2.  Free radical damage has been implicated in a
wide variety of age related chronic diseases
such as atherosclerosis, diabetes, ophthalmic
and neurodegenerative disease as well as
being involved in the ageing process.
 Obesity has been described as a state of
chronic oxidative stress.

4.  A free radical is any species capable of
independent existence that contains one or
more unpaired electrons.
 They are unstable, very reactive and short-
lived as they tend to catch an electron from
other molecules.

5.  This definition includes the hydrogen atom
and most of the transition metal ions.
 It also includes the oxygen molecule, which
is biradical since its outer two electrons are in
different orbitals and have parallel spins (they
are not paired).
 Free radicals may be electrically neutral or
either positively or negatively charged.

6.  They attack sites of increased electron
density such as:
 the nitrogen atom present in proteins and DNA
predominantly
 and carbon-carbon double bonds present in
polyunsaturated fatty acids and phospholipids
 to produce additional free radical, often
reactive, intermediates.

7.  The presence of low concentrations of free
radicals is important for normal cellular redox
status, immune function and intracellular
signaling.
 However, excessive production can damage
lipids, proteins and DNA and compromise cell
function leading to cell death by necrosis or
apoptosis.

8.  The first organic free radical identified was
triphenylmethyl radical, by Moses
Gomberg in 1900.
 Historically, the term radical has also been
used for bound parts of the molecule,
especially when they remain unchanged in
reactions. These are now called functional
groups.
 For example, methyl alcohol was described
as consisting of a methyl "radical" and a
hydroxyl "radical". Neither are radicals in
the modern chemical sense.

9.  Homolysis of covalent bonds
 Addition of a single electron to a neutral
atom
 Loss of a single electron from a neutral atom

10.  As a rule, a radical needs to pair its unpaired electron with
another, and will react with another molecule in order to
obtain this missing electron. If a radical achieves this by
"stealing" an electron from another molecule, that other
molecule itself becomes a radical (Reaction 1), and a self
propagating chain reaction is begun (Reaction 2).
 If a radical pairs its unpaired electron by reacting with a
second radical, then the chain reaction is terminated, and
both radicals "neutralize" each other (Reaction 3).

11.  Most common free radicals are reactive oxygen
(ROS) and reactive nitrogen (RNS) species such as:

12.  Certain non-radical molecules have been included in this
definition. These are oxidizing agents or molecules that can
be easily converted into radicals.

15.  Superoxide dismutase (SOD)
 Cu-Zn SOD (in cytosol, SOD1)
 Mn-SOD (in mitochrondria, SOD2)
 EC-SOD (Cu-Zn SOD, SOD3)
 Glutathione peroxidase (GPX)
 Glutathione reductase (GR)
 Catalase (CAT)
 Glutathione (GSH)
 Glucose-6-phosphate dehydrogenase

16.  GSSG-S-transferase
 Proteindisulphide isomerase
 Thioredoxin
 Thioredoxin reductase
 Glutaredoxin
 Peroxiredoxin
 Heme oxygenase
 Methionine sulphoxide reductase
 Paroxonase (PON-1)
 Thiol-disulphide oxidoreductase

17.  Vitamin C (ascorbate)
 Vitamin A (α, β and γ carotenes)
 Vitamin E (8 different isomers)
 α-lipoic acid
 Phytochemicals (flavanoids, lignans, phenols)
 Selenium (for GPX activity)
 Copper (for Cu-Zn SOD activity)
 Zinc (for Cu-Zn SOD activity and protects S-H
groups)
 Manganese (for Mn-ZN SOD activity)

18.  Transition metals are tightly bound to various
proteins that prevent them from reacting with
peroxides to form free radicals. These include:
 Ceruloplasmin
 Lactoferrin
 Metallothionein
 Transferrin
 Haemoglobin
 Myoglobin
 Cytochrome oxidases
 Ferritin

19.  Some commonly measured analytes have
antioxidant activities:
 Gamma GT
 Uric acid
 Bilirubin
 HDL
 CK
 Myoglobin

20.  Organisms respond to oxidative stress by
upregulating a large number of genes that
encode for proteins involved in antioxidant
reactions.
 In the yeast (Saccaromyces cerevisiae)
exposure to H2O2 leads to the altered
transcription of ~70 genes.

