Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st year (First Year) Biochemistry ppt lectures Topic 60 Free Radical Notes. - biochemistry notes pdf, biochemistry mbbs 1st year notes pdf, biochemistry mbbs notes pdf, biochemistry lecture notes, paramedical biochemistry notes, medical biochemistry pdf, biochemistry lecture notes 2022 ppt, biochemistry pdf.
Free radical
Case report
? A 2 months old girl, born at a gestational age of 38 weeks was referred
for investigations regarding persistent pneumonia. The girl suffered
from several episodes of recurrent infections such as neonatal sepsis
and gastroenteritis, since the 6th day of her life. At the time of
admission, she had received three recent course of antibiotics from
the previous hospital including cefepime, clindamycin and imipenem
due to persistent pneumonia. On admission, physical examination
revealed mild chest retraction and rales in both lung fields. She was
alert and responsive, with no neurological deficit. Her height and
weight were at the 25th-50th percentile and the 50th-75th percentile for
her age respectively.
? A chest X-ray confirmed the presence of bilateral pulmonary
consolidation and atelectasis. Diagnostic bronchoalveolar lavage (BAL)
was performed to obtain specimens for cytology and culture. A
computed tomography (CT) of the thorax showed a left sided
consolidation and evidence of hematogenously disseminated
multifocal pneumonia.
? She had no family history related to primary immunodeficiency. Her total
leucocyte count was 10,500/?L, with 34% neutrophils and 52%
lymphocytes. The ESR was 12 mm/hr, and CRP 3.54 mg/dL. Aspartate
aminotransferase (AST) and alanine aminotransferase (ALT) levels were
raised (174 IU/L and 398 IU/L, respectively). Electrolyte levels and the
results of renal function tests were normal. Investigations for her
immunologic work-up showed Ig G, Ig A, Ig M, Ig E, C3, C4, and CH50 levels
to be within her age specific reference range. Lymphocyte subset analysis
revealed normal B cells and T cells for her age.
? She had negative neutrophil nitroblue tetrazolium (NBT) slide test and
abnormal neutrophil respiratory burst activity test.
? What could be the possible diagnosis ?
What is its pathophysiology?
Learning objectives
? Definition ? free radicals, reactive oxygen
species
? Generation
? Damage
? Clinical significance
? Scavenger system
4
Free radical
? Free radical: is a molecule or an atom with an
unpaired electron in its outer most orbital. It is
highly reactive and has an extremely short half
life of microseconds. It reacts indiscriminately
with any molecule that comes in its contact to
achieve stability by either accepting an
electron or donating an electron.
ROS& oxidative damage
? Normal y the oxygen is completely reduced to
water and successive reduction with four
electrons. However incomplete reduction
produces ROS such as superoxide, ?O2
-, hydroxyl, ?OH, and perhydroxyl, ?O2H.
H2O2 is not a radical by definition but is a highly
reactive ROS and produces other species of ROS.
? Tissue damage caused by oxygen radicals is often
cal ed oxidative damage
ROS encountered in living cells
? Sources of ROS in cell
1. Leakage from electron transport chain in
mitochondria
2. Cytochrome P450 in Endoplasmic Reticulum
3. Degradation of
Purine nucleotides
to Uric acid
4. Interaction with metal
5. Peroxisome
? Hydrogen peroxide produced during oxidation
of long chain FA in a FAD dependent reaction
in peroxisome.
13
4
6. Respiratory burst
? Neutrophils, eosinophils, monocytes and
macrophages produces superoxide anion by
respiratory burst during phagocytosis by
NADPH oxidase.
15
NADPH oxidase
Activation of
macrophages and
consumption of
oxygen by the cell
is increased
drastically called
as respiratory
burst
16
Other exogenous sources
? Drugs and chemicals: Acetaminophen : Hgh
dose cause liver damage
? Peroxidation catalyzed by lipo-oxygenase in
platelets and leukocytes.
? H2O -------(gamma, UV radiation)---- e- + OH?
? Oxygen photolysis by light produced singlet oxygen
? Cigarate smoking
? Environmental pollution
18
FORMATION OF FREE RADICALS
19
Damage caused by free radicals
1. Proteins
2. Lipid peroxidation
3. Nucleic acids
20
Cellular damage caused by ROS
? Protein
? Aminoacids like proline, histidine, arginine,
methionine are particularly susceptible to
oxidative damage .
? Protein fragmentation
? Protein aggregation
? Protein-protein cross linking
Lipid Peroxidation
In vitro, peroxidation would lead to rancidity of fats
and oils.
In vivo, the membrane lipids are more liable to attack
by free radicals and produce damage to integrity of
the membrane.
22
Lipid peroxidation
Damages to DNA
? Damage to DNA
? in germ line cells in ovaries and testes ? heritable
mutation
? in somatic cells ? cancers.
