present with all
B. ω Oxidation except:
C. α Oxidation A. 106
D. γ Oxidation B. 33 A. Hypoglycemia
1.C; 2.A; 3.A; 4.A; 5.A; 6.B; 7.A; 8.B; 9.A; 10.C;11.B;.
4. Rate limiting enzyme of

C. 26 B. Ketosis
D. 16 C. Hyperammonemia
8. Number of ATP gener-

fatty acid oxidation: D. Dicarboxylic aciduria
A. Carnitine Acyl Transferase ated in the liver by complete

I oxidation of Stearic acid is:
B. Carnitine Acyl Transferase
II A. 106
C. Acyl CoA dehydrogenase B. 120
D. Thiolase C. 30
D. 26
14 >>>
LIPOPROTEIN
METABOLISM
FUNCTION & STRUCTURE OF LIPOPROTEINS
• Lipoprotein is used to transport lipid in This helps them to hold the non polar
blood lipids within themselves.
• To transport any non polar substance • Lipoproteins have a lipid particle attached
in a polar medium, we prefer to have an to apoprotein
amphipathic substance. When an amphi- • Lipid particle has an amphipathic outer
pathic substance is dropped in a polar layer made up of phospholipid and cho-
medium, it orients itself in such a way that lesterol. The inner non polar lipid core is
the polar groups face the exterior, this en- made up of triacyl glycerol and cholester-
ables them to soluble in a polar medium. ol ester.
The non polar groups face the interior.
BASIC STRUCTURE OF A LIPOPROTEIN

APOPROTEIN
1. Ligands for receptors
2. Regulators for enzymes
AMPHIPATHIC CHOLESTEROL
OUTER LAYER PL
INNER NON POLAR CHOLESTEROL

LIPID CORE ESTER
TAG
FUNCTIONS OF APOPROTEINS
Ligands of receptors concerned with Regulators of enzymes concerned with
lipoprotein metabolism: lipoprotein metabolism:
• Apo E is accepted by remnant receptor • ApoCII activates Lipoprotein Lipase
and LDL receptor • Apo CIII inhibits lipoprotein lipase
• Apo B100 is accepted by LDL receptor • Apo A I activates LCAT or Lecithin Choles-
terol Acyl Transferase
15
CHYLOMICRON
METABOLISM
• Transports exogenous or dietary TAG • ApoCII of functional chylomicron activates
from intestine to extrahepatic tissues LPL
• Formed by enterocytes and released as • LPL converts TAG to glycerol and fatty ac-
nascent chylomicron. It is called as Nas- ids. Glycerol reaches liver (the only organ
cent chylomicron because it has only which can use glycerol is liver). Fatty acid
ApoB48. enter into extrahepatic tissues
• Nascent chylomicron becomes functional • Action of LPL converts functional chylo-
chylomicron with the help of HDL micron to Chylomicron remnant

• HDL functions as a repository of all apo- • Chylomicron remnant is accepted by liver
proteins through LDL receptor or remnant recep-
• HDL donates apo CII and apo E to acti- tor
vate nascent chylomicron as functional
chylomicrons
VLDL METABOLISM
• Transports endogenous or dietary TAG • Nascent VLDL becomes functional VLDL
from liver to extrahepatic tissues with the help of HDL
• Formed by hepatocytes and released • Active apo CII activates LPL
as nascent VLDL. Apoproteins present • Action of LPL converts TAG in functional
in nascent VLDL are Apo B100, inactive VLDL to form glycerol and fatty acids.
apoCII and inactive Apo E • Glycerol is used by liver and fatty acids
enter into extrahepatic tissues.
TWO FATES OF VLDL
• If VLDL loses its apo CII immediately to • If VLDL does not lose its apo CII, retains
HDL, it is called as VLDL remnant its apo CII, goes through multiple cycles
• VLDL remnant is accepted by liver of LPL activity, loses its major chunk of
through LDL receptor or remnant recep- TAG, and then loses its apoCII, now its size
tor is reduced and its density is increase, This
is called as IDL
16 >>>
TWO FATES OF IDL
• IDL is accepted by liver through LDL re- converting IDL to a lipoprotein rich in
ceptor or remnant receptor cholesterol ester
or • IDL gives back apo E to HDL
If IDL comes across a HDL involved in re- • Thus, IDL gets converted to LDL
verse cholesterol transport, two exchange • LDL is accepted by liver through LDL
reactions happen between IDL and HDL receptors
• Cholesterol Ester Transfer Protein trans- or
fers cholesterol ester from HDL to IDL, LDL is accepted by extrahepatic tissues
through LDL receptors
HDL METABOLISM
• Transports Cholesterol ester and phos- when they are released into the circula-
pholipids from extrahepatic tissues to tion.
liver • Phospholipid and cholesterol are amphi-
• Formed by hepatocytes and enterocytes pathic and because there is no non polar
• Intestinal HDL gets apo CII and apo E lipid to form a core, they take up a dis-
from liver HDL oidal HDL and hence called as Discoidal
• Both intestinal and liver HDL have only HDL
a layer of phospholipid and cholesterol,
ACTION OF LCAT & ABCI
• LCAT converts cholesterol in discoidal extrahepatic tissue membranes and is

HDL to form cholesterol ester added to HDL3.
• Cholesterol ester is non polar and hence • This converts HDL3 to HDL 2
Discoidal HDL becomes functional HDL3 • HDL2 reaches liver and is accepted by
with the help of LCAT SRB1 and is released as HDL3
• Apo A I of HDL3 activates ABCI of extra- • This is HDL cycle
hepatic tissue membranes. ABCI extracts • Pre β HDL is the most active form of HDL
cholesterol ester and phospholipids from
17
DISORDERS OF HDL METABOLISM

• LCAT DEFICIENCY • ABCI MUTATION: Tangier’s disease
• Partial LCAT deficiency – Fish eye disease • Hepatosplenomegaly
• Complete LCAT deficiency • Greyish Orange Tonsils
• Hemolytic anemia and renal failure • Mononeuritis multiplex
• LpX detected on electrophoresis
MCQS
1. The main function of lipoproteins is to 5. The apoprotein which activates Lipo-
protein lipase is:

A. Activate fatty acid synthesis
B. Transport lipids to kidney for excretion A. Apo B48
C. Stimulate lipolysis B. Apo B100
D. Transport lipids in blood between tissues C. Apo CII
2. Apo AI activates:
D. Apo A1
6. The apoprotein present in nascent chy-

A. Lipoprotein Lipase lomicron is:
B. Lecithin Cholesterol Acyl Transferase
C. Hormone Sensitive Lipase A. apo B100
D. LRP B. apo B48
3. Apo CII activates:

C. apo CII
D. apo E
A. Lipoprotein Lipase 7. True regarding chylomicron are all

B. Lecithin Cholesterol Acyl Transferase except:
C. Hormone Sensitive Lipase
D. LRP A. HDL is involved in activation of chylomi-
4. Chylomicron transports triacylglycerol

cron
B. LPL helps in the conversion on chylomi-
from intestine to: cron to chylomicron remnant
C. Chylomicron remnant reaches the extra-
A. Liver hepatic tissues
B. kidney D. Chylomicron reaches the extrahepatic
C. Extrahepatic tissues tissues
D. Brain
18 >>>
8. Inhibition of which of the following pro- 11. Which of the following helps in the
teins can increase HDL? conversion of discoidal to Spheroidal
HDL?
A. LPL
B. LCAT A. LPL
C. ABCA1 B. LCAT
D. CETP C. ABCA I
9. Remnant receptor accepts lipoproteins

D. CETP
with which of the following apoproteins? 12.Regarding HDL all are true, EXCEPT :
A. Apo E A. HDL is synthesized by Liver and intestine
B. Apo B100 B. Pre β HDL is the most active form
C. Both C. Mutation in ABC-1 cause Tangiers disease
D. None D. Both liver and intestinal HDL have Apo C
10. LDL receptor accepts lipoproteins

and Apo E
with which of the following apoproteins?
A. Apo E
B. Apo B100
C. Both
D. None
1.; 2.B; 3.A; 4.C; 5.C; 6.B; 7.C; 8.D; 9.A; 10.C; 11.B; 12.D
19
HYPERLIPOPROTEINEMIA
HYPERLIPOPROTEINEMIAS
Friedrickson’s Classification » Type I » Type III

• Six types » Type IIa » Type IV
» Type IIb » Type V
• Isolated Hypercholesterolemia – This • Isolated Hypertriglyceridemia – This cate-

presents with tendon xanthomas and gory presents with recurrent pancreatitis,
accelerated atherosclerosis. Tendon xan- eruptive xanthomas and lipemia retinalis.

thomas are tiny eruptive lesions present The plasma is lipemic (milky white) and
along the line of attachment of tendons. turbid and hence causes clogging of
They are present along the knuckles and capillaries – the explanation for recurrent
ankles. This type Includes Type II A Hy- pancreatitis. It includes Type I, IV and V
perlipoproteinemia. It is called as Familial • Both Hypercholesterolemia and Hyper-
Hypercholesterolemia. It is caused by a triglyceridemia – This category includes
defect of LDL receptor. It is an autosomal Type IIb and Type III
dominant condition.
ERUPTIVE
XANTHOMAS
TENDON
XANTHOMAS
20 >>>
TYPE IIA HYPERLIPOPROTEINEMIA

• It is otherwise called as Familial Hyper- • It is characterised by tendon Xanthomas
cholesterolemia and accelerated atherosclerosis
• It is caused by a defect of LDL receptor • It is an autosomal dominant condition
with gene dose effect
TYPE I HYPERLIPOPROTEINEMIA
• It is otherwise called as Familial Chylomi- is to be obtained, as LPL is clinging to

cronemia syndrome the vessel wall held in place by heparan
• It is caused by a defect of either Apo CII sulphate. If the LPL activity in the post
or LPL defect heparinised sample happens to be low,
• As in either case, chylomicron metabolism a mixing study is to be performed to find
is affected, chylomicron accumulates. out if in a patient, LPL is defective or apo
As chylomicron carries predominantly CII is defective
triglycerides, it presents as Hypertriglyc- • A mixing study is done for this purpose.
eridemia Equal volume of the patient’s plasma and
• Hence the patient presents with recurrent pooled normal plasma are mixed and LPL
pancreatitis, eruptive xanthomas and on activity is re estimated.
examination we observe Lipemia retinalis • If the repeat LPL activity normalises, it is
• To make a presumptive diagnosis of this apo CII defect. If the repeat LPL activity
condition, LPL activity should be measure does not normalise. It is LPL defect. Apo
and should be observed to be low, for CII defect responds to fresh frozen plas-
which a post heparinised blood sample ma administration.
TYPE III HYPERLIPOPROTEINEMIA

• It is caused by Apo E defect is called as BROAD BETA DISEASE or as
• Apo E is an apoprotein of remnants. FAMILIAL DYSBETALIPOPROTEINEMIA
Hence it is called as REMNANT DISEASE • It is caused by. Homozygous E2/E2 muta-
• Remnants carry both cholesterol and tion
triglyceride and hence this condition is • The two pathognomonic features of this
characterised by ELEVATION OF BOTH type are Palmar eruptive xanthoma and
CHOLESTEROL & TGL Xanthoma palmaris striae
• A broad beta band is seen on lipoprotein
electrophoresis and hence this condition
21
PALMAR ERUPTIVE PALMARIS

XANTHOMA XANTHOMA STRIAE
LIPOPROTEIN ELECTROPHORESIS
• It is run on a glass slide with agarose as • The lipoprotein which moves the fastest is
the support medium. Towards one end of HDL, as HDL has the highest phospholipid
the glass slide, the serum is applied using and protein content and hence the high-
a micropipette. The slide is placed in an est negative charges. Hence HDL forms
electrophoretic tank filled with an alkaline the alpha band
buffer. Alkaline buffer provides negative • In contrast the lipoprotein which hardly
charges to all the proteins. The tank is moves is chylomicron, as chylomicron
connected to an electrical supply in such size is the largest. As fasting sample is
a way that close to the point of applica- usually run, no band is seen at the point
tion is connected to negatively charged of application. If a band is seen at the
electrode, the opposite side is connected point of application, consider the follow-
to positively charged electrode. When the ing possibilities
electric current is switched on, all lipopro- » Non fasting sample
teins start moving towards the positively » Familial chylomicronemia syndrome
charged electrode » LpX due to LCAT deficiency or
• The two factors which affect mobility are
» Number of negative charges
» Size
22 >>>
LIPOPROTEIN ELECTROPHORESIS
Chylomicron/ ALPHA BAND

LpX / HDL
BROAD BETA/ Pre BETA

TYPE III BAND /VLDL
- +
MCQS
1. A 25 year old male presents with an 2. A 38 year old male presents with
episode of pancreatitis. His cholesterol is yellowish discoloration of palmar creases
190mg/dL and Triglyceride level is 1180mg/ as shown in the image. His cholesterol is
dL. His post heparinised Lipoprotein lipase 300mg/dL and Triglyceride level is 280mg/
activity is low. On mixing study, the Lipopro- dL. What is expected in his lipoprotein elec-
tein lipase activity normalises. Which of the trophoresis?
following is true about his condition?
A. A band at the site of application
A. He will respond to fresh frozen plasma B. Broad alpha band
administration C. Broad beta band
B. It is a defect of Lipoprotein lipase D. Absent alpha band
C. It is a defect of apo CIII
D. It is an autosomal dominant condition
1.B; 2. C
23
GENETICS
NUCLEOTIDE STRUCTURE
& LINKAGES
INTRODUCTION – CHROMOSOME,
GENE ORGANISATION
» The organization begins with nitrogenous » Two polynucleotide chains get associat-
base which becomes a nucleoside. Nu- ed to form a double stranded DNA
cleoside is converted to a mono phos- » dsDNA on condensation forms a chromo-
phate nucleotide some
» When multiple monophosphate nucleo- » Every chromosome has multiple genes,
tides are linked, it becomes a polynucleo- which can code for a protein or RNA
tide chain.
TYPES OF NITROGENOUS BASES

Two types » The significance of minor purine base
xanthine is that caffeine, theophylline
• PURINES and theobromine are derivatives of
» Purine ring has 2 rings and 9 atoms xanthine. Caffeine is 1,3,7 trimethyl
» The two types of purine bases are xanthine. Theophylline is 1,3 dimethyl
major purine bases and minor purine Xanthine, Theobrimine is 3,7 dimethyl
bases Xanthine
» Major purine bases include Adenine » All purines on catabolism give rise to
and Guanine uric acid
» Minor purine bases include Hypoxan-
thine, xanthine and Uric acid • PYRIMIDINES
» Adenine and Guanine are present in a » Pyrimidine ring has 1 ring and 6 atoms
polynucleotide chain » Pyrimidine ring include Cytosine, Uracil
and Thymine
25
BIOCHEMISTRY I A COMPLETE COURSE I Part 02 @dr.c.shanmugapriya Dr. C. Shanmugapriya BIOCHEMISTRY
NUCLEOSIDE
• When a purine or a pyrimidine base is » Purine nucleosides include Adenosine,
attached to a ribose or a 2 deoxyribose Guanosine, Inosine (a nucleoside of
sugar, a nucleoside is formed hypoxanthine)
• The first hydroxyl group of a ribose or a » Pyrimidine nucleosides include cyti-
deoxyribose is attached to N9 of purine dine, thymidine and uridine. Pseudou-
or N1 of pyrimidine through beta N gly- ridine is a nucleoside present in one
cosidic linkage form a nucleoside. of the arms of tRNA. In pseudouridine,
the ribose sugar instead of getting
attached to N1, gets attached to C5
RIBOSE & DEOXYRIBOSE
N9 of N1 of
Purine Pyrimidine
26 >>>
STRUCTURE OF NUCLEOSIDE
β N GLYCOSIDIC
LINKAGE
NUCLEOTIDE
• When a nucleoside is attached to a phos- • Acid anhydride linkages are rich in en-
phate group, a nucleotide is formed ergy. Hence triphosphate nucleotides
• The phosphate group is attached to 5’ with 2 acid anhydride linkages act as an
hydroxyl group of a nucleoside through energy currency and diphosphate nucle-
a phosphoester linkage to form a otides with 1 acid anhydride linkage can
monophosphate nucleotide. act as a source of energy
• When an additional phosphate group • Multiple monophosphate nucleotides get
gets attached to the already existing linked though 3’5’ phosphodiester linkag-
phosphate group through acid anhydride es to form a polynucleotide chain.
linkage to form a diphosphate nucleotide.
STRUCTURE OF A NUCLEOTIDE
PHOSPHOESTER
LINKAGE
27
ADENOSINE DIPHOSPHATE
ACID ANHYDRIDE
LINKAGE
2 ACID ANHYDRIDE
LINKAGES
POLYNUCELOTIDE FORMATION
3’5’ PHOSPHODIESTER
LINKAGE
28 >>>
ds DNA – WATSON & CRICK MODEL

» When two polynucleotide chains interact like fashion. The backbone of the ladder
with each other with hydrogen bonds in is made up of 3’5’ phosphodiester link-
between, it is called as ds DNA ages and deoxyribose sugar. The steps
» The structure of dsDNA is explained by are made up of hydrogen bonds. There
Watson and crick model. According to are two hydrogen bonds between A and
Watson and crick, the two strands are T and three hydrogen bonds between
exactly complementary and antiparallel. G and C. Based on this model, there are
The two strands are oriented in a ladder seven conformations of DNA
B TYPE: A TYPE: Z TYPE:
» It is the most common » This type is found when- » GC rich sequences
physiological form ever DNA undergoes » Left handed helix
» It is a right handed helix dehydration or whenever » Every full turn has 12
» Every full turn has 10 bas- there is a DNA RNA hy- bases
es. Each base pair rises brid, RNA RNA hybrid
the height by 3.4 A0 and » Right handed helix
one full turn with 10 base » Every full turn has 11
pairing measures 34 A0. bases
The Width is 20 A0.Every » All grooves will be of the
full turn has a major and a same dimension
minor groove
29
BIOCHEMISTRY I A COMPLETE COURSE I Part 02 @dr.c.shanmugapriya Dr. C. Shanmugapriya BIOCHEMISTRY
DNA & CHROMOSOMES

