Alterations in serum levels of selected markers of oxidative imbalance in adult celiac patients with extraintestinal manifestations: a pilot study

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1 ORIGINAL ARTICLE Altertions in serum levels of selected mrkers of oxidtive imblnce in dult celic ptients with extrintestinl mnifesttions: pilot study Agnieszk Piątek Guziewicz 1, Pweł Zgrodzki 2,3, Pweł Pśko 3, Mirosłw Krośnik 3, Agt Ptk Belowsk 4, Mgdlen Przybylsk Feluś 5, Tomsz Mch 5, Młgorzt Zwolińsk Wcisło 5 1 Deprtment of Gstroenterology nd Heptology, The University Hospitl, Krków, Polnd 2 The Henryk Niewodniczński Institute of Nucler Physics, Krków, Polnd 3 Deprtment of Food Chemistry nd Nutrition, Jgiellonin University Medicl College, Krków, Polnd 4 Deprtment of Physiology, Jgiellonin University Medicl College, Krków, Polnd 5 Unit for Clinicl Dietetics, Deprtment of Gstroenterology, Heptology nd Infectious Diseses, Jgiellonin University Medicl College, Krków, Polnd KEY WORDS celic disese, glutthione peroxidse, nitric oxide, oxidtive stress, vitmin E Correspondence to: Młgorzt Zwolińsk Wcisło, MD, PhD, Zkłd Dietetyki Klinicznej, Ktedr Gstroenterologii, Heptologii i Chorób Zkźnych, Uniwersytet Jgielloński, Collegium Medicum, ul. Śnideckich 5, Krków, Polnd, phone: , e mil: mzwcislo@su.krkow.pl Received: Februry 20, Revision ccepted: My 4, Published online: My 5, Conflict of interest: none declred. Pol Arch Intern Med. 2017; 127 (7-8): doi: /pmw.4020 Copyright by Medycyn Prktyczn, Krków ABSTRACT INTRODUCTION Oxidtive stress is considered to be one of the mechnisms responsible for gluten toxicity, but its role in celic disese (CD) remins uncler. OBJECTIVES The im of the study ws to evlute oxidtive imblnce in the pthomechnism of CD by determining the concentrtions of nitric oxide (NO) nd selected ntioxidnt prmeters. PATIENTS AND METHODS The study involved 197 dult ptients: 53 ptients with untreted ctive CD, 92 celic ptients on gluten free diet (GFD), nd 52 controls. The serum levels of ntioxidnts (uric cid, bilirubin, ferritin, lbumin), celic ntibodies, NO, glutthione peroxidse 3 (GPx3), nd vitmin E were mesured. A histopthologicl study of duodenl biopsy ws performed. RESULTS Celic ptients hd higher uric cid concentrtions thn controls (P <0.001). NO levels were higher in ptients with ctive CD thn in controls (P <0.01) nd were correlted with the degree of mucosl dmge (r 2 = 0.04; P = 0.01). Vitmin E levels were decresed in celic ptients (P <0.01), nd GPx3 ctivity ws reduced in ptients with ctive CD compred with controls (P <0.001). CONCLUSIONS Oxidtive imblnce my be involved in the pthomechnism of CD in dults. GFD only prtilly reduces oxidtive stress. Serum NO levels seem to be mrker of the effectiveness of tretment. Uric cid my ct s n ntioxidnt in CD. INTRODUCTION Celic disese (CD) is common heritble chronic condition, in which the ingestion of the gluten frction of whet or the dequte proteins from rye nd brley cuses chronic inflmmtion of the smll intestine. 1 The clinicl presenttion of CD is diversified nd vries with the ge of ptients, durtion of illness, nd severity of the disese, nd extrintestinl mnifesttions my be present. 2 The clssic presenttion of CD s predomintely peditric disese is chrcterized by symptoms such s chronic dirrhe, bloting, nd growth filure. Adult celic POLISH ARCHIVES OF INTERNAL MEDICINE 2017; 127 (7-8) ptients hve presented with different clinicl fetures, including the nonclssic, subclinicl, or symptomtic form of the disese. The pthogenesis of CD is complicted nd still not fully explined. Besides genetic predisposition, the immunologicl mechnism plys the min role in the disese development. Recent studies hve indicted the direct cytotoxic effect of gluten on enterocytes. 3 It hs been ssumed tht oxidtive stress (OS), becuse of n increse in the concentrtion of rective oxygen species (ROS) nd decrese of ntioxidnt cpcity, is

