Thi ol s ,Mal onal de hyde and Tot al Ant i oxi dant St at us i n t he Tur ki s h Pat i e nt s wi t h Type 2 Di abe t e s Me l l i t us

Thi ol s ,Mal onal de hyde and Tot al Ant i oxi dant St at us i n t he Tur ki s h Pat i e nt s wi t h Type 2 Di abe t e s Me l l i t us

B ELGI N S ̈SLEYICI U D UMAN ,M ELEK ̈ O _{ZTU ̈RK} ,S ELMA Y I LMAZER and H ̈SREV U

H ^ATEMI

Depar tment of Medical Biology and Genetics, Kadir Has Univer sity Faculty of Medicine, Istanbul, Tur key,

Depar tment of Medical Biology, Istanbul Univer sity Cer r ahpasa Faculty of Medicine, Istanbul, Tur key,

Tur kish Diabetes Hospital, Istanbul, Tur key, and

Depar tment of Endocr inology and Metabolism, Istanbul Univer sity, Cer r ahpasa Faculty of Medicine, Istanbul, Tur key

D ^UMAN ,B. S. ,O ̈ _{ZTU ̈RK} ,M. ,Y ^{I LMAZER} ,S.and H ^ATEMI ,H. Thiols, Malonalde- hyde and Total Antioxidant Status in the Tur kish Patients with Type 2 Diabetes Mellitus. Tohoku J .Exp.Me d. ,2 0 0 3 ,2 0 1( 3 ) ,1 4 7 -1 5 5 Non- i ns ul i n- de pe nde nt ( Type 2 )di abe t e s me l l i t us( NI DDM)i s a r i s k f ac t or f or c ar di ovas c ul ar di s e as e s ( CVD) . Oxi dat i ve s t r e s s me c hani s ms ar e of t e n r e por t e d t o be i mpl i e d i n t ype 2 di abe t e s me l l i t us . I n or de r t o de t e r mi ne t he i r c l i ni c al r e l e vanc e ,we i nve s t i gat e d s e ve r al pl as ma i ndi c at or s i n t he Tur ki s h pat i e nt s wi t h NI DDM:( i )homoc ys t e i ne ( Hc y)and c ys t e i ne( Cys )whi c h c ont r i but e t o i nc r e as e t he r i s k of at he r os c l e r os i s dur i ng NI DDM, ( i i )gl ut at hi one( GSH)and c ys t e i nyl gl yc i ne( Cys Gl y)r e s ul t i ng f r om GSH de gr adat i on c at al yz e d by γ- gl ut amyl c ys t e i ne t r ans f e r as e ( GGT) ,( i i i ) mal onal de hyde( MDA)as a mar ke r f or l i pi d pe r oxi dat i on,and( i v)t ot al ant i ox- i dant s t at us( TAS) . Our mai n r e s ul t s we r e e val uat e d bas e d on s e x and di abe t i c s t at us . I n f e mal e pat i e nt s ,pl as ma c onc e nt r at i ons of MDA and Hc y we r e s i gni f i - c ant l y hi ghe r t han i n c ont r ol s ,whi l e GSH l e ve l s we r e s i gni f i c ant l y l owe r . I n mal e s ,a di f f e r e nc e be t we e n c ont r ol and di abe t i c gr oups was not i c e d onl y f or Hc y, l e ve l s be i ng al s o hi ghe r i n pat i e nt s . I n t he di abe t i c gr oup,i nc r e as e i n s e r um gl uc os e c onc e nt r at i on was s i gni f i c ant l y c or r e l at e d wi t h i nc r e as e d GGT ac t i vi t y.

I n bot h c ont r ol s and di abe t i c pat i e nt s ,GGT ac t i vi t y was c or r e l at e d wi t h a r ai s e d Cys c onc e nt r at i on and a de c r e as e d GSH l e ve l . I n bot h c ont r ol s and di abe t i c pat i e nt s ,t he r e we r e s i gni f i c ant pos i t i ve c or r e l at i ons be t we e n Cys and Hc y and be t we e n GSH and Hc y. We c onc l ude d t hat GSH and MDA l e ve l s ar e c l i ni c al i ndi c at or s f or an oxi dat i ve pr oc e s s l i nke d t o t ype 2 di abe t e s me l l i t us ,e s pe c i al l y i n wome n. NI DDM;oxi dat i ve s t r e s s ;l i pi d pe r oxi dat i on;s e x

Ⓒ 2 0 0 3 Tohoku Uni ve r s i t y Me di c al Pr e s s Tohoku J.Exp.Med. ,2003,201,147-155

Recei ved May 26,2003;r evi s i on accept ed f or publ i cat i on Sept ember 25,2003.