» A ds DNA measures a few metres long, forms loops on chromosome scaffold to
which can not be packed inside a nucleus form a highly condensed chromosome.
of few microns in diameter. Hence, the ds » A chromosome exists in a highly con-
DNA is condensed with the help of pro- densed form, when the chromosome need
teins called as histones to form a chromo- not perform its functions of replication
somes and transcription. As unwinding is the first
» Histones are basic proteins. They are step of both replication and transcription,
positively charged. dsDNA is negatively chromosomes undergo uncondensation
charged. So, a segment of ds DNA gets before replication and transcription
wound around histones to give the first
level of compactness.
» There are five types of Histones – H1,
H2A, H2B, H3, H4. All except H1, remain-
ing 4 form dimers , which is called as a
Histone octamer. A segment of ds DNA
surrounds the Histone octamer to form a
nucleosome. Several such nucleosomes
exist, which are connected by a linker
fragment. H1 is present on the linker
fragment. This forms a 10 nm fibril. When
a segment of 10nm fibril gets folded on it-
self, it is called as 30 nm fibril. 30 nm fibril
30 >>>
GENE, TRANSCRIPTION,
MODIFICATIONS
» Gene or transcription unit is a segment of

chromosome which can code for a protein
or an RNA
» Gene contains both coding and non cod-
ing sequences or introns
» The immediate product of transcription of
gene is called as a primary transcript or
heternonuclear RNA
» Primary transcript undergoes 3 post tran-
scriptional modifications in nucleolus to
form functional mRNA
» 7 methyl guanosine cap addition, Poly A
tail addition and splicing are the 3 post
transcriptional modifications
» These post transcriptional modifications
are not universal. Histone mRNA is an
exception. Histone genes do not have
introns. Hence no splicing. They don’t un-
dergo poly A tail addition, instead they are
attached to a stem loop structure
TYPES OF CHROMATIN
There are two types of chromatin: highly condensed, and hence stains
» Euchromatin more densely.
» Heterochromatin » The two types of heterochromatin are
• Euchromatin: » Constitutive Heterochromatin – A part
» It is the transcriptionally active form of of the chromosome which is transcrip-
a chromosome. As it is transcriptionally tionally inactive always eg., centromere
active, it is uncondensed. As it is un- and telomeric ends
condensed, it stains less densely » Facultative heterochromatin – Occa-
• Heterochromatin: sionally active eg., Female X chromo-
» It is the transcriptionally inactive region some
of a chromosome. As it is inactive, it is
31
CHROMATIN
EUCHROMATIN HETEROCHROMATIN
Active
Inactive
Uncondensed
condensed
Less densely
More densely stained
stained
TYPES OF HETEROCHROMATIN
CHROMATIN
EUCHROMATIN HETEROCHROMATIN
CONSTITUTIVE FACULTATIVE
Occasionally
Always inactive
active
e.g., Centromere
e.g., X
& Telomeres
chromosome
DENATURATION
» Denaturation is a process by which a ds unwinding to form two single strands. It
DNA is unwound to form 2 single strands. is directly proportional to GC%, inversely
This is accomolished by breaking hydro- proportional to AT%. It is also directly pro-
gen bonds. Hydrogen bonds are broken portional to salt concentration
at higher temperatures. » Formamide can bring about denaturation
» Tm or melting temperature is the temper- by removing the salt concentration
ature at which 50% dsDNA undergoes
32 >>>
CELL CYCLE
» The sequence of phases of cell cycle is chromosomes should be mediated, for
G0--> G1 →S → G2 → M. In M phase, one which the chromosome has to be uncon-
cell divides into two. The resultant daugh- densed.
ter cells either get ready and get into the » In S phase or synthetic phase, replication
next cell cycle ot they enter into a resting happens and hence the chromosomes
phase should be highly condensed. So in G1, S
» In G0 or resting phase, neither replication and G2 phases otherwise called as Inter-
nor transcription occurs. Hence to ena- phase, chromosomes should be uncon-
ble package, the chromosome is highly densed.
condensed in G0 phase. » Though chromosomes are condensed
» In M phase, there is a phase called as with the help of histones, as histones get
metaphase, where in mitotic spindle is at- acetylated in the nucleus, they tend to
tached to the centromere and when the uncondense. They are retained in a con-
mitotic spindle contracts the centromere densed manner in G0 and M phase with
breaks. For chromosomal breakage and the help of retinoblastoma protein and
segregation to be mediated, the chromo- histone deacetylase.
some has to be highly condensed in M » The transition from condensed form of
phase chromosome in G0 phase to uncon-
» In G1 phase or in presynthetic phase densed form of chromosome in M phase
and in G2 or postsynthetic or premitot- is done by Cyclins.
ic phase, proof reading and repair of
33
MCQS
1. All of the following are purine bases 5. The linkage present between individual
except nucleotides in a polynucleotide chain is
A. Adenine A. 3’5’ phosphodiester linkage

B. Uric acid B. β N glycosidic linkage
C. Hypoxanthine C. Phosphoester linkage
D. Uracil D. 5’3’ phosphodiester linkage
2. The linkage present in a nucleoside is 6. Regarding DNA structure true is,

A. α N glycosidic linkage A. The double helical structure is stabilised

B. β N glycosidic linkage by covalent bonds
C. Phosphoester linkage B. The individual strands are stabilized by
D. Acid anhydride linkage 5’3’ phosphodiester linkage
3. The linkage present in a monophos-

C. The individual strands are stabilized by
3’5’ phosphodiester linkage
phate nucleotide is D. The term 5’ end indicates that the 5’ end
is linked to kinetochore.
7. The histone that is not present is a nu-

A. α N glycosidic linkage
B. β N glycosidic linkage
C. Phosphoester linkage cleosome is
D. Acid anhydride linkage
4. The linkage present between

A. H1
B. H2A
phosphate groups in a triphosphate C. H2B
nucleotide is D. H3
A. α N glycosidic linkage 8. Barr body is an example of

B. β N glycosidic linkage
C. Phosphoester linkage A. Euchromatin
D. Acid anhydride linkage B. Constitutive heteochromatin
C. Facultative heterochromatin
D. Hypersensitive heterochromatin
34 >>>
9. The most common form of DNA is, 13. Replication occurs in

A. B DNA A. Go Phase
B. Z DNA B. G1 Phase
C. A DNA C. S Phase
D. E DNA D. M phase
10. The features of B DNA include, all 14. The phase in which chromosomes
except are uncondensed is
A. It is a right handed helix A. Go Phase

B. One full turn of DNA has 10 nuceotides B. G1 Phase
and measure 34A0, width 20A0 C. Metaphase
C. Major groove is equal to minor groove in D. M phase
15. Which of the following is true?

terms of width
D. It is the form that is present under physio-
logical conditions.
11. The left handed helix is seen in,

A. To break AT bonds, high Tm is required
B. To break GC bonds, low Tm is required
C. To break AT bonds, low Tm is required
A. DNA D. AT bonds and GC bonds need the same
B. Z DNA Tm
C. A DNA
D. E DNA
12. Denaturation of DNA is done by all

1.D;2.B;3.C; 4.D; 5.A; 6.C; 7.A; 8.D; 9.A; 10.C; 11.B; 12.B; 13.C; 14.B; 15.C;
except,
A. Increasing the temperature

B. Increasing the salt concentration
C. Decreasing the salt concentration
D. Formamide
35
HUMAN GENOMIC
SEQUENCES
FACTS ABOUT HUMAN GENOMIC SEQUENCES
» Number of bp per human haploid ge- » Largest gene is RBFox1
nome is 3.3 X109 » Largest protein is Titin
» Total number of genes identified 20 to » Gene with the largest exon is Titin
25000 » Gene with maximum number of exons is
» Average size of a gene is 27000bp Titin
» Average size of mRNA 2000 nucleotides
TYPES OF HUMAN GENOMIC SEQUENCES

Three types • CLASS II – 45% -TRANS- » SINES or Short Inter-
POSONS OR JUMPING spersed sequences (100
• CLASS I – 30% - GENE GENES OR MODERATELY to 200 bp in length)
SEQUENCES REPETITIVE SEQUENCES
» 1.5% are coding exons (repeated between 1000 • CLASS III – 25% - MIS-
» 28.5% are non coding and lakh times) CELLANEOUS SEQUENC-
intervening sequences or » LINES or Long Inter- ES
introns spersed Sequences (6 to » 3% is called as satellite
8 KB in length) DNA
HUMAN GENOME
SEQUENCES
GENE TRANSPOSONS MISCELLENEOUS
EXONS INTRONS HIV

LINES SINES SATELLITE LSD ???
like
36 >>>
APPLICATIONS OF SATELLITE DNA

» It has Forensic application. It can be used father, to prove that X is the biological
to clear off paternal dispute cases. father of the child, satellite DNA or VNTR
» Suppose there is a mother with a child analysis is used.
and the mother claims person X as a
STEPS INVOLVED IN PATERNAL DISPUTE

CASES
» Blood sample is collected from mother, times in the child’s DNA and it is repeated
child and the suspected father 11/8 in the mother, the inference is that
» DNA is extracted from all 3 blood sam- one of the 8s of the child has come from
ples the mother, hence it is imperative that
» In all the three DNA samples, we analyse the other 8 comes from the father. If the
the inheritance of atlease 5 microsatellite father’s DNA shows a repeat other than
repeats at 5 loci each 8, then the conclusion is that this person
» Suppose at a particular locus, a specif- can not be the father of the child!
ic microsatellite repeat is repeated 8/8
CHILD’S DNA – 8/8
MOTHER’S DNA – 11/8 FATHER’S DNA 11/10
NOT THE BIOLOGICAL FATHER
I. Alu family belongs to, II A paternal dispute case was filed by a

mother. The forensic centre checked 5 VNTR
A. Unique non repetitive sequence systems. Which of the following repeats
B. Moderately repetitive LINE sequence should be observed in the putative father’s
C. Moderately repetitive SINE sequence DNA for him to be considered as the biologi-
D. Highly repetitive sequence cal father of the child?
INDIVIDUAL VNTR1 VNTR2 VNTR3 VNTR4 VNTR5

CHILD 9/10 8/11 10/12 9/8 6/7
MOTHER 7/9 8/9 11/10 9/11 6/9
A. 7/8, 9/11, 11/12, 8/10,6/8

B. b. 7/8,9/11,10/12, 8/10, 6/8
C. c. 10/11, 11/10, 12/11, 8/7, 7/9
I.C; II.C
D. d. 10/11,11/10,11/10, 11/9, 9/10

37
REPLICATION
INTRODUCTION
» Replication is a process by which we syn- » REQUIREMENTS OF DNA POLYMERASES:
thesise two ds DNA from a single dsDNA • Template strand in 3’5’ direction
» It is a semiconservative process, as in • Primers
the daughter DNA, only one of the two • All 4 deoxynucleotide triphosphates
strands is new and the other one is old • Magnesium or Manganese to act as a
and is partially conserved. catalyst

» It is a bidirectional process – During repli- • Buffer to maintain the pH
cation, a replication bubble is formed and » All DNA polymerases have proof reading
all enzymes start acting from the centre. and repair activity. To enable proof read-
From the centre, one set of enzymes ing and repair activity, they are all pro-
act in one direction and the other set of vided with 3’5’ exonuclease activity. DNA
enzymes act in the other direction polymerase I has both 3’5’ exonuclease
» Replication is done by DNA polymerases. activity and 5’3’ exonuclease activity
All DNA polymerases have 5’3’ polymer-
ase activity
REPLICATION – SEMICONSERVATIVE PROCESS
38 >>>
REPLICATION - BIDIRECTIONAL
DNA POLYMERASES
TEMPLATE STRAND
STEPS OF REPLICATION
» Replication is initiated by identification of initiated, as the two strands have comple-
Ori. Ori or Origin of replication is a seg- mentary bases, they have a tendency to
ment of the genome, which is rich in AT reanneal. Single stranded Binding pro-
sequences. Along AT sequences, mediat- teins bind to both the strands and that
ing unwinding is easier. prevent them from reannealing. This is
» Ori Binding proteins bind to Ori and that how a replication bubble is formed
initiates unwinding. Though unwinding is
39
» All enzymes act from the centre of the » Primase of the replication fork synthe-
replication bubble. It is a bidirectional sizes primer . DNA polymerase III syn-
process and hence two sets of enzyme thesizes DNA strand. DNA polymerase
act from the centre. The strand in3’5’ III synthesizes the DNA strand, until it
direction from the centre is treated as comes across an RNA primer, which was
leading. DNA polymerase III synthesizes synthesized by the previous replication
the new strand continuously against the fork. DNA polymerase III leaves the loca-
leading strand tion recruiting DNA polymerase I. DNA
» The other strand in 5’3’ direction from polymerase I removes the RNA primer
the centre is treated as lagging strand. as it has 5’3’ exonuclease activity. DNA
Against lagging strand, new strands can polymerase I fills the gap by extending
be synthesized only discontinuously from the previous strand. DNA ligase unites
the angle of separation. To reach the an- the ends.
gle of separation, helicase and primase, » The short fragments with RNA primers
join to form primosome and move along attached to DNA are called as Okazaki
the strand. In the angle of separation, hel- fragments. Okazaki fragments are syn-
icase, primase, DNA polymerase III and thesized by DNA polymerase III. They are
single stranded binding proteins form the joined together by DNA ligases
replication fork.
40 >>>
POINTS TO REMEMBER ABOUT OKAZAKI

FRAGMENTS
» RNA primers attached to DNA are called » They are synthesized by DNA polymerase III
as Okazaki fragments » They are joined together by DNA ligases
» They are synthesized against lagging
strands
FUNCTIONS OF PROKARYOTIC DNA

POLYMERASES
S.No DNA POLYMERASE FUNCTIONS
1 DNA Polymerase I
2 DNA Polymerase II
3 DNA Polymerase III
1 DNA Polymerase I Removes RNA primer and fills the gap

during lagging strand synthesis
2 DNA Polymerase II Proof reading and repair
3 DNA Polymerase III Leading strand synthesis and Okazaki

fragment synthesis
41
DIFFERENCES BETWEEN PROKARYOTIC &

EUKARYOTIC GENOME
S.No PROKARYOTIC GENOME EUKARYOTIC GENOME
2
S.No PROKARYOTIC GENOME EUKARYOTIC GENOME
1 Smaller Larger
Multiple Ori s
2 Closed circular Open linear

Telomeric end shortening
TELOMERIC END SHORTENING
42 >>>
» Following replication, in both the daugh- » Telomeric end shortening beyond a criti-
ter DNAs, in both the ends, one of the cal length causes neoplasia
two strands is replaced by RNA, which is » Telomeric end shortening is avoided by
lost. This causes telomeric end shorten- Telomerase
ing » Telomerase is a reverse transcriptase
» A minimal length of telomere is necessary » Increased telomerase activity causes
to maintain the stability of a chromosome neoplasia
» Telomeric end shortening causes aging
and death
FUNCTIONS OF EUKARYOTIC DNA

POLYMERASES
1 DNA Polymerase α
2 DNA Polymerase β
3 DNA polymerase ε
4 DNA polymerase γ
5 DNA Polymerase δ
1 DNA Polymerase α RNA Primas
2 DNA Polymerase β Proof reading and repair
3 DNA polymerase ε Leading strand synthesis - Initiation
4 DNA polymerase γ Mitochondrial DNA synthesis
5 DNA Polymerase δ Completion of leading, Okazaki fragment synthesis,

Removes RNA primer and fills the gap during lagging
strand synthesis
43
MCQS
1. Which of the following is true about 5. Function of DNA polymerase δ,
replication?
A. Gap filling
A. Conservative process B. Leading strand synthesis
B. Bidirectional C. Okazaki fragments synthesis
C. Non conservation D. RNA primase
6. DNA polymerase with repair mechanism is:

D. Unidirectional
2. DNA polymerase requires all except,

A. DNA polymerase I
A. RNA primer B. DNA polymerase II
B. 3’ to 5’ strand to act as a template C. DNA polymerase III
C. d NTP D. DNA polymerase α
7. True about telomerase are all except:

D. 5’ to 3’ strand as a template
3. Replication fork includes all except,

•
a. Helicase A. They are present at the ends of eukaryot-

ic chromosome
A. Helicase B. Increased telomerase activity is associat-
B. Primase ed with malignancy
C. DNA polymerase III C. DNA dependent RNA polymerase
D. DNA polymerase I. D. DNA polymerase
4. Replication along lagging strand, true

is,
1.A; 2. 2.D; 3.D; 4.B; 5.D; 6.B; 7.C;
A. Polymerase I synthesizes along 3’ to 5’

direction
B. Helicase and primase join to form primo-
some and moves along the lagging strand
C. Okazaki fragments are joined together by
DNA helicase
D. DNA polymerase III removes RNA primer
44 >>>
ERRORS OF REPLICATION &

DNA REPAIR MECHANISMS
ERRORS OF DNA
The four types of errors of DNA are • Mismatch Error: This is the most com-
» Base Excision error mon error that happens during replica-
» Mismatch error tion as DNA polymerases are not error
» Pyrimidine Pyrimidine dimer proof
» Ds DNA break error • Pyrimidine Pyrimidine Dimer: UV
light has a propensity to cause adjacent
• Base Excision Error: The most com- pyrimdines to dimerise.
mon error of DNA is base excision, be- • Ds DNA break: Breaking. The 3’5’ phos-
cause the β N glycosidic linkage is ther- phodiester linkages by hydrolysis can
molabile and bases get lost cause ds DNA break. This occurs in the
presence of ionising radiation
FOUR REPAIR MECHANISM

1. Base Excision Repair : This is mediated ase makes a nick at the nearest GATC se-
by apurinic or apyrimidinic endonucle- quence. The exposed end is removed by
ase. This enzyme makes a nick at a site DNA polymerase epsilon or beta and they
close to where a base has been lost. This use their polymerase activity to substi-
creates an end. DNA polymerase epsilon tute a new strand. A defect of mismatch
or beta use their exonuclease activity to repair causes Hereditary Non Polyposis
remove the defective strand and they Colon Cancer or Lynch Syndrome. The
use their polymerase activity to substi- most common mutation causative of HN-
tute a new strand PCC is human Mutant S Homology 2.
2. Mismatch repair: The effectiveness 3. Nucleotide Excision repair: This is
of the repair mechanism depends upon brought out whenever UV light causes
how efficiently the defective strand is Pyrimidine pyrimidine dimerization.. The
identified. DNA methylases methylate the repair is initiated by a helicase, which
normal strand, so that the other strand is removes hydrogen bond in the adjacent
identified as defective. GATC endonucle- regions. UV specific endonuclease or
45
excinuclease makes two nicks close to mechanism than HDR, however this
where the defect has happened and it causes loss of gene sequences.
excises a nucleotide away and hence the » A defect of this repair mechanism
name. DNA polymerase epsilon or beta causes Ataxia Telangiectase, Bloom
use their polymerase activity to substi- Syndrome and Severe Combined Im-
tute a new strand and ligase unites the munodeficiency
ends. A defect of replication coupled NER • Homologous DNA repair (HDR):
cause Xeroderma Pigmentosa and a de-
» This is done with the help of homol-
fect of transcription coupled NER causes
gous DNA sequences. DNA polymer-
Cockayne syndrome
ase epsilon or beta removes a part
4. Ds DNA break repair: This is done by of one of the two strands, so that it
two ways: creates an overhanging edge in the
• Non Homologous End Joining (NHEJ): other strand. The overhanging edge
» This is done by Ku helicase, which is has higher strand invasion ability and
a dimer. Each of the two units bind to it invades the normal homologous
both the ends and they cause un- sequence, which is used as a template
winding and approximation of both and the break is repaired.

the ends. Approximation continues » A defect of homologous DNA repair
until they find base pairing. Excess causes Fanconi’s anemia and Human
strands ger removed and ligase unites Breast and Ovarian Cancer.
the ends. This is more common repair
MISMATCH ERROR
DEFECTIVE
STRAND
HNPCC
GATC
ENDONUCLEASE
CH3 CH3 CH3
hMSH2 DNA
POLYMERASE ε
&β
DNA LIGASE
46 >>>
UV MEDIATED DAMAGE
XERODERMA HELICASE
PIGMENTOSA
UV SPECIFIC
COCKAYNE ENDONUCLEASE
OR
SYNDROME
EXCINUCLEASE
TRANSCRIPTION DNA
COUPLED NER POLYMERASE
DEFECT ε/ β
NHEJ - Ku HELICASE
KU HELICASE
NHEJ RESULTS IN
LOSS OF GENE
SEQUENCES
47
HOMOLOGOUS DNA REPAIR

DEFECTS OF ds DNA BREAK REPAIR

» ATAXIA TELANGIECTASIA
NHEJ SCID
» BLOOM’S SYNDROME
» FANCONI’S ANEMIA
HDR
» HBOC (Human Breast and Ovarian Cancer)
MCQS MUST KNOW

