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1 Nutrition Journl BioMed Centrl Reserch Copper Chperone for Cu/Zn Superoxide Dismutse is sensitive iomrker of mild copper deficiency induced y modertely high intkes of zinc Monic Iskndr, Eleonor Swist, Keith D Trick, Bingtun Wng, Mry R L'Aé nd Jesse Bertinto* Open Access Address: Nutrition Reserch Division, Food Directorte, Helth Products nd Food Brnch, Helth Cnd, 2203C Bnting Reserch Centre, Ottw, ON, K1A 0L2, Cnd Emil: Monic Iskndr - monic_iskndr@hc-sc.gc.c; Eleonor Swist - eleonor_swist@hc-sc.gc.c; Keith D Trick - keith_trick@hc-sc.gc.c; Bingtun Wng - ingtun_wng@hc-sc.gc.c; Mry R L'Aé - mry_l'e@hc-sc.gc.c; Jesse Bertinto* - jesse_ertinto@hc-sc.gc.c * Corresponding uthor Pulished: 24 Novemer 2005 Nutrition Journl 2005, 4:35 doi: / This rticle is ville from: Received: 23 Septemer 2005 Accepted: 24 Novemer Iskndr et l; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Astrct Bckground: Smll increses in zinc (Zn) consumption ove recommended mounts hve een shown to reduce copper (Cu) sttus in experimentl nimls nd humns. Recently, we hve reported tht copper chperone for Cu/Zn superoxide dismutse (CCS) protein level is incresed in tissues of overtly Cu-deficient rts nd proposed CCS s novel iomrker of Cu sttus. Methods: Wenling mle Wistr rts were fed one of four diets norml in Cu nd contining norml (30 mg Zn/kg diet) or modertely high (60, 120 or 240 mg Zn/kg diet) mounts of Zn for 5 weeks. To egin to exmine the clinicl relevnce of CCS, we compred the sensitivity of CCS to mild Cu deficiency, induced y modertely high intkes of Zn, with conventionl indices of Cu sttus. Results: Liver nd erythrocyte CCS expression ws significntly (P < 0.05) incresed in rts fed the nd/or diet compred to rts fed norml levels of Zn (). Erythrocyte CCS expression ws the most sensitive mesure of reduced Cu sttus nd ws le to detect decrese in Cu nutriture in rts fed only twice the recommended mount of Zn. Liver, erythrocyte nd white lood cell CCS expression showed significnt (P < 0.05) inverse correltion with plsm nd liver Cu concentrtions nd ceruloplsmin ctivity. Unexpectedly, rts fed the highest level of Zn (Zn- 240) showed overll etter Cu sttus thn rts fed lower level of elevted Zn (). Improved Cu sttus in these rts correlted with incresed duodenl mrna expression of severl Zntrfficking proteins (i.e. MT-1, ZnT-1, ZnT-2 nd ZnT-4). Conclusion: Collectively, these dt show tht CCS is sensitive mesure of Zn-induced mild Cu deficiency nd demonstrte dose-dependent iphsic response for reduced Cu sttus y modertely high intkes of Zn. Pge 1 of 10 (pge numer not for cittion purposes)

2 Nutrition Journl 2005, 4:35 Bckground Zinc (Zn) nd copper (Cu) ply vitl roles s structurl nd ctlytic components of metlloenzymes nd re essentil nutrients required for growth nd development [1-3]. It is well recognized tht consuming lrge quntities of Zn for extended periods of time cuses severe Cu deficiency nd cn led to the development of nemi nd other normlities [4-8]. Of potentilly greter significnce, however, re studies showing tht even smll increses in Zn consumption ove recommended mounts depress Cu sttus in experimentl nimls [9,10] nd humns [11,12]. This strong ntgonism of excess Zn to Cu sttus ws the sis for setting the Tolerle Upper Intke Levels (ULs) for Zn [1]. At present, the mechnism y which Zn impedes Cu sorption is not known. In humns, overt Cu deficiency is uncommon nd usully only seen in specific situtions [13]. However, mild Cu deficiency from consuming diets indequte in Cu [14,15] or high in Zn [16] my e of concern. Notly, studies hve shown tht lrge proportion of young children hve Zn intkes exceeding the ULs nd much of the Zn consumed y these children is from Zn-fortified foods [16-18]. Although overt Zn toxicity hs not een reported in these children, excessive Zn intke my cuse smll ut potentilly hrmful reductions in Cu lnce, stressing the need for sensitive iomrkers le to detect mild Cu deficiency. Insertion of Cu into Cu/Zn superoxide dismutse (SOD1) requires the copper chperone for SOD1 (CCS). We hve previously shown tht CCS protein is up-regulted in liver nd erythrocytes of Cu-deficient rts [19] nd Cu regultes the degrdtion of CCS y the 26 S proteosome [20]. Another group hs since reported similr increse in CCS in tissues of Cu-deficient rts nd mice [21,22]. CCS is promising iomrker of reduced Cu sttus, s CCS protein level is more responsive to Cu deficiency thn reduction in SOD1 ctivity [19], the endpoint used to set the ULs for