21.  GAPDH has been identified as a target of oxidative
modification in many cellular systems and it may have a
regulatory role as a sensor of oxidative stress conditions.

25.  Other oxidases and oxygenases
 Cytochrome P450
 Auto-oxidation reactions eg:
 FMH2, FAD, catecholamines, tetrahydropteridines (such
as tetrahydrobiopterin) and thiol compounds such as
cysteine.
 Auto-oxidation greatly accelerated by transition metal ions
especially manganese, iron and copper ions.
 Haem proteins: estimated that ~3% of Hb in erythrocytes
undergoes oxidation every day exposing these cells to a
constant flux of O2.-

26.  In the electron transport chain, electrons are
passed through a series of proteins via
oxidation-reduction reactions, with each
acceptor protein along the chain having a
greater reduction potential than the previous.
 The last destination for an electron along this
chain is an oxygen molecule.

27.  95-99% of oxygen consumed is reduced into water
catalyzed by coenzyme Q (CoQ):
 However 1-5% will form the superoxide radical
where CoQ is turned into a superoxide generator:

28.  Immune system: neutrophils and macrophages use
ROS to destroy engulfed microorganisms.

29.  Can serve as second messengers or modify
oxidation-reduction (redox) states.
 Involved in some enzyme activation.
 Involved in drug detoxification.
 Play an essential role in muscle contraction.

30.  Because of their reactive nature free radicals
can provoke inflammation or altered cellular
functions through:
 lipid peroxidation
 protein modification
 DNA modification

32.  Lipid peroxidation
 Protein modification
 DNA modification

34.  Lipid peroxidation is a complex process and a
wide range of products are formed in variable
amounts.
 The major products are:
 α,β-unsaturated reactive aldehydes
▪ 4-hydroxy-2-nonenal (HNE)
▪ malondialehyde (MDA)
▪ 2-propenal (acrolein)
 isoprostanes

35.  The lipid aldehydes are relatively stable, but reactive, and
can diffuse within or escape from the cell and attack targets
far from their site of formation. They can be regarded as
“second cytotoxic messengers”.
 They can react with various biomolecules including protein,
DNA and phospholipids generating stable end-products.
 They react with amino acids, mainly Cys, His and Lys, to
modify protein structure and function.
 They can cross-link lipids in cell membranes interrupting
structure and fluidity.
 They can react with DNA to form a number of DNA
adducts having mutagenic and carcinogenic effects.

36.  Isoprostanes resemble the prostaglandins
but are formed in vivo by the non-enzymatic
free radical-catalyzed peroxidation of
polyunsaturated fatty acids with three or
more double bonds:
 linolenic acid (C18:3 ω3 )
 arachidonic acid (C20:4 ω6 )
 eicosapentanoic acid (C20:5 ω3).

37.  Vasoconstriction, seemingly by activation of
receptors, analogous or identical to those for
thromboxane.
 Adduct formation with thiol groups forming
glutathione.
 Adduct formation with lysine groups on
proteins and induce cross-links.

38.  Proteins are major targets of free radical
attack because of their high abundance and
because they are primarily responsible for
most functional processes within cells.
 Protein modification may alter every level of
protein structure from primary to quaternary
causing major structural changes.

39.  Oxidative damage is induced either directly or
indirectly:
 by reaction of secondary products leading to peptide
backbone cleavage or fragmentation, cross-linking,
altered susceptibility to proteolytic enzymes, and/or
modification of the side chains of virtually every
amino acid.
 by the formation of new reactive groups (from tyr,
cys, his, pro, lys, arg, trp, phe, val) or the formation of
protein carbonyls.