MCQ
1. The most com- 2. Mismatch repair 3. UV light damage 4. Double

mon error in a DNA is defect causes , to the DNA leads to: stranded DNA break
repair defect causes
A. Base Excision A. HNPCC A. Purine dimers all except
B. Pyrimidine dimer B. Xeroderma pig- formed
C. Mismatch mentosa B. DNA hydrolysis A. Hereditary Non
D. Ds DNA break C. Fanconi’s anemia occurs polyposis Colon
D. Ataxia Telangiec- C. Specific endonu- Cancer
1.A; 2. A; 3.C; 4.A.
tasia clease recognises B. Ataxia Telangiec-

the damage tasia
D. Double stranded C. Bloom’s syndrome
DNA break occurs. D. Fanconi’s anemia
48 >>>
RNA, TRANSCRIPTION
AND POST
TRANSCRIPTIONAL
MODIFICATIONS
TYPES OF RNA
• rRNA • mRNA • tRNA • sRNA
>> rRNA
• Four types – 5,5.8,18 and 28srRNA
• 18srRNA is associated with 40s ribo-
some
• A common gene codes for all the rRNAs.
This gene on transcription provides a
larger primary transcript, which is 45sr-
RNA. This during post transcriptional
modifications will be cleaved to provide
all type of rRNAs except 5srRNA. 5srRNA
is transcribed separately from a different
gene.
• 28srRNA is a ribozyme. The enzymatic
activity it exhibits is peptidyl transferase.
Peptidyl transferase shifts the growing
polypeptide chain from P site to A site tRNA - STRUCTURE
and it forms a peptide linkage
49
FACTS ABOUT tRNA
• tRNA or transfer RNAs are so called » Acceptor arm accepts the aminoac-
because they transfer aminoacids from id. Acceptor arm has a triplet nucleo-
aminoacid pool to ribosome, the trans- tide CCA attached to the 3’ end
lation machinery. » Tψ C arm is for ribosomal attachment
• To code for 21 aminoacids, we have 50 » D arm is for the attachment of ami-
to 100 tRNAs. Each of these are coded noacyl tRNA synthetase
by a unique gene
• Function of aminoacyl tRNA synthetase
• Every tRNA is clover leaf shaped be- » It picks up the specific aminoacid,
cause it has four stable arms and one
activates the aminoacid using two high
variable arm
energy phosphates and attaches the
• The four stable arms of tRNA are: activated aminoacid to itself forming
» Anticodon arm has an anticodon aminoacyl tRNA
that is complementary to the codon of » Fidelity of gene is conferred by ami-
mRNA noacyl tRNA synthetase
>> snRNA
» Six types – U1 to U7 except U3 » U7 helps in stem loop structure attach-

» All except U7 help in splicing. So snR- ment of histone mRNA
NAs are examples of ribozymes
>> siRNA
» Small interference RNAs mediate tween, they recruit RNA trsnacription

interference of gene expression at the silencing complex. One of the proteins
translation level. These siRNAs have of this complex is an endonuclease,
their own sequences and if they find which makes multiple nicks on the
complementary sequences on any mRNA. This mRNA is not translated
of the fully formed mRNAs, they hy- » Interference phenomenon acts as one
bridise with those mRNAs and form a of the mechanisms of regulation of
double stranded RNA structure. When gene expression
ribosome translates the mRNA with a » As it down regulates gene expression,
double stranded RNA structure in be- it causes “gene knock down”
50 >>>
MCQS
1. m RNA is characterized by, all except 5. Which of the following is the functions
of D arm of tRNA
A. The nucleotide bases of mRNA are
grouped in three to form a codon. A. Ribosomal attachment
B. Mature mRNA has a 7-methyl guanosine B. Attachment of aatRNA synthetase
cap and poly A tail C. Aminoacid attachment
C. hn RNA is the form that is present in cyto- D. Anticodon arm
6. The function of Sn RNA is

plasm
D. hn RNA has introns in it
2. Fidelity of gene is conferred by, A. Formation of hn RNA

B. Formation of t RNA
A. r RNA C. Splicing
B. m RNA D. Formation of ribosomes.
7. siRNA causes
C. t RNA
D. Sn RNA
3. Fidelity of gene is conferred by, A. Gene knock down

B. Gene knock in
A. r RNA C. Gene Knock out
B. aatRNA synthetase D. Gene conversion
C. t RNA .
D. Sn RNA
4. The amino acid is attached to which

.
arm of tRNA
A. D arm
B. T ψ C arm
C. Acceptor arm
1.C; 2.C; 3.B; 4.C; 5.B; 6.C; 7.A.
D. Anticodon arm
51
TRANSCRIPTION
INTRODUCTION
» Transcription is a process by which a sin- acts as a template, based on which we
gle stranded primary transcript or hetero- synthesise a new RNA which is comple-
nuclear RNA is synthesized from gene or mentary and antiparallel. This 3’5’ strand
transcription unit is called as a template strand.
» In both replication and transcription, the » The other strand in 5’3’ direction, which
parent ds DNA unwinds but in replication, we get when we unwind the parent dsD-
each of the two strands acts as a tem- NA or gene in this case, does not take
plate, based on which we synthesise two part in transcription. Though it does not
new strands. In transcription, after un- take part in transcription, we call it as a
winding only that strand in 3’5’ direction coding strand.
FINDING OUT RNA SEQUENCES

» If template strand is provided, to find the polarity of the strand is not specified, it
RNA sequences, the following steps need is considered as a 5’3 strand
to be followed » If the coding strand is provided, to find
• Write the template strand sequence in the RNA sequences, retain the polarity,
3’5’ direction and write complemen- retain the sequence, just replace T by U
tary sequences, mark it as 5’3’. If the
DIFFERENCES BETWEEN DNA & RNA

POLYMERASES
» RNA polymerases do not need a primer
» RNA polymerases need NTP
» RNA polymerases do not have proof read-
ing and repair activity
» RNA polymerase I – rRNA except 5srRNA
» RNA polymerase II – mRNA & miRNA
» RNA Polymerase III – tRNA, snRNA, 5srRNA
52 >>>
TRANSCRIPTION CONTROL REGIONS

>> All gene sequences are denoted as • Fidelity promotors – Fidelity promotors
coding strand sequences. In a coding strand, fix the +1 site to RNA polynerases. Eg.,
the first nucleotide of the coding sequence TATA box, Inr, DPE
is marked as +1, and anything downstream • Frequency promotors – Frequency
from there (towards the 3’ end) will be promotors control the number of time
marked as + and anything upstream (towards the given gene will be transcribed un-
the 5’ end) is marked as -. Note: For any der basal conditions eg., GC box and
gene +1 +2 +3 are ATG, as AUG is the initia- CAAT box
tion codon » Regulated expression control regions or
>> Transcription control regions are se- regulators – they control regulated ex-
quences which are present either within the pression. They are of two types
gene or outside the gene and they help in • Inducers or Enhancers – sequences
controlling transcription. They are of two which increase the number of times
types the given gene is transcribed
» Basal Expression control regions or pro- • Repressors – sequences which de-
motors – they control basal expression. crease the number of times the given
They are of two types gene has to be transcribed
TRANSCRIPTION CONTROL REGIONS
PROMOTORS REGULATORS
FIDELITY FREQUENCY REPRESSORS INDUCERS
DIFFERENCES BETWEEN PROMOTOR AND REGULATORS
S.No PROMOTOR REGULATOR
53
S.No PROMOTOR REGULATOR
1 Sequence specific- TATA Box is No sequence specificity

active in human genome only when
it is TATAAAG
2 Position specific – TATAAAG No position specificity. A regulator can

sequence is to be present at -25th act as a regulator even if it is separated
position in the coding strand for it to by a few 100 nucleotides from the coding
be active sequence
3 Orientation specific – TATAAAG is No orientation specificity

to be read that way when it is read
from the 5’ end to the 3’ end in the
coding strand
4 Highly conserved, to some extent Not conserved

species specific
5 Not gene specific Tissue specific
RNA EDITING
» It is one of the post transcriptional modifi- of the protein formed gets altered in some
cations. Here we edit some of the nucleo- tissues. This explains tissue specific regula-
tide sequences of the fully formed mRNA tion of gene expression
in specific tissues, so that the aminoacids » Example is Apo B48 formation
Apo B48 formation

» Apo B48 is an apoprotein of chylomicron, hepatocyte. But both are products of the
which is formed in enterocyte. Apo B100 is same Apo B gene.
an apoprotein of VLDL, which is formed in
54 >>>
» In both enterocytes and hepatocytes, the there is premature termination of protein

apo B gene undergoes the same transcrip- synthesis in enterocytes and a truncated
tion and the same set of modifications until protein is produced which has only 48% of
they all reach the cytoplasm. the aminoacids found in the whole protein
» In the cytoplasm of enterocyte, Cytidine of hepatocytes. Hence the whole protein
deaminase deaminates cytidine of CAA is called as apo B100 and the truncated
to form UAA, which is a stop codon, so protein is called as apo B 48
MCQS
1. Template strand is 5’ 3. A person admitted with 5. True regarding regula-
CGTTATTTACTA3’. If this is the complaints of nausea and tors is,
transcribed by RNA polymer- vomiting following intake of
ase, the sequence of the new mushroom in a party an hour A. They are required for ba-
RNA will be before. A sample of mush- sal expression
room was obtained an it was B. They are orientation specific
A. 5’GCAATAAATGAT3’ found to be contaminated C. They should be located
B. 5’GCAAUAAAUGAU3’ with amanita phalloides. The within a particular dis-
C. 5’UAGUAAAUAACG3’ RNA polymerase type tance, to be active on a
D. 5’CGUUAUUUACUA3’ inhibited by this mushroom, particular gene
2. Regarding transcription
which is responsible for these D. They are cell specific
6. Posttranscriptional
symptoms is,
true is,
A. RNA polymerase I modification of mRNA include
A. The two strands of DNA B. RNA polymerase II all except
are transcribed stimulta- C. RNA polymerase III
neously D. RNA polymerase IV A. 7- methyl guanosine cap-
4. True regarding
B. RNA primer is first formed . ping
C. RNA polymerase needs B. Poly A tail
the template strand in 5’ promoter is, C. Complete removal of
to 3’ direction introns
D. RNA polymerase synthesis A. It is gene specific D. RNA editing
7.
the primary transcript in 5’ B. It is orientation non-specific
to 3’ direction C. It is highly conserved spe- apo B 48 formation is an
cies specific example of
1.C;2.D; 3.B; 4.C5.D; 6.C; 7.B
D. TATA Box is the only pro-

moter so far discovered A. Truncation of protein
B. RNA editing
C. Rearrangement of glyco-
sidic bond
D. Modification by methyla-
tion
55
GENETIC CODE
PROPERTIES OF GENETIC CODE
» Degeneracy: More than one codon » Unambiguous: Not more than one ami-
can code for a single nucleotide. This is noacids can be coded by a single codon.
called as degeneracy. Exceptions methio- » Non overlapping: Ribosomes while
nine and Tryptophan. This is explained reading the mRNA do not overlap one
by wobble phenomenon. According to codon with another codon.
wobble phenomenon, there is a reduced » Not punctuated: There is no punctua-

stringency when the 3rd nucleotide pair tion after every 3 nucleotides. This is the
is formed between codon and anticodon explanation for frameshift mutation
or the 3rd nucleotide of codon of mRNA
» Universal: The same codon is applicable
is not specific or the 1st nucleotide of
for all the cells. Exception is mitochondri-
anticodon of tRNA is not specific
al DNA.
TYPES OF MUTATION
» Mutation is classified at three levels: » Insertion
» Based on its effect on nucleotide » Deletion
sequence » Based on its effect on aminoacid
• Point mutation or substitution sequence
» Transition – If a purine is replaced by a • Silent: Because of a mutation, if a
purine or a pyrimidine is replaced by a codon coding for an aminoacid is
pyrimidine, it is called as transition replaced by another codon coding
» Transversion – If a purine is replaced for the same aminoacid, it is called as
by a pyrimidine or viceversa, it is silent mutation
called as transversion • Missense : Because of a mutation, if
• Frame shift mutation a codon coding for an aminoacid is
replaced by a codon coding for an-
» Whenever there is a loss or a gain of
other aminoacid, and if the aminoacid
nucleotides, the entire frame of read-
sequence is changed, it is called as
ing gets shifted from the place, that is
missense mutation
called as frameshift mutation. It is of
two types
56 >>>
• Nonsense: Because of a mutation if bin Bristol, if it is replaced by Glutamic

a codon coding for an aminoacid is acid, it is called as Hb Milwauke, if it is
replaced by a stop codon and if there replaced by Alanine, it is called as Hb
is a premature termination of protein Sydney. In all these cases, oxygen car-
synthesis and if a truncated protein rying capacity of Hb is normal. Hence
is produced, it is called as non sense they are examples of acceptable muta-
mutation tion
» Based on its effect on the function • Partially acceptable: If there is a
of protein change in nucleotide sequence, and
• Acceptable: If there is a change in the if there is a change in the aminoacid
nucleotide sequence, and if there is a sequence and if the function of the
change in the aminoacid sequence and protein is affected but if its not life
if the function of protein is unaffected, threatening, it is called as partially ac-
it. Is called as acceptable mutation. ceptable mutation. Eg., HbS
Eg., Hemoglobin Milwauke, Hemoglobin • Unacceptable: If there is a change in
Bristol and Hemoglobin Sydney. Usual- nucleotide sequence, and if there is a
ly the 67th aminoacid of beta glo0bin change in the aminoacid sequence and
chain is valine, and if that is replaced if the function of the protein is affected
by Aspartic acid, it is called Hemoglo- but if its life threatening, it is called as
partially acceptable mutation. Eg., HbM
MCQS
1. No of possible 3. Codon consists 5. All the following 7. Wobble phenom-
codons of: are properties of ge- enon explains which
A. 3 base pairs netic code except, of the following,
A. 64 B. 2 base pairs
B. 61 C. 5 base pairs A. Degenerate A. Degeneracy
C. 20 D. 3 nucleotides B. Ambiguous B. Unambiguity
4. Stop codon
D. 31 . C. Nonoverlapping C. Ambiguity
2. No of codons
D. Universal D. Punctuation
which code for an A. UAG 6. The amino acid

aminoacid B. UCA which does not follow
1.A; 2;B; 3.D; 4.A; 5.B; 6.C; 7.A.
C. UAC degeneracy of codon

A. 64 D. AUG is,
B. 61
C. 20 A. Glycine
D. 31 B. Glutamine
C. Tryptophan
D. Tyrosine.
57
TRANSLATION
STEPS OF TRANSLATION
» Translation is a process by which nucle- » It involves many translation factors, which
otide sequences of mRNA get translated are named as eIF or eEF depending upon
as aminoacid sequences of a polypeptide whether they are a part of initiation phase
chain. It is done by ribosomes. Eukaryotic or elongation phase
ribosome is an 80 s unit, which is disso-
ciated to form 40s and 60s subunits. 60s

subunit has P site and A site.
INITIATION FACTORS & FUNCTIONS

» eIF
tRNA Methionine » eIF2C

complex formation
Stabilise 40s ribosome » eIF3 & 1A
Guides mRNA cap to » eIF 4G/4A

ribosom
ATP dependent helicase » eIF4A/4B
Removes 3 & 1A » eIF5
STEPS OF INITIATION
» Initial few steps of initiation do not need P » 40s ribosome is converted to 43 s PIC by
and A set attachment of a ternary complex made
» Hence initiation of translation begins with up of the following components
dissociation of 80s into 40 and 60s. But • imet tRNA
they have a tendency to reassociate, to • eIF2C
prevent which 40s has to be stabilised • GTP
» 40s ribosome is stabilised by eIF3 and 1A
58 >>>
» mRNA cap is guided by eIF4G/4A » Now the mettRNA and AUG should be at-
» Hairpin loops of mRNA are removed by tached to P site, which is a component of
the ATP dependent helicase eIF4A/4B 60s, which is kept aside. To reassociate
» imettRNA identifies the initiation AUG 60s back, 3 and 1A have to be removed
codon with the help of shine Dalgarno » eIF5 removes eIF3 and eIF1A and then
sequence in prokaryotes or Kozak Con- 60 s is reassociated with 40s
sensus sequences in eukaryotes » imettRNA is attached to P site of 60s
» In Kozak consensus sequences -3 and +4 ribosome
should be purines » This is the end of initiation!!
STEPS OF ELONGATION
» Corresponding to A site, there is a codon is already activated by aminoacyl tRNA
on the mRNA, depending upon which a synthetase
complementary anticodon containing » EF2 with the help of GTP translocates
tRNA is recruited, which comes with an the ribosome ahead and hence now the
aminoacid. polypeptide chain is on the P site and A
» Peptidyl transferase shifts the growing site is free
polypeptide chain from P site to A site, » Thus 4 high energy phosphates are re-
and forms a peptide linkage. This step quired for attaching every aminoacid to a
does not need energy, as the aminoacid growing polypeptide chain
STEPS OF TERMINATION
» Ribosome keeps moving ahead on the » This recruits a Releasing factor, which uses
mRNA until it meets a stop codon on the one GTP to release the polypeptide chain
mRNA attached to the last tRNA on the P site
ENERGETICS OF TRANSLATION
S.No STEP Number of ATPs
59
S.No STEP Number of ATPs

1 Activation of aminoacid 2
2 Attachment of aatRNA to A site 1
3 Translocation step 1
TOTAL: 4
DIFFERENCES BETWEEN imet tRNA & OTHER tRNAs
S.No imet tRNA aa tRNA

1
S.No imet tRNA aa tRNA

1 Guided by eIF2C Guided by eEF1A
2 Attached to P site of 60 s ribosome Attached to A site of 60 s

ribosome
3 3 high energy phosphates are required 4 high energy phosphates

are required
60 >>>
TRANSLATION
INHIBITORS
INHIBITORS LIST
S.No INHIBITOR MECHANISM OF ACTION
61
S.No INHIBITOR MECHANISM OF ACTION
1 Aminoglycosides All steps – freeze initiation, inhibit

polysome formation
2 Tetracycline EF1A – inhibits attachment of aminoacyl

tRNA to A site
3 Chloramphenicol 23srRNA or prokaryotic peptidyl

transferase
4 Cycloheximide & Ricin Eukaryotic peptidyl transferase
5 Macrolide and clindamycin Prokaryotic translocation
6 Diphtheria and Pseudomonas toxin Eukaryotic translocation

7 Puromycin aatRNA analogue causes premature

termination of protein synthesis
INHIBITORS LIST - MNEMONIC
» BUY AT 30 AND CCEL AT 50 • Chloramphenicol
• Aminoglycosides • Clindamycin
• Tetracycline • Erythromycin
• Linezolid
MCQS
1. P and A sites are compo- 2. The function of shine 3. The translation factor
nents of Dalgarno sequence is to which guides aatRNA to A site
is:
A. 80s A. Identify the termination A. eIF1A
B. 40s signal B. eEF4A
C. 60s B. Help in guiding mRNA to C. eEF1A
D. 30s ribosome D. eIF3
C. Identify initiation codon .
D. Dissociate ribosome
62 >>>
4. The total number of

8.
A. Peptide bond formation
ATPs required for attaching Peptidyl transferase B. Termination
every aminoacid to a growing true are all except C. Translocation
polypeptide chain is D. Initiation
13. In Iron deficiency

A. It is a protein
A. 1 B. It is an RNA
B. 2 C. It does not require high anemia,
C. 3 energy phosphate
D. 4 D. It is inhibited by chlor- A. Iron binds to Iron re-
5.
maphenicol sponse element and does
9.
The total number of not cause translation of
ATPs required for forming Tetracycline inhibits sTfR
a polypeptide chain with n B. Iron does not bind to Iron
aminoacids is A. IF1A response element and
B. EF1A hence there is no transla-
A. 4n-1 C. IF2C tion of sTfR
B. 4n+1 D. EF2C C. Iron binds to Iron re-
10.
C. 3n sponse element and
D. 4n Ricin inhibits causes translation of sTfR
6. mettRNA is attached to
D. Iron does not bind to Iron
A. aatRNA synthetase response element and
1.C; 2.C; 3.C;4.D; 5.D; 6.C; 7.C; 8.A; 9.B; 10.B;11.D; 12.C13.D.
B. Peptidyl transferase hence there is translation
A. P site of 40s ribosome C. Termination of sTfR
B. A site of 40s ribosome D. Initiation
11. Diphtheria toxin inhib-

C. P site of 60s ribosome
D. A site of 60s ribosome
7. The step which does not

its
require high energy phos- A. IF1A

phate is, B. EF1A
C. IF2C
A. met tRNA synthesis D. EF2
B. Aminoacyl tRNA synthesis
C. Peptide bond formation
12. Macrolide inhibitss

D. Termination
REGULATION OF TRANSLATION OF IRON RELATED PROTEINS
• L ferritin, H ferritin, ALAS2 +

• DMT1, stfR - IRON
AVAILABILITY
63
• IRE • Iron binding to IRE increases translation

