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ROS and T2DM-2
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7/18/2019 ROS and T2DM-2
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ORIGINAL ARTICLES
Antioxidant and oxidative stress status in type 2 diabetes and diabetic
foot ulcer
Eliabet! "osede "ola#o$o% &ensese Sontin 'ossanda% (rancis Adeniyi% Olubayo A$inosun% Adeso#i (asan)ade% 'p!o
'oropane
Objective. Oxidative stress (OS) has been implicated in the with the NC subjects. This increase in both parameters was
aetiology and progression o diabetic complications including greater or !" subjects# $%.&' and '.*+ respectively.
diabetic oot ulcer. ,n this study- the levels o lipid peroxides SO! activities were signiicantly reduced in ! (+/.$&)
(01O) and $2hydroxy2&32deoxyguanosine ($2O4d5) as well as and !" (/.%*) subjects in contrast with elevated activities
the en6ymatic antioxidant activities o superoxide dismutase o 51x observed in ! (&+.$7) and !" (&%.*/) subjects.
(SO!) and glutathione peroxidase (51x) in type & diabetes 5lycated haemoglobin8asting plasma glucose (4b9+c8"15)
mellitus and diabetic oot ulcer subjects were assessed correlated positively with 01O- $2O4d5 and 51x- whereas aand compared with apparently healthy normal subjects to negative correlation was observed or SO!.
understand the involvement o OS in the subjects. Conclusion. ,ncreased oxidation subse:uent to diabetic
Method . The abovementioned OS mar;ers were measured in conditions induces an over2expression o 51x activity
% subjects or each o the ollowing groups# type & diabetes suggesting a compensatory mechanism by the body to
mellitus (!)- diabetic oot ulcer (!") and non2diabetic prevent urther t issue damage in the subjects.
control (NC).
Results# Signiicant elevated values o 01O ('*.$<) and S Afr Med J &%%$= *$#
$2O4d5 (/.') were ound in ! subjects compared
9 body o evidence exists concerning the involvement o
oxidative stress (OS) in the aetiology o diabetes and its later
complications- o which diabetic oot ulcer is one.+ OS arises
in cells and tissues through the increased production o
reactive oxygen species (>OS) and8or rom decreases in the
antioxidant deence system.& Several mechanisms seem to be
involved
in the generation o OS in the presence o elevated glucose
concentrations= they include glucose auto2oxidation-
enhanced glucose lux through the polyol pathway- and non2
en6ymatic and progressive glycation o proteins with
conse:uent increased ormation o glucose2derived advanced
glycosylation end products (95?s).'
@nder normal physiological conditions- a widespread
antioxidant deence system protects the body against the
adverse eects o >OS generation. The deence
mechanism3s
Department of Chemical Pathology !niversity of "impopo Medunsa Campus
#aRan$u%a
&ensese Sontin 'ossanda% 1h!
Eliabet! "osede "ola#o$o% Sc
Department of Anatomical Pathology !niversity of "impopo Medunsa Campus
'p!o 'oropane% ATech
Department of Chemical Pathology !niversity of &badan &badan 'igeria
*+, (rancis Adeniyi% 1h!
Olubayo A$inosun% A AS
Department of Medicine !niversity of &badan
Adeso#i (asan)ade% A AS
Corresponding author: ( Mossanda )$mossan*ul+ac+,a-
eiciency is altered in diabetes and
the ineective scavenging o ree
radicals may thereore play a crucial
role in determining tissue damage in
these subjects./
The present investigation was
carried out to assess the levels o
lipid peroxides (01O) (a mar;er o
lipid peroxidation) and $2hydroxy2
&′2deoxyguanosine ($2O4d5) (a
mar;er o !N9 damage)- as well as
the en6ymatic antioxidant activities
o superoxide dismutase (SO!) and
glutathione peroxidase (51x) in
normal control (NC) type & diabetes
mellitus (!) and diabetic oot
ulcer (!") subjects- to understand
the involvement o OS in diabetic
oot ulcers.
