Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 16 The Ability of Nutrient Antioxidants to Influence Oxidative Stress and Lower the Dose of Prednisolone in Patients with Alopecia Areata AshwaqN. Al- Jaff * , Salim A.Humadi * , Saleh.A. Wohaieb** Rece ived 9-2-2003 Accepted 14-12-2003 ABSTRACT Alope cia area ta is a co mmon dis ord er, hypothesized to b e autoimmune in etio lo gy. Cortis one ta ke n ora lly ma y s timula te new hair growth. Pre dnis one (orally a dministered s te ro id ) ha s p ro ved effe ctive for p atie nts with a lo pe cia areata , but its p otential side e ffec ts include we ight gain, metab olic ab normalities , a cne and me ns trua l prob le ms. This c linica l s tudy wa s des igne d to a ss es s the clinica l significa nce o f the nutrie nt a ntioxida nts (vita min A, vitamin E a nd vita min C) in re duc ing the d os e o f cortic oste roids (prednisolone), and a s a co nse quenc e, their side e ffec ts in pa tie nt with alope cia. The res ults of this stud y re ve al the p otential clinical s ignific anc e of the the ra py for two months with thes e antioxid ants in re duc ing the do se of prednis olone from 10 0mg to 10 mg administe re d eac h othe r da y and imp ro ving the rate of hair gro wth by attenuating free rad ic als d ama ging effect o n immune system, thereby de crea sing the immune co mplex de po sition. Ac cording to the res ults of this s tudy, the use of nutrient antioxid ants ma y ha ve an importa nt role in protecting the immune system, and d ec rea sing the d os e and s id e effe cts that re sult from the use o f high d os e of c ortico steroids . الخالصة عي الثعلبة يعتبر داء منا ه إلى الجهاز ال ي يعزى أسباب و الت حفيز .من األمراض الشائعة طريق الفم إلى ت ن يؤدي الكورتيزون المعطى ع رضى داء ن لم ة البردنزولو ت فعليا فعالي و الشعر حيث اثب ة نم علب وزن الث مع تأثيره الجانبي المؤثر والذي يتضمن زيادة ال زئي الج ت األيضية اضطر, عمليا ة,ابات في ال وب و اضطراب الدورة الشهري حب .انتشار ال مين سدة فيتا عات األك مان سريرية للعالج لمدة شهرين ب همية ال ة لتقيم األ ري ري ة الس ي تقليل جرعة ) ج ,ه ,أ( صممت هذه الدراس ف ة المعطاة ره الجانبي الناتج من الجرعة العالي ولون وبالتالي تقليل تأثي ردنز .لثعلبةللمرضى المصابين بداء االب ن من ة البردنزولو عات األكسدة في تقليل جرع مان ة ل ة السريرية العالي ة األهمي هذه الدراس ج رت نتائ وم 10ملغ إلى 100أضه ملغ بين ي عي و بالتالي تقليل منا رة على الجهاز ال ر الح ر الهدام للجذو و الشعر عن طريق إنهاء التأثي وتحسين سرعة نم ر معقدات و آخ رسب ال ت ى جرع . المناعية حاجة إل ما يؤدي إلى تقليل ال مناعي م جهاز ال حماية ال ي مانعات األكسدة ف همية دور ن أ ة تبي ك فأن هذه الدراس عا لذل وتب ورتيزونات ن الك ولون( عالية م ردنز ع العالية) الب .و بالتالي تقليل التأثير الجانبي الناتج من الجر INTRODUCTION Alo pecia area ta is a common, unpre dictab le , no n-scarring form of hair loss (1,2,3,4). This dis order a ffec ts a ll age gro ups , with a highe r prevalence in c hild re n and adolescents (4). The cause is unknown but i t is associated with a n a lte ra tion in the immuno lo gical sys tem (5,6). Current trea tment is not, a t this po int , d irec ted at the etio lo gy of alopecia area ta but rathe r at the res ulting inflamma tory infiltra te a nd (p re sumably) the growth inhib itory fa ctors p roduced by this res po nse (5,6) The use of nutrient antioxid ants in a lo pecic pa tients re vea led a s ignific ant decrease in basa l and H2 O2 induced MDA (b io marke r of oxida tive stress ) level in RBC a nd plasma , increase glutathione lev el ( major a ntioxida nt) in both RBC a nd plasma , increase to ta l p ro te in and finally increase ca ta lase activity. The se e ffec ts s ugges t the imp orta nt role of nutrie nt antio xidants in protec ting the b od y( immune system fro m the o xida tive d ama ge prod uce d b y the d isease ) a nd may influe nce the se ve rity of the disease(7). Res earch has s hown that the d isease responds to a v arie ty of i mmunomod ulating trea tme nts , that pa tie nts with alopecia a re ata may have a highe r inc id ence o f c ircula ting antibo dies a ga inst other bod y organs or tis sues , and that family me mbe rs have a higher incidence o f a utoimmune disease (3, 5, 6). *Departments o f clinical pha rmacy, College of p har mac y, university o f Baghdad, Baghda d- Iraq **Departmen t of Pharmcology and Toxico lo gy,Co lle ge of Pha rmacy, Univers ity o f Baghdad, Baghda d – Iraq . Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 17 Res ea rc h ha s shown that the disease re spo nds to a variety of i mmu nomo dulating tre atmen ts , that p atients with a lopecia areata ma y hav e a higher incidence of circ ulating a ntib odies agains t othe r bo dy orga ns or tiss ue s , and that family me mbe rs have a highe r incide nce of a utoimmune disease (3, 5, 6). Sys temic s te ro ids are reserved for use in ra pidly progress ive or exte ns ive a lo pec ia area ta (8,9,10). Systemic steroids , particularly a short co urse (4-8 wee ks ) of ta pering dose s, are ofte n us ed eithe r alone or in co mbina tion with to pica l agents. A high do se up to 100 mg pred nisolone da ily has been reco mmended . In this se tting a cne and weight ga in are co mmonly se en s ide effe cts (11). Pre dnisolone doses as low as 20 mg p er da y ma y b e associated with s ep tic necrosis of the hip or se ve re gas trointes tinal b le ed ing ( 12,13). This s tudy was de signe d to inves tigate the role of nutrient a ntio xidants (A, E, &C) in red ucing the do se of prednisolone and as a c onseq ue nce their s ide effe cts in pa tient with alopecia. SUBJECTS and METHODS 1- Subjec ts A-Study Gro up: co mpris ed o f total of 84s ub je cts, 3 0 no rmal co ntro ls (mean age 25.9 7± 8.09 years) and 54 c ase s with alope cia (me an age 25.20 ±7.05 yea rs ). Pa tie nts invo lve d in this stud y were under a de rma to lo gist sup ervision who determine d the se verity o f the d is eas e ac co rding to numbe r of the p atches they hav e, and a cco rd ing to progres sion of dise as e (2) they were non- smo kers , no n-alcoholic s and free from ap parent o ther dise as es. The d uratio n of dise ase ra nge d from (20 da y- 18 ye ars). B-Pa tie nts: Fifty-four patients a ged 10 -4 0 ye ars (26 fema le s, 2 8 ma le s) with alop ecia( with no previous trea tment) were includ ed in this study. [Twenty s eve n of them re ce iv ed co rticos teroids (100 mg prednisolone) ea ch othe r day, and the o ther twenty s eve n re ce iv e (10 mg p re niso lo ne) eac h othe r day]. Treatme nt s che dule s a lso includ ed a co mbination of antioxid ants [vita min A (500 0 I.U./day), vita min E (10 0 mg/da y) a nd vitamin C (50 0 mg/day)] giv en to b oth gro up s. The trea tment with nutrient antio xidants fo r alop ecic p atie nts inc luded in this stud y co ntinued for tw o months . C-S ample s : he pa rinize d ve nous bloo d sa mples we re c olle cted from alope cic pa tie nts as well a s fro m controls using pla stic dispo sa ble syringe s. Fresh blood s amp le we re use d fo r MDA and GSH mea surme nts. 2-Methods  Erythrocy te s Malondialde hyde (MDA) As say: Me as ureme nts o f e rythrocyte and p la sma MDA (which is a by prod uct of lip id p eroxid atio n), b ase d on the reac tio n of thio barbituric a cid (TBA) forming TBA-MDA a dd uct, we re ca rried out us ing the mo dified method of S to cks and Dormandy(14) as d es crib ed by Gilbe rt e t a l(15). The res ults we re e xp re ss ed as nmole/g Hb and µmo l/ L p la sma b as ed on the molar e xtinction co efficient of MDA is 1.56 ×10 5 M-1.CM-1.  Glutatione Assay: Erythro cyte s and p la sma GSH co ntents w ere de termined ac co rd ing to the method of Godin et al.