• Stem loop structures of mRNAs if IRE is present in the 5’ end
• UTR • Iron binding to IRE decreases translation
• 5’ end of L ferritin, H ferritin, ALAS2 of mRNAs if IRE is present in the 3’ end
• 3’ end of DMT1, stfR
CASE BASED MCQs

1. A 45 year old male is detected to have 2. A 25 year old male presents with a skin
fasting and postprandial infection with yellowish crusts on the face.
hyperglycemia in the diagnostic range of dia- Impetigo was diagnosed and was prescribed
betes. To understand his long term glycemic Mupirocin ointment. Mupirocin acts by
control, he is asked to estimate his HbA1C.
Chromatogram detects an abnormal Hemo- A. Inhibiting the binding of RNA polymerase

globin peak, corresponding to Hb Bristol. to +1 site
His oxygen carrying capacity is normal. Hb B. Causing premature termination of mRNA
Bristol is an example of synthesis
C. Causing premature termination of protein
A. Silent Mutation synthesis
B. Acceptable mutation D. Inhibiting translocation of ribosomes
C. Partially acceptable mutation
D. Non sense mutation
IMAGE BASED MCQs

3. Name X shown in the blue box, given
the clue that it is an antibiotic, which inhibits
the blue circle shown in the image.
A. Puromycin
B. Macrolide
C. Clindamycin
D. Chloramphenicol
1.B; 2.
64 >>>
CLONING
» Cloning is production of identical copies. • DNA polymerases
Here, we are producing identical cop- • Primer
ies of DNA. It has many applications like • All 4 dNTPs
genetic engineering purposes. For exam-
• Magnesium or Manganese
ple to produce Insulin on a large scale,
we need multiple copies of Insulin gene. • Buffer
It has forensic applications too. At the » Depending upon what we depend upon,
crime site, we get a hair follicle or a blood for a supply of all these raw materials,
spot. In either case, we get one or two there are two types of cloning:
copies of DNA. Before performing any • Cell based cloning or recombinant
DNA fingerprinting techniques, we ampli- DNA technology
fy the DNA multiple times. As a process, • Enzyme based cloning or Polymer-
it is replicating DNA multiple times. There ase Chain Reaction
are few requirements for replication:
PCR
PCR is In vitro amplification of a desired » Elongation: This is the step in which
fragment of DNA. We synthesise the desired a thermostable DNA polymerase
fragment of DNA by constructing primers, in Taq DNA polymerase elongates new
such a way that they are complementary to strands. This step is carried out at 720C
flanking sequences of desired fragments • The equipment used for PCR is thermo-
• STEPS OF PCR: cycler.
» Denaturation: This is the step where • Sometimes to avoid using thermocycler,
the parent ds DNA gets unwound to a isothermal amplification technique or
form two strands by breaking hydreogen LAMP assay is used. All steps are carried
bonds. This is carried out at 94 or 950C. out at 600C. The enzyme used for this
» Annealing: In this step we add two prim- purpose is Bst or Bsm DNA polymerase
ers which are complementary to the 3’ – Bacillus stearothermophilus or Bacillus
flanking sequences of both the strands. Smithii DNA Polymerase.
This step is carried out at (Tm-5) 0C
STEPS OF MOLECULAR DIAGNOSTICS OF KNOWN MUTATION SITE
• STEP 1: BLOOD SAMPLE COLLECTION » Centrifuge the blood sample. The

• STEP 2: DNA EXTRACTION sample gets segregated into 3 layers.
The top plasma layer, the middle layer
65
is buffy coat is made up of WBCs and which is complementary to the flank-

the bottom layer is RBCs. For DNA ing sequence of the fragment which
extraction, we need the buffy coat, so has the mutation site. Here, PCR helps
that is transferred to another tube. 6M in only obtaining the segment and is
sodium chloride or Guanidium Isothi- not used for diagnosis directly
ocyanate is used to cause cell lysis • STEP 4: SEQUENCING OR RFLP
and nuclear lysis and then the DNA is » To find out if the mutation site is nor-
released. The extracted DNA has all 46 mal or abnormal sequencing is direct
chromosomes with all 25000 genes. answer but because of availability
For diagnosing a disorder of known issues and cost issues, we use RFLP
mutation site, we need to get a specif-
» RFLP or Restriction Fragment Length
ic fragment of the gene which includes
Polymorphism: This technique uses
the mutation site. This is achieved by
a restriction enzyme which cuts at a
subjecting the extracted DNA to PCR
specific site. Depending upon whether
• STEP 3: PCR the PCR product is cut or uncut after
» The extracted DNA is subjected to incubation with the restriction enzyme,
PCR by using a specific set of primers the diagnosis is done
RT PCR OR REVERSE
TRANSCRIPTION PCR
STEPS INVOLVED IN MOLECULAR DIAGNOSIS OF SARS
COV2 INFECTION
» Step 1: Oropharyngeal and Naso- » Step 4: In the lab, RNA is extracted

pharyngeal Swab sample collection from the sample
» Step 2: The swab sample is transport- » Step 5: The RNA is subjected to Re-
ed in a Viral Transport Medium (VTM), verse Transcriptase –PCR (to convert
which is a Hanks Balanced salt solu- RNA to DNA)
tion, Calcium and Magnesium and heat » Step 6: The DNA is then subjected
inactivated bovine serum albumin, to Real Time PCR or Quantitative PCR
Gentamycin and AmB (semiquantitative in many cases) to
» Step 3: The swab sample in VTM is quantify the nucleic acid of SARS
then transported to the laboratory in CoV2.
cold chain at 2 to 80C
66 >>>
REAL TIME PCR

» Real Time PCR or Quantitative PCR is much, as the number of template DNA
used to quantify nucleic acid in the available initially is less
exponential phase of PCR. For this pur- » LINEAR PHASE
pose, it uses Taq Man Probe » There is a parallel increase in the num-
» BACKGROUND OF PCR KINETICS: ber of copies of DNA as the number
• PCR Kinetics goes through three phases of template DNA and the raw materials
» EXPONENTIAL PHASE OR LAG PHASE: are optimally available
• Initially inspite of there being an in- » PLATEAU PHASE:
crease in the number of cycles, the » There is no further increase in the
number of products do not increase number of copies of DNA as all raw
materials get used up before this
TAQ MAN PROBE AND ITS ROLE IN REAL TIME PCR

A Taq Man Probe has two fluroscent dyes sequence. The oligonucleotide sequence is
connected by an oligonucleotide sequence. complementary to the mid sequences of nu-
One is a reporter dye, the fluroscence of cleotide of interest. In real time PCR, we add
which is captured by the equipment. The Taq Man probe along with the usual require-
other is a quencher dye, which quenches ments of PCR. So the primer binds to flanking
the fluroscence of the reporter dye, as long sequences and the probe binds to the mid
as they are connected by an oligonucleotide sequences, as shown below
67
When Taq polymerase elongates from the reporter dye molecules that get detached
primer, it comes across the oligonucleotide from the quencher dye is directly propor-
sequences of the Taq Man probe and it uses tional to the Taq Polymerase activity, which
its exonuclease activity to remove the oli- in turn is directly proportional to the number
gonucleotide. Now that the reporter dye is of template DNA available. A graph is drawn
detached from the quencher dye, it starts with fluroscent intensity along they axis and
giving fluorescence. The intensity of fluros- number of cycles along the X axis. Ct is the
cence is directly proportional to the number number of cycles at which the fluroscence
of reporter dye molecules that get detached intensity crosses the threshold intensity. Low-
from the quencher dye. The number of er Ct means higher viral load.
ONE LINERS
The instrument used for PCR is Thermocycler
The enzyme used in conventional PCR is Taq DNA polymerase
The enzyme used in LAMP PCR is Bst or Bsm DNA polymerase
Real Time PCR quantifies in the exponential phase of PCR
MCQS
1. SARS CoV2 diagnosis is 2. The temperature of 3. Annealing temperature
done by all except? Denaturation step in PCR is? in PCR is?
A. RT PCR A. 60 degrees A. Variable

B. Real Time PCR B. 72 degrees B. 72 degrees
C. Southern Blotting C. 55 degrees C. 55 degrees
D. Immunoassays D. 95 degrees D. 95 degrees
68 >>>
4. All the following are requirements of 5. Which of the following is true about mo-
PCR except? lecular diagnosis of SARS CoV2 infection?
A. DNA polymerase A. Higher ct value implies higher viral load

B. dNTP B. Always involves an RNA extraction step
C. Restriction enzyme C. Necessitates the use of a thermocycler
D. Magnesium D. Involves reverse transcription
CASE BASED MCQS

6. A 21 year old man wants a molecular
2. Sample collectionFISHRFLP
3. Cytogenetics
diagnosis of sickle cell anemia as three of 4. Conventional PCR
his maternal cousins are diagnosed with 5. DNA extraction
sickle cell anemia. The intern knows few
steps involved in molecular diagnostics in a A. 2,7,1,3
jumbled way. Help him choose appropriate B. 2,7,6,4
steps and arrange in the right sequence. C. 7,2,1,3
1. RT-PCR D. 2,6,7,3
IMAGE BASED MCQS

7. Three Individuals A,B and C of a
family of Sickle cellanemia wants to get
their molecular diagnosis done. With the
provided RFLP data, choose the genotype
of persons A, B and C.
1.B; 2.D; 3.A; 4.C; 5.B; 6.B; 7.D.
A. Carrier, normal and disease

B. Disease, normal and carrier
C. Normal, disease and carrier
D. Normal, carrier and disease
69
LAC OPERON
FACTS ABOUT lac OPERON
• Lac Operon is used for lactose utilisation • Lac operon is a linear array of genes
in E.Coli of all the three enzymes (lacZ coding
• For E coli to utilise lactose, it needs 3 for beta galactosidase, lac Y coding for
enzymes lactose permease and lac Z coding for
» Beta galactosidase thiogalactosyl transacetylase) and they all
share a common promotor and a com-

» Lactose permease
mon operator
» Thiogalactosyl transacetylase
P O Lac Z Lac y Lac A
β GALACTOSIDASE LACTOSE THIOGALACTOSYL

PERMEASE TRANSACETYLASE
LINEAR ARRAY OF ALL GENES INVOLVED IN A METABOLIC PATHWAY

WITH A COMMON PROMOTOR & A COMMON OPERATOR
PREFERRED FUEL FOR E.Coli

• To understand how Lac operon functions, • In the presence of glucose, even if lac-
we need to understand the preferred fuel tose is also provided, it continues using
of E.coli. glucose.
• Glucose is the preferred fuel for glucose. • Only when E.coli is completely deprived
off glucose, it goes through a lag phase
and then E.coli starts utilising lactose.
Requirements for Lac operon to be transcribed

• The two requirements for lac Operon to locus for the RNA polymerase to be
be transcribed are recruited
» cAMP saturated Catabolite Activator » For RNA polymerase to reach the +1
Protein should bind to the promotor site, the operator locus has to be free.
70 >>>
» However, there is a constitutively • Positive regulator of lac operon is cAMP

expressed lac I gene, which produces saturated CAP
repressor protein. This tetramerises • Negative regulator of lac Operon is lac I
and blocks the operator locus product repressor
LAC OPERON
cAMP SATURATED RNA POLYMERASE

CAP
+1
Lac I P O Lac Z Lac y Lac A

cAMP SATURATED CAP SHOULD BIND TO THE PROMOTOR LOCUS
OPERATOR LOCUS HAS TO BE FREE
SCENARIOS
The three scenarios to be considered hibits adenylyl cyclase and hence cAMP
regarding utilisation of fuel by E.Coli: is low. Hence cAMP saturated CAP is not
>> I. When only glucose is available: available.
• Lac I gene product repressor protein te- • As only one of the two requirements is
tramerises and blocks the operator locus met, lac Operon is not transcribed and
hence E.Coli can not utilise lactose in the
• Glucose is utilised by E.Coli and the
presence of glucose
energy level is high. High energy inhibits
adenylyl cyclase and hence cAMP is low. >> III. When lactose is available in the ab-
Hence cAMP saturated CAP is not availa- sence of glucose:
ble. • Lac I gene product repressor protein
• As both the requirements are not availa- tetramerization is inhibited and hence the
ble, lac Operon is not transcribed operator locus is free
>> II. When both glucose and lactose are • As Glucose is not available, the energy
available: level is low. Low energy activates ade-
nylyl cyclase and hence cAMP saturated
• Lac I gene product repressor protein
CAP is available.
tetramerization is inhibited and hence the
operator locus is free • As both the requirements are met, lac
Operon is transcribed and hence E.Coli
• However, Glucose is utilised by E.Coli and
starts utilising lactose in the absence of
the energy level is high. High energy in-
glucose
71
LAC OPERON – IN THE PRESENCE OF GLUCOSE

& NO LACTOSE
RNA POLYMERASE
cAMP SATURATED
CAP
+1

IN THE PRESENCE OF GLUCOSE & NO LACTOSE

E.Coli utilises Glucose
HIGH ENERGY REPRESSOR

TETRAMER BLOCKS
OPERATOR LOCUS
INHIBITS ADENYLYL
CYCLASE
Lac OPERON IS NOT
LOW cAMP TRANSCRIBED
LOW cAMP SATURATED

CAP
LAC OPERON – IN THE PRESENCE OF GLUCOSE

& LACTOSE
RNA POLYMERASE
cAMP SATURATED
CAP
+1

72 >>>
IN THE PRESENCE OF GLUCOSE & NO LACTOSE
E.Coli utilises Glucose
HIGH ENERGY REPRESSOR

TETRAMERISATION IS
BLOCKED
INHIBITS ADENYLYL
CYCLASE
Lac OPERON IS NOT
LOW cAMP TRANSCRIBED
LOW cAMP SATURATED

CAP
IN THE ABSENCE OF GLUCOSE AND IN THE

PRESENCE OF LACTOSE
RNA POLYMERASE
cAMP SATURATED
CAP
+1

IN THE ABSENCE OF GLUCOSE - PROVIDING

LACTOSE
No Glucose
RNA polymerase is
recruited
Low ENERGY
REPRESSOR
TETRAMERISATION IS
ACTIVATION OF BLOCKED
ADENYLYL CYCLASE
Lac OPERON IS NOT
High cAMP TRANSCRIBED
cAMP SATURATED CAP
73
LAC OPERON IN RECOMBINANT

DNA TECHNOLOGY
» In recombinant DNA technology, we
link the gene of interest with a vector
to form a recombinant vector, and is
then introduced into E.Coli. Whenever
the vector replicates we get multiple
copies, whenever E.coli multiplies we
get multiple copies.
» Formation of recombinant vector and
introduction of recombinant vector into
E.coli are two difficult steps in recom-
binant DNA technology. So after the in-
troduction of recombinant vector, there

should be a way of identifying E.Coli
colonies with recombinant vector.
This is accomplished by using marker
genes. The most widely used marker
gene is lac Z of E.Coli
BLUE AND WHITE TECHNIQUE

» To detect transformed colonies » The medium is mixed with IPTG (allo-
» E.coli with a mutated lac Z operon is lactose which is an inducer of lac
used (produces only an N terminal operon) and x-gal (5 Bromo 4 chloro 3
truncated omega subunit of Beta Indoxyl galactoside)
galactosidase, which does not let the » If the colony is transformed with the
enzyme tetramerise) vector, beta galactosidase acts on
» The alpha subunit coding segment of x-gal and gives blue colour because of
beta galactosidase (with restriction alpha complementation
site) is incorporated as a marker gene » As the restriction site is placed within
in the vector the alpha subunit coding segment,
» So only when E.Coli is transformed f the colony is transformed with the
with the vector carrying the alpha sub- recombinant vector, no alpha comple-
unit, alpha complementation happens mentation occurs and hence it pro-
and the E.Coli produces a functional duces white colony
tetrameric beta galactosidase
74 >>>
FUNDAMENTALS OF ALPHA COMPLEMENTATION
Bromo 4 chloro Blue Colour

OMEGA SUBUNIT 3 Indoxyl colony
ALPHA SUBUNIT
RESTRICTION
SITE
ALPHA ACTIVE BETA

COMPLEMENTATION GALACTOSIDASE
BLUE & WHITE TECHNIQUE
WHITE COLONY BLUE COLONY
FAILURE OF ALPHA ALPHA

COMPLEMENTATION COMPLEMENTATION
RECOMBINANT NO RECOMBINANT
VECTOR VECTOR
MCQS
1. Lac A in lac operon 2. The positive regulator 3. The substrate that is
codes for: of lac operon is: used in blue and white tech-
nique is:
A. Beta galactosidase A. CAP
B. Lactose permease B. cAMP saturated CAP A. Lactose
C. Thiogalactose C. Lac I product B. Isopropylthiogalactosyl
1.B; 2.B; 3;D.
transacetylase D. Lac A product pyranoside

D. Isopropyl thiogalactosyl C. Allolactose
pyranoside D. 5 Bromo 4 chloro 3 In-
doxyl galactoside
75
CRISPR CAS SYSTEM

• There are two components • CRISPR is abbreviated as Clustered
» CAS is an endonuclease which makes Regularly Insterspersed Short Palindro-
nicks at specific sites, which are com- mic repeats. The sequence has many
plementary to the guide RNA present CAS genes upstream, followed by a
within them leader sequence, followed by a clus-
» The guide RNA is complementa- ter of 50 to 100 palindromic repeats,
ry to the spacer sequences found which are interspersed by spacer
in CRISPR sequences sequences.
CAS
ENZYME
GUIDE RNA
SIGNIFICANCE IN PROKARYOTES
• They are present in bacteria and arche- as CAS enzyme and they quickly make
ae. They act as a form of immunological nick at bacteriophage DNA
memory, protecting them from future viral • Discovered by a Japanese scientist
infections Yoshizumi Ishino in 1987
• During the first infection, cas enzyme • Effectively repurposed for gene editing
cuts viral nucleic acids close to PAM (Pro- by Emmanuel Charpentier in 2020 by en-
tospacer Adjacent Motifs) and acquire gineering cas9 to use a single guide RNA
those fragments as spacer sequences instead of crRNA and TranscrRNA
• During a subsequent infections, the spac-
er sequence gets transcribed as guide
RNA and the CAS genes get transcribed
76 >>>
CLINICAL SIGNIFICANCE
• CRISPR CAS system make double strand- • Following a ds DNA break caused by
ed DNA break at specific sites CRISPR CAS system, if the repair mech-
• Hence, they result in either gene knock anism is done by Non Homologous End
out or knock in Joining, it cause gene knock out
• Gene Knock out is when the defective • Following a ds DNA break caused by
gene is removed CRISPR CAS system, if the repair mecha-
• Gene knock in is when the defective nism is done by Homologous DNA repair,
gene is replaced with a normal gene it cause gene knock in
MCQS
1. Which is true about CRISPR CAS sytem?: 2. Following CRISPR mediated gene nicks,
which of the following can result in gene
A. Bacteriophages exhibit extensive CRISPR knock in?
CAS system
B. The repeat sequences code for the guide A. Non Homologous End Joining
RNA B. Homologous DNA repair
C. Emmanuel Charpentier received a nobel C. Interference
prize to identify the first CRISPR system D. Ku helicase mediated repair
D. PAM sequences help in spacer acquisition 1.D; 2.B;
77
AMINOACID
AND PROTEIN
CHEMISTRY
CLASSIFICATION OF
AMINOACIDS
INTRODUCTION
• Aminoacids are structures which have tached is called as α carbon atom. Based
both an amino group and a carboxyl on the R group that is attached, aminoac-
group as functional groups. The carbon ids are classified as polar and non polar
atom to which the functional groups is at- aminoacids.
CLASSIFICATION OF AMINOACIDS
POLAR AMINOACIDS » Positively charged or basic aminoacids