2 TABLE 1 Ptient groups Chrcteristics of the study groups (n = 53) Treted CD (n = 92) Controls (n = 52) Age, y, men (SD) 35.9 (11.7) 42.6 (15.1) 39.6 (12.7) Sex, femle/mle, n 44/9 77/15 44/8 Celic ptients on gluten free diet Abbrevitions: CD, celic disese one of the processes possibly involved in glidin toxicity. 1 Toxic oligopeptides collected in the smll intestine my led to toxic effects in geneticlly susceptible individuls. 4 However, lthough OS is considered to be one of the mechnisms responsible for gluten (glidin) toxicity, its role in ptients with CD hs not been fully explored. Oxidtive imblnce induced by glidin peptides in enterocytes leds to the ctivtion of the trnscription of proinflmmtory cytokines nd enzymes such s inducible nitric oxide synthse (inos), which in turn leds to incresed production of nitric oxide (NO) metbolites promoting OS. 5 NO tkes prt in the pthogenesis of vrious inflmmtory disorders, such s Crohn disese, ulcertive colitis, nd in certin studies, higher NO levels in ptients with inflmmtory bowel disese hve been shown. 6,7 Some studies hve reported tht rective nitrogen species lso tke prt in the pthogenesis of CD. The constitutive enzyme inos is expressed in humn enterocytes, with incresed ctivity in ptients with untreted CD nd with prtil correction in celic ptients on gluten free diet (GFD). 8,9 It is possible tht incresed concentrtion of fsting plsm NO is consequence of incresed inos expression in the smll intestine. Excessive production of NO my led to incresing mucosl permebility due to dmge to gut brrier function. 10 In norml conditions, the hrmful effects of ROS re opposed by the ntioxidnt defense system consisting of ntioxidnt enzymes (glutthione peroxidse [GPx], glutthione reductse [GR], superoxide dismutse [SOD], nd ctlse), nonenzymtic ntioxidnts (such s glutthione [GSH], lbumin, bilirubin, ceruloplsmin, nd uric cid [UA]), s well s nutritionl ntioxidnts (crotenoids nd vitmins A, C, nd E). 11 The reduced ntioxidnt defenses my mke the inflmed mucos more sensitive to oxidtive tissue dmge nd my disrupt the recovery nd integrity of the mucos. Using thiobrbituric cid rective substnces s mrker of OS, Odetti et l 12 showed tht redox equilibrium is impired in ptients with CD. They lso observed decresed serum α tocopherol levels in group with silent CD in comprison with controls. Erlier studies lso showed tht the ctivity of SOD is mrkedly incresed in peditric ptients with CD, while the ctivity of GPx is significntly decresed. 13 Interestingly, studies concerning the ctivity of GPx, s most studies on the role of OS in CD, hve been conducted so fr only in children with clssic clinicl symptoms of mlbsorption syndrome nd villous trophy. Studies on oxidtive imblnce in dult celic ptients with extrintestinl mnifesttions re sprse. To the best of our knowledge, there is lck of reserch on nturlly occurring nonenzymtic ntioxidnts nd their role in CD. Therefore, we imed to evlute the involvement of OS in the pthomechnism of CD nd to monitor ntioxidnt defense in dult celic ptients with extrintestinl mnifesttions. For tht purpose, we exmined the levels of fsting plsm nitrte s mrker of endogenous NO production nd monitored individul components of ntioxidnt cpcity: GPx3 ctivity nd serum levels of UA, ferritin, lbumin, bilirubin, nd vitmins D nd E. PATIENTS AND METHODS The study included 197 outptients nd inptients of the Deprtment of Gstroenterology nd Heptology of the University Hospitl in Krków, Polnd (TABLE 1). The first group comprised 53 ptients with ctive CD (newly dignosed ptients s well s ptients with CD not dhering to GFD, with positive celic ntibody titers). The second group included 92 ptients with treted CD who were on GFD for t lest 2 yers (men [SD] disese durtion, 10.4 [8.1] yers). The third group included 52 ptients with functionl disorders of the gstrointestinl trct, without bnormlities on upper gstrointestinl endoscopy or on