Effect of TCN2 776C G on vitamin B 12 cellular availability. end-stage renal disease patients

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1 Kidney Interntionl, Vol. 64 (2003), pp Effect of TCN2 776C G on vitmin B 12 cellulr vilbility in end-stge renl disese ptients MANUELA FÖDINGER, MARIO VEITL, SONJA SKOUPY, JADWIGA WOJCIK, CLAUDIA RÖHRER, WOLFGANG HAGEN, HEIDI PUTTINGER, ANNA-CHRISTINE HAUSER, ANDREAS VYCHYTIL, nd GERE SUNDER-PLASSMANN Institute of Medicl nd Chemicl Lbortory Dignostics; nd Division of Nephrology nd Dilysis, Deprtment of Medicine III, University of Vienn, Austri Effect of TCN2 776C G on vitmin B 12 cellulr vilbility in end-stge renl disese ptients. Bckground. Trnscoblmin II is serum protein tht trnsports vitmin B 12 from the intestine to the tissues. This complex, holo-trnscoblmin II, my reflect vitmin B 12 vilbility in the body. Conflicting dt exist with regrd to the effect of polymorphism in the gene coding for trnscoblmin II, TCN2 776C G, on trnscoblmin II levels in the generl popultion, which in turn my ffect holo-trnscoblmin II, vitmin B 12, s well s totl homocysteine (thcy) plsm levels. The effect of TCN2 776C G on vitmin B 12 cellulr vilbility in dilysis ptients is unknown. Methods. We exmined the effect of TCN2 776C G on holotrnscoblmin II, vitmin B 12, nd thcy plsm concentrtions in 120 dilysis ptients. Results. Holo-trnscoblmin II levels were norml or suprnorml in ll ptients nd showed liner ssocition with lbumin (r 0.205, P 0.025) nd with vitmin B 12 (r 0.778, P 0.001), but not with ge, cretinine, body mss index, thcy, ln-thcy, vitmin B 6, plsm folte, nd red blood cell folte concentrtion. TCN2 776C G showed no effect on holo-trnscoblmin II, vitmin B 12, nd thcy concentrtion [one-wy nlysis of vrince (ANOVA), post-hoc Scheffe test]. Multiple liner regression nlyses showed tht lbumin nd B 12 re independently ssocited with holo-trnscoblmin II, wheres TCN2 776C G nd MTHFR 677C T hd no effect. Independent predictors of ln-thcy included cretinine, red blood cell folte, nd the MTHFR 677TT genotype. There ws lso n effect of the TCN2 776CC genotype on ln-thcy levels in this multivrite nlysis, however, tht deserves cutious interprettion becuse there ws no effect of TCN2 genotypes by ANOVA nd Scheffe test [medin ln-thcy concentrtions ccording to TCN2 genotypes ( mol/l): CC, 3.22; CG, 3.30; GG, 3.23]. Conclusion. TCN2 776C G does not influence holo-trnscoblmin II or vitmin B 12 levels, nd hs no mjor effect on thcy concentrtions of end-stge renl disese ptients. Key words: hemodilysis, peritonel dilysis, TCN2, MTHFR, polymorphism, holo-trnscoblmin II, folte, vitmin B 12, totl homocysteine. Received for publiction Jnury 24, 2003 nd in revised form April 17, 2003 Accepted for publiction My 6, by the Interntionl Society of Nephrology Hyperhomocysteinemi is n estblished risk fctor for crdiovsculr disese [1]. In the cell, 5-methyltetrhydrofolte nd vitmin B 12 (coblmin) re required for remethyltion of homocysteine (Hcy) to methionine, which is medited by methionine synthse nd methionine synthse reductse. Therefore, intrcellulr vilbility of vitmin B 12 influences totl Hcy (thcy) plsm concentrtions. Three proteins re involved in vitmin B 12 trnsport in the body. In the stomch, vitmin B 12 binds to intrinsic fctor, which is essentil for the intestinl vitmin uptke. In the plsm, 20% to 30% of vitmin B 12 is bound to trnscoblmin II (TC II). The complex of vitmin B 12 bound to trnscoblmin II is termed holo-trnscoblmin