Effect of Vitamin D Supplementation on C reactive Protein in Patients with Nonalcoholic Fatty Liver

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1 Effect of Vitmin D Supplementtion on C rective Protein in Ptients with Nonlcoholic Ftty Liver Mhdi Foroughi 1,2, Zhr Mghsoudi 1,2, Rez Ghisvnd 1,2,3, Bijn Irj 4, Gholmrez Askri 1,2, 1 Deprtment of Community Nutrition, School of Nutrition nd Food Sciences, Food Security Reserch Center, Isfhn University of Medicl Sciences, Isfhn, Irn, 2 Deprtment of Nutrition nd Food Sciences, Metbolic Liver Diseses Reserch Center, Isfhn University of Medicl Sciences, Isfhn, Irn, 3 Deprtment of Community Nutrition, Metbolic Liver Diseses Reserch Center, School of Nutrition nd Food Science, Isfhn University of Medicl Science, Isfhn, Irn, 4 Deprtment of Nutrition nd Food Sciences, Isfhn Endocrine nd Metbolism Reserch Center, Isfhn University of Medicl Sciences, Isfhn, Irn Correspondence to: Dr. Gholmrez Askri, Deprtment of Community Nutrition, Metbolic Liver Diseses Reserch Center, School of Nutrition nd Food Science, Isfhn University of Medicl Sciences, Isfhn, Irn. E-mil: skri@mui.c.ir Dte of Submission: Dec 31, 2013 Dte of Acceptnce: Jun 4, 2014 How to cite this rticle: Foroughi M, Mghsoudi Z, Ghisvnd R, Irj B, Askri G. Effect of Vitmin D Supplementtion on C rective Protein in Ptients with Nonlcoholic Ftty Liver Int J Prev Med 2014;5: ABSTRACT Bckground: Nonlcoholic ftty liver disese (NAFLD) is the most common chronic liver disese in the worldwide. It is ssocited with chronic disorders such s dibetes nd hert diseses. Inflmmtion is one of the bsic cuses of metbolic diseses. Severl studies hve shown tht Vitmin D cn reduce inflmmtion. The purpose of this study ws to investigte the effect of Vitmin D supplementtion on inflmmtion in ptients with NAFLD. Methods: This study involved 60 NAFLD ptients, divided eqully into two intervention nd plcebo groups. During 10 weeks, ptients in the intervention group receive Vitmin D (cpsules contining 50,000 IU vitmin D), weekly. Vitmin D levels, C rective protein (CRP), triglyceride (TG), sprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) levels were mesured t the beginning nd end of the study. Dt were nlyzed using nlysis of covrince tests. Anlyses were done using SPSS softwre (version 16) (SPSS Inc., Chicgo, USA). P < 0.05 set s significnt level. Results: Vitmin D supplementtion resulted in n increse of serum 25(OH) D concentrtions in inter group (P < 0.05) nd intr group (P < 0.05) in intervention group. At the end of the study, in the intervention group, TG nd CRP reduced significntly compre with bseline (P < 0.05). A significnt increse ws seen in clcium serum in the intervention group in comprison with bseline (P < 0.05) nd compred with the plcebo group (P < 0.05). After djusting for bseline vlues, level of ALT, AST, TG, nd CRP were reduced in the intervention group compred with the plcebo group, but this decrese ws not significnt between the two groups. Conclusions: Vitmin D supplementtion hd no effect on CRP nd other vribles in the intervention group compred with the plcebo group. Further studies with strong design nd more smple must conduct to demonstrte the effect of Vitmin D supplementtion on inflmmtion in ptients with NAFLD. Keywords: Inflmmtion, nonlcoholic ftty liver, vitmin D Originl Article 969

2 INTRODUCTION Nonlcoholic ftty liver disese (NAFLD) is one of the most common chronic liver diseses in the worldwide nd it is ssocited with chronic diseses such s dibetes nd hert disese. NAFLD encompsses wide spectrum of liver dmge. [1] This rnge is include from simple estetosis to estetoheptitis nd ultimtely cirrhosis. [2] NAFLD is ssocited with insulin resistnce nd is defined s ft ccumultion in liver, exceeding 5% of liver weight, in the bsence of ethnol intke >10 g/dy. [3,4] Prevlence of NAFLD is estimted t round 34% in the dult popultion. Two theories exist bout the development of NAFLD. The first one is bout the development of insulin resistnce tht explins the effect of insulin resistnce in NAFLD. The second theory is incresed inflmmtion which cn led to NAFLD. In study conducted in Jpn, significnt reltionship ws observed between the level of serum C rective protein (CRP) nd risk of NAFLD. [5] However, in nother study, no significnt correltion