'aterials and )et!ods
-atients
The study population comprised
% type & ! subjects and % !"
subjects with Bagner3s grade ,,
ulcer classiication (i.e. ulcer
without abscess or osteomyelitis).
ales and non2pregnant8non2
lactating emales between the ages
o /% and <% years with 4b9+c
<. were recruited rom the
medical ward o @niversity
College 4ospital- ,badan-
Nigeria- as Dtest3 groups. ,n
addition- % age2matched
healthy non2diabetic subjectso both genders with 4b9+c
E<. were selected
as a normal control group
rom among the sta o the
same university. ,normed
consent was sought and
obtained rom each subject
beore recruitment into this
study- which was approved
by the ?thical Committee o
the @niversity o ,badan
and the @niversity College
4ospital ,nstitutional>eview Committee
(@,8@C4 ,>C) (approval
number @,8 ,>C8%'8%%*<).
9ugust
&%%$- Fol.
*$- No. $ SA'.
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ORIGINAL ARTICLES
"lood
sa)plin/
Ten2millilitre
ali:uots o
venous blood
drawn ater a
+%2hour
overnight
ast were
collected in
heparinised
?!T9 or
luoridesample tubes
and
centriuged
at ' %%% rpm
or +%
minutes.
1lasma and
haemolysate
were stored at
G$%HC until
the day o
analysis.
9ntioxidant
en6ymes
(SO!- 51x)
were
measured in
heparinised
whole blood-
whereas
oxidative
status
parameters
(01O- $2
O4d5) were
assessed in
?!T9
plasma.
"asting
plasma
glucose
("15) and
glycated
haemoglobin
9+c (4b9+c)
were
measured in
plasma rom
sodium
luoride and
?!T9
samples
respectively-
on the day o
collection
using
standard
laboratory
techni:ues.
Analyses
Chemical
reagents o the
highest :uality
were
purchased
rom Sigma2
9ldrich-
5ermany. 01O
concentrations
were measured
spectrophotom
etrically at <%
nm using the
errous
oxidation with
xylenol orange
("OI
F?>S,ON ,,)
assay
according to
the method o
Nouroo62
Jadeh et al .
This method is
based on the
principle o
rapid peroxide2
mediated
oxidation o "e&
K to "e'
K
under acidic
conditions.
1lasma
levels o
$2O4d5
were
measured
at /% nm
on a
microplat
e plate
reader
using a
commerci
al ;it
rom the
Lapan
,nstitute
or the
Control
o 9ging
("u;uroi-
Lapan).
The method
is based on a
competitive
in vitro
en6yme2
lin;ed
immunosorbe
nt assay or
:uantitative
measurement o
this !N9
metabolite in
tissue- serum
and plasma.<
?rythrocyte
SO! activity was
determined by
the method o
9rthur and
Aoyne-7 using a
commercial ;it
obtained rom
>andox
0aboratories-
@M. This method
uses xanthine
and xanthine
oxidase (IO!)
to generate
superoxide
radicals which
react with &2(/2
iodophenyl)2'2
(/2nitrophenol)2
2
phenyltetra6oliu
m chloride (,NT)
to orm a red
orma6an dye.
SO! activity is
then measured by
the degree o
inhibition o this
reaction
spectrophotometr
ically at % nm.
The
determination o
erythrocyte 51x
activity was based
on modiication o
the method o
1aglia and
Falentine-$ using a
commercial ;it
obtained rom
>andox
0aboratories- @M.
This method
involves the
oxidation o
glutathione (5S4)
by cumene
hydroperoxide
catalysed by 51x.
,n the presence o
glutathione
reductase (5>)and N9!14- the
oxidised
glutathione
(5SS5) is
immediately
converted to the
reduced orm with
a concomitant
7/18/2019 ROS and T2DM-2
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oxidation o
N9!14 to
N9!1K. The
decrease in
absorbance is
then measured
spectrophotom
etrically at '/%
nm.