? dialdehydes formed as a result of radical-induced
lipid peroxidation in cell membranes can also
modify bases in DNA
? Chain break
24
Tissue damage by radicals
Role of free radicals in autoimmune
disease
? Modification of amino acids in the protein ? by
direct radical action or by reaction with
products of lipid peroxidation.
? Protein recognized as non-self by the immune
system.
? Production of antibodies ? cross react with
normal tissue proteins ? initiating autoimmune
disease.
26
Total body radical burden can be
estimated by measuring products of lipid
peroxidation:-
? FOX (Ferrous Oxidation in Xylenol) Assay
? Estimation of dialdehydes
? Measurement of Pentane and Methane in
Exhaled air
F OX assay
? Lipid peroxides can be measured by the ferrous
oxidation in xylenol orange (FOX) assay.
? Acidic conditions fe++
fe+++
? Which form chromophore with xylenol orange.
28
TBARS assay
? The dialdehydes (malondialdehyde) formed
from lipid peroxides can be measured by
reaction with thiobarbituric acid, they form red
fluorescent product - generally reported as
TBARS (thiobarbuturic acid reactive
substances).
29
Pentane and Ethane assay
? Peroxidation of n-6 PUFA form ? pentane
? Peroxidation of n-3 PUFA form ? ethane
? Both can be measured in exhaled air.
30
Clinical significance of free radicals
? Chronic inflammation ? rheumatoid arthritis
due to free radicals released by neutrophils
? ROS induced tissue damage ? ulcerative colitis,
chronic glomerulonephritis
? Acute inflammation ? macrophages produce
free radicals at inflammatory site
? Respiratory burst ? increase activity of NADPH
oxidase in macrophages and neutrophils
31
In chronic granulomatous disease (CGD),
NADPH oxidase is absent.
? So, bacteria are ingested normally but they
cannot be destroyed.
32
Respiratory disease
? Breathing of 100% oxygen for more than 24 hr
produce destruction of endothelium and lung
edema ? due to free radicals release
? Premature newborn ? high oxygen exposure
for prolonged period causes ?
bronchopulmonary dysplasia
? Adult ? ARDS due to pulmonary edema
caused by free radicals
33
Disease of eyes
? ROP (Retrolental fibroplasia) ?
? Premature newborn treated with pure oxygen for
long time.
? Free radicals causing thromboxane release,
sustained vascular contracture and cellular injury.
? Cataract - related with aging and photochemical
generation of free radicals
34
Atherosclerosis and MI
? LDL deposited in the endothelial cells,
undergo oxidation by free radicals.
? Attracts macrophages ? form foam cells ?
formation of plaque ? atherosclerosis.
35
Skin disease
? Plant products like psoralens are administered
in the treatment of psoriasis and leucoderma.
? Drug applied to affected skin and irradiated by
UV light
? Singlet oxygen is produced with clinical
benefit
36
Carcinogenesis and treatment
? Cancer is treated by radiotherapy
? Irradiation produced reactive oxygen species
in the cells which trigger the cell death
37
? Antioxidant
Antioxidants
? Substances which protect against cellular
injury by ROS or other oxidants by scavenging
them are called antioxidants
? In normal healthy state a balance is
maintained between FRs & antioxidants.
? Moreover we can as well supplement these
from outside (in vitro Antioxidants).
Free radical scavenging system
? Preventive antioxidant: reduce the rate of chain
initiation
? Glutathione peroxidase
? Catalase
? Chain breaking antioxidant: Interfere with chain
propagation
? SOD
? Uric acid
? Vit E
? Enzymes
? SOD
? Catalase
? Glutathione peroxidase
? Non enzymes
? Transferrin and ferritin
? Cerruloplasmin
? Albumin
? Glutathione
? Uric acid
? Bilirubin
? Ubiquinone
? Vit E
? Vit C
? Beta carotene
? Selenium
Superoxide dismutase
2O . -
2 + 2H+ H O
2 2+ O 2
? SOD - is present in all oxygen-metabolizing cells,
different cofactors (metals)
? SOD contain Mn, Cu and Zn as co-factor.
? mitochondrial SOD is Mn dependent.=SOD2=Chr 6=
idiopathic cardiomyopathy, premature aging
? Cytoplasmic SOD is Cu and Zn dependent= SOD1= Chr
21.
? Defect in SOD1 ? amyotrophic lateral sclerosis(Lou
Gehring's
? SOD 3= extracellular SOD [Cu-Zn]=Chr 4= disease
associated Asbestosis
43
SOD
? Increase concentration of SOD during exercise
? Because aerobic metabolism - increases ROS
generation during exercise
? This is turn stimulates the cell to synthesize
more enzymes.
? The net effect is increase protection during
the post-exercise or rest periods.
44
ANTIOXIDANTS
Catalase
2 H 2O
2
2 H 2O + O2
?