• To be soluble in water, they should be – His, Lys and Arg
either charged or they should have polar » Negatively charges or acidic aminoac-
groups like OH or SH or NH groups which ids – Asp, Glu
can form hydrogen bonding with water • Uncharged polar aminoacids are classi-
• Hence polar aminoacids are classified as fied as
» Charged aminoacids » OH group containing aminoacids eg., Ser,
» Uncharged aminoacids Thr, Tyr
• Charged polar aminoacids are classified as » SH group containing aminoacids eg., Cys
» NH group containing aminoacids eg., Asn,
Glm
79
NON POLAR AMINOACIDS • Aromatic aminoacids absorb UV light at

• Non polar aminoacids are classified as 280nm
» Aliphatic aminoacids - Gly, Ala, Val, Leu,, Ile • Pro has one hydrogen lesser, hence it can
and Met not effectively take part in hydrogen bond-
ing. Hence it disrupts alpha helix
» Aromatic aminoacids – Phe, Try
• Pka of His is 6 which is close to the physio-
» Iminocids – Pro
logical pH of 7.4
→ FACTS TO REMEMBER ABOUT AMINOACIDS
• Asp has an additional beta carboxyl group
• Gly is the simplest aminoacid with H as the
• Glu has an additional gamma carboxyl
R group. It does not have any asymmetric
group
carbon atom. Hence it is optically inactive
MNEMONICS - AMINOACIDS • TIA – Tryptophan has Indole ring and it an-

• HIP – Histidine has Imidazole ring and it swers Aldehyde test
answers Pauly’s test • AGS – Arginine has Guanidium ring and it
answers Sakaguchi test
AMINOACIDS
POLAR NON POLAR
CHARGED UNCHARGED Gly, Ala, Val,

ALIPHATIC
Leu, Ile, Met
BASIC ACIDIC OH SH NH2

AROMATIC Phe, Try
His, Ser,
Glu, Asn,
Lys, Thr, Cys
Asp Gln IMINO Pro
Arg Tyr
PEPTIDE LINKAGE
• Peptide linkage is formed between alpha
carboxyl group of an aminoacid and al-
pha aminogroup of an aminoacid, with the
removal of water
• There are four atoms in peptide linkage –
C,O,N and H
• All four atoms are coplanar
• At a physiological pH of 7.4, nitrogen will try
to get a valency of 4, to satisfy the fourth
valency, nitrogen pulls the shared pair of
electron between C and O to itself and this
gives a partial double bond character
80 >>>
FACTS ABOUT GLUTATHIONE

• Glutathione is a tripeptide
• It is gamma glutamyl cysteinyl glycine
• It is made up of glutamic acid, cysteine and
glycine
α
• It is represented as GSH
• It is an antioxidant and is a conjugating β
agent
• It has a pseudopeptide linkage, as the link- γ
age. Is formed between gamma carboxyl
group of Glu and alpha amino group of Cys,
hence the prefix Gamma in its name
PSEUDOPEPTIDE
LINKAGE
PROTEIN STRUCTURE
INTRODUCTION
Protein structure is defined at five levels quencing, Edman’s sequencing and reverse
» Primary structure sequencing
» Secondary structure • Sanger’s reagent is 2,4 dinitrobenzene
» Super secondary structure • Edman’s reagent is phenylisothiocyanate
» Tertiary structure >> SECONDARY STRUCTURE
» Quaternary Structure • It is defined by how the contiguous seg-
ments of a polypeptide chain get organ-
>> PRIMARY STRUCTURE
ized to form ordered units
• It is defined by the number and sequence
of aminoacids which are linked by peptide
• The two forms of secondary structure
are:
linkages
» Alpha helix
• The linkage which stabilizes primary structure
» Beta pleated sheets
of a protein is peptide linkage
• Methods used for studying secondary
• Methods used for studying primary structure structure are optical rotatory dispersion
or for sequencing a protein are Sanger se- and ocular dichroism
81
DIFFERENCES BETWEEN ALPHA HELIX AND BETA PLEATED SHEET
S.No ALPHA HELIX BETA PLEATED SHEET
1 Compact (1.5Ao) Extended (3.5Ao)
2 Intra chain hydrogen bonding Interchain hydrogen bonding
3 Only right handed alpha helix Parallel and antiparallel beta pleated
sheets are found
4 Proline and glycine disrupt -
SUPERSECONDARY STRUCTURE
• These are segments of polypeptide chain • They are stabilised by intrachain hydro-
which link adjacent secondary structures gen bonds
• The two forms of supersecondary struc- • Proline and glycine are found among
tures are turns or bends and loops supersecondary structures
• They often form functional domains
TERTIARY STRUCTURE
• This is defined by how a polypeptide dominantly by hydrophobic interactions.
chain gets organised in three dimension- However, all non covalent interactions
al space to form functional domains. In stabilise tertiary structure
short it is about protein folding. As the • Methods used to study tertiary structure
major driving force for protein folding are X ray crystallography, UV spectrosco-
is protein solubility, it is stabilised pre- py and NMR spectroscopy
QUATERNARY STRUCTURE
• Not all proteins exhibit quaternary structure. • Based on quaternary structure, two struc-
Only those proteins which have more than tures are possible – Taut Structure and
polypeptide chains can have quaternary Relaxed structure
structure and it is defined by how individual • Examples of proteins which can exhibit
polypeptide chains interact with each other. quaternary structure – Hemoglobin, LDH,
• It is stabilized by disulphide bridges. CK, Insulin
• Examples of proteins which do not exhibit
quaternary structure – Myoglobin, Hexokinase
82 >>>
PROTEIN STRUCTURE
STRUCTURE LINKAGE METHOD
Primary Peptide Sanger’s sequencing, Edman’s

sequencing, reverse sequencing
Secondary Hydrogen bond Optical rotatory dispersion,

Ocular Dichorism
Supersecondary Hydrogen bond X ray crystallography, UV

structure spectroscopy & NMR spectroscopy
Tertiary Structure Hydrophobic

interaction
Quartenary Structure Disulphide bridges Reducing type of SDS PAGE
1. Which of the following 4. Primary structure of a 7. Which of the following

is a polar but uncharged protein is stabilized by is a physiological buffer?
aminoacid?
A. Peptide linkage A. It is stabilised by inter
A. BSerine B. Hydrogen bond chain hydrogen bonds
B. Glutamate C. Hydrophobic interaction B. It is extended
C. Arginine D. Disulphide bridges C. Parallel beta pleated
5.
D. Tryptophan sheets are found
2.
Secondary structure D. Antiparallel beta pleated
Which of the following of a protein is studied by sheets are never possible
8.
is a physiological buffer?
A. Sanger’s method Tertiary structure of a
A. BSerine B. Optical Rotatory dispersion protein is studied by
B. Glutamate C. X ray crystallography
C. Arginine D. NMR Spectroscopy A. Sanger’s method
6.
D. Histidine B. Optical Rotatory dispersion
3.
All the following are C. X ray crystallography
All the following are true about Alpha helix D. NMR Spectroscopy
true about Glutathione except
1.A; 2.D; 3.B; 4.A; 5.B; 6.D; 7.D; 8.C
except?
A. It is stabilised by intra
A. It is a tripeptide chain hydrogen bonds
B. It has 3 peptide linkages B. It is compact
C. It has a pseudo peptide C. Proline disrupts alpha
linkage helix
D. It is an antioxidant D. Left handed alpha helices
are common
83
AMINOACID
METABOLISM
AMINOACID BREAKDOWN
FACTS ABOUT AMINOACID BREAKDOWN

• All aminoacids during breakdown • Aminoacids which neither undergo
lose their aminogroup first and then transamination nor deamination are
the carbon skeleton undergoes » Lysine
breakdown. This is done by » Threonine
» Transamination » Proline
» Deamination » Hydroxyproline
FACTS ABOUT TRANSAMINATION REACTIONS
• All transaminases use PLP as a coen- with a ketoacid, which is most commonly
zyme. alpha ketoglutarate. The aminoacid gives
• Suppose an aminoacid undergoes break- off it’s amino group to alpha ketogluta-
down by transamination reaction, it reacts rate, converting it into glutamate. This
way the aminoacid becomes a ketoacid.
EXAMPLES:
AST/ SGOT
• Aspartate + α ketoglutarate↔OAA + Glutamate
ALT/ SGPT
• Alanine + α ketoglutarate↔Pyruvate + Glutamate
FACTS ABOUT OXIDATIVE DEAMINATION
• It uses L Aminoacid oxidase. It uses FAD FADH2 is linked to ETC, for every FADH2
as a coenzyme. The enzyme converts to be oxidised as FAD, Oxygen becomes
aminoacid to ketoacid and ammonia. As it Hydrogen Peroxide
is done by oxidation, hydrogen atoms are • Two limitations of oxidative deamination:
removed from aminoacid, and it is used » Hydrogen peroxide generation
to convert FAD to form FADH2. As this
» Ammonia generation
85
L- Aminoacid
Oxidase
• L - Aminoacid + FAD↔ Ketoacid + Ammonia + FADH2
H2O2 Oxygen
AMMONIA DETOXIFICATION
• Ammonia is generated in all tissues by • The most common non toxic form of
oxidative deamination reactions. But ammonia is glutamate. In all tissues, as
ammonia is detoxified as urea only in and when ammonia is generated, it reacts
liver. Hence from all the production sites, with alpha ketoglutarate and NADH to
ammonia has to be transported in a non form NAD and glutamate. This happens
toxic form to reach liver. in the presence of glutamate dehydroge-
nase enzyme.
Glutamate
Dehydrogenase
Ammonia + α KG + NADH↔Glutamate + NAD
• Exceptions are neurons and muscle. In • The most common non toxic form of
neurons glutamine is formed and in mus- ammonia is glutamate. In all tissues, as
cle, it forms alanine and when ammonia is generated, it reacts
• An understanding of why glutamine and with alpha ketoglutarate and NADH to
alanine are formed in neurons and mus- form NAD and glutamate. This happens
cles depends upon an understanding of in the presence of glutamate dehydroge-
why ammonia is toxic! nase enzyme.
• Ammonia is generated in all tissues by • Ammonia is detoxified in the form of
oxidative deamination reactions. But » Glutamic acid – most common
ammonia is detoxified as urea only in » Glutamine – Neurons
liver. Hence from all the production sites, » Alanine - Muscle
ammonia has to be transported in a non
toxic form to reach liver.
86 >>>
WHY IS AMMONIA TOXIC?

» Ammonia has a high Pka (9). Hence it stimulated and the person hyperventi-
senses the intracellular pH as acidic. To lates causing respiratory alkalosis
neutralize the acidic pH, it accepts hydro- » As it sequestrates hydrogen ions, elec-
gen ions from the surrounding, convert- tron transport chain is inhibited, this
ing ammonia to ammonium ions. These inhibits ATP production
ammonium ions are osmotically active » As ammonia reacts with alpha ketogluta-
and they attract water. Hence ammonia rate, it causes alpha ketoglutarate defi-
is called as an osmolyte. This neuronal ciency. This inhibits TCA cycle and hence
swelling causes respiratory centre to be ATP production
FACTS ABOUT GLUTAMINE FORMATION IN NEURONS

» If one of the mechanisms by which am- utilise alpha ketoglutarate. Hence, glu-
monia exhibits toxicity is that it uses tamine is formed, this way two ammonia
alpha ketoglutarate, in a principal cell of get detoxified, but we use only one alpha
our body, neurons, we try to optimally ketoglutarate
Glutamate
Dehydrogenase
• Ammonia + α KG + NADH↔Glutamate + NAD
Glutamine
synthetase
• Glutamate + Ammonia↔ Glutamine
FACTS ABOUT ALANINE FORMATION IN MUSCLE
» In muscle alanine is formed, as this this way alpha ketoglutarate is regen-

way we don’t only detoxify ammonia, erated
we also detoxify pyruvate. Additionally,
Glutamate
Dehydrogenase
• Ammonia + α KG + NADH↔Glutamate + NAD
ALT
• Glutamate + Pyruvate↔ αKetoglutarate + Alanine
87
FACTS ABOUT UREA
» Urea is the best non toxic form of ammonia

» It is formed in liver
Ammonia Aspartate
» Urea has 2 nitrogens – N1 and N3
» Donor of N1 is ammonia
N1 N2
» Donor of N2 is Aspartate
AMMONIA IN LIVER
» After ammonia is transported in the form ammonia, which enters into urea cycle
of glutamate, glutamine and alanine from and forms N1 of urea
various tissues to the liver, all the forms » The remaining 50% glutamate undergo
get converted to glutamate transamination reaction with oxaloace-

» 50% glutamate go through reversal of tate to form Aspartate, which forms N2 of
glutamate dehydrogenase step to form urea
FACTS & STEPS OF UREA CYCLE

» Urea cycle happens in cytoplasm and in » Citrulline leaves mitochondria and reach-
mitochondria es cytoplasm through ornithine citrulline
» The first two steps catalysed by carba- transporter
moyl phosphate synthetase I and Ornith- » In the cytoplasm, citrulline reacts with
ine transcarbamoylase happen in mito- aspartate in the presence of argininosuc-
chondria. The remaining steps happen in cinate synthetase and two high energy
cytoplasm phosphates to form arginine succinate
» In the first step, carbon dioxide reacts » Argininosuccinate in the presence of
with ammonia and ATP react in the pres- argininosuccinate lyase gets converted
ence of another ATP, which acts as a to arginine and fumarate.
source of energy to form carabamoyl » Fumarate enters into TCA cycle, where it
phosphate in the presence of carbamoyl is converted to malate and oxaloacetate.
phosphate synthetase I This oxaloacetate undergoes transami-
» In the next step, carbamoyl phosphate nation to form Aspartate, which acts as a
reacts with ornithine in the presence source of N2.
of ornithine transcarbamoylase to form » Arginine is acted upon by Arginase to
citrulline form urea and ornithine is got back. Orni-
thine formation closes the cycle. Hence,
ornithine is the catalyst of urea cycle.
88 >>>
DIFFERENCES BETWEEN CPS I AND CPS II

S.No PROPERTY CPS I CPS II
S.No PROPERTY CPS I CPS II
1 Pathway Urea Cycle Pyrimidine Synthesis
2 Suborganelle Mitochondria Cytoplasm
3 Nitrogen Source Ammonia Glutamine
4 Regulation Stimulated by NAG* Inhibited by C,U,T
*The signal for high ammonia generation is glutamate. This glutamate reacts with acetyl CoA in the presence of
N Acetyl Glutamate synthetase to form NAG, which stimulates detoxification of ammonia.
In fatty acid oxidation defects, acetyl coA is not formed and hence CPSI is not Stimulated. This causes
hyperammonemia in fatty acid oxidation defects.
89
FACTS ABOUT UREA CYCLE

» Involves mitochondria and cytoplasm » Every cycle uses 4 ATPs and regenerates
» Two enzymes present in mitochondria 2.5 ATPs through TCA cycle
include » Net ATP utilised in every Urea cycle is 1.5
• Carbamoyl phosphate synthetase I » Urea cycle and citric acid cycle are linked
• Ornithine Transcarbamoylase through fumarate
» In every cycle 2 ammonia are detoxified
UREA CYCLE DISORDERS
S.No ENZYME NAME OF THE

BIOCHEMICAL FEATURES
DEFECT CONDITION
1
90 >>>
S.No ENZYME NAME OF THE

BIOCHEMICAL FEATURES
DEFECT CONDITION
1 CPS I Type I High ammonia, High glutamate, glutamine
hyperammonemia
2 OTC Type II High ammonia, High glutamate, glutamine,

hyperammonemia orotic acid and uracil and low citrulline
3 Argininosuccinate Citrullinemia High ammonia, High glutamate, glutamine,

synthetase orotic acid and uracil and high citrulline
4 Argininosuccinate Argininosuccinic High ammonia, High glutamate, glutamine,

lyase aciduria or orotic acid and uracil and high citrulline,
Trichorrhexis high argininosuccinic acid
nodosa
5 Arginase Argininemia High ammonia, High glutamate, glutamine,

orotic acid and uracil and high citrulline,
argininosuccinic acid, arginine
6 Ornithine HHH syndrome Hyperammonemia, hyperornithinemia,

Citrulline hyper homocitrullinemia
Transporter
MCQS
1. Aspartate on 2. Alanine on transamina- 3. All of the following are
transamination forms: tion forms true about aminoacid oxi-
dase except
A. BPyruvate A. Pyruvate
B. Alpha ketoglutarate B. Alpha ketoglutarate A. FAD is used as a coen-
C. Alanine C. Aspartate zyme
D. Oxaloacetate D. Oxaloacetate B. Linked to ATP production
C. Source of oxidative stress
D. Releases toxic ammonia
91
4. The most common non- 9. The second nitrogen 13. A 6 months old
toxic form of ammonia is donor for urea formation is, female infant began to vom-
it occasionally and ceased
A. Alanine A. Glutamine to gain weight. The child
B. Glutamine B. Ammonia became habitually drowsy,
C. Glutamate C. Aspartate temperature raised and her
D. Alphaketo glutarate D. Glutamate liver was enlarged. The EEG
5. The non-toxic form of 10. All of the following

was abnormal. When the milk
feeding was avoided and
ammonia formed in neuron is are true about urea cycle was started on glucose, the
except condition improved. Urine
A. Alanine analysis showed abnormally
B. Glutamine A. It involves mitochondria high glutamine, uracil, orotic
C. Glutamate B. To detoxify two molecules acids. Blood showed high
D. Alphaketo glutarate of ammonia, it requires 4 ammonium concentration.
6. The non-toxic form of

ATPs Citrulline level was normal.
C. It is stimulated by N – The child has defective:
ammonia formed in muscle is acetyl Glutamate
D. It is linked to citric acid A. CPS I
A. Alanine cycle B. b. Cps II
11. Urea cycle and citric

B. Glutamine C. Ornithine trans carbo-
C. Glutamate moylase
D. Alphaketo glutarate acid cycle are linked through D. Arginino succinate syn-
7. The aminoacid
thase
A. Aspartate
which does not undergo B. Fumarate
transamination or oxidative C. Malate
deamination is D. Pyruvate
1.D; 2.A; 3.B; 4.C; 5.B; 6.A; 7.C; 8.B; 9.C;10.B; 11.B; 12.C; 13.C.
A. Serine 12. Which of the follow-
B. Cysteine ing is true about CPSI
C. Threonine
D. Tryptophan A. Present in cytoplasm
8. The first nitrogen donor

B. Involved in Pyrimidine
synthesis
for urea formation is, C. Stimulated by NAG
D. Glutamine is the nitrogen
A. Glutamine source
B. Ammonia
C. Asparate
D. Glutamate
92 >>>
CARBON SKELETON CATABOLISM

» Based on the products of catabolism of » Leu & Lys are purely ketogenic
the carbon skeleton of aminoacids, ami- » Phe, Tyr, Try, Ile are the aminoacids which
noacids are classified as are both ketogenic and glucogenic
• Purely ketogenic
• Both ketogenic and Glucogenic
• Purely glucogenic
GLUCOGENIC AMINOACIDS
» Glucogenic aminoacids on catabolism » Alphaketoglutarate is formed from Glu,
give rise to a glycolytic intermediate – Gln, His, Pro, Arg
pyruvate or one of the four TCA Cycle » Succinyl CoA is formed from Val, Ile and
intermediates – alpha ketoglutarate, suc- Met
cinyl CoA, fumarate and Oxaloacetate » Fumarate is formed from Phe, Tyr
» Pyruvate is formed from Ala, Gly, Ser, Thr, » Oxaloacetate is formed by Asp, Asn
HyP and Cys
14. All of the following on catabolism 16. All of the following on catabolism
give rise to pyruvate except give rise to Succinyl CoA except
A. Glycine A. Leucine
B. Valine B. Isoleucine
C. Cysteine C. Valine
D. Serine D. Methionine
15. All of the following on catabolism 17. All of the following on catabolism give
give rise to acetyl CoA except rise to αketoglutarate except
A. Leucine A. Proline
B. Isoleucine B. Arginine
14.B; 15.C. 16.A; 17.D.
C. Histidine C. Histidine
D. Tyrosine D. Aspartate
93
INDIVIDUAL AMINOACID
METABOLISM
>> GLYCINE
SOURCES OF GLYCINE
» Reversal of glycine cleavage system: » Serine: Serine in the presence of serine
Gly is cleaved by Glycine cleavage sys- hydroxymethyl trasferase gets converted
tem to form Carbon dioxide, ammonia, to glycine. In this step, we get N5, N10
and N5, N10 methylene THFA. As this methylene THFA
reaction is reversible, it helps in the for- » Threonine: Threonine in the presence
mation of glycine. of threonine aldolase gets converted to
glycine
GLYCINE CLEAVAGE SYSTEM - SUBUNITS
» T – THFA dependent Aminomethyl transferase

