Download File

INTRODUCTION TO BIOMOLECULES AND METABOLISM:
Bio-molecule
Are the biological molecules that particitipates in the physiological functioning of the living
things. They are either organic or inorganic in nature. They may be classified as monomeric or
polymeric compounds.
They include; Carbohydrates, Lipids, Proteins and amino acids, Enzymes, Plasma proteins,
nucleic acids, Immunoglobulin, Biological oxidation, Haemoglobin and haemoglobinopathies,
Vitamins, Prostaglandins.
Metabolism:
These are the sequences of the biochemical reaction that degrade, synthesize, or interconvert
small molecules inside a cell. This is for the understanding both normal functioning and
metabolic basis of human diseases, molecular basis of drug action, drug interaction and many
genetic diseases that are caused by the absence of a particular proteins or enzyme.
The metabolic pathways include, gluconeogenesis, glycolysis, TCA cycle, β oxidation of fatty
acids e.t.c
Metabolism is divided into two
a. Catabolism:
The degradation process concerned with breakdown of complex molecules to simpler
ones, with a corresponding release of energy in the form of ATP i.e. glycolysis TCA
cycle , oxidation of fatty acids
b. Anabolism:
This is the biosynthesis reaction involving the synthesis or the formation of complex
molecules from simple precursor i.e. gluconeogenesis, glycogenesis
Food and fuels found in the body:
There are three kinds of fuels found in the body. That is
1. Amino acids and proteins
2. Carbohydrates
3. Fatty acids and ketones
These fuels undergoes the inter conversion process to yield or utilize energy in the form of ATP
The inter conversion requires ATP as the high energy compound. The process by which the ATP
is formed or generated and utilized is known as ATP-ADP cycle
The dietary fuel being the carbohydrates, proteins and fats the can undergoes the digestion and
absorbed and the products of digestion undergoes circulation in the blood and enter into various
tissues and are eventually taken up by cells and get oxidized to produce energy and carbon
dioxide and water molecules and oxygen is required for oxidation process to take place.
The fuels are stored in the body as;
Fats: triglycerides in the adipose tissues
Carbohydrates: glycogen in muscles, liver and other cells
Protein: in muscles
The oxidation of fuels is also referred to respiration process. It requires the utilization of oxygen
to yield carbon dioxide and water. Several pathways are there which involves generation of the
ATP from the fuels and the oxidation of glucose, fatty acids, and the amino acids to acetyl CoA a
substrate for the TCA cycle. In the TCA cycle they are completely oxidized, electrons are
transferred to oxygen by the electron transport chain and the energy is used to generate ATP (
formed conversion of the ADP and Pi to ATP by the process known as Oxidative
phosphorylation.
TOPIC: BIOENERGETICS
Bioenergetic is the study of the energy transfer and utilization in biochemical reaction. It is also
known as biochemical thermodynamics.
Bioenergetics is classified into two:
 Endergonic reaction: this involves energy utilization
 Exergonic reactions: this involves energy release.
Bioenergetics generally deals with the initial and the final state of the reactants and not
mechanism of reaction.
Free Energy: (G)
This is the utilizable or energy available to do work which can be utilized (absorbed) or released
and is denoted by (G). A decrease in free energy leads to spontaneous reaction.
Enthalpy ( H): is a measure of change in heat between reactants and the products.
Entropy( S): represents a change in randomness or disorder of reactants and products which
attains its maximum as the reactions approaches equilibrium.
Relationship between Free energy, entropy and enthalpy:
FREE ENERGY (G) = ENTHALPY ( H) –T ENTROPY ( S) ~ T=absolute temperature
in Kelvin ( K=273+oC)
Standard free energy (G0) is the free energy change when reactants or products are at
concentration of 1 mol/l at pH 7.0.
Negative and Positive free Energy (G):
Negative free energy (-G): is known as exergonic reaction referring to loss of energy leading to
spontaneous reaction.
Example:
ATP +H2O
ADP +Pi (G0 =-7.3 Cal/Mol)
Positive free energy (+G): is known as endergonic reaction referring to utilization or supply of
energy hence non-spontaneous reaction.
ADP +Pi
ATP
(G0 =7.3 Cal/Mol)
Free energy change is zero when the reaction is at equilibrium hence at a constant temperature
and pressure, change in free energy (G) depends on the concentration of product and
concentration of reactant. Example; In the conversion of A to B,
Mathematically;
G =G0 +RT ln [B]
[A]
Whereby;
G0 =Standard free energy change
R=gas constant (1.987 Cal/mol)
T=Absolute temperature (273+0C)
Ln =Natural Logarithm
[B]=concentration of products
[A]= concentration of reactants
G0 Is related to equilibrium constant in case of reactions; example; conversion of A
B while free energy is 0.Hence;
G =0 =G0 +RT ln [B]Eq
[A]Eq
Hence =G0 =-RTln Keq
HIGH ENERGY COMPOUND:
These are the substances which posses sufficient free energy to liberate at least 7 cal/mol at PH
of 7.0 i.e. ATP
High energy compounds can be classified as;
1. Pyrophosphates i.e ATP
2. Acylphosphates i.e. 1,3 bisphosphoglycerates
3. Enol phosphates i.e. phosphoenolpyrophosphate
4. Thioether e.g acetyl COA
5. Phosphoguanidines e.g. phosphocreatine.
High energy compounds: are compounds that possess acid anhydride bonds (phosphoanhydride
bonds) whish are formed by the condensation of two acidic groups or related compounds.
Lipmann suggested the symbol ~ to represent high energy bond hence ATP can be written as
AMP~P~P
SNO
COMPOUND
HIGH ENERGY PHOSPHATES
1. Phosphoenol pyruvate
2. Carbamoyl phosphate
3. Cyclic AMP
4. 1,3 bisphosphoglycerate
5. Phosphocreatine
6. Acetyl phosphate
7. S-Adenosylmethionine*
8. Pyrophosphates
9. Acetyl COA**
G0 (Cal/Mol)
-14.8
-12.3
-12.0
-11.8
-10.3
--10.3
-10.0
-8.0
-7.7
10. ATP
-7.3
LOW ENERGY COMPOUNDS
11. ADP
-6.6
12. Glucose 1-phosphate
-5.0
13. Fructose 1-phosphate
-3.8
14. Glucose 6 phosphate
-3.3
15. Glycerol 3 phosphate
-2.2
 *Sulfonium compounds
 ** thioether
ATP-ADP CYCLE:
The hydrolysis of ATP is associated with the release of large amount of energy.
ATP +H2O
ADP +Pi (G0 =-7.3 Cal/Mol)
The energy which is liberated can be utilized by various process like muscle contraction, active
transport, e.t.c
ATP can also act as donor of high energy phosphates to low energy compounds making them
rich and on the other hand ADP can accept high energy phosphates from compounds possessing
higher free energy contents to form ATP.
ATP serves as an immediately available energy currency of a cell which is constantly utilized
and regenerated bearing a high turn over
ATP acts as an energy link between the catabolism (degradation of molecules) and anabolism
(synthesis) in the biological system.
SYNTHESIS OF ATP:
ATP can be synthesized in two ways;
1. Oxidative phosphorylation
Is an aerobic major source of energy which is linked with mitochondrial ETC
2. Substrate level phosphorylation
ATP synthesis occurring directlysubstrate oxidation bearing high energy compounds like
phosphoenolpyruvate and 1,3 bisphosphoglycerate (intermediate in glycolysis), succinyl COA in
TCA cycle which are involed in the transfer of phosphate energy to produce ATP
High energy compounds are stored in vertebrates as phosphocreatine or creatine phosphate in
muscles and brains and in inverterbrates is stored as phosphoarginine or arginine phosphate.
ORGANIZATION OF ELECTRON TRANSPORT CHAIN:
METABOLIC PATHWAYS
TOPIC: CARBOHYDRATE METABOLISM
GLYCOLYSIS
A
B
4
4
3
C
5
KEY:
A, B, C are allosteric regulation steps of glycolysis
 3- bromohydroxyacetone phosphate inhibitor
 4- iodoacetate and arsenate inhibitor
 5-fluoride inhibitor
Glylycosis pathway is also known as Embden- Meyerhof pathway and it is a catabolic
reaction.
Glycolysis pathway Occur in two ways:
1. Aerobic: in the presence of oxygen and the end product is pyruvate and later it is oxidized
to carbon dioxide and water
2. Anaerobic: in the absence of oxygen and the end product is Lactate and it is summarized as
Glucose +2ADP +2Pi
2 Lactate +
2ATP
It occurs in the tissue which are lacking mitochondria i.e erythrocytes, cornea, lens and it is
essential source of energy for brains.
REACTIONS OF GLYCOLYSIS:
A. Energy investment phase or priming phase
B. Splitting phase
C. Energy generation phase
Energy investment phase:
 Glucose gets phosphorylated to glucose 6 phosphate by hexokinase or glucokinase
depending on ATP ang Mg2+
NB: glucose 6-phosphate is impermeable into cell membrane hence its metabolic fates are
glycolysis, glycogenesis, gluconeogenesis, and pentose phosphate pathway (Hexose
Monophosphate Shunt pathway-HMP)
 Isomerization of glucose 6 phosphate to fructose 6 phosphate in the presence of
phosphohexose isomerase and Mg2+
 Fructose 6 phosphate gets phosphorylated tofructose 1-6-bisphosphate by
phosphofructokinase (PFK). PFK is an allosteric and regulatory step in glycolysis
Splitting Phase:
 Fructose 1,6 biphosphate gets split into glyceraldehydes 3 phosphate and
dehydroxyacetone phosphate by the enzyme aldolase (Fructose 1,6 biphosphate
aldolase).
 Glyceraldehydes 3 phosphate and dehydroxyacetone phosphate get interconverted by
phosphotriose isomerase enzyme.
It gets inhibited by bromohydroxyacetone
phosphate.
Energy generation phase:
 Oxidative phosphorylation occurs when Glyceraldehydes 3 phosphate get converted into
1,3 bisphosphoglycerate in the presence of Glyceraldehydes 3 phosphate
dehydrogenase in the precence of NAD+ which undergoes reduction by accepting
Hydrogen ions to NADH and addition of phosphate to Glyceraldehydes 3 phosphate. 1,3
bisphosphoglycerate is a high energy compound formed. (NADH has 3 ATP hence 3X2=
6 ATPs formed). The reaction is Inhibited by iodoacetate and arsenate
 Substrate level phosphorylation occurs when1,3 bisphosphoglycerate is converted
into3-phosphoglycerate hence synthesis of ATP in the presence of phosphoglycerate
kinase.
 3 phosphoglycerate is converted into 2 phosphoglycerate in the presence of
phosphoglycerate mutase and Mg2+.
 Phosphoenol pyruvate is formed from 2-phosphoglycerate in the presence enolase
enzyme and Mg2+ or Mn2+ which is inhibited by fluoride.
 Pyruvate is formed from phosphoenol pyruvate in the presence of pyruvate kinase
which requires K+and Mg2+ or Mn2+ with release of ATP.
NB: Total number of ATP Generated: 8 ATPs -2ATP Utilized= 6 ATPs
REGULATION OF GLYCOLYSIS:
Regulation of glycolysis is done by three enzymes, hexokinase or
phosphofructokinase and pyruvate kinase due to its irreversible reactions.
glucokinase,
1. Hexokinase is inhibited by glucose 6 phosphatase accumulation due to product
inhibition.
2. Phosphofructokinase (PFK) which is allosteric enzyme and is regulated by allosteric
effectors, ATP, citrate and H+ ions (low PH). Fructose 2,6 bisphosphate,AMPand Pi are
allosteric activators.
3. Pyruvate kinase is inhibited by ATP and activated by F16BP. Pyruvate kinase is active in
dephosphorylated state and inactive in phosphorylated state which is brought about by
cAMP dependent protein i.e. hepatic glucagon
ROLE OF HORMONES IN METABOLISM
1. INSULIN:
 Secreted in the pancreas by islets of langerhans
 It is made up of amino acids, cystein,proline, isoleucine
 The hormone requires zinc (metal)for its crystallization
Functions:

Increases glucose uptake ,glycolysis, conversion of pyruvate to acetyl CoA and fatty acid
synthesis
 It stimulates glycogenesis
 It decreases lipolysis
2. GLUCAGON:
 Secreted in the pancreas by islets of langerhans
 It is made of amino acids like tyrosine methionine and tryptophan.
 It does not require metal ions for its crystaization
Functions:


Mobilization of hepatic glycogen to give glucose by glycogenolysis
It increases glycogenolysis, gluconeogenesis and lipolysis hence regulating decreased
amount of blood glucose.
3. Hormones of hypophylysis
 Thyrotropin i.e. thyroid stimulating hormone (TSH)
Functions:
 Stimulates the synthesis of thyroid hormones
 Increases the DNA contents
 Stimulates glycolysis
CONVERSION OF PYRUVATE TO ACETYL COA
Pyruvate is converted to Acetyl CoA by the process of Oxidative decarboxylation.
This reaction is catalyzed by the enzyme pyruvate dehydrogenase complex (PDH) found in the
mitochondria, cardiac muscles and kidney.
The co-factor (co-enzymes) required are:
TPP, lipoamide, FAD, Coenzyme A and NAD+
The overall reaction of PDH is:
Pyruvate +NAD+ + CoA
Reaction:
PDH
Acetyl Coa + CO2 +NADH +H+
TCA CYCLE
TRI CARBOXYLIC ACID is also known as KREBS CYCLE or CIRTIC ACID CYCLE
It involve the oxidation of Acetyl Coa to caron dioxide and water and it is the common oxidative
pathway to carbohydrates,fats, and amino acids.
It intermediates include:amino acids, heme, glucose.
Occurance: mitochondria
In triarboxylic acid cycle it involves combination of 2 carbon atom ACETYL CoA and 4
carbon atom OXALOACETATE to form a 6 carbon atom tricarboxilic acids CITRATE
OXALOACETATE plays a major catalytic role in TCA cycle
Function: To burn the acetyl-CoA made from fat, glucose, or protein in order to make ATP in
cooperation with oxidative phosphorylation.
Location: All cells with mitochondria.
Connections: From glycolysis through acetyl-CoA. Pyruvate makes oxaloacetate and malate
through the anaplerotic oxidation reactions through acetyl-CoA. Amino acid degradation through
acetyl-CoA and various intermediates of the cycle.
Regulation: Supply and demand of TCA cycle. Availability of NAD- and FAD as substrates.
Inhibition by NADH High-energy signals turn off. Low-energy signals turn on. ATP yield:
Pyruvate 15ATP Acetyl-CoA 12ATP
Equations:
COMPONENTS OF ELECTRON TRANSPORT CHAIN
SOURCES OF CARBOHYDRATES:
GLYCOGENOLYSIS:
This is the process of the breakdown of glycogen in liver and tissues to replenish glucose in
blood.
The process of glycogenolysis requires enzyme Glycogen Phosphorylase. The first step in
glycogenolysis is catalyzed by glycogen phosphorylase, commonly called phosphorylase. This
enzyme cleaves the a-1,4 glycosidic bonds of glycogen.
Glycogen
+ HPO4 2- glycogen phosphorylase
glucose 1 -phosphate + glycogen
(Glucose residues)
(- 1 glucose residues)
The reaction catalyzed by glycogen phosphorylase is physiologically irreversible and is the
regulated step in glycogen mobilization. Different isozymes of glycogen phosphorylase are
present in muscle and liver, permitting organ-specific regulation of glycogenolysis.
The debranching enzyme:
α- 1,6-glucosidase catalytic site of the debranching enzyme then hydrolyzes the a- 1,6 glycosidic
bond of the remaining glucose unit, releasing free glucose and producing a long, unbranched a1,4 glycosidic chain that is a suitable substrate for continued glycogenolysis by glycogen
phosphorylase
Fate of glucose 6 phosphate obtained from glycogenolysis:
The glucose molecules obtained from glycogenolysis is either utilized in the liver to regulate
blood glucose or it undergoes Glycolysis to yield energy that can be utilized in Brain and
muscles
Blood sugar level and its significance:
Normal values: the normal fasting or absorptive glucose taken atleast three hours after the last
meal is:
 As per glucose oxidase method (“true glucose”) 60 mg to 100mg %
 As per “ folin and wu’s method” is 80mg to 120 mg %
Abnormalities in blood glucose level:
Increased in blood glucose level above the normal is called “hyperglycemia”
Decreased in blood glucose below the normal values is called “hypoglycemia”
A: HYPERGLYCEMIA
Causes of hyperglycemia:
1. Diabetes meritus »fasting blood glucose varying from the normal to more than 500 mg %
depending on severity of the disease.
2. Hyperactivity of the thyroid, pituitary and adrenal glands
3. Emotional stress
4. Diffuse diseases of the pancreas i.e. pancreatitis and carcinoma of the pancreas readily
increases blood glucose.
5. Sepsis
6. Intracranial diseases like meningitis, encephalitis, intracranial tumors and hemorrhage.
7. Anaesthesia increases blood sugar depending on the degree and duration.
B: Hypoglycemia:
Causes of hypoglycemia:
This is a state in which the blood glucose is below 40 mg % (“true “ glucose) through glucose
oxidase test.
It can be caused by
1. Overdosage of insulin hormone in a case of treatment of diabetes mellitus.
2. Hypoactivity of thyroid hormones (myxoedema and creatinism),hypopituitarism
(symmond’s diseases) and hypoadrenalism (addison’s diseases) can cause the fasting
glucose to reduce.
3. Severe liver diseases
4. Glycogen storage diseases (GSD) i.e. von gierk’s diseases which is due to liver
phosphorylase enzyme deficiency hence impaired glucagon synthesis from glucose.
5. In children spontaneous hypoglycemia and leucine sensitive hypoglycemia is
experienced.
GLYCOGENESIS
 It is the formation of glycogen from glucose.
Sites:
 Occurs in the liver and skeletal muscles, but it can occur in every tissue to some extent.
Limitations of storage:
 In humans, the liver may contain as much as 4 to 6 per cent of glycogen as per weight of
the organ, when analysed shortly after a meal, high in carbohydrate. After 12 to 18 hours
of fasting, the liver becomes almost totally depleted of glycogen.
Reactions of Synthetic Pathway
 Glucose is first phosphorylated to glucose-6-P by Glucokinase (or hexokinase), common
to first reaction of glycoltic pathway.
 Glucose-6-P is converted to glucose-1-P by the enzyme phosphoglucomutase.
 Glucose-1-P then reacts with one molecule of uridine- triphosphate (UTP) to form the
“active” nucleotide uridine-diphosphate-glucose (UDP-Glc), under the influence of the
enzyme UDP-Glc pyrophosphorylase with elimination of a molecule of “pyrophosphate”
(PPi). The subsequent hydrolysis of inorganic pyrophosphate (PPi) by inorganic
pyrophosphatase drives the reaction to the right.
 The enzyme glycogen synthase acts on C-1 of the activated glucose of UDP-Glc to form
a glycosidic bond with the C-4 of a terminal glucose residue of glycogen primer,
liberating UDP.
 A pre-existing glycogen molecule or primer must be present to initiate this reaction.The
glycogen primer is formed on a protein primer known as glycogenin. Glycogenin is a 37
kDa protein that is glycosylated on a specific tyrosine residue by UDPGlc. Further
glucose residues are attached in the 1- 4 position to form a short chain forming a substrate
for glycogen synthase.
 An existing glycogen chain can be repeatedly extended by one glucose unit at a time.
 In each extension, 2 ATP molecules are expended:
• One in Phosphorylation of glucose to form G-6-P.
• Another in the regeneration of UTP.
 Glycogen synthase requires glucose-6-P as an activator.
The addition of a glucose residue to a pre-existing glycogen chain occurs at the “nonreducing” outer end of the molecule, so that the particular branch of the glycogen tree
becomes elongated as successive 1→ 4 linkages occur.
 Glycogen synthase is the principal key enzyme which regulates the glycogen formation.
When the chain has been lengthened to a minimum of 11 glucose residues, a second
enzyme Branching enzyme (Amylo-1, 4 → 1, 6-transglucosidase) comes into play and
transfers a part of a 1 → 4 chain, minimum length 6 glucose residues, to a neighbouring
chain to form a 1→ 6 linkage thus establishing a branch point in the molecule.
 The branches grow by further additions of a1 → 4 glucosyl units and further branching.
Regulation of glycogenesis:
 Glycogen synthase is the key enzyme which regulates the process of glycogenesis.
It is present as ‘active’ and ‘inactive’ forms, which are interconvertible.
• ‘Active’ form is: GS ‘a’ (previously called as GS-I)
• ‘Inactive’ form is: GS ‘b’ (previously called as GS-D)
1. ‘Active’ GS-‘a’ is converted to ‘inactive’ GS-‘b’ by phosphorylation, which is modulated
by Cyclic—AMP dependant protein kinase.
When glycogen synthase is converted to ‘inactive’ form glycogenesis is inhibited.
2. ‘Inactive’ GS-b is converted to ‘active’ GS ‘a’ by dephosphorylation of serine residue in
enzyme protein molecule catalysed by the enzyme Protein phosphatase-1.
When glycogen synthase is converted to ‘active’ form glycogenesis occurs.
The interconversion of active to inactive and vice-versa is controlled by substrate level,
end-product and hormones.
Role of cyclic-AMP dependant protein kinases:
 Protein kinases exist in cells, the enzyme is of wide specificity and c-AMP dependant.
Activation and Inactivation of Protein Kinases:
 Protein kinases are “tetramer”, consisting of 2 pairs of subunits. Each pair consists of
regulatory subunit(R) and a ‘catalytic’ subunit (C), the latter contains the “active site”.
Inactive Protein kinase is R2C2.
 When c-AMP level in cells increases, 2 molecules of c-AMP bind to each of the
regulatory (R) units, and thus releases the ‘active’ catalytic (C) units.
Active Protein kinase is C2 thus,
 Active protein kinase C2 formed due to increased cyclic AMP in the cell has two effects
on glycogenesis:
• It brings about phosphorylation of glycogen synthase with the help of ATP and converts
“active” GS ‘a’ (dephosphorylated) to ‘inactive’ GS ‘b’ (phosphorylated). Thus glycogen
synthesis is inhibited.
• At the same time, ‘active’ protein kinase (C2), stimulates a protein factor called inhibitor-1
(inactive) and phosphorylates it to form “active” Inhibitor-1-P, which in turn inhibits Protein
phosphatase-1 thus conversion of inactive GS-‘b’ to active GS-‘a’ does not take place. Hence,
glycogenesis in the cells is inhibited.
GLYCOGENOLYSIS
 The breakdown of glycogen to glucose is called as glycogenolysis.
 It is initiated by the action of a specific enzyme phosphorylase, which brings about
Phosphorolytic cleavage of a 1→ 4 linkage to yield glucose-1-P.
 The liver and muscle contain the enzyme phosphorylase. The enzyme can be in ‘active’
and “inactive” forms and both are interconvertible.
Steps of Glycogenolysis
1. Phosphorylase is the rate-limiting and key enzyme in glycogenolysis. With proper activation
and in the presence of inorganic phosphate (Pi), the enzyme breaks the glucosyl α-1→4 linkages
and removes by phosphorolytic cleavage the 1 →4 glucosyl residues from outermost chains of
the glycogen molecule until approximately four (4) glucose residues remain on either side of a α1→6 branch (Limit dextrin).
Note:
By phosphorylase activity glucose is liberated as glucose-1-P and not as free glucose.
2. When four glucose residues are left from the branch point, then another enzyme, α -1, 4 → α1, 4 glucan transferase transfers a trisaccharide unit from one side to the other thus exposing α 1
→ 6 branch point.
3. The hydrolytic splitting of α-1 → 6 glucosidic linkage requires the action of a specific
debranching enzyme (Amylo-1→ 6-glucosidase). As the α-1→ 6 linkage is hydrolytically split,
one molecule of free glucose is produced, rather than one molecule of glucose-1-P as is the case
with phosphorolytic cleavage with the enzyme phosphorylase.
4. Fate of Glucose-1-P
Phosphoglucomutase enzyme converts glucose-1-P to glucose-6-P. The reaction is reversible.
• A specific enzyme, glucose-6- phosphatase present in the liver and kidney, removes PO4 from
glucose-6-P, enabling “free glucose” to form and diffuse from the cells to extracellular spaces
including blood. This is the final step in hepatic glycogenolysis, which is reflected by a rise in
blood glucose.
•The enzyme glucose-6-phosphatase is absent in muscles. Hence glucose-6-P enters into
glycolytic cycle and forms pyruvate and lactic acid. Muscle glycogenolysis does not contribute
to blood glucose directly. But indirectly, lactic acid can go to glucose formation in liver.
Regulation of Glycogenolysis
Cyclic-AMP dependant protein kinase has dual effects, on glycogenolysis regulation.
1. Activation of phosphorylase enzyme:
Catecholamines, glucagon and thyroid hormones bring about glycogenolysis, by increasing the
cyclic AMP level in cells, which in turn activates protein kinase.