ws seen between serum CRP nd NAFLD. [3] Vitmin D is ft soluble vitmin tht exists in number of food products such s oils nd diry products. Vitmin D is trditionlly known s regultor of clcium nd phosphorus metbolism, but in recent yers significnt reltionship hs been observed between serum levels of Vitmin D nd risk of chronic diseses such s dibetes nd crdiovsculr diseses. [6] Vitmin D receptors re present in more thn 38 tissues nd its receptors ffect genes controlling oxidtive stress nd inflmmtion. [7] Moreover, mcrophges nd dendrite cells hve Vitmin D receptors. [8,9] There re severl lines of evidence demonstrte n ssocition between Vitmin D nd liver disese. [10,11] However, in severl studies, there were no ssocition between Vitmin D concentrtion nd severity of ftty liver. [12,13] The effect of Vitmin D on its receptor in these cells regultes nd blnces the inflmmtion nd oxidtive stress. [14,15] Moreover, severl studies hve confirmed the reltionship between serum Vitmin D levels nd inflmmtion. [16 18] Due to the role of inflmmtion in NAFLD, therefore probbly Vitmin D supplementtion cn reduce inflmmtion. Hence, the purpose of this study ws to investigte the effect of Vitmin D supplementtion on inflmmtion in ptients with NAFLD. 970 METHODS Prticipnts chrcteristics A rndomized double blind plcebo controlled clinicl tril ws conducted on 60 ptients (30-70 yers) with NAFLD. Smples were clculted with confidence intervl of 95% nd 80% power of the test by below formul [19] (S = 1.7, d = 0.7). 2 ( ) ( ) Z + Z S 2 1-α/ 2 1-β 1 S n = 2 ( d) This study ws conducted in Metbolic Liver Disese Reserch Center in Isfhn University of Medicl Sciences, Isfhn, Irn. The Study ws performed with the pprovl of Isfhn University of Medicl Sciences Locl Ethics Committee. Informed consent ws obtined from prticipnts. Inclusion criteri were tht NAFLD ws confirmed by ultrsound test. Norml rnge lnine minotrnsferse (ALT) nd sprtte minotrnsferse (AST) ws <31 IU/L in this study. Exclusion criteri in our study were defined s cute illnesses, chronic kidney disese, hyperprthyroidism, hypoprthyroidism, chronic hert filure, heptitis C or heptitis B, Wilson syndrome, history of chronic liver diseses, or disorders tht ffect gllbldder nd bile ducts, pregnncy or history of tking ny drugs ffecting levels of ALT including (vlproic cid, tmoxifen, 3 hydroxy 3 methylglutryl coenzyme A reductse inhibitors, metformin, ngiotensin converting enzyme 1 nd ngiotensin converting enzyme relted 1). Furthermore, subjects should not follow ny especil diet. Subjects should not intke orl Vitmin D/clcium/multivitmin supplementtion in the previous study. Prticipnts were divided into two groups using permuted block rndomiztion: () 30 ptients in Vitmin D group (cpsules contining 50,000 IU Vitmin D), (b) 30 ptient in the plcebo group. cpsules were in the sme color, odor nd tste ppernce. Intervention period followed for 10 weeks nd ptients received vitmin supplements or plcebo cpsules every week. To evlute the cceptbility of Vitmin D supplementtion, serum 25 hydroxy Vitmin D level ws mesured t the beginning nd end of the study. Dietry records were collected every 2 weeks, nd intkes

3 were determined bsed on estimted vlues in household mesurements. To obtin nutrient intkes of prticipnts on the bsis of these 5 d food diries, Nutritionist IV softwre (version 7.0; N squred Computing, Slm, OR, USA), which ws modified for Irnin food items were used. 5 dy physicl ctivity records tken (one per 2 weeks). Physicl ctivity levels estimted s metbolic equivlent of tsk hours per week (MET hr/wk). MET hr/wk for ech exercise progrm clculted (dys per week hours of exercise ech time MET equivlent of exercise) nd summed of ll MET min/wk vlues for ech ptient evluted. Anthropometric mesurements Height nd body weight were mesured while the subjects were in stnding position t bseline nd 10 th week. Body mss index ws mesured by the formul (weight (kg) /height 2 (meter) ). Biochemicl mesurements Fsting blood smples were tken t the beginning nd end of the study. 