Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 27 The Effects of Melatonin On The Oxidative Stress , Protein Glycation , Microalbuminuria and Lipid Profile In Type II Diabetes Mellitus Hasan M.H. Al–Mhbashy*; Saad A. Hussain* ;Nawfal A.M. Numan* and Majee d A. Saed** Received 31-12-2002 Accepted 13-6-2004 ABSTRACT P re vious s tudies ind ic ated tha t suppleme ntation with antio xidants has a protec tive e ffec ts a ga inst oxid ativ e stre ss –induce d dama ge in type 2 diabe te s. In this s tudy we e valua ted the a ntio xidant e ffec ts of me la to nin on the oxid ativ e s tre ss pa ra meters and mic roa lbuminuria in typ e 2 DM pa tients. 30 pa tients w ith type 2 DM were tre ated with 3 mg/da y melatonin for 9 0 days. Erythro cyte s and p la sma MDA a nd gluta thio ne , fa sting plas ma gluc os e, %Hb AIC, microa lb uminuria, to ta l plas ma protein and lipid p rofile w ere mea sured e ac h 30 da ys a nd c ompa re d with tho se o btaine d from 20 healthy c ontrols. A dec re as e in MDA lev els as soc iated with the elev atio n in GSH leve ls were obs erve d, c omp ared with the pre–trea tme nt lev els. F as ting p la sma glucos e, glyca te d hemoglob in a nd microalbuminuria we re significantly d ecrea se d, a ss ociate d w ith an improv eme nt in the total cho le sterol, HDL–C and LDL– C le ve ls , with res pec t to the pretre atment va lues .In co nclus ion , tre atment of type 2 DM p atients with melatonin may ha ve p rote ctiv e effe cts agains t the o xidative s tres s–ind uc ed da mage during the c ourse of type 2 DM. Ke y wo rds: Diabe te s Me llitus , Ox idativ e Stre s s, Me la to nin,Mic ro albuminuria. الخالصة  هور ماً في ظ مه وراً عب د ويل ماً به ٬ سل كري أمرًا م من داء الس ى النوع الثاني كسدي لدى مرض الجهاد التأ هرة ازدياد ا ت ظا لقد أصبح مرض عفات ال جة . مضا عال سة م را ه فقد تم في هذه الد علي ة ) 30(و مي جرع يو ري ب من داء السك وع الثاني غم 3( مريضاً بالن وم / مل من ) ي ومًا ) 90(لميالتونين ولمدة مادة ا ة . ي :وتم قياس المتغيرات التالي ز كي خضاب الدم (GSH)و (MDA)تر وزة رجة كل ود الزما الدم وز في ب كلوك مقدار ال وخاليا الدم و ما ومين (HbIAC)في البالز وااللب ي البالز ة ف وم المختلف راكيز صور الشح وت ما روتينات البالز جمالي ب وا ول ٬ ي الب كل الدقيق ف عالج و عالج 30ما ٬ قبل البدء بال من ال ماً يو مدة مًا 90ول .يو ول وتقليل ن طرح االلبومين في الب والتقليل م سدي هاد التأك ي تقليل فرط االج ونين قد ساهم ف ميالت حليل النتائج بأن استخدام ال من ت ح اتض ي سن واضـح ـف ى تح كما أدى إل زة خضاب الدم ٬ وكلو ز في الدم كو ة مقدار الجلو ة عالـي مي ات الشـح روتيـن ول والب كولسـتر ويات ال سـت م الج ها قبل البدء بالع كثافة مقارنة بمستويات واطئة ال ة . و ماـي ي ح الً ـف اع رًا ـف ن دو وني الستنتاج بان للميالـت كن ا هذه النتائج يم من خالل و كري من داء الس ي وع الثان ي لدى مرضى الن كسد الجهاد التأ رط ا عن ف ج رر النات من الض .الجسم INTRODUCTION The re is c urre ntly a gre at interes t in the pote ntia l co ntribution o f the increa se d oxid ativ e stre ss to diab etes me llitus (DM)(1, 2). Different abnormal p athwa ys that are s trictly re la ted to chronic hype rglyce mia o f DM, s uc h as non– enzymatic glyca tion (3), po lyol p athwa y (4) a nd gluc ose a utooxid atio n (5). During the progres s of DM, the re is a c ontinuous eve nts of metab olic s tre ss , tis sue dama ge a nd ce ll de ath that could lea d to b oth increa se d free radic als production , and c ompromise d to ta l plasma antioxid ant c apa city (6). Chro nic co mplic atio ns , whic h ma y oc cur , like re tinopa thy , ne phrop athy and dyslip id emia, are among the dangerous conse que nc es that us ually a cco mpa ny d ia be te s Although the mechanis ms be hind the ir p atho genes is rema in poo rly unde rs to od, re ce nt s ev eral mechanis ms, including glycation of protein, o xidation of lipo pro tein, gro wth fa ctors