» P – PLP dependent glycine decarboxylase
» L – Lipoamide dependent dehydrogenase
» H – electron transfer system
94 >>>
MEASURES TO REDUCE AMMONIA

» In any patient with hyperammonemia, Even if ammonia is generated by these
ammonia can be reduced by either microorganisms, ammonia absorption is
removing the source of ammonia or by inhibited by providing lactulose
scavenging ammonia » Scavenging ammonia is done by admin-
» Ammonia source can be reduced by two istration of Sodium Benzoate or Phenyl
ways – inhibiting protein degradation or Acetate
by inhibiting urease producing microor- » Sodium benozoate reacts with glycine to
ganisms form hippuric acid. To form glycine am-
» Protein degradation is avoiding by ensur- monia is used
ing adequate calorie intake. » Phenylacetate is conjugated with gln to
» Urease producing microorganisms are form phenyl acetyl glutamine. Glutamine
inhibited by using low dose neomycin. is formed from 2 ammonia molecules.
GLYCINE USES
» Inhibitory neurotransmitter » Creatine
» Conjugating agent » Heme synthesis
» Glutathione » Purine ring – C4, C5 and N7
» Collagen » One carbon pool
SYNTHESIS OF CREATINE
Glycine Arginine
Kidney Amidotransferase
Guanidoacetate
Liver Methionine
Creatine Creatine Phosphate

Muscle
ONE CARBON POOL

» Includes a pool of one carbon derivatives » N5, N10 Methylene THFA is used for the
of THFA, their sources and their special- convertion of uridine to thymidine
ised products » N5, N10 methylene THFA is converted to
» Glycine Cleavage system and serine hy- methenyl THFA. Methenyl THFA is used
droxymethyl transferase act as a source for forming C8 of purine ring
of N5, N10 Methylene THFA
95
» N5, N10 Methylene THFA goes through » Methyl Folate trap:

an irreversible chemical reaction cart- • BACKGROUND: In B12 deficiency,
alysed by a reductase to form methyl methyl B12 is unavailable and hence,
THFA. Methyl THFA has a low group methionine synthase is inactive, this
transfer potential and it can donate its causes deficiency of creatinine, cho-
methyl group only to B12 forming me- line and epinephrine. To generate
thyl B12. Methyl B12 acts as a coenzyme these products, all THFA get converted
for methionine synthase or homocyst- to methyl THFA and gets trapped as
eine methyl transferase, which converts methyl THFA.
homocysteine to methione. Methione • DEFINITION: In B12 deficiency, there
is converted to SAM, which is used for is a functional deficiency of THFA, as
transmethylation reactions. The trans- THFA get trapped as methyl THFA due
methylation products obtained are cre- to the inactivity of methionine syn-
atine, choline and epinephrine thase
• EFFECT: B12 deficiency presents with
macrocytic anemia
TRY IN ONE CARBON POOL

• Try in the presence of Try Pyrrolase • The formyl group is transferred to
gets converted to formyl kynenurine. THFA, forming N10 formyl THFA
• Formyl Kynenurine gets converted to • N10 formyl THFA is used for forming
Kynenurine by formyl transferase. C2 of purine ring
TRYPTOPHAN IN ONE CARBON POOL
TRYPTOPHAN
TRYPTOPHAN
PYRROLASE
FORMYL
THFA KYNENURINE
FORMYL
C2 OF FORMYL TRANSFERASE
PURINE RING THFA KYNENURINE
HIS IN ONE CARBON POOL

• Histidine in the presence of Histidase lase becomes FIGLU or formimino
gets converted to urocanate glutamate
• Urocanate in the presence of Uro- • If the person has THFA, the formimino
canase becomes Imidazole propionic group is transferred to THFA to form
acid, which in the presence of hydro- Formimino THFA, glutamate becomes
96 >>>
alpha ketoglutarate, which enters into • If you suspect Folate deficiency, esti-
TCA cycle mate FIGLU levels in urine after His
• In folate deficiency FIGLU accumu- load
lates
HISTIDINE IN ONE CARBON POOL
HISTIDINE
HISTIDASE
UROCANATE
UROCANASE
IMIDAZOLE
PROPIONIC
ACID
HYDROLASE
THFA FIGLU
FITHFA GLU
ONE CARBON POOL
HOMOCYSTEINE
B12 METHYL B12
METHIONINE
METHYL
Glycine THFA
& Serine
REDUCTASE
THFA METHYLENE
THYMIDINE
THFA
TRYPTOPHAN
C8 OF PURINE
METHENYL THFA RING
N10
FORMYL THFA
FORMIMINO THFA HISTIDINE
C2 OF PURINE
RING
97
THFA DERIVATIVES, SOURCES AND

SIGNIFICANCE
S.No THFA DERIVATIVE SOURCE SIGNIFICANCE
2
S.No THFA DERIVATIVE SOURCE SIGNIFICANCE
1 N5, N10 Methylene Glycine, Serine dUMP → dTMP

THFA
2 N10 formyl THFA Tryptophan C2 of purine ring
3 N5, N10 methenyl N5, N10 Methylene THFA C8 of purine ring

THFA
ONE LINERS
The aminoacid necessary for heme synthesis is Glycine
The most common inhibitory neurotransmitter of brain is GABA
The most common inhibitory neurotransmitter of spinal cord is Glycine
N5 N10 methylene THFA is used for the formation of Thymidine
C2 of purine ring is formed from N10 formyl THFA
C8 of purine ring is formed from N5, N10 methenyl THFA

98 >>>
MCQS
1. All of the following are aminoacids 5. FIGLU test is done after a load of :
required for creatine synthesis except:
A. Tyrosine
A. Glycine B. Tryptophan
B. Alanine C. Histidine
C. Arginine D. Glycine
6.
D. Methionine
2.
Glycine acts as a source of:
All of the following act as sources of
atoms of purine ring except : A. N5, N10 methylene THFA
B. N5, N10 methenyl THFA
A. Glycine C. N10 formyl THFA
B. THFA D. Formimino THFA
7.
C. Arginine
D. Aspartate All the following enzymes take part in
3.
one carbon pool except:
All of the following are specialised
products of Glycine except : A. Glycine
B. Tryptophan
A. Collagen C. Histidine
B. Creatine D. Proline
8.
C. Glutathione
D. Pyrimidine All of the following are products of
4.
Glycine Cleavage system except:
Tryptophan acts as a source of:
A. CO2
A. N10 formyl THFA B. Ammonia
B. N5, N10 methylene THFA C. N5, N10 Methylene THFA
1.B; 2.C; 3.D; 4.A; 5.C; 6.A; 7.D; 8.D.
C. N5, N10 Methenyl THFA D. NADPH

D. N5 formyl THFA
99
>> AROMATIC AMINOACID METABOLISM
1. PHENYL ALANINE & TYROSINE

METABOLISM
FATE OF PHENYL ALANINE

• Major fate of phe is that gets converted • Phenyl Pyruvate is reduced to phenyl
to Tyr by phenyl alanine hydroxylase. lactate
BH4 is used as a coenzyme which is con- • Phenyl pyruvate is oxidatively decarboxy-
verted to BH2. lated to form phenylacetate

• Minor fate of phe is that it is acted upon • Phenylacetate is converted to phenyl
by a transaminase to form phenyl pyru- acetyl glutamine
vate (phenyl ketone).
SPECIALISED PRODUCTS FROM TYROSINE

• Specialised products obtained from ty- » Thyroid hormones
rosine are • Neurotransmitters including Dopamine,
» Neurotransmitters – Dopamine, Norepi- Norepinephrine and epinephrine are ob-
nephrine and epinephrine tained by the action of tyrosine hydroxy-
» Melanin lase on tyrosine
100 >>>
• Tyrosine hydroxylase forms Dihydroxy- Dopamine Beta hydroxylase is Vitamin C

phenyl alanine or DOPA dependent
• DOPA undergoes decarboxylation to for • Norepinephrine undergoes methylation
Dopamine. This step needs PLP to form epinephrine
• Dopamine in the presence of Dopamine
beta hydroxylase forms Norepinephrine.
TYROSINE DOPA DOPAMINE
Tyr DOPA
Hydroxylase decarboxylase DOPAmine β
Hydroxylase
EPINEPHRINE NOREPINEPHRINE
• Melanin by the action of tyrosinase. a layer of follicular cells. Thyroglobulin is

Tyrosinase converts Tyrosine to DOPA, secreted by follicular cells into colloid.
which goes through a series of non The Tyr residues of Thyroglobulin under-
enzyme catalysed chemical reactions to go iodination and coupling to give rise to
form melanin thyroid hormones
• Thyroid gland is organised in the form of
follicles. Every follicle is surrounded by
PHENYLKETONURIA
• It is caused by the deficiency of phenyl » Hypopigmentation – Tyrosine deficien-
alanine hydroxylase. cy causes melanin deficiency
• It is characterised by » Screening tests
» Mental retardation • Fecl3 test is positive in urine
• Tyrosine deficiency causes defi- • Guthrie’s test positive in blood –
ciency of neurotransmitters this is based on the fact that an
• Accumulated phenyl ketones com- organism Bacallus subtilis needs
pete with neutral aminoacids to phenyl ketones for its growth
cross the BBB • Both the above tests are obsolete
» Mousy odour – phenyl acetate accu- • High Performance Liquid Chroma-
mulation is the cause tography with Tandem Mass Spec-
trometry is recommended
101
TYROSINE METABOLISM
TYROSINE P hydroxy phenyl
Homogentisic acid
pyruvate
TYROSINE DIOXYGENASE
TRANSAMINASE
HOMOGENTISATE
OXIDASE
Fumarate
Fumaryl Maleyl
acetoacetate acetoacetate
FUMARYL CIS TRANS
ACETOACETATE ISOMERASE
Acetoacetate HYDROLASE
TYPE III
TYROSINEMIA
TYROSINE P hydroxy phenyl

Homogentisic acid
pyruvate
TYROSINE DIOXYGENASE
TRANSAMINASE
TYPE II TYROSINEMIA
HOMOGENTISATE
ALKAPTONURIA OXIDASE
Fumarate
Fumaryl Maleyl
acetoacetate acetoacetate
FUMARYL CIS TRANS
ACETOACETATE ISOMERASE
Acetoacetate HYDROLASE
NITISINONE
TYPE I TYROSINEMIA
TYPE I TYROSINEMIA
• Type I Tyrosinemia is caused by the de- • Liver damage presents as Jaundice,
fect of Fumaryl acetoacetate Hydrolase. hepatomegaly, hypoglycemia, cirrhosis,
• It is called as Hepatorenal syndrome hepatocellular carcinoma
• Succinylacetone is toxic to renal and liver • Renal damage presents as Fanconi syn-
parenchymal cells drome and Renal failure
• Succinylacetone inhibits ALA dehy-
dratase and hence it mimics porphyria
102 >>>
TYPE II TYROSINEMIA
• It is caused by the defect of Tyrosine • It is characterised by Painful corneal ero-
Transaminase sions and palmar hyperkeratosis
• It is otherwise called as Richner Hanhart
syndrome or Oculocutaneous syndrome
TYPE III TYROSINEMIA

• It is caused by the defect of p-hydroxy- • Gain of function mutation of p-hydroxy-
phenyl pyruvate dioxygenase. phenyl pyruvate dioxygenase causes
• It is characterised by Intermittent ataxia Hawkinsinuria , which is characterised by
and seizures swimming pool odour of urine
• Partial defect of p-hydroxyphenyl pyru-
vate dioxygenase causes Transient Tyros-
inemia of neonates
ALKAPTONURIA
• It is caused by the defect of Homogenti- the nose, thenar and hypothenar emi-
sate Oxidase. nences
• Homogentisic acid on oxidation forms • Pigmentation of mucous membranes
benzoquinone acetate, on polymerisation causes Osler’s sign, which is nothing but
forms melanin like fibrils pigmentation of sclera along the attach-
• Accumulation of melanin like fibrils caus- ment of medial and lateral rectus
es cartilage destruction and that causes • Urine turning dark on standing – first
Oochronosis symptom
• Accumulation of melanin like fibrils in the
skin causes Pigmentation of pinna, tip of
NITISINONE
• Acts by inhibiting p hydroxyohenylpyru- • Contraindicated in Type II and Type III
vate dioxygenase Tyrosinemia
• Used for treating Type I tyrosinemia,
Hawkinsinuria and Alkaptonuria
103
URINE ODOUR - DISORDER
• Mousy odour
• Fruity Odour
• Cabbage odour
• Boiled cabbage odour
• Oasthouse odour
• Swimming pool
• Sweaty feet odour
URINE ODOUR IN DISORDERS

S.No URINE ODOUR DISORDER DEFECT
104 >>>
S.No URINE ODOUR DISORDER DEFECT
1 Mousy odour Phenyl ketonuria PAH
2 Fruity odour DKA
3 Cabbage odour Type I Tyrosinemia FAH
4 Boiled cabbage Hypermethioninemia Methionine Adenosyl Transferase

odour
5 Swimming pool Hawkinsinuria P-Hydroxy phenyl pyruvate

dioxygenase (GOF)
6 Maple Syrup MSUD BCKAD
7 Sweaty feet Isovaleric acidemia Isovaleryl CoA dehydrogenase
8 Rotten fish Trimethylaminuria Flavin Monooxygenase 3
105
2. TRYPTOPHAN
The two specialized products obtained from Kynenurine, which in the presence of
Typtophan are niacin and serotonin formyl transferase becomes kynenurine
• SYNTHESIS OF SEROTONIN FROM » Kynenurine undergoes hydroxylation
TRYPTOPHAN: to form 5 hydroxy kynenurine, which in
» Try undergoes hydroxylation in the the presence of kynenurinase becomes
presence of Try Hydroxylase to form 5 5 hydroxy anthranilic acid. Kynenuri-
hydroxy tryptophan nase is PLP dependent
» 5 hydroxy tryptophan undergoes de- » 5 hydroxyanthranilic acid goes through
carboxylation to form serotonin a series of non enzyme catalysed
• SYNTHESIS OF NIACIN FROM chemical reactions to form Quinolinate
TRYPTOPHAN: » Quinolinate in the presence of QPRTAse
» Tryprophan in the presence of Tryp- become Niacin mono nucleotide
tophan pyrrolase becomes formyl » NMN becomes NAD and NADP
SYNTHESIS OF NIACIN
TRYPTOPHAN
TRYPTOPHAN
PYRROLASE
FORMYL
THFA KYNENURINE
FORMYL NAD
FORMYL TRANSFERASE
THFA
KYNENURINE QPRTASE
HYDROXYLASE NMN
3 OH
KYNENURINE QPRTASE
PLP KYNENURINASE
3 OH
QUINOLINATE
ANTHRANILIC
ACID
CAUSES OF PELLAGRA
» Niacin deficiency in the diet » Carcinoid syndrome
» Tryptophan deficiency in the diet – » B6 deficiency
Maize based diet » Leucine pellagra – sorghum based diet
» Tryptophan malabsorption – Hartnup’s (leucine inhibits QPRTase)
disease
106 >>>
TRYPTOPHAN
MALABSORPTION TRYPTOPHAN SEROTONIN
TRYPTOPHAN
PYRROLASE
FORMYL
THFA KYNENURINE
FORMYL NAD
FORMYL TRANSFERASE
THFA QPRTASE
KYNENURINE
HYDROXYLASE NMN
3 OH
KYNENURINE LEUCINE PELLAGRA QPRTASE
PLP
KYNENURINASE
3 OH
QUINOLINATE
ANTHRANILIC
ACID
>> OTHER AMINOACIDS METABOLISM
HOMOCYSTEINE METABOLISM
CYSTATHIONINE
BETA SYNTHASE
HOMOCYSTEINE
+ SERINE
B6
CYSTATHIONINE
CYSTATHIONINASE
HOMOSERINE CYSTEINE
METHIONINE SYNTHASE
HOMOCYSTEINE METHIONINE
Methyl
B12
B12
THFA Methyl
THFA
107
HOMOCYSTEINURIA
CYSTATHIONINE
BETA SYNTHASE
HOMOCYSTEINE
+ SERINE
B6
CYSTATHIONINE
CYSTATHIONINASE
TYPE I
HOMOCYSTEINURIA
HOMOSERINE CYSTEINE
METHIONINE SYNTHASE
HOMOCYSTEINE METHIONINE
Methyl
B12
B12
TYPE II
HOMOCYSTEINURIA
THFA Methyl
THFA
TYPES OF HOMOCYSTEINURIAS
TYPE I HOMOCYSTEINURIA TYPE II HOMOCSYTEINURIA
» Defective enzyme : Cystathionine beta » Defective enzyme : Methionine syn-
synthase thase
» Biochemical changes: High homocyst- » Biochemical changes: High homocyst-
eine, low cysteine and high methionine eine, high cysteine and low methionine
» Responds to B6 administration » Responds to B12 administration
CYSTINURIA & CYSTINOSIS

CYSTINURIA CYSTINOSIS
» Defective neutral aminoacid transport- » Defective efflux of Cysteine from Lys-
er which transports Cys, Orn, Lys and osome
Arg along PCT » Fanconi Syndrome
» Cystine stones » Photophobia
» Hexagonal stones » Sequin like crystals
108 >>>
PLP COENZYME ROLES

>> PLP is necessary as a coenzyme for the ALA. ALA synthase is PLP depenedent.
following reactions Hence B6 deficiency causes anemia.
» Transamination » Homocysteine metabolism
» Decarboxylation • PLP is necessary as a coenzyme for
• Example Glutamate undergoes de- cystathionine Beta synthase. Cys-
carboxylation in the presence of Glu- tathione Beta synthase is necessary
tamate decarboxylase to form GABA. for the conversion of Homocysteine to
This is PLP dependent. Hence in B6 cysteine. Hence B6 deficiency causes
deficiency, excesss Glu accumulates Homocysteinuria
in the synapse, which being an excita- » Niacin synthesis
tory neurotransmitter causes seizures • Kynenurinase is dependent on B6 and
and refractory seizures respond to B6 hence B6 deficiency caused pellagra
administration >> PLP dependent clinical states
• Gly reacts with succinyl CoA in the • Seizures
presence of ALA synthase to form • Anemia
• Homocysteinuria
• Pellagra
MCQS
1. All of the following 3. Type I Tyrosinemia 5. Melanin is synthesized
are features of is characterized by all from which of the follow-
phenylketonuria, except: except: ing amino acid?
A. Mental retardation A. Hepatocellular carcinoma A. Tyrosine

B. Hyperpigmentation B. Renal failure B. Tryptophan
C. Mousy odour C. Porphyria C. Leucine
D. Guthrie’s test +ve D. Hyperglycemia D. Phenylalanine
2. Guthrie’s test is based 4. Type II Tyrosinemia

on the growth of which of is characterized by all
the following organisms: except ::
A. Bacillus subtilis A. Palmar hyperkeratosis

B. Thermus aquaticus B. Corneal ulcer Dopamine,
C. Bacillus diphtherium C. Porphyria Norepinephrine and
D. Corynebacterium diph- D. Oculocutaneous syn- Epinephrine, Thyrpid
hormone
therium drome
• Niacin and Serotonin
109
6. Gyrate atrophy of GYRATE ATROPHY OF RETINA

retina is associated with,
defect in catabolism of ARGINASE
OAT
A. Histidine ARGININE ORNITHINE
B. Arginine
C. Glycine B6 GAMMA
D. Proline GLUTAMATE
SEMIALDEHYDE
7. Tryptophan acts as a source of : 11. A 4 years old child presents with men-
tal retardation, charley chaplin gait, ectopia
A. N10 formyl THFA lentis. Plasma homocysteine, methionine
B. N5, N10 methylene THFA level was raised, plasma cysteine is mark-
C. N5, N10 Methenyl THFA edly reduced. There is increased excretion
D. N5 formyl THFA of homeocystine, Methionine in urine. The

diagnosis is:
8. Mousy odour of urine is a feature of A. Homocystinuria type I

B. Homocystinuria type II
A. Phenylketonuria C. Homocystinuria type III
B. Type I Tyrosinemia D. Hypermethioninemia
12. Pyridoxal deficiency causes all ex-