Statistical
analysis
9ll data were
presented as
mean
standard
deviation (S!)
or % subjects
in each group.
The Statistical
1ac;age or
SocialSciences
(S1SS)
(version +')
was used or
statistical
evaluation.
Signiicance o
dierences
was
determined
using one2way
analysis o
variance
(9NOF9).
1earson3s
correlation
coeicient was
determined
within groups.
Statistical
signiicance
was set at p2
values E%.%.
9ugust &%%$-Fol. *$- No. $
SA'.
Results
1e
ars
on
3s
co
rre
lat
io
n
an
al
ysi
s
o
allth
e
gr
ou
ps
re
ve
ale
d a
str
on
g
co
rre
lat
io
n
o
"1
5
an
d
4
b
9
+c
with
01
O-
$2
O
4
d
5
an
d
5
1x
-
in
co
ntr
ast
to
a
ne
ga
tive
corre
latio
n
with
SO!
. 9s
expe
cted-
NC2
4b9
+c
corre
lated
posit
ively
with
NC2
"15
(r%.
$**).
Thiscorre
latio
n
beca
me
stron
ger
in
the
!
and
!"
grou
ps-
with
a
corre
latio
n
coe
icien
t o
%.*$
&
and
%.*%+
respectively- as
shown in
Table ,
( pE%.%).
The
negative
correlati
on
between
"15 and
SO!
(rG
%.$7*) inthe NC
group is
shown in
"ig. +.
The
mean
values o
7/18/2019 ROS and T2DM-2
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"15
and
4b9+
c
were
respe
ctivel
y
&+/.$
/<.
&<
mg8dl
and
7.*7
&.
or
the
!
group
. "or
the
!"group
- they
were
&&<.*
'+&7
.+%
mg8dl
and
$./%
+.*+
respe
ctively-
comp
ared
with
*+.<*
*.<
mg8dl
and
/.%$
%.7
or
the
NC
group
- as
show
n in
"ig.
&a.
9
hi
g
h
le
v
el
o
0
1
O
w
as
o
bs
erv
ed
in
the
pla
sm
a
o
the
!"
gr
ou
p
(
<.<
+
+7.
'/
P
)
-co
mp
are
d
wit
h
the
!
gr
ou
p
(/'.*'
+<./<
P)
and NC
group
('+./+
+.*
P).
The
extent
o
oxidati
on was
relecte
d in the
concent
ration
o
!N9
adduct-
Ta
ble
I0
Co
rre
lati
on
ana
lysi
s of
-(
G
an
d
1b
A+
c
it!
oxida
nt
and
antio
xidan
t
eny
)es
it!in
NC%
3'
and
3(
/rou
ps
7/18/2019 ROS and T2DM-2
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1earson correlation
Correlation between test parameters coeicient ( pE%.%)
NC "15 NC 01O %.*+*
NC "15 NC $2O4d5 %.*77
NC "15 NC SO! G%.$7*
NC "15 NC 51x %.*7*
NC "15 NC 4b9+c %.*%%
! "15 ! 01O %.*$
! "15 ! $2O4d5 %.*$&
! "15 ! SO! G%.*+
! "15 ! 51x %.*&<
! "15 ! 4b9+c %.*$&
!" "15 !" 01O %.$/&
!" "15 !" $2O4d5 %.*&$
!" "15 !" SO! G%.$/*
!" "15 !" 51x %.$&'
!" "15 !" 4b9+c %.*%+
NC 4b9+c NC 01O %.*7
NC 4b9+c NC $2O4d5 %.*+*
NC 4b9+c NC SO! G%.7+%
NC 4b9+c NC 51x %.*%$
! 4b9+c ! 01O %.*<
! 4b9+c ! $2O4d5 %.*7 *+4! 4b9+c ! SO! G%.*+
! 4b9+c ! 51x %.*%<
!" 4b9+c !" 01O %.$/&
!" 4b9+c !" $2O4d5 %.*&$
!" 4b9+c !" SO! G%.$/*
!" 4b9+c !" 51x %.$&'
pg614-617.indd 615
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ORIGINAL ARTICLES
.ig
+ /+
Rel
atio
nsh
ip
bet
%ee
n
.P
#and
SO
D
in
'C
gro
up+
Coe
ffici
ent
of
det
erm
inat ion
)R&
-0 1
2+3
345
cor
rela
tion
coe
ffici
ent
)r-0
1
2+6
37
) p8
2+2
9-+
$2
O4d5
#
/*.%+'.