40.  Most damage is irreparable and may have a wide
range of downstream consequences affecting
the function of receptors, enzymes, transport
proteins etc. and may generate new antigens
provoking an immune response.
 In turn it can result in secondary damage to
other biomolecules such as inactivation of DNA
repair enzymes and loss of fidelity of damaged
DNA polymerase in replicating DNA.

41.  Free radicals induce several types of DNA damage
including
 strand breaks,
 DNA-protein cross-links
 and a large range of base and sugar modifications.
 Of the free radicals the highly reactive hydroxyl
radical (.OH) is the most prominent in the
development of base and sugar modifications.
 DNA damage also occurs through reactive nitrogen
species undergoing mainly nitration and deamination
of purines

42.  The net result is an increased risk of
mutagenesis and carcinogenesis.
 Oxidative damage to DNA is repaired by
cellular repair systems and DNA base damage
is thought to be repaired mainly by base-
excision repair.

43.  Direct measurement
 Biomarkers of free radical damage

44.  Electron spin resonance (ESR)
 detects the presence of unpaired electrons.
 By itself it only detects fairly unreactive radicals as
reactive radicals do not accumulate to any significant
degree.
 The method can be modified by adding “trapping”,
agents that intercept reactive radicals and react with
them to form stable radicals, which can then be
measured by ESR.
 However, the technique is not suitable for clinical
laboratories because of the need for expensive
equipment, expertise and time.

45.  Measurement of free radical mediated
damage on:
 Total Antioxidant Activity
 Lipids
 Proteins
 DNA

46.  is a measure of the total capacity of a plasma sample to
quench an oxidative burst of a “free radical” (peroxynitrate).
 The peroxynitrate is neutralised by antioxidants in the
sample and this “activity” is measured by its reaction with
Phalosin, a photoprotein that emits an intense luminescence
upon oxidation.
 The luminescence is read off a standard curve made using an
antioxidant (Trolox, a vitamin E analogue).

47.  The TAC assay does not measure total antioxidant activity.
Generally measure the low molecular weight antioxidants
and exclude the contribution of antioxidant enzymes and
metal binding proteins.
 The major contributor to the TAC assay is urate, often
accounting for >50% of the activity. But urate is of limited
importance as an antioxidant in vivo.
 A number of other compounds exist that can react but are
not antioxidants.
 Different TAC assays may not correlate with each other
because the various antioxidants react differently in different
assays.

48.  Numerous markers are available for
measurement of DNA modification.
 The most used marker is 8-hydroxy-2’-
deoxyguanine (8-OHdG), produced by free
radical induced guanine oxidation and used
as a marker of “whole body” oxidative DNA
damage.

49.  The oxidized DNA is continually repaired, and oxidized
nucleotides such as 8-OHdG are excreted via blood and
urine.
 Assayed by HPLC and MS techniques.
 However, 8-OHdG can also arise from degradation and
oxidation of guanine in the DNA precursor pool.
 There are many more products of oxidative DNA damage
thus 8-OHdG is a partial measure of damage to guanine
residues in DNA and may not truly reflect rates of oxidative
damage to DNA.
 Also likely is artefactual production of 8-OHdG by auto-
oxidation during and after sample extraction.

50.  Glutathione and S-glutathionylated proteins
 Tyrosine oxidation, nitration and
halogenation
 Carbonylated proteins

51.  Protein carbonyls (C=O) may be generated by
the oxidation of several amino acids (lys, arg,
pro and thr).
 Carbonyls can arise as a result of protein
glycation by sugars, by binding of aldehydes
such as lipid peroxidation products and by
direct oxidation of amino acid side chains by
free radicals

52.  Protein carbonyl content (PCC) is the most widely
used marker of oxidative modification of proteins.
 There are several methodologies for the
quantitation of PCC; in all of them 2,4 dinitrophenyl
hydrazine is allowed to react with the protein
carbonyls to form the corresponding hydrazone.
 Measured readily by spectrophotometric and
immunoassay techniques.