25 hydroxy Vitmin D ws ssessed by using commercil ELISA kit (Immuno Dignostic Systems). t the beginning nd the end of the study. Serum clcium ws mesured by commericl kit (Prs Azmun). Triglycerides (TGs) were mesured by enzymtic methods. AST nd ALT serum levels were mesured through enzymtic photometric method (IFCC) with sensitivity of 2 U/L nd coefficient of vrition of 14% (ALT, AST, nd lkline phosphtse (ALP) were mesured by commercilly vilble enzymtic regents (Prs Azmoon) on BT-3000 (Biotechinic) utonlyzer. CRP serum ws mesured by high sensitivity enzyme (test Prs Tehrn, Tehrn, Irn). Degree of ft ccumultion in liver Level of liver estetosis ws ssessed by using ultrsonogrphy with Esote Medicl ultrsound mchine (convex 3.5 MHz) t the beginning nd the end of the study. US performed by the sme expert rdiologist with the sme instrument in the begging nd the end study. Heptic ultrsonogrphy ws conducted by someone who is unwre of the objectives of the study. Ptients for ultrsound should be fsting for 8 h. Ultrsonogrphy is performed in the supine position. Right nd left lobes of the upper nd lower surfces re studied. Echogenicity the liver, the presence or bsence of bulky tumors cystic or solid nd clcifiction ws ssessed. Intrheptic bile ducts, portl vein nd heptic rtery were evluted. Sttisticl nlysis Normlity of studied vribles ws evluted using Kolmogorov Smirnovtest or probbilityprobbility plot. For nonnorml vribles, log trnsformtion ws used. Independent smples student s t test ws used to detect differences in generl chrcteristics nd dietry intkes between two groups. In this nlysis, Vitmin D nd plcebo tretment ws regrded s between subjects fctor. Further nlyses were conducted to investigte between group comprisons by using nlysis of covrince. Chi squre test for comprison the grdes of ftty liver between two groups of plcebo nd intervention used nd pired t test used to ssess the within group comprison of quntittive vribles. P <0.05 ws considered to be significnt level. All sttisticl nlyses conducted using Sttisticl Pckge for the Socil Sciences (SPSS), version 16 (SPSS Inc., Chicgo, USA). RESULTS The study flow chrt shows the screening, rndomiztion nd follow up of the prticipnts [Figure 1]. In this study, 29 men nd 31 women prticipted. Men ge of prticipnts ws 48.5 yers. Bseline subclinicl chrcteristics of 60 ptients re given in Tbles 1 nd 2. Dt shows tht 23.6% nd 10.9% of our subjects hve bnorml ALT nd AST levels t bseline, respectively. Complince with the tretments ws good in both groups nd no side effects were presented. On the bsis of 5 d dietry intke nd physicl ctivity records, no significnt differences were seen between the two groups [Tble 3]. Vitmin D supplementtion resulted in n increse of serum 25(OH) D concentrtions in inter group (P < 0.05) nd intr group (P < 0.05). At the end of study, in the intervention group, TG nd CRP reduced significntly compre with bseline (P < 0.05). A significnt increse ws seen in clcium serum in the intervention group in comprison with bseline (P < 0.05) nd compred with the plcebo group (P < 0.05). 971

4 Figure 1: Study digrm Tble 1: The comprison of subclinicl chrcteristics t bseline nd fter intervention of study Chrcteristic 972 Vitmin D supplementtion P vlue c BMI (kg/m 2 ) Before 31.2±4 32.4± After 30.78± ±5.78 P vlue b AST (IU/L) Before 25.88± ± After 21.48± ±17.3 P vlue b ALT (IU/L) Before 31.93± ± After 29.53± ±21.56 P vlue b Triglyceride (mg/ dl) Before ± ± After ± ±16.86 P vlue b 0.04* 0.06 CRP (mg/dl) Before 0.91± ± After 0.8± ±0.09 P vlue b 0.04* Clcium (mg/dl) Before 9.5±3 12.9± After 13±1 9.7±1 P vlue b 0.03* Vitmin D serum (ng/dl) Contd... Tble 1: Contd... Chrcteristic Vitmin D supplementtion After djusting for bseline vlues, levels of ALT, AST, TG nd CRP were reduced in the intervention group in comprison with the plcebo group, but these chnges were not significnt between two groups fter intervention. DISCUSSION P vlue c Before 49±1 47± After 117± ±0.44 P vlue b 0.001* All vlues re mens±sds, All vlues re mens±ses djusted for bseline vlues. b Obtined from independent smple t test. c Significnt levels t <0.05. ALT=Alnine mino trnsferees, AST=Asprtt mino trnsferees, CRP=C rective protein This study ws the first one tht investigted the effect of Vitmin D supplementtion on inflmmtion in ptients with NAFLD. In this study, Vitmin D supplementtion did not reduce inflmmtion in ptients with NAFLD in the intervention group compred with the control group. In ddition, Vitmin D supplementtion did not significnt effect on serum TG levels nd liver enzymes.

