Vitamin K 2 Improves Femoral Bone Strength without Altering Bone Mineral Density in Gastrectomized Rats

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1 J Nutr Sci Vitminol, 60, 71 77, 2014 Vitmin K 2 Improves Femorl Bone Strength without Altering Bone Minerl Density in Gstrectomized Rts Jun Iwmoto 1, Yoshihiro Sto 2 nd Hideo Mtsumoto 1 1 Institute for Integrted Sports Medicine, Keio University School of Medicine, Tokyo , Jpn 2 Deprtment of Neurology, Mitte Hospitl, Fukuok , Jpn (Received August 13, 2013) Summry Gstrectomy (GX) induces osteopeni in rts. The present study exmined the skeletl effects of vitmin K 2 in GX rts. Thirty mle Sprgue-Dwley rts (12 wk old) were rndomized by the strtified weight method into the following three groups of 10 nimls ech: shm opertion (control) group; GX group; nd GX1orl vitmin K 2 (mentetrenone, 30 mg/kg, 5 d/wk) group. Tretment ws initited t 1 wk fter surgery. After 6 wk of tretment, the bone minerl content (BMC), bone minerl density (BMD), nd mechnicl strength of the femorl diphysis nd distl metphysis were determined by peripherl quntittive computed tomogrphy nd mechnicl strength tests, respectively. GX induced decreses in the BMC, BMD, nd ultimte force of the femorl diphysis nd distl metphysis. Vitmin K 2 did not significntly influence the BMC or BMD of the femorl diphysis or distl metphysis in GX rts, but ttenuted the decrese in the ultimte force nd incresed the stiffness of the femorl diphysis. The present study showed tht dministrtion of vitmin K 2 to GX rts improved the bone strength of the femorl diphysis without ltering the BMC or BMD, suggesting effects of vitmin K 2 on the corticl bone qulity. Key Words gstrectomy, corticl bone, bone strength, bone minerl density, vitmin K Gstric surgery is mostly needed for the tretment of gstric cncer. Gstrectomy (GX) in ptients results in mlnutrition nd bone diseses, including not only osteoporosis but lso osteomlci or mixed pttern of osteoporosis/osteomlci with secondry hyperprthyroidism (1, 2). The risk of frctures is incresed in GX ptients (3 8). This risk is sustined even when ptients re cured of their disese (9). Since GX induces impirment of clcium nd vitmin D metbolism (5, 10), clcium nd vitmin D supplementtion my ply n importnt role in mintining bone helth (7). However, vitmin K insufficiency s well s vitmin D insufficiency nd deficiency owing to mlnutrition re speculted to be prtly ssocited with the incresed risk of frcture in GX ptients, similr to the cse for ptients with neurologicl diseses (11 13). Vitmin K insufficiency hs been suggested to increse the risk of hip frctures in elderly people (14 17). Met-nlyses hve demonstrted the nti-frcture efficcy of vitmin K 2, which is widely used for tretment of osteoporosis in Asi, in ptients with n incresed risk of frcture (18, 19). Thus, it is surmised tht vitmin K 2 therpy could be useful for mintining bone helth in GX ptients. An niml model of corticl nd cncellous osteopeni cn be creted by performing GX in rts. Totl GX nd resection of the cid-producing prt of the stomch (fundectomy) induce gstrinemi nd mlnutrition (20, 21), thereby inititing trbeculr nd corticl osteopeni in rts (22 26). The hypothesis of the present preclinicl study ws tht vitmin K 2 would improve the E-mil: jiwmoto@8.keio.jp bone strength in GX rts. Thus, experiments were conducted to determine the skeletl effects of vitmin K 2 in GX rts. The primry endpoint ws the bone strength t sites rich in trbeculr or corticl bone. The secondry endpoints included the bone weight, bone minerl content (BMC), nd bone minerl density (BMD). MATERIALS AND METHODS Hndling of nimls. Thirty mle Sprgue-Dwley rts (11 wk old) were purchsed from Chrles River Jpn (Kngw, Jpn). The rts were housed in locl niml room with temperture of 24 C, humidity of 50%, nd 12-h/12-h light/drk cycle, nd were llowed free ccess to wter nd stndrd pellet diet contining 1.25% clcium nd 0.9% phosphorus (CRF-1; Orientl Yest Co., Ltd., Tokyo, Jpn). After 1 wk of dpttion to this environment, the 12-wk-old rts were sorted into strt by body weight nd then rndomized by the strtified weight method