“Active” Protein kinase (C) in turn phosphorylates ‘inactive’ phosphorylase kinase ‘b’ and
converts it
to active phosphorylase Kinase ‘a’. ‘Active’ Phosphorylase kinase ‘a’ now with the help of ATP
phosphorylates ‘inactive’ Dephosphophosphorylase ‘b’ to ‘active’ Phosphophosphorylase ‘a’,
which brings about phosphorolytic cleavage of a-1→ 4 glucosidic linkages as stated above.
Glycog en storage diseases (GSDs):
These are a group of inherited disorders associated with glycogen metabolism, familial in
incidence and characterized by deposition of normal or abnormal type and quantity of glycogen
in the tissues.
Type I: Von Gierke’s Disease
•Enzyme deficiency: Glucose-6-phosphatase.
The enzyme is absent in liver cells and also in intestinal mucosa.
Inheritence:
Autosomal recessive
• Liver cells, intestinal mucosa and cells of renal tubular epithelial cells are loaded with glycogen
which is normal in structure but metabolically not available.
CLINICAL FEATURES
1. Since very little glucose is derived from the Liver, children with this disease tend to develop
hypoglycemia.
Glucose-1-P cannot be converted to glucose-6-P due to deficiency of the enzyme and it is
“locked” in the cells.
2. Fat is utilised as energy source, which leads to Lipaemia, Acidaemia and Ketosis.
3. Excess acetyl-CoA is diverted for cholesterol synthesis resulting to increase in cholesterol
level, which may produce Xanthomas.
4. Increased FA synthesis can produce fatty infiltration of liver.
5. Persistent hypoglycaemia can have two effects:
• Hypoglycaemia inhibits insulin secretion which in turn inhibits protein synthesis which causes
stunted growth (dwarfism).
• Hypoglycaemia stimulates secretion of catecholamines, which cause muscle glycogen to
breakdown producing Lactic acid and lactic acidosis.
6. Increased blood Lactic acid competes with urate excretion by kidneys leading to increased
blood uric Acid level. There is also evidence that there is increase in uric acid synthesis in those
children who develop symptoms of gout.
• Prognosis:
Although many of these children die young, a number of them have survived for adolescence,
when for some unknown reason much improvement can occur.
Biochemical change in type-1 Von Gierke’s disease and its correlation with clinical
manifestations.
Type-II: Pompe’s Disease
•Enzyme deficiency: Acid Maltase. Enzyme is present in lysosome and catalyses break down of
oligosaccharides.
Inheritance: Autosomal recessive.
•Glycogen structure is normal: Generalised involvement of organs seen including heart, liver,
smooth and striated muscles. Nearly all tissues contain excessive amount of normal glycogen.
•Clinically:
Enl argement of heart (cardiomegaly) seen.
Muscle hypotonia leading to muscle weakness. No hypoglycaemia.
• Prognosis:
Infants usually die of cardiac failure and bronchopneumonia.
Death usually occurs before 9 months. A few cases, milder form survived up to 2½ years.
Type-III:
Limit Dextrinosis (Forbe’s Disease)
• Enzyme deficiency: Debranching Enzyme
• Inheritance: Autosomal recessive.
• Glycogen structure: Limit dextrin type, abnormal, short or missing outer chains.
Organs involved are liver (18%), heart, and muscle (6%).
Clinically and biochemically:
Hepatomegaly, moderate hypoglycaemia, acidosis, progressive myopathy.
Similar to type1, but glycogen is abnormal and runs a milder course.
Enzyme deficiency can be demonstrated in leucocytes (diagnostic).
•Prognosis: Survives well to adult life.
Type-IV:
Amylopectinosis (Andersen’s Disease)
Enzyme deficiency: Branching Enzyme
• Inheritence: Not definitely known.
• Glycogen deposited is abnormal type, few branch points and very long inner and outer
unbranched chains (looks similar to “amylopectin”).
• Main organs affected are: Liver (mainly affected), others are Heart, Muscle and kidney.
Deposit occurs in RE system.
•Clinically and biochemically:
Hepatomegaly, splenomegaly, ascites, moderate hypoglycaemia, nodular cirrhosis of liver and
hepatic failure.
Enzyme deficiency can be demonstrated in Leucocytes and Liver.
•Prognosis:
Usually fatal. Longest survival reported as 4 years.
Type-V:
McArdle’s Disease
• Enzyme deficiency: Muscle Phosphorylase
• Inheritence: Autosomal recessive.
• Glycogen deposited is normal in structure; organs involved skeletal muscle (excess of normal
glycogen).
• Clinically:
Muscle cramps on exercise, pain, weakness and stiffness of muscles. No lactate is formed.
Muscles recover on rest, due to utilisation of FA. for energy.
• Epinephrine test: After administration of epinephrine/or glucagon, rise in blood glucose occurs
which shows that hepatic phosphorylase activity is normal.
Type-VI:
Her’s Disease
• Enzyme deficiency: Liver phosphorylase
• Glycogen deposited is normal in structures; organs affected are mainly liver and also
leucocytes.
•Clinically and biochemically: Hepatomegaly, mild to moderate hypoglycaemia, mild acidosis;
presents like mild case of type-1. The condition has also been reported to occur in association
with Fanconi’s syndrome.
THE HEXOSE MONOPHOSPHATE PATHWAY (HMP SHUNT)
It is also known as the pentose phosphate pathway or 6 phosphogluconate pathway.
Occurs in the cytosol of the cell.
It includes two reactions,
a. Irreversible oxidative reactions.
b. Followed by a series of reversible sugar–phosphate interconversions.
No ATP is directly consumed or produced in the cycle.
 Carbon 1 of glucose 6-phosphate is released as CO2, and two NADPH are produced for
each glucose 6-phosphate molecule entering the oxidative part of the pathway. The rate
and direction of the reversible reactions of the pentose phosphate pathway are determined
by the supply of and demand for intermediates of the cycle.
 The pathway provides a major portion of the body’s NADPH, which functions as a
biochemical reductant.
 It also produces ribose 5-phosphate, required for the biosynthesis of nucleotides and
provides a mechanism for the metabolic use of five-carbon sugars obtained from the diet
or the degradation of structural carbohydrates in the body.
IRREVERSIBLE OXIDATIVE REACTIONS
 The oxidative portion of the pentose phosphate pathway consists of three reactions that
lead to the formation of ribulose 5-phosphate, CO2, and two molecules of NADPH for
each molecule of glucose 6-phosphate oxidized.
 This portion of the pathway is particularly important in the liver, lactating mammary
glands, and adipose, which are active in the NADPH-dependent biosynthesis of fatty
acids (testes, ovaries, placenta and adrenal cortex), are active in the NADPH-dependent
biosynthesis of steroid hormones and in erythrocytes, which require NADPH to keep
glutathione reduced.
A. Dehydrogenation of glucose 6-phosphate
 Glucose 6-phosphate dehydrogenase (G6PD) catalyzes an irreversible oxidation of
glucose 6-phosphate to 6-phosphogluconolactone in a reaction that is specific for NADP+
as its coenzyme. The pentose phosphate pathway is regulated at the G6PD reaction.
 NADPH is a potent competitive inhibitor of the enzyme, and under most metabolic
reactions the ratio of NADPH/NADP is sufficiently high to substantially inhibit enzyme
activity. However, with increased demand for NADPH, the ratio of NADPH/NADP
decreases. Flux through the cycle increases in response to the enhanced activity of G6PD.
Insulin up regulates expression of the gene for G6PD. Flux through the pathway increases
in the well fed state.
B. Formation of ribulose 5-phosphate
 6-Phosphogluconolactone is hydrolyzed by 6-phosphogluconolactone hydrolase. The
reaction is irreversible and not rate-limiting.
 The oxidative decarboxylation of the product, 6-phosphogluconate is catalyzed by 6phosphogluconate dehydrogenase. This irreversible reaction produces a pentose sugar
phosphate (ribulose 5-phosphate), CO2 (from carbon 1 of glucose), and a second
molecule of NADPH.
REVERSIBLE NONOXIDATIVE REACTIONS
 The nonoxidative reactions of the pentose phosphate pathway occur in all cell types
synthesizing nucleotides and nucleic acids.
 These reactions catalyze the interconversion of sugars containing three to seven carbons.
These reversible reactions permit ribulose 5-phosphate to be converted either to ribose 5phosphate (needed for nucleotide synthesis, or to intermediates of glycolysis—fructose 6phosphate and glyceraldehydes 3-phosphate). For example, many cells that carry out
reductive biosynthetic reactions have a greater need for NADPH than for ribose 5phosphate.
 Transketolase (transfers two-carbon units in a thiamine pyrophosphate (TPP)-requiring
reaction) and transaldolase (transfers three-carbon units) convert the ribulose 5-phosphate
produced as an end product of the oxidative reactions to glyceraldehydes 3-phosphate and
fructose 6-phosphate, which are intermediates of glycolysis.
 In contrast, under conditions in which the demand for ribose for incorporation into
nucleotides and nucleic acids is greater than the need for NADPH, the nonoxidative
reactions can provide the biosynthesis of ribose 5-phosphate from glyceraldehydes 3phosphate and fructose 6-phosphate in the absence of the oxidative steps.
USES OF NADPH
The coenzyme NADP+ differs from NAD+ only by the presence of a phosphate group on one of
the ribose units. This small change in structure allows NADP+ to interact with NADP specific
enzymes that have unique roles in the cell.
For example, in the cytosol of hepatocytes the steady-state ratio of NADP /NADPH is
approximately 0.1, which favors the use of NADPH in reductive biosynthetic reactions. This
contrasts with the high ratio of NAD+ /NADH, which favors an oxidative role for NAD+.
This section summarizes some important NADP+ or NADPH-specific functions.
A. Reductive biosynthesis
 NADPH can be thought of as a high-energy molecule, much in the same way as NADH.
However, the electrons of NADPH are destined for use in reductive biosynthesis, rather
than for transfer to oxygen as is the case with NADH. Thus, in the metabolic
transformations of the pentose phosphate pathway, part of the energy of glucose 6phosphate is conserved in NADPH—a molecule with a negative reduction potential that,
can be used in reactions requiring an electron donor.
B. Reduction of hydrogen peroxide
 Hydrogen peroxide is one of a family of reactive oxygen species (ROS) that are formed
from the partial reduction of molecular oxygen.
These compounds are formed continuously as by-products of aerobic metabolism,
through reactions with drugs and environmental toxins, or when the level of antioxidants
is diminished, all creating the condition of oxidative stress. The highly reactive oxygen
intermediates can cause serious chemical damage to DNA, proteins, and unsaturated
lipids, and can lead to cell death.
 These ROS have been implicated in a number of pathologic processes, including
reperfusion injury, cancer, inflammatory disease, and aging. The cell has several
protective mechanisms that minimize the toxic potential of these compounds.
1. Enzymes that catalyze antioxidant reactions:
 Reduced glutathione, a tripeptide-thiol (γ-glutamylcysteinylglycine) present in most cells,
can chemically detoxify hydrogen peroxide. Then it is catalyzed by the seleniumrequiring glutathione peroxidase, forms oxidized glutathione, which no longer has
protective properties.
 The cell regenerates reduced glutathione in a reaction catalyzed by glutathione reductase,
using NADPH as a source of reducing equivalents. Thus, NADPH indirectly provides
electrons for the reduction of hydrogen peroxide.
Note:
Erythrocytes are totally dependent on the pentose phosphate pathway for their supply of NADPH
because, unlike other cell types, erythrocytes do not have an alternate source for this essential
coenzyme.
Additional enzymes, such as superoxide dismutase and catalase, catalyze the conversion of other
toxic oxygen intermediates to harmless products. These enzymes serve as a defense system to
guard against the toxic effects of reactive oxygen species.
2. Antioxidant chemicals:
 A number of intracellular reducing agents, such as ascorbate, vitamin E and ß-carotene,
are able to reduce and detoxify oxygen intermediates in the laboratory.
 Consumption of foods rich in these antioxidant compounds has been correlated with a
reduced risk for certain types of cancers, as well as decreased frequency of certain other
chronic health problems.
C. Cytochrome P450 monooxygenase system
 Monooxygenases (mixed function oxidases) incorporate one atom from molecular
oxygen into a substrate (creating a hydroxyl group), with the other atom being reduced to
water. In the
cytochrome P450 monooxygenase system, NADPH provides the reducing equivalents required by
this series of reactions. This system performs different functions in two separate locations in
cells. The overall reaction catalyzed by a cytochrome P450 enzyme is:
R-H + O2 + NADPH + H+→ R-OH + H2O + NADP
Where R may be a steroid, drug, or other chemical.
[Note:
Cytochrome P450s (CYPs) are actually a superfamily of related, heme-containing
monooxygenase enzymes that participate in a broad variety of reactions. The name P450 reflects
the absorbance 450 nm by the protein.
D. Phagocytosis by white blood cells
 Phagocytosis is the ingestion by receptor-mediated endocytosis of microorganisms,
foreign particles, and cellular debris by cells such as neutrophils and macrophages
(monocytes).
 It is an important body defense mechanism, particularly in bacterial infections.
Neutrophils and monocytes are armed with both oxygen-independent and oxygendependent mechanisms for killing bacteria.
1. Oxygen-independent mechanism:
 Uses pH changes in phagolysosomes and lysosomal enzymes to destroy pathogens.
2. Oxygen-dependent system:
 This includes the enzymes, NADPH oxidase and myeloperoxidase (MPO) that work
together in killing bacteria.
 This system is the most potent of the bactericidal mechanisms. An invading bacterium is
recognized by the immune system and attacked by antibodies that bind it to a receptor on
a phagocytic cell. After internalization of the microorganism has occurred, NADPH
oxidase, located in the leukocyte cell membrane, is activated and reduces molecular
oxygen from the surrounding tissue into superoxide (O2–•), a free radical. The rapid
consumption of molecular oxygen that accompanies formation of superoxide is referred
to as the respiratory burst.
GLUCOSE 6-P DEHYDROGENASE DEFICIENCY
 This is an inherited disease characterized by hemolytic anemia caused by the inability to
detoxify oxidizing agents.
 G6PD deficiency is the most common disease-producing enzyme abnormality in humans,
affecting more than 400 million individuals worldwide.
 This deficiency has the highest prevalence in the Middle East, tropical Africa and Asia,
and parts of the Mediterranean.
 G6PD deficiency is X-linked, and is, in fact, a family of deficiencies caused by more than
400 different mutations in the gene coding for G6PD. Only some of these mutations
cause clinical symptoms.
Note:
(In addition to hemolytic anemia, a clinical manifestation of G6PD deficiency is neonatal
jaundice appearing 1–4 days after birth.
The jaundice, which may be severe, typically results from increased production of unconjugated
bilirubin).
 The life span of individuals with a severe form of G6PD deficiency may be somewhat
shortened as a result of complications arising from chronic hemolysis.
 This negative effect of G6PD deficiency has been balanced in evolution by an advantage
in survival—an increased resistance to falciparum malaria shown by female carriers of
the mutation.
[Note: Sickle cell trait and ß-thalassemia minor also confer resistance.]
A. Role of G6PD in red blood cells
 Diminished G6PD activity impairs the ability of the cell to form the NADPH that is
essential for the maintenance of the reduced glutathione pool. These results in a decrease
in the cellular detoxification of free radicals and peroxides formed within the cell.
 Glutathione also helps maintain the reduced states of sulfhydryl groups in proteins,
including hemoglobin. Those sulfhydryl groups lead to the formation of denatured
proteins that form insoluble masses (called Heinz bodies) that attach to the red cell
membranes.
 Additional oxidation of membrane proteins causes the red cells to be rigid (less
deformable), and they are removed from circulation by macrophages in the spleen and
liver.
 Although G6PD deficiency occurs in all cells of the affected individual, it is most severe
in erythrocytes, where the pentose phosphate pathway provides the only means of
generating NADPH.
 Other tissues have alternative sources for NADPH production (such as NADP+ dependent malate dehydrogenases), that can keep glutathione reduced. The erythrocyte
has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, red blood
cells are particularly vulnerable to enzyme variants with diminished stability.
B. Precipitating factors in G6PD deficiency
 Most individuals who have inherited one of the many G6PD mutations do not show
clinical manifestations, that is, they are asymptomatic. However, some patients with
G6PD deficiency develop hemolytic anemia if they are treated with an oxidant drug,
ingest fava beans, or contract a severe infection.
1. Oxidant drugs:
 Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency
are best remembered from the mnemonic AAA—Antibiotics (for example, sulfa
methoxazole and chloramphenicol), Antimalarials(eg , primaquine but not quinine), and
Antipyretics (eg, acetanilide but not aceta minophen).
2. Favism:
 Some forms of G6PD deficiency, for example the Mediterranean variant, are particularly
susceptible to the hemolytic effect of the fava (broad) bean, a dietary staple in the
Mediterranean region.
 Favism, the hemolytic effect of ingesting fava beans, is not observed in all individuals
with G6PD deficiency, but all patients with favism have G6PD deficiency.
3. Infection:
 Infection is the most common precipitating factor of hemolysis in G6PD deficiency. The
inflammatory response to infection results in the generation of free radicals in macro
phages, which can diffuse into the red blood cells and cause oxidative damage.
C. Properties of the variant enzymes
 Almost all G6PD variants are caused by point mutations in the gene for G6PD. Some
mutations do not disrupt the structure of the enzyme’s active site and, hence, do not affect
enzymic activity.
 However, many mutant enzymes show altered kinetic properties, eg , variant enzymes
may show decreased catalytic activity, decreased stability, or an alteration of binding
affinity for NADP, NADPH, or glucose 6-phosphate. The severity of the disease usually
correlates with the amount of residual enzyme activity in the patient’s red blood cells.
G6PDA– is the prototype of the moderate (Class III) form of the disease.
 The red cells contain an unstable but kinetically normal G6PD, with most of the enzyme
activity present in the reticulocytes and younger erythrocytes.
 The oldest cells, have the lowest level of enzyme activity, and are preferentially removed
in a hemolytic episode.
 G6PD Mediterranean is the prototype of a more severe (Class II) deficiency in which the
enzyme has decreased stability resulting in decreased enzymic activity. Class I mutations
(rare) are the most severe and are associated with chronic nonspherocytic anemia, which
occurs even in the absence of oxidative stress.
REGULATION OF BLOOD GLUCOSE
(Homeostasis)
Blood glucose level is maintained within physiological limits of 60 to 100 mg% (“true” glucose)
in fasting state and 100 to 140 mg% following ingestion of a carbohydrate containing meal, by a
balance between two sets of factors:
(A) Rate of glucose entrance into the blood stream, and
(B) Rate of its removal from the blood stream.
(A) RATE OF SUPPLY OF GLUCOSE TO BLOOD:
Blood glucose may be derived directly from the following sources:
• By absorption from the intestine
• Breakdown of glycogen of Liver (Hepatic glycogenolysis)
• By gluconeogenesis in Liver, source being glucogenic amino acids, lactate and pyruvate,
glycerol and propionyl-CoA
• Glucose obtained from other carbohydrates, e.g. fructose, galactose, etc.
The kidney contributes a minor amount.
(B) RATE OF REMOVAL OF GLUCOSE FROM BLOOD
• Oxida tion of glucose by the tissues to supply energy
•Glycogen formation from glucose in Liver (Hepatic glycogenesis)
• Glycogen formation from glucose in muscles (Muscle glycogenesis)
• Conversion of glucose to fats (lipogenesis) especially in adipose tissue
• Synthesis of compounds containing carbohydrates—blood glucose is utilised, e.g.
 Formation of fructose in seminal fluid, formation of lactose (sugar of milk) in
lactating mammary gland, synthesis of glycoproteins and glycolipids,
 Formation of ribose sugars from glucose required for nucleic acid synthesis.
Excretion of glucose in urine (glycosuria), when blood glucose level exceeds the renal threshold.
All the above processes are under substrate, end- product, nervous and hormonal control.
1. CONDITION OF BLOOD GLUCOSE IN POST-ABSORPTIVE STATE
What is Post-Absorptive State?
 This is fasting state, approx. 12 to 14 hours after last meal. There is practically no
intestinal absorption.
 It is not prolonged starvation, as there are no metabolic abnormalities. It is the condition
of a subject between 8 to 10 A.M, if he had his dinner previous evening about 8 P.M and
had taken nothing thereafter.


Under Such a Situation only Source of Glucose is Liver Glycogen. At rest, tissues utilise
approximately 200 mg of glucose per minute from blood. Glycogen stores of Liver is
limited approx. 4 to 6 per cent = 72 to 108 gm, if we take liver weight as 1800 gm. The
above store of liver glycogen can supply for 8 hours approximately as per rate of
utilization stated above.
Muscle glycogen store is approx. 0.7 per cent of the weight of muscle mass. Muscle
glycogen cannot provide blood glucose by glycogenolysis due to lack of the enzyme
Glucose-6-phosphatase. It can supply indirectly by gluconeogenesis from the pyruvates
and lactates, the products of muscle glycogenolysis. Thus muscle glycogen can provide
indirectly blood glucose approx. less than 25 hours.
2. CONDITION OF BLOOD GLUCOSE IN POSTPRANDIAL STATE
 Condition following ingestion of food is called postprandial state.
 Absorbed monosaccharides are utilised for oxidation to provide energy. Remaining in
excess is stored as glycogen in Liver and muscles.
 In well-nourished individuals, glycogen stores are fairly saturated. About 40 per cent of
absorbed glucose is used for lipogenesis and some for synthesis of glycoproteins,
glycolipids etc, when load of glucose is very high renal mechanisms operate. Tm G is
250 to 350 mg/mt, which is maximum renal tubular reabsorption.
 When blood glucose rises more than 160 to 180 mg%, glucose appears in urine
(glycosuria). This is an abnormal state. In normal intestinal absorption such situation does
not occur. It can take place with an IV load or disease processes.
AUTOREGULATION (Fundamental Regulatory Mechanisms)
Processes of hepatic glycogenesis, glycogenolysis and tissue utilization of glucose are sensitive
to relatively slight deviation from the normal blood sugar concentration.
1. As blood sugar tends to increase:
•Glycogenesis is accelerated, and Utilisation of glucose by tissues is increased, resulting to fall in
blood glucose level. The reverse occurs as the blood glucose level tends to fall.
Normal balance between production and utilization of blood glucose, at a mean level of
circulating glucose of approximately 80 mg % is, therefore, dependant upon the sensitivity of
these processes to variations above and below this concentration.
This level of sensitivity is determined to a considerable extent by the balance between:
•Insulin in one hand, and
• Hormones of adrenal cortex and anterior pituitary on the other hand.
 Overall effect of Insulin is to lower the blood glucose level and adrenocortical and
growth hormone to raise it.
 In as much as these two sets of factors are mutually antagonistic to each other. It is the
ratio between them rather than their absolute amounts that is of Prime importance in this
connection.





The processes of hepatic glycogenesis, glycogenolysis, and glucose utilisation and also
the blood glucose concentration are continually exposed to disturbing influences under
physiological conditions.
These include absorption of glucose from intestine, physical and mental activity,
emotional states, etc.
Primary effect of majority of these is to cause a rise in blood glucose.
This results in:
 Decrease in delivery of glucose by Liver, and
 Acceleration of utilisation by tissues.
Simultaneous increase in insulin secretion, stimulated by elevated blood glucose
concentration results in increase in ratio of insulin/adrenocortical hormones and
growth hormone. This change in ratio and hormonal balance results in:
• Increased hepatic glycogenesis
• Decreased gluconeogenesis
• Decreased output of glucose from Liver and
• Increased utilisation of glucose.
As a result of above, the blood glucose concentration tends to fall.
2. As blood sugar tends to decrease:
A drop in blood glucose concentration below the normal resting level causes:
•Decrease in secretion of insulin, resulting to decrease in ratio of insulin/glucocorticoids and GH,
• Increased production of blood glucose mainly by gluconeogenesis, leading to decreased
glucose utilisation .
The above actions lead to rise of blood glucose.
If it falls to hypoglycaemic levels, additional emergency mechanisms come into play: ie,
• Stimulation of secretion of catecholamines by hypoglycaemia resulting in hepatic
glycogenolysis and rise in blood glucose.
• The increase in catecholamines may also stimulate production of ACTH, hence of
adrenocortical hormones, causing increased gluconeogenesis.
The blood glucose concentration in normal health regulates itself. Efficient operation of this
autoregulation at physiological levels, requires a normal balance between
(a) Insulin and
(b) The carbohydrate active, adrenocorticoids and anterior pituitary hormones, and also to
normal responsiveness of the pancreatic islet cells to variation in blood glucose
concentration. This constitutes the “autoregulation” or central regulatory mechanism.
HORMONAL INFLUENCES: (ENDOCRINE INFLUENCES) ON CARBOHYDRATE
METABOLISM
There are two categories of endocrine influences:
(a) Those which exert a regulatory influence, their normal function being essential for normal
carbohydrate metabolism, eg, hormones of pancreatic islet cells especially Insulin and hormones
of adrenal cortex and anterior pituitary.
(b) Those which influence carbohydrate metabolism, but are not essential for its autoregulation
under normal physiological conditions, e.g. hormones of adrenal medulla and hormones of
thyroid gland.