High affinity to H2O2 : peroxisomes,
mitochondria, cytoplasm of erytrocytes
45
Glutathione peroxidases
? It is selenium dependant enzyme
? It catalyses reduction of hydrogen peroxide
and lipid peroxide by glutathione
? Sulfhydryl groups of reduced glutathione
(GSH) serves as a electron donor.
? It is oxidized to the disulphide form (GS-SG)
which is reduced back by reductase enzyme
using NADPH
46
Glutathione reductase
? Is flavo-protein enzyme using FA D as a co-
factor to reform the reduced glutathione from
oxidized glutathione
? The reducing equivalent donor is NADPH
? NADPH comes from HMP shunt.
47
48
49
Non-enzymatic antioxidants
? Metal binding proteins ? Transferrin, Ferritin,
Ceruloplasmin.
? Albumin ? bind with various oxidizing
substances.
? Glutathione
? Bilirubin ? principal antioxidants in plasma.
One molecule of bilirubin scavenges
two hydroperoxy radicals and gets
oxidized to biliverdin.
50
? Uric acid
? Capability of scavenging free electron and
prevents the propagation of FR damage in plasma.
51
Vitamin E
?
Fat-soluble antioxidant
?
Absorbed in Small Intestines
?
Primary defender against effects of free
radicals in the body
?
Stored in liver and fat cells.
?
Protects components of the cell and their
membrane from destruction
52
Vitamin E Is the Major Lipid-Soluble
Antioxidant in Cell Membranes & Plasma
Lipoproteins
Vitamin E
? Lipid peroxides are neutralized by glutathione
peroxidase
? Thus vitamin E acts synergistical y with glutathione
peroxidase enzyme which contains selenium.
? So sparing effect on dietary requirement of selenium
by supplementing the activity of this enzyme.
? Organel es in the cells exposed to the highest
amounts of oxygen like mitochondria seem
to
have the highest amount of Vitamin E.
? Evidence suggests that it protects LDL against
oxidation, which in turn protects us against heart
disease.
54
ANTIOXIDANTS
Vitamin C
? Gives up electrons very easily when they are needed.
? Helps to reactivate Vitamin E, glutathione, urate and beta
carotene.
?Having the ability to recycle themselves over and over
again.
?Protects oxygen and iron from oxidation.
? Helps protect arteries against oxidative damage.
?It works in aqueose environment by breaking chain reaction
and can scavenge physiologically important reactive oxygen
and nitrogen species
55
ANTIOXIDANTS
Vitamin A and Beta carotene
? Beta Carotene is a water soluble precursor of Vitamin A,
and is an antioxidant in itself;
? It is chain breaking antioxidant by trapping peroxy
radicals in tissues at low partial pressure of oxygen.
Found In:
Corn, squash and carrots, egg yolk, and other
pigmented fruits and vegetables. This is what
helps give them their yellow color.
56
ANTIOXIDANTS
Selenium
?
An essential trace mineral,
RDA of 70 ug/day.
?
Found in Glutathion peroxidase which is a free
radical scavenging enzyme that contains
selenium.It destroys peroxides and thus protects
lipid membranes as does Vitamin E.
57
ANTIOXIDANTS
BHA (Butylated Hydroxyanisole)
?
Generally made in the lab as an antioxidant
?
Used as a food preservative.
?
Protects the oxidation of fats or oils.
?
Naturally found in Rosemary
?
Encourages the development of tumors
?
May enlarge liver and bladder
58
ANTIOXIDANTS
Phytochemicals
?Compounds found in plant-derived foods that have biological
activity in the body.
?Contribute to food taste, aromas, colors and other characteristics.
?Act as antioxidants, suppressing the development of diseases.
?Work better when combined
with other phytochemicals.
?May help keep cholesterol in
check
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Food
Phytochemical(s)
Allium vegetables
Phytochemicals
Allyl sulfides
(garlic, onions, chives, leeks)
Most Commonly Studied Phytochemicals
Cruciferous vegetables
(broccoli, cauliflower,
Indoles/glucosinolates
cabbage, Brussels sprouts,
Sulfaforaphane
kale, turnips, bok choy,
Isothiocyanates/thiocyanates
kohlrabi)
Thiols
Solanaceous vegetables
(tomatoes, peppers)
Lycopene
Umbelliferous vegetables
Carotenoids
(carrots, celery, cilantro,
Phthalides
parsley, parsnips)
Polyacetylenes
Compositae plants (artichoke)
Silymarin
Citrus fruits
Monoterpenes (limonene)
(oranges, lemons, grapefruit)
Carotenoids
Glucarates
Other fruits (grapes, berries,
Ellagic acid
cherries, apples, cantaloupe,
Phenols
watermelon, pomegranate)
Flavonoids (quercetin)
Beans, grains, seeds
Flavonoids (isoflavones)
(soybeans, oats, barley, brown
Phytic acid
rice, whole wheat, flax seed)
Protease inhibitors
Saponins
Herbs, spices (ginger, mint,
rosemary, thyme, oregano,
Gingerols
There are also hundreds more
sage, basil, tumeric, caraway,
Flavonoids
phytochemicals existing and in need
fennel)
Monoterpenes (limonene)
of discovery!