7* P
and
/<.'$+$.
%' P-
correspon
ding to an
increase
o
'.*+
and
/.'
respective
ly or the
!" and
!
groups-
compared
with the
NC group
('+.$7+
+.$ P)
("ig. &b).
9
slight
increase
in 51x
activity
was
observed
in the
! and
!"
groups
(&+.$7
and
&%.*/
respective
ly) in
contrast
with
a
substant
ial
decreas
e in
SO!
activity
in the
!"
(/.%*)
and
especial
ly in the
!
group
with adecreas
e o
+/.$&
( pE%.%
) ("ig.
&c).
3iscu
ssion
!uring
diabetes-
persist
ent
hyperg
lycae
mia
leads
to an
increas
ed
produc
tion o
>OSthroug
h the
glucos
e
autoxi
dation-
60.00
) o f a c t u a l v a l u e s
50.00 *226.93 * 8.40
214.84* *7.79
40.00
30.00
P e r c e n t a g e s ( %
4.0891.69
20.00
10.00
*+*0.00
FPG(mg/dl) HbA1C(%)
.ig+ :a+ .asting plasma glucose).P#-and glycatedhaemog lobin A/c);bA/c - intype : DMand D. groups
compar ed %iththe 'C group+<al=uesare statistically significant at> p82+2
7/18/2019 ROS and T2DM-2
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/+ ?hetype : DMand D. groups%erecompared%iththenon=diabet ic 'C group+?he percentagesofactualvaluesofeach parameter%erecalcul atedand plottedagainst the parameter foreaseofcomparison+
60.00
P e r c e n t a g e s ( % ) o f a c t u a l v a l u e s
50.00
43.93*40.00
30.0031.41
20.00
10.00
0.00
LPO(µM)
.ig+ :b+ "evels oflipid pero@ides)"PO- and 6=hydro@y=:=deo@yguanosine )6=O;d#- in type : DM and D. subjects incomparison %ith 'C sub=jects+<alues are statistically significant at> p82+2/+ ?he type : DM and D. groups%ere compared %iththe non=diabetic 'C group+ ?he percent=
ages of actualvalues of each parameter %erecalculated and plotted against the parameter for easeof comparison+
60.00
) o f a c t u a l v a l u e s
50.00
40.00 4408.683755.11
Q
**
30.00
P e r c e n t a g e s ( %
20.00
10.00
0.00
SOD(U/gHb)
.ig+ :c+ ?heen,ymatic activitiesof SOD and #P@ in
type : DM and D. subjects compared %ith 'C subjects+<alues are statistically significant at
> p82+2/ andB p82+29 andnot significant)ns- p2+29+ ?hetype : DM and D. groups %erecompared %iththe non=diabetic 'C group+ Percent=age ofactual values ofeach parameter%ere calculatedand plottedagainst the parameter forease ofcomparison+
sorbitol
pathway and
non2en6ymatic
protein
glycation.
Oxidative stress
arises when the
production o
>OS (which
include both
oxygen radicals
such as
superoxide
(O&R)- al;oxyl
(>O)- peroxyl
(>OO) and
hydroxyl (4O)
radicals- and
non2radical
derivatives o
oxygen- namely
hydrogen
peroxide)
exceeds the
capacity o the
available
antioxidant
deence system.