Addr es s f or r epr i nt s :Dr .Bel gi n Sus l eyi ci Duman,Depar t ment of Medi cal Bi ol ogy and Genet i cs , Kadi r Has Uni ver s i t y Facul t y of Medi ci ne,Vef abey St r eet ,No: 5 80810,Gayr et t epe- I s t anbul ,Tur key.

e- mai l :bs us l eyi ci ＠khas . edu. t r

147

One of t he pr i nc i pal c aus e s of mor bi di t y and mor t al i t y i n t he i ndi vi dual s wi t h di abe t e s me l l i t us i s at he r os c l e r os i s and r e s ul t i ng c ar di o- vas c ul ar di s e as e s( CVD)( Boe mi e t al .1 9 9 3 ) . Type 2 di abe t e s me l l i t us wi t h an i nc r e as i ng i nc i de nc e wor l d wi de i s c har ac t e r i z e d by an i nc r e as e d r i s k f or t he de ve l opme nt of ne ur o- pat hi c ,mi c r o-and mac r o- vas c ul ar c ompl i c a- t i ons( As s mann e t al .1 9 9 9 ) . Se ve r al e xpe r i - me nt al ,e pi de mi ol ogi c and c l i ni c al s t udi e s s up- por t t he not i on t hat oxi dat i ve s t r e s s pl ays a s i gni f i c ant r ol e i n t ype 2 di abe t e s me l l i t us( De Zwar t e t al .1 9 9 9 )and i n t he de ve l opme nt of CVD( J ac ob and Bur r i 1 9 9 6 ;Gar e wal 1 9 9 7 ) .

The pl as ma t hi ol s me as ur e me nt appe ar s t o be a s ui t abl e par ame t e r t o i nve s t i gat e t he nume r ous pat hol ogi c al di s e as e s t hat ar e r e l at e d t o oxi dat i ve s t r e s s pr oc e s s e s . Hype r homoc ys - t e i ne mi a has be e n s ugge s t e d t o pr omot e t he pr oduc t i on of hydr oxyl r adi c al s ,l i pi d pe r oxi da- t i on i ni t i at or s ,t hr ough homoc ys t e i ne ( Hc y) aut ooxi dat i on and t hi ol act one f or mat i on ( St aml e r e t al .1 9 9 3 ) . Mor e ove r ,Hc y r e pr e - s e nt s a s t r ong and i nde pe nde nt r i s k f ac t or f or CVD ( Ue l and e t al .1 9 9 2 ;Bous he y e t al .1 9 9 5 ; Gr aham e t al .1 9 9 7 ) . The i nc i de nc e of CVD i nc r e as e s wi t h i nc r e as i ng pl as ma or s e r um Hc y c onc e nt r at i ons( Ue l and e t al .1 9 9 2 ;Bous he y e t al .1 9 9 5 ) . Fur t he r mor e ,hype r homoc ys t e i ne mi a has be e n de s c r i be d i n di abe t i c pat i e nt s( Muns hi e t al .1 9 9 6 ;Hooge ve e n e t al .1 9 9 8 ) . Though l e s s r e ac t i ve t han Hc y,c ys t e i ne( Cys )e xhi bi t s s ome of i t s c he mi c al pr ope r t i e s t hr ough t he s ul f hydr yl gr oup ( St ampe r and Sl i vka 1 9 9 6 ) . The pot e nt i al noxi ous e f f e c t s of Cys may have be e n ove r l ooke d s i nc e f e w s t udi e s anal ys e d t he r e l at i ons hi p be t we e n Cys and CVD( Ar aki e t al . 1 9 8 9 ;Mans oor e t al .1 9 9 5 ;Ve r hoe f e t al .1 9 9 6 ) . GSH pl ays an i mpor t ant r ol e as a s c ave nge r of oxi dant s pe c i e s and f r e e r adi c al s( Me i s t e r and Tat e 1 9 7 6 ) . The pr e c ur s or s i nvol ve d i nt o GSH bi os ynt he s i s ,i . e .Cys and γ- gl ut amyl c ys t e i ne ( γ- Gl uCys ) ,and c ys t e i nyl gl yc i ne( Cys Gl y)r e s ul t - i ng f r om t he r e ac t i on c at al ys e d by γ- gl ut amyl t r ans f e r as e( GGT E. C. 2 . 3 . 2 . 2 ) ,ar e al s o

of i nt e r e s t t o e val uat e t he GSH home os t as i s i n t he bi ol ogi c al s ys t e m,e . g. ,c e l l s ,t i s s ue s ,bl ood or pl as ma( St aml e r and Sl i vka 1 9 9 6 ) . Mor e ove r , Cys Gl y i s a hi ghl y r e ac t i ve me t abol i t e and c an pr oduc e r e ac t i ve oxyge n s pe c i e s( ROS)i n pr e s - e nc e of t r ans i t i on me t al s( Pompe l l a 1 9 9 7 ;Dr oz - dz e t al .1 9 9 8 )t he r e by l e adi ng t o l i pi d pe r oxi da- t i on ( LPO) . Se r um γ- gl ut amyl c ys t e i ne t r ans - f e r as e( GGT)i s di r e c t l y i nvol ve d i n GSH me t ab- ol i s m and al s o modul at e s Cys and Cys Gl y l e ve l s . Pe r r y e t al .( 1 9 9 8 )s ugge s t e d t hat an i nc r e as e d s e r um GGT l e ve l i s an i nde pe nde nt r i s k f ac t or f or CVD i n NI DDM. Se r um GGT l e ve l mi ght be a s i mpl e and r e l i abl e mar ke r of he pat i c i ns ul i n r e s i s t anc e .