serologicl nd histologicl exmintions, who served s controls. The dignosis of CD ws bsed on clinicl symptoms, positive celic ntibody titers (ntitissue trnsglutminse ntibodies [TGA] nd/or ntiendomysil ntibodies [EmA]) nd the chrcteristic histologic fetures of smll intestinl biopsies. Celic ptients showed nonclssic signs nd symptoms including extrintestinl mnifesttions such s nemi, iron deficiency without gstrointestinl symptoms, chronic bdominl pin without typicl mlbsorption syndrome, osteoporosis, osteopeni, s well s symptomtic disese. Ptients with Duhring disese, dibetes, inflmmtory bowel disese, current infectious disese, history of cncer, chronic heptobiliry disese, chronic renl impirment, lcohol buse, or those receiving chronic nonsteroidl nti inflmmtory drugs, ntioxidnt supplements, orl contrceptives, or immunosuppressive nd immunostimultory drugs were excluded from the study. All ptients were nonsmokers. All prticipnts underwent upper gstrointestinl endoscopy, nd t lest 4 duodenl specimens were obtined for microscopic exmintion. The degree of intestinl mucosl dmge ws then clssified in ccordnce with the Mrsh clssifiction. 14 On the dy of gstroscopy, blood ws collected by venipuncture to ssess serum levels of TGA nd/or EmA, NO, nd ntioxidnts: vitmins D nd E, GPx3, UA, lbumin, ferritin, nd bilirubin. The TGA titer ws evluted using commercil enzyme linked immunosorbent ssy (ELISA) kit ORIGINAL ARTICLE Selected mrkers of oxidtive imblnce in dult celic ptients 533

3 (Aesku Dignostics GmbH, Wendelsheim, Germny). A concentrtion higher thn 15 U/ml ws considered positive. The EmA titer ws ssessed with the immunofluorescence method. A titer higher thn 1:10 ws regrded s positive. All study prticipnts provided written informed consent to prticipte in the study. The study protocol ws pproved by the Ethicl Committee of Jgiellonin University Medicl College (No. KBET/174/B/2013) nd conducted ccording to the Declrtion of Helsinki. Blood smple collection Venous blood smples were obtined in the fsting stte. The levels of lbumin, UA, ferritin, bilirubin, nd vitmin D were evluted on the sme dy. For GPx3, NO, nd vitmin E ssys, blood smples were centrifuged t 1000 g for 15 minutes t temperture of 4 o C, nd the serum ws collected nd stored t temperture of 80 o C until further ssy. Nitric oxide, vitmin E, nd glutthione peroxidse-3 ssys The levels of NO nd vitmin E were evluted using commercilly vilble ELISA kits ccording to the mnufcturer s protocol. NO levels were determined with the Prmeter Totl Nitric Oxide nd Nitrte/Nitrite KGE001 kits (R&D Systems, Minnepolis, Minesott, United Sttes) nd vitmin E levels with Generl Vitmin E Elis Kit E0922Ge (EIAb Science, Wuhn, Chin). A spectrophotometric microplte reder (Stt Fx 2100 Awreness Technology Inc., Plm City, Florid, United Sttes) ws used to determine the opticl density t 540 nm nd 450 nm, respectively. The levels of NO nd vitmin E were clculted from stndrd curve. The nitrite concentrtion in the smple ws determined by subtrcting the endogenous nitrite concentrtion from the totl nitrite concentrtion. The plsm GPx3 level ws evluted with hydrogen peroxide s the substrte, s described previously. 15 Sttisticl nlysis For ll prmeters, descriptive sttistics were clculted. The normlity of the distribution of prmeters ws checked by the Kolmogorov Smirnov test. Comprisons between the ptient groups were performed using either the nlysis of vrince with the Tukey post hoc test for prmeters with norml distribution nd homogenous vrinces or the Kruskl Wllis test with the Dunn post hoc test for ll other prmeters. Differences with P vlue of less thn 0.05 were considered significnt. Sttisticl clcultions were done using the commercilly vilble pckges STATISTICA PL v.10 (Stt Soft, Tuls, Oklhom, United Sttes) nd Grph Pd Prism v.3.02 (GrphPd Softwre, Sn Diego, Cliforni, United Sttes), while the correltion weights were clculted using the softwre delivered