The excess >OS
tends to react
with virtually
all cell
components-
resulting in lipid
membrane peroxidation-
protein
denaturation
and !N9
damage.*
,n this
study- a high
level o
4b9+c and
"15 was
ound in both
type & !and !"
groups- with
the latter
group having
9ugust &%%$-
Fol. *$- No. $
SA'.
pg614-
617.indd
616 7/14/08 1:35:53 PM
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ORIGINAL ARTICLES
a greater
increase as a
conse:uence
o poorly
controlled
glycaemia in
these
subjects. The
correlation
coeicient
results
obtained in
this study
revealed a
strong
association
between
these two
parameters
("15 and
4b9+c) and
the level o
oxidants and
the activity
o
antioxidant
en6ymes in
diabetic
subjects with
and without
oot ulcers as
well as non2
diabetic
subjects.
One o the
characteristic
eatures o
chronic
diabetes is
lipid
peroxidation
resulting rom
excessive
reactions o
ree radicals
with
polyunsaturat
ed atty acids
(1@"9s) in
cell
membranes.
This lipid peroxidatio
n in turn
leads to
elevated
production
o ree
radicals.+%
0ipid
peroxide2
mediated
damage has
been
observed in
both types o !.
Be have
indeed
conirmed in our
study the
indings o
Santini et al .++
on the increase
o plasma 01O
levels in diabetic
subjects-
compared with
those o controlsubjects. ,n
addition to these
observations- a
substantially
elevated level o
this parameter
was ound in !"
subjects- which
may be due to
their increased
production o
>OS.
9s with
other
biomolecules-
deoxyribonucle
ic acid (!N9)
is also
susceptible to
damage
induced by ree
radicals. 9n
elevated level
o $2O4d5 (a
mar;er used
or assessing
the extent o
!N9 oxidative
damage by ree
radicals') was
indeed
observed in
type & ! and
!" subjects
(/.' and
'.*+
respectively)-
compared with
the NC group.
This inding is
in agreementwith the study
by !andona et
al .-+&
who
reported
greater
oxidative
damage to
!N9 with
more increased
generation o
>OS in both type
+ and type & !
patients than
normal controls.
Speciic
en6ymatic
antioxidant
deence systems
have evolved to
deal with
individual >OS.
SO! scavenges
superoxide
radicals by
converting them to
hydrogen peroxide
and molecular
oxygen- while 51x
converts hydrogen
peroxide to
oxygen and
water.+'
Controversial
reports on
changes in
erythrocyte
activity o SO!in both type +
and type &
diabetes have
been published.
,n some- a
decrease* in the
activity was
observed-
whereas in
others an
increase+/
or no
change+
was
reported. ,n this
study- however- asubstantial
decrease in SO!
activity (+/.$&=
pE%.%) in !
and a slight
reduction (/.%*=
p%.%) in !"
groups were
observed-
compared with
the NC group.
The reduction in
SO! activity
observed in this
study is
comparable to the
wor; o Ahatia et
al .-* who reported
a signiicant
reduction in SO!
activity in !
7/18/2019 ROS and T2DM-2
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subjects. The
observed
decrease in
SO! activity
could have
resulted rom
glycation o
the en6yme-
which has
been reported
to occur in
diabetes with
poor
glycaemic
control.l'
,n addition-
heterogeneous
results o
unchanged-+/
decreased+
or
elevated+<
activity o
erythrocyte
51x have
been
9ugust &%%$-
Fol. *$- No. $
SA'.
reported.
,n this
study- asigniica
ntly
elevated
51x
activity
was
observed
in !
(&+.$7)
and !"
subjects
(&%.*/)
-
compared with
the NC
subjects.
These
indings
accord
with the
wor; o
5upta
and
Chari+<
in both
types odiabetes-
where an
increase
was also
reported.
The
increase
in the
activity
o this
alternativ
e
antioxida
nten6yme
may
constitut
e a
compens
atory
mechanis
m to
prevent
urther
tissue
damage
in
diabetic
subjects.