Zn [1]. Further, CCS is prticulrly ppeling given tht commonly used mesures of Cu sttus re ffected y vrious common conditions unrelted to Cu nutriture [14,15,23-25] nd thought to e insensitive to mrginl deficiency [14,15,26]. The ltter is underscored y experiments in rts showing tht diets mrginlly low in Cu induce normlities in hert morphology nd function, ut miniml chnges in conventionl indices of Cu sttus [27,28]. Severl reports hve estlished incresed CCS protein in tissues of overtly Cu-deficient rts nd mice [19-22], however, it is not known whether CCS is responsive to smll reductions in Cu sttus. The ojectives of this study were two-fold; (1) to exmine the effects of grded levels of modertely high dietry Zn on Cu sttus nd (2) to evlute the ility of CCS to detect smll reductions in Cu nutriture, induced y excess Zn, s first step in determining the usefulness of CCS s iomrker tht cn e used in clinicl setting. Methods Animls nd Test Diets Wenling (21-dy-old) mle Wistr rts (n = 12/diet group) (Chrles River Cnd, St. Constnt, Cnd) hd free ccess to one of 5 test diets [29] nd deminerlised drinking wter. Food consumption nd ody weight were mesured weekly. After 5 weeks of consuming the diets, rts were killed y exsnguintion while nesthetised with 3% isoflurne. Blood ws withdrwn from the dominl ort. The intestine ws extrcted nd intestinl contents removed y wshing with isotonic sline. Extrcted intestine, liver nd kidneys were frozen until nlysis. The Helth Products nd Food Brnch Animl Cre Committee of Helth Cnd pproved the experimentl protocol. Rts were treted in ccordnce with the guidelines of the Cndin Council on Animl Cre. Test diets were prepred y dding pproprite mounts of Cu (cupric cronte) nd Zn (zinc cronte) from cornstrch premixes. Other thn differences in Cu nd Zn Tle 1: Totl food consumption, ody weight nd Zn content in tissues of rts fed diets differing in Zn nd Cu 1,2 Test Diets (mg/kg diet) Totl Food Consumption (g) Body Weight week 5 (g) Liver Zn (µg/g dry weight) Kidney Zn (µg/g dry weight) Mucosl Zn (µg/g dry weight) Zn Cu ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.71 c 5.77 ± ± ± ± ± ± ± 0.80 d 5.78 ± ± ± ± ± ± 6.77 c ± 2.54 e 1.07 ± ± ± ± ± ± Vlues in column without common letter differ, P < Vlues re mens ± SEM, n = 12/diet group. Pge 2 of 10 (pge numer not for cittion purposes)

3 Nutrition Journl 2005, 4:35 Tle 2: Tissue Cu concentrtions nd SOD1 ctivity 1,2 Liver Cu (µg/g dry weight) Kidney Cu (µg/g dry weight) Mucosl Cu (µg/g dry weight) Liver SOD1 Activity U/mg protein Erythrocyte SOD1 Activity U/mg H ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.82 c ± 0.32 c 5.50 ± 0.49 c ± ± Vlues in column without common letter differ, P < Vlues re mens ± SEM, n = 12/diet group. content, composition of the diets were similr to those descried previously [19]. Zn nd Cu content in smples of ech test diet were determined y flme tomic sorption spectrophotometry (AAS) (Perkin-Elmer 5100 PC; Perkin Elmer Cetus Instruments, Norwlk, CT) s descried [30]. Blood Frctiontion Blood smples were collected in EDTA tues nd seprted into its components y centrifugtion. Plsm ws frozen in liquots. White lood cells (WBCs) were crefully removed (trying to void contmintion with erythrocytes), wshed with isotonic sline nd centrifuged. The procedure ws repeted 3 4 times until WBC preprtion with no visile contmintion with erythrocytes ws otined. Erythrocytes were wshed 3 times with sline prior to freezing. Hemtologicl Mesurements Blood smples were collected in vcutiner K 3 EDTA tues nd shipped to VITA-TECH (Mrkhm, Cnd) for nlysis of hemtologicl prmeters (See dditionl file 1: Tle 1). Western Blotting Liver protein extrcts were prepred y homogenizing in ice-cold 0.5% (v/v) Triton-X-100 uffer contining protese inhiitor cocktil (Roche, Lvl, Cnd). WBCs nd erythrocytes were lysed in Triton-X-100 uffer or GSH regent (5 mmol/l KH 2 PO 4 /K 2 HPO 4, 2 mmol/l glutthione, ph 7.0), respectively. Extrcts (40 µg totl protein or hemogloin) were seprted over 8 16% Tris-Glycine grdient gels (Invitrogen, Burlington, Cnd) under denturing nd reducing conditions. Gels for ech tissue were simultneously electrolotted onto single nitrocellulose memrne. The memrne ws locked for 1 h t room temperture (RT) in TBS-Tween [20 mmol/l Tris, 500 mmol/l NCl, 0.1% Tween 20 (v/v), ph 7.5] supplemented with 5% (wt/v) nonft dry milk (BioRd, Hercules, CA). Memrnes were proed with CCS ntiody (FL-274; Snt Cruz Biotechnology, Snt Cruz, CA) t finl concentrtion of 0.6 mg/l (for liver nd erythrocytes) or 