1. Insulin
Administration of insulin leads to a fall in blood glucose concentration to hypoglycaemic levels,
if adequate amounts are given. This results from:
(A) Net decrease of delivery of glucose to systemic blood by Liver, and
(B) Increase in the rate of utilisation of glucose by tissue cells.
(A) Diminished supply of glucose to blood is due to:
• Decreased hepatic glycogenolysis
•Increased hepatic glycogenesis by its direct action on Protein phosphatase-1, thus converting
glycogen synthase ‘b’ to glycogen synthase ‘a’
• Decreased gluconeogenesis
• The liver glycogen tends to increase although this may be obscured by the hypoglycaemia,
which itself tends to accelerate hepatic glycogenolysis.
(B) Increase in the rate of utilisation of glucose by tissue cells:
Glucose is removed from the blood more readily and is utilised more actively for:
•Oxidation for energy production
• Increases lipogenesis, and
• For glycogenesis.
The overall effect of insulin is antagonistic to that of adrenal glucocorticoids and growth
hormone. Its primary action in extrahepatic tissues is to facilitate entrance of glucose into the
cells. In the liver, which is freely permeable to glucose, insulin exerts a regulatory influence
upon the activity of glucokinase.
2. Adrenocortical hormones: Adrenal cortex produces steroid hormones, of which the
“glucocorticoids” are important in carbohydrate metabolism. The predominant glucocorticoid in
man is cortisol.
Glucocorticoids
(a) Increases blood glucose level: By gluconeogenesis, as a result of:
• Increased protein catabolism in the peripheral tissues, so that more amino acids are available.
• Increased hepatic uptake of amino acids increasing the activity of transaminases and all the
enzymes concerned with gluconeogenesis, e.g. Pyruvate carboxylase, PEP-carboxykinase,
fructose-1,6-biphosphatase and glucose-6-phosphatase.
• Diminishing peripheral uptake and utilisation of glucose.
(b) Increases liver glycogen: Attributable in part to increased activity of glycogen synthase, ‘b’
to ‘a’ conversion.
3. Anterior Pituitary Gland
Secretes hormones that tend to elevate the blood glucose level and therefore, antagonise the
effect of insulin. Growth hormone secretion is stimulated by hypoglycaemia.
• Growth hormone decreases glucose uptake in certain tissues, e.g. muscles. Some of this effect
may not be direct since it mobilises FFA from adipose tissue and long-chain fatty acids inhibit
glucose utilisation.
• Liver: There is increase in liver glycogen due to increased gluconeogenesis.
Chronic administration of GH leads to Diabetes. By producing hyperglycaemia, leads to
stimulation of secretion of insulin, which eventually produces exhaustion of ß -cells.
4. Catecholamines
These are hormones produced by adrenal medulla:
•Produces an increase in blood glucose level and also blood Lactic acid level.
It stimulates glycogen breakdown (glycogenolysis) in Liver as well as in muscle and is
accompanied by a decrease in glycogen content. The action in liver is mediated through cyclic
AMP dependant protein kinase.
• In muscle due to absence of glucose-6-pase, glycogenolysis does not directly contribute to
blood glucose.
It increases the pyruvate and lactate. Pyruvate and Lactates diffuse into the blood and
5. Glucagon
Glucagon is a protein hormone, a Polypeptide
cells of islets of Langerhans. It is also known as HGF (hyperglycaemic glycogenolytic factor).
• In response to hypoglycaemia, α-cells produce glucagon which produces rapid glycogenolysis
in Liver. It activates phosphorylase enzyme, by increasing cyclic AMP level, which mediates its
action through cyclic AMP mediated protein kinase. Active protein kinase, activates
phosphorylase kinase which in turn activates the enzyme phosphorylase.
Note: Glucagon cannot produce glycogenolysis in muscle as it lacks the receptor.
• Glucagon also enhances “gluconeogenesis” from amino acids, pyruvates and lactates.
6. Thyroid Hormones
Thyroxine accelerates hepatic glycogenolysis, with consequent rise in blood glucose. This may
be due to:
• Increased sensitivity of the tissues to catecholamines, and
• In part to accelerated destruction of insulin.
• Thyroid hormones may also increase the rate of absorption of hexoses from the intestine.
• Increased hepatic glucose-6-phosphatase activity.
• Rate of protein catabolism is increased by excessive thyroid hormones and thus increases
gluconeogenesis from amino acids.
There is experimental evidence that thyroxine has a diabetogenic action and that thyroidectomy
inhibits the development of diabetes.
BLOOD SUGAR LEVEL AND ITS CLINICAL SIGNIFICANCE
1. Normal values: The range for normal fasting or Post absorptive blood glucose taken at least
three hours after the last meal is:
• As per glucose-oxidase method (“true” glucose) 60 to 100 mg%, some authorities give as 60 to
95 mg%.
• As per “Folin and Wu’s method”: 80 to 120 mg%.
2. Abnormalities in Blood Glucose Level
• Increase in blood glucose level above normal is called hyperglycaemia.
• Decrease in blood glucose level below normal is called hypoglycaemia.
(a) Hyperglycaemia
Causes of Hyperglycaemia
• Most common cause is Diabetes mellitus in which the highest values for fasting blood glucose
is obtained, in which it may vary from normal to 500 mg% and over, depending on the severity
of the disease.
 Hyperactivity of the thyroids, pituitary, and adrenal glands. Except in DM, fasting blood
glucose rarely exceeds 200 mg%. There may be increased incidence of DM in
hyperthyroidism and hyperpituitarism.
 Emotional ‘stress’ can increase the blood glucose level.
 In diffuse diseases of pancreas, e.g. in pancreatitis and carcinoma of pancreas some
increase in fasting blood glucose may occur.
 In sepsis and in a number of infectious diseases.
 In some intracranial diseases such as meningitis, encephalitis, intracranial tumors and
haemorrhage.
 Anaesthesia: Can also increase blood glucose, depending on the degree and duration of
anaesthesia.
 Asphyxia may also increase blood sugar level.
 Increase in blood sugar, rarely exceeding 150 to 180 mg% may be seen in convulsions
and in the terminal stages of many diseases.
b) Hypoglycaemia
Causes of hypoglycaemia:
Hypoglycaemia may be considered to be present when the blood glucose is below 40 mg%
(“true” glucose value by glucose oxidase method).
 Overdosage of Insulin in treatment of diabetes mellitus.
Insulin-secreting tumor (Insulinoma) of pancreas produces a severe hypoglycaemia in
which blood glucose is very low or may be almost completely absent. It is extremely rare.
 Fasting blood glucose may be reduced in hypoactivity of thyroids (Myxoedema, and
cretinism), hypopituitarism (Simmond’s disease) and hypoadrenalism (Addison’s
disease).
 Severe liver diseases: Low blood glucose levels are often found. In childhood, an
idiopathic hypoglycaemia, due to sensitivity to the amino acid leucine has been
recognized (Leucine-sensitive hypoglycaemia).
An acquired Leucine-sensitivity has also been stated to exist.
 Spontaneous hypoglycaemia in childhood may be due to deficiency of glucagon
production. Severe exercise may produce hypoglycaemia due to depletion of liver
glycogen.
 In some of the Glycogen storage diseases (GSDs), e.g. in Von Gierke’s disease, Liver
phosphorylase deficiency—Due to impaired ability to produce glucose from glycogen.
Impaired absorption of glucose in some types of steatorrhoea. The blood glucose may be
in the lower part of the normal range, it is rare to be subnormal.
 Transient post-prandial hypoglycaemia (“reactive” hypoglycaemia), may occur in an
occasional case,some one and a half to three hours after taking food and more commonly
in patients with partial gastrectomy.
 In cases of alcohol ingestion.
 In a variety of tumors of nonendocrine origin, particularly, retroperitoneal fibrosarcoma
may produce hypoglycaemia by secreting insulin-like hormones.
GLYCOSURIA
Introduction
 Under ordinary dietary conditions, glucose is the only sugar present in the free State in
blood plasma in demonstrable amounts.
 Normal urine contains virtually no sugar; under certain circumstances, glucose or other
sugars may be excreted in the urine. This condition is called melituria (excretion of sugar
in urine).
 The ter ms glycosuria, fructosuria, galactosuria, lactosuria and pentosuria are applied
specially to the urinary excretion of glucose, fructose, galactose, lactose, and pentose
respectively.
RENAL THRESHOLD FOR GLUCOSE AND MECHANISM OF GLYCOSURIA
 Glucose is present in the glomerular filtrate in the same concentration as in the blood
plasma. Under normal conditions, it undergoes complete reabsorption by the renal tubular
epithelial cells and is returned to the blood stream.
 In normal subjects, a very small amount less than 0.5 gm of glucose may escape
reabsorption by tubules and be excreted by urine. But this amount is not detected by
Benedict’s qualitative test.
 Rate of glucose absorption is expressed as TmG (tubular maximum for glucose) which is
350 mg/mt.
 When the blood levels of glucose are elevated, the glomerular filtrate may contain more
glucose than can be reabsorbed, the excess passes in urine to produce “glycosuria”.
 In normal individuals, glycosuria occurs when the venous blood glucose exceeds 170 to
180 mg/100 ml. This level of the venous blood glucose is termed as the renal threshold
for glucose. Since the maximal rate of reabsorption of glucose by the tubule (TmG—the
tubular maximum for glucose) is a constant, it is a more accurate measurement than the
renal threshold, which varies with changes in the GFR.
What is glycosuria?
It is the excretion of glucose in urine which is detectable by Benedict’s Qualitative test.
MECHANISM OF GLYCOSURIA
Excretion of abnormal amounts of glucose in the urine may be due to two types of
abnormalities:
(a) Increase in the amount of glucose entering in the tubule/mt.
(b) Decrease in the glucose reabsorption capacity of the renal tubular epithelium.
(a) The quantity of glucose entering the tubules is the product of:
• The minute volume of glomerular filtrate, and
•The concentration of glucose in the filtrate, i.e. in the arterial blood plasma.
Glomerular filtration is rarely increased markedly; glycosuria of this type is due to an increase in
the blood glucose concentration above the “renal threshold level” and called as hyperglycaemic
glycosuria.
(b) Reabsorption of glucose by renal tubular epithelium:
It is accomplished mainly by an “active transport” mechanism, by transport carrier protein. The
capacity for reabsorption may be diminished by:
• ‘Hereditary’ cause: Absence of ‘carrier protein’ or defective carrier protein.
• Acquired: Due to certain types of kidney diseases especially involving the tubules or damage of
tubules by chemicals/poisons.
• Induced: Experimental glycosuria, e.g. by administration of glycoside “phloridzin”.
The above type of glycosuria is called as renal glycosuria. Blood glucose level in this type is
normal or even may be subnormal.
TYPES OF GLYCOSURIAS
There are two main groups:
A. Hyperglycaemic glycosuria
B. Renal glycosuria
A. Hyperglycaemic Glycosuria
1. Alimentary glycosuria:
 When a large carbohydrate diet is taken, blood sugar rises and may cross renal threshold
in occasional case and may produce glycosuria. This condition does not seem to be a
normal process, as homeostatic control is so efficient in normal healthy person that such
glycosuria should not occur.
 Alimentary glycosuria is only possible in those subjects in whom the power of glucose
utilisation is impaired and such people should be kept under observation and should be
screened regularly for diabetes.
2. Nervous or “emotional” glycosuria:
 Stimulation of the sympathetic nerves to the Liver or of the splanchnic nerves,
breakdown of liver glycogen occurs and produces hyperglycaemia and glycosuria.
 Nervous stimulation mentioned above causes:
 Glycogenolysis directly, and
 Increased secretion of catecholamines, producing glycogenolysis.
Anything that stimulates sympathetic system such as excitement, stress, etc. may produce
glycosuria.
3. Glycosuria due to endocrine disorders:
Deranged function of a number of endocrine glands produces hyperglycaemia which may
result in glycosuria.
Examples are:
Diabetes mellitus (clinical): In this case ß-cells of islets of Langerhans fail to secrete adequate
amount of insulin, producing absolute or relative deficiency of insulin. Lack of insulin produces
hyperglycaemia and glycosuria.
o Hyperthyroidism: Hyperactivity of thyroid is always attended with low sugar
tolerance, hyperglycaemia and may be glycosuria. In 25 to 35 per cent of cases,
hyperthyroidism and diabetes mellitus can coexist.
o Epinephrine: Increased secretion of epinephrine or prolonged administration
through subcutaneous route can increase the breakdown of liver glycogen leading
to hyperglycaemia and glycosuria.
o Hyperactivity of anterior pituitary: Hyperactivity of anterior pituitary as in
Acromegaly is attended with hyperglycaemia and glycosuria (20 to 30% cases),
due to increased secretion of GH and adrenocortical hormones.
o Adrenal cortex: Hyperactivity of adrenal cortex as in Cushing’s
syndrome/disease may cause hyperglycaemia and glycosuria. Glucocorticoids
stimulate gluconeogenesis and increased resistance to insulin (glucose uptake by
peripheral tissues inhibited).
o Glucagon: Increased secretion of glucagon by α-cells of islets of Langerhans can
cause glycogenolysis producing hyperglycaemia and glycosuria.
4. Experimental Hyperglycaemic Glycosurias
(a) “Piqure” glycosuria: Certain injuries to the nervous system can cause
hyperglycaemia and glycosuria.
 Claude Bernard found that Puncture of a particular spot in the floor of the IV ventricle
of rabbits produces hyperglycaemia and glycosuria. (Puncture Diabetes). This
glycosuria persists for 24 hours or more and is accompanied by marked hyperglycaemia.
Mechanism:

It is suggested that experimental procedure stimulates a group of nerve cells at the
floor of IV ventricle which sends impulses through the splanchnic nerves to adrenal
medulla and liver, increasing glycogenolysis by increased secretion of catecholamines.
(b).‘Alloxan’ diabetes and glycosuria: Injection of alloxan to an experimental animal
like dog, a substance related chemically to “pyrimidine” bases, produces permanent
diabetes. Diabetes is due to degeneration, necrosis and resorption of β-cells of islets
of Langerhans. The α-cells and acinar cells are unaffected. The alloxan acts directly,
promptly and specifically on β -cells. Its effect can be prevented by administration of
cysteine, glutathione, BAL, or thioglycolic acid immediately before or within a few
minutes after injection of the alloxan. This protective action is due apparently to the –SH
content of these compounds, the alloxan being probably reduced to an inactive substance.
B. Renal Glycosuria
1. Hereditary:
 A milder glycosuria occurs spontaneously, as hereditary familial traits, persisting
throughout life, due to absence of “carrier Protein” or altered kinetics of the carrier
system due to failure of development.
2. Acquired
 Diseases of renal tubules: In some cases of kidney diseases, the renal tubules may be
grossly affected, thus they fail to reabsorb glucose producing glycosuria.
 Due to heavy metal poisoning: The heavy metals like Lead (Pb), Cadmium (Cd),
Mercury (Hg), etc. can damage the renal tubules thus interfering with the reabsorption of
glucose, resulting to glycosuria.
3. Lowering of renal threshold:
 15 to 20 per cent cases of Pregnancy may be associated with physiological glycosuria
with advancement of pregnancy, due to lowering of renal threshold. But pregnancy may
be associated with Diabetes mellitus in which there will be hyperglycaemic glycosuria.
These two can be differentiated by performing a fasting blood sugar level.
Renal glycosuria:
 It may occur in association with evidences of other renal tubular transport defects, e.g.
aminoacidurias, Renal tubular acidosis, hyperphosphaturia as in Fanconi syndrome.
5. ‘Experimental’ renal glycosuria: “phloridzin” glycosuria:
 Phloridzin is a glycoside found in roots of apple tree; when hydrolysed it gives glucose
and aglycone
Phloretin. When phloridzin is administered subcutaneously, it gives rise to intense glycosuria.
The dose given to dogs is 1 gm/day, in oil, SC. certain other glycosides, such as ‘Arbutin’ have
similar effects.
Mechanism: Phloridzin displaces sodium from the sodium binding site from “Carrier protein”
and hence glucose cannot be bound to the glucose binding site, thus inhibiting glucose
reabsorption.
DIABETES MELLITUS
 Is a chronic disease due primarily to a disorder of carbohydrate metabolism caused by the
deficiency or diminished effectiveness of insulin, resulting in hyperglycaemia and
glycosuria.
 Secondary changes may occur in the metabolism of proteins, fats, water and electrolytes
and in tissues/organs sometimes with grave consequences.
 It is a common disease in man. A predisposition to the disease is probably inherited as an
autosomal recessive trait. About 25 per cent of relatives of diabetics show abnormal
glucose tolerance curves as compared to 1 per cent in the general population.
STAGES OF DIABETES MELLITUS
“Overt” diabetes is seen till after the age of 40, there must be a stage of Pre-diabetes which
dates from the time of conception.
American Diabetes Association has divided the stages into four stages.
The four stages and findings are shown above in tabular form in the box.
CLINICAL TYPES AND CAUSES
There are two main groups:
(a) Primary (Idiopathic):
It constitutes the major group. Exact cause is not known, metabolic defect is insufficient
insulin which may be absolute or relative.
(b) Secondary:
Constitute minor group where it can be secondary to some disease process.
(a) Primary (Idiopathic)
There are two clinical types:
1. Juvenile-onset diabetes: Now called as Type-I (Insulin dependent)—IDDM.
It requires insulin to control the hyperglycemia.
2. Maturity onset diabetes: Type-II NIDDM— (Noninsulin Dependent).
Differences
Other Factors
1. Heredity: In both types, familial tendency is noted.
 Genetic factors more important in those who develop after 40.
 Juvenile type, susceptibility is associated with particular HLA phenotype. RISK is two to
three times more in those who are HLA phenotype B8 or BW.
2. Autoimmunity: is the system of immune responses of an organism against its own cells and tissues.
 Insulin-dependent juvenile type maybe an autoimmune disorder and has been found to
co-exist with other autoimmune disorders. Evidences in favour of autoimmunity:
•Lymphocytic and plasma cells infiltrations in pancreas.
• Detection of autoantibodies by immunofluorescence.
3. Infections:
 Certain viral infections may precipitate Juvenile type. Incidence is high after mumps.
Antibodies to coxsackie B4 virus have been found in young Juvenile type.
4. Obesity:
 Majority of middle aged maturity-onset diabetics are obese, stress like pregnancy may
precipitate.
5. Diet:
 Over-eating and under activity are also predisposing factors in elderly middle aged
maturity onset diabetes.
6. Insulin antagonism:
 In maturity onset diabetes, the deficiency of insulin is relative and glucose induced
insulin secretion may be greater and more prolonged than normal. This deficiency may be
due to insulin antagonism, exact cause for the same is not known but various factors have
been incriminated from time to time. They are:
 Synalbumin of Vallence-Owen in plasma, dialyzable, thermostable substance.
 β1- lipoprotein factor: Another similar factor found in β-lipoprotein fraction of plasma in
diabetics.
 Insulin antibodies.
 Secretion of abnormal and less active insulin or altered insulin.
 A tissue barrier to the transport of insulin to the cells, probably receptor deficiency.
 Lack of cellular response to insulin.
(b) Secondary:
This forms a minor group. Diabetes is secondary to some other diseases.
1. Pancreatic diabetes:
 Pancreatitis
 Haemochromatosis
 Malignancy of Pancreas.
2. Abnormal concentrations of antagonistic hormones:
 Hyperthyroidism
 Hypercorticism: like Cushing’s disease and syndrome
 Hyperpituitarism: Like acromegaly
 Increased glucagon activity.
3. Iatrogenic:
In genetically susceptibles, may be precipitated by therapy like corticosteroids, thiazide
diuretics.
CLINICAL FEATURES AND BIOCHEMICAL CORRELATIONS
• Large amounts of glucose may be excreted in urine (may be 90 to 100 G/day in some cases).
Loss of solute produces osmotic diuresis thus large volume of urine (polyuria).
•Loss of fluid leads to thirst and polydypsia.
• Polyphagia: Eats more frequently, more fond of sweets.
The above symptoms may persist for many months in maturity-onset diabetes. In juvenile onset
type-1, further symptoms develop if treatment is not started.
• Tissues including muscles received liberal supply of glucose but cannot use glucose due to
absolute or relative deficiency of insulin/ or transport defect to cells. This causes weakness and
tiredness.
• As glucose cannot be used for fuel, fat is mobilized leading to increase FFA- in blood and liver.
• Increased acetyl-CoA is diverted for cholesterol synthesis—Hypercholesterolaemia and
Atherosclerosis.
• Increased ketone bodies lead to acidosis, which leads to hyperventilation (air-hunger).
• If ketosis is severe, acetone will be breathed out, giving characteristic “fruity” smell in breath.
There may be excessive breakdown of tissue proteins. Deaminated amino acids are catabolised
to provide energy, which accounts for Loss of weight.
• Due to ketosis, the subject develops anorexia, nausea, and vomiting. Continued loss of water
and electrolytes increases dehydration.
• Ketoacidosis produces increasing drowsiness, leading to diabetic coma in untreated cases.
METABOLIC CHANGES IN DIABETES MELLITUS
1. Hyperglycaemia:
 Occurs as a result of decreased and impaired transport and uptake of glucose into muscles
and adipose tissues.
 Repression of key glycolytic enzymes like Glucokinase, phosphofructokinase and
pyruvate kinase takes place.
 Derepression of key gluconeogenic enzymes like Pyruvate carboxylase, phosphoenol
pyruvate, carboxykinase, fructose biphosphatase and glucose-6-phosphatase occur,
promoting gluconeogenesis in Liver. This further contributes to hyperglycaemia.
 Elevated amino acid level in the blood particularly alanine provides fuel for
gluconeogenesis in Liver.
2. Amino Acids Level
 Transport and uptake of amino acids in peripheral tissues is also depressed causing an
elevated circulating level of amino acids, particularly alanine. Glucocorticoid activity
predominates having catabolic action on peripheral tissue proteins, releasing more amino
acids in blood.
 Amino acids breakdown in Liver results in increased production of urea N.
3. Protein synthesis: Protein synthesis is decreased in all tissues due to:
 Decreased production of ATP
 Absolute or relative deficiency of Insulin.
4. Fat Metabolism
 Decrease extramitochondrial de Novo synthesis of FA and also TG synthesis due
to decrease in acetylCoA from carbohydrates, ATP, NADPH and glycero-(p) in
all tissues.
 Stored lipids are hydrolysed by increased Lipolysis liberating free fatty acids
(FFA).
 Increased FFA interferes at several steps of carbohydrate phosphorylation in
muscles, further contributing to hyperglycaemia.
Effects of Increased FFA Level
 FFA reaching the Liver in high concentration inhibits further FA synthesis by a
feedback inhibition at the acetyl-CoA carboxylase step.
 Fats are mobilised for energy; increased fatty acid oxidation increases acetylCoA level, which in turn activates Pyruvate carboxylase, stimulating the
gluconeogenic pathway required for conversion of amino acids C-skeletons to
glucose.
 FA also stimulates gluconeogenesis by entering TCA cycle and increasing
production of citrates. Citrate in turn inhibits glycolysis at phosphofructokinase
level. Eventually FA inhibits TCA cycle at the level of citrate synthase and
possibly pyruvate dehydrogenase complex and Isocitrate dehydrogenase level.
5. Effect on glycogen synthesis:
Glycogen synthesis is depressed as a result of:
 Decreased glycogen synthase activity due to deficiency of insulin.
 By activation of phosphorylase producing glycogenolysis through the action of
epinephrine and/or glucagon (antagonistic) hormones.
 By increased ADP: ATP ratio.
COMPLICATIONS OF DIABETES MELLITUS
1. Immediate:
 Diabetic ketoacidosis and coma is one of the most important and dreaded
complication especially in Type-I.
II. Late complications:
 Other complications are late to appear and are due to changes in blood vessels.
These are of two types:
 Involvement of large vessels
 Involvement of small vessels.
(a) Large vessels involvement:
 Atherosclerosis and its effects:
 Involvement of coronary vessels can produce myocardial infarction.
 Involvement of cerebral vessels can produce stroke.
(b) Small vessels changes involve:
 Thickening of basement membrane
 Microvascular changes.
1. Diabetic retinopathy (70%):
 Tiny haemorrhages, punctate or flame-shaped, exudates. Haemorrhage in vitreous
humour can cause sudden blindness.
2. Diabetic cataract: Is due to:
 Non-enzymatic glycosylation of lens protein, α-crystallin;
 Osmotic damage to lens protein due to accumulation of sorbitol.
3. Diabetic nephropathy (50% cases):
Characterised by
(a) Proteinuria,
(b) Hypertension and
(c) Oedema.
4.
5.
6.
7.
The triad is called as Kimmelsteil-Wilson syndrome. Microscopic lesions are called as
‘Kimmelsteil-Wilson lesions/disease’. Lesions are often present when syndrome is not
developed. Sometimes kidney lesions may be shown as:
 Papillary necrosis: A dangerous complication.
 Pyelonephritis: When secondary infections occur.