by MP System Sp. z o.o. (Chrznów, Polnd). RESULTS Blood tests in controls nd celic ptients The men ferritin level ws lower in ptients with untreted ctive CD thn in controls (P <0.001). The medin ferritin level in ptients with untreted CD ws lower thn tht in treted ptients (P <0.01). Serum lbumin levels did not differ between the celic groups (men [SD], 43.0 [4.3] g/l in ptients with untreted ctive CD nd 43.9 [3.0] g/l in ptients with treted CD), while they were lower in the control group (P <0.01). Serum UA concentrtions were elevted only in celic ptients: in 4 ptients (7.5%) with ctive CD nd in 4 ptients (4.3%) on GFD. UA levels were higher in the celic groups thn in controls (P <0.001), while bilirubin levels were lower in ptients with ctive CD thn in controls (P <0.05). Reduced vitmin D levels were reported in 37 ptients (69.8%) with ctive CD, in 61 ptients (66.3%) with treted CD, nd in 18 controls (34.6%). Moderte vitmin D deficiency (10 19 ng/ml) ws reported in 24 ptients (45.3%) with untreted CD nd 21 ptients (22.8%) with treted CD; severe deficiency (<10 ng/ml) ws reported in 8 celic ptients (5.5%) nd in none of the controls. The men vitmin D level ws lower in ptients with ctive CD thn in controls or treted celic ptients (P <0.001 nd P <0.05, respectively), nd ws lower in treted celic ptients thn in controls (P <0.05). The results of biochemicl tests re presented in TABLE 2. Serum nitric oxide levels The serum NO level ws higher in ptients with ctive CD compred with controls (men [SD], 86.4 [61.4] μmol/l vs 56.8 [37.3] μmol/l, P <0.01). The men serum NO level ws lower in ptients on GFD thn in untreted celic ptients nd higher thn in controls, but the differences were not significnt (TABLE 2 nd FIGURE 1). The degree of intestinl mucosl dmge correlted with serum NO levels in celic ptients (r 2 = 0.04; P = 0.01). Serum vitmin E levels Serum vitmin E levels were lower in untreted celic ptients nd in treted celic ptients thn in controls (men [SD], 41.1 [36.8] μmol/l vs 48.1 [20.8] μmol/l nd 37.3 [32.1] μmol/l vs 48.1 [20.8] μmol/l, respectively, P <0.01). Vitmin E deficiency, defined s the levels lower thn 16.2 μmol/l, 20 ws detected in over 60% of celic ptients nd in 3.7% of controls. Optiml vitmin E levels (>30 μmol/l) required for protection ginst crdiovsculr disese nd cncer were reported in less thn 40% of celic ptients nd in more thn 96% of controls (TABLE 2 nd FIGURE 2). Serum glutthione peroxidse-3 levels We observed decresed ctivity of GPx3 in celic groups compred with controls. The difference ws significnt between ctive celic ptients nd controls (men [SD], [107.2] U/l vs [89.1] U/l, P <0.001). Treted celic ptients showed higher ctivity of GPx3 compred with untreted celic 534 POLISH ARCHIVES OF INTERNAL MEDICINE 2017; 127 (7-8)

4 TABLE 2 Results of blood tests in the study groups (only the tests with sttisticlly significnt results re presented) Vrible Controls (n = 52) Celic ptients (n = 145) Treted CD (n = 92) (n = 53) Men (SD) Medin Men (SD) Medin Men (SD) Medin Albumin, g/l 42.0 (3.8) (3.0) c (4.3) 43.0 Bilirubin, µmol/l 10.0 (3.8) (4.4) (5.3) e 7.7 Ferritin, µg/l 53.9 (24.5) (81.9) (163.2) 22.0 d,f GPx3, U/l (89.1) (90.4) (107) d NO, µmol/l 56.8 (37.3) (41.8) (61.4) c 75.8 Uric cid, µmol/l (41.8) (49.6) d (55.2) d Totl vitmin D, ng/ml 28.1 (5.5) (10.8) e (10.3) b,d 18.8 Vitmin E, µmol/l 48.1 (20.8) (32.1) c (36.8) c 30.3 Celic ptients on gluten free diet; b P <0.05 vs treted CD; c P <0.01 vs controls; d P <0.001 vs controls; e P <0.05 vs controls; f P <0.01 vs treted CD Abbrevitions: GPx3, glutthione peroxidse 3; NO, nitric oxide; others, see TABLE 1 FIGURE 1 Serum concentrtions of nitric oxide (NO) in controls, ptients on gluten free diet (treted CD), nd ptients with the ctive form of celic disese P <0.01 (compred with the control group) NO, µmol/l Control Treted CD FIGURE 2 Serum concentrtions of vitmin E in controls, ptients on gluten free diet (treted CD), nd ptients with the ctive form of celic disese P <0.01 (compred with the control group) Vitmin E, µmol/l Control Treted CD ORIGINAL ARTICLE Selected mrkers of oxidtive imblnce in dult celic ptients 535