I n t he s ame way,t he c apac i t y of a s ubj e c t t o r e s i s t oxi dat i ve s t r e s s i s i ndi c at e d by t he t ot al ant i oxi dant s t at us( TAS)( Mi l l e r e t al .1 9 9 3 ; Maxwe l l e t al .1 9 9 7 ) . I n s e r um,s e ve r al ant i ox- i dant c ompounds c ont r i but e t o TAS mai nl y al bumi n,ur i c ac i d,α- t oc ophe r ol ,as c or bi c ac i d and bi l i r ubi n( Cao and Pr i or 1 9 9 8 ;Di az e t al . 1 9 9 8 ) .

De t e r mi nat i on of GSH,MDA ( as a f r e e r adi c al - me di at e d LPO pr oduc t )and TAS i s t he r e f or e of pr ac t i c al and c l i ni c al i mpor t anc e i n or de r t o as s e s s t he i mpl i c at i on of f r e e r adi c al s i n t ype 2 di abe t e s . I n t he pr e s e nt s t udy i nvol vi ng Tur ki s h s ubj e c t s ,t he mai n obj e c t i ve was t o de t e r mi ne whe t he r t he r e was a l i nk be t we e n c l i ni c al par ame t e r s and( i )t he s t at us of t hi ol s and GGT ac t i vi t y i n t ype 2 di abe t e s ,( i i )t he i nc r e as e of f r e e r adi c al ac t i vi t y,me as ur e d as MDA,and( i i i )t he TAS i n di abe t i c and he al t hy s ubj e c t s .

M ATERI ALS AND M ETHODS

Subjects

Pat i e nt s wi t h t ype 2 di abe t e s me l l i t us( 5 2 me n and 5 5 wome n)we r e e nr ol l e d. Al l pat i e nt s we r e t aki ng or al ant i di abe t i c dr ugs . The di ag- nos i s of t ype 2 di abe t e s me l l i t us was bas e d on t he c r i t e r i a of The Expe r t Commi t t e e on t he di agnos i s of di abe t e s me l l i t us( 2 0 0 0 ) . Di abe t i c pat i e nt s we r e r e c r ui t e d f r om t hos e c ons e c ut i ve - B. S.Duman et al .

148

l y at t e ndi ng t he Tur ki s h Di abe t e s Hos pi t al , I s t anbul f or t he i r r out i ne c l i ni c al e xami nat i on ( e ve r y 1 -2 mont hs )and i nc l ude d i n t he s t udy,i n agr e e me nt wi t h Tur ki s h l oc al e t hi c c ommi t t e e . Non di abe t i c c ont r ol s ubj e c t s we r e r e c r ui t e d f r om t hos e at t e ndi ng t he i r r out i ne he al t h s c r e e n- i ng i n t he Ce nt r al Labor at or y of Ce r r ahpas a Me di c al Fac ul t y( I s t anbul Uni ve r s i t y,Tur ke y) . As c ont r ol s ,9 9 he al t hy s ubj e c t s( 4 6 me n and 5 3 wome n)we r e s t udi e d. No pat i e nt s i n t he s t udy we r e r e l at e d. Al l di abe t i c pat i e nt s had nor mal he pat i c and e ndoc r i ne f unc t i ons and we r e r e l a- t i ve l y we l l c ont r ol l e d wi t h gl yc os yl at e d he mo- gl obi n ( HbA ) ≦6 -7 % ( nor mal r ange ≦8 %) . Tobac c o and al c ohol c ons umpt i ons we r e c ol - l e c t e d by us i ng s t andar di s e d que s t i onnai r e . The pat i e nt s wi t h mac r o-and mi c r oangi opat hi c c ompl i c at i ons we r e e xc l ude d f r om t hi s s t udy.

De t e r mi nat i on of bas e l i ne c ar di ovas c ul ar r i s k f ac t or s i nc l ude d age ,body mas s i nde x ( BMI ) ( Ange l i c o e t al .1 9 9 0 ) ,al c ohol c ons umpt i on and s moki ng s t at us .

Blood sampling

Af t e r an ove r ni ght f as t ,bl ood was dr awn by ve ni punc t ur e at t he ant e c ubi t al ve i n f r om t he s ubj e c t s be t we e n 8: 0 0 and 1 0: 0 0 a. m. The s ampl e s we r e c ol l e c t e d i n t wo vac uum t ube s : one t ube wi t h EDTA, one t ube wi t hout ant i c oagul ant . Bl ood was c e nt r i f uge d pr ompt - l y at 1 0 0 0 g f or 1 5 mi nut e s at＋4 C f or pl as ma and s e r um.