Peripheral neuritis (neuropathy):
Manifestated by loss of sensation and tingling. Biochemically probably the cause is
myoinositol deficiency. Sometimes there may be associated myopathies, weakness of
muscles.
Diabetic gangrene:
Occurs due to diminished blood supply due to atherosclerotic changes in blood vessels.
Also associated tissue hypoxia due to formation of HbA (glycosylated Hb), less oxygen
carrying capacity.
Skin lesions:
Prone to infections: boils/ulcers and carbuncles. There may be necrosis of skin,
Necrobiosis diabeticorum.
 May be punctate depigmented atrophy
 Wound healing is delayed.
Pulmonary tuberculosis:
Susceptible to pulmonary tuberculosis.
GLUCOSE TOLERANCE TEST (GTT)
What is Carbohydrate Tolerance?
The ability of the body to utilise carbohydrates may be ascertained by measuring its
carbohydrate tolerance. It is indicated by the nature of blood glucose curve following the
administration of glucose.
Glucose tolerance is a valuable diagnostic aid. A 70 kg man can ingest approx. 1500 gm/ day.
Decreased Glucose Tolerance
• In Diabetes mellitus
•In hyperactivity of anterior pituitary and adrenal cortex
• In hyperthyroidism.
Increased Tolerance
• Hypopituitarism
• Hyperinsulinism
• Hypothyroidism
• Adrenal cortical hypofunction (such as Addison’s disease)
• Also if there is decreased absorption, like sprue, caeliac disease.
TYPES OF GLUCOSE TOLERANCE TEST
This is of two types:
(A) Standard oral glucose tolerance test
(B) IV glucose tolerance test.
(A) Standard Oral GTT
Indications
• In patients with transient or sustained glycosuria, who have no clinical symptoms of Diabetes
with normal fasting and PP blood glucose.
• In patients with symptoms of Diabetes but with no glycosuria and normal fasting blood glucose
level.
• In persons with strong family history but no overt symptoms.
• In patients with glycosuria associated with thyrotoxicosis, infections/sepsis, Liver diseases,
Pregnancy, etc.
• In women with characteristically large babies 9 lbs or individuals who were large babies at
birth.
• In patients with neuropathies or retinopathies of undetermined origin.
• In patients with or without symptoms of DM, showing one abnormal value.
Pre-requisites:
Precautions to be taken on the day prior to the test:
• The individual takes usual supper at about 2000 hours and does not eat or drink anything after
that. Early morning if so desires, a cup of tea/or coffee may be given without sugar or milk. No
other food or drink is permitted till the test is over.
 Should be on normal carbohydrate diets at least for three days prior to test (approx
300 G daily), otherwise false high curve may be obtained. FOUR
 Complete mental/and physical rest.
 No smoking is permitted.
 All samples of blood should be venous preferably. If capillary blood from ‘finger prick’
is used, all samples should be capillary blood.
Procedure
1. A fasting sample of venous blood is collected in flouride bottle (fasting sample)
2. The bladder is emptied completely and urine is collected for qualitative test for glucose and
ketone bodies (fasting urine).
3. The individual is given 75 Gm of glucose dissolved in water about 250 ml to drink. Lemon
can be added to make it palatable and to prevent nausea/vomiting. Time of oral glucose
administration is noted.
4. A total of five specimens of venous blood and urine are collected every ½ hour after the oral
glucose viz. ½ hour, 1 hour, 1½ hour, 2 hour and 2½ hour.
5. Glucose content of all the six (including fasting sample) samples of blood are estimated and
corresponding urine samples are tested qualitatively for presence of glucose and ketone bodies.
A curve is plotted which is called as Glucose tolerance curve.
Explanation and Significance of a Normal Curve
1. A sharp rise to a peak, averaging about 50 per cent above the fasting level within 30 to 60
minutes. Extent of the rise varies considerably from person to person, but maximum should not
exceed 160 to 180 mg% in normal subjects.
Reason
• Rise is due directly to the glucose absorbed from the intestine, which temporarily exceeds the
capacity of the Liver and tissues to remove it.
• As the blood glucose concentration increases, regulatory mechanisms come into play.
• Increased insulin secretion due to hyperglycaemia, Hepatic glycogenesis is increased,
• Hepatic glycogenolysis is decreased, and Glucose uptake and utilization in tissues increase.
2. A sharp fall to approximately the fasting level at the end of 1½ to 2 hours.
Reason:
Glucose now leaves the circulation faster than it is entering. This is due to:
• Continuing stimulation of the mechanisms stated above, i.e. increased utilisation and hepatic
glycogenesis, and
• To slowing or completion of glucose absorption from the intestines.
3. Hypoglycaemic “dip”:
Continued fall to a slightly sub fasting (10 to 15 mg lower than fasting value) at 2 hours and
subsequent rise to fasting level at 2½ to 3 hours.
Reason:
The hypoglycaemic ‘dip’ is due to “inertia” of the regulatory mechanisms. The decreased
output of glucose by liver and increased utilisation induced by the rising blood glucose are not
reversed as rapidly as the blood sugar falls.
Characteristics of Different Types of GTC
(a) A Normal GTC
1. Fasting blood glucose within normal limits of 60 to 100 mg% (“True” glucose)
2. The highest peak value is reached within one hour.
3. The highest value does not exceed the renal threshold, i.e. 160 to 180 mg%
4. The fasting level is again reached by 2½ hour
5. No glucose or Ketone bodies are detected in any specimens of urine.
A normal GTC:
(b) Diabetic Type of GTC
1. Fasting blood glucose is raised 110 mg% or more (“True” Glucose).
2. The highest value is usually reached after 1 to 1½ hour.
3. The highest value exceeds the normal renal threshold.
4. Urine samples always contain glucose except in some chronic diabetics or nephritis who may
have raised renal threshold (Dangerous type), hyperglycaemia but no glycosuria. Urine may or
may not contain ketone bodies depending on the type of Diabetes and severity.
5. The blood glucose does not return to the fasting level within 2½ hours. This is the most
characteristic feature of true DM.
According to severity, it may be:
(a) Mild Diabetic curve,
(b) Moderately severe Diabetic curve, and
(c) Severe Diabetic curve
(b) Renal glycosuria curve:
Glucose appears in the urine at levels of blood glucose much below 170 mg%.
Patients who show no glycosuria when fasting may have glycosuria when the blood glucose is
raised.
The condition may be:
• Idiopathic without any pathological significance
• Occasionally occurs in certain renal diseases and in pregnancy.
• May be found in case of “early” Diabetes with low renal threshold
• It has been reported in children of diabetic parents.
These cases should be reviewed from time to time (every six months).
(d) ‘Lag’ Curve (or Oxyhyperglycaemic Curve):
1. Fasting blood glucose is normal but it rises rapidly in the ½ to 1 hour and exceeds the renal
threshold so that the corresponding urine specimens show glucose.
2. The return to normal value is rapid and complete.
This type of GTC may be obtained in:
• Hyperthyroidism
• After gastroenterostomy
• During pregnancy
• Also in “early” diabetes.
A patient showing “lag curve” should be reviewed from time to time after every six months.
GTC of arterial (capillary) blood differs from GTC of venous blood as follows:
1. Rise begins somewhat earlier.
2. Peak is usually reached at 30 to 45 minutes.
3. The level in capillary blood may be 20 to 70 mg (average 30) higher than the venous blood.
4. The return to the fasting level at 1½ to 3 hours is not as rapid as in the case of venous blood.
TOPIC: PROTEIN METABOLISM
GLUCOGENIC AND KETOGENIC AMINO ACIDS:
Glucogenic amino acids:
These are the amino acids that are the precursor for the synthesis of carbohydrates i.e. arginine,
phenylalanine e.t.c
Ketogenic amino acid:
These are the amino acids that are precursor for the synthesis of ketone compounds like acetyl
CoA, Acetoacetate e.t.c. which are the precursor for the synthesis of biomolecules like lipids.
METABOLISM OF AMINO ACIDS,SPECIALIZED PRODUCTS
METABOLIC DISORDER.
AND THEIR
GLYCINE:
 A nonessential, glycogenic amino acid.
 Actively involved in the synthesis of many specialized product i.e. Heme, purines,
creatine
 Incorperated in the body for the synthesis of proteins,synthesis of serine and glucose,
particitipate in one carbon metabolism.
Synthesis of glycine:
 Synthesized from serine in the presence of hydroxymethyl transferase which is
tetrahydrofolate dependent
 Can be obtained from threonine catalysed by threonine aldolase
 Glycine can be synthesized from one carbon compoun (N5N10-methylene THF),CO2 and
NH3 in the precence of glycine synthase.
SYNTHESIS OF SPECIALIZED PRODUCT:
1. Formation of purine ring: utilized in the formation of position 4 and 5 opf carbon and
position 7 nitrogen in the purine ring.
2. Synthesis of heme (porphyrine ): glycine condences with succinyl CoA to form δ-amino
levulinate which is the precursor for Heme synthesis.
Succinyl CoA +glycine
δ-amino levulinate (ALA)
3. Biosynthesis of creatinine:glycine mmethinine and arginine are required for the synthesis
of creatinine
 Transfer of guanidino group of arginine to glycine in the presence of arginine
transamidase to produce guanidoacetate (glycocyamine).
 S-Adenosylmethionine (active methionine) transfers methyl group from
glycocyamine to produce creatine which occurs in the liver.
 Creatine is phosphorylated to phosphocreatine (cratine phosphate) in the presence
of creatine kinase and stored in the muscles as a high energy compound.
Clinical importance:
Serum: creatine 0.2-0.6 mg/dl creatinine 0.6-1mg/dl
Urine:creatine 0-50mg/day creatinine 1-2g.day
Metabolic disorders of glycine metabolism.
1. GLYCINURIA:
A state in which there is high excreation of glycine in urine above 0.5-1 g/day due to defective
rena reabsorbtion.
Characterized by increased tendency for the formation of oxalate real stones.
2. Primary hyperoxaluria:
Defect due to glycine transaminase impairment in glyoxalate oxidation to formate
Charactcetrized by increased urinary oxalate leading oxalate stoness.(oxalosis) depositing of
oxalate is observed in various tissues.
PHENYLALANINE AND TYOSINE :
They are aromatic amino acid,phenylalanine is essential amino acids while tyrosine is nonessential amino acids.
Metabolism of phenylalanine occurs through tyrosine
Tyrosine can be incorperated into various proteins like epinephrine, norepinephrine,
dopamine(catecholamine), thyroid hormones and pigments like melanine.
Conversion of phenylalanine to tyrosine:
Under normal circunstances degradation of phenyalanine occurs through tyrosine.
Phenylanalanine is hydroxylated by phenylalanine hydroxylase (present in the liver)to produce
tyrosine.
This
is
irreversible
reaction
and
requires
bio[pterine
as
the
coenzyme(tetrahydrobiopterine) which is oxidised to dihydrobiopterine which is then
regenerated by NADPH dependent dihydrobiopterine reductase. The failure or blockage of
phenyalanine hydroxylase may result in phenylketonuria.
Degradation of tyrosine or phenylalanine:
1. Phenylalanine is converted to tyrosine
2. Tyrosine undergoes transamination to give p-hydroxyphenypyruvate in the precence of
tyrosin transaminase (PLP sdependent).
3. p-hydroxyphenypyruvate undergoes oxidative decarboxylase in presence of phydroxyphenypyruvate hydroxylase or dioxygenase (copper containing enzyme) to
produce homogentisate
4. Homogentisate is
converted to 4-maleylacetoacetate a reaction catalysed by
homogentisate oxygenase which requiresmolecular oxygen to break the aromatic ring.
5. Isomerization of 4-maleylacetoacetate to 4 fumarylacetoacetate reaction catalysed by
maleylacetoacetate isomerase.
6. 4 fumarylacetoacetate undergoes hydrolysis to yeild fumarate and acetoacetate which are
the precursor in lipid synthesis ,TCA cycle and glucose synthesis.
SYNTHESIS OF MELANIN:
Synthesis of melanin occors in the melanosomes present in the melanocytes.
Tyrosin is the precusor for melaniin and tyosinase(copper containing) is the primary enzyme that
is involved in the synthsis if the pigment.
Tyrosine gets concerted to3,4 –dihydroxyphenylalanine (DOPA) in the reaction catalysed by
tyrosinase.DOPA gets converted to dopaquinone and in subsequent reactions itforms
leucodopachrome followed by 5,6 dihydroxyindole. Oxidation process takes place,Tyrosinse
converts the 5,6 dihydroxyindole .melanochrome is synthesized from indole quinone which on
polymerization we get melanin.
Alternatively, dopaquinone iscondenced with cysteine and red melanin is generated.
BIOSYNTHESIS OF THYROIOD HORMONES:
Thyroid hormones are synthesized from tyrosine. Tetraiodothyronine(thyroxine) and
triiodothyronine homones.
Iodinization of tyrosine ring occurs to produce mono and diiodotyrosine from which
triiodothyronine (t3)and thyroxine (T4) are synthesized
Protein thyroglobulin undergoes proteolytic breakdown to release the free T3and T4 hormones.
Disorders of tyrosine and phenylalanine metabolism:
PHENYLKETONURIA:
Due to hepatic enzyme phenylalanine hydroxylase this leads to accumulation of phenylalanine in
tissues and blood. And in urine there is elevated amount of phenylpyruvate and other keto acids.
Biochemical manifestation:
1. Effects on central nervous system-mental retardation,failure to walk,failure to grow
tremor.failureto transport other aromatic amino acid like tryptophan and tyrosine,and
leads to defectin myelin formation.
2. Effect on pigmentation-inhibition of tyrosinase leads albinism
Diagnosis:
Done byand Guthrie test and ferric chloride test and green colour is obtained. Normal values in
phenylalanine in plasma PKU20-65mg/dl
Treatment:
Dietary intake of phenylalanine should be measured in plasma levels and adjusted.
TYROSENEMIA TYPE II: (RICHNER-HANART SYNDROME)
Due to blockage of tyrosine transaminase hence accumulation and excreation of tyrosine and its
metabolites
Characterised in skkin(dermatitis) and eye lession
Neonatal tyrosenemia:
Due to p-hydroxyphenylpyruvatedioxygengase. Can be controlled byascorbic acid
ALKAPTONURIA:
Defect on the enzyme homogentisate oxidase hence accumulation of homogentisate in blood and
tissues. Alkapton is the pigment produced incase of accumulation and deposit occurs in the
connective tissues,bones and various organs resulting in ochronosis
Diagnosis:
Benedict’s test,carry out ferric chloride and silver nitrate test in urine
Treatment:
Consumption of proteins with low phenylalanine contents
Tyosinisis or tyrosinemia type I:
Due to deficiency of Fumarylacetoacetate hydroxylase and maleylacetoacetate isomerase which
may lead to liver failure, rickets, renal tibular dysfunction and polyneuropathy
Treatment:
Recommended diets with low tyrosine, phenylalanine and methionine
ALBINISM:
Causes:
1. Deficiency or lack of enzyme tyrosinase
2. Secrease in melanosomes of melanocytes
3. Impairment in melanin polymerization
4. Lack of protein matrix in melanosomes
5. Limitation of substrate (tyrosine) availability
6. Presence of inhibitors of tyrosinase
The common cause of albinism is defect on tyrosinase enzyme responsible for the synthesis of
melanin.
Clinical manifestation:
Melanin protect the body from sun rays hence albinos havesensitive to sunlight and susceptible
to skin cancer(carcinoma). Photophobia is associated with lack of the pigment in the eyes.
UREA CYCLE:
Site of metabolism is liver and urea is the end product of protein metabolism.
Metabolism discovered or elucidated by Hans Kreb and Kurt Henseleit in 1932.
Urea has two amino groups one from ammonia and other from aspartate and carbon is supplied
from carbon dioxide.
Catalitic enzymes:
Two enzymes are found in mitochondria and others in cytosol.
Steps in urea cycle:
1. Synthesis of carbomoyl phosphate:
Synthesized by condesation carbon dioxide(CO2) with ammonium ions (NH4+) to form
carbomyl phosphate in the presence of carbomyl phosphate synthetase a rate limiting
enzyme requiring N-Acetylglutamate.
2. Formation of citrulline:
Citrulline is synthesized when ornithine is condence with carbomyl phosphate in the
presence of ornithine transcarbomylase. The citrulline synthesized at this point is
transported into cytosol. Ornithine and citrulline are basic amino.
3. Synthesis of arginosuccinate:
Citrulline is condensed by aspartate in the presence arginosuccinate synthetase to yeild
arginosuccinate. This is a step for second amino group incorperation and ATP is
requirded and is cleaved to yeild AMP and PPi
4. Cleavage of arginosuccinate:
Arginosuccinate is cleaved by arginosuccinase to give fumarate and arginine. Arginine is
immediate precursor for urea synthesis. Fumarate enters into TCA cycle, gluconegenesis.
5. Formation of urea:
Arginase leaves arginine to yield urea and ornithine. Ornithine enters into
mitochondriafor re-use.
Arginase is activated by Mn2+
CLINICAL SIGNIFICANCE OF UREA
A moderately active man consuming about 300 gm carbohydrates, 100 gm of fats and 100 gm of
proteins daily must excrete about 16.5 gm of N daily. 95 per cent is eliminated by the kidneys
and the remaining 5 per cent,for the most part as N, in the faeces.
1. Normal Level
The concentration of urea in normal blood plasma from a healthy fasting adult ranges from 20 to
40mg%.
2.Increase of Levels
Increases in blood urea may occur in a number of diseases in addition to those in which the
kidneys are primarily involved. The causes can be classified as:
Prerenal
Most important are conditions in which plasma vol/ body-fluids are reduced:








Renal
Salt and water depletion,
Severe and protracted vomiting as in pyloric and intestinal obstruction,
Severe and prolonged diarrhoea,
Pyloric stenosis with severe vomiting,
Haematemesis,
Haemorrhage and shock; shock due to severe burns,
Ulcerative colitis with severe chloride loss,
In crisis of Addison’s disease (hypoadrenalism).
The blood urea can be increased in all forms of kidney diseases:
 In acute glomerulonephritis.
 In early stages of type II nephritis (nephrosis) the blood urea may not be increased, but in
later stages with renal failure, blood urea rises.
 Other conditions are malignant nephrosclerosis,
 chronic pyelonephritis and mercurial poisoning.
 In diseases such as hydronephrosis, renal tuberculosis; small increases are seen but
depends on extent of kidney damage.
Postrenal Diseases
These lead to increase in blood urea, when there is obstruction to urine flow. This causes
retention of urine and so reduces the effective filtration pressure at the glomeruli; when
prolonged, produces irreversible kidney damage.
Causes:
 Enlargement of prostate,
 Stones in urinary tract,
 Stricture of the urethra,
 Tumours of the bladder affecting urinary flow.
Decreased levels:
Decreases in blood urea levels are rare. It may be seen:
 In some cases of severe liver damage,
 Physiological condition: Blood urea has been seen to be lower in pregnancy than in
normal nonpregnant women.
REGULATION OF UREA CYCLE:
The rate limiting enzyme is carbamoyl phosphate synthase. Requires NAG (N-acetyl
glutamate) synthesized from acetyl CoA and Glutamate. Increase of NAG increases synthesis of
urea in the liver.
Carbamoylphosphate synthase I and Glutamate dehydrogenase located in the nitochondria
coordinate the synthesis of NH3 for the synthesis of cabamoyl phosphate.
The remaining four enzymes particitipate in the synthesis of urea depending on the concentration
of the substrates.
INTERGRATION OF UREA CYCLE WITH TCA CYCLE:
 The formation of fumarate in urea cycle is the intergrating point to TCA cycle.
 Oxaloacetate undergoes transamination to produce aspartate which enters urea cycle as
the second source of amino group to synthesis urea.
 ATP (12) generated in the TCA cycle while 4 ATP are utilized in urea cycle.
 CO2 and H2O are the end product that are formed on complete oxidation of various
metabolites whareby CO2 generated is utilized in the urea synthesis.
Metabolic disorders of urea cycle:
Hyperammonemia- a disorder of increased amount of ammonia in blood.
Disorder
Enzyme involved
Hyperammonemia type 1
Carbamoyl phosphate synthase I
Hyperammonemia type II
Ornithinetranscarbamoylase
Citrullinemia
Arginosuccinate synthase
Arginosuccinic
Arginosuccinase
Hyperargininemia
Arginase
Blood urea clinical significance:
 Pre-renal-associated with increased protein breakdown hence negative nitrogen balance
observed during diabetic coma, thyroxicosis, leukemiableeding disorders
 Renal-increased in cases of patients suffering from acute glomerulonephritis, chronic
nephritis, nephrosclerosis,polycystic kidney.
 Post renal-elevated in cases of urinary tract obstraction i.e. tumor,stones
TOPIC: METABOLISM OF NUCLEIC ACIDS
Nucleotides consist of a nitrogenous base, a pentose and a phosphate. The pentose sugar is Dribose in RNA and 2-deoxy D-ribose in DNA. They participate in almost all the biochemical
processes, either directly or indirectly. They are structural components of nucleic acids,
coenzymes and are involved in the regulation of several metabolic reactions.
Steps of Biosynthesis
1. Formation of 5-phosphoribosyl-1-pyrophosphate (PRPP):
The process begins with D-Ribose-5'-P obtained from HMP-shunt pathway. PRPP is synthesized
by the enzyme PRPP synthase from D- ribose-5'-P and ATP.
2. Formation of 5'-phosphoribosyl-1 amine (PRA):
The amide group of glutamine is transferred to C of PRPP by the enzyme Glutamine PRPP
amido transferase. Phosphate group is replaced by –NH group. This gives the N-9 of purine ring.
3. Formation of glycinamide ribotide, GAR ( 5'-Phosphoribosylglycinamide):
Glycine condenses with PRA using ATP as energy source to form glycinamide ribotide (GAR).
The reaction is catalysed by the enzyme Glycinamide kinosynthase. This provides C-4, C-5 and
N-7 of the Purine ring.
4. Formation of formylglycinamide ribotide, FGAR ( 5'-phosphoribosyl-N-formylglycinamide):
The amino nitrogen of glycinamide is formylated by N10-formyl tetrahydrofolate catalysed by the
enzyme formyl transferase. The formyl carbon becomes C-8 of the purine ring.
5. Formation of a-N-formylglycinamidine ribotide, FGAM: ( 5'-phosphoribosyl-Nformylglycinamidine):
Another amide group of glutamine is transferred to FGAR to form FGAM.
Phosphoribosylglycinamidine synthase is the enzyme that catalyses the reaction and ATP
provides the energy. The reaction contributes N-3 of Purine ring.
6. Formation of 5-Aminoimidazole riboside, AIR ( 5'-phosphoribosyl-5-aminoimidazole):
This reaction is catalysed by the enzyme aminoimidazole ribosyl phosphate synthetase (AIRPsynthetase), which brings about the dehydratative closure of the ring, by removal of a molecule
of H2O. ATP is required for the reaction.
7. Formation of 5-aminoimidazole-4-carboxylic acid ribotide, C-AIR (also called 5'phosphoribosyl-5aminoimidazole-4 carboxylate):
This reaction uses CO2 to carboxylate AIR. It contributes to C-6 of the purine nucleus. Neither
Biotin nor ATP is required for carboxylation.
8. Formation of 5-aminoimidazole-N-succinyl carboxamide ribotide, 5-AISCR.
(5-'phosphoribosyl-5-aminoimidazole-4-N succino carboxamide):
This reaction is catalysed by the enzyme succinyl carboxamide synthetase. ATP is used to
condense Aspartic acid with aminoimidazole carboxylate-5-Phosphate. This contributes to N-1
of the purine nucleus.
9. Formation of 5-aminoimidazole-4-carboxamide ribotide, 5-AICAR. ( 5'-Phosphoribosyl
5- aminoimidazole-4-carboxamide):
5-AISCR under-goes cleavage by the cleaving enzyme adenylosuccinate lyase to form 5-AICAR
and fumarate.
10. Formation of 5-formamidoimidazole-4-carboxyamide ribotide, 5-FICR. (5'phosphoribosyl-5-formamidoimidazole-4 carboxamide):
Carbon-2 (C-2) the final carbon of the Purine ring is donated by N10-formyl tetrahydrofolate in a
reaction catalysed by the enzyme formyl transferase and forms 5-FICR.
11. Formation of inosinic acid (IMP):
5-FICR undergoes a dehydrative ring closure, by elimination of one molecule of water.
The reaction is catalysed by the enzyme IMP cyclohydrolase to form Inosinic acid (IMP).
Formation of Other Purine Nucleotides
1. Formation of AMP from IMP
This is brought about in 2 steps:
 The enzyme adenylosuccinate synthetase catalyses the condensation of Aspartic acid with
IMP to form adenylosuccinate. GTP provides the required energy.
 Adenylosuccinate is then cleaved by the cleaving enzyme adenylosuccinate lyase to form
AMP and fumarate.
Formation of GMP from IMP: This is brought about in 2 steps:
 The enzyme IMP dehydrogenase oxidises IMP to xanthosine monophosphate or xanthylic
acid (XMP).