0.2 mg/l (for WBCs) overnight t 4 C. After wshing with TBS-Tween, memrnes were incuted with n nti-rit (0.16 mg/l) HRP-conjugted secondry ntiody (BioRd) in locking solution for 2 h t RT. Antiody-ound proteins were detected y enhnced chemiluminescence nd exposure to film. Memrnes were stripped with stripping uffer (100 mmol/l 2-mercptoethnol, 2% (wt/v) SDS, 62.5 mmol/l Tris-HCl, ph 6.8) nd re-proed with n ntiody ginst Actin [(I-19)- R, Snt Cruz Biotechnology] or GAPDH (MCA-1D4, Encor Biotech, Alchu, FL) t 0.5 mg/l concentrtion or t 1:1000 dilution, respectively. Film ws scnned nd nd intensity determined using Scion Imge softwre (Scion Corportion, Frederick, MD). Bnd intensities were determined t exposures within the liner response rnge of the film. Minerl Anlyses in Tissues Cu nd Zn content in liver nd kidney smples were determined y flme AAS s descried [30]. Plsm Cu concentrtion ws mesured y grphite furnce AAS using SIMAA 6000 (Perkin-Elmer Cetus Instruments) with Zeemn ckground correction. To determine Zn nd Cu levels in the intestinl mucos, mucosl cells were gently scrped with glss cover slide from 10 cm intestinl segment strting 10 cm cudl to the pyloric sphincter. Mucosl scrpings were dried, dissolved in concentrted HNO 3 nd microwve digested using CEM (Mrs5) microwve (CEM Corportion, Mtthews, NC). Zn nd Cu in digested smples were determined y flme AAS nd grphite furnce AAS, respectively. Quntittive PCR A segment (1 cm in length; strting 8 cm cudl to the pyloric sphincter) of frozen intestine ws excised nd totl RNA isolted using QIAGEN's RNesy mini kit (QIAGEN, Mississug, Cnd). All RNA smples were treted with RNse-free DNse (QIAGEN). cdna from ech smple ws generted using n oligo (dt) primer (Amion, Austin, TX). Sequences for genes (MT-1, ZnT-1, ZnT-2 nd Pge 3 of 10 (pge numer not for cittion purposes)

4 Nutrition Journl 2005, 4:35 A Plsm Cu (µg/l) B Ceruloplsmin Activity (U/L) Sctter ctivity Figure of plots 1 rts of fed (A) diets plsm differing Cu concentrtion in Zn nd Cund (B) Cp Sctter plots of (A) plsm Cu concentrtion nd (B) Cp ctivity of rts fed diets differing in Zn nd Cu. Ech solid circle corresponds to one rt (n = 12/diet group). The horizontl line cross ll diet groups represents the nonresponse men of rts nd tht of the non-responders from other diet groups (see Methods). The dshed line within ech diet group signifies the response men for tht diet group. The non-response men (diet group ) nd the response mens (diet groups,, nd ) were compred. Diet groups without common letter differ (P < 0.05). c c d c ZnT-4) were otined from GenBnk (See dditionl file 2: Tle 2 QPCR primers). To otin the rt Zip4 sequence, primers specific for the mouse Zip4 gene (Forwrd: 5'-ACT GGA CGG CCT GTT AAA TAC GCT-3'; Reverse: 5'-TAC TCC GAC TGC TAG AGC CAC GTA-3') were used to mplify the rt Zip4 cdna from totl RNA. The sequence of the PCR product ws ligned ginst the rt genome nd the mouse Zip4 gene to confirm mplifiction of the rt Zip4 cdna. All primers used for QPCR (See dditionl file 2: Tle 2 QPCR primers) were designed using PrimerQuest (Integrted DNA Technologies, Skokie, IL). QPCR ws performed using n Mx4000 Multiplex Quntittive PCR System (Strtgene, L Joll, CA). Rections were performed in triplicte using the Brillint SYBR Green QPCR Core Regent kit (Strtgene). To ensure mplifiction of single homogeneous product, post-mplifiction dissocition curves were performed. All primer sets produced single product of expected size (See dditionl file 3: Figure 1 PCR frgments of genes nlysed y QPCR). Chnges in gene expression were determined using the stndrd curve method with β-actin s the normlizing gene. Enzyme Assys Liver nd erythrocyte SOD1 ctivity ws mesured y the cytochrome c reduction ssy [31] modified for nlysis with microplte reder. Plsm ceruloplsmin (Cp) ctivity ws mesured s descried [32]. Sttisticl Anlyses Dt were nlysed y one-wy ANOVA nd differences etween mens were determined y Fisher's lest significnt difference test. Dt re reported s men ± SEM. Liner regression nlyses were performed to exmine the ssocition etween conventionl indices of Cu sttus nd tissue CCS expression. Person's correltion coefficient (r) ws clculted to mesure the goodness of fit of the dt. To test for differences in the response mens nd the non-response men for plsm Cu nd Cp ctivity (Figure 1), dt were nlysed using n ANOVA model with diet group s the min effect. The men levels were compred using Tukey's Studentized Rnge Test. Becuse of the prtil response ehviour, these dt were fitted using mximum likelihood methods with the diet groups fitted with the sme men s the group for the nonresponders nd seprte mens for the responders, ech with common vrince nd proportion of responders. The response mens nd the non-response men were compred using the log-likelihood difference nd grouped when not significntly different. Becuse the mximum likelihood pproch does not clssify individul oservtions, cut-off vlue of 2 stndrd devitions from the men of rts ws used to chrcterise individul rts s responders nd non-responders (Tle 3). Fisher exct test ws used to determine differences in the Pge 4 of 10 (pge numer not for cittion purposes)