Assays of biochemical par ameter s

Tot al c hol e s t e r ol( TC) ,t r i gl yc e r i de( TG) and gl uc os e we r e me as ur e d i n s e r um on a Cobas - Mi r a anal yz e r ( Roc he Di agnos t i cs , Me yl an,Fr anc e ) . Se r um we r e anal ys e d wi t h- out pr e t e at me nt and di l ut e d i n doubl e - di s t i l l e d wat e r whe n l i pi d l e ve l s e xc e e de d r e f e r e nc e val ue s . I nt e r - as s ay c oe f f i c i e nt s of var i at i on of t hi s me t hod we r e 4 . 7 %,2 . 1 % and 1 . 0 %,f or TG, TC and gl uc os e ,r e s pe c t i ve l y. Bl ood gl uc os e val ue s we r e me as ur e d f r om ve nous bl ood s e r um.

GGT ac t i vi t y was me as ur e d i n pl as ma

s ampl e s us i ng a s pe c t r ophot oc ol or i me t r i c as s ay ki t( Gr anut e s t 2 5,Me r c k)on a Cobas - Mi r a anal ys e r . I nt r a-and i nt e r - as s ay c oe f f i c i e nt s of var i at i ons we r e 2 . 0 % and 2 . 4 %,r e s pe c t i ve l y.

Se r um TAS was de t er mi ned on a Cobas - Far a anal ys e r( Roc he Di agnos t i c s )wi t h a Tot al Ant i oxi dant St at us ki t( Randox Labs . , Cr uml i n,UK) . The as s ay pr i nc i pl e r e l i e s upon t he abi l i t y of ant i oxi dant s wi t hi n bi ol ogi c al f l ui ds t o que nc h t he abs or banc e( me as ur e d at 6 0 0 nm)of t he r adi c al c at i on f or me d by t he r e ac t i on of a c hr omoge n ( 2 , 2 ′ - az i no- di ［e - t hyl - be nz t hi az ol i ne s ul f onat e ］;ABTS)wi t h a pe r - oxi das e and H O ( Mi l l e r e t al .1 9 9 3 ) . The s ys t e m c al i br at or i s Tr ol ox,a wat e r - s ol ubl e vi t ami ne E anal ogue . Re s ul t s ar e e xpr e s s e d as μmol Tr ol ox/l . I nt r a-and i nt e r - as s ay c oe f f i - c i e nt s of var i at i ons we r e 1 . 9 % and 3 . 5 %,r e s pe c - t i ve l y.

Tot al pl as ma l ow mol e c ul ar mas s t hi ol s ( al l f or ms i nc l udi ng di s ul f i de s ,mi xe d di s ul f i de s , and f r e e t hi ol s ) ,i . e . Cys ,Cys Gl y,Hc y and GSH,we r e me as ur e d s i mul t ane ous l y us i ng a hi gh- per f or mance l i qui d chr omat ogr aphy ( HPLC)me t hod i nc l udi ng a pr e c ol umn de r i vat - i z at i on ( wi t h 7 - f l uor o- 2 , 1 , 3 - be nz oxadi az ol e - 4 - s ul f onami de ;ABD- F)and f l uor e s c e nc e de t e c - t i on( Sal az ar e t al . 1 9 9 9 ) . I nt r a-and i nt e r - as s ay c oe f f i c i e nt s of var i at i ons me as ur e d wi t h a pl as ma pool was 2 . 2 % and 7 . 2 % f or Cys ,1 . 7 % and 3 . 1 % f or Cys Gl y,1 . 9 % and 4 . 2 % f or Hc y and 7 . 2 % and 1 0 % f or GSH at c onc e nt r at i ons of 1 7 8 . 0 ,2 7 . 8 ,7 . 9 and 3 . 4μmol /l i t e r ,r e s pe c t i ve l y.

The HPLC me t hod f or pl as ma MDA me a-

s ur e me nt s( Young and Tr i mbl e 1 9 9 1 )c ons i s t s i n

t he f ol l owi ng s t e ps :( i )pr e c i pi t at i on of pr ot e i ns

by phos phor i c ac i d i n or de r t o e l i mi nat e t he

i nt e r f e r e nc e by t he wat e r - s ol ubl e s ubs t anc e s

t hat r e ac t wi t h t he t hi obar bi t ur i c ac i d( TBA) ,

( i i )f or mat i on of t he TBA- MDA adduc t and

( i i i )s e par at i on i n a HPLC s ys t e m wi t h f l uor e s -

c e nc e de t e c t i on. I nt r a-and i nt e r - as s ay c oe f f i -

c i e nt s of var i at i ons we r e 5 . 1 % and 1 0 . 5 %,

r e s pe c t i ve l y.