• In the second stage, the amide group of glutamine is transferred to C-2 of Xanthine
Monophosphate catalysed by the enzyme. GMP synthetase to form GMP.
A. Purine Salvage Pathways
Two pathways are available.
1. One-step Synthesis
• Formation of GMP and IMP: Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase)
catalyses the one-step formation of the nucleotides from either guanine or hypoxanthine, using
PRPP as the donor of the ribosyl moiety.
Regulation:
The enzyme HGPRTase is regulated by the competitive inhibition of GMP and IMP respectively.
• Formation of AMP: The enzyme Adenine phosphoribosyl transferase (APRTase) in similar
way catalyses the formation of AMP from adenine, ribosyl moiety is donated by PRPP.
Regulation:
The enzyme APRTase is regulated by the competitive inhibition of AMP.
2. Two-Step Synthesis
(Nucleoside phosphorylase-nucleoside kinase pathway)
Under some conditions, it is possible for purine bases to be salvaged by a two-step process as
under:
Nucleoside Phosphorylase is an enzyme that brings about nucleoside breakdown. But the
reaction is readily reversible and can form back ‘nucleoside’ which is rather a favourable
pathway. Once the nucleoside is formed, a kinase enzyme may phosphorylate it to the 5'nucleotide.
Formation of AMP
Adenine is the only purine that may be salvaged by the two-step pathway. Guanosine and inosine
kinases have not been detected in animal cells.
PURINE SALVAGE CYCLE
This is a cycle in which GMP and IMP as well as their deoxyribonucleotides are converted into
their respective nucleosides by a purine 5'nucleotidase enzyme. The nucleosides formed can be
hydrolytically cleaved producing the corresponding sugar phosphates and setting free the Nbases. The guanine and hypoxanthine then can be phosphoribosylated again to complete the
cycle.
Regulation of Purine Synthesis
• PRPP synthetase enzyme regulates purine synthesis. It is allosterically inhibited by the
feedback effects of PRPP and a number of purine nucleotides such as AMP, GMP, ADP, GDP,
NAD and FAD.
• Glutamine PRPP amidotransferase enzyme is for the rate-limiting step of purine synthesis. It is
regulated by feedback inhibitory effects of AMP and GMP.
•A proper balance between the Adenine and guanine concentration is maintained by
adenylosuccinate synthetase and IMP dehydrogenase respectively.
CATABOLISM OF PURINES
Purines are Catabolised to Uric Acid
An average of 600 to 800 mg of uric acid is excreted by human beings most of it is found in
urine. Guanine and Adenine nucleotides have their separate enzymes until the formation of a
common product xanthine. The final reaction is the conversion of xanthine to uric acid by
xanthine oxidase.
A. Adenine Nucleotide Catabolism
1. In the Liver and Heart Muscle
 An enzyme purine-5’-nucleotidase hydrolyses adenylate (AMP). As a result a nucleoside,
 Adenosine is obtained.
 Adenosine deaminase removes ammonia from adenosine and gives inosine. SECTION
THREE
 Purine nucleoside phosphorylase phosphorolyses inosine to ribose-1-P and hypoxanthine.
 Xanthine oxidase then converts hypoxanthine to xanthine and xanthine to uric acid.
Molecular
 oxygen is reduced at each stage to the superoxide (O2–) which is converted to H2O2 by
superoxide dismutase.
2. In the Skeletal Muscle
 Adenylate deaminase converts AMP into inosine monophosphate (IMP).
 Inosine monophosphate is hydrolysed by purine-5-nucleotidase to inosine.
 Inosine is changed to uric acid as mentioned above. GMP inhibits adenylate deaminase to
reduce the catabolism of AMP.
B. Guanine Nucleotide Catabolism
1. In the Liver, Spleen, Kidneys, Pancreas
 GMP is hydrolysed by purine-5’-nucleotidase into guanosine.
 Purine nucleoside phosphorylase phosphorolyses guanosine into Ribose -1-Phosphate and
guanine.
 Guanine deaminase deaminates guanine to xanthine and produces NH3.
 Oxidation of xanthine to uric acid is brought about by xanthine oxidase.
2. In the Liver Mainly
 Guanosine deaminase deaminates guanosine into xanthosine.
 Xanthosine is then phosphorolysed to Ribose -1-Phosphate and xanthine by purine
nucleoside phosphorylase.
 Xanthine is then oxidised to uric acid by xanthine oxidase.
Further Catabolism of Uric Acid (Nonprimates)
o In many nonprimate animals uric acid may be oxidized and decarboxylated by
uricase, a hepatic copper containing enzyme to allantoin.
o Some fishes carry uricase as well as allantoinase. This converts allantoin into
allantoic acid.
o Amphibians and other such animals contain allantoinase which converts allantoic
acid into ureidoglycolate. Ureidoglycolate is further cleaved by ureidoglycolase
into urea and glyoxylate.
o Urea is further converted to NH3 and CO2 in crustaceans by an enzyme urease
found in their liver.
URIC ACID METABOLISM AND CLINICAL DISORDERS OF PURINE
The main site of uric acid formation is the liver from where it is carried to the kidneys.
o Miscible Pool
It is the quantity of uric acid present in body water.
In normal subjects an average of 1130 mg of uric acid is present. Plasma contains higher
concentration of uric acid compared to other body compartments containing water.
o Turnover
This is the rate at which uric acid is synthesized and lost from the body. Normally, 500–600 mg
of uric acid is synthesised. Not all is excreted in urine; some uric acid is excreted in bile. Some is
converted to urea and ammonia by the intestinal bacteria.
Distribution:
It is very irregularly distributed in the body. Serum contains 3 to 7 mg/dl. Average values
are slightly higher in males. Red cells contain half as much uric acid as serum. Muscles also
contain less amounts compared to blood.
o Dietary effects:
Uric acid excretion continues at a rather steady rate during starvation and during a purine-free
diet owing to the so-called endogenous purine metabolism. The ingestion of foods high in
nucleoproteins such as glandular organs produces a marked increase in urinary uric acid. Diets
like milk, eggs and cheese, with low purine contents causes practically no increase in urinary
uric acid.
o Effect of hormones:
Administration of the glucocorticoid hormones and ACTH increases the excretion of uric acid in
urine.
o Excretion of uric acid:
Uric acid in the plasma is filtered by the glomeruli but is later partially reabsorbed by the renal
tubules. Glycine is believed to compete with uric acid for tubular reabsorption. Certain uricosuric
drugs such as salicylates, block reabsorption of uric acid. Lactic acid competes with uric acid in
its excretion.
Hence in lactic acidosis uric acid is retained, and can produce gout. There is now conclusive
evidence for tubular secretion of uric acid by kidney. Thus uric acid is cleared both by
(a) glomerular filtration; (b) by tubular secretion.
Clinical disorders
Uric acid is the end product of purine metabolism in humans. Normal concentration in the serum
of adults is 3-7 mg/dl.
Hyperuricemia refers to the elevation in the serum uric acid concentration. L DISORDERS:
CLINICAL ASPECTS
Gout:
It is a metabolic disease associated with overproduction of uric acid.
It is a chronic disorder characterised by:
o Excess of uric acid in blood (Hyperuricemia).
o Deposition of sodium monourate in alveolar and non alveolar structures
producing so called tophi.
o Recurring attacks of acute arthritis. These are due to deposition of monosodium
urate in and around the structures of the affected joints.
Types:
There are two main types of gout:
1. Primary gout,
2. Secondary gout.
1. Primary Gout
Here the hyperuricaemia is not due to increased destruction of nucleic acid. The essential
abnormality is increased formation of uric acid from simple carbon and nitrogen compounds
without intermediary incorporation into nucleic acids.
a. Primary metabolic gout:
It is due to inherited metabolic defect in purine metabolism leading to excessive rate of
conversion of glycine to uric acid.
X-linked recessive defects enhancing the de novo synthesis of purines and their catabolism can
also lead to hyperuricaemia. For example, defects of PRPP synthetase may make it feedback
resistant. X-linked recessive defects of hypoxanthine guanine phosphoribosyl transferase may
reduce utilisation of PRPP in the salvage pathway. Increased intracellular PRPP enhances de
novo purine synthesis.
b. Primary renal gout:
It is due to failure in uric acid excretion.
2. Secondary Gout
a. Secondary metabolic gout:
It is due to secondary increase in purine catabolism in conditions like leukaemia, prolonged
fasting and polycythemia.
b. Secondary renal gout:
Occurs due to defective glomerular filtration of urate due to generalised renal failure.
Treatment of Gout:
Consist of:
(a) Palliative Treatment and
(b) Specific Treatment
(a) Palliative treatment:
This involves;
a. Bed rest in acute stage,
b. Diet—Purine free diet,
c. Restricting alcohol consumption.
• Anti-inflammatory Drugs
1. Colchicine:
One of the nonspecific anti-inflammatory drugs.
It has no effect on urate metabolism or excretion.
Colchicine therapy is instituted during acute attack.
Mechanism:
Suppresses the synthesis and secretion of the chemotactic factor that is produced in urate crystalinduced inflammation.
Dosage:
Available as 0.5 mg tablet.
In acute gout:
One tablet hourly till symptoms are relieved or diarrhoea occurs.
Long-term management: One tablet 3 to 4 times a week.
2. NSAIDS:
Drugs like: Indomethacin, Diclofenac, Naproxen, Piroxicam, Fenoprofen, Flurbiprofen,
Ibuprofen, Rofecoxabin, etc.
These drugs inhibit the synthesis of prostaglandins which are important mediators of the
inflammatory response. These drugs have been found effective in treating patients having
recurrent attacks of acute gout and also for terminating acute attack of gout.
(b) Specific Treatment
Aim: To lower the uric acid level of blood.
Methods: The above can be achieved in three ways:
o By increasing the renal excretion of uric acid (uricosuric drugs).
o By decreasing the synthesis of uric acid using enzyme inhibitor.
o By increasing oxidation of uric acid.
1. Uricosuric Drugs
A uricosuric agent is one that enhances the renal excretion of uric acid probably by specific
inhibition of its tubular reabsorption or secretion.
Drugs used are:
o Salicylates
Effects vary with dosage. In low dosage of 1 to 2 gm/day, salicylates cause uric acid retention
but in higher dosage 5 to 6 gm/day it has uricosuric effect. Longterm therapy with high dosage is
not desirable due to the side effects.
o Probenecid (Benemide)
– It is an efficient and harmless uricosuric drug.
– Lowers the uric acid level. Fall is immediate and sustained.
Dose: Available as 500 mg tablet. ½ tablet twice daily for the first week and then one tablet
twice daily. Not recommended for children. Therapy is continued for 10 to 12 weeks and patients
can return to normal activities.
o Halofenate:
The drug has good uricosuric effect. It also has a hypolipaemic effect. It liberates urates from
urate binding sites of proteins of plasma and removes uric acid by normal excretion. The drug
can be safely used for short-term and long-term therapy.
2. Enzyme Inhibitor
• Allopurinol (zyloprin) drug of choice: It has similar structure like hypoxanthine. Acts by
competitive inhibition on “xanthine oxidase” and thus uric acid synthesis is impaired. The drug
causes a rapid fall in serum uric acid level and an increase in concentration of hypoxanthine and
xanthine in blood. Both xanthine and hypoxanthine are more soluble and so are excreted easily in
urine. Allopurinol is acted upon by Xanthine oxidase and converted to alloxanthine.
Dosage: Available as 100 mg tablet.
Maintenance: 200 to 600 mg daily. Not recommended in children.
• The drug can be used in secondary hyperuricaemia.
• Allopurinol also has an inhibitory action on the enzyme tryptophan pyrrolase.
2. Drugs Increasing Uric Acid Oxidation
• Urate oxidase: The drug can be used in lowering uric acid level by oxidising uric acid.
Dosage: 10,000 IU daily for 10 days. It shows a significant decrease in uric acid level. Can be
used in severe gout with renal involvement and secondary hyperuricaemia.
3. Lesch-Nyhan Syndrome
Affects only males due to the defect of hypoxanthine-guanine phosphoribosyltransferase. The
enzyme is almost absent and leads to increased purine salvage pathway from PRPP. This can
result in severe gout, renal failure, poor growth, spasticity and tendency for self-mutilation.
4. Xanthinuria:
An autosomal recessive deficiency of xanthine oxidase, blocks the oxidation of hypoxanthine
and xanthine to uric acid. It can cause xanthine lithiasis and hypouricaemia.
5. Adenosine deaminase deficiency:
An autosomal recessive deficiency of adenosine deaminase. It is associated with severe
immunodeficiency and both T cells and B cells (lymphocytes) are deficient. There occurs an
accumulation of deoxyribonucleotides which inhibit further production of precursors of DNA
synthesis especially dCTP. Hypouricaemia occurs which is due to defective breakdown of purine
nucleotides. Recently Gene replacement therapy has been used successfully in a few cases.
6. Nucleoside phosphorylase deficiency:
An autosomal recessive deficiency of purine nucleoside phosphorylase, causes the urinary
excretion of guanine and hypoxanthine nucleosides. There is reduced production of uric acid.
There is severe deficiency of cell mediated and humoral immunity.
c. In von-Gierke’s disease:
Deficiency of G-6-phosphatase leads to elevated rate of pentose formation in HMP. This
acts as a good substrate for PRPP synthetase and enhances the synthesis of purines followed by
their catabolism to uric acid.
Increase lactic acid competes with uric acid excretion resulting to retention of uric acid.
YNTHESIS OF PYRIMIDINES
SYNTHESIS OF PYRIMIDINES
The synthesis of pyrimidines is much simpler compared to purines.
Aspartate and glutamine (amide group) and CO2 contibute to atoms in the formation of
pyrimidine ring.
Materials required for pyrimidine synthesis;yrimidines
o Carbamoyl phosphate: Synthesised from CO2 and glutamine.
o PRPP: 5-phosphoribosyl-1-pyrophosphate
o
Various enzymes: Carbamoyl phosphate synthetase II, Transcarbamoylase,
Dihydro-orotase, Dehydrogenase, Transferase and Decarboxylase. ATP: For
energy, amino acid: Aspartic acid and Cofactors: FAD+, NAD+, and Mg++.
Steps of Synthesis
1. Synthesis of carbamoyl phosphate:
The synthesis of pyrimidine ring begins with the formation of carbamoyl phosphate from
glutamine, CO2 and ATP, catalysed by the enzyme carbamoyl phosphate synthetase II. The
enzyme is present in cytosol and does not require N-acetyl glutamate (NAG). The reaction is
controlled by the feed-back inhibitory effect of pyrimidine nucleotide UTP.SECTION THREE
2. Formation of carbamoyl aspartic acid (CAA):
The enzyme apartate transcarbamoylase then transfers carbamoyl group from carbamoyl
phosphate to aspartic acid to form carbamoyl aspartic acid (CAA).
Committed and rate limiting step.
• Enzyme is oligomeric.
• Enzyme is inhibited (“feedback” allosteric inhibition) by CTP and UTP.
• Inhibition can be reversed by ATP.
3. Formation of dihydro-orotic acid:
The reaction is catalysed by the enzyme dihydro-orotase, which removes a molecule of water
(dehydration) from carbamoyl aspartic acid and brings about the closure of the ring to produce
dihydro-orotic acid.
4. Formation of orotic acid:
The next step is oxidation of dihydro-orotate which is brought about by the enzyme dihydroorotate dehydrogenase. The enzyme carries Fe, S, FMN and FAD in its prosthetic group. NAD+
is required as a coenzyme.
5. Formation of orotidylic acid:
Under the influence of the enzyme Orotate phosphoribosyl trans-ferase, 5phosphoribosyl group
from 5-phospho-ribosyl-1pyrophosphate (PRPP) is transferred to orotic acid to produce
orotidylic acid. Mg++ ions are required in the reaction.
6. Formation of uridylic acid (UMP):
Orotidylic acid undergoes decarboxylation, catalysed by the enzyme Orotidylate decarboxylase,
and forms uridylic acid (UMP).
• UMP is the first pyrimidine nucleotide to be formed.
• Other pyrimidine nucleotides viz. UDP, UTP, CTP and d-UDP are synthesised from UMP.
• UMP is the “feedback” inhibitor of the decarboxylase enzyme.
Formation of Other Pyrimidine Nucleotides
1. Formation of UDP and UTP
• UMP is phosphorylated by ATP to form UDP, catalysed by the enzyme nucleoside
monophosphokinase
• UDP can be further phosphorylated by ATP to form UTP.
2. Formation of CTP
CTP can be synthesised from UTP, catalysed by the enzyme CTP synthetase. The reaction
requires ATP for energy and glutamine.
3. Formation of dUDP (Deoxyuridine Diphosphate).
dUDP is formed from UDP by the action of the enzyme Ribonucleoside reductase. The reaction
requires Thioredoxin (Iron-sulphur protein) and NADPH.
Synthesis of Thymine Deoxyribonucleotides
 dTMP is synthesised from dUMP. dUMP may arise from the hydrolysis of dUDP by
phosphatase. Alternatively, dCMP may be produced by phosphorylation of circulating
deoxycytidine with the help of ATP. The enzyme is deoxycytidine kinase.
Thymidylate synthetase methylates dUMP into dTMP by transferring methyl group from N5, N10
methylene H4 folate, the C5 of the uracil moiety.
Thymidylate synthetase is covalently inhibited by folate antagonists like aminopterin and
amethopterin which competitively inhibits DH2-folate reductase.
 dTMP can be changed to dTDP by transphosphory-lation with the help of ATP, Mg++
using thymidylate kinase. This is the synthesis of thymine and pseudouridine ribonucleotides.
This occurs in TψU loop of each t-RNA molecule. UMP residue is precursor.
Synthesis of Pyrimidine Deoxyribonucleotides
Reduction of ribose to deoxyribose is achieved by ribonucleotide reductase. This enzyme
transfers electrons from iron-sulphur containing protein called thioredoxin to the 2’-C of ribose
moiety of purine or pyrimidine nucleotide diphosphate. NADPH reduces the oxidized
thioredoxin to its reduced state by thioredoxin reductase.
The enzyme contains FAD as its prosthetic group. Rate limiting step in dTTP synthesis. dTDP is
then phosphorylated to dTTP by thymidine diphosphokinase using ATP and Mg++.
CATABOLISM OF PYRIMIDINES
1. Cytosine and Uracil
o The first step of the catabolism of pyrimidines is dephosphorylation to the
nucleosides by 5’- nucleotidases. Pyrimidine nucleosides are then phosphorolysed
into free pyrimidines and pentose 1 phosphate with the help of Pi and nucleoside
phosphorylases.
o Uracil, is then reduced to 5,6-dihydrouracil by dihydrouracil dehydrogenase using
NADPH.
o Cytosine will form uracil by deaminase.
o The hydrolysis of 5,6-dihydrouracil is the next step.
This is done hydrolytically by hydropyrimidine hydrase to produce ß-ureidopropionic acid.
The next step is further hydrolysis by ureidopropionase into CO2, NH3 and β-alanine.
CLINICAL DISORDERS OF PYRIMIDINE METABOLISM
Orotic Aciduria:
It is of 2 types:
 Type I orotic aciduria: It is an autosomal recessive genetic disorder of a protein acting as
both orotate phosphoribosyltransferase and OMP decarboxylase. Orotate fails to be
converted to uridylate. This results in accumulation of orotate in blood elevating its level,
Growth retardation and megaloblastic anaemia.
Type II orotic aciduria:
It is autosomal recessive, affecting OMP decarboxylase and is characterized by megaloblastic
anaemia and the urinary excretion of asididine in higher concentrations than orotate.
Other Causes of Orotic Aciduria
o Orotic aciduria that accompanies Reye Syndrome:
Probably it is a consequence of the inability of severely damaged mitochondria to utilise
carbamoyl phosphate, which then becomes available for cytosolic overproduction of orotic acid.
o Associated with deficiency of urea cycle enzyme:
Increased excretion of orotic acid, uracil and uridine, sometimes accompanies a deficiency of
liver mitochondrial ornithine transcarbamoylase. Excess carbamoyl-P passes to cytosol where it
stimulates pyrimidine nucleotide biosynthesis resulting to mild orotic aciduria, which may get
aggravated by high N2 foods.
o Drug Related Orotic Aciduria
Allopurinol: Allopurinol an alternative substrate for orotate phosphoribosyl transferase competes
with orotic acid. The resulting nucleotide product also inhibits orotidylate decarboxylase
resulting in orotic aciduria and orotidinuria.
o 6-Azauridine:
6-Azauridine, following its conversion to 6-azauridylate, also competively inhibits orotidylate
decarboxylase resulting to increased excretion of orotic acid (orotic aciduria) and orotidine
(orotidinuria).
TOPIC LIPID METABOLISM:
OXIDATION OF FATTY ACIDS:
The oxidation of fatty acids occurs in the mitochondria and liver.
o ACC: acetyl-CoA carboxylase (ACC)
o PKA: cAMP-dependent protein kinase (PKA),
Fatty Acids Are Activated Before Being Catabolized. Fatty acids must first be converted to an
active intermediate before they can be catabolized. This is the only step in the complete
degradation of a fatty acid that requires energy from ATP. In the presence of ATP and coenzyme
A, the enzyme acyl-CoA synthetase (thiokinase) catalyzes the conversion of a fatty acid (or free
fatty acid) to an “active fatty acid” or acyl-CoA, which uses one high-energy phosphate with the
formation of AMP and PPI. The PPi is hydrolyzed by inorganic pyrophosphatase with the loss of
a further high-energy phosphate, ensuring that the overall reaction goes to completion. Acyl-CoA
synthetases are found in the endoplasmic reticulum, peroxisomes, and inside and on the outer
membrane of mitochondria.
Carnitine plays a major role in the transport of long-chain fatty acids through the inner
mitochondrial membrane. Long-chain acyl-CoA cannot pass through the inner mitochondrial
membrane, but its metabolic product, acylcarnitine, can.
General reactions of fatty acid oxidation
Beta oxidation of palmitic acid:
1. The fatty acid is activated from free palmitic acid to palmitoyl CoA
2. It undergoes oxidation to yield 8 successful acetyl C0A that enters the tricarboxylic acid
cycle.
Utilization and excretion of the ketone bodies:
Under metabolic conditions associated with a high rate of fatty acid oxidation, the liver produces
considerable quantities of acetoacetate and D(_)-3-hydroxybutyrate (ß-hydroxybutyrate).
Acetoacetate continually undergoes spontaneous decarboxylation to yield acetone. These three
substances are collectively known as the ketone bodies (also called acetone bodies or
[incorrectly*] “ketones”). Acetoacetate and 3-hydroxybutyrate are interconverted by the
mitochondrial enzyme D()-3-hydroxybutyrate dehydrogenase; the equilibrium is controlled by
the mitochondrial [NAD+]/[NADH] ratio, ie, the redox state.
Transport of ketone bodies from the liver and pathways of utilization and oxidation occurs in
extra hepatic tissues.
Hypolipidemic drugs and possible mechanism of action
DISORDERS OF FATTY ACID OXIDATION:
Refsum’s diseases:
Is a genetically inherited disorder (autosomal recessive) due to phytanate α- oxidase deficiency
which converts phytanic acid to pristanic acid hence accumulation in tissues and blood (indicated
by an increase in 20% of the total fatty acids). It affects both the ages from childhood to adults. It
s clinical manifestations are:
 Neurological symptoms like neuropathy with distal muscular atrophy and progressive
paresis of the distal parts of extremeties.
 Sensory disturbances which may include parenthesiae and severe knee pains.
 Eye manifestation like night blindness, concentric narrowing of visual fields and typical
pigmentary retinitis.
 Increased CSF proteins whilst cell count is usually normal.
Treatment:
 Reduction or omitting the intake of phytols diet which is a precursor of phytanic acids.
TOPIC: ENDOCRINOLOGY INTRODUCTION
Most glands of the body deliver their secretions by means of ducts. These are called exocrine
glands.
There are few other glands that produce chemical substance that they directly secrete into the
bloodstream for transmission to various target tissues. These are ductless or endocrine glands.
The secretions of endocrine glands are called as hormones.
They regulate many functions, including growth, sex drive, hunger, thirst, digestion, metabolism,
fat burning and storage, blood sugar and cholesterol levels, and reproduction
Definition of Hormones
It is a chemical substance which is produced in one part of the body, enters the circulation
and is carried to distant target organs and tissues to modify their structures and functions.
Hormones are stimulating substances and act as body catalysts. The word hormone is derived
from Greek word hormacin meaning to excite. The hormones catalyse and control diverse
metabolic processes, despite their varying actions and different specificities depending on the
target organ.
Similarities of Hormone and Enzyme
The hormones have several characteristics in common with enzymes:
• They act as body catalysts resembling enzymes in some aspect.
• They are required only in small quantities.
• They are not used up during the reaction.
Dissimilarities of Hormone and Enzyme
They differ from enzymes in the following ways:
• They are produced in an organ other than that in which they ultimately perform their action.
• They are secreted in blood prior to use.
• Thus the circulating levels of hormones can give some indication of endocrine gland activity
and target organ exposure. Because of the small amounts of the hormones required, blood levels
of the hormones are extremely low. In many cases it is ng/μg or mIU, etc.