5 Nutrition Journl 2005, 4:35 Tle 3: Effect of dietry Zn nd Cu on plsm Cu concentrtion nd Cp ctivity of rts Animls Plsm Cu Concentrtion 1,2 Cp Activity 3,4 No. of non-responders No. of responders c c Totl Non-responders hd rnge of µg/l. 2 Responders (rnge < µg/l) were those nimls hving plsm Cu concentrtion lower thn 2SD elow the men plsm copper concentrtion of nimls in the diet group (; men ± SD = ± µg/l). 3 Non-responders hd rnge of U/L. 4 Responders (rnge < U/L) were those nimls hving Cp ctivity lower thn 2SD elow the men Cp ctivity of nimls in the diet group (; men ± SD = ± 23.9). 5 No. of responders were compred using the Fisher exct test. For ech diet group, No. of responders without common letter differ, P < numer of responders etween diet groups. Sttisticl significnce ws set t P < Dt were nlysed using Sttistic 7 softwre (SttSoft, Tuls, OK). Results Test diets were prepred y dding 30, 60, 120 nd 240 mg Zn/kg diet, which corresponds to norml mounts of Zn or 2, 4 or 8 times the AIN recommended mount [33], respectively. An dditionl group of rts ws fed Cu-deficient diet contining norml mounts of Zn nd served s positive control for Cu deficiency. Precise Zn nd Cu content of ech test diet is shown in Tle 1. All test diets contined higher mounts of Zn thn wht would e expected from mounts dded to the diet preprtions. Anlysis of individul dietry components reveled tht csein ws the mjor contriutor to the dditionl Zn in the finl diet preprtions (dt not shown). Totl food consumption of rts from ech diet group ws similr (Tle 1). Rts fed the nd diet ccumulted lrger (P < 0.05) mounts of Zn in the liver compred to control rts fed norml mounts of Zn (Zn- 30) (Tle 1). Only rts fed the lrgest mount of Zn (Zn- 240) showed significnt (P < 0.05) increse in kidney Zn content. Zn in the intestinl mucos of rts fed the or the diet ws incresed (P < 0.05). Zn content in the liver, kidney nd intestinl mucos of rts fed the Cudeficient diet () ws similr to tht of rts fed dequte Cu (), indicting tht the Cu-deficient diet did not ffect Zn ccumultion in these tissues. Rts consuming the diet contining the lrgest mount of Zn () showed significnt (P < 0.05) increse in ody weight t week 4 (dt not shown) nd 5 (Tle 1) of the study compred to rts fed norml Zn. Conversely, ody weight of rts fed the, or diet did not differ from tht of rts (Tle 1). Hemogloin (H) levels nd other hemtologicl prmeters of rts fed elevted Zn or the diet were similr to those of rts fed the diet (See dditionl file 1: Tle 1). Rts fed 4 times the norml level of Zn () showed smll decrese (P < 0.05) in liver Cu compred to rts fed norml Zn (Tle 2). Surprisingly, liver Cu did not decrese further in rts fed the highest mount of Zn (Zn- 240). In fct, Cu levels did not differ from those of rts. A similr trend ws oserved for kidney Cu content. Diets high in Zn did not lter Cu content in the intestinl mucos when compred to rts fed norml Zn. However, Cu content in the intestinl mucos ws higher (P < 0.05) in compred to rts. Cu levels in the liver, kidney nd intestinl mucos of rts fed the diet were mrkedly decresed (P < 0.05) compred to rts fed the control diet. Liver nd erythrocyte SOD1 ctivity ws unffected (P > 0.05) in rts fed high Zn (Tle 2). In contrst, rts fed the diet showed significnt (P < 0.05) decrese in SOD1 ctivity in liver nd erythrocytes. Sctter plots of plsm Cu levels clerly showed tht while some rts responded to incresed dietry Zn with reduction in plsm Cu other rts hd norml levels (Figure 1A), indicting n ll or none response. The numer of rts tht responded incresed from 2 rts when fed the Zn- 60 diet to 7 rts when fed the diet (Tle 3). Consistent with liver, kidney nd intestinl mucos Cu content indicting improved Cu sttus in rts fed the diet compred to rts fed the diet, only 3 rts fed the diet were chrcterised s responders. The response men for plsm Cu ws lso higher in compred to rts (Figure 1A). Plsm Cu of rts ws mrkedly decresed, consistent with dt from previous study from our lortory [10]. As expected, given tht most plsm Cu is ssocited with Cp, Cp ctivity prlleled very closely plsm Cu levels nd showed similr ll or none response ehviour (Figure 1B, Tle 3). A strong positive correltion (r = 0.984) ws found etween plsm Cu nd Cp ctivity (dt not shown). Pge 5 of 10 (pge numer not for cittion purposes)