Statistical analysis

St at i s t i c al anal ys e s we r e pe r f or me d us i ng t he BMDP s t at i s t i c al s of t war e( UCLA,Los Ange l e s ,CA,USA) . Val ue s we r e e xpr e s s e d as me an± ^S . ^D . Compar i s on be t we e n gr oups was pe r f or me d us i ng St ude nt t - t e s t or ANOVA.

As c e ndant s t e pwi s e mul t i pl e r e gr e s s i on was pe r f or me d t o anal yz e i nt e r r e l at i ons hi ps be t we e n par ame t e r s of i nt e r e s t( t hi ol s ,MDA and TAS l e ve l s )and t he f ol l owi ng f ac t or s :age ,s e x,BMI , t obac c o c ons umpt i on, al c ohol c ons umpt i on, s e r um c hol e s t e r ol ,t r i gl yc e r i de s ,apo AI ,apo B and gl uc os e c onc e nt r at i on,HbA l e ve l ,and GGT ac t i vi t y. St at i s t i c al s i gni f i c anc e f or al l t e s t s was ac c e pt e d at p≦0 . 0 5 l e ve l .

R ^ESULTS

The mai n c har ac t e r i s t i c s of c ont r ol s and

pat i e nt s ar e s hown i n Tabl e 1 . I n f e mal e s , pat i e nt s wi t h NI DDM had hi ghe r TC, apo B and l owe r apo A1 c onc e nt r at i ons t han c ont r ol s , di f f e r e nc e f or apo A1 l e ve l be i ng onl y bor de r l i ne s i gni f i c ant . I n mal e s ,c har ac t e r i s t i c s di d not di f f e r s i gni f i c ant l y,e xc e pt f or gl uc os e ,be t we e n c ont r ol s and pat i e nt s . I n f e mal e pat i e nt s , pl as ma c onc e nt r at i ons of MDA and Hc y we r e s i gni f i c ant l y hi ghe r t han i n c ont r ol s ,whi l e GSH l e ve l s we r e s i gni f i c ant l y l owe r . I n mal e s ,a di f f e r e nc e be t we e n di abe t i c and c ont r ol gr oups was not i c e d onl y f or Hc y,l e ve l s be i ng hi ghe r i n t ype 2 di abe t i c s .

The as s oc i at i on be t we e n oxi dat i ve s t r e s s i ndi c at or s and t he i r c l i ni c al or bi ol ogi c al de t e r - mi nant s i s s hown i n Tabl e 2 . Gl uc os e c onc e n- t r at i on,de s c r i bi ng t he di abe t i c s t at us ,i s s i gni f i - c ant l y c or r e l at e d wi t h a r e duc e d s e r um TAS.

Eve n wi t hout s i gni f i c ant di f f e r e nc e of GGT T

1. Main char acter istics, TAS, and, concentr ations of MDA and thiol in contr ols (n＝

99) and type 2 diabetic patients (n＝107) Men Cont r ol ( n＝46)

Di abet es ( n＝52)

Women Cont r ol

( n＝53)

Di abet es ( n＝55) Age( year s ) 55. 9±14. 5 57. 8±11. 8 55. 2±9. 1 57. 9±8. 25 Body mas s i ndex( kg/m ) 21. 6±3. 2 21. 8±3. 1 23. 4±3. 5 23. 8±3. 9 Smoki ng( pack- year ) 1. 9±1. 2 2. 1±0. 9 1. 1±0. 8 0. 9±0. 7 Al cohol( g/week) 0. 07±0. 26 0. 02±0. 15 ― ― Ser um gl ucos e( mmol /l i t er ) 3. 35±0. 56 9. 07±3. 83 3. 54±0. 52 9. 08±3. 80

HbA ( %) ND 6. 7±1. 9 ND 6. 9±2. 2

Ser um t r i gl ycer i des( mmol /l i t er ) 1. 48±0. 55 2. 18±2. 12 1. 65±0. 67 1. 70±0. 95 Ser um chol es t er ol( mmol /l i t er ) 4. 89±1. 46 5. 17±1. 05 5. 30±1. 10 5. 86±1. 04 Apo A1( g/l i t er ) 1. 28±0. 25 1. 27±0. 24 1. 54±0. 29 1. 44±0. 22 Apo B ( g/l i t er ) 1. 11±0. 26 1. 11±0. 31 1. 07±0. 25 1. 18±0. 25 GGT act i vi t y( U/l i t er ) 20. 9±2. 8 25. 0±3. 1 20. 0±1. 7 21. 9±2. 4 MDA ( μmol /l i t er ) 0. 36±0. 15 0. 43±0. 19 0. 37±0. 12 0. 47±0. 23 TAS( μmol /l equi v Tr ol ox ) 1. 40±0. 12 1. 43±0. 14 1. 39±0. 14 1. 42±0. 12 Pl as ma cys t ei ne( μmol /l i t er ) 279±42 291±53 269±57 282±57 Pl as ma cys t ei nyl gl yci ne( μmol /l i t er ) 41. 1±7. 7 40. 6±13. 5 37. 0±8. 3 37. 9±10. 8 Pl as ma homocys t ei ne( μmol /l i t er ) 11. 2±3. 6 14. 6±5. 7 9. 4±2. 7 11. 4±3. 7 Pl as ma gl ut at hi one( μmol /l i t er ) 7. 7±2. 1 7. 3±3. 1 7. 8±2. 7 6. 4±3. 4

Val ues ar e means ±

.