5 FIGURE 3 Serum concentrtions of glutthione peroxidse (GPx3) in controls, ptients on gluten free diet, nd ptients with the ctive form of celic disese P <0.01 (compred with the control group) GPx3, U/l Control Treted CD TABLE 3 Clinicl chrcteristics of the study groups: serum levels of celic ntibodies nd the degree of intestinl mucosl dmge Controls (n = 52) Treted CD (n = 92) (n = 53) Antibody titer b 0 52 (100) 89 (96.7) 2 (3.8) (3.3) 14 (26.4) (34) (35.8) Men (SD) (0.2) 2.02 (0.9) d,e Medin Degree of intestinl mucosl dmge c Norml mucos 0 51 (96) 31 (33.7) 3 (5.7) Mrsh (3.8) 31 (33.7) 11 (20.8) Mrsh Mrsh (15.2) 11 (20.8) Mrsh 3b (15.2) 15 (28.3) Mrsh 3c (2.2) 13 (24.5) Men (SD) 0.1 (0.2) 1.5 (1.6) 3.2 (1.7) Medin c 4.0 d,e Dt re presented s number (percentge) of ptients unless indicted otherwise. Celic ptients on gluten free diet b Antibody titer: 0, negtive; 1, norml (TGA <3 ULN; EmA [+]); 2, high (3 ULN < TGA <10 ULN; EmA [++]); 3, very high (TGA >10 ULN; EmA [+++]) c The degree of intestinl mucos dmge ws clssified in ccordnce with Mrsh prmeters, nd ech stge ws given score from 0 (norml mucos) to 5 (totl villous trophy); dt re presented s number (percentge) of ptients. d e P <0.001 vs controls P <0.001 vs treted CD Abbrevitions: EmA, ntiendomysil ntibodies, TGA, ntitissue trnsglutminse ntibodies; ULN, upper limit of norml; others, see TABLE 1 ptients, but the difference ws not significnt (TABLE 2 nd FIGURE 3). Serum levels of celic ntibodies nd the degree of intestinl mucosl dmge The serum levels of celic ntibodies were negtive in controls. Significntly higher levels were observed in untreted celic ptients. In treted celic ptients, the levels of ntibodies were significntly lower thn in untreted celic ptients. The degree of intestinl dmge ws the lowest in controls, while it ws higher in ptients with treted CD nd the highest in ptients with untreted ctive CD. The differences between the groups were significnt (TABLE 3). DISCUSSION The pthogenesis of CD hs not been fully explined. Becuse of n increse of ROS nd the reduced ntioxidnt protection, inflmmtion nd OS seem to prticipte in the pthomechnisms of the disese. Most studies concerning the pthomechnism of CD involved children with clssic clinicl symptoms of mlbsorption syndrome. However, mlbsorption lone does not explin the pthophysiology nd clinicl course of numerous extrintestinl mnifesttions nd nonclssic symptoms tht predominte in dult ptients with CD. Other possible mechnisms include gluten toxicity with oxidtive imblnce nd utoimmunity. To the best of our knowledge, our study is the first to hve nlyzed the serum levels of the prmeters of oxidtive imblnce in dult celic ptients with extrintestinl mnifesttions. NO is produced by NO synthse, n enzyme found in number of cell types. NO hs different functions in the gstrointestinl trct, both physiologicl nd pthologicl, nd NO synthse hs 2 forms: constitutive nd inducible. 16 The ltter produces NO in response to pthologicl impulses such s inflmmtory process. 17 Some uthors noted incresed levels of NO in the serum 536 POLISH ARCHIVES OF INTERNAL MEDICINE 2017; 127 (7-8)