6 Nutrition Journl 2005, 4:35 A Liver CCS Expression (Aritrry Units) c Liver CCS ws incresed (P < 0.05) >1.7-fold in rts fed the diet compred to rts fed norml Zn (Figure 2A). CCS expression ws lower (P < 0.05) in rts compred to rts. Erythrocyte CCS ws incresed (P < 0.05) >1.5-fold in nd rts (Figure 2B). The diet induced lrger increse in CCS content in liver nd erythrocytes (>2.5-fold). WBC CCS expression ws not significntly (P > 0.05) incresed in rts fed elevted Zn, ut ws incresed (P < 0.05) >3-fold in rts (Figure 2C). Rts fed high Zn nd chrcterised s responders for plsm Cu hd higher (P < 0.05) liver nd WBC CCS expression thn non-responders or rts (Tle 4). Erythrocyte CCS expression of responders ws higher (P < 0.05) thn tht of rts, ut not significntly (P > 0.05) different from non-responders. B C Erythrocyte CCS Expression (Aritrry Units) c Person liner correltions reveled strong inverse ssocition etween liver CCS nd liver (r = ) nd plsm (r = ) Cu nd Cp ctivity (r = ) (Tle 5). A significnt (P < 0.001) ssocition ws lso oserved when rts from the diet group were omitted from the nlyses. Liver CCS only showed moderte inverse correltion with liver nd erythrocyte SOD1 ctivity when ll rts were used in the nlyses. A significnt correltion ws not found (P > 0.05) when rts were omitted from the nlyses, consistent with the lck of significnt decrese in SOD1 ctivity in rts fed elevted Zn (Tle 2). Erythrocyte CCS expression ws lso significntly (P < 0.001) correlted with liver nd plsm Cu nd Cp ctivity (Tle 5). WBC CCS expression showed strong inverse correltion with liver (r = ) nd plsm (r = ) Cu nd Cp ctivity (r = ). WBC CCS Expression (Aritrry Units) (A) fed Figure diets Liver, 2differing (B) erythrocyte in Zn nd nd Cu(C) WBC CCS content of rts (A) Liver, (B) erythrocyte nd (C) WBC CCS content of rts fed diets differing in Zn nd Cu. CCS expression in liver is expressed reltive to tht of β-actin. CCS expression in erythrocytes nd WBCs is expressed reltive to tht of GAPDH. Brs signify the men ± SEM, n = 12 (for liver nd erythrocytes) or 7 (for WBCs)/diet group. Diet groups without common letter differ (P < 0.05). Given tht rts fed the highest mount of Zn () showed etter Cu sttus thn rts fed lower level of excess Zn (), it prompted us to evlute chnges in expression of duodenl Zn-trfficking proteins in response to these levels of Zn. Messenger RNA for MT-1 nd Zn trnsporters ZnT-1, ZnT-2 nd ZnT-4 ws significntly (P < 0.05) incresed only in rts when compred to rts (Figure 3). MT-1 (~5-fold) nd ZnT-2 (~3-fold) showed the lrgest increses followed y ZnT-1 nd ZnT-4. Zip4 expression ws unchnged in rts fed elevted Zn compred to rts fed norml Zn. Collectively, these dt indicte tht mrna expression of MT-1 nd severl ZnT trnsporters in the duodenum of rts is refrctory to smll increses in dietry Zn, ut responds to higher level of Zn intke. Discussion This is the first study to demonstrte tht CCS protein expression responds to mild Cu deficiency induced y modertely high dietry Zn, underscoring the potentil usefulness of CCS s iomrker in clinicl setting. In contrst to rts nd mice used in previous studies Pge 6 of 10 (pge numer not for cittion purposes)

7 Nutrition Journl 2005, 4:35 Tle 4: Comprison of tissue CCS expression etween rts nd responders nd non-responders for plsm Cu 1,2 Animls Non-responders 3 Responders 4 CCS Expression Liver Erythrocyte WBC 1.00 ± 0.14 (n = 12) 1.18 ± 0.11 (n = 24) 1.97 ± 0.12 (n = 12) 1.00 ± 0.18 (n = 12) 1.44 ± 0.16 (n = 24) 1.86 ± 0.20 (n = 12) 1.00 ± 0.20 (n = 7) 0.95 ± 0.11 (n = 17) 2.02 ± 0.54 (n = 4) 1 Vlues in column without common letter differ, P < Vlues re mens ± SEM. 2 n vlues re in prentheses. 