5 Tble 2: Adjusted chnges in metbolic vribles in NAFLD who received either Vitmin D supplements or plcebo n=30 (%) P vlue b Tble 3: Dietry intkes nd physicl ctivity of NAFLD who received either Vitmin D supplements or plcebo throughout the study Group P vlue b Vitmin D group 3 Vitmin D (ng/ml) 68±12 (138.5) Clcium (mg/dl) 4±0.4 (42) BMI (kg/m 2 ) 0.42±0.14 ( 1.3) Triglyceride (mg/dl) 7.43±34.65 ( 3.7) CRP (mg/dl) 0.11±0.27 ( 12) ALT (IU/L) 2.4±1.6 ( 7.5) AST (IU/L) 4.4±1.9 ( 17) group 1.9±2.44 ( 4.04) 3.2±1 ( 24) 0.17±0.22 ( 0.52) 2.92±22.3 ( 1.9) 0.084±0.14 (8.5) 0.8±2 ( 2.1) 2.8±2.2 ( 9.25) All vlues re mens±ses. b Obtined from ANCOVA djusted for (bseline vlue, ge, sex, BMI nd physicl ctivity). ALT=Alnine minotrnsferse, AST=Asprtte minotrnsferse, SEs=Stndrd errors, CRP=C rective protein, NAFLD=Nonlcoholic ftty liver disese, BMI=Body mss index Tble 2b: Ftty liver grdes in NAFLD who received either Vitmin D supplements or plcebo Grde ftty liver Vitmin D Group (30) P vlue Obtined from Chi squre test. NAFLD=Nonlcoholic ftty liver disese In severl studies, significnt reltionship ws seen between NAFLD nd inflmmtion. [3] Inflmmtion ws considered in the pthogenesis of NAFLD. Severl studies hve shown significnt reltionship between low serum Vitmin D nd increse inflmmtory mrkers. In study conducted by Grossmnn et l., 12 weeks of Vitmin D supplementtion cused significnt decrese in the levels of inflmmtory fctors in ptients with cystic fibrosis. [20] In nother study tht conducted by Chndler et l. on 328 Africn Americn ptients, Vitmin D supplementtion significntly decresed systemic inflmmtion in these ptients. [21] In nother Vitmin D Energy (kcl/dy) ± ± Crbohydrte (% clorie/dy) Protein (% clorie/dy) Ft (% clorie/dy) Cholesterol (mg/dy) 236± ± Dietry fiber (g/dy) 24±5 19± Vitmin D (mg/dy) 4±0.3 3± Physicl ctivity score (MET h/week) 33.2± ± All vlues re mens±sds, b Obtined from independent smples t test. SD=Stndrd devition, MET=Metbolic equivlent, NAFLD=Nonlcoholic ftty liver disese study, the effect of Vitmin D supplementtion ws investigted on inflmmtory mrkers in ptients with type 2 dibetes. In this study, Vitmin D supplementtion significntly decreses systemic inflmmtion nd CRP concentrtion in ptients with type 2 dibetes. [22] However, in severl studies, Vitmin D supplementtion hd no effect on systemic inflmmtion nd CRP. [23] Vitmin D my reduce inflmmtion through severl mechnisms. [24] There re Vitmin D receptors in >37 tissues in the body. [4] Vitmin D ffects through receptors on these orgns nd regultes pro inflmmtory nd systemic inflmmtion in the body. [7] Vitmin D receptors re locted in the nucleus of the mcrophges. Numerous mcrophges produce cytokines, prticulrly tumor necrosis fctor lph (TNF α). [25,9,10] TNF α expression depends on the effect of nucler fctor κb (NF κb), significntly. Vitmin D increses expression of the inhibitory protein NF κb. Vitmin D reduces the expression of NF κb nd through TNF α level is reduced. [26,27] In ddition, Vitmin D with binding to its receptors in monocytes cn reduce pro inflmmtory cytokines. It lso suppresses expression of NF κb gene. Thereby Vitmin D reduces the production CRP nd systemic inflmmtion. [28 30] In this study, we hd severl limittions. The first one ws use of ultrsound method for dignosis of ftty liver disese, while for ccurte dignosis of ftty liver, liver biopsy should be used. The second 973