alteratio ns , changes in pe rme ab ility and v as cular c ha nge s, have bee n p ro pos ed to cla rify the link be tween diabe te s and thes e ab normalitie s (7). Me la to nin, N–ac etyl–5 –me thoxytryp ta mine, is a pine al sec re tory product that a ffec t rep ro duc tive functions, mo dula te s immune sys tem ac tivitie s, inhibits oxid ativ e s tres s (8), a nd regulates circ adian rhythms . Re cent s tudies have exa mined the effica cy of melatonin as an antine oplas tic a ge nt (9, 10, 11) a nd a s an antioxida nt (12, 13). * De par tme nt of Phar mac ology and Tox ic ology, Colle ge o f Ph ar ma cy, Un iver sity of Bag hda d,Bagh dad–I raq . ** Na tion al Diab etes Ce nter , Al–Ya rmoo k Tea ching Ho spital, Ba ghd ad–Ir aq. Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 28 Howe ver, de spite the wid es pread us e of melatonin, the re is minimal information on its us e a s a pharmac ologica l ap pro ac h in the trea tment of DM and its co mplic ations . There is a lso minimal information on the toxicology of high pha rma co lo gical dos es (14). Ba sed o n LD50 v alue s in animals, it ap pea rs that melatonin has an e xtre mely low ac ute to xicity(15). This stud y de termined whether trea tme nt with melatonin a ltered the clinical and bioc hemica l a bno rma litie s as so ciated with DM in patients with type II d ia betes mellitus . PATIENTS and METHODS This study was c arried o ut o n 30 p atie nts who ha ve type II DM for at le as t 5 ye ars, w ith age ra nge (40 –80 years ) at the Na tio nal Dia bete s Cente r, Al–Mustansiria University. All the se le cted pa tie nts were pre viously maintaine d on o ra l hypoglyce mic d rugs a nd diet re stric tion, but with p oor glyce mic co ntro l. The y are tre ated with 3 mg/da y of me la tonin in a ca psule dos age form, p re pa re d spe cially for this purpo se , give n orally a t be d time for thre e months. Twe nty he althy subjects, with the sa me age ra nges as tha t of p atie nts were se le cted and se rv ed as controls for c omp aris on. Fas ting b lo od sa mples w ere c olle cted from all subjec ts b y vein puncture, before starting trea tment with melatonin (as bas e line sa mples ), and the n afte r e ac h 3 0 d ays of trea tment to follow the cha nges in the stud ie d pa ra meters. All bloo d samp le s were c olle cted in he parinized tubes . Erythro cytes were se parated by c entrifugatio n at 3 000 r.p.m. for 1 0 min. at 4C, after the remov al o f the puffy coa t, the erythroc ytes were washed tw ic e with ice – co oled sa line conta ining 2.5 mM so dium azid e to inhib it c atalas e a ctivity (16). Urine sa mples we re c olle cted from all s ubjec ts at the ea rly morning before starting treatme nt and ev ery 30 da ys for the eva luation of microalbuminurea (17). Erythrocytes a nd plasma malondialdehyd e (MDA) le vels we re a nalyzed a cc ord ing to the metho d o f Stocks and Do rma ndy (197 1)(17). Erythrocytes and plas ma glutathione lev els were meas ured ac co rding to the method of God in e t al. (198 8) (18). P la sma gluco se leve ls were e valua ted using a read y ma de kit (LABKIT, Sp ain) a cco rd ing to the me thod of Barha n and Trindo er (19 72)(19), and glyca te d he moglob in (HbAIC) lev el was dete rmine d ac cording to the me thod o f Sushil (20 00) (20). Total p la sma pro tein co nc entratio ns were de te rmine d acc ording to the Reinhold (19 53) metho d (21), while hemoglo bin (Hb) lev els were e stimated ac cording to the me thod of Dra pkin a nd Aus tin (19 35) (22). Plasma lipid profile wa s eva luated through the meas ureme nt of to ta l p la sma cho le sterol acc ording to the metho d of Richmond (19 74) (23), and triglycerides lev els acc ording to the metho d of F oss ati and Principe (1 982 )(24), while