(16 ). Kno wn amo unts o f GS H we re a ss ayed by the same me thod and use d for ca lc ulation of GSH quantities in e rythro cyte s. The statistic al significa nce o f the diffe re nce in me an wa s teste d by student t-te st. RESULTS A- Ba sa l plas ma a nd erythroc yte MDA leve ls in bo th gro ups o f patients we re s ignifica ntly highe r than tho se in controls. Tre atment with either 10 o r 1 00 mg prednis olone plus antioxida nts no rma lize d MDA lev els in bo th plasma (Ta ble 1) and e rythro cyte s (Table 2 ) as early as 1 mo nth after treatme nt. Furthermore, tota l plasma GS H c ontent was signific antly higher than co ntrols (Tab le 3 ), and tre atment of p atie nts with bo th d os es of pre dnis olone p lus a ntioxida nts slightly increas ed GS H p atie nts, but did no t no rma lize these v alue s. On the othe r ha nd, erythrocyte GSH co ntent wa s s ignifica ntly lower in patients c omp ared to c ontrols, and that trea tme nt with bo th dos es o f pred nisolone plus antioxida nts did significa ntly elev ate GS H conte nt in pa tients afte r 1 month o f trea tment, and normalized the se v alue s after 2 mo nth’s of trea tme nt (Ta ble 4). B- Clinic ally there is lowe r inc id ence of pre dnis olone s id e e ffec ts( ac ne and we ight gain) amo ng those pa tients taking pre dnis olone d os e 10 mg ea ch o ther d ay than those ta king 10 0 mg ea ch othe r da y ( Ta ble 5 and 6 ). Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 18 Ta ble (1): Effec t of the a ddit io n of n utrie nt antioxida nts (A, E, &C) to pre dniso lo ne the rapy (10& 100 mg ) on p la sma MDA le ve ls in patie nts with alopec ia a re ata. Group MDA ( mo le /L) Contro l N=3 0 Patie nts with alope cia Pre-tre atment N=2 7 Months a fter treatment 1 N=2 7 2 N=27 I- Antioxidants + 10 mg pre dniso lo ne 0.72 ±0.30 3.17±1.67* 0.87± 0 .4 5† 0.66±0.27† II- Ant ioxida nts + 100mg pre dniso lo ne 0.72 ±0.30 2.73±1.66* 0.86± 0 .4 6† 0.67±0.26† Value s are ex pre ssed as mea ns  SD. * Signif ic antly diffe re nt from co ntrol (p< 0.05). † Signif ic antly diffe re nt from pre trea tme nt value s (p<0.05 ). N Numbe r of s ubje cts Ta ble (2): Effec t of the addit ion of nutrie nt antioxida nts (A, E, &C) to pre dniso lo ne the rapy (10& 100 mg ) on e rythroc ytes MDA le ve ls in patie nts with a lo pe c ia a re ata. Group MDA (n mo le /g Hb) Contro l N=3 0 Patie nts with alope cia Pre-tre atment N=2 7 Months a fter treatment 1 N=2 7 2 N=27 I- Antioxidants + 10 mg pre dniso lo ne 5.981.04 28 .3818.60 * 6 .252 .95† 5.442.48† II- Ant ioxida nts + 100mg pre dniso lo ne 5.981.04 28 .4018.84 * 6 .273 .09† 5.462.60† Value s are ex pre ssed as mea ns  SD. * Signif ic antly diffe re nt from co ntrol (p< 0.05). † Signif ic antly diffe re nt from pre trea tme nt value s (p<0.05 ). N Numbe r of s ubje cts Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 19 Table (3): Effec t of the addition of nutrie nt antiox idants (A, E, &C) to pre dnis olone the ra py (1 0& 10 0 mg) o n plas ma gluta thio ne leve ls in patie nts with a lo pe cia areata. Group GSH ( mol/L) Contro l N=3 0 Patie nts with alope cia Pre-tre atment N=2 7 Months a fter treatment 1 N=2 7 2 N=27 I- Antioxidants + 10 mg pre dniso lo ne 0.90 ±0.20 1.21±0.35* 1 .27±0 .55* 1.54±0.77 * II- Ant ioxida nts + 1 00 mg pre dnisolone 0.90 ±0.20 1.23±0.38* 1 .27±0 .57* 1.58±0.79 * Value s are ex pre ssed as mea ns  SD. * Signif ic antly diffe re nt from co ntrol (p< 0.05). N Numbe r of s ubje cts Ta ble (4): Effec t of the addit ion of nutrie nt antioxida nts (A, E, a ndC) to pre dniso lo ne the rapy (10and 100 mg) o n e rythro cy te s glutathione le ve ls in patie nts with a lo pe c ia areata Group GSH ( mo le /gm Hb.) Contro l N=3 0 Patie nts with alo pec ia Pre -tre atmen t N=27 Months after tre atmen t 1 N=2 7 2 N=27 I- Ant io xidants + 1 0 mg pre dnis olone 6.53 ±0.83 3.95±1.32 * 5.43±1.39*† 5.98 ±1.25† II- Ant io xidants + 100 mg pre dnis olone 6.53 ±0 .83 4.06±1.39 * 5.44 ±1 .42*† 5.99 ±1.29† Value s are ex pre ssed as mea ns  SD. * Signif ic antly diffe re nt from co ntrol (p< 0.01). † Signif ic antly diffe re nt from pre trea tme nt value s (p<0.01 ). N Numbe r of s ubje cts Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 20 Ta ble (5): body we