53.  Oxidized amino acids can be absorbed from the
diet.
 The general lack of knowledge in oxidized amino
acid kinetics, as they may decompose in complex
ways (eg. fragmentation, crosslinking and
unfolding) which may accelerate or hinder
proteolytic and proteosome mediated turnover.
 Only a small number of proteins may be
carbonylated.

54.  4-hydroxy-2-nonenal (HNE)
 Malondialdehyde (MDA)
 2-propenal (acrolein)
 Isoprostanes
 Expired pentane, ethane or hexane

55.  Common measurement based on the reaction of
malondialdehyde (MDA) with thio-barbituric acid
(TBA);
 forming a MDA-TBA2 adduct that absorbs strongly
at 532 nm (TBARS assay).
 Limitations of TBARS assay:
 Most of the TBA reactive material in body fluids are not
related to lipid peroxidation.
 The TBARS assay can be used as a general marker of lipid
peroxidation but noting that it may over-estimate lipid
peroxidation.

56.  MDA is only one of the many aldehydes formed during
lipid peroxidation and MDA can also arise from free
radical attack on sialic acid and deoxyribose.
 The concentration of free MDA is probably low in vivo as
they readily conjugate to proteins.
 Lipid aldehydes undergo further metabolism by cells.
 Lipid peroxides can also be absorbed from the diet.

57.  Isoprostanes are specific end-products of free radical
peroxidation of unsaturated acids.
 Of the isomers the most studied are the F2-isoprostanes
from arachidonic acid, specifically 8-iso-PGF2a.
 Isoprostanes are measured by immunoassay or LCMS.
 At present the measurement of 8-iso-PGF2a is regarded as
one of the most reliable approaches to the assessment of
free radical-mediated lipid peroxidation in vivo.
 A tissue that does not contain isoprostanes has yet to be
reported.

58.  They are minor end-products of lipid peroxidation.
 They are chemically stable in vivo and ex vivo but
once they are released into the circulation they are
rapidly metabolized undergoing hydrolysis by
various phospho-lipases and then by β-oxidation .
 They are also present in plasma in two forms:
esterified to lipids and as free acids, with the
esterified form being the most abundant. In urine
they exist as only as the free.

59.  The free-radical theory of aging (FRTA) states that
organisms age because cells accumulate free
radical damage over time.
 For most biological structures, free radical damage
is closely associated with oxidative damage.
 Earlier, this theory was only concerned with free
radicals such as superoxide (O2- ), but it has since
been expanded to encompass oxidative damage
from ROS such as H2O2, or OH-.

60.  Denham Harman first proposed the free radical theory
of aging in the 1950s, and in the 1970s extended the idea
to implicate mitochondrial production of reactive
oxygen species.
 In later years, the free radical theory was expanded to
include not only aging per se, but also age related
diseases.
 Free radical damage within cells has been linked to a
range of disorders including cancer, arthritis,
atherosclerosis, Alzheimer’s disease and diabetes.

61.  Mutant strains of the roundworm that are more susceptible
to free radicals have shortened lifespan, and those with less
susceptibility have longer lifespan.
 In some model organisms, such as yeast and Drosophila,
there is evidence that reducing oxidative damage can extend
lifespan.
 In mice, interventions that enhance oxidative damage
generally shorten lifespan.
 However, in roundworms, blocking the production of the
naturally occurring antioxidant (superoxide dismutase) has
recently been shown to increase lifespan.

62.  Consumption of high levels of antioxidants, which should increase
lifespan under the theory, may extend average but not maximum
lifespan in mice. The effect, if present, is weak and only
inconsistently observed.
 In one laboratory, Phenybutylnitrone (PBN) was shown to produce
about a 10% extension of maximum lifespan in experimental
animals. However, this finding has not been reproduced by other
laboratories.
 Antioxidant supplementation has not been conclusively shown to
produce an extension of lifespan in a mammal.
 Whether reducing oxidative damage below normal levels is
sufficient to extend lifespan remains an open and controversial
question.