II (holo-tc II). The cellulr supply with vitmin B 12 is, therefore, relted to the concentrtion of holo-tc II in the circultion. Consequently, low holo-tc II concentrtions my be n erly indictor of vitmin B 12 deficiency. The remining 70% to 80% of vitmin B 12 is bound to hptocorrin, the role of which is not completely understood, but which my ct s storge protein. Previously, TC II ws suggested to be n cute-phse protein becuse its levels my increse during certin disorders (e.g., prolifertive histiocytosis) [2]. However, more recent literture tends to tke the opposite view, s studies hve found no correltion between levels of TC II nd C-rective protein [3]. Holo-trnscoblmin II hs never been promoted s n cute-phse protein [4]. Nevertheless, ptients with myocrdil infrction showed elevted levels of holo-tc, which were ssumed to be relted to mjor tissue injury (Lrs Orning, personl communiction, 2003). A polymorphism (TCN2 776C G; P259R) in the TC II gene (TCN2) hs n effect on the concentrtion of blood TC II [5]. In helthy Cucsin individuls with the TCN2 CC genotype, TC II concentrtions were higher compred to heterozygotes or individuls homozygous for the G llele. Furthermore, heterozygotes showed higher thcy plsm concentrtions compred to both homozy- 1095

2 1096 Födinger et l: TCN2 polymorphism in ESRD gous genotypes [5], suggesting tht TCN2 776C G interferes with vitmin B 12 cellulr delivery nd Hcy metbolism. By contrst, others observed no mjor effect of TCN2 776C G on plsm thcy nd plsm vitmin B 12 concentrtion, s well s decrese of holo-tc II concentrtions, rising the question of the impct of TCN2 776C G on vitmin B 12 cellulr delivery nd thcy concentrtions [6 8]. Even lower thcy concentrtions hve been observed in individuls in the upper qurtile of vitmin B 12 levels with the TCN2 776GG genotype [9]. Hyperhomocysteinemi is present in the mjority of dilysis ptients nd is influenced by vitmin deficiencies nd genetic fctors [10, 11]. The effect of TCN2 776C G on vitmin B 12 cellulr vilbility in ptients with renl filure is unknown. We therefore exmined the impct of TCN2 776C G on thcy, vitmin B 12, nd holo-tc II concentrtions mong chronic dilysis ptients. METHODS Study design This cross-sectionl study ws conducted in 120 endstge renl disese ptients who prticipted in two studies relted to hyperhomocysteinemi in renl disese [12, 13]. None of the peritonel dilysis ptients received routine folic cid or B vitmin supplementtion. The hemodilysis ptients received multivitmin contining 0.16 mg of folic cid nd 10 mg of vitmin B 6, besides other vitmins, but not vitmin B 12 (Dreisvit-Filmtbletten, GRY-Phrm GmbH, Kirchzrten, Germny), nd which hs no effect on thcy plsm levels [14]. The ethicl review bord t the University of Vienn pproved the study. All ptients gve written informed consent ccording to the Declrtion of Helsinki. Biochemicl methods Blood ws drwn fter n overnight fst in peritonel dilysis ptients nd pre-dilysis (fter the two-dy intervl) in hemodilysis ptients, nticogulted with ethylenediminetetrcetic cid (EDTA), immeditely plced on crushed ice, nd protected from light. Blood liquots for extrction of DNA were snp frozen t 70 C. Blood liquots for determintion of red blood cell folte concentrtion were hemolyzed with 0.2% scorbic cid (1:21) for two hours nd frozen t 70 C. Red blood cell folte ws determined using rdiossy (SimulTRAC-SNB, ICN Phrmceuticls, Inc., Cost Mes, CA, USA) ccording to the instructions of the mnufcturer [red