ight of c ontro l and age matche d alopecic pa tie nts .. Group Bo dy we ig ht (Kg) Control N=30 Patie nts with alo pec ia Pre -tre atmen t N=27 Months after tre atmen t 1 N=2 7 2 N=27 I- Ant io xidants + 10 mg pre dnis olone 6 9.69 ±10.79 66 .0 9±12.00 68.88±12 .27 7 1.22±12 .63 II- Ant io xidants + 100 mg pre dnis olone 6 9.69 ±10.79 66 .7 9±12.76 72.56±12 .62 8 0.88±12 .23 Value s are ex pre ssed as mea ns  SD. N Numbe r of s ubje cts Ta ble (6): se verity of ac ne appe a ra nce in c ontro l and age matche d alope cic pa tie nts . Group Prese nce of acne Control N=30 Patie nts with alo pec ia Pre -tre atmen t N=27 Months after tre atmen t 1 N=2 7 2 N=27 I- Antioxida nts + 10 mg pre dniso lo ne nega tive ne gative + + II- Ant ioxida nts + 100 mg pre dniso lo ne nega tive ne gative ++ ++++ Se ve rity of the prese nce of a cne dete rmine d by de rmalog is ts . N Numbe r of s ubje cts DISCUSION Cortic os tero ids are pa rt o f the trea tment of ma ny diso rd ers in which inflamma tion is thought to be c aused by e xcess ive or inappropriate activity of the i mmune sys tem like in Alopecia areata (17, 18, 19, 20, 21). Giv en in high d oses, co rtic os te ro id drugs red uce infla mma tio n by blocking the action of pros ta glandins re spons ible for triggering the infla mma tory respo nse (16). They also te mpo ra rily depress the i mmune sys tem b y re duc ing the ac tivity of c erta in typ es of white blood cells . The extent of hair loss and the age of the p atie nt a re used to selec t an appropria te trea tme nt for patie nts with a lopec ia a reata (3). For tho se with mo re tha n fi fty perce nt s ca lp hair loss one may c ons ider the use of s ys te mic cortic os te ro ids but the c oncern ab out lo ng-term use a nd s ide effects o f systemic corticosteroids must be taken into co ns ideration. The present study revealed the presence of end oge no us oxidative s tress in b oth groups of patients , as manifes ted by the increased MDA Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 21 levels and dec reased GS H conte nts in erythroc ytes . This oxidative stress ma y result from phagocyte s de rived free rad icals a nd the associated lip id pe ro xida tion (22). Da ta o f the present study a ls o indica te d that, despite the difference in o ra l pred niso lo ne dose (1 0 vs. 100 mg) be tween the two groups, addition of nu trie nt a ntio xida nts to pred niso lo ne therap y resulted in compa ra ble and significa nt d ecrease in MDA lev els and correction of GSH co ntent in blood , as well as simila r improve ment in the ra te o f ha ir growth with less side e ffec t (ac ne, weight ga in a nd ga stro intestina l disturba nces) re gardless the do se of p re dnisolone. Previous study in our la b showed that, witho ut antioxida nt the ra py, the effe ct of 1 00 mg prednis olone was more effe ctiv e than lower doses of prednis olone in improving ha ir growth in a lo pecic area ta patients (7). The re fo re , the a dd ition of nutrie nt antioxida nts to corticosteroids attenuate d the negativ e effects o f oxid ative stress on immune sys tem and dec reas ed the nee d fo r high dos e of cortic os te ro id ; thereb y de creased the unwanted side effec ts a ssocia ted with the prolonge d use of hig h doses . Free rad icals → Affe ct i mmune system → Pha go cytos is → Incre ase fre e ra dica l ( Imp ro bab le action) p ro duc tion Antioxida nts Prdnis olone An tioxid ants REFRENCES: 1. A.J.Pa padop oulos , R.A. Schwartz and Krys ic ka Janniger.alope cia a re ata: eme rging once pts .dermatovenerologica.200 0, vol 9, nu mbe r 3. 2. Sharma VK, Da wn G, Ku mar B. Profile of alopec ia a reata in northern Ind ia . Intern J Derma to l 1996; 35: 22 -7. 3. Sharma VK, Ku mar B, Daw n G. A clinical study o f c hild hoo d alopecia area ta in Chand igarh, Ind ia . Ped Derma to l 1996; 13: 372-7. 4. Safavi KH, Muller S A, Suman VJ , et a l. 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