.

Tes t on l og- t r ans f or med val ues .

p ≦0. 05, p ≦0. 01, p ≦0. 001:St udents t - t es t bet ween cont r ol s and pat i ent s orχ t es t ND,not det er mi ned.

B. S.Duman et al .

150

ac t i vi t y be t we e n t he c ont r ol and di abe t i c gr oups ,i n bot h c ont r ol s and di abe t i c pat i e nt s GGT ac t i vi t y i s r e l at e d wi t h a r ai s e d Cys c on- c e nt r at i on and a de c r e as e d GSH l e ve l . Ne ve r - t he l e s s ,no pot e nt i al ac t i vi t y of GGT on GSH was de t e c t e d af t e r t he bl ood upt ake :GSH c on- c e nt r at i on di d not c hange dur i ng s ampl e t r e at - me nt i n t he r ange of GGT ac t i vi t i e s me t i n t he pr e s e nt s t udy( dat a not s hown) .

The r e l at i ons hi ps be t we e n oxi dat i ve s t r e s s i ndi c at or s ar e s ummar i z e d i n Tabl e 3 . I n bot h c ont r ol s and di abe t i c pat i e nt s ,t he r e ar e s i gni f i - c ant pos i t i ve c or r e l at i ons be t we e n Cys and Hc y and be t we e n GSH and Hc y.

D I SCUSSI ON

Pat i e nt s wi t h NI DDM have i nc r e as e d mor - t al i t y and mor bi di t y c ompar e d wi t h non- di abe t i c s and ar e mor e l i ke l y t o de ve l op CVD ( Boe mi e t al .1 9 9 3 ) . The hi gh r i s k f or vas c ul ar di s e as e c an onl y par t l y be e xpl ai ne d by t he t r adi t i onal r i s k f ac t or s f or t he ge ne r al popul a- t i on s uc h as s moki ng,hype r t e ns i on and r ai s e d c hol e s t er ol . I t has been pr opos ed t hat oxi dat i ve s t r e s s may be as s oc i at e d wi t h t he pat hoge ne s i s of t he c ompl i c at i ons of NI DDM ( De Zwar t e t al .1 9 9 9 ) ,par t i c ul ar l y CVD( J ac ob and Bur r i 1 9 9 6 ;Gar e wal 1 9 9 7 ) .

One i s s ue t hat has not pr e vi ous l y be e n addr e s s e d i s t he i nt e r r e l at i ons hi p be t we e n t hi ol s T

2. Deter minants of TAS, MDA and thiol levels in (A) contr ols (n＝99) and (B) type 2

diabetic patients (n＝107) (both males and females) A (Contr ols)

TAS ( μmol /l )

MDA ( μmol /l )

Cys ( μmol /l )

Cys Gl y ( μmol /l )

Hcy ( μmol /l )

GSH ( μmol /l ) Age( year s ) ― ― 1. 27( 0. 49) ― ― 0. 067( 0. 028)

Sex ― ― ― ― −3. 11( 1. 16) ―

BMI( kg/m ) ― ― ― ― ― ―

Ser um chol es t er ol

( mmol /l ) ― ― 14. 5( 4. 9) ― ― ―

GGT act i vi t y( U/l ) ― ― 47. 9( 22. 8) ― ― −2. 67( 1. 25)

I nt er cept ― ― 70. 8 ― 17. 66 7. 72

R ― ― 0. 238 ― 0. 095 0. 161

B (Diabetic patients)

TAS ( μmol /l )

MDA ( μmol /l )

Cys ( μmol /l )

Cys Gl y ( μmol /l )

Hcy ( μmol /l )

GSH ( μmol /l ) Age( year s ) ― ― 2. 54( 0. 62) ― 0. 134( 0. 036) ―

Sex ― ― ― ― −2. 13( 0. 68) ―

BMI( kg/m ) ― 0. 015( 0. 006) ― ― ― ―

Ser um gl ucos e

( mmol /l ) 0. 0093( 0. 0036) ― ― ― ― ―

GGT act i vi t y( U/l ) 0. 178( 0. 053) ― 60. 6( 21. 8) ― ― 4. 61( 1. 31) I nt er cept 1. 28 0. 876 56. 4 ― 5. 60 12. 66

R 0. 175 0. 077 0. 243 ― 0. 224 0. 134

Regr es s i on coef f i ci ent( s t andar d er r or ) . Log- t r ans f or med val ues .

NS not s i gni f i cant , p ≦0. 05, p ≦0. 01, p ≦0. 001.