6 limittion ws smll smple size of prticipnts, especilly in grdes one nd three. Vitmin D supplementtion hd no effect on CRP nd other vribles in the intervention group compred with the plcebo group. Further studies with strong design nd more smple must conduct to demonstrte the effect of Vitmin D supplementtion on inflmmtion in ptients with NAFLD. ACKNOWLEDGMENT This rticle ws extrcted from Msc. disserttion which ws pproved by School of Nutrition nd Food Sciences, Isfhn University of Medicl Sciences with code We thnk ll the prticipnts nd Collegue of Food Security Reserch Center nd Deprtment of Community Nutrition, School of Nutrition nd Food Science, Isfhn University of Medicl Sciences, Isfhn, Irn. REFERENCES 1. Ahmed MH, Byrne CD. Obstructive sleep pne syndrome nd ftty liver: Assocition or cusl link? World J Gstroenterol 2010;16: Zivkovic AM, Germn JB, Snyl AJ. Comprtive review of diets for the metbolic syndrome: Implictions for nonlcoholic ftty liver disese. Am J Clin Nutr 2007;86: Hukelnd JW, Dmås JK, Konopski Z, Løberg EM, Hlnd T, Goverud I, et l. Systemic inflmmtion in nonlcoholic ftty liver disese is chrcterized by elevted levels of CCL2. J Heptol 2006;44: Prdhn A. Obesity, metbolic syndrome, nd type 2 dibetes: Inflmmtory bsis of glucose metbolic disorders. Nutr Rev 2007;65:S Uchihr M, Izumi N. High sensitivity C rective protein (hs CRP): A promising biomrker for the screening of non lcoholic stetoheptitis (NASH). Nihon Rinsho 2006;64: Flores M. A role of vitmin D in low intensity chronic inflmmtion nd insulin resistnce in type 2 dibetes mellitus? Nutr Res Rev 2005;18: Jmes WP 22 nd Mrbou Symposium: The chnging fces of vitmin D. Nutr Rev 2008;66: Melmed ML, Michos ED, Post W, Astor B. 25 hydroxyvitmin D levels nd the risk of mortlity in the generl popultion. Arch Intern Med 2008;168: Plomer X, González Clemente JM, Blnco Vc F, Muricio D. Role of vitmin D in the pthogenesis of type 2 dibetes mellitus. Dibetes Obes Metb 2008;10: Rhee EJ, Kim MK, Prk SE, Prk CY, Bek KH, Lee WY, et l. High serum vitmin D levels reduce the risk for nonlcoholic ftty liver disese in helthy men independent of metbolic syndrome. Endocr J 2013;60: Jblonski KL, Jovnovich A, Holmen J, Trgher G, McFnn K, Kendrick J, et l. Low 25 hydroxyvitmin D level is independently ssocited with non lcoholic ftty liver disese. Nutr Metb Crdiovsc Dis 2013;23: Li L, Zhng L, Pn S, Wu X, Yin X. No significnt ssocition between vitmin D nd nonlcoholic ftty liver disese in Chinese popultion. Dig Dis Sci 2013;58: Ktz K, Brr PC, Prekh N, Liu YH, Weitzmn M. Suspected nonlcoholic Ftty liver disese is not ssocited with vitmin d sttus in dolescents fter djustment for obesity. J Obes 2010;2010: Mrtini LA, Wood RJ. Vitmin D sttus nd the metbolic syndrome. Nutr Rev 2006;64: Hussler MR, Hussler CA, Brtik L, Whitfield GK, Hsieh JC, Slter S, et l. Vitmin D receptor: Moleculr signling nd ctions of nutritionl lignds in disese prevention. Nutr Rev 2008;66:S Sigmundsdottir H, Pn J, Debes GF, Alt C, Hbtezion A, Soler D, et l. DCs metbolize sunlight induced vitmin D3 to progrm T cell ttrction to the epiderml chemokine CCL27. Nt Immunol 2007;8: Fritsche J, Mondl K, Ehrnsperger A, Andreesen R, Kreutz M. Regultion of 25 hydroxyvitmin D3 1 lph hydroxylse nd production of 1 lph, 25 dihydroxyvitmin D3 by humn dendritic cells. Blood 2003;102: vn Etten E, Mthieu C. Immunoregultion by 1,25 dihydroxyvitmin D3: Bsic concepts. J Steroid Biochem Mol Biol 2005;97: Brchett I, Angelico F, Del