ROS and T2DM-2

7/18/2019 ROS and T2DM-2

http://slidepdf.com/reader/full/ros-and-t2dm-2 1/11

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'.

7/18/2019 ROS and T2DM-2


pg614-617.indd 614

7/18/2019 ROS and T2DM-2


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


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


"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


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

7/18/2019 ROS and T2DM-2


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


/+ ?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

7/18/2019 ROS and T2DM-2


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


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

7/18/2019 ROS and T2DM-2


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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+'. 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 &%%%=

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*+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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