TAS,Tot al ant i oxi dant s t at us ;MDA,Mal onal dehyde;GSH,Gl ut at hi one.

( i . e . ,Cys ,Cys Gl y,Hc y and GSH)s t at us and t ype 2 di abe t e s . Ac c or di ng t o a pr e vi ous s t udy ( Chi c o e t al .1 9 9 8 ) ,di abe t i c pat i e nt s had hi ghe r Hc y l e ve l s t han c ont r ol s ubj e c t s . Mor e ove r , t he obs e r ve d s e x- r e l at e d di f f e r e nc e( Hc y c onc e n- t r at i ons l owe r i n f e mal e t han mal e pat i e nt s and c ont r ol s )i mpl i e s t hat Hc y me t abol i s m may not di f f e r be t we e n di abe t i c pat i e nt s and c ont r ol s ubj e c t s . Das e t al .( 1 9 9 9 ) s ugge s t e d t hat NI DDM may s t r ongl y pr omot e oxi dat i ve s t r e s s and e nhanc e CVD i n pat i e nt s who have c oe xi s - t e nt hype r homoc ys t e i ne mi a. De s pi t e no s i gni f i - c ant var i at i on of Cys and Cys Gl y c onc e nt r at i ons was obs e r ve d i n our s t udy,t he i nc r e as e of oxi dat i ve s t r e s s i s c onf i r me d,f or wome n,i n di abe t i c pat i e nt s wi t h l owe r GSH t han i n c on- t r ol s( Sundar am e t al .1 9 9 6 )and wi t h a GGT ac t i vi t y r e l at e d t o oxi dat i ve s t r e s s i n t ype 2 di abe t e s( Pe r r y e t al .1 9 9 8 ) . The r e l at i ons hi p be t we e n GGT ac t i vi t i e s and i t s pr o- oxi dant e f f e c t has be e n de mons t r at e d i n vi t r o( Dr oz dz e t al .1 9 9 8 ;Enoi u e t al .2 0 0 0 )and i n c l i ni c al s t udi e s by t he i nc r e as e i n l i pi d pe r oxi dat i on( Pompe l l a

1 9 9 7 ;Pe r r y e t al .1 9 9 8 ) . Our f i ndi ngs we r e i n agr e e me nt wi t h Sundar am e t al .( 1 9 9 6 )s t r e s s i ng t o an i nc r e as e i n l i pi d pe r oxi dat i on i n pat i e nt s wi t h t ype 2 di abe t e s me l l i t us . Us i ng t he f e r r ous - oxi dat i on of xyl e nol or ange as s ay t o de t e r mi ne l i pi d hydr ope r oxi de s ,Nour ooz - Zade h e t al .( 1 9 9 7 )s ugge s t e d t hat c hange s i n oxi dat i ve s t r e s s we r e r e l at e d t o t he unde r l yi ng me t abol i c abnor mal i t i e s i n t ype 2 di abe t e s me l l i t us r at he r t han t o t he appe ar anc e of c ompl i c at i ons . Thi s i s s uppor t e d by t he f i ndi ng of r ai s e d LPO pr od- uc t s i n pat i e nt s wi t h i mpai r e d gl uc os e t ol e r anc e , e ve n be f or e t he ons e t of f r ank di abe t e s( Vi - j ayal i ngam e t al .1 9 9 6 ) . I n our s t udy,a r ai s e d LPO was de mons t r at e d i n f e mal e di abe t i c pat i e nt s wi t h hi ghe r MDA c onc e nt r at i ons t han i n c ont r ol s . The hi ghe r l e ve l of oxi dat i ve s t r e s s i n wome n mi ght be be c aus e of t he i r hi ghe r BMI l e ve l s . The ave r age pl as ma c on- c e nt r at i ons of MDA me as ur e d i n our he al t hy adul t s we r e s i mi l ar t o t hos e f ound by Car bon- ne au e t al .( 1 9 9 1 ) ,and hi ghe r val ue s obs e r ve d i n di abe t i c pat i e nt s we r e de s c r i be d by Langl e y e t T

3. Cor r elation coefficients between TAS, MDA

and thiol levels in (A) contr ols (n＝99) and (B) type 2 diabetic patients (n＝107) (both males and females) adjusted for age and sex A (Contr ols)

TAS MDA Cys Cys Gl y Hcy

MDA −0. 012 ― ― ― ―

Cys −0. 185 0. 034 ― ― ― Cys Gl y 0. 241 0. 143 0. 350 ― ― Hcy 0. 152 −0. 143 0. 332 0. 272 ― GSH 0. 085 −0. 156 0. 027 0. 157 0. 298

B (Diabetic patients)