6 nd urine of children with CD, with positive correltion between the concentrtion of NO nd incresed concentrtion of inos in the smll intestine. 10,18-20 Murry et l 17 showed higher plsm NO concentrtions in dult ptients with CD thn in treted ptients with CD on GFD nd those with other upper gstrointestinl disorders. Ertekin et l 19 reported tht serum NO levels decresed fter 1 yer GFD in children with CD, nd there ws significnt correltion between the degree of intestinl mucosl dmge nd serum NO levels. Higher production of NO metbolites, nd, in consequence, nitrostive stress, promote the impirment of tight junctions in the smll intestine of CD ptients, perhps by downregulting the expression of zonul occludens These observtions re in line with the results of our study. We lso reported significnt correltion between the degree of intestinl mucosl dmge nd the serum NO level. Spencer et l 22 showed tht plsm NO levels decresed just fter the introduction of GFD in dults with CD nd correlted with intestinl chnges t dignosis but not fter 6 months of tretment with GFD. All these results my imply tht oxidtive injury induced by NO does not depend on the clinicl form but is ssocited with histologic chnges. Incresed serum levels of NO re probbly mrker of n ongoing inflmmtion in the smll intestine in untreted dult celic ptients in the sme wy s in untreted children with CD with clssic mnifesttions. The persistent significnt elevtion of serum NO levels despite dietry complince my suggest refrctory CD nd indicte the need for further study, including control endoscopy. Hence, it seems to be mrker of the effectiveness of tretment. Elevted OS with incresed serum levels of NO hs lso been observed in neurodevelopmentl conditions such s ttention deficit hyperctivity disorder nd utism spectrum disorders. 23 Although the reltionship between these disorders nd CD is not well estblished, some uthors believe tht the elevted concentrtion of NO could be useful in identifying the ptients who my derive the gretest therpeutic benefit from GFD. 23 It is interesting tht ptients on GFD in our study hd lso elevted NO serum levels despite significnt decline in ntibody levels. This my indicte tht nitrostive stress in CD ptients persists despite GFD, serologicl nd clinicl remission, nd my be responsible for persistent histopthologicl chnges. On the other hnd, it my point to the difficulty in complete elimintion of ll sources of gluten in modern diet; perhps the NO concentrtion is more sensitive mrker thn TGA in the detection of trce mounts of gluten in diet. The min ntioxidnt enzymes re SOD, GPx, nd ctlse. GPx is involved in elimintion of lipid peroxides using GSH s reducing fctor. 24 Ståhlberg et l 25 reported decresed expression of GPx in smll intestinl mucos in children with totl villous trophy. Stojiljković et l 26 showed reduction of GSH levels nd decresed GPx nd GR ctivity in the peripherl blood of celic children. In subsequent ppers, the sme uthors reported tht GPx ctivity in the smll intestine ws lso significntly lower in children with untreted nd silent CD thn in controls, nd they showed positive correltion between GPx ctivity nd both GR nd GSH concentrtions. 13,27 To the best of our knowledge, no previous reserch hs investigted serum GPx3 levels in dult celic ptients. In greement with the bove dt, our results demonstrted decresed ctivity of this ntioxidnt enzyme in the celic group when compred with controls. A significnt decrese in GPx3 expression ws observed in ptients with the ctive form of the disese, which mens tht ntioxidnt cpcity in such ptients my be reduced. In ptients on GFD, the men ctivity of GPx3 ws slightly incresed compred with the untreted group, but ws lower thn in controls. The pttern of the observed GPx3 ctivity in serum seems to be very similr to tht reported by other uthors in intestinl mucos, suggesting tht these chnges my be systemic. The ctivity of GPx depends on the vilbility of GSH nd selenium, the depletion of which ws reported in celic ptients. 13,26,27 It ppers tht reduction in GSH nd selenium levels is followed by decrese in GPx ctivity. Dietry ntioxidnts such s vitmin E help mintin oxidtive blnce in wy similr to tht of other ntioxidnts. Vitmin E ensures stbility of biologicl membrnes, thus protecting from hrmful cellulr effects of ROS, including the deleterious effects of lipid peroxidtion. 28 Numerous disorders re relted to chnges in vitmin E levels, but it is uncler whether this is the result or the cuse of the disese. There hve been numerous reports on the insufficiency of ft soluble vitmins in dult subjects with gluten enteropthy. 