3 Rts from diet groups, nd chrcterised s non-responders for plsm Cu. 4 Rts from diet groups, nd chrcterised s responders for plsm Cu. [19,21,22], rts used in this study hd norml hemtology nd did not show ny indictions of the development of nemi, indicting tht these rts were not overtly Cu deficient. Further, rts did not show impired growth, phenotype ssocited with severe Cu deficiency in growing rts [19]. Erythrocyte CCS expression ws determined to e the most sensitive index of reduced Cu nutriture nd ws le to detect decrese in Cu sttus in rts fed only twice the AIN recommended mount of Zn [33]. This sensitivity my lso prtly ccount for the sence of significnt difference in erythrocyte CCS expression etween responders nd non-responders for plsm Cu, s CCS my hve incresed in some rts fed high Zn ut chrcterise s non-responders. Notly, erythrocyte CCS expression in non-responders ws higher thn in rts nd this difference ws pproching sttisticl significnce (P = 0.09). Liver CCS lso responded to elevted dietry Zn nd ws incresed in rts fed 4 times the recommended mount. These re importnt findings, s these sme rts did not show significnt decrese in SOD1 ctivity, the iomrker of reduced Cu sttus used to set the ULs for Zn. Rts fed elevted Zn showed n ll or none response for decresed plsm Cu nd Cp ctivity, indicting tht these prmeters re not idel for dignosing smll reductions in Cu sttus nd providing n ccurte ssessment of precise Cu nutriture. These dt re consistent with previous study showing similr ll or none response for Cp ctivity in rts fed elevted levels of Zn [9]. This response likely reflects the presence of strong homeosttic mechnisms tht mintin plsm Cu levels in the norml rnge until liver stores hve een pprecily depleted. Although gold stndrd for ssessing reductions in Cu sttus is lcking, decresed liver Cu content is regrded s one of the most ccurte nd sensitive mesures in experimentl nimls, while plsm Cu nd Cp ctivity re the most common indictors used in clinicl situtions. The strong inverse correltion of liver CCS expression with these mrkers indictes tht incresed CCS expression ws specific for reduced Cu sttus nd is good predictor of chnges in common mesures of Cu deficiency. Erythrocyte CCS showed weker ssocition with these indictors tht my e explined, in prt, y the slow turnover of erythrocytes, which my not ccurtely reflect the current Cu sttus of the rts. This is in contrst to plsm nd liver Cu levels nd Cp ctivity tht likely respond more rpidly to chnges in Cu vilility. Tle 5: Correltion etween CCS content in tissues nd liver nd plsm Cu concentrtion, Cp ctivity nd liver nd erythrocyte SOD1 ctivity 1,2 Tissue CCS s 3, n = 60 s 4, n = 48 Liver Cu Plsm Cu Cp Liver SOD1 Erythrocyte SOD1 Liver Cu Plsm Cu Cp Liver SOD1 Erythrocyte SOD1 Liver Erythrocyte WBC (<0.005) (<0.05) (<0.005) (<0.01) (<0.005) (<0.05) (<0.05) (<0.005) (<0.05) (<0.005) (NSD) (NSD) (NSD) (NSD) (NSD) (<0.05) 1 Person's correltion coefficients. 2 P vlues re in prentheses; NSD = no sttisticl difference. 3 Diet groups,,,, nd. 4 Diet groups,,, nd. 5 n = n = 28. Pge 7 of 10 (pge numer not for cittion purposes)

8 Nutrition Journl 2005, 4:35 8 Reltive Expression (Aritrry Units) MT-1 ZnT-1 ZnT-2 ZnT-4 Zip4 QPCR Figure nlysis 3 of duodenl mrna expression of Zn-trfficking proteins in rts fed diets differing in Zn nd Cu QPCR nlysis of duodenl mrna expression of Zn-trfficking proteins in rts fed diets differing in Zn nd Cu. MT-1, ZnT-1, ZnT-2, ZnT-4 nd Zip4 mrna content is expressed reltive to β-actin expression. Brs represent the men ± SEM, n = 4/diet group. For ech gene, diet groups without common letter differ (P < 0.05). During our experiments we discovered tht CCS protein in WBCs is more susceptile to degrdtion thn CCS in erythrocytes, requiring tht WBCs e isolted quickly from lood smples. Smples showing degrdtion of CCS were excluded from our nlyses. Of the 7 rts per diet group nlysed, we noticed tht the mjority of rts fed high Zn were chrcterised s non-responders for decresed plsm Cu nd Cp ctivity. This over smpling of rts showing norml plsm Cu nd Cp ctivity my ccount for the sence of significnt increse in WBC CCS in rts fed elevted Zn. Nonetheless, WBC CCS expression ws incresed in rts fed the Cu-deficient diet nd ws highly correlted with conventionl mesures of Cu sttus. Further, rts chrcterised s responders for plsm Cu (which represents rts with poorer Cu sttus) hd higher WBC CCS levels thn non-responders or Zn- 30 rts, indicting tht WBC CCS responds to Cu deficiency induced y excess zinc. Becuse of the slow turnover of erythrocytes, WBC CCS my hve vlue s n indictor of erly reductions in Cu sttus. Interestingly, we found tht the sl expression level of CCS in WBCs is much higher thn in other tissues such s liver, hert nd erythrocytes in rts (dt not shown). A mjor finding of this pper is tht rts fed 8 times the recommended mount of Zn () showed overll etter Cu sttus thn rts fed 4 times the recommended mount (). This ws consistently oserved with severl iomrkers. Remrkly, Cu sttus indictors of most rts fed the diet were indistinguishle from rts fed diet norml in Zn. Importntly, liver CCS ws significntly lower in compred to rts, further demonstrting the exquisite sensitivity of CCS to chnges in Cu sttus. At this point, we cnnot offer definite explntion s to why other studies from our lortory [9,34] filed to detect this iphsic response, other thn to speculte tht differences in the iovilility of the Zn source, other dietry components or durtion of the studies my ccount for this discrepncy. Presently, the mechnism y which Zn interferes with Cu sorption is unknown, lthough it is elieved to occur t the site of the intestinl enterocyte. It ws thought tht incresed metllothionein (MT) levels sequestered Cu in the enterocyte leding to Cu deficiency [35,36]. However, MT-null mice fed elevted Zn ecome Cu deficient indicting tht MT induction is not the primry cuse of the Cu deficiency [37]. Results presented here re consistent with these dt, s rts fed the nd diet showed reduced Cu sttus in the sence of incresed duodenl MT-1 trnscript. Becuse MT cn ind Cu, however, it would e interesting to determine whether higher Pge 8 of 10 (pge numer not for cittion purposes)

9 Nutrition Journl 2005, 4:35 MT-1 expression in rts plyed ny role in incresing Cu in the intestinl mucos nd improving Cu sttus of these rts compred to rts. Experiments with Cco-2 cells hve indicted tht elevted levels of Zn ffect Cu trnsport nd reduce Cu efflux t the solterl side [38], suggesting tht Zn my lock sorption of Cu y ffecting the ctivity of Cu trnsporter. Thus, improved Cu sttus of compred to rts my reflect secondry effect of the higher Zn diet on the ctivity of Cu trnsporter. It is known tht distinct pools of Zn exist within cells nd chnges in expression of Zn-trfficking proteins cn lter the intrcellulr distriution of Zn [39]. For exmple, incresed expression of ZnT-2 likely promotes the ccumultion of Zn within vesicles, decresing cytoplsmic Zn levels [40]. In ddition, incresed MT levels result in more Zn ound to MT. Given tht trnscripts of severl Zn-trfficking proteins were incresed in the duodenum of rts fed the Zn- 240 ut not the diet, it is possile tht incresed expression of one or more of these Zn-trfficking proteins ltered the distriution of Zn within sorptive enterocytes of rts in mnner tht llevited the lock in ctivity of Cu trnsporter. Lstly, we report n interesting oservtion tht rts fed the highest level of Zn hd incresed ody weight. Although it is well recognized tht Zn deficiency cn result in impired growth [41], it is not estlished tht consuming lrger quntities of Zn, ove recommend mounts, cn enhnce growth. Given tht lower doses of Zn ove the AIN recommended mount clerly depressed Cu sttus of rts, the enefit of consuming this higher level of Zn is presently uncler. Conclusion In this study we report two mjor discoveries. Firstly, we show tht CCS is sensitive iomrker of Zn-induced mild Cu deficiency in rts. Given this finding, further studies evluting CCS s iomrker in humns re necessry, s CCS my prove to e etter mesure of reduced Cu sttus thn conventionl indictors nd impct studies imed t setting more ccurte Dietry Reference Intkes for Zn nd Cu. Secondly, we demonstrte dose-dependent iphsic response for the reduction of Cu sttus y modertely high intkes of Zn. Although it is not known whether humns respond to excess Zn in similr mnner to rts, this finding offers cution when interpreting dt from studies reporting no reduction in Cu sttus tht hve used single dose of supplementl Zn nd emphsizes the importnce of using dose-response pproch. Competing interests The uthor(s) declre tht they hve no competing interests. Authors' contriutions MI performed the QPCR nd sttisticl nlyses. MI nd ES crried out the tissue minerl nlyses nd the determintion of tissue CCS expression. ES performed the enzyme ssys. KT prepred the test diets nd prticipted in the niml phse of the study. BW isolted the lood components. ML prticipted in plnning the study nd ws involved in the interprettion of the results. JB wrote the mnuscript nd ws involved in plnning nd coordinting the study nd interpreting the dt. All uthors red nd pproved the finl mnuscript. Additionl mteril Additionl file 1 White lood cells (WBC), erythrocytes (ERCS), hemogloin (H), hemtocrit (HCT), men corpusculr volume (MCV), men corpusculr hemogloin (MCH), men corpusculr hemogloin concentrtion (MCHC), reticulocytes (RTC), pltelet count (PLT) nd red cell