Licorice root
Green tea
Glycyrrhizin Catechins
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Polyphenols
ANTIOXIDANTS
Flavanoids
? Sometimes referred to as "Super Antioxidants."
? Shown to have: antiviral, antiallergic, anti-inflammatory,
antithrombogenic and anticarcinogenic effects.
? Over 4000 flavanoids have been found, fall in four different
groups: flavones, flavanones, catechins, and anthocyanins.
?Found in: certain fruits, flowers, roots, stems, tea, wine,
grains and vegetables.
61
ANTIOXIDANTS
Flavanoids
?
20 times stronger antioxidant then Vitamin C
and 50 times stronger then Vitamin E.
?
Water soluble
?
Ability to attach to cells and their proteins for
up to 72 hours protecting them from oxidation
and free radical damage.
?
Able to cross the blood-brain barrier
62
FREE RADICALS AND ANTI-OXIDANTS
? The Antioxidant Paradox -
Prooxidant
The Antioxidant Paradox--Antioxidants
Can Also Be Pro-Oxidants
Vitamin C
? these pro-oxidant actions require relatively
high concentrations of ascorbate, which are
unlikely to be reached in tissues
? once the plasma concentration of ascorbate
reaches about 30 mmol/L, the renal threshold
is reached,
? at intakes above about 100 to 120 mg/d the
vitamin is excreted in the urine quantitatively
with intake.
-carotene.
? carotene is protective against lung and other cancers.
? two major intervention trials in the 1990s showed an
increase in death from lung (and other) cancer
? -carotene is a radical-trapping antioxidant:
under conditions of low partial pressure of oxygen,
? Under high partial pressures of oxygen (as in the lungs)
and especial y in high concentrations, -carotene is an
autocatalytic pro-oxidant,
? can initiate radical damage to lipids and proteins.
vitamin E
? vitamin E is protective against atherosclerosis and
cardiovascular disease.
? meta-analysis of intervention trials with vitamin E
shows increased mortality among those taking
(high dose) supplements.
? vitamin E acts as an antioxidant by forming a
stable radical that persists long enough to
undergo metabolism to nonradical products.
? radical also persists long enough to penetrate
deeper in to the lipoprotein, causing further
radical damage,
Summary
? Free radicals are highly reactive molecular specie
? They can modify, proteins, nucleic acids and fatty
acids in cel membranes and plasma lipoproteins.
? Radical damage to lipids and proteins in plasma
lipoproteins is a factor in the development of
atherosclerosis and coronary artery disease;
? radical damage to nucleic acids may induce
heritable mutations and cance
? radical damage to proteins may lead to the
development of autoimmune diseases.
? Oxygen radicals arise as a result of exposure to ionizing
radiation, nonenzymic reactions of transition metal
ions, the respiratory burst of activated macrophages
? Protection against radical damage is afforded by
enzymes that remove superoxide ions and hydrogen
peroxide, enzymic reduction of lipid peroxides linked to
oxidation of glutathione, nonenzymic reaction of lipid
peroxides with vitamin E
? Except in people who were initial y deficient,
intervention trials of vitamin E and -carotene have
general y shown increased mortality among those
taking the supplements
? at higher concentrations of oxygen carotin is an
autocatalytic pro-oxidant
? Vitamin E forms a stable radical penetrating
further into lipoproteins and tissues, so
increasing radical damage.
? Radicals are important in cel signaling for
apoptosis of DNA damaged cel s
? high concentrations of antioxidants, may quench
the signaling radicals, so increasing, the risk of
cancer development.
MCQ 1
? Which one of the following is NOT a source of
oxygen radicals?
A. Action of superoxide dismutase
B. Activation of macrophages
C. Nonenzymic reactions of transition metal ions
D. Reaction of -carotene with oxygen
E. Ultraviolet radiation
MCQ 2
? Which one of the following best explains the antioxidant action
of vitamin E?
A. It forms a stable radical that can be reduced back to active
vitamin E by reaction with vitamin C.
B. It is a radical, so that when it reacts with another radical a
nonradical product is formed.
C. It is converted to a stable radical by reaction with vitamin C.
D. It is lipid soluble and can react with free radicals in the blood
plasma resulting from nitric oxide (NO) formation by vascular
endothelium.
E. Oxidized vitamin E can be reduced back to active vitamin E by
reaction with glutathione and glutathione peroxidase.
This post was last modified on 05 April 2022