C. Hypermethioninemia
D. Isovaleric acidemia
9. Type I Homocysteinuria responds to the

cept:
administration of? A. Pellagra
1.B; 2.A; 3.D; 4.C; 5. A; 6.B; 7.A; 8.A; 9.A; 10.D; 11.A; 12.B; 13.B.
B. Methyl folate trap
A. B6 C. Seizures
B. B12 D. Anaemia
13. A 12 year old boy presented with

C. Folate
D. Thiamine
10. Cystinuria is characterized by?

photophobia, short stature, polyuria and
polydipsia. Slit lamp examination of the
cornes is shown in the image. The probable
A. Fanconi‘s syndrome diagnosis is
B. Photophobia
C. Porphyria A. Cystinuria
D. Renal stones B. Cystinosis
C. Homocystinuria
D. Hartnup’s
disease
110 >>>
VITAMINS
VITAMIN A
VITAMIN A FORMS
» Retinol: This is form in which Vitamin A is differentiation of epithelium. 13 cis retinoic
absorbed, transported and stored in liver acid suppresses keratinisation of epithe-
» Retinal: This is the form in which Vitamin A lium, stimulates apoptosis of sebaceous
is form in rhodopsin. Rhodopsin until stimu- glands and stimulates NGAL. Hence vitamin
lated by light is 11 cis retinal and Opsin A deficiency is characterised by hyperkera-
» Retinoic acid: There are two forms – all totic conditions like phrynoderma, conjunc-
trans retinoic acid and 13 cis retinoic acid. tival xerosis, Bitot’s spots and keratomala-
All trans retinoic acid supports growth and cia. 13 cis retinoic acid is used for resistant
acne and for Harlequin icthyosis
WALD’S VISUAL CYCLE

» This cycle includes all the steps that hap- LRAT (Lecithin retinol acyl transferase) to
pen when light falls on retina. retinyl esters. RPE65 or isomerohydrolase
» The cycle starts with 11 cis retinal undergo- then converts all trans retnyl ester to 11 cis
ing a transformation to form all transretinal retinol
when light falls, following this transfor- » 11 cis RDH converts 11 cis retinol to 11 cis
mation, it gets detached from opsin. This retinal and is incorporated into rhodopsin
is called as Photobleaching. It ends with » Light causes photoisomerization of 11 cis
the regeneration of rhodopsin after pho- retinal to all trans retinal, which is detached
tobleaching from rhodopsin. This causes photobleaching.
» The cycle involves RPE and rod cells » Regeneration of rhodopsin can happen in
» The four facts to remember are 2 ways
• Rhodopsin is 11 cis retinal + Opsin » If there is adequate vitamin A in the body,
• Circulatory form is all trans retinol 11 cis retinal will be readily available in RPE
• RPE has LRAT, RPE65 (isomerohydro- » If not, 11 cis retinal has to be regenerated
lase), 11 cis RDH by RPE
• Rods and cones have all trans RDH » All trans retinal is converted by all trans
» All trans retinol transported in the circula- RDH to form all trans retinol. This reached
tion reaches RPE, where it is acted upon by RPE. This is Wald’s visual cycle
112 >>>
VITAMIN A - RDA
» Adult men : 1000ug/day » Lactation: 950ug/day

» Adults women: 850ug/day » Children:400 to 650ug/day
» Pregnancy: 900ug/day
VITAMIN A - SOURCES
» Animal sources: Milk, butter, cream, » Plant sources: Carrot, papaya, mango,
cheese, egg yolk and liver, fish liver oils pumpkins and green leafy vegetables
VITAMIN A - DEFICIENCY
CAUSES: • Liver cirrhosis

» Primary: Dietary deficiency • Nephrotic syndrome
» Secondary • Chronic alcoholism
• Obstructive Jaundice
113
VITAMIN A – DEFICIENCY MANIFESTATIONS
» Xerophthalmia » Xerophthalmia
» Bitot’s spots » Bitot’s spots
» Keratomalacia
» Corneal opacity
» Hyperkeratosis – phrynoderma
VITAMIN A - TOXICITY
» Anorexia » High ICT – pseudotumor cerebri
» Irritability, headache » Bony exostosis
» Vomiting » Hepatomegaly
VITAMIN E & K
CHAIN BREAKING ANTIOXIDANTS
» Chain breaking antioxidants are molecules • Lipid phase chain breaking antioxidant -
which can donate an electron or accept α tocopherol reacts with peroxyl radical
electron from unstable intermediates of to form tocopheroxyl radical
lipid peroxidation converting them into • Aqueous phase chain breaking antiox-
stable intermediates. idant – Vitamin C regenerates tocoph-
» They are of two types erol, polyphenol flavinols like epitachin
gallate
114 >>>
OTHER FACTS ABOUT VITAMIN E

• Vitamin E requirement is based on fat • Lactation 12mg/day
intake • Major source is Vegetable oil
• Selenium reduces the requirements of • Deficiency presents as hemolytic anemia –
Vitamin E uncommon
• RDA in males 10mg/day • Hypervitaminosis presents as hemorrhagic
• Females 8mg/day tendency
• Pregnancy 10mg/day
VITAMIN K STRUCTURE
• It has NAPHTHOQUINONE RING with a
polyisoprenoid side chain, depending
upon the chain length of the isoprenoid
side chain, there are two forms of Vitamin
K – Vitamin K1, which has a 20carbon side
chain and Vitamin K2 with a 30 C side
chain. This isoprenoid side chain is respon-
sible for its non polar nature
• In menadione, there is no isoprenoid side
chain, which makes it polar. Hence me-
nadione can be used to treat Vitamin K
deficiency caused by fat malabsorption
VITAMIN K FUNCTION
• Vitamin K is necessary as a coenzyme for • The form of Vitamin K necessary to act
Gamma carboxylase. Gamma carboxylase as a coenzyme for gamma carboxylase is
is necessary for attaching an additional hydroquinone form
carboxyl group to the gamma carbon atom • During the reaction, hydroquinone form
of Glutamate residues of those proteins gets converted to epoxide form. For re-
which need to adsorb calcium ions. generation of hydroquinone form, epoxide
• Vitamin K dependent clottind factors in- reductase is required.
clude • Epoxide reductase is inhibited by Warfarin
» Clotting factors II, VII, IX and X and hence it acts as an anticoagulant
» Protein C and S
» Osteocalcin
115
ONE LINERS
• The form in which Vitamin A is stored is retinol
• The RDA of Vitamin A in adult male is 1000ug/day
• Vitamin E is a lipid phase chain breaking antioxidant
• The RDA of vitamin E in males is 10 mg/day
• Vitamin E deficiency causes Hemolytic anemia
• Hypervitaminosis E causes Hemorrhagic tendency
• Vitamin K is required as a coenzyme for gamma carboxylase
MCQS
1. Rhodopsin until 4. True about Wald’s 6. Warfarin causes in-

stimulated by light is Visual Cycle is creased concentration of
which of the following?
A. 11 cis Retinol A. Rhodopsin is all trans
B. 11 cis retinal retinal + Opsin A. Menadione
C. All trans Retinal B. Retinal pigment epitheli- B. Hydroquinone
D. All trans retinol um converts 11 cis retinal C. Epoxide form
2.
to all trans retinal D. Factor II
7.Vitamin A deficiency
The form in which vita- C. Light converts 11 cis reti-
min A is transported is nol to all trans retinol
D. RPE65 helps in the forma- leads to?
A. 11 cis retinol tion of 11 cis retinol
5.
B. All trans Retinoic acid • A.Xerophthalmia
C. All trans retinal Chain breaking antioxi- B.Beri-Beri
1.B; 2.D; 3.C; 4.D; 5.D; 6.C; 7.A; 8.C; 9.D; 10.C 11.A;
D. All trans retinol dants are all except C.Pellagra
3.
D.Neuropathy
Retinoic acid plays a A. Tocopherol
WHO GRADING OF
role in all except, B. Ascorbic acid
VITAMIN A DEFICIENCY
C. Polyphenolic flavinols
XN - Nyctalopia
A. Growth D. Superoxide dismutase
X1A – Conjunctival Xerosis
B. Differentiation of epithe-
X1B – Bitot’s spots
lium
X2 – Corneal Xerosis
C. Reproduction
X3A – Keratomalacia (<1/3)
D. Stimulates production of
X3B – Keratomalacia (>1/3)
NGAL
XS – Corneal Scar
XF – Xerophthalmic Fundus
116 >>>
CASE BASED MCQS

8. A 27-year-old sailor presenting with 9. A woman on antidepressants presents
chief complaints of flushing, headache, with bleeding. She gives a history of bulky
nausea, and joint pain. He had consumed stools which stick to the pan. Which of the
800 g of grilled ocean perch liver the day following vitamin deficiencies can cause
before and had experienced numbness bleeding in this condition?
shortly after. Two days later he presented
with exfoliation of skin, persisting, head- A.Vitamin A
ache and vomiting. On examination his BP B.Vitamin D
was high and heart rate was low. Imaging C.Vitamin E
studies did not detect any SOL in the D.Vitamin K
brain. The most probable cause is?
A.Sea sickness
B.Stroke
C.Vitamin A toxicity
D.Vitamin D toxicity
IMAGE BASED MCQS

10. A 7 year old girl presents with bi- 11. Which of the following vitamin deficien-
laterally symmetrical discretempapules cies causes the condition shown in the
with central keratinous plug localized to image?
elbows as shown in the image. The proba-
ble cause is deficiency of A. Vitamin A
B. Vitamin D
A. Retinol C. Vitamin E
B. Retinal D. Vitamin K
C. Retinoic acid
D. Vitamin D
117
VITAMIN D
BIOACTIVATION OF VITAMIN D
• Vitamin D is synthesized by the action of 25 hydroxylase. 25 hydroxycholecalciferol
UV light on 7 dehydrocholesterol, which is is converted to 1,25 dihydroxycholecalcif-
present in the malphigian layer of skin epi- erol by 1 alpha hydroxylase in kidney
dermis. UV light opens up one of the rings • The active form of Vitamin D is 1,25 dihy-
of cholesterol, and hence it gets converted
doxycholecalciferol
to cholecalciferol (hence vitamin D is not a • 1 alpha hydroxylase is the rate limiting
strict steroid, it is a secosteroid) enzyme of vitamin D synthesis, which is
• Cholecalciferol reaches liver, where it is stimulated by parathormone
converted to 25 hydroxycholecalciferol by
SYNTHESIS OF ACTIVE FORM OF VITAMIN D
CHOLECALCIFEROL
25 ALPHA
LIVER
HYDROXYLASE
25 HYDROXY
CHOLECALCIFEROL
1 ALPHA
KIDNEY
HYDROXYLASE
1,25 DIHYDROXY
CHOLECALCIFEROL
ACTIONS OF VITAMIN D
• Vitamin D has receptors in intestine, liver Whenever osteoblasts get activated, in
and kidney related to calcium and phos- parallel osteoclasts also get activated and
phate balance hence it stimulates osteoclasts indirectly,
• In intestine, it increases calbindin expres- • In kidney it increases calcium and phos-
sion, which causes an increase in calcium phate reabsorption
absorption • Summary, Vitamin D increases both calci-
• In bone it has receptors on osteoblasts and um and phosphorus levels in blood
thereby helps in mineralization of the bone.
118 >>>
REGULATION OF VITAMIN D SYNTHESIS

• Vitamin D synthesis levels are regulated • Hypercalcemia indirectly inhibits 1 alpha
based on calcium and phosphorus levels. hydroxylase by inhibiting parathormone
• Hyperphosphatemia directly inhibits 1 secretion
alpha hydroxylase • 1,25 DHCC inhibits 1 alpha hydroxylase
and it stimulates 24 hydroxylase
PTH
+ -
CALCIUM
1αHYDROXYLASE
25OHVITD 1,25DHVITD
PHOSPHATE
-
NORMAL VITAMIN D LEVEL

• 30 to 80ng/mL • Total 25 OH Vitamin D • 25 OH Vitamin D2 and D3
BIOCHEMICAL FEATURES OF VITAMIN D DEFICIENCY
• Vitamin D deficiency causes hypocalcemia • Calcium reabsorption normalizes low calci-

and hypophosphatemia. um. Hence, in vitamin D deficiency, calcium
• Hypocalcemia causes secondary hyper- is low normal or near normal
parathyroidism • Phosphate secretion causes aggravation of
• Parathormone increases calcium reabsorp- hypophosphatemia
tion and phosphate secretion • ALP level is high
Near normal calcium Hypophosphatemia
Secondary
Hyperparathyroidism
Increases Calcium
Phosphate secretion
Reabsorption
Biochemical Features of 2. Hypophosphatemia

Vitamin D deficiency 3. High PTH
1. Normal Calcium 4. High ALP
119
DIFFERENCE BETWEEN VITAMIN D2 AND D3

• Vitamin D2 is Ergocalciferol – artificially » 30 to 80ng/mL
synthesised – double bond in the side chain » Total 25 OH Vitamin D
• Vitamin D3 is cholecalciferol formed nat- » 25 OH Vitamin D2 and D3
urally. Both are functionally similar. Hence • Significance of estimation of 1,25 DHCC
differentiating Vitamin D2 and D3 does
» Chronic Kidney Disease
not provide any clinical significance.
» Hypoparathyroidism
• Normal Vitamin D level – The parameter
estimated is Total 25 hydroxy vitamin D » Sarcoidosis
SIGNIFICANCE OF 1,25 DH VITAMIN D

» Chronic Kidney Disease – low 1,25 DHCC » Sarcoidosis – granulomatous tissues ex-
» Hypoparathyroidism – low 1,25 DHCC press 1 alpha hydroxylase activated and
hence sarcoidosis presents with hyper-
calcemia
CHRONIC KIDNEY DISEASE – CALCIUM AND

PHOSPHORUS BALANCE
» As kidneys fail to filter phosphate, hyper- » Hence serial estimation of PTH and ALP is
hosphatemia is observed. essential
» As 1,25 DHCC is not synthesized, hypoc- » To avoid secondary hyperparathy-
alcemia is a feature of CKD roidism:
» Hyperphosphatemia and hypocalcemia • Calcium supplementation
stimulate parathormone stimulation and this • 1,25 DHCC supplementation
causes secondary hyperparathyroidism • Calcimimetic drugs like cinacalcet and
» Secondary hyperparathyroidism causes re- Etelcacetide
nal osteodystrophy and this causes patho-
logical fractures
ONE LINERS
• The active form of Vitamin D is 1,25 Dihydroxycholecalciferol
• The rate limiting enzyme of vitamin D synthesis is 1 alpha hydroxylase
• 1 alpha hydroxylase is stimulated by parathormone
• Normal serum Vitamin D is 30 to 80 ng/mL
120 >>>
MCQS
1. True about Vitamin D action is 3. 1,25 Dihydroxy Vitamin D is increased in
A. It decreases calcium absorption along A. Chronic Kidney Disease

intestine B. Hypoparathyroidism
B. It stimulates osteoblasts indirectly C. Pseudohypoparathyroidism
C. It stimulates osteoclasts directly D. Sarcoidosis
4.
D. It increases serum calcium and phosphorus
2.
True about Vitamin D deficiency is
True about Vitamin D synthesis and
regulation are all except A. High Calcium
B. High Phosphorus
A. 1 alpha hydroxylase is the rate limiting C. High PTH
enzyme D. Low ALP
B. 1 alpha hydroxylase is stimulated by PTH
C. 1 alpha hydroxylase is directly inhibited
by high phosphate
D. 1 alpha hydroxylase is directly inhibited
by high calcium
CASE BASED MCQS IMAGE BASED MCQS
5. A known patient of chronic kidney 6. X,Y, A and B respectively are

disease presents with low serum
calcium, high serum phosphorus, A. 25 α hydroxylase,
high ALP. Which of the following is 1 α hydroxylase, CHOLECALCIFEROL
true? liver and kidney
B. 25 β hydroxylase, X A
A. His 1,25 DHCC is expected to be high 1 β hydroxylase, 25 HYDROXY

B. His PTH is expected to be low liver and kidney CHOLECALCIFEROL
C. Cinacalcet is recommended C. 25 α hydroxylase,

Y B
1D; 2.D; 3.D; 4.C; 5.C; 6.A.
D. He is prone to develop hypoparathy- 1 α hydroxylase,

roidism kidney and liver 1,25 DIHYDROXY
CHOLECALCIFEROL
D. 25 β hydroxylase,
1 β hydroxylase,
kidney and liver
121
WATER SOLUBLE VITAMINS
>> VITAMIN B1
THIAMINE
• It is a sulphur containing vitamin • PDH

• It is present in the aleurone layer of germs • αKGDH
and hence polishing causes loss of thia- • Branched chain ketoacid dehydrogenase
mine » Transketolase
• Coenzyme roles of thiamine: • RDA of thiamine is 0.5mg/1000C
» Oxidative decarboxylation • Thiamine deficiency causes beriberi.
BERIBERI
Dry beriberi (CNS involvement) Wet beriberi (CVS involvement)
• Thiamine is necessary for the aerobic utili- • Thiamine is necessary for PDH complex
zation of glucose • Defect of PDH complex causes pyruvate to
• Neurons use glucose aerobically be converted to lactate
• Hence thiamine deficiency causes dry • Lactate is a vasodilator and hence it causes
beriberi hypotension and tachycardia
ALCOHOLISM AND THIAMINE

• Wernicke’s encephalopathy – Acute thia-
mine Deficiency - GOA
• Korsakoff syndrome- Chronic thiamine defi-
ciency – Amnesia and confabulation
122 >>>
>> RIBOFLAVIN
COENZYME ROLES
COENZYME FORMS: » Acyl CoA dehydrogenase uses FAD as a

• FMN coenzyme
• FAD • In Aminoacid metabolism
BIOCHEMICAL ROLES: » L – Aminacid oxidase uses FAD as a
• In Electron Transport chain coenzyme
• In Carbohydrate metabolism • Citric acid cycle
» PDH has FAD as a coenzyme » Succinate dehydrogenase uses FAD as a
• In Lipid metabolism coenzyme
RIBOFLAVIN DEFICIENCY
» Earliest manifestation: Circumcorneal
neovascularization
» Diagnostic investigation: RBC Glutathione
reductase
>> VITAMIN B12
COENZYME ROLES OF B12

>> Methyl B12: Homocysteine methyl trans- yl CoA. D methyl malonyl CoA in the pres-
ferase or methionine synthase : ence of racemase forms L methylmalonyl
» Involved in the conversion of Homocyst- CoA. L methyl malonyl CoA in the presence
eine to methionine of methyl malonyl CoA mutase forms succi-
» Hence deficiency of B12 causes homocyst- nyl CoA.
einuria and methyl folate trap » In B12 deficiency methylmalonyl CoA accu-
» Functional deficiency of THFA causes mac- mulates causing methyl malonic aciduria.
rocytic anemia » Methyl malonyl CoA gets incorporated into
>> Adenosyl B12: Methyl malonyl CoA mutase fatty acid causing abnormal myelin forma-
» Odd chain fatty acids on oxidation forms tion. This causes demyelination and hence
propionyl CoA. Propionyl CoA in the pres- the neurological manifestations of B12
ence of carboxylase forms D methyl malon- deficiency.
123
METHYL MALONIC ACIDURIA IN B12 DEFICIENCY
ADENOSYL B12
METHYL MALONYL
SUCCINYL COA
COA
METHYL MALONYL
METHYL MALONIC COA MUTASE
ACIDURIA
?? B12 DEFICIENCY
ODD CHAIN FATTY
PROPIONYL COA
ACID OXIDATION
OVERNIGHT
FAST
FACTORS AFFECTING VITAMIN B12