distriution width (RDW) of wenling mle Wistr rts fed diets with differing mounts of Zn nd Cu for 5 weeks (Tle 1). Click here for file [ Additionl file 2 QPCR primers (Tle 2). Click here for file [ Additionl file 3 PCR frgments of genes nlysed y QPCR (Figure 1). Click here for file [ Acknowledgements We thnk the Animl Resource Division of Helth Cnd for ssistnce in cre of the rts. We would lso like to cknowledge Stephen Hywrd for ssistnce with the sttisticl nlyses. Bingtun Wng is Ntionl Institute of Nutrition (NIN) Post-doctorl Fellow. This study ws funded y the Bureu of Nutritionl Sciences, Helth Cnd. This is puliction no. 607 of the Bureu of Nutritionl Sciences. References 1. Institute of Medicine: Dietry reference intkes for vitmin A, vitmin K, rsenic, oron, chromium, copper, iodine, iron, mngnese, molydenum, nickel, silicon, vndium, nd zinc. Wshington, DC Schumnn K, Clssen HG, Dieter HH, Konig J, Multhup G, Rukguer M, Summer KH, Bernhrdt J, Bieslski HK: Hohenheim consensus workshop: copper. Eur J Clin Nutr 2002, 56: McCll KA, Hung C, Fierke CA: Function nd mechnism of zinc metlloenzymes. J Nutr 2000, 130:1437S-46S. Pge 9 of 10 (pge numer not for cittion purposes)

10 Nutrition Journl 2005, 4:35 4. Botsh AS, Nsc J, Duowy R, Weinerger HL, Oliphnt M: Zincinduced copper deficiency in n infnt. Am J Dis Child 1992, 146: Pore TJ, Belmont JW, Mhoney DH Jr: Zinc-induced nemi nd neutropeni in n dolescent. J Peditr 2000, 136: Broun ER, Greist A, Tricot G, Hoffmn R: Excessive zinc ingestion. A reversile cuse of siderolstic nemi nd one mrrow depression. JAMA 1990, 264: Hein MS: Copper deficiency nemi nd nephrosis in zinc-toxicity: cse report. S D J Med 2003, 56: Willis MS, Monghn SA, Miller ML, McKenn RW, Perkins WD, Levinson BS, Bhushn V, Kroft SH: Zinc-induced copper deficiency: report of three cses initilly recognized on one mrrow exmintion. Am J Clin Pthol 2005, 123: L'Ae MR, Fischer PW: The effects of dietry zinc on the ctivity of copper-requiring metlloenzymes in the rt. J Nutr 1984, 114: L'Ae MR, Fischer PW: The effects of high dietry zinc nd copper deficiency on the ctivity of copper-requiring metlloenzymes in the growing rt. 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Am J Clin Nutr 2003, 78: Bilostosky K, Wright JD, Kennedy-Stephenson J, McDowell MA, Johnson CL: Dietry intkes of mcronutrients, micronutrients, nd other dietry constituents: United Sttes, Vitl Helth Stt , 245: Hunt CD, Mechm SL: Aluminum, oron, clcium, copper, iron, mgnesium, mngnese, molydenum, phosphorus, potssium, sodium, nd zinc: concentrtions in common western foods nd estimted dily intkes y infnts; toddlers; nd mle nd femle dolescents, dults, nd seniors in the United Sttes. J Am Diet Assoc 2001, 101: Bertinto J, Iskndr M, L'Ae MR: Copper deficiency induces the upregultion of the copper chperone for Cu/Zn superoxide dismutse in wenling mle rts. J Nutr 2003, 133: Bertinto J, L'Ae MR: Copper modultes the degrdtion of copper chperone for Cu,Zn superoxide dismutse y the 26 S proteosome. J Biol Chem 2003, 278: West EC, Prohsk JR: Cu,Zn-superoxide dismutse is lower nd copper chperone CCS is higher in erythrocytes of copper-deficient rts nd mice. 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J Inorg Biochem 1979, 11: Fischer PW, Giroux A, L'Ae MR: Effects of zinc on mucosl copper inding nd on the kinetics of copper sorption. J Nutr 1983, 113: Reeves PG: Copper metolism in metllothionein-null mice fed high zinc diet. J of Nutr Biochem 1998, 9: Reeves PG, Briske-Anderson M, Johnson L: Physiologic concentrtions of zinc ffect the kinetics of copper uptke nd trnsport in the humn intestinl cell model, Cco-2. J Nutr 1998, 128: Liuzzi JP, Cousins RJ: Mmmlin zinc trnsporters. Annu Rev Nutr 2004, 24: Plmiter RD, Cole TB, Findley SD: ZnT-2, mmmlin protein tht confers resistnce to zinc y fcilitting vesiculr sequestrtion. EMBO J 1996, 15: Brown KH, Peerson JM, Allen LH: Effect of zinc supplementtion on children's growth: met-nlysis of intervention trils. Bil Nutr Diet 1998, 54: Pulish with BioMed Centrl nd every scientist cn red your work free of chrge "BioMed Centrl will e the most significnt development for disseminting the results of iomedicl reserch in our lifetime." Sir Pul Nurse, Cncer Reserch UK Your reserch ppers will e: ville free of chrge to the entire iomedicl community peer reviewed nd pulished immeditely upon cceptnce cited in PuMed nd rchived on PuMed Centrl yours you keep the copyright BioMedcentrl Sumit your mnuscript here: Pge 10 of 10 (pge numer not for cittion purposes)