into the following three groups of 10 nimls ech: shm opertion control (CON) group; GX group; nd GX1vitmin K 2 group. Totl GX ws performed under generl nesthesi with mg/kg of pentobrbitl (Kyoritsu Seiyku Co. Ltd., Tokyo, Jpn) injected intrperitonelly together with 2 3% isoflurne (Myln Inc., Tokyo, Jpn) delivered vi Tble Top Lbortory Animl Anesthesi System (V1 Type VetEquip Inc., Plesnton, CA). A longitudinl incision ws mde on the bdomen to expose the stomch. The whole stomch ws excised, nd n nstomosis of the esophgus nd duodenum ws performed. Tretment ws initited t 1 wk fter surgery. In the CON nd GX groups, vehicle ws dministered by gvge five times 71

2 72 Tble 1. Body weights nd femorl lengths nd weights. Iwmoto J et l. CON GX GX1vitmin K 2 Body weight (g) At surgery At bseline After 6 wk of tretment Femorl length (mm) b Femorl weight (g) Dt re expressed s the men6sd. ANOVA with Fisher s PLSD test ws used to compre dt mong the three groups. Significnt difference vs. CON, b significnt difference vs. GX. CON: control, GX: gstrectomy. week. In the GX1vitmin K 2 group, vitmin K 2 (menquinone-4, mentetrenone; Eisi Co. Ltd., Tokyo, Jpn) ws suspended in ftty cid preprtion (Miglyol 812; Mitsub Trding Co. Ltd., Tokyo, Jpn) t dosge of 30 mg/ml/kg nd dministered by gvge five times week. Although the dosge my be considered to be very high, tretment with vitmin K 2 t 30 mg/kg five times week hs previously been reported to mintin bone helth in rts without cusing ny dverse events (27, 28). The weight of the rts ws monitored weekly nd the durtion of the tretment ws 6 wk. The study ws crried out t the lbortory of Hmri Co. Ltd. (Ibrki, Jpn), which hs been pproved by the Assocition for the Assessment nd Accredittion of Lbortory Animl Cre (AAALAC) Interntionl. The experimentl protocol ws pproved by the Institutionl Animl Cre nd Use Committee (IACUC) of Hmri Co. Ltd. Preprtion of specimens. At 6 wk fter the strt of tretment, the nimls were euthnized by exsnguintion under generl nesthesi with 2 3% isoflurne using the bove-described Tble Top Lbortory Animl Anesthesi System. The bilterl femor were hrvested from ech niml. The left femur ws used for mesurements of the length with dil cliper nd the weight on n electronic blnce (A&D Compny, Tokyo, Jpn). This femur ws then preserved in sline, stored in freezer (260 C), nd processed for the mechnicl strength tests. The right femur ws preserved in 70% ethnol nd processed for peripherl quntittive computed tomogrphy (pqct). pqct of the femur. The distl metphysis nd diphysis of the femur were scnned by pqct (XCT-Reserch SA1; Strtec Medizintechnik GmbH, Pforzheim, Germny) in 70% ethnol/sline. The bones were plced horizontlly in polypropylene tube nd scnned t voxel size of 0.12 mm. The scn line ws djusted using the scout view, nd sites t 3 nd 14 mm proximl to the distl growth plte were scnned. For nlysis, threshold of 395 mg/cm 3 in contour mode 2 ws used to seprte the bone tissue from the mrrow in the distl metphysis. The totl nd trbeculr BMC, BMD, nd re were evluted. A constnt threshold of 690 mg/ cm 3 in contour mode 1 ws used to seprte the corticl bone from the trbeculr bone in the diphysis. The BMC, BMD, re, thickness, nd periostel nd endocorticl perimeters were evluted. Mechnicl strength test of the femur. Ech specimen ws submerged for 1 h before testing to llow temperture equilibrtion. The mechnicl strength of the femorl diphysis ws evluted by three-point bending test. A lod ws pplied to the bone midwy between two supports plced 18 mm prt. The femur ws positioned so tht the loding point ws t the center of the femorl diphysis, nd bending occurred t the medil lterl xis. Lod-displcement curves were recorded t crosshed speed of 5 mm/min using mteril-testing mchine (MZ-500S; Mruto Instrument Co. Ltd., Tokyo, Jpn). The prmeters nlyzed were the stiffness, work to filure, nd ultimte force. Just fter the three-point bending test of the femorl diphysis, the distl metphysis of the femur ws isolted over length of 10 mm from the joint surfce of the femorl condyle. The mechnicl strength of this segment ws then mesured by compression test. A