blood cell folte (nmol/l of pcked red blood cells) (hemolyste folte concentrtion) 21/(hemtocrit in deciml nottion); norml: 272 nmol/l]. The remining EDTA-nticogulted blood smples were centrifuged within 30 minutes t 2000g t 4 C (20 minutes), nd plsm liquots for determintion of thcy, folte, vitmin B 6, vitmin B 12, nd holo-tc II were snp-frozen nd stored t 70 C. Holo-trnscoblmin II plsm levels (reference rnge, 30 to 160 pmol/l) were mesured by rdioimmunossy (HoloTC RIA; Axis-Shield, Oslo, Norwy). The thcy plsm concentrtion ws determined by fluorescence polriztion immunossy (IMx nlyzer; Abbott Lbortories, Abbott Prk, IL, USA). Hyperhomocysteinemi ws defined s thcy plsm level bove 15 mol/l [15]. Folte (norml, 3.4 nmol/l) nd vitmin B 12 (norml, 118 pmol/l) plsm level were mesured with rdiossy (Simul TRAC-SNB; ICN Phrmceuticls, Inc., Cost Mes, CA, USA). Vitmin B 6 plsm level (norml, 20 nmol/l) ws determined with rdioenzymtic ssy (Bühlmnn Lbortories AG, Allschwil, Switzerlnd). Identifiction of the TCN2 776C G polymorphism ws performed using n mplifiction refrctory muttion system [5]. Identifiction of the 677C T trnsition in MTHFR ws performed s previously described [16]. Sttisticl nlyses Continuous dt re given s men stndrd devition or s medin nd qurtiles. Ctegoricl dt re given s bsolute counts nd percentges. Becuse thcy concentrtion ws positively skewed, we used logrithmic trnsformtion to normlize the distribution. We nlyzed liner ssocitions using Person s correltion. The effect of the different genotypes of TCN2 776C G ormthfr 677C T on vitmin B 12, holo-tc II, nd thcy plsm levels ws ssessed in one-wy nlysis of vrince (ANOVA) nd post-hoc Scheffe test. Multiple liner regression nlyses with ll vribles tht showed liner ssocition (P 0.1) with ln-thcy or holo-tc II including polymorphisms (genotypes coded s dummy vribles) with significnt effect in the onewy ANOVA models were estblished to ssess the independent effects on ln-thcy or holo-tc II. In second set of multiple liner regression nlyses, both polymorphisms were included in models exmining the effects on ln-thcy nd on holo-tc II plsm concentrtions. The third set of nlyses included interction terms of the genotypes with n effect on ln-thcy or holo-tc II. All nlyses were performed using SPSS (Windows version ; Mcintosh version ; SPSS, Inc., Chicgo, IL, USA). To detect n increse in thcy of 8 mol/l from 25 to 33 mol/l in homozygous versus heterozygous ptients with type 2 error of 0.05 nd power of 80%, 25 subjects were needed in ech group. RESULTS Ptients nd genotypes The chrcteristics of ll ptients re indicted in Tble 1. The llele frequency of TCN2 776G in our study

3 Födinger et l: TCN2 polymorphism in ESRD 1097 Tble 1. Ptient chrcteristics Gender femle/mle 53/67 Age yers Body mss index kg/m Durtion of dilysis tretment yers Peritonel dilysis/hemodilysis N 54/66 ws 0.4, which is comprble to the frequency of 0.42 nd 0.47 observed by others [5 9]. Among our ptients, 18.3% were homozygous for the TCN2 775G llele nd 42.5% were heterozygotes. Vitmin levels nd thcy plsm concentrtions The plsm levels of thcy, holo-tc II, vitmin B 12, foltes nd vitmin B 6, nd of other biochemicl vribles re indicted in Tble 2 nd 3 ccording to TCN2 nd MTHFR genotypes. Low plsm levels (less then 30 pmol/l) of holo-tc II were observed in none of our ptients, plsm levels were bove 160 pmol/l in 38 ptients. Hyperhomocysteinemi ws present in 112 ptients. Vitmin B 12 levels of less thn 118 nmol/l were mesured in 