Conclusion
"inding
s in this
study are
compatibl
e with the
hypothesi
s that
persistent
hyperglyc
aemia
leads to
increased
productio
n o
oxidants
(01O and
$2O4d5)
in
diabetic
subjects.
The
increase
is more
pronounced in
subjects
with !".
,ncreased
oxidation
subse:ue
nt to
diabetic
condition
s induces
an over2
expressio
n o 51xactivity-
suggestin
g a
compensa
tory
mechanis
m by the
body to
prevent
urther
tissue
damage
in such
subjects.
Be
grateully
ac;nowle
dge the
inancial
support
o Third
Borld
Organi6at
ion or
Bomen
inSciences
(TBOB
S) and
the
technical
assistanc
e o the
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Chem
ical
1athol
ogy
!epar
tment
o the
Natio
nal
4ealt
h
0abor
atory
Servic
es
(N40
S) o
South
9rica.
References
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Aoston# Mluwer 9cademic 1ublishers- &%%%# +2$.
&. 5umienic6e; 9- 4op;ala 4- Bojtowich J- Ni;olaju; L. Changes in antioxidant status o heartmuscle tissue in experimental diabetes in rabbits.
'. Aonneont2>ousselot !- Aastard L1- Laudon C- !elattre L. Conse:uences o the diabetic
status on the oxidant8antioxidant balance.
/. Aetteridge !L. Bhat is oxidative stress
. Nouroou62Jadeh L- Tajaddini2Sarmadi L- cCarthy S- A etteridge !L- Bol S1. ?levated levels
o authentic plasma hydroperoxides in N,!!.
<. Toyo;uni S- Tana;a T- 4attori U-
$2hydroxy22 2deoxyguanosine by monoclonal N/.+- its application to erric nitrilotriacetate2
induced renal carcinogenesis model.
7. 9rthur L>- Aoyne >. Superoxide dismutase and glutathione peroxidase activities in
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$. 1aglia !?- Falentine BN. 5lutathione peroxidase determination.
+$2+<*.
*. Ahatia S- Shu;la >- adhu SF- 5ambhir LM- 1rabhu M. 9ntioxidant status- lipid
peroxidation and nitric oxide end products in patients o type & diabetes mellitus with
nephropathy. Clin iochem &%%'= '<(7)# 72<&.
+%. 0evy @- Jalt6ber 4- Aen29mot6 9- Manter U- 9viram . Β2carotene aects antioxidant status
in non2insulin2dependent diabetes mellitus. Pathophysiol +***= <# +72+<+.
++. Santini S9- arra 5- 5iardina A- et al . !eective plasma antioxidant deenses and enhanced
susceptibility to lipid peroxidation in uncomplicated ,!!. Diabetes +**7= /<(++)# +$'2+$$.
+&. !andona 1- Thusu M- Coo; S- et al . Oxidative damage to !N9 in diabetes mellitus. "ancet
+**<= '/7($***)# ///2//.
+'. Melly "L. @se o antioxidants in the prevention and treatment o disease. J &nt .ed Clin Chem
+**$= +%(+)# &+2&'.
+/. 9ydin 9- Orhan 4- Sayal 9- O6ata - Sahin 5- ,simer 9. Oxidative str ess and nitric oxide
related parameters in type ,, diabetes mellitus# eects o glycemic control. Clin iochem &%%+=
'/# <27%.+. Mesavulu - 5iri >- Mameswara >A- 9pparao C. 0ipid peroxidation and antioxidant
en6yme levels in type & diabetics with microvascular complications. Diabetes Metab &%%%=
&<()# '$72'*&.
*+5+<. 5upta - Chari S. 0ipid peroxidation and antioxidant status in patients with diabetic
retinopathy. &nd J
Physiol Pharmacol &%%= /*(&)# +$72+*&.
Accepted 42 May:226+
pg614-617.indd 617
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