Burs te in e t al. (197 0) (25) me thod was utilized for the me asure ment of high d ensity lipo pro tein–c holes terol (HDL–c ) leve ls fro m which the plas ma conce ntra tions of the lo w density lipo protein–c ho le sterol (LDL–c) le vels were c alcula te d ind irec tly us ing Burtis and Ashwood formula (19 99) (26). Statistic al ana lysis o f d ata wa s d one by two –way comp aris on o f mea n va lues , utilizing Stude nt’s t– te st, P –va lues les s than 0.05 was conside red signific ant. RESULTS Comb ination of the routine ly used meas ures for glyce mic c ontrol with 3 mg/day melatonin, produce d s ignifica nt red uction in MDA lev els in plas ma (a fter 6 0 d ays), and erythrocyte s (a fter 3 0 d ays) Ta ble (1). The le vel o f re duc tion in MDA reac he d v alues which a re c ompa ra ble, or e ven lowe r tha n that fo und in co rres ponding c ontrols afte r 90 d ays of treatme nt. Table (1): Effe cts of treatme nt with 3 mg/day me latonin on p la sma a nd e rythro cy te s MDA leve ls in ty pe 2 dia be tic patie nts. Re s ults re pre s e nt me an  s ta nda rd e rror. n = numbe r o f subje cts. * Significantly diffe re nt with re s pe c t to control ( P < 0.05 ). ** S ig nifica ntly diffe re nt with re spe ct to z e ro time ( P < 0 .0 5 ). T re atme nt pe riod n Pla sma MD A mo l/L Erythrocy te s MD A nmol/g Hb Control 2 0 1 .36  0.1 16 .9 8  0 .86 Ze ro time 3 0 1 .98  0.2* 19 .8 9  0 .98 * 30 days 3 0 1 .54  0.11 15 .6 3  1 .19 ** 60 days 3 0 1 .21  0.15** 12 .8 9  0 .8* * 90 days 3 0 0 .97  0.09** 12 .5 5  0 .62 ** Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 29 Tab le (2) showed tha t the o xidant stre ss – induced d epletion of GSH in the p la sma wa s impro ve d a fter 3 0 d ays o f tre atment with 3 mg/da y o f melatonin (58 %), a nd continuous trea tme nt led to furthe r eleva tion in p la sma GSH le vels , which nea rly ma tc he d their v alues in normal c ontrols after 9 0 da ys of trea tment. Ta ble (2): Effec ts of tre atme nt with 3 mg /day me la tonin on plas ma and e ry throcytes g luta thio ne (GSH) le ve ls in ty pe 2 dia be tic patie nts Re sults re prese nt mean  s ta nda rd e rror. n = nu mbe r of s ubjec ts . * Sig nifica ntly d iffe re nt with re spect to c ontro l ( P < 0 .0 5 ). ** Significa ntly diffe re nt with re s pe ct to ze ro time ( P < 0.05 ). Howe ver, e rythro cyte s GSH le ve ls whic h are se verely de pleted d ue to DM–ind uce d oxid ant stress , s howe d a no n–s ignific ant increa se (P > 0.05 ), e ven a fter 9 0 d ays o f trea tme nt with melatonin. As a re sult of treatme nt with 3 mg/day melatonin for 9 0 days, fa sting plas ma gluc ose (FP G) a nd glyc ated Hemoglo bin (HbAIC) leve ls were s ignifica ntly red uce d (26 % and 1 1% re spe ctiv ely) compared w ith pretrea tme nt le vels (Tab le 3 ). Ta ble (3): Effec ts o f tre atme nt with 3 mg /day me latonin o n fa sting blood gluco se (FPG) and Gly ca te d he mo globin (Hb AIC) le ve ls in type 2 diabe tic patie nts. Re sults re pre se nt me an  standard e rror. n = numbe r of s ubje c ts . * Sig nific antly diffe re nt with re spe ct to c ontro l ( P < 0.0 5 ). * * Signific antly diffe re nt with re s pe c t to z e ro time ( P < 0 .0 5 ). As ind ic ated in Tab le ( 4 ) , microalbuminurea and total pla sma protein le ve ls we re s ev erely affe cted b y DM–ind uce d oxid ant stre ss , whe re 5 2% increas e and 1 6% dec re as e in both pa ra meters were obs erved (Ta ble 4) res pec tiv ely, a nd we re s ignifica ntly different co mpa re d to c ontrols . Ta ble (4): Effec ts o f tre atme nt with 3 mg /day me latonin o n the mic ro albuminurea a nd total plasma p ro te in leve ls in ty pe 2 diabe tic patie nts. Re sults re pre se nt me an  standard e rror. n = numbe r of s ubje c ts . * S ig nifica