• Structurally they are not always proteins. Few hormones are protein in nature, few are small
peptides. Some hormones are derived from amino acids while some are steroid in nature.
The major hormone secreting glands are:
• Pituitary • Thyroid • Parathyroid • Adrenal • Pancreas • Ovaries • Testes. Several other
glandular tissues are considered to secrete hormones, viz.:
• JG cells of kidney: May produce the hormone erythropoietin which regulates erythrocyte
maturation, erythropoiesis.
• Thymus: This produces a hormone that circulates from this organ to stem cells in lymphoid
organ inducing them to become immunologically competent lymphocytes.
• Pineal gland: It produces a hormone that antagonises the secretion or effects of ACTH. It also
produces factors called glomerulotrophins that regulates the adrenal secretion of aldosterone.
• GI tract: Few hormones are also produced by certain specialised cells of GI tract and they are
called GI Hormones.
Classification of Hormones: Hormones can be classified chemically into three major groups:
1. Steroid hormones: These are steroid in nature such as adrenocorticosteroid hormones,
anqdrogens, estrogens and progesterone.
2. Amino acid derivatives: These are derived from amino acid tyrosine, e.g. epinephrine,
norepinephrine and thyroid hormones.
3. Peptide/Protein hormones: These are either large proteins or small or medium size peptides,
e.g. Insulin, glucagon, parathormone, calcitonin, pituitary hormones, etc.
Factors Regulating Hormone Action
Action of a hormone at a target organ is regulated by four factors:
1. Rate of synthesis and secretion: The hormone is stored in the endocrine gland.
2. In some cases, specific transport systems in plasma.
3. Hormone-specific receptors in target cell membranes which differ from tissue to tissue, and
4. Ultimate degradation of the hormone usually by the liver or kidneys.
Several other glandular tissues are considered to secrete hormones, viz.:
• JG cells of kidney: May produce the hormone erythropoietin which regulates erythrocyte
maturation, erythropoiesis.
• Thymus: This produces a hormone that circulates from this organ to stem cells in lymphoid
organ inducing them to become immunologically competent lymphocytes.
• Pineal gland: It produces a hormone that antagonises the secretion or effects of ACTH. It also
produces factors called glomerulotrophins that regulates the adrenal secretion of aldosterone.
• GI tract: Few hormones are also produced by certain specialised cells of GI tract and they are
called GI Hormones. Classification of Hormones: According to Li, the hormones can be
classified chemically into three major groups:
1. Steroid hormones: These are steroid in nature such as adrenocorticosteroid hormones,
androgens, estrogens and progesterone.
2. Amino acid derivatives: These are derived from amino acid tyrosine, e.g. epinephrine,
norepinephrine and thyroid hormones.
3. Peptide/Protein hormones: These are either large proteins or small or medium size peptides,
e.g. Insulin, glucagon, parathormone, calcitonin, pituitary hormones, etc.
Biochemical mechanisms of hormonal actions.
Although the exact site of action of any hormone is still not well understood, the following
mechanisms of actions of a hormone have been proposed.
1. Interaction with nuclear chromatin (nuclear action): Steroid hormones act mostly by
changing the transcription rate of specific genes in the nuclear DNA. The steroid hormone has a
specific soluble, oligomeric receptor protein (mobile receptor) either in the cytosol and/or inside
the nucleus. This brings about conformational changes and also changes in the surface charge of
the receptor protein to favour its binding to the nuclear chromatin attached to nuclear matrix. The
receptor-steroid complex is translocated to the nuclear chromatin and binds to a steroid
recognising acceptor site called the hormone-responsive element (HRE) of a DNA strand on the
upstream side of the promoter site for a specific steroid responsive gene.
2. Membrane receptors:
 certain molecules cannot enter target cells through the membrane lipid bilayer.
 This is achieved by the specific receptor molecules present on the surface of the plasma
membrane. Many hormones seen specifically involved in the transport of a variety of
substances across cell membrane.
 These hormones specifically bind to the receptors on cell membrane. They cause rapid
secondary metabolic changes in the tissue but have little effect on metabolic activity of
membrane-free preparations.
 Most protein hormones and catecholamines activate transport of membrane enzyme
systems by direct binding to specific receptors on the membrane.
3. Stimulation of enzyme synthesis at the ribosomal level:
 Activity at the level of translation of information is carried by the mRNA on the
ribosomes for the production of enzyme.
 Ribosomes taken from growth hormone treated animal have a modified capacity to
synthesise protein in the presence of normal mRNA.
Mechanism of action of steroid hormones through interaction with nuclear chromatin.
4. Direct activation at the enzyme level:
 Although the direct effect of a hormone on a pure enzyme is difficult to demonstrate,
treatment of the intact animal or of isolated tissue with some hormones results in a
changeof enzyme activity, not related to de novo synthesis.
5. c-AMP and hormone action:
 3’-5’ c-AMP plays a unique role in the action of many protein hormones.Its level may be
decreased or increased by hormonal action as the effect varies depending on the tissue.
 The hormones such as glucagon, catecholamines, PTH, etc. act by influencing a change
in intracellular c-AMP concentration through the adenylate cyclase c-AMP system. The
hormone binds to a specific membrane receptor.
 Different types of these receptors remain associated with either Gs or Gi type of
GTPdependent trimeric nucleotide regulatory complexes of the membrane. Both Gs and
Gi are made up of 3 subunits: Gs contains αs βγ while Gi contains αi βγ.
 Formation of the receptor-hormone complex promotes the binding of GTP to the α
subunit of either Gs or Gi.
 When αs-GTP is released it binds to adenylate cyclase located on the cytoplasmic surface
of the membrane and changes its conformation to activate it.
6. Role of polyphosphoinositol and diacylglycerol in hormone action:
 Just like c-AMP other compounds such as 1, 4, 5 inositol triphosphate (ITP) and
diacylglycerol (DAG) act as second messengers. This is specially found in case of
vasopressin, TRH, GnRH, etc.
 These hormones activate the phospholipase C-polyphosphoinositol system to produce
ITP and DAG.
 By binding with the specific receptor protein on cell membrane, the hormone activates a
trimeric nucleotide regulatory complex.
 The complex in turn activates phospholipase C on the inner surface of the membrane.
Inositol triphosphate enhances the mobilisation of Ca++ into the cytosol from
intracellular Ca++ pool from mitochondria, calcium ions then act as tertiary messenger.
While DAG activates the Ca++ phosphatidyl-serine-dependent protein kinase C located
on the inner surface of the membrane, by lowering its Km for Ca++.
 This enzyme then phosphorylates specific enzymes and other proteins in the cytosol to
modulate their activities.
7. Role of calcium in hormone action:
 The action of most protein hormones is inhibited in absence of calcium even though
ability to increase or decrease c-AMP is comparatively unimpaired.
 Thus calcium may be more terminal signal for hormone action than c-AMP. It is
suggested that ionised calcium of the cytosol is the important signal.
 The source of this calcium may be extracellular fluid or it may arise from mobilisation of
intracellular tissue bound calcium.
8. Role of c-GMP in hormone action:
 Hormones suchas insulin and growth hormone affect the guanylate cyclase c-GMP
system. This will increase the intracellular conc. of c-GMP and activate c-GMPdependent protein kinases.

The active c-GMP-protein kinase would in turn bring about phosphorylation of specific
cellular proteins to change their activities, leading to relaxation of smooth muscles,
vasodilatation and other effects.
 It is likely that Ca++ may act as a second messenger to activate guanylate cyclase and
thereby increasing the conc. of c-GMP inside the cell.
9. Role of phosphorylation of tyrosine kinase:
 In fact asecond messenger for insulin, growth hormone, prolactin, oxytocin, etc. has not
been identified so far.
 However binding of them to their respective membrane receptors activates a specific
protein kinase called tyrosine kinase which phosphorylates tyrosine residue of specific
proteins. This may bring about some metabolic changes.
Mechanism of hormone action.
REGULATION OF HORMONE SECRETION.
Hormone secretion is strictly under control of several mechanisms.
A. Neuroendocrinal control mechanism:

Nerve impulses control some endocrine secretions. Cholinergic sympathetic fibres
stimulate catecholamine secretion from adrenal medulla.
 Centres in the midbrain, brainstem, hippocampus, etc. can send nerve impulses which
react with the hypothalamus through cholinergic and bioaminergic neurons.
 At the terminations of these neurons they release acetylcholine and biogenic amines to
regulate the secretions of hypophysiotropic peptide hormones from hypothalamic
peptidergic neurons.
 Some of the endocrine releases are controlled by either stimulatory or inhibitory
hormones from a controlling gland, e.g. corticosteroids are controlled by corticotropin
and thyroid hormones are controlled by thyrotropin from anterior pituitary.
 The tropins are further regulated by hypothalamic releasing hormones.
B. Feedback control mechanism:
 It is due mainly to negative feedback that such control is brought about. When there is a
high blood level of target gland hormones, it may inhibit the secretion of the tropic
hormone stimulating that gland.
 Adrenal cortex secretes a hormone called cortisol which brings about the inhibition of
secretion of corticotropin from anterior pituitary and corticotropin releasing hormone
from the hypothalamus by a long-loop feedback. This leads to reduction in cortisol
secretion.
C. Endocrine rhythms:
 There are certain cyclic rhythms associated with the secretion of hormones over a period
of time. When there is a cyclic periodicity of 24 hours, it is called as circadian rhythm.
 However, if it is more than 24 hours, it is named as infradian rhythm and when it is
less than 24 hours it is called as ultradian rhythm. Due to such rhythms, the highest and
lowest conc. of corticotropin is normally found in the morning and around midnight.
 Growth hormone and prolactin rise in the early hours of deep sleep. Cortisol peak is
found between 4 AM and 8 AM. Endocrine rhythms result from cyclic activities of a
biological clock in the limbic system, supplemented by the diurnal light dark and sleep
activity cycles and mediated by the hypothalamus.
Thyroid hormones.
THYROID GLAND AND ITS HORMONES
The thyroid gland consist two major cells that secrete thyriod hormones:
1. Follicular cells:
2. Parafollicular C-cells:
The principal hormones secreted by the follicular cells of thyroid are:
 Thyroxine (T4)
 Tri-iodo thyronine (T3) and‘Reverse’ T3
Chemistry of Thyroid Hormones:
 The hormones T4, T3 and “reverse” T3 are iodinated amino acid tyrosine.
 The iodine in thyroxine accounts for 80 per cent of the organically bound iodine in
thyroid venous blood.
 Small amounts of ‘reverse’ tri-iodo thyronine, Monoiodotyrosine (MIT) and other
compound are also liberated.
 Two raw materials (substrates) required by thyroid gland to synthesise the thyroid
hormones are:
I.
Thyroglobulin
II.
Iodine
A. Thyroglobulin
Thyroid hormones are synthesised by the iodination of tyrosine residues of a large protein
called “thyroglobulin”
Chemistry of thyroglobulin.
 Thyroglobulin is a dimeric glycoprotein, 19S in type (a macroglobulin) with a molecular
weight of 660,000.
 The receptor tyrosine molecules are present in this macroglobulin protein, each molecule
containing 115
tyrosine residues.
 Carbohydrates account for 8 to 10 per cent of the weight of thyroglobulin and iodide for
about 0.2 to 1 per cent, depending on the iodine content of the diet. The carbohydrates
are N-acetyl glucosamine, mannose, glucose, galactose, fucose and sialic acid.
 About 70 per cent of the iodide in thyroglobulin exists as inactive precursors ‘monoiodo-tyrosine’ (MIT)
Thyroid aciner cells have three functions:
1. They synthesise thyroglobulin and stores as colloid in follicles.
2. They collect and transport I2 for synthesis of the hormones in the colloid (see below), and
• They remove T3 and T4 from thyroglobulin secreting the hormones into the circulation.
Proteins other than thyroglobulin:
Besides thyroglobulin, some other albumin-like and hormonally inactive two other 4S
iodoproteins of uncertain function are also found in thyroid.
Note: The above can appear in circulating blood and can contribute to the measured protein
bound iodine (PBI).
Synthesis of thyroglobulin: Thyroglobulin is synthesised in acinar cells and stored in colloid.
Steps:
1. Thyroglobulin is translated by the polysomes on the granular endoplasmic reticulum (ER) of
thyroid acina cells. The polypeptide portion is synthesised as two subunits and later aggregated
to form the dimer.
2. The glycosylation of the molecule starts in the smooth endoplasmic reticulum (SER) with the
incorporation of mannose and is completed in the Golgi cisternae where the other sugars as Nacetyl glucosamine, galactose, fucose and sialic acid are added in the oligosaccharide chains
(Studied by EM auto-radiography).
3. The glycoproteins formed as above are packaged into small vesicles. Electron-lucent
membrane bound vesicles containing thyroglobulin are then pinched off from the Golgi
cisternae.
4. The vesicles then move towards the apical plasma membrane and fuse with it, releasing their
contents into the colloid of thyroid follicles.
B. Iodine
 The other substrate required for thyroid hormone synthesis is iodine.
Iodine metabolism: Vegetables and fruits grown and obtained from sea-shore and also
sea fishes are rich in iodine.
 Vegetables and fruits in hilly regions lack iodine (for people residing in hilly regions,
table salt should be iodinated).
 Ingested dietary iodine is converted to iodide and absorbed from the gut. Of a total of 50
mg of iodine in the body about 10 to 15 mg are in thyroids.
 The normal daily intake of iodide is 100 to 200 μg. Minimum requirement is 25 μg.
 This iodide is absorbed mainly from small intestine and is transported in plasma in loose
attachment to protein; can also be absorbed from lungs, other mucous membranes and
skin.
 Small amounts of iodide are secreted by the salivary glands, stomach, and small intestine
and traces in milk. About 2/3 (40-80%) of the ingested iodide is excreted by the kidneys,
the remaining 1/3 is taken up by the thyroid glands for synthesis of thyroid hormones.
 Thyroid-stimulating hormone (TSH) of anterior pituitary gland stimulates iodide
uptake by the thyroid gland.
 Inorganic iodide in plasma and cells varies from 0.3 to 1.0 μg per cent. Part of the
“circulating pool” is iodide liberated from thyroid hormones broken down in the tissues
and to a minor extent in the thyroid itself. In the kidneys, 97 per cent of the filtered iodide
is reabsorbed, so that the loss from the body by this route, at normal plasma iodide levels
is about 15 μg/day.
Steps of Iodine Incorporation in Thyroid Gland for Synthesis of Thyroid Hormones
1. Iodine trapping
The thyroid concentrates iodide by “actively” and “selectively” transporting it from the
circulation to the colloid. The transport mechanism is called as “iodidetrapping” mechanism or
“iodide-pump”.
The I2 trapping is done:
• Against electrical gradient
Biosynthesis of thyroid hormones
Steroid hormones.
Steroids derived from cholesterol in animals include five families of hormones (the `androgens,
estrogens, progestins, glucocorticoids and mineralocorticoids) and bile acids.
 Androgens such as testosterone and estrogens such as estradiol mediate the
development of sexual characteristics and sexual function in animals.
 The progestins such as progesterone participate in control of the menstrual cycle and
pregnancy.
 Glucocorticoids (cortisol, for example) participate in the control of carbohydrate,
protein, and lipid metabolism, whereas the mineralocorticoids regulate salt (Na+, K+,
and Cl_) balances in tissues.
 The bile acids (including cholic and deoxycholic acid) are detergent molecules secreted
in bile from the gallbladder that assist in the absorption of dietary lipids in the intestine.
Therapeutic use of anabolic steroids:
 Bone marrow stimulation (lukemia, kidney failure)
 Stimulation of growth (for growth failures) .
 Stimulation of appetite and increase in muscle mass, for patients with chronic wasting
conditions such as AIDS.
 Hormone replacement therapy, for treating men with low levels of testosterone
Testosterone derivatives.
Sex Hormone Structure
groups and alkene features.
The hypophysis( pituitary gland).
HORMONES OF THE ANTERIOR PITUITARY
The hormones secreted by the anterior lobe of the pituitary gland are:
• Growth hormone, and Pituitary tropic hormones such as prolactin, gonadotropins FSH and
LH, Thyrotropic hormones (TSH) and Adrenocorticotropic Hormone (ACTH). The pituitary
tropins are under the positive and negative control of peptide factors from hypothalamus. Further
the tropic hormones are usually subject to feedback inhibition at the pituitary or hypothalamic
level by hormone product of the final target gland. Prolactin (mammotropin), TSH (or
thyrotropin), FSH and LH (gonadotropins), ACTH (corticotropin) are the tropic hormones
secreted by the pituitary gland.
Growth hormone.

Growth hormone (GH) or somatotropin (STH) controlled by Hypothalamic Releasing
Factors.

Control of hormone secretion from the pituitary is in part modulated by regulating factors
or hormones from the hypothalamus.
 The median eminence of the hypothalamus is connected directly to the pituitary stalk.
Within this stalk is a portal system of blood vessels required to maintain normal secretory
activity of the pituitary gland.
 The activities of the cells of the anterior lobe are controlled by the nerve cells of the
hypothalamus which send axons to the capillary beds.
 The nerve endings liberate chemical substances, hypothalamic releasing factors or
hormones.
Hypothalamic Hormone or Factor Abbreviation
• Corticotropin (ACTH) releasing hormone
CRH or CRF
• Thyrotropin (TSH) releasing hormone
TRH or TRF
• Follicle stimulating hormone
(FSH) FSH-RH or
releasing hormone
FSH-RF
• Luteinizing Hormone (LH) releasing
LH-RH or
Hormone
LH-RF
• Growth-hormone (GH)
GH-RH or
releasing hormone
GH-RF
• Growth-hormone release
GH-RIH or
inhibiting hormone
GIF
• Prolactin (PL) release
PL-RIH or
inhibiting hormone
PL-RIF
• Prolactin (PL) releasing hormone
PRH or PRF
• Melanocyte stimulating hormone
MSH-RIH or
(MSH) release inhibiting hormone
MSH-RIF
• Melanocyte stimulating hormone
MSH-RH or
(MSH) releasing hormone
MSH-RF
Gland/Tissue Hormones Major Functions Structure
Hypothalamus -Thyrotropin-releasing hormone (TRH) Stimulates secretion of TSH and prolactin
Peptide
Corticotropinreleasing hormone (CRH)
Causes release of ACTH
Peptide
Growth hormone–releasing hormone (GHRH) Causes release of growth hormone
Peptide
Growth hormone inhibitory hormone (GHIH)
Inhibits release of growth hormone Peptide
(somatostatin)
Gonadotropin-releasing hormone (GnRH)
Causes release of LH and FSH
Dopamine or prolactin-inhibiting factor (PIF)
Inhibits release of prolactin Amine
Anterior pituitary Growth hormone
Stimulates protein synthesis and overall growth
Peptideof most cells and tissues
Thyroid -stimulating hormone (TSH)
Stimulates synthesis and secretion of thyroid
Peptide
hormones (thyroxine and triiodothyronine)
Adrenocorticotropic hormone (ACTH)
Stimulates synthesis and secretion of adrenocortical
Peptide
Hormones
(cortisol, androgens, and aldosterone)
Prolactin Promotes development of the female breasts and Peptide
secretion of milk
Follicle-stimulating hormone (FSH)
Causes growth of follicles in the ovaries and sperm
Peptide
maturation in Sertoli cells of testes
Luteinizing hormone (LH)
Stimulates testosterone synthesis in Leydig cells of
Peptide
testes; stimulates ovulation, formation of corpus
luteum, and estrogen and progesterone synthesis
in ovaries
Posterior pituitary Antidiuretic hormone (ADH) (causes Increases water reabsorption by the
kidneys and
Peptide
(vasopressin) causes vasoconstriction and increased blood
pressure
Oxytocin Stimulates milk ejection from breasts and uterine
Peptide
contractions
Thyroid Thyroxine (T4) and triiodothyronine (T3) Increases the rates of chemical reactions in
most Amine
cells, thus increasing body metabolic rate
Calcitonin -Promotes deposition of calcium in the bones and Peptide
decreases extracellular fluid calcium ion concentration
Adrenal cortex -Cortisol Has multiple metabolic functions for controlling
Steroid
metabolism of proteins, carbohydrates, and fats;
also has anti-inflammatory effects
Aldosterone
Increases renal sodium reabsorption, potassium
Steroid
secretion, and hydrogen ion secretion
Adrenal medulla Norepinephrine, epinephrine Same effects as sympathetic stimulation Amine
Pancreas Insulin (b cells) Promotes glucose entry in many cells, and in
Peptide
(Chapter 78) this way controls carbohydrate metabolism
Glucagon (a cells) Increases synthesis and release of glucose from
Peptide
the liver into the body fluids
Parathyroid Parathyroid hormone (PTH) Controls serum calcium ion concentration Peptide
increasing calcium absorption by the gut and
kidneys and releasing calcium from bones
Testes Testosterone Promotes development of male reproductive
system and male secondary sexual characteristics
Ovaries Estrogens Promotes growth and development of female
reproductive system, female breasts, and female
secondary sexual characteristics
Progesterone Stimulates secretion of “uterine milk” by the uterine
endometrial glands and promotes development of
secretory apparatus of breasts
Steroid
Steroid
Steroid
Placenta Human chorionic gonadotropin (HCG) Promotes growth of corpus luteum and secretion
of
Peptide
estrogens and progesterone by corpus luteum
Human somatomammotropin Probably helps promote development of some
Peptide
fetal tissues as well as the mother’s breasts
Estrogens See actions of estrogens from ovaries
Progesterone See actions of progesterone from ovaries
Kidney Renin Catalyzes conversion of angiotensinogen to
angiotensin I (acts as an enzyme)
Steroid
Steroid
Peptide
1,25-Dihydroxycholecalciferol Increases intestinal absorption of calcium and bone Steroid
mineralization
Erythropoietin
Increases erythrocyte production
Peptide
Heart Atrial natriuretic peptide (ANP) Increases sodium excretion by kidneys, reduces
Peptideblood pressure
Stomach Gastrin Stimulates HCl secretion by parietal cells
Peptide
Small intestine Secretin Stimulates pancreatic acinar cells to release
Peptide
bicarbonate and water
Cholecystokinin (CCK) Stimulates gallbladder contraction and release of
Peptide
pancreatic enzymes
Adipocytes
Leptin Inhibits appetite, stimulates thermogenesis
Peptide
Metabolic role:
Growth hormone has a variety of effects on different tissues. The hormone acts slowly
requiring from 1-2 hours to several days before its biological effects are detectable. This
slow action and its stimulatory effects on RNA synthesis suggest that it is involved in protein
synthesis. The hormone acts by binding to specific membrane receptors on its target cells. But its
exact mechanism of action and the second messenger are not yet known.
1. Protein synthesis:
Growth hormone brings about positive nitrogen balance by retaining nitrogen. It stimulates
overall protein synthesis with an associated retention of phosphorus probably by increasing
tubular reabsorption. Blood amino acid and urea level are decreased.
It facilitates the entry of amino acids into the cell. In addition, growth hormone facilitates
protein synthesis in muscle tissue by a mechanism independent of its ability to provide amino
acids. Thus protein synthesis carries on even if the amino acid transport is blocked.
• Growth hormone increases DNA and RNA synthesis.
• It increases the synthesis of collagen.
2. Lipid metabolism:
Growth hormone brings about lipolysis in a mild way by mobilising fatty acids from adipose
tissue by activating the hormone sensitive triacylglycerol lipase. Thus it increases circulating free
fatty acids.
3. Carbohydrate metabolism:
Growth hormone is a diabetogenic hormone, antagonises the effect of insulin. Hypersecretion
of GH can result in hyperglycaemia, poor sugar tolerance and glycosuria. Growth hormone
produces:
• Hyperglycaemia by increasing gluconeogenesis.
• It reduces insulin sensitivity and thereby decreases the hypoglycaemic effect of insulin.
• It brings about glycostatic effect, i.e. increases liver glycogen. It can also increase muscle and
cardiac glycogen level probably by reducing glycolysis.
4. Effect on growth of bones and cartilages:
Growth hormone when secreted in abnormally high concentration prolongs the growth of
epiphyseal cartilages to cause overgrowth of long bones.
Prolactin: PRL or Leuteotropic Hormone (LTH).
This is a monomeric simple protein (MW 23,000). It contains 199 amino acids with three –S–S–
linkages.
Metabolic role
• The main function of PRL is to stimulate mammary growth and the secretion of milk. By
acting through specific glycoprotein receptors on plasma membraneof mammary gland cells, it
stimulates mRNA synthesis. This ultimately leads to enlargement of breasts during pregnancy.
This is called as mammotropic action.
• The synthesis of milk proteins such as lactalbumin, and casein takes place after parturition such
an effect is called as lactogenic action.
• Estrogens, thyroid hormones and glucocorticoids increase the number of prolactin receptors on
the mammary cell membrane.
• Progesterone has the opposite effect.
Thyrotropic Hormone or Thyroid Stimulating Hormone (TSH)
This is produced by basophil cells of anterior pituitary and is glycoprotein in nature. Its
molecular weight is approximately 30,000. This consists of a and b subunits.