7 ptients. Folte plsm levels, s well s red blood cell folte concentrtions, were not decresed in ny ptient. There ws liner ssocition of holo-tc II with lbumin (r 0.205, P 0.025) nd with vitmin B 12 (r 0.778, P 0.001), but not with cretinine (P 0.058), ge, body mss index (BMI), ln-thcy, vitmin B 6, plsm folte, nd red blood cell folte concentrtion. We observed liner ssocition of ln-thcy with cretinine (r 0.474, P 0.001), lbumin (r 0.284, P 0.002), serum folte (r 0.338, P 0.001), nd red blood cell folte (r 0.472, P 0.001). There ws no effect of ge (P 0.051), vitmin B 12 (P 0.087), BMI, vitmin B 6, nd holo-tc II. There ws no effect of TCN2 776C G on plsm levels of vitmin B 12, holo-tc II, nd plsm thcy/ln-thcy concentrtions (one-wy ANOVA, post-hoc Scheffe test) (Tble 2). The MTHFR 677C T genotypes showed significnt effect on plsm ln-thcy levels, but not on holo-tc II or vitmin B 12 levels (one-wy ANOVA, posthoc Scheffe test) (Tble 3). Independent effects of TCN2 nd MTHFR genotypes on holo-tc II nd ln-thcy concentrtions Multiple liner regression nlysis including relevnt vribles (cretinine, lbumin, vitmin B 12 ) showed n independent ssocition of lbumin nd vitmin B 12 with holo-tc II levels (Tble 4, Model 1). Including MTHFR (Tble 4, Model 2), TCN2 (Tble 4, Model 3), or both polymorphisms (Tble 4, Model 4) in the nlysis reveled no effect on holo-tc II concentrtions. There ws no interction of genotypes tht showed n effect on ln-thcy (TCN2 776CC x MTHFR 677TT) (Tble 4, Model 5). We found significnt independent effect of cretinine, red blood cell folte, nd MTHFR 677TT genotype on ln-thcy plsm concentrtions (Tble 5, Model 1), wheres ge, lbumin, vitmin B 12, serum folte, nd MTHFR 677CC, or CT genotype showed no effect by multiple liner regression nlysis. Including TCN2 genotypes in this nlysis disclosed n independent effect of TCN2 776CC genotype on ln-thcy concentrtions (Tble 5, Model 2). This effect remined when MTHFR nd TCN2 were both included in the nlysis (Tble 5, Model 3), but disppered following the inclusion of n interction term TCN2 776CC MTHFR 677TT (Tble 5, Model 4). DISCUSSION None of the end-stge renl disese ptients included in this nlysis showed decresed holo-tc II plsm levels, which is in line with previous observtions in renl filure ptients [17]. There ws liner ssocition of holo-tc II with lbumin nd vitmin B 12, but not with ge or ln-thcy plsm concentrtion. Our study shows tht the 776C G polymorphism of TCN2 hs no mjor effect on holo-tc II, vitmin B 12 levels, nd ln-thcy plsm concentrtions. There ws lso no effect of MTHFR 677C T on holo-tc II or vitmin B 12 levels. By liner multiple regression nlysis, however, TCN2 showed minor independent effect on ln-thcy plsm concentrtions tht ws probbly due to somewht lower ln-thcy plsm concentrtions in the TCN2 776CC genotype compred to CG/GG genotypes (Tble 2). The lck of effect of the TCN2 776C G polymorphism on holo-tc II levels in our study is in contrst to two studies reporting holo-tc II decresing effect of TCN2 776C G in subjects without renl disese [7, 18]. For determintion of holo-tc II plsm concentrtions we hve used commercilly vilble rdioimmunossy. Becuse different methods hve been used for determintion of holo-tc II in vrious studies, it cnnot be excluded tht the observed discrepncies re due to different test systems. Alterntively, the decresing effect of TCN2 776C G on holo-tc II is not present in renl filure. Interestingly, in one of the studies reporting holo-tc II