ntly diffe re nt with re s pe ct to co ntrol ( P < 0.05 ). * * Signific antly diffe re nt with re spe ct to z e ro time ( P < 0.05 ). T re atme nt pe riod n Plas ma GS H mo l/L Erythrocyte s GSH  mol/g Hb C ontrol 20 0 .36  0 .07 8 .29  0 .1 Ze ro time 30 0 .13  0.01* 6.26  0.43* 3 0 da ys 30 0.21  0.01* * 6 .79  0.57 6 0 da ys 30 0.27  0.02* * 6 .8  0.4 2 9 0 da ys 30 0.29  0.02* * 7 .0  0.5 1 Tre atment perio d n FPG mg /d l Hb AIC % Contro l 20 94  4.6 5.4  0.28 Zero time 30 1 90  2 2* 9.46  0.6* 3 0 da ys 30 153  15 - 6 0 da ys 30 173  17 8.7  0.59 9 0 da ys 30 135  11** 7.2  0 .4** Tre ame nt pe riod n Microalbu minure a mg/L Total plasma pro te in g /dl Co ntrol 20 169  4.1 8.55  0 .3 4 Ze ro time 30 258  38 * 7.23  0.12* 30 day s 30 169  43 7.47  0 .0 9 60 day s 30 1 57  41** 7.35  0 .0 8 90 day s 30 1 40  30** 8.9  0.1** Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 30 Melatonin trea tment res ulte d in s ignific ant re ductio n in microa lb uminure a (3 9%) a fter 6 0 da ys, with further d ecrea se afte r 90 d ays (4 2%). Howe ver, total p la sma p ro te in lev els started to show significant e le vation (P < 0.05 ) after 9 0 days of trea tme nt only (Tab le 4 ). Table (5 ) clea rly demo ns trated tha t, the ab norma l lip id p rofile in DM s ta te , res pond ve ry well to melatonin tre atment, where the eleva te d tota l c ho le sterol va lues sta rted to dec re as e signific antly (20%, P < 0.05) afte r 6 0 days, rea ching a le vel which was lowe r than that obs erved in controls a fter 90 days. Triglyc erid e le vels showe d (22 %) d ec re ase afte r 90 da ys of treatme nt, but this wa s still non–significa nt c ompa re d with pre–trea tme nt value s. Table (5): Effe cts of tre atme nt with 3 mg/day me la to nin on the lip id profile in t he plas ma of ty pe 2 diabe tic pa tie nts Re s ults re pre s e nt me a n  s tandard e rror. n = numbe r o f subje cts. * Signific antly diffe re nt with re s pe c t to c ontrol ( P < 0.05 ). ** Significa ntly diffe re nt with re s pe ct to z e ro time ( P < 0.05 ). HDL– c le vels sta rted to inc re as e significantly after 30 da ys of trea tme nt with melatonin (15 %, P < 0.05 ). Maximum inc re as e in HDL–c v alue wa s ob serve d after 9 0 days of trea tment (30 %), whic h is higher than that obs erve d for c ontrols (Ta ble 5 ). Ho weve r, LDL– c leve ls starte d to dec re as e as a re spo ns e fo r melatonin treatme nt o nly afte r 60 da ys of trea tment (3 6%, P < 0 .0 5). Afte r 90 days, 4 6% de creas es in LDL–c v alues we re ob se rv ed, which is highly significant c ompa re d with p re – trea tment va lues , and eve n lowered tha n thos e obs erve d in c ontrols. DISCUSSION Me la to nin has be en sugge sted to ha ve potent antioxid ant pro pe rtie s that ma y preve nt the de velop ment of ca nce r, athe ro scle ros is, and o ther co ns equences of aging, howe ver, the se hypo thetic al e ffe cts are unprove n (27). Thus conclus iv e stud ie s re ga rding the relev ance of antioxid ant prope rties of melatonin in the prev ention of dise ase s, like d ia be te s me llitus, a nd the ir co mplic atio ns a re not wid ely ava ilab le . In this stud y, we e valua te d the pos sible antioxid ant ac tivity of melatonin in ame liorating the oxid ant stre ss s tate during DM, and the res ults shown in table (1) v ery well indica ted tha t this lo w d ose o f melatonin res ulted in s ignific ant re ductio n in MDA productio n in pla sma and erythroc ytes o f DM patients . This re sult clearly indica te d the promising antioxida nt activity, e sp ecially whe n co rrelated with the o bs erved elev atio n of the solub le antioxida nt, the glutathio ne (ta ble 2 ). Higher dos es may be req uire d to a chie ve more imp ro veme nt in the antioxid ant