compressive lod ws pplied by rectngulr prllelepiped crosshed (length, 2 cm; width, 2 cm; height, 1 cm) to the femorl distl metphysis from the lterl spect to the medil spect. The specimens were positioned so tht the loding point ws t the center of the femorl lterl condyle. Lod-displcement curves were recorded t crosshed speed of 5 mm/min nd compression depth of 2.5 mm using mteril-testing mchine (MZ500D; Mruto Instrument Co.). The prmeters nlyzed were the stiffness, work to filure, nd ultimte force. Sttisticl nlysis. Dt were expressed s the men6stndrd devition (SD). Comprisons mong the groups were performed by nlysis of vrince (ANOVA) using Fisher s protected lest significnt difference (PLSD) test. All sttisticl nlyses were crried out using the Stt View j-5.0 progrm on Windows computer nd significnce ws ccepted t p,0.05. RESULTS Body weight, femorl length, nd femorl weight As shown in Tble 1, the initil body weights t surgery did not differ significntly mong the three groups. The body weights t bseline (1 wk fter surgery, but

3 Vitmin K 2 for Gstrectomized Rts 73 Tble 2. pqct dt for the femorl diphysis. CON GX GX1vitmin K 2 BMC (mg/mm) BMD (mg/cm 3 ) 1, , , Are (mm 2 ) Thickness (mm) Periostel perimeter (mm) Endocorticl perimeter (mm) Dt re expressed s the men6sd. ANOVA with Fisher s PLSD test ws used to compre dt mong the three groups. Significnt difference vs. CON. CON: control, GX: gstrectomy, pqct: peripherl quntittive computed tomogrphy, BMC: bone minerl content, BMD: bone minerl density. Tble 3. pqct dt for the femorl distl metphysis. CON GX GX1vitmin K 2 BMC (mg/mm) Totl Trbeculr BMD (mg/cm 3 ) Totl Trbeculr Are (mm 2 ) Totl b Trbeculr Dt re expressed s the men6sd. ANOVA with Fisher s PLSD test ws used to compre dt mong the three groups. Significnt difference vs. CON, b significnt difference vs. GX. CON: control, GX: gstrectomy, pqct: peripherl quntittive computed tomogrphy, BMC: bone minerl content, BMD: bone minerl density. before tretment) nd fter 6 wk of tretment were lower in the GX group thn in the CON group. Vitmin K 2 dministrtion to GX rts did not significntly influence the body weight. After 6 wk of tretment, the femorl lengths did not differ significntly between the CON nd GX groups. However, the femorl length ws lower in the GX1vitmin K 2 group thn in the CON nd GX groups. The femorl weight ws lower in the GX group thn in the CON group. Vitmin K 2 dministrtion to GX rts did not significntly influence the femorl weight. pqct nlysis of the femur Tble 2 shows the results of the pqct nlysis of the femorl diphysis fter 6 wk of tretment. GX cused decreses in the BMC, BMD, re, nd thickness nd n increse in the endocorticl perimeter, but did not cuse ny significnt chnge in the periostel perimeter. The men GX-induced decrese rtes of the BMC nd BMD were 22.9% nd 3.2%, respectively. Vitmin K 2 dministrtion to GX rts did not significntly improve ny of these prmeters. Tble 3 shows the results of the pqct nlysis of the femorl distl metphysis fter 6 wk of tretment. GX cused decreses in the totl BMC, nd totl nd trbeculr BMD nd increses in the trbeculr BMC nd re, but did not cuse ny significnt chnge in the totl re. The men GX-induced decrese rtes of the totl BMC nd BMD were 33.3% nd 32.4%, respectively. The corresponding chnge rtes of the trbeculr BMC nd BMD were 140.2% nd 226.4%, respectively. Vitmin K 2 dministrtion to GX rts did not significntly improve the totl or trbeculr BMC nd BMD or trbeculr re, but decresed the totl re. Mechnicl strength of the femur Figure 1 shows the results of the mechnicl strength tests of the femorl diphysis nd distl metphysis fter 6 wk of tretment. In the femorl diphysis, GX induced decrese in the ultimte force, but did not cuse ny significnt chnges in the stiffness or work to filure. Vitmin K 2 dministrtion to GX rts ttenuted the decrese in the ultimte force nd incresed the stiffness, but did not significntly chnge the work to filure. In the femorl distl metphysis, GX induced decreses in the stiffness, work to filure, nd ultimte force. Vitmin K 2 dministrtion to GX rts did not significntly improve ny of these prmeters.