decresing effect of TCN2 776C G, this observtion ws not ssocited with n increse of thcy concentrtions, lthough methylmlonic cid concentrtions were significntly elevted [7]. Mesurement of methylmlonic cid levels represents tool to dignose vitmin B 12 deficiency [19]. We hve not mesured methylmlonic cid levels becuse methylmlonic cid ccumultes in renl filure ptients [20, 21], but it my be useful for monitoring of vitmin B 12 supplementtion in such ptients [22]. The lck of effect of TCN2 776C G on vitmin B 12 concentrtion is in line with ll but one study [6 8],

4 1098 Födinger et l: TCN2 polymorphism in ESRD Tble 2. Ptient chrcteristics ccording to TCN2 776C G genotype TCN2 776C G CC CG GG Dilysis ptients N Plsm holo-tc II pmol/l 130 ( ) 117 (89 163) 143 ( ) Plsm vitmin B 12 pmol/l 248 ( ) 252 ( ) 266 ( ) Plsm thcy lmol/l 25 ( ) 27.2 ( ) 25.3 ( ) Plsm ln-thcy lmol/l 3.22 ( ) 3.30 ( ) 3.23 ( ) RBC folte nmol/l 1346 ( ) 1180 ( ) 1280 ( ) Plsm folte nmol/l 17 ( ) 16 ( ) 18.6 ( ) Plsm vitmin B 6 nmol/l 44.2 ( ) 34.3 ( ) 26.5 ( ) Albumin g/dl 40.8 ( ) 39 ( ) 35.1 ( ) Cretinine mg/dl 8.6 ( ) 8.1 ( ) 7.4 ( ) Hemoglobin g/dl 11.6 ( ) 12 ( ) 11.3 ( ) RBC is red blood cells. Medins, percentiles 25 nd 75 re shown in prentheses. No effect of genotype by one-wy nlysis of vrince; no effect of genotypes by post-hoc Scheffe test Tble 3. Ptient chrcteristics ccording to MTHFR 677C T genotype MTHFR 677C T CC CT TT Dilysis ptients N Plsm holo-tc II pmol/l 143 (98 185) 117 (84 163) 125 ( ) Plsm vitmin B 12 pmol/l 276 ( ) 234 ( ) 195 ( ) Plsm thcy lmol/l b 24.7 ( ) 27.1 ( ) 31.9 ( ) Plsm ln-thcy lmol/l c 3.21 ( ) 3.30 ( ) 3.46 ( ) RBC folte nmol/l 1288 ( ) 1154 ( ) 1343 ( ) Plsm folte nmol/l 17.1 ( ) 16.0 ( ) 15.5 ( ) Vitmin B ( ) 33.2 ( ) 47.1 ( ) Albumin g/dl 38.0 ( ) 38.6 ( ) 41.7 ( ) Cretinine mg/dl 8.1 ( ) 8.5 ( ) 8.0 ( ) Hemoglobin g/dl 11.5 ( ) 11.9 ( ) 12.7 ( ) RBC is red blood cell. Medins, percentiles 25 nd 75 re shown in prentheses. No effect of genotype by one-wy nlysis of vrince (ANOVA); no effect of genotype by post-hoc Scheffe test b P by ANOVA; TT vs. CC P by post-hoc Scheffe test c P by ANOVA; TT vs. CC P 0.001, CT vs. CC P by post-hoc Scheffe test Tble 4. Independent predictors of holo-tc II plsm concentrtions (multiple liner regression nlyses) Model P P P P P Vitmin B Cretinine Albumin MTHFR 677CC b b MTHFR 677CT b b b b MTHFR 677TT TCN2 776CC TCN2 776CG b b b b b b TCN2 776GG TT 776CC Vrible not included in the model; b Vrible excluded by regression procedure supporting the concept tht this muttion does not influence vitmin B 12 cellulr delivery. Serum vitmin B 12 tends to run spuriously high in some ptients with renl filure, nd low in others, often not reflecting vitmin B 12 sttus well. Even holo-tc II tends to run high in renl filure [17]. In this context, the kidney ctively clers TC II [23], nd perhps even synthesizes nd releses some holo-tc II [24]. Indeed, the holo-tc II results of the present study consist of mny elevted vlues, wheres none of the ptients presented with lev-