• The α-subunit of TSH, LH, HCG and FSH are nearly identical.
• The biological specificity of thyrotropin must therefore be in β-subunit. The α-subunit
consists of 92 amino acids while β-subunit has 112 amino acids. Both α and β have several
disulfide bridges. Its carbohydrate content is 21 per cent and its α and β chains bear two and one
oligosaccharide chains linked by N-glycosidic linkages to specific asparagine residues. The
chains are synthesised separately by separate structural genes and later undergo posttranslation
modifications and glycosylations separately.
METABOLIC ROLE OF THYROID HORMONES
1. Effects on Protein Metabolism:
• In hypothyroid children and in physiological doses, thyroid hormones when given in small
doses, favour protein anabolism, leading to N-retention (+ve N-balance), because they stimulate
growth.
• Large, unphysiological doses of thyroxine, cause protein catabolism, leading to –ve N-balance.
2. Effects on Carbohydrate Metabolism:
Net effect on carbohydrate metabolism:
• Increase in blood sugar ↑ (hyperglycaemia), and glycosuria
• Increased glucose utilisation, and decreased glucose tolerance. Thyroid hormones are,
therefore, antagonistic to insulin.
• Thyroid hormones increase the rate of absorption of glucose from intestine.
• Decreased glucose tolerance may be contributed to also by acceleration of degradation of
insulin.
Note: Diabetes mellitus is aggravated by coexisting thyrotoxicosis or by administration of
thyroid hormones.
• Increased hepatic glycogenolysis, because they enhance the activity of Glucose-6-phosphatase.
• In addition there is increased sensitivity to catecholamines, they potentiate the glycogenolytic
effect of epinephrine by increasing the β-adrenergic receptors on hepatic cell membrane.
• Stimulate glycolysis as well as oxidative metabolism of glucose via TCA cycle and also
increasing Hexosemonophosphate pathway (HMP-Shunt). Thyroxine increases the activity of
“G-6-PD” enzyme in liver.
• Thyroid hormones cause a decrease of glycogen store in liver and to a lesser extent, in the
myocardium and skeletal muscle.
• At the same time, thyroid hormones increase hepatic gluconeogenesis by increasing the
activities of pyruvate carboxylase and PEP carboxykinase.
3. Effects on Lipid Metabolism:
• Increases lipolysis in adipose tissue thus increasing plasma FFA. This effect is rather indirect
in the sense it increases sensitivity to catecholamines, potentiates the lipolytic effect of
epinephrine, by increasing the β-adrenergic receptors on adipocyte cell membrane.
• They may stimulate, at the same time, lipogenesis ↑ by increasing the activities of malic
enzyme, ATP citrate lyase and G-6-PD.
• Cholesterol: Despite the fact that hepatic synthesis of cholesterol and PL is depressed
following thyroidectomy and is increased in thyrotoxicosis, the concentration of cholesterol and
to a lesser extent PL in plasma is increased in hypothyroidism and decreased in hyperthyroidism.
Decreased value in hyperthyroidism is explained as follows:
Although thyroid hormones increase the rate of biosynthesis of cholesterol, they increase:
• The rate of degradation
• Increases the formation of bile acids (cholic acid/deoxycholic acid) and
• Increases biliary excretion, to a greater extent accounting for the lowered blood concentration.
• Lipoproteins: The concentration of plasma lipoproteins of Sf 10 to 20 class (LDL) is
frequently increased in hypothyroidism and decreased in thyrotoxicosis, or following
administration of thyroid hormones to normal subjects.
4. Calorigenic Action: Thyroid hormones increases considerably O2 consumption and oxygen
coefficient of almost all metabolically active tissues. Exceptions are: Brain, testes, uterus,
lymph nodes, spleen and anterior pituitary. There is increase in heat production and BMR. This
effect is due to:
• Induction of glycerol-3-P-dehydrogenase and other enzymes involved in mitochondrial
oxidation.
• More important is increased activity and increased units of Na+-K+ ATP-ase pump. It
hydrolyses ATP for transmembrane extrusion of Na+, leading to enhanced heat production, O2
consumption and oxidative phosphorylation.
There are glycoprotein receptors on the thyroid cell membrane which bind to the receptor
binding site on β-subunit of TSH. The complex then activates adenylate cyclase which catalyses
the formation of c-AMP which nacts as the second messenger for most TSH actions as follows:
• The TSH stimulates the synthesis of thyroid hormones at all stages such as Iodine uptake,
organification and coupling.
• It enhances the release of stored thyroid hormones.
• It increases DNA content, RNA and translation of proteins, cell size.
• It stimulates glycolysis, TCA cycle, HMP and phospholipid synthesis. Stimulation of last two
does not involve c-AMP.
• It activates adipose tissue lipase to enhance the release of fatty acids (lipolysis).
Adrenocorticotropic Hormone (ACTH) or Corticotropin
Metabolic role
• The principal actions of corticotropin are exerted on the adrenal cortex and extraadrenal tissue.
ACTH increases the synthesis of corticosteroids by the adrenal cortex and also stimulates
their release from the gland. Profound changes in the adrenal structure, chemical composition
and enzymatic activity are observed as a response to ACTH. Total proteinsynthesis is found to be
increased. Thus, ACTH produces both a tropic effect on steroid production and tropic effect on
adrenal tissue.
ACTH also stimulates the synthesis and secretion of
glucocorticoids.
• ACTH is found to increase the transfer of cholesterol from plasma lipoproteins into the
fasciculata cells.
• The ACTH induces rise in c-AMP, brings about phosphorylation and activation of cholesterol
esterase. The enzyme action ultimately makes a large pool of free cholesterol.
• Corticotropin promotes the binding of cholesterol to mitochondrial cytochrome P450 required
for hydroxylating cholesterol.
• It activates the rate limiting enzyme for conversion of cholesterol to pregnenolone.
• It activates dehydrogenases of HMP to increase the conc. of NADPH required for
hydroxylation.
• By activating adenylate cyclase of adipose tissue it increases intracellular c-AMP which in turn
activates hormone sensitive lipase. This enzyme is involved in lipolysis which increases the level
of free fatty acids.
• It leads to increased ketogenesis and decreased RQ.
Metabolic Role of FSH
It brings about its action by specific receptor binding and c-AMP.
In females:
• It promotes follicular growth
• Prepares the Graafian follicle for the action of LH and
• Enhances the release of estrogen induced by LH.
In males:
• It stimulates seminal tubule and testicular growth, and
• Plays an important role in maturation of spermatozoa.
Role of FSH in Spermatogenesis
The conversion of primary spermatocytes into secondary spermatocytes in the seminiferous
tubules is stimulated by FSH. In absence of FSH, spermatogenesis cannot proceed. However,
FSH by itself cannot cause complete formation of spermatozoa. For its completion,
testosterone is also required. Thus, FSH seems to initiate the proliferation process of
spermatogenesis, and testosterone is apparently necessary for final maturation of
spermatozoa. Since the testosterone is secreted under the influence of LH, both FSH and LH
must be secreted for normal spermatogenesis.
Metabolic Role of LH
This hormone is also known as interstitial cells stimulating hormone (ICSH).
In females:
• It causes the final maturation of Graafian follicle and stimulates ovulation.
• Stimulates secretion of estrogen by the theca and granulosa cells.
• It helps in the formation and development of corpus luteum for luteinisation of cells.
• In conjunction with luteotropic hormone (LTH), it is concerned with the production of estrogen
and progesterone by the corpus luteum.
• In the ovary it can stimulate the nongerminal elements, which contain the interstitial cells to
produce the androgens, androstenedione, DHEA and testosterone.
Action of LH in Ovulation: Ovulatory surge for LH
It is necessary for final follicular growth and ovulation.Without this hormone, even though large
quantities of FSH is available, the follicle will not progress to the stage of ovulation.
LH acts synergistically with FSH to cause rapid swelling of the follicle shortly before
ovulation. It is worth noting that especially large amount of LH called ovulatory surge is
secreted by the pituitary during the day immediately preceding ovulation.
Regulation of Testosterone Secretion by LH
Testosterone is produced by the interstitial cells of Leydig only when the testes are stimulated by
LH from the pituitary gland, and the quantity of testosterone secreted varies approximately in
proportion to the amount of LH available. Thus in males, LH stimulates the development and
functional activity of Leydig cells (interstitial) and consequently testicular androgen.
The adrenals and the pancreas.
Glucagon, insulin, somatostatin
Hormonal deficiency diseases ,
Chemical Hyperthyroidism: In rare subjects with chemical hyperthyroidism, in whom
circulating level of bound and free T4 is normal, the T3 concentration is elevated and accounts
for the thyrotoxic state. In these patients, significant amount of T3 probably arises by deiodination of T4 at peripheral level.
The catabolic response in skeletal muscle, in cases of hyperthyroidism, is sometimes so severe
that muscle weakness is a prominent symptom and creatinuria is marked, called thyrotoxic
myopathy. The K+ liberated during protein catabolism appears in urine and there is an increase
in urinary hexosamine and uric acid excretion.
In hypothyroidism, these complexes accumulate promoting water retention, which produces
characteristic puffiness of the skin; when thyroxine is administered, the proteins are mobilised
and diuresis continues until the puffiness (myxoedema) is cleared.
Iodide therapy is sometimes done by surgeons to hyperthyroid patients for a short interval to
prepare the patient for surgery (subtotal thyroidectomy).
Hormornal assay.
Measurement of Hormone Concentrations in the Blood is known as hormone assay
Methods of hormonal assay.
Most hormones are present in the blood in extremelyminute quantities; some concentrations are
as low as one billionth of a milligram (1 picogram) per milliliter.
Therefore, it was very difficult to measure these concentrations by the usual chemical means.
An extremely sensitive method, however, was developed about 40 years ago that revolutionized
the measurement of hormones, their precursors, and their metabolic end products.
This method is called radioimmunoassay
Radioimmunoassay
The method of performing radioimmunoassay is as follows.
 First, an antibody that is highly specific for the hormone to be measured is produced.
 Second, a small quantity of this antibody is (1) mixed with a quantity of fluid from the
animal containing the hormone to be measured and (2) mixed simultaneously with an
appropriate amount of purified standard hormone that has been tagged with a
radioactive isotope.
However, one specific condition must be met:
 There must be too little antibody to bind completely both the radioactively tagged
hormone and the hormone in the fluid to be assayed.
 Therefore, the natural hormone in the assay fluid and the radioactive standard hormone
compete for the binding sites of the antibody.
 In the process of competing, the quantity of each of the two hormones, the natural and the
radioactive, that binds is proportional to its concentration in the assay fluid.
 Third, after binding has reached equilibrium, the antibody-hormone complex is separated
from the remainder of the solution, and the quantity of radioactive hormone bound in this
complex is measured by radioactive counting techniques.
 If a large amount of radioactive hormone has bound with the antibody, it is clear that
there was only a small amount of natural hormone to compete with the radioactive
hormone, and therefore the concentration of the natural hormone in the assayed fluid was
small.
 Enzyme-Linked Immunosorbent
Assay (ELISA)
 Enzyme-linked immunosorbent assays (ELISAs) can be used to measure almost any
protein, including hormones.
 This test combines the specificity of antibodies with the sensitivity of simple enzyme
assays. often performed on plastic plates that each have 96 small wells.
 Each well is coated with an antibody (AB1) that is specific for the hormone being
assayed.
 Samples or standards are added to each of the wells, followed b a second antibody (AB2)
that is also specific for the hormone but binds to a different site of the hormone
molecule.A third antibody (AB3) is added that recognizes AB2 and is coupled to an
enzyme that converts a suitable substrate to a product that can be easilydetected by
colorimetric or fluorescent optical methods.
 Because each molecule of enzyme catalyzes the formation of many thousands of product
molecules, even very small amounts of hormone molecules can be detected.
 In contrast to competitive radioimmunoassay methods, ELISA methods use excess
antibodies so that all hormone molecules are captured in antibody-hormone complexes.
Therefore, the amount of hormone present in the sample or in the standard is
proportional to the amount of product formed.
 The ELISA method has become widely used in clinical laboratories because (1) it does
not employ radioactive isotopes, (2) much of the assay can be automated using 96wellplates, and (3) it has proved to be a cost-effective and accurate method for assessing
hormone levels.
TOPIC : CANCER
Thus the cancer-forming process, called oncogenesis or tumorigenesis, is an interplay between
genetics and the environment. Most cancers arise after genes are altered by carcinogens or by
errors in the copying and repair of genes.
Even if the genetic damage occurs only in one somatic cell, division of this cell will transmit the
damage to the daughter cells, giving rise to a clone of altered cells.
Rarely, however, does mutation in a single gene lead to the onset of cancer. More typically, a
series of mutations in multiple genes creates a progressively more rapidly proliferating cell type
that escapes normal growth restraints, creating an opportunity for additional mutations.
Mutations in two broad classes of genes have been implicated in the onset of cancer: protooncogenes and tumorsuppressor genes. Proto-oncogenes are activated to become oncogenes by
mutations that cause the gene to be excessively active in growth promotion. Either increased
gene expression or production of a hyperactive product will do it. Tumorsuppressor genes
normally restrain growth, so damage to them allows inappropriate growth. Many of the genes in
both classes encode proteins that help regulate cell birth (i.e., entry into and progression through
the cell cycle) or cell death by apoptosis; others encode proteins that participate in repairing
damaged DNA.
Thus the cancer-forming process, called oncogenesis or tumorigenesis, is an interplay between
genetics and the environment. Most cancers arise after genes are altered by carcinogens or by
errors in the copying and repair of genes. Even if the genetic damage occurs only in one somatic
cell, division of this cell will transmit the damage to the daughter cells, giving rise to a clone of
altered cells.
Rarely, however, does mutation in a single gene lead to the onset of cancer. More typically, a
series of mutations in multiple genes creates a progressively more rapidly proliferating cell type
that escapes normal growth restraints, creating an opportunity for additional mutations.
Normal cells are restricted to their place in an organ or tissue by cell-cell adhesion and by
physical barriers such as the basal lamina, which underlies layers of epithelial cells and also
surrounds the endothelial cells of blood vessels.
Cancer cells have a complex relation to the extracellular matrix and basal lamina. The cells must
degrade the basal lamina to penetrate it and metastasize, but in some cases cells may migrate
along the lamina.
Many tumor cells secrete a protein (plasminogen activator) that converts the serum protein
plasminogen to the active protease plasmin. Increased plasmin activity promotes metastasis by
digesting the basal lamina, thus allowing its penetration by tumor cells.
In order for most oncogenic mutations to induce cancer, they must occur in dividing cells so that
the mutation is passed on to many progeny cells. When such mutations occur in non dividing
cells (e.g., neurons and muscle cells), they generally do not induce cancer, which is why tumors
of muscle and nerve cells are rare in adults.
Nonetheless, cancer can occur in tissues composed mainly of non dividing differentiated cells
such as erythrocytes and most white blood cells, absorptive cells that line the small intestines,
and keratinized cells that form the skin. The cells that initiate the tumors are not the
differentiated cells, but rather their precursor cells.
Fully differentiated cells usually do not divide. As they die or wear out, they are continually
replaced by proliferation and differentiation of stem cells, and these cells are capable of
transforming into tumor cells.
What is Cancer?
Cancer is a cellular tumour that, unlike benign tumour cells, can metastasize and invade the
surrounding and distant tissues.INTRODUCTION
Cancer has been a major cause of death in the USA for the past few decades, being second only
to cardiac diseases. Approximately 20 per cent of all deaths in America are due to cancer.
There are at least fifty different types of malignant tumours being identified. More than 50 per
cent of the newly diagnosed cancers occur in five major organs:
(i) Lungs, (ii) Colon/Rectum, (iii) Breast, (iv) Prostate and (v) Uterus.
Cancers of the lungs, colon/rectum and prostate are the principal leading causes of deaths in
males and in females, breast, colorectal and uterine cancers are the most common.
Environmental factors play a very important part. In Japan, death rate from cancer of stomach is
about seven times more than that in the USA. Other examples are:
• Increased risk of certain cancers with occupational exposures to asbestos, naphthylamine, etc.
• Association of cancers of oropharynx, larynx, oesophagus and lungs with tobacco chewing and
cigarette smoking.
PROPERTIES OF CANCER CELLSSECTION SIX
Cancer cells are characterised by three important
1. Diminished or unrestricted control of growth.
2. Capability of invasion of local tissues, and
3. Capable of spreading to distant parts of body by metast
Changes shown by cultured cells undergoing malignant transformation in vitro have been
studied.
(a) Morphological Changes
• Have usually rounded shape, larger than normal cells.
• Cells show nuclear and cellular pleomorphism, hyperchromatism, altered nuclear:
cytoplasmic ratio, abundant mitosis, sometimes tumour giant cells.
• Transformed cells often grow over one another and form multilayers.
• Can grow without attachment to the surface in vitro, diminished adhesion.
(b) Biochemical Changes
• Increased synthesis of DNA and RNA.
• Show increased rate of glycolysis both aerobic and anaerobic.
Show alterations of permeability and surface charge.
• Changes in composition of glycoproteins and glycosphingolipids on cell surfaces.
• Alterations of the oligosaccharide chains.
Increased activity of ribonucleotide reductase and decreased catabolism of pyrimidines.
• Secretion of certain proteases and protein kinases.
• Alterations of isoenzyme patterns often to a foetal pattern and synthesis of foetal proteins,
e.g. carcinoembryonic antigen (CEA), α-fetoprotein AFP), etc.
• Appearance of new antigens and loss of certain antigens.
ETIOLOGY OF CANCER
(Carcinogenesis)
(a) Predisposing Factors
1. Age: Cancer can develop in any age, though it is most common in those over 55 years of age.
Certain cancers are particularly common in children below 15 years of age, viz.
• Retinoblastomas
• Neuroblastomas
• Wilms’ tumours
• Certain tumours of haemopoietic tissues as lymphomas and leukaemias.
• Sarcomas of bones and skeletal muscles.
2. Heredity: Heredity plays an important role in carcinogenesis. Certain precancerous
conditions are inherited.
Examples are:
• Susceptibility to childhood retinoblastomas is inherited as an autosomal dominant trait and
approximately 40 per cent of retinoblastomas are familial.
• Susceptibility to multiple colonic polyposis is inherited as autosomal dominant trait and almost
all cases develop into adenocarcinomas in later life.
• Chromosomal DNA instability may be inherited as an autosomal recessive trait. Conditions are
characterised by some defect in DNA repair.
• In xeroderma pigmentosa, a skin condition, the affected individuals develop carcinomas of
skin in areas exposed to UV rays of sunlight.
3. Environmental factors: Statistically it has been shown that 80 per cent of human cancers
are caused byenvironmental factors, principally chemicals, viz.
• Lifestyle: Cigarette smoking, tobacco chewing.
• Dietary: Groundnuts and other foodstuffs infected with fungus like Aspergillus produce
aflatoxin-B
Which is carcinogenic.
• Occupational: Asbestos, benzene, naphthylamines, beryllium, etc.
• Iatrogenic: Certain therapeutic drugs may be carcinogenic.
4. Acquired precancerous disorders: Certain clinical conditions are associated with increased
risk of developing cancers. Examples are:
• Leukoplakia of oral mucosa and genital mucosa developing into squamous cell carcinomas.
• Cirrhosis of liver: A few cases can develop hepatoma (hepatocellular carcinoma).
• Ulcerative colitis: Can produce adenocarcinoma of colon.
• Carcinoma in situ of cervix: Can produce squamous cell carcinoma of cervix.
(b) Carcinogenic Agents (Agents Causing Cancer):
Carcinogens that cause cancer can be divided into three main broad groups:
1. Physical: Radiant energy
2. Chemicals: Variety of chemical compounds can cause cancer. Some of these can act directly
and others can act as procarcinogens
3. Biological: Oncogenic viruses.I. RADIANT ENERGY (RADIATIONS):
Radiations can cause cancer mainly in two ways:
1. Direct Effect
By producing damage to DNA, which appears to be the basic mechanism but the details are not
clear.
Radiations like X-rays, γ-rays or UV rays are harmful to DNA of cells and they can be
mutagenic and carcinogenic. Damages to DNA brought about by radiations may be as
follows:
• Single or double strand breaks.
• Elimination of purine/pyrimidine bases.
• Cross-linking of strands.
• Formation of pyrimidine dimers.
2. Indirect Effects
In addition to direct effects on DNA as stated above, radiations like γ-rays and X-rays produce
free radicals, viz. OH, superoxide and others which may interact subsequently with DNA and
other macromolecules leading to molecular damage.
UV rays: Natural UV rays from sun can cause skin cancer. Fair-skinned people living in places
where sunshine is plenty are at greatest risk. Carcinomas and melanomas of exposed skin are
particularly common in Australia and New Zealand.
UV rays produce:
Damage to DNA by formation of pyrimidine dimers.
• Secondly by immunosuppression.
Ionising Radiations
The ability of ionising radiations to cause cancer lies in their ability to produce mutations
(mechanisms discussed). Particulate radiations such as x-rays,
-particles and neutrons are more carcinogenic than electromagnetic radiations like X-rays
and uv-rays.
Evidences in favour of carcinogenicity of ionising radiations.of carcinogenic chemicals is given
below
Class Nature of Chemicals Compound
1. Polycyclic aromatic
• Benzpyrene hydrocarbon • Dimethyl-benzanthracene
Note: Aromatic hydrocarbons are present in cigarette smoke and they are thus relevant in
pathogenesis of lung cancer.
3. Azodyes(Aromaticamines•-Naphthylamine•N-methyl-4-aminoazobenzene
•
2acetylaminofluorine
Note: ß-naphth ylamine, an aniline azo dye used in the rubber industries has been held
responsible for bladder cancers in exposed workers.
4. Nitrosamines and amides • Dimethylnitrosamine • Diethylnitrosamine
Note: Nitrosamines and amides can be synthesised in GI tract from ingested nitrites or derived
from digested proteins and may contribute to induction of gastric cancer.
5. Naturallycompoundsoccurring
Aflatoxin B produced by the fungus, Aspergillus flavus.
Note: The fungus grows on groundnuts, peanuts and other grains in congenial environmental
conditions. It produces “aflatoxin B ” which is a potent hepatocarcinogen. This is believed to
be responsible for high incidence of liver cell carcinoma in Africa, where the contaminated foods
are eaten.
6. Various Drugs • Alkylating and acylating agents, e.g. Cyclophosphamide and busulfan.
Note: The drugs are used in cancer treatment and also as imm unosuppressants. Patients
receiving such therapy are at a higher risk for developing cancer.
• Diethylstilbestrol, oestrogen.• Nitrogen mustard.• -propiolactone,
Mechanisms of Chemical Carcinogenesis
As discussed above, chemical carcinogens may be:
• Direct acting
• Procarcinogens
(a) Direct Acting
A few chemical carcinogens like alkylating agents, e.g. cyclophosphamide, busulfan, etc. can
interact directly with target molecules.
(b) Procarcinogens
Vast majority of the chemicals act as procarcinogens. Procarcinogens are not chemically
reactive. In the body, after metabolism they are converted to “ultimate carcinogens” which are
highly carcinogenic.
Enzymes involved: The enzyme systems involved in metabolic activation are cytochrome P
species present in the endoplasmic reticulum of cells. Recently a particular mono-oxygenase
species cytochrome P450 (AHH-Aromatic hydrocarbon hydroxylase) has been incriminated
in the metabolism of polycyclic aromatic hydrocarbons.
Molecular Targets of Chemical Carcinogens
DNA is the primary and most important target of chemical carcinogens. Hence chemical
carcinogens are mutagens.
Damage to DNA can be:
• Binding covalently with DNA, (also to RNA and proteins).
• Interaction with the purine, pyrimidine and phospho- diester groups of DNA.
Oncogenes and Proto-oncogenes
• Oncogenes
They are genes whose products are associated with neoplastic transformation (V-onc).
• Proto-oncogenes
They are normal cellular genes that affect growth and differentiation (V-onc proto-oncogene).
Proto-oncogenes are converted into oncogenes before they can be carcinogenic by: Transduction
into retroviruses (V-oncs) or
• Changes in situ that affect their expression and function thereby converting them into cellular
oncogenes (c-oncs).
II. Biochemistry of Metastasis
Definition of metastasis:
Metastasis is the spread of cancer cells from the primary site of origin to other tissues, both
neighbouring and distant, where they grow as the secondary tumours.NTRODUCTION
1. Benign tumours can grow very rapidly and attain big sizes and may be sometimes lifethreatening but they do not metastasize.
2. It is the malignant tumours, cancerous ones, invade surrounding tissues and send out cells to
begin new tumours at distant sites. The spread may be bloodborne/or through lymphatics.
3. This colonisation at distant sites is metastasis and is the major cause of death from human
malignancies.
4. Tumour cells must attach to degrade and penetrate the “extracellular matrix” (ECM) at several
steps of metastasis. Thus metastasis biochemically is a multistep process.