4 74 Iwmoto J et l. (N/mm) A. Femorl diphysis Stiffness (N mm) (N) Work to filure Ultimte force b b (N/mm) B. Femorl distl metphysis Stiffness (N mm) (N) Work to filure Ultimte force Fig. 1. Mechnicl strength of the femorl diphysis nd distl metphysis. Dt re expressed s the men6sd. ANOVA with Fisher s PLSD test ws used to compre dt mong the three groups. : significnt vs. CON, b: significnt vs. GX. CON: control, GX: gstrectomy, GX1K: gstrectomy1vitmin K 2. DISCUSSION The present study exmined the skeletl effects of vitmin K 2 in GX rts. The key findings were: 1) influences of GX on the BMC, BMD, nd bone strength of the femorl diphysis nd distl metphysis in rts; 2) lck of significnt influences of vitmin K 2 dministrtion to GX rts on the BMC nd BMD of the femorl diphysis nd distl metphysis; nd 3) effects of vitmin K 2 dministrtion to GX rts on the bone strength of the femorl diphysis. Gstric cid is thought to fcilitte the intestinl bsorption of ingested clcium by mobilizing clcium from insoluble complexes in the diet (22). After GX in humns, pigs, nd rts, serum gstrin decreses, clcium bsorption is impired, serum clcium nd 25(OH) D decrese, nd 1,25(OH) 2 D increses (5, 10, 29 31). GX in rts induces osteopeni (reduced BMC nd BMD) (21, 25, 26, 32 34). However, trbeculr bone loss ppers to be greter thn corticl bone loss (25, 35), nd the inner dimeters of the tubulr bones (femur nd tibi) re gretly incresed (36). In the present study, GX decresed the BMC nd BMD of the femorl diphysis nd distl metphysis, with greter decreses in the femorl distl metphysis thn in the femorl diphysis. The endocorticl perimeters of the femorl diphysis nd the trbeculr re of the femorl distl metphysis were incresed. These findings re consistent with those in previous studies (21, 25, 26, 32 35). The increse in the trbeculr BMC of the femorl distl metphysis my be ttributble to the increse in the trbeculr re. A preclinicl study showed tht bisphosphonte incdronte prevented GX-induced osteopeni nd osteomlci in rts (29). Theoreticlly, however, bisphosphontes my be contrindiction for osteomlci (37), nd not only clcium nd vitmin D, but lso drugs tht increse bone minerliztion, my be suitble therpies for bone disorders fter GX. Vitmin K is known to be cofctor of g-crboxylse, which converts glutmic cid (Glu) residues in osteoclcin molecules to g-crboxyglutmic cid (Gl) residues nd is therefore essentil for g-crboxyltion of osteoclcin (38 40). Without this modifiction, osteoclcin lcks structurl integrity nd the bility to bind to the hydroxyptite minerl. Thus, vitmin K 2 is likely to ply role in minerliztion of bone nd cn be good cndidte for the tretment of bone disorders fter GX. In the present study, however, vitmin K 2 improved the ultimte force nd stiffness of the femorl diphysis without ltering the BMC or BMD in GX rts, suggesting tht it hd effects on the corticl bone qulity.