TAS MDA Cys Cys Gl y Hcy

MDA −0. 004 ― ― ― ―

Cys 0. 108 0. 078 ― ― ― Cys Gl y 0. 258 0. 159 −0. 101 ― ― Hcy 0. 293 0. 171 0. 275 0. 431 ― GSH −0. 201 0. 092 −0. 266 0. 531 0. 291

p ≦0. 10,p ≦0. 05,p ≦0. 01,p ≦0. 001

B. S.Duman et al .

152

al .( 1 9 9 3 )i n i l l s ubj e c t s c onc e r ne d by s uc h oxi dat i ve s t r e s s r e l at e d pat hol ogi e s . Mor e - ove r ,i n he al t hy i ndi vi dual s ,oxi dat i ve damage was r e por t e d t o be mor e e xt e ns i ve i n wome n t han i n me n i n a s t udy pe r f or me d by Bl oc k e t al .( 2 0 0 2 ) ,whe r e as I de e t al .( 2 0 0 2 )f ound e nhan- c e d oxi dat i ve s t r e s s i n me n c ompar e d t o wome n.

The TAS s houl d r e f l e c t t he abi l i t y of an i ndi vi dual t o r e s i s t oxi dat i ve s t r e s s . Low s e r um ant i oxi dat i ve ac t i vi t y i n NI DDM may be r e l at e d t o an i nc r e as e d t e nde nc y t o LPO.

Ac c or di ng t o our r e s ul t s ,t he r e was no s i gni f i - c ant di f f e r e nc e i n s e r um TAS be t we e n di abe t i c pat i e nt s and c ont r ol s ubj e c t s ,whi c h i s i n c on- t r as t t o ot he r r e por t s s howi ng a de c r e as e d ant i oxi dat i ve ac t i vi t y i n di abe t i c pat i e nt s ( Ce r i e l l o e t al .1 9 9 7 ;Maxwe l l e t al .1 9 9 7 )but i n c onc or danc e wi t h Le i none n e t al .( 1 9 9 8 ) . How- e ve r ,t he r e l e vanc e of t he Randox- TAS has be e n r e c e nt l y di s c us s e d i n c ompar i s on wi t h ot he r ant i oxi dant as s ays,i . e . ,t he oxyge n r adi - c al abs or banc e c apac i t y( ORAC)and t he f e r r i c r e duc i ng abi l i t y of pl as ma( FRAP)as s ays( Cao and Pr i or 1 9 9 8 ) :a l ow c or r e l at i on was poi nt e d out be t we e n TAS and t he t wo ot he r me t hods . Ne ve r t he l e s s ,TAS i s al s o an e as i e r appr oac h t han s pe c i f i c me t hods s uc h as hydr oxyl at i on of as pi r i n( ge ne r at i on of hydr oxyl r adi c al )al r e ady us e d i n NI DDM ( Ghi s e l l i e t al .1 9 9 2 ) . Se ve r al s t udi e s have c ons i s t e nt l y de mons t r at e d de f i - c i e nc y i n i ndi vi dual ant i oxi dant s i n NI DDM pat i e nt s( Sundar am e t al .1 9 9 6 ;Vi j ayal i ngam e t al .1 9 9 6 ;Ce r i e l l o e t al .1 9 9 7 ;Nour ooz - Zade h e t al .1 9 9 7 )wi t h r e duc e d c onc e nt r at i ons of GSH, vi t ami n C and t he ant i oxi dant e nz yme s ,i . e . , s upe r oxi de di s mut as e and c at al as e ,as we l l as a de c r e as e i n t ot al r adi c al t r appi ng ant i oxi dant par ame t e r ,s ugge s t i ng a r e duc e d t ot al ant i ox- i dant de f e nc e . Ove r al l ,t he s e s t udi e s as s ume t hat oxi dat i ve s t r e s s and i mpai r e d ant i oxi dant de f e nc e i s a f e at ur e of t ype 2 di abe t e s me l l i t us t hat i s pr e s e nt e ar l y i n t he di s e as e and may c ont r i but e t o i t s pr ogr e s s i on and e ve n t o t he de ve l opme nt of c ompl i c at i ons .

I n t he pr e s e nt s t udy,we have c onf i r me d t hat t ype 2 di abe t i c wome n pat i e nt s de ve l ope d an oxi dat i ve s t r e s s by t he de c l i ne i n ant i oxi dant de f e ns e i nc l udi ng a l owe r e d GSH ( as a maj or ant i oxi dant )l e ve l and an i nc r e as e d MDA ( as a LPO pr oduc t )c onc e nt r at i on. The s e f i ndi ngs ar e of par t i c ul ar i nt e r e s t i n vi e w of t he f ac t t hat wome n mi ght be at a gr e at e r r i s k of di abe t e s t han me n. The i nc r e as e d oxi dat i ve damage t hat we have poi nt e d out may t he r e f or e pr e di s - pos e t o t he de ve l opme nt of CVD. Pr of i l i ng t he oxi dat i ve s t r e s s par ame t e r s s t at us i n s uc h a pat hol ogy may c ont r i but e t o pr ot e c t i on f r om CVD i n t he f e mal e popul at i on.

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