Download MBBS Biochemistry PPT 60 Free Radical Lecture Notes

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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

59

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

60

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