5 Vitmin K 2 for Gstrectomized Rts 75 Severl preclinicl studies hve suggested effects of vitmin K 2 on bone strength or bone qulity (mtrix volume, osteocyte density, nd collgen cross-links), but not on bone mss, in rts (41 47). Cliniclly, Tnk et l. (48) reported tht the osteoclcin/deoxypyridinoline nd osteopontin/clcium rtios of corticl bone were lower in ptients with femorl neck frctures thn in those with osteorthritis of the hip. Iwmoto et l. (18) reported the nti-frcture efficcy of vitmin K 2 ginst frctures of the hip, skeletl site rich in corticl bone, in ptients with neurologicl diseses. These reports support significnt influence of osteoclcin nd beneficil effect of vitmin K 2 on corticl bone qulity nd/ or strength. Vitmin K 2 decresed the femorl length nd femorl distl metphysel totl re in GX rts. These results suggest the retrdtion of bone growth, which could be limittion of high dose vitmin K 2 dministrtion. Further studies re needed to determine whether there ws toxicity of high dose vitmin K 2 on the skeleton. The body weight mrkedly decresed fter GX in rts, suggesting n induction of mlnutrition by GX. It is well known tht GX cuses impirment of clcium bsorption nd vitmin D insufficiency or deficiency. However, vitmin K insufficiency could coexist in GX rts owing to mlnutrition. Nevertheless, vitmin K nutritionl sttus fter GX hs rrely been studied. Serum levels of vitmin K 1, vitmin K 2, nd undercrboxylted osteoclcin, which increses ccording to vitmin K insufficiency becuse of impirment of g-crboxyltion of osteoclcin, pper to be good indices of vitmin K nutritionl sttus. Thus, the ssessment of these prmeters could revel the existence of vitmin K insufficiency fter GX nd the beneficil effect of vitmin K 2 on the skeleton in GX rts. Further studies re needed to clrify these issues. The present study hd severl limittions. First, we did not evlute serum or urinry clcium or phosphorus, serum 25(OH)D, 1,25(OH) 2 D, prthyroid hormone (PTH), or bone turnover mrkers. The effects of vitmin K 2 on these biochemicl prmeters my be of importnce for trnslting the results of our preclinicl study into clinicl prctice. Second, we used young mle rts becuse previous studies hve reported the influence of GX on bone prmeters in young mle rts. Thus, the results of the present study my be relevnt to GX in men. GX is ssocited with significnt risk of hip frctures in men (4). However, ovriectomized ged rts my be more interesting to study from the point of view of the incresed burden of osteoporosis mong elderly women fter GX, lthough it my not be esy for ged rts to survive fter GX. Thus, further studies re needed to estblish the skeletl effects of vitmin K 2 in rt model of elderly women with osteoporosis fter GX. In conclusion, the present study showed tht GX induced decreses in the BMC, BMD, nd ultimte force (bone strength) of the femorl diphysis nd distl metphysis, nd tht dministrtion of vitmin K 2 to GX rts improved the ultimte force nd stiffness of the femorl diphysis without ltering the BMC or BMD. These findings suggest effects of vitmin K 2 on the qulity nd strength of corticl bone. It will be of interest to determine whether vitmin K 2 cn prevent frctures in GX ptients by conducting clinicl trils. Disclosures All the uthors stte tht they hve no conflicts of interest. Acknowledgments We thnk Drs. Kiichi Nonk nd Kori Shindo (ELK Corportion, Tokyo, Jpn) for the pqct nlyses, nd Dr. Tsuyoshi Ishii (Mruto Instrument Co. Ltd., Tokyo, Jpn) for the mechnicl strength tests. REFERENCES 1) Gennri C, Mrtini G, Nuti R Secondry osteoporosis. Aging (Milno) 10: ) Tovey FI, Hll ML, Ell PJ, Hobsley M A review of postgstrectomy bone disese. 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