5. Approximately 50 per cent of patients who develop malignant tumours can be cured with
various therapies, viz. surgical removal, radiation therapy and chemotherapy. Of the remaining
50 per cent, majority die because of metastasis. Hence, in a real sense, if metastasis could be
controlled, cancer could be controlled and for the most part, cured.
Biochemical Basis of Metastasis
Metastasis is an active process of invasion.
Metastasis require:
• Specific surface receptors
• Requires enzymes
• The process uses energy and
• Requires protein synthesis
Note
Theoretically, if any of the above is blocked, invasion can be prevented. As stated above,
metastasis is not a passive pheno- menon. It is an active process.
The phenomenon of metastasis involves:
• A metastatic cell has to penetrate the extracellular matrix (ECM) that surrounds the tumour.
• Travels through the tissue till it reaches a blood vessel/or a lymphatic.
• In case of blood borne metastasis, the tumour cell then attaches to the blood vessel wall,
dissolves a portion of the wall and propels itself through into the circulating blood.
• Metastatic cells often travel in the circulation as small clumps of cells, called emboli.
• At a distant site, the tumour cell again re-attaches to the blood vessel wall and repeats the
process, travelling as much as two or three cell diameters into the invaded tissue before it settles
down and begins to form a new tumour.L WITH EXTRACELLULAR MATRIX
Composition of Extracellular Matrix
Extracellular matrix (ECM) can be divided into two major categories:
• Basement membrane (BM)
• Interstitial connective tissue (ICT)
Important Constituents of ECM
• Collagen: Basement membrane contains type IV collagen and interstitial connective tissues
type I and type III collagen.
• Adhesion-promoting proteins: Basement membrane contains laminin and interstitial
connective tissue fibronectin.
– Both laminin and fibronectin are large multi- functional molecules that can bind to other ECM
components such as collagen, proteoglycans and to cells.
– Attachment of cells to laminin and fibronectin is brought about by distinct and specific cell
surface receptors.
Interaction with basement membrane: Basement membrane (BM) is the first tough elastic
barrier that surrounds both tissues and blood vessels. Hence, an invading cancer Cell must pass
this barrier several times, in order to establish metastatic colonies in distant tissues.
Stages
The above interaction of cancer cell under three steps.
I. Step 1: Attachment of the invading metastatic cell to basement membrane (BM).
II. Step 2: Dissolution of the basement membrane (BM), so that the cell can pass through it.
III. Step 3: Migration of tumour cells. It has been shown that specific biochemicals are required
for the tumour cell to complete the process.
Note
To the histopathologist an intact BM is an important criterion to differentiate benign and
malignant tumours.
Benign tumours are always surrounded by an intact BM whereas in invading malignant
tumours, the membrane becomes thin and broken and often entirely lacking.
I. Step 1
• The tumour cell binds to one of the membrane’s glycoproteins, a cross-shaped molecule called
as laminin
• Binding sites for laminin on the surfaces of certain types of cancer cells have been
demonstrated.
• Laminin appears to serve as a bridge between receptors on the surface of the invading cancer
cell and the BM itself.
Note
Monoclonal antibodies, if prepared against the receptor protein, can be used to block these
receptors on metastatic cells. When the antibodies bind to receptorsof metastatic cells, the
cells will not be able to bind to laminin and metastasis can be prevented.
II. Step 2
Once the tumour cell is attached to laminin, the invasive tumour cell secretes certain
proteolytic enzymes that degrade the BM. Several such proteolytic enzymes have been
incriminated:
1. Collagenases: A collagen degrading enzyme that acts specifically on type IV collagen, the
principal structural component of membrane has been isolated from highly metastatic cells.
Properties
• It is a metalloenzyme,
• Secreted as zymogen latent form and is clipped to form the “active” enzyme by a second
enzyme cathepsin B.
• Active enzyme has a molecular wt of 60,000.
Note: Type IV collagenases cleave BM collagen, whereas “interstitial collagenases” cleave type
I and type III collagen.
2. Heparanase
Many metastatic cells also produce an enzyme called “heparanase” that degrades heparan
SO4, the predominant proteoglycan of the basement membrane. Many other components of
the membrane attach to heparan SO4 including laminin and fibronectin.
Note
• Heparanase enzyme is also produced by monocytes. But it has been claimed that heparanase
produced by malignant metastatic cells is slightly different than the comparable enzyme
produced by monocytes and even normal cells.
• The enzyme produced by tumour cells appears to clip the heparan SO4 chain at a slightly
different position and produce different fragments than the enzyme produced by non-malignant
cells.
3. Cathepsin B: A lysosomal protease.
• Cathepsin B activates “Latent” collagenases. It clips type IV collagenase to its 60,000
molecular weight ‘active’ form.
• This enzyme has been found in metastatic cancer cells in high concentration.
4. Plasmin: Degrades several non-collagenous extracellular matrix proteins.
III. Step 3
Factors that favour migration of tumour cells in the passage created by the degradation of EC
matrix including BM are not well understood.
Implicated in this process are:
•Autocrine motility factors called as migration factors.
• Tumour cells induced degradation of the interstitial matrix produces fragments of ECM that are
attractive to the tumour cells and cause it to move forward.
Summary
1. Process of metastasis is selective.
2. It is not a passive diffusion, but an active process and requires specific receptors, enzymes,
protein synthesis and energy.
3. Not every cell in a malignant tumour is capable of forming metastasis.
4. The classic oncogenes cause tumours to grow, but they do not necessarily cause metastasis
and invasion.
5. It has been postulated that there can be new special types of genes different from the classic
oncogenes.
6. Ability to metastasise can be transformed genetically to cells.
7. The vast majority of metastatic cells that enter the bloodstream are destroyed “en route”.
Natural killer cells (NKC), a part of host’s immune system have been claimed to be
reasonably efficient in destroying metastatic cells in bloodstream.
8. Natural killer cells (NKC) are also present in tissues, but they are much less efficient at
destroying tumour cells outside the bloodstream.
9. It is claimed that cells populating a metastatic colony are more capable of metastasising than
the cells populating the parent tumour.
10. Monoclonal antibodies if produced can be used to block the receptors on metastatic
cells so that they can not bind to “Laminin” and “fibronectin” and thus can prevent
metastasis.
ACTIVATION OF PROTO-ONCOGENES TO ONCOGENES: BIOCHEMICAL
MECHANISMS
At least five mechanisms are known that alter the structure/expression of proto-oncogenes and
convert them to “oncogenes” which produces cancer.
1. Single Point Mutation
• Proto-oncogene can be converted to oncogene by a single-point mutation, in that it differs
from each other in one base only resulting in an amino acid substitution.
• Murine retrovirus oncogene V-ras produces a protein called P21, having molecular wt of
21,000. Functionally it is related to G-protein, which is acted upon by the enzyme ‘GTPase’, and
modulates the activity of adenylate cyclase and thus the cyclic-AMP level in the cell.
• Proto-oncogene of C-ras from normal human cells and C-ras of oncogene from a cancer differ
only in one base, resulting to an amino acid substitution at position 12 of P21 protein.
• This mutational change brings about diminished activity of the enzyme GTPase. Lowered
GTP-ase activity can result in chronic stimulation of adenylate cyclase; which normally is
decreased when GDP is formed from GTP. The resulting increased activity of adenylate cyclase
increases cyclic AMP level, which produces a number of effects on cellular metabolism. It
affects the activities of various cyclic AMP-dependent protein kinases which brings about
phosphorylations of various proteins. Such altered cellular metabolism favours transformation.
2. Gene Amplification
• Gene amplifications can be seen in certain tumour cells. The amplified genes may be detected
as a “homogeneously stained regions” on a specific chromosome.
• Activation of certain “C-onc” is brought about by amplification.
• Increased amount of products formed from certain oncogenes may help in progression of a
tumour cell to malignant (cancerous) form.
• N-myc amplification, 3 to 300 copies, seen in neuroblastomas. A strong correlation observed
amongst ‘N-myc’ amplification, advanced stage and poor prognosis.
3. Promoter Insertion
• It has been seen that certain retroviruses, e.g. Avian leukaemia virus do not have ‘V-oncs’.
They can cause cancer over a longer period of time as compared to retroviruses which possess Voncs, called slow-transforming retroviruses.
• When such a retrovirus infects cells, a DNA copy, called cDNA is synthesised from the RNA
genome of the virus by reverse transcriptase. The cDNA, thus formed, is integrated in the host
genome. The integrated double-stranded cDNA is called as a “provirus”.
• The cDNA copies thus formed are usually flanked at both ends by ‘long terminal repeats’
(Jumping genes) which acts as a ‘promoter’ resulting in transcription.
Example
• Normal chicken B lymphocytes have “myc” gene on its chromosome which is inactive.
• Infection of such a cell with Avian leukaemia virus (ALV) produces cDNA copy (Provirus)
which becomes integrated.
• The ‘myc’ gene is activated in an upstream, by its right hand long terminal repeat (LTR), which
is a strong promoter and results in transcription of myc mRNA.
4. Enhancer Insertion
In this mechanism, the cDNA copy (Provirus) gets inserted ‘downstream’ from the gene and
hence cannot act as a promoter. Instead, a certain portion of proviral sequence acts as an
“enhancer” element leading to activation of upstream gene and its transcription.
5. Translocations
Most of the tumour cells show chromosomal abnormalities. Translocation is the most
commonly seen chromosomal abnormality, in which a piece of one chromosome is split off
and then is joined to another chromosome (direct translocation).
Reciprocal translocation: In this the second chromosome donates the piece to the first.
Examples
• Burkitt’s lymphoma, a fast growing cancer of human B lymphocytes is an example of
“reciprocal translocation”.
• Usually chromosomes 8 and 14, (t 8:14) are involved. In 90 per cent of the patients with
Burkitt’s lymphoma, there is an exchange of genetic material between chromosomes 8 and
14. Chromosome 14, in humans, bears the genes of the H-chain locus.
• In 10 per cent of the patients with Burkitt’s lymphoma, there is exchange between
chromosomes 8 and 2 or 22 (t 8:2 or t 8:22), which in the human genome carry the “kappa” and
lambda” immunoglobulin genes respectively.
• Chronic granulocytic leukaemia: In chronic granulocytic leukaemia, the Philadelphia
chromosome involves chromosomes 9 and 22. It is an example of “chromosomal direct
translocation”. There is fusion of the gene with new genetic sequences (t 9:22). Translocation
relocates the ‘C-abl’ gene from chromosome 9 to the ‘bcr’ locus on chromosome 22. The “Cablbcr” hybrid gene (Philadelphia chromosome) codes for a chimeric protein that exhibits the
‘tyrosine kinase’ activity.
CANCER SUPPRESSOR GENES (GROWTH SUPPRESSOR GENES)
• Antioncogenesis
Recently it has been shown that genes other than oncogenes can play a role in etiology of certain
types of cancer. These are called cancer suppressor genes (or growth suppressor genes or
antioncogenes). Mechanism of action is quite different here. A loss or inactivation of such genes
removes certain mechanisms of growth control.
Example
An important model for understanding is the tumour known as retinoblastoma which occurs in
children. Retinoblasts are precursor cells of cones, the photoreceptor cells in retina. 40 per cent
of retinoblastomas are familial; and the remaining are sporadic. Rb gene is located on
chromosome 13q14 which is the cancer suppressor gene and exerts an inhibitory effect.
Hence both copies of normal Rb gene has to be inactivated for the tumour to develop. Two-hit
hypothesis has been proposed as follows:
• Both normal alleles of the Rb locus must be inactivated (two hits) for the development of
retinoblastoma.
• In “familial” cases, the children inherit one defective Rb gene and the other is normal.
Retinoblastoma develops only when the normal Rb gene is lost in the retinoblasts as a
result of mutation.
• In sporadic cases, both normal Rb alleles are lost by somatic mutation.
Other examples: Loss of antioncogenes producing cancer has been incriminated also in the
following:
• Wilms’ tumour of kidney
• Small cell carcinoma of lung (Oat-cell carcinoma)
• One type of breast cancer.
Note
Patients with familial retinoblastomas have been found to have increased risk of developing
osteosarcomas and soft tissue tumours.
Mechanism of action of anti-oncogenes: Mechanism of action of Rb gene and other
antioncogenes is not very clear. Their products are nuclear proteins and they may act as
repressors of DNA synthesis and modulate gene expression.
A Cancer Causing Tumour Suppressor Gene Discovered
Recently two research teams have simultaneously discovered a gene that plays an
important role in aggressive brain tumour like Glioblastoma multiforme.n The gene is also
incriminated in other types of cancers viz., breast, prostate and renal cancers and highly
malignant skin tumour melanoma.
• The gene has been designated:
– P-TEN (Phosphatase and tension homolog) by one group and
– MMAC 1 (Mutated multiple advanced cancers) by other group.
• The gene appears to be located on chromosome 10 and appears to be an important “tumour
suppressor” and when mutated it allows the cells to grow out of control and become
malignant.
• Studies have shown that an area of genetic code corresponding to gene on chromosome 10 is
lost when a glioma, a benign tumour of brain progresses into glioblastoma multiforme, a more
aggressive and spreading form of the disease.
Pathogenesis of Cancer and Mechanisms of Action of Oncogenes
• Cancer is a genetic disease; changes in the genome of somatic cells may be brought about by
genetic factors.
• It can be acquired environmental factors, viz. radiations, chemicals, and viral oncogenes.
• Tumour cells can gain growth autonomy by either.
1. Activation of growth-promoting antigens, or
2. Loss of growth inhibitory ‘cancer suppressor genes’ (antioncogenes).
• Activated oncogenes may promote growth by various mechanisms:
1. May encode membrane proteins critical to signal transduction and may act on key intracellular
pathways involved in growth control.
2. May encode growth factors, or
3. May encode for growth factor receptors that are either defective or amplified.
GROWTH FACTORS
A variety of growth factors have recently been isolated and identified:
• Are usually polypeptides.
• Can initiate cell migration, differentiation and tissue remodelling.
• May be involved in various stages of wound healing.
• Can affect many different types of cells, viz. haemopoietic cells, epithelial tissues, nervous
system and mesenchymal tissues.
• Play a major role in regulating differentiation of stem cells to form various types of mature
cells.
• Are mitogenic to target cells.
• Products of several oncogenes are either growth factors or parts of receptors for growth factors.
• Some growth factors may be inhibitory to growth of certain cells, e.g. TGF-β .
• Chronic exposure of target cells to increased amounts of a growth factor or to decreased
amounts of a growth inhibitory factor may alter the balance of cellular growth (growth
autonomy).
Mechanism of Action of Growth Factors
Growth factors may act in the following 3 ways:
(a) Endocrine action: Similar to hormone action. May be synthesised in one place and then
carried by bloodstream to target cells where they exert their effects.
(b) Autocrine action: Synthesised and act on the same cells.
(c) Paracrine action: Synthesised in certain cells and secreted from them to affect the
neighbouring cells.
Action of Growth Factors at Molecular Level
1. Most of the growth factors have high affinity protein receptors on the membranes of target
cells.
2. Genes for receptors of EGF and IGF have been extensively studied. Structurally they are
found to have short membrane spanning segments and external and cytoplasmic domains of
varying lengths.
3. Most of the receptors, e.g. of PDGF, EGF, etc. have been found to exhibit protein tyrosine
kinase activity which is located in cytoplasmic domain.
4. The kinase activity brings about autophosphorylation the receptor protein and also
phosphorylation of other proteins of target cells.
5. Growth factors interact with the specific membrane receptor and transmits the message across
the plasma membrane to the interior of the cells by transmembrane signal transduction, which
finally affect one or more processes involved in mitosis of the cells.
6. Molecular event is similar to ‘src’ gene and has been studied with PDGF. Phospholipase c is
stimulated in cells exposed to PDGF resulting in hydrolysis to form “second messengers”,
Inositol-tri-P (ITP) and Di-acylglycerol (DAG). IT-P releases intracellular bound calcium
increasing cellular Ca++ ions. DAG stimulates protein kinase c; these events in turn
phosphorylate a number of proteins in target cells, affecting cell metabolism.
7. In addition, subsequent hydrolysis of Diacylglycerol (DAG) by Phospholipase A2 releases
arachidonic acid which results in increased production of PG’s and leukotrienes which in turn
exert their biological effects.
8. Growth factors like PDGF can bring about rapid activation of certain cellular “protooncogenes”.
Naturally Occurring Anticancer Substances
It is worthwhile to note that there are number of substances occurring in nature have anticancer
activity. Some of them are:
1. Vitamin A and β-carotene: (antioxidant action, mops up free radicals)
2. Vitamin E (Tocopherols)—antioxidant
3. Lycopene: A carotenoid present in ripe red tomatoes (antioxidant action)
4. Ascorbic acid vitamin C: (antioxidant)
5. Selenium: A trace element has anticancer activity (Refer on Metabolism of Minerals and
Trace Elements)
6. Zinc (a trace element)
7. Quercetin a flavonoid present in apple which is anticancer, shown to prevent growth of
prostatic cancer cells.
8. Glucosinolates: Present in bitter Brussel sprouts. It contains:
• Sinigrin: Suppresses development of precancerous cells
• Glucoraphanin: It breaks down into a compound called sulphoraphane which neutralises
carcinogens.
9. Epigallocatectin Gallate (EGCg)
EGCg is a compound found in green tea leaves also to a lesser extent in black tea leaves. This
compound has been shown to inhibit the activity of an enzyme called tumorassociated quinol
oxidase (tNox), which is an overactive form of an enzyme known as NOX: The ‘NOX’ enzyme
is found on the surface of cells, and plays a key role in growth of both normal and cancerous
cells. Normal cells produce the “NOX” enzyme only when hormonal signals prompt the cells to
divide. But cancerous cells appear to be able to produce the abnormal form “tNOX”, all the time.
The “tNOX” protein has been found on many types of cancer cells including the breasts, prostate
and colonic cancer cells. Drugs that inhibit ‘tNOX’ have been shown to block cancer cell growth
in the laboratory.
The compound ‘EGCg’ is present in ten to hundred times more in green tea leaves as compared
to black tea leaves.Thus tea has been found to have anticancer activity.
Some Drugs that are used in Cancer Chemotherapy
Various drugs that have been used in chemotherapy of cancer are classified as follows:
• Polyfunctional alkylating agents: – Nitrogen mustard, busulfan, chlorambucil, triethylenemelamine.
• Hormones
– Sex hormones: Oestrogens – Corticosteroids: Prednisone
• Antimetabolites – Folate antagonist: Methotrexate – Purine antagonist: Mercaptopurine –
Pyrimidine antagonist: 5-fluorouracil
• Antibiotics – Actinomycin D – Doxorubicin
• Miscellaneous agents – Vinblastine – Vincristine – Cisplatin
– Retine—a nontoxic anticancer agent, a natural constituent of body cells.
• Enzyme: L-Asparaginase
Resistance of cancer cells to drugs and multidrug resistance (MDR)
The drugs are effective initially to cancer chemotherapy, but after several months of treatment,
the drugs become ineffective by the mechanisms developed by the tumour cells producing
acquired resistance to drugs. This is called drug resistance.
Multidrug Resistance (MDR)
In multidrug resistance (MDR), the resistance is not only to a particular drug but also to other
structurally unrelated anti-cancer agents.
Role of P-glycoprotein in MDR: Phosphorylated glycoprotein (P-glycoprotein) present in the
plasma membrane. It contains 1280 amino acids and has a molecular weight of 12,000. It acts as
energy-dependent efflux pump expelling a variety of drugs and thus mediating MDR.
Telomeres and Telomerase: Role in Ageing and Cancer
A. Telomeres
The ends of each chromosome contain structures called telomeres. They are composed of DNA
and proteins that are located at the ends of chromosomes of lower and higher eukaryotes. They
consist of short repeat TG-rich sequences. In humans, telomeres consist of many (1000 or more)
arrays of TTAGGG repeats at the terminals of the 3' ending strands. These repeats are
maintained in germline cells by the action of a special enzyme called Telomerase.
Significance and Importance of Telomeres
Telomeres confer stability on the ends of chromosomes and are necessary because the RNA
Primers at the 5'-end of a completed lagging strand cannot be replaced with DNA since the
primer would have no place to bind. This would inevitably lead to shortening of the
chromosomes at each replication of DNA with resultant loss of important genes.
B. Telomerase
Telomerase or called as “Telomere terminal transferase” a ‘reverse transcriptase’ an
enzyme responsible for telomere synthesis and thus for maintaining the length of telomere.
Telomerase is a ribonucleoprotein and in humans contains an RNA molecule that has one
segment that is complementary to the “TTAGGG” repeat. This is used as a template for the
replication of the telomeric sequences using the appropriate deoxynucleotide triphosphates.
COMMONLY USED TUMOUR MARKERS
A. Carcinoembryonic Antigen (CEA)
CEA is one of the oncofoetal antigens used most frequently and widely as a tumour marker in
clinical oncology. It was originally described by Gold and Freedman as a tumour specific
antigen present only in cancer cells, in the circulation of patients with gastrointestinal
malignancy and in the normal epithelial cells of foetal GI tract, hence it was named as CEA
because of its presence in both carcinoma and embryonic tissue. It was discovered in 1965 by
raising antiserum against a colon cancer.
Properties of CEA and Chemical Composition
• It is a glycoprotein.
• Molecular weight varies from 150,000 to 300,000 (average 185,000).
• Protein Part
– A single polypeptide chain (monomeric unit) consisting of 30 amino acids with lysine at Nterminus.
– By EM, it appears as a twisted rod.
– Protein content is 46 to 75 per cent.
• Carbohydrate Component
• Carbohydrates surround the protein and constitutes 45 to 57 per cent . On analysis of
carbohydrates, it is found to contain fucose, mannose and galactose.
• N-acetyl galactosamine is low whereas large amount of N-acetyl glucosamine is present.
• Sialic acid varies significantly.
PHYSIOLOGY AND METABOLISM
1. Sites: CEA is chiefly present in:
• Endodermally derived tissues, viz. GI mucosa, lungs and pancreas.
• Also may be in nonendodermally derived tissues
(?), conclusive evidences lacking. It has been detected in GI tract of foetuses as early as three
months of gestation. Also found in embryonic liver, pancreas and lungs. CEA has been detected
in free brush border of normal mucosal cells and also in cytoplasm of colonic carcinoma cells.
7. Metabolism: Not known exactly. CEA is probably broken down in liver. It disappears
from circulation in 3 to 4 weeks after removal of CEA-producing tumour.
CLINICAL USES AND REMARKS
1. CEA has been reported to be most useful as tumour marker in colorectal Cancer.
2. It is elevated also in other malignancies. Found to be useful in:
• Breast cancer
• Bronchogenic carcinoma of lung specially small cell carcinoma of lungs (SCCL)
• Other malignancies where the value is raised are:
– Pancreatic carcinoma
– Gastric carcinoma
– Cancer of urinary bladder
– Prostatic cancer, neuroblastomas, ovarian cancer and carcinoma of thyroids.
3. Value in Colorectal Cancer
• Most valuable, has been used as an aid in diagnosis.
Value of CEA as a tumour marker is greatest in colorectal cancer.
• Has been useful in staging. Found to be elevated in 28 per cent of patients with stage A
colorectal cancer and in 45 per cent of patients with stage B colorectal cancer.
• Most important use of CEA has been monitoring the response of colorectal cancer to
treatment.
• Patients with colorectal cancer who initially had elevated CEA show return of CEA values to
normal after complete and successful surgical removal. Values of CEA remain normal as long
as remission persists (serial assays are helpful). A rise again in postsurgical patients is
definite indication of relapse/recurrence.
• Prognostic usefulness: Patients of colorectal carcinoma with near normal pretreatment CEA
levels had a lower incidence of metastasis. On the other hand, majority of patients having high
CEA pretreatment levels developed metastasis.
Note
CEA is considered as best available noninvasive tumour marker for the postoperative
monitoring of surgically
treated patients with colorectal cancer.
4. Value in Other Malignant Tumours
• Reports are conflicting.
• A role of CEA in monitoring the therapeutic response in patients with gastric carcinoma and
lung carcinoma is not proven.
• But in small cells lung cancer (SCLC) it is claimed that chemotherapy may show dramatic,
short lived responses, monitoring the CEA level may be of value.
REFERENCES:
 Lehniger’s principles of Biochemistry (2nd edn 2000) by D.L.Nelson and M.M Cox,
Macmillan, Worth Pub Inc, NY.
 Biochemistry (4th edn 1992) by Lubert Stryer WH Freeman & co., NY
 Harper’s Biochemistry (25th edn) by R.K.Murray and others, Appleton and Lange,
Stanford.
 Fundamentals of Biochemistry (1999) by Donalt Voet, Judith G Voet and Charlotte W
Pratt, John Willey & Sons, New York.
 Medical Biochemistry by M.Chattergen & Rana Shinde. T.Palwer 2002 K.Wilson and
J.Walker 2002 5th Edition.
 Biochemistry A problems Approach by Wood.Wilson. Benbow.Hood. 2nd Edition.
 Textbook of Biochemistry by D M Vasudevan and Srikumari
 Essentials of Medical Biochemistry by R.C. Gupta
 Lippincot’s illustrated reviews