Ben M, Broni MG, Pozzilli P, Morini S, et l. Strong ssocition between non lcoholic ftty liver disese (NAFLD) nd low 25(OH) vitmin D levels in n dult popultion with norml serum liver enzymes. BMC Med 2011;9: Grossmnn RE, Zughier SM, Liu S, Lyles RH, Tngprich V. Impct of vitmin D supplementtion on mrkers of inflmmtion in dults with cystic fibrosis hospitlized for pulmonry excerbtion. Eur J Clin Nutr 2012;66: Chndler PD, Scott JB, Drke BF, Ng K, Mnson JE, Rifi N, et l. Impct of vitmin D supplementtion on inflmmtory mrkers in Africn Americns: Results of four rm, rndomized, plcebo controlled tril. Cncer Prev Res (Phil) 2013;168: Pitts AG, Hrris SS, Strk PC, Dwson Hughes B. The effects of clcium nd vitmin D supplementtion on blood glucose nd mrkers of inflmmtion in nondibetic dults. Dibetes Cre 2007;30:980 6.

7 23. Mitri J, Murru MD, Pitts AG. Vitmin D nd type 2 dibetes: A systemtic review. Eur J Clin Nutr 2011;65: von Hurst PR, Stonehouse W, Cod J. Vitmin D supplementtion reduces insulin resistnce in South Asin women living in New Zelnd who re insulin resistnt nd vitmin D deficient rndomised, plcebo controlled tril. Br J Nutr 2010;103: Hotmisligil GS, Shrgill NS, Spiegelmn BM. Adipose expression of tumor necrosis fctor lph: Direct role in obesity linked insulin resistnce. Science 1993;259: Chiu KC, Chu A, Go VL, Sd MF. Hypovitminosis D is ssocited with insulin resistnce nd bet cell dysfunction. Am J Clin Nutr 2004;79: Jblonski KL, Chonchol M, Pierce GL, Wlker AE, Sels DR. 25 Hydroxyvitmin D deficiency is ssocited with inflmmtion linked vsculr endothelil dysfunction in middle ged nd older dults. Hypertension 2011;57: Peterson CA, Heffernn ME. Serum tumor necrosis fctor lph concentrtions re negtively correlted with serum 25(OH) D concentrtions in helthy women. J Inflmm (Lond) 2008;5: Ngo DT, Sverdlov AL, McNeil JJ, Horowitz JD. Does vitmin D modulte symmetric dimethylrginine nd C rective protein concentrtions? Am J Med 2010;123: Dobnig H, Pilz S, Schrngl H, Renner W, Seelhorst U, Wellnitz B, et l. Independent ssocition of low serum 25 hydroxyvitmin D nd 1,25 dihydroxyvitmin d levels with ll cuse nd crdiovsculr mortlity. Arch Intern Med 2008;168: Source of Support: Nil, Conflict of Interest: None declred. Author Help: Reference checking fcility The mnuscript system ( llows the uthors to check nd verify the ccurcy nd style of references. The tool checks the references with PubMed s per predefined style. Authors re encourged to use this fcility, before submitting rticles to the journl. The style s well s bibliogrphic elements should be 100% ccurte, to help get the references verified from the system. Even single spelling error or ddition of issue number/month of publiction will led to n error when verifying the reference. Exmple of correct style Shehn P, O lery G, Lee G, Fitzgibbon J. Cystic cervicl metstses: Incidence nd dignosis using fine needle spirtion biopsy. Otolryngol Hed Neck Surg 2002;127: Only the references from journls indexed in PubMed will be checked. Enter ech reference in new line, without seril number. Add up to mximum of 15 references t time. If the reference is correct for its bibliogrphic elements nd punctutions, it will be shown s CORRECT nd link to the correct rticle in PubMed will be given. If ny of the bibliogrphic elements re missing, incorrect or extr (such s issue number), it will be shown s INCORRECT nd link to possible rticles in PubMed will be given. 975