profile, since animal stud ie s s ho wed that, for me la tonin to sho w a potent a ntio xidant a ctiv ity in vivo , ve ry high d aily dos es may b e required (10 mg/kg/da y)(28). Res ea rc hers ha ve disco ve re d melatonin to ha ve the mo st p owe rful antioxida nt p ro pertie s, it s cav enges the mo st dama ging fre e rad ic al, the hydroxyl ra dical, five time s be tter tha n GSH, a nd is twice as effe ctiv e in dea ctiv ating the pe ro xyl radica l as vita min E (8). Montilla e t al. (1 998 ) demons trated that melatonin s how a ma rked protec tiv e effe ct aga inst o xidant stre ss resulte d fro m hyp erglyce mia a nd protein glyco sylation in exp erime ntal a nimals, two p atho genic corne rs to ne s indica tive o f diab etic comp lica tions (29). The res ults p re sented in table (3 ), indicated a s ignifica nt red uction in FPG and HbAIC leve ls a fte r 90 days of treatme nt with melatonin. The pres ente d data a nd the pre limina ry findings of o thers (30) suggest that melatonin is pro te ctive against DM–induced dama ge , eve n at p hys iologic le ve ls . Eve n if only pharmaco lo gica lly re le va nt, the findings hav e imp orta nt implications in tha t melatonin has no kno wn to xicity a nd re adily abs orbed when a dministe re d by oral ro ut. T re atmen t p eriod n TC mg/dl TG mg/dl HD L–c mg/dl LDL–c mg /dl Co ntro l 20 1 84  5.8 1 14  18 .1 3 7  0 .95 1 25  5.8 Ze ro time 30 2 11  16 .7 2 11  33 .7* 3 3  0 .83 * 1 32  14 .2 30 days 30 1 70  15 .1 2 01  29 .9 3 8  1 .9* * 1 00  11 .1 60 days 30 1 68  9.4 ** 1 83  22 .8 4 2  3 .5* * 8 4  8 .2** 90 days 30 1 47  11 ** 1 64  18 .9 4 3  3 .3* * 7 1  9 .2** Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 31 Antioxida nts no w a da ys are fo und in many clinical trials, to p la y a ro le in the amelioratio n of the mic ro vas cula r changes during diab etic ne phrop athy, a nd this was clearly de mons tra te d b y Naka mura et al. (199 9)(31), who ob se rv ed the be neficial e ffec ts of Vit. E on mic ro albuminuria in DM nep hropa thy. Tab le (4) clea rly de mons tra te d the effect of a 3 mg/da y melatonin in the red uction of microalbuminuria after 60 d ays, and this co nse quently re flec te d on the improv ement of to tal plas ma protein after 90 d ays , and thes e re sults see ms to be co mpa tible with thos e obs erve d by Ha e t al. (19 99), where trea tment with me la tonin de crea se the inc id enc e of early glome rulo pathy in d ia betic ra ts(32). The ab ility o f melatonin to inhib it the impairment of lipid profile in DM patients wa s stud ie d, a nd the res ults p re se nted in ta ble (5 ) showed that treatme nt with 3 mg/da y melatonin improv es the lipid profile through the red uction of TC and LDL–c le vels, as soc ia ted with signific ant elev ation in HDL– c le ve ls . It is po stulated tha t inhib ition of LDL– c oxid atio n by antioxid ants might protect against the de velop ment of a theros clerosis (33), and it is noted that, in bo th huma n and animal stud ie s, res istance of LDL–c to oxid atio n ha s be en a ss ociate d with de cre as ed v as cular co mplic atio ns (34), and protec tio n b y antioxid ants de crea ses s us ce ptib ility to va scula r d is turb anc es d ue to other d is ea ses like DM (35). Rec ent studies s ugges te d that HDL–c has a n antioxid ant effe ct o n LDL–c , thus Klimov et al. (199 3) rep orte d that HDL–c was p rote ctiv e against LDL– c oxida tion, and this e ffe ct wa s co nce ntra tion dep end ent(36). Therefore, the us e of me la tonin alone or with other antioxida nts, may co ntributed to the protec tio n o f LDL– c oxid atio n and re store the e ndo genously pres ent HDL– c molecule s to p la y the protec tive role effe ctively . 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