29,30 The plsm or serum concentrtions of α tocopherol exceeding 16.2 μmol/l re considered s sufficient; the levels rnging from 11.6 to 16.2 indicte low vitmin E levels, nd the levels of less thn 11.6 μmol/l suggest deficiency. Recently, it hs been proposed tht the dequte plsm concentrtion of α tocopherol to prevent neoplsm nd crdiovsculr disorders is more thn 30 μmol/l. 31 In contrst to Hozysz et l, 32 who reported tht in untreted ptients the levels of plsm tocopherol were significntly lower compred with those on GFD, 32 we showed significntly decresed serum concentrtions of vitmin E in both celic groups regrdless of complince with diet. Noteworthy, the GFD did not increse the level of this vitmin. Our observtions my indicte tht oxidtive imblnce persists despite the exclusion of gluten nd suggests the need for dditionl supplementtion of dietry ntioxidnts. We explored, for the first time, the serum levels of UA s nonenzymtic plsm ntioxidnt in dult ptients with CD. Hyperuricemi is included in metbolic syndrome, nd numerous ORIGINAL ARTICLE Selected mrkers of oxidtive imblnce in dult celic ptients 537

7 uthors hve reported positive correltion between the prevlence of metbolic syndrome nd incresed UA concentrtions. 33,34 In the sme wy, Do et l 34 reported high rte of metbolic syndrome mong ptients with gout. To our knowledge, there hve been no studies reporting n incresed incidence of gout in celic ptients. In our study, we excluded ptients with dibetes nd chronic renl impirment, nd none of the celic ptients hd been dignosed with gout or severe dyslipidemi. Hence, it does not seem tht the observed increse in serum UA concentrtions resulted from metbolic disorders or GFD. UA is considered mrker of oxidtive imblnce s well s n ntioxidnt with protective feture. 35,36 Elevtion of the serum UA concentrtion occurs s physiologic response to incresed OS. 37 It is possible tht high level of UA reflects the specific mechnisms for the prevention or correction of oxidtive dmge. Our results my indicte tht higher levels of UA in celic ptients compred with controls re consequence of incresed OS, nd tht UA my function s n ntioxidnt in this cse. The role of UA in disorders relted to OS remins uncler. Glntzounis et l 38 reported tht UA cts s n ntioxidnt in vivo. UA lso functions s prooxidnt by enhncing the levels of free rdicls nd inducing endothelil injury, inflmmtory process, bnormlities in NO concentrtions, nd therosclerosis. 38 More studies re required to explin the role of UA in CD nd to exmine its function s mrker of OS s well s n ntioxidnt. Similrly to α tocopherol, bilirubin is n ntioxidnt tht blocks vsculr cell dhesion molecule 1 expression in vitro. 39 Significntly lower bilirubin levels were reported in severe sthm in n Austrlin study. 40 This my suggest tht bilirubin nd ntioxidnt vitmins ffected the indequte control of inflmmtion in ptients with sthm. This observtion is consistent with our result, suggesting n ltered concentrtion of bilirubin s result of OS. However, the role of bilirubin in oxidtive imblnce in CD ptients requires further reserch. Ferritin protects ginst OS by cheltion with free iron in conditions of excessive OS. 41 Dt on OS nd the ntioxidnt defense system in ptients with sideropenic nemi re limited nd debtble Akç et l 45 showed incresed OS in peditric ptients with iron deficiency nemi nd reported its normliztion following tretment. 45 Potczek et l 46 showed tht iron deficiency is ssocited with n incresed rte of venous thromboembolism, nd one of the postulted mechnism behind tht finding ws reduced ntioxidnt defense due to iron deficiency nd reduced GPx ctivity. 