5 Födinger et l: TCN2 polymorphism in ESRD 1099 Tble 5. Independent predictors of ln-thcy plsm concentrtions (multiple liner regression nlyses) Model P P P P Vitmin B Cretinine Albumin Age Plsm folte Red blood cell folte MTHFR 677CC MTHFR 677CT b b b b b b MTHFR 677TT TCN2 776CC TCN2 776CG b b b b b b TCN2 776GG TT 776CC Vrible not included in the model; b vrible excluded by regression procedure els below the reference rnge. By crude clcultion using the mens, the percentge of the totl vitmin B 12 tht is bound to trnscoblmin is round 50%. This is in contrst to the literture, which sys tht this percentge is usully round 20% to 30%. This observtion suggests tht renl disese nd/or dilysis ffects totl B 12 nd/or holo-tc II levels in wy tht hs not been previously recognized, nd thus my potentilly confound the results of this study. Furthermore, there lso my be reltion to n impirment of cellulr uptke of holo-tc II in renl filure (W. Herrmnn nd R. Obeid, Hmburg, Germny, personl communiction, 2003). There ws no effect of TCN2 776C G on plsm lnthcy concentrtions in our study of dilysis ptients by one-wy ANOVA (Tble 2). By multiple liner regression nlysis, red blood cell folte, cretinine, nd the MTHFR 6777TT genotype were independent predictors of ln-thcy concentrtions. Inclusion of the TCN2 genotypes in the multivrite nlysis showed n independent effect of the CC genotype tht ws probbly due to somewht lower ln-thcy levels in TCN2 776CC genotype ptients compred to CG/GG genotypes. The interprettion of the discrepncies of univrite nd multivrite nlyses, with regrd to the effect of TCN2 on ln-thcy levels in this study, deserves some criticl pprisl. First, the effect in the multivrite nlysis my hve occurred by chnce. Second, lrger study popultion would probbly clerly disclose n effect of TCN2 776CC genotype in renl filure ptients. There is lso hint from the literture tht the effect of TCN2 776C G on thcy my be restricted to individuls of younger ge [25]. Together, our finding of slight effect of TCN2 on lnthcy by multivrite nlysis is in contrst to dt of helthy elderly individuls nd dementi ptients [6, 7], but compres well with results obtined in studies of helthy individuls [5, 8, 9], vsculr disese ptients [9], nd mothers of children with neurl tube defects [8], reporting wek thcy elevting effect of TCN2 776C G. These divergent observtions could be due to investigtion of different study popultions showing divergent ge, genetic bckground, nd vitmin sttus influencing thcy levels. Finlly, there ws tendency for different vitmin B 6 plsm levels mong the three TCN2 genotype groups in our study, however, which ws not significnt by onewy ANOVA nd my be relted to the multivitmin tht contined 10 mg of vitmin B 6 nd ws prescribed to hemodilysis ptients. CONCLUSION TCN2 776C G does not predict holo-tc II or vitmin B 12 plsm concentrtions nd is no mjor dditionl determinnt of hyperhomocysteinemi of end-stge renl disese ptients. Reprint requests to Gere Sunder-Plssmnn, M.D., Division of Nephrology nd Dilysis, Deprtment of Medicine III, University of Vienn, Währinger Gürtel 18-20, A-1090 Wien, Austri. E-mil: Gere.Sunder-Plssmnn@univie.c.t REFERENCES 1. The Homocysteine Studies Collbortion: Homocysteine nd risk of ischemic hert disese nd stroke. A met-nlysis. JAMA 288: , Fehr J, De Vecchi P: Trnscoblmin II: A mrker for mcrophge/histiocyte prolifertion. Am J Clin Pthol 84: , Nexo E, Christensen AL, Petersen TE, Fedosov SN: Mesurement of trnscoblmin by ELISA. Clin Chem 46: , Crmel R: Mesuring nd interpreting holo-trnscoblmin (holotrnscoblmin II). Clin Chem 48: , Nmour F, Olivier JL, Abdelmouttleb I, et l: Trnscoblmin codon 259 polymorphism in HT-29 nd Cco-2 cells nd in Cucsins: Reltion to trnscoblmin nd homocysteine concentrtion in blood. Blood 97: , McCddon A, Blennow K, Hudson P, et l: Trnscoblmin polymorphism nd homocysteine. Blood 98: , 2001

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