ABSORPTION
I. As Vitamin B12 sources are non vegetarian receptors. Cobalophilin receptors bind to
and hence they are attached to proteins. B12 only after it is attached to IF. Hence IF
To remove proteins proteolytic enzymes of is essential
pancreas is essential III. Intestinal microorganisms utilise B12
II. For Vitamin B12 to be absorbed in terminal IV. Health of terminal Ileum
ileum, it should be attached to cobalophilin
CAUSES OF VITAMIN B12 MALABSORPTION

• Pancreatic insufficiency • Blind loop syndrome
• IF deficiency • Crohn’s disease
SCHILLING’S TEST
PURPOSE: • Step 1: To treat Vitamin B12 and folate
» To identify the presence of Vitamin B12 deficiency
malabsorption • Step 2: Oral radiolabelled Vitamin B12 is
» To identify the cause of Vitamin B12 malab- provided
sorption • Step 3: IM unlabelled vitamin B12 is admin-
STEPS: istered to saturate all Vitamin B12 receptors
present in the liver
124 >>>
• Step 4: 24 hours urine is collected and » Inference: If less than 10% of orally ad-
labelled vitamin B12 is estimated ministered Vitamin B12 is found in urine
• Inference: If less than 10% of orally adminis- – Blind loop syndrome is excluded
tered Vitamin B12 is found in urine – malab- • Step 7: Test is repeated after 2 days of
sorption is confirmed pancreatic enzymes
• Step 5: Test is repeated after oral IF » Inference: If less than 10% of orally ad-
» Inference: If less than 10% of orally administered Vitamin B12 is found in urine
ministered Vitamin B12 is found in urine – pancreatic insufficiency is excluded
– Autoimmune gastritis is excluded • Inference 2: Probably crohn’s disease or
• Step 6: Test is repeated after 3 weeks celiac sprue
antibiotic
B6 DEFICIENCY DETECTION
??? B SIX
TRYPTOPHAN
DEFICIENCY
TRYPTOPHAN
PYRROLASE
FORMYL XANTHURENIC
KYNENURINE ACID
THFA FORMYL
TRANSFERASE
FORMYL
KYNENURINE TRYPTOPHAN
THFA
XANTHURENIC HYDROXYLASE
ACID 3 HYDROXY
KYNENURINE
PLP KYNENURINASE
3 HYDROXY
ANTHRANILIC
ACID
NAD
FOLATE DEFICIENCY DETECTION
??? FOLATE HISTIDINE

DEFICIENCY
FIGLU UROCANATE
HISTIDINE
IMIDAZOLE
PROPIONIC ACID
HIGH
FIGLU
FIGLU
THFA
FORMIMINO THFA
TCA
GLUTAMATE CYCLE
125
VITAMIN DEFICIENCY DIAGNOSIS

SUSPECTED VITAMIN
INVESTIGATION LOAD
DEFICIENCY
VITAMIN B SIX
FOLATE
VITAMIN B12
SUSPECTED VITAMIN
INVESTIGATION LOAD
DEFICIENCY
VITAMIN B SIX Xanthurenic acid Tyrptophan load
FOLATE FIGLU Histidine load
VITAMIN B12 Methyl malonic acid Overnight fast
ONE LINERS
• Schilling’s test is used to diagnose Vitamin B12 malabsorption
• Vitamin K form used to treat Vitamin K deficiency due to fat malabsorption is Menadione
• Vitamin E RDA is dependent on Fatty acid intake
• RDA of Vitamin B1 is dependent on carbohydrate intake
• Tryptophan load test is done to detect B6 deficiency

• Histidine load test is done to detect folate deficiency
126 >>>
MCQS
1.
3. 5.
Thiamine deficiency is
diagnosed by measuring Methylfolate trap in All the following are
which of the following? Vitamin B12 deficiency is PLP dependent states
because of the inhibition except?
A. RBC Glutathione reduc- of?
tase activity A. Anemia
B. RBC Transketolase activity A. Methyl Malonyl CoA mu- B. Seizures
C. RBC G6PD activity tase C. Pellagra
D. RBC Glutathione peroxi- B. Methylmalonyl CoA D. Albinism
dase activity isomerase
2.
C. Methionine synthase
Riboflavin deficiency D. Cystathionine Beta syn-
is diagnosed by measur- thase
4.
ing which of the follow-
ing?: PLP is necessary as a
coenzyme for all except?
A. RBC Glutathione reduc-
tase activity A. Transaminases
B. RBC Transketolase activity B. Glutamate decarboxylase
C. RBC G6PD activity C. Methionine synthase
D. RBC Glutathione peroxi- D. Cystathionine Beta syn-
dase activity thase
CASE BASED MCQS

6. A chronic alcoholic presents with an A. RBC transketolase activity
episode of hypoglycemia. He is treated B. RBC Glutathione reductase activity
with dextrose infusion and thiamine C. Urine methyl malonic acid level
injection. Before discharge, he is advised D. Urine Xanthurenic acid level
about effects of chronic alcoholism and
he wants to check his thiamine availability
status. What is the investigation you
would prescribe?
127
7. A known patient of Tuberculosis is 8. A person, who is on mixed

on INH and he presents with microcytic balanced diet. presented with tiredness
hypochromic anemia. His serum ferritin ,weakness, macrocytic anemia and
and transferrin saturation are normal. A subacute combined degeneration.
vitamin deficiency is suspected. Which He was given radiolabelled B12 orally
vitamin deficiency can cause microcytic and then intramuscular unlabelled
hypochromic anemia and what is the B12 was injected. 24 hours urinary
investigation of choice for diagnosing this radiolabelled B12 was less than 10% of
condition? oral administered dose. After oral IF, 24
hours urinary labelled B12 was more than
A. B12, methylmalonic acid in urine 10% oral administered dose. Which of the
B. Folate, FIGLU level in urine following is the probable cause?
C. B2, Glutathione reductase activity in RBCs
D. PLP, Xanthurenic acid in urine A. Dietary B12 deficiency
B. Methyl Folate trap
C. Pancreatic insufficiency
D. Type A gastritis
IMAGE BASED MCQS
9. A 65 year old man with alcohol

use disorder, presented with insomnia,
irritability and confusion. His son
attributed the confusion to a probable
dehydration caused by diarrhoea. On
examination, there were erythematous
non itchy skin lesions in the sun exposed
areas as shown in the image. The
probable cause of confusion is
A. Wernicke’s encephalopathy
B. Delerium tremens
C. Korsakoff syndrome
D. Pellagra
128 >>>
PREVIOUS YEAR QUESTIONS

10. Which of the following plays a role in
collagen maturation?
A. Copper and Zinc

B. Copper and Ascorbic acid
C. Proline
D. Phenylalanine
COLLAGEN SYNTHESIS
• Triple helix
• 1000 aminoacids : Gly –
Procollagen
X–Y
Intracellularly
• Hydroxyproline and Hy-
droxylysines
• Prolyl or lysyl hydroxylase
- Vitamin C
• Glycosylation
• Triple helix is formed
COLLAGEN SYNTHESIS EXTRACELLULAR

• N terminal and C terminal ends of the • Quarter staggered alignment
triple helix is cleaved - tropocollagen
Lysyl Oxidase – Copper dependent
129
11. A patient presented to the OPD with A. Copper

liver damage. The picture depicts the B. Glucose
patient having their eyes examined. C. Galactose
Which of the subsequent factors is the D. Mannose
cause of this condition?
SNOWFLAKE
OILDROP CATARACT –
SUNFLOWER CATARACT CATARACT – Juvenile

GALACTOSEMIA
Diabetes
WILSON’S DISEASE
• ATP7B mutation
Ceruloplasmin less than 0.2g/L
• Copper secretion into bile – copper
toxicity – liver damage and lenticular
Urinary copper more than
nucleus
100ug/24hours
• Copper incorporation into ceruloplas-
min – low ceruloplasmin, low serum
copper and high urinary copper Liver copper more than 250ug/g
12. Breastfeeding is contraindicated in 13.. A patient presented to the OPD with

which of the following conditions? complaints of Diarrhea. The patient was
later diagnosed with dementia and photo-
A. Galactosemia sensitive dermatitis in sun-exposed areas.
B. CMV Which Vitamin deficiency is responsible
C. Hepatitis B for this condition?
D. West Nile Virus
A. Vitamin B6
B. Niacin
C. Ascorbic acid
D. Biotin
130 >>>
14. A known patient of Tuberculosis, on INH STRUCTURAL ANALOGUE OF

for six months presents with tingling sensa-
PYRIDOXAL
tion and paraesthesia. Deficiency of which
of the following vitamins is the most proba-
ble cause? COMPETITIVE INHIBITOR OF
PYRIDOXAL KINASE
A. Vitamin B6
B. Vitamin B3
C. Vitamin B12 NO FORMATION OF
PYRIDOXAL PHOSPHATE
D. Vitamin B7
15. Which of the following enzyme activi-

ties can be estimated in RBCs to diagnose
Vitamin B2 deficiency?
A. Transketolase
B. Glutathione reductase
C. Kynenurinase
D. Pyruvate Dehydrogenase
1.B; 2.A; 3. C; 4.C; 5.D; 6.A; 7.D; 8.D; 9.D; 10.B; 11.A; 12.A; 13.B; 14.A; 15.B.
131
ALL ABOUT ABG
INTERPRETATION IN
AN HOUR
INTERPRETATION OF ABG REPORT

• Can be done in 5 steps: • Step 5: If increased anion gap metabolic
• Step 1: Check pH acidosis, calculate delta delta ratio to find
• Step 2: Identify the primary acid base dis- hidden normal anion gap metabolic acido-
order sis or metabolic alkalosis
• Step 3: Calculate compensation to find • Step 6: If normal anion gap metabolic aci-
hidden acid base disorder dosis, calculate urinary anion gap to find if
its gI loss or urinary loss of bicarbonate
• Step 4: If the primary acid base disorder is
metabolic acidosis, calculate anion gap to
identify the cause of metabolic acidosis
STEP 1: CHECK PH
• Normal values: • If pH is less than 7.36, it is acidosis, if pH is
» pH = 7.36 to 7.44 more than 7.44, it is alkalosis
» PCO2 =36 to 44 mmHg • Normal pH does not exclude an acid base
» HCO3 = 21 to 27 mEq/L disorder, it can be a mixed acid base disorder
STEP 2: CHECK BICARBONATE AND PCO2
• Purpose is to identify if it is a respiratory or • If high pH is accompanied by high bicarbo-

metabolic disorder nate, it is metabolic alkalosis
• If low pH is accompanied by low bicarbo- • If high pH is accompanies by low pCO2 it is
nate, it is metabolic acidosis respiratory alkalosis
• If low pH is accompanied by high PCO2, it is
respiratory acidosis
STEP 3: CALCULATE COMPENSATION
• Purpose is to identify hidden acid base » In respiratory disorders, there is an acute

disorders compensation caused by the shift in
• BASICS OF COMPENSATION: equilibrium and a chronic compensation
» Compensation is parallel, if the primary caused by kidneys
is a change in bicarbonate, the compen- • Formulae for compensation:
sation is a change in Carbon dioxide and » Metabolic acidosis - Expected pCO2 =
the change is parallel. Decrease in bicar- 1.5XHCO3 + 8
bonate is compensated by a decrease in » Metabolic alkalosis - Expected pCO2 =
PCO2 and viceversa 0.9XHCO3 + 16
133
FORMULAE
>> RESPIRATORY DISORDERS
DISORDER ACUTE CHRONIC
RESPIRATORY 1 3.5
ACIDOSIS 10mmHg 1mEq/L 3.5mmEq/L
PCO2 HCO3 HCO3
RESPIRATORY 2 5
ALKALOSIS 10mmHg 2mEq/L 5mEq/L
PCO2 HCO3 HCO3
STEP 4: IF METABOLIC ACIDOSIS, CALCULATE ANION GAP

» Purpose is to identify the cause of meta- an abnormal acid eg., ketoacidosis, lactic
bolic acidosis acidosis, methanol, ethanol or ethylene
» Anion gap = [Na] – {[cl] + [HCO3]} glycol poisoning
» Normal anion » If the anion gap is normal, the metabolic
» If the anion gap is high, the metabolic acidosis is caused by gastrointestinal or
acidosis is caused by the production of renal loss of bicarbonate
METABOLIC ACIDOSIS
ANION GAP
INCREASED ANION
NORMAL ANION GAP
GAP
1. DKA 1. GI LOSS
2. LA 2. Type I RTA
3. METHANOL/ETHANOL 3. Type II RTA
4. SALICYLATE 4. Type IV RTA
STEP 5: IF HIGH ANION GAP METABOLIC ACIDOSIS,

CALCULATE DELTA DELTA RATIO
» Delta Delta ratio = Anion gap -12

24 – Bicarbonate
134 >>>
» If delta delta ratio is between 1 and 2, it » If delta delta ratio is less than 1, it is
is only increased anion gap metabolic increased anion gap metabolic acidosis
acidosis + normal anion gap metabolic acidosis
» If delta delta ratio is more than 2, it is
increased anion gap metabolic acidosis
+ metabolic alkalosis
STEP 6: IF NORMAL ANION GAP METABOLIC ACIDOSIS,

CALCULATE URINE ANION GAP
» Urinary anion gap = (Na + K) – Cl » If urinary anion gap is positive, it is renal
» If urinary anion gap is negative, it is gut loss of bicarbonate
loss of bicarbonate (diarrhea)
MCQS
1. Give your opinion regarding the acid 3. Interpret the ABG report
base status of a blood sample that was Blood pH : 7.30
taken from a person, who was acutely pCO2 : 29 mmHg
hysterical. Plasma HCO3 :14 mEq/L
Blood pH: 7.55 Na+: 130 meq/L
pCO2 :20 mmHg Cl-: 90 meq/L
Plasma HCO3 :20 mEq/L
A. Compensated Increased anion gap meta-
A. Respiratory acidosis bolic acidosis
B. Respiratory alkalosis B. Uncompensated Increased anion gap
C. Metabolic acidosis metabolic acidosis
D. Metabolic alkalosis C. Compensated Normal anion gap metabol-
2.
ic acidosis
Interpret the ABG report D. Uncompensated Normal anion gap meta-
Blood pH : 7.30 bolic acidosis
pCO2 : 29 mmHg
Plasma HCO3 :14 mEq/L
A. Compensated metabolic acidosis

B. Uncompensated metabolic acidosis
C. Compensated respiratory acidosis
D. Uncompensated respiratory acidosis
135
4. Interpret the following Venous ABG Cl: 109mEq/L

report and identify a probable cause after Spot Urine:
considering the spot urine values: Na: 20mEq/L
pH:7.411 K: 5.6mEq/L
PO2: 72.7mmHg Cl: 22mEq/L
PCO2: 23.7mmHg pH:7
Base Excess: -7.7mmol/L
Base Excess (ECF): -9.9mmol/L A. Diarrhea
cHCO3: 17.5mmol/L B. Distal Renal Tubular acidosis
cHCO3 (std): 20.1mmol/L C. Proximal renal tubular acidosis
Na: 138mEq/L D. Lactic acidosis
K: 2.56mEq/L
METABOLIC NORMAL ANION

RESPIRATORY
ACIDOSIS WITH GAP METABOLIC URINARY ANION ALKALOSIS WITH
RESPIRATORY ACIDOSIS GAP IS POSITIVE METABOLIC
COMPENSATION COMPENSATION COMPENSATION
5. Identify the acid base disorder in a 7. A 7 week old baby was brought by
patient with the following values. the mother with complaints of repeated
pH – 7.2 projectile vomiting and pellet stools. The
PO2 – 90mmHgpCO2 – 80mmHg probable metabolic disturbance is
Bicarbonate – 35mEq/L
A. Normal anion gap Metabolic acidosis
A. Metabolic alkalosis B. Hypochloremic hypokalemic Metabolic
B. Metabolic acidosis alkalosis
C. Respiratory alkalosis C. Hyperchloremic hypokalemic Metabolic
D. Respiratory acidosiss alkalosis
6.
D. Respiratory acidosis
8.
60 year old diabetic patient presented
with repeated vomiting following a recent A hyperventilating hysterical woman
1.B; 2.A; 3.A; 4. B; 5.D; 6.A; 7.B; 8.C.
dine out. Her blood pressure was 90/60. presents with carpopedal spasm. The
pH was 7.3. HCO3: 18mEq/L, PCO2: cause is
35mmHg. Identify the acid base disorder.
A. High Total calcium
A. Metabolic acidosis B. Low total calcium
B. Metabolic alkalosis C. Alkalosis
C. Respiratory alkalosis D. Acidosis
D. Respiratory acidosis
136 >>>

Biochemistry - PART II - Day 4

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Biochemistry - PART II - Day 4

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HDL Metabolism 17 Protein structure 81

Hyperlipoproteinemia 20 AMINOACID METABOLISM 84

RNA, Transcription and post Vitamin K 115

HMG CoA MEVALONATE

Squalene Lanosterol Zymosterol Desmosterol

REGULATION OF HMG COA REDUCTASE

• Allosteric Regulation • It is activated by dephosphorylation

1.D; 2.A; 3.B.

FATTY ACID OXIDATION

DIFFERENCE BETWEEN MITOCHONDRIAL AND

S.No PROPERTY MITOCHONDRIA PEROXISOME

1 Type of fatty acids

S.No PROPERTY MITOCHONDRIA PEROXISOME

2 Type of oxidation β oxidation β oxidation

3 Products n/2 acetyl CoA n/2 acetyl CoA

4 ATP + No ATP, H2O2 peroxidation

HIGH LOW Acetyl CoA

PHASES OF FATTY ACID OXIDATION

STEPS OF EVERY CYCLE OF BETA OXIDATION

β HYDROXY ACYL CoA

DETAILS OF PHASE I – EVERY CYCLE

• Acyl CoA 4 Acetyl CoA

FORMULA FOR CALCULATION OF ENERGETICS OF FATTY

METABOLIC CHANGES IN DKA

KETONE BODY SYNTHESIS

1 Acetyl CoA Acetyl CoA β HYDROXYBUTYRATE

Acetyl CoA HMG CoA

HMG CoA Acetoacetate

FACTS ABOUT KETONE BODY METABOLISM

CAUSES OF DEFECTS OF BETA OXIDATION

>> Congenital: >> Acquired:

BIOCHEMICAL FEATURES OF FATTY ACID

Dicarboxylic aciduria tion). Peroxisomes can not oxidise very

2. Very short chain fatty

3. Very long chain fatty

7. Number of ATP gener-

11. Fatty acid oxidation

4. Rate limiting enzyme of

8. Number of ATP gener-

A. Carnitine Acyl Transferase ated in the liver by complete

BASIC STRUCTURE OF A LIPOPROTEIN

INNER NON POLAR CHOLESTEROL

chylomicron with the help of HDL micron to Chylomicron remnant

TWO FATES OF VLDL

TWO FATES OF IDL

ACTION OF LCAT & ABCI

• LCAT converts cholesterol in discoidal extrahepatic tissue membranes and is

DISORDERS OF HDL METABOLISM

protein lipase is:

6. The apoprotein present in nascent chy-

3. Apo CII activates:

A. Lipoprotein Lipase 7. True regarding chylomicron are all

4. Chylomicron transports triacylglycerol

9. Remnant receptor accepts lipoproteins

10. LDL receptor accepts lipoproteins

with which of the following apoproteins?

Friedrickson’s Classification » Type I » Type III

• Isolated Hypercholesterolemia – This • Isolated Hypertriglyceridemia – This cate-

accelerated atherosclerosis. Tendon xan- eruptive xanthomas and lipemia retinalis.

TYPE IIA HYPERLIPOPROTEINEMIA

• It is otherwise called as Familial Chylomi- is to be obtained, as LPL is clinging to

TYPE III HYPERLIPOPROTEINEMIA