47 Iron deficiency in celic ptients my hve n dditionl effect on the severity of OS; therefore, n dequte tretment of this deficiency my be importnt to enhnce ntioxidnt defenses. In our study, vitmin D deficiency ws observed in celic ptients despite the bsence of clssic clinicl mlbsorption syndrome. Inflmmtion my led to vitmin D deficiency. It is likely tht proinflmmtory cytokines, such s tumor necrosis fctor α, contribute to conversion of 25(OH)D to 1,25(OH) 2 D in the intestine, hence lowering the serum concentrtion of 25(OH)D. In ddition, 1,25(OH) 2 D is involved in the inhibition of the production of proinflmmtory cytokines by type-1 helper T cells, thus reducing inflmmtion. 48 This inverse reltionship between the ctivity of CD nd serum vitmin D levels ws reported in our current study. Choleclciferol (vitmin D 3 ) nd its ctive metbolite were found to be membrne ntioxidnts. 48 Their ntioxidnt properties re rther newly recognized nd less well studied. Vitmin D 3 probbly contributes to the stbility of biologicl membrnes nd protects them from the products of lipid oxidtion. 48 Vitmin D 3 exerts its ntioxidnt functions lso by ffecting the ntioxidnt enzymes. 49 Our study showed decresed serum ctivity of GPx3 in the study groups with reduced serum vitmin D 3 levels. Inflmmtion with overexpressed tumor necrosis fctor α my led to reducing serum vitmin D 3 levels, nd on the other hnd, reduced concentrtions of vitmin D 3 seem to be one of the cuses leding to the impirment of ntioxidnt defense. This observtion indictes tht erly dignosis of vitmin D 3 deficiency is very importnt in ptients with CD, prticulrly in those who do not comply with GFD. It seems to be importnt not only for bone metbolism but lso for effective tretment of intestinl dmge by reducing OS. This is especilly importnt becuse, ccording to lrge scle Polish study, 50 only very limited percentge of the urbn popultion (9.1%) hve dequte 25(OH)D levels, which is consistent with the Europen nd Americn reports on the vitmin D 3 sttus. The effect of the vitmin D 3 sttus on OS in ptients with CD requires further investigtion. A limittion of our study is tht we did not ssess the intestinl levels of GPx, NO, nd vitmin E to compre the pttern of concomitnt ltertions in intestinl mucos. In conclusion, oxidtive imblnce ppers to be one of the min pthomechnisms of CD by ffecting intestinl dmge, the disese course, nd perhps extrintestinl disorders. The observed ltertions in the serum concentrtions of the bove prmeters of OS nd ntioxidnts my suggest tht these chnges in oxidtive imblnce re systemic nd cn contribute to extrintestinl mnifesttions. Our results indicte tht OS persists even in treted ptients, lthough to lesser extent, nd tht GFD is only prtilly ble to improve oxidtive imblnce. The serum NO level seems to be mrker of the effectiveness of tretment, but further studies re necessry to clrify this issue nd to elucidte the potentil role of UA in CD s mrker of OS nd its potentil therpeutic role s n ntioxidnt. Considering tht OS is involved in the moleculr mechnisms of CD, the dditionl effect of such 538 POLISH ARCHIVES OF INTERNAL MEDICINE 2017; 127 (7-8)

8 ntioxidnts s vitmin E on oxidtive imblnce my prove to be n effective djuvnt therpy, besides rigorous GFD. Acknowledgments This study ws supported by grnt from the specific subsidy for holding the reserch cpcity of the Ministry of Science nd Higher Eduction (grnt no. K/ZDS/ ; to MZ W). The funding body hd no role in this study or its publiction. Contribution sttement AP G nd MZ W conceived the ide of nd designed the reserch; AP G nd MP F conducted the literture serch nd study selection; MZ W ssessed the qulity of the included studies; AP G, AP B, PP, PZ, nd MK performed the reserch; PP nd PZ nlyzed the dt; AP G nd MZ W wrote the pper; PZ nd TM revised the mnuscript for finl submission. REFERENCES 1 Ferretti G, Bcchetti T, Mscingelo S, Sturni L. Celic disese, inflmmtion nd oxidtive dmge: nutrigenetic pproch. Nutrients. 2012; 4: Esteve M, Rosinch M, Fernández Bñres F, et l. 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