Effect of isoflavone-enriched whole soy milk powder supplementation on bone metabolism in ovariectomized mice

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1 Nutrition Reserch nd Prctice 2018;12(4): c2018 The Koren Nutrition Society nd the Koren Society of Community Nutrition Effect of isoflvone-enriched whole soy milk powder supplementtion on one metolism in ovriectomized mice So Mi Kim 1*, Hyun Sook Lee 2*, Je In Jung 1, Su-Min Lim 1, Ji Hoon Lim 3, Wng-Hyun H 3, Chng Le Jeon 3, Je-Yong Lee 1,4 nd Eun Ji Kim 1 1 Center for Efficcy Assessment nd Development of Functionl Foods nd Drugs, Hllym University, 1 Hllymdehk-gil, Chuncheon, Gngwon 24252, Kore 2 Deprtment of Food Science & Nutrition, Dongseo University, Busn 47011, Kore 3 Institute of Food Processing Technology, Uwell Bio Co. Ltd., Gngwon 25451, Kore 4 Deprtment of Biochemistry, College of Medicine, Hllym University, Gngwon 24252, Kore BACKGROUND/OBJECTIVE: There is intense interest in soy isoflvone s hormone replcement therpy for the prevention of postmenopusl osteoporosis. A new kind of isoflvone-enriched whole soy milk powder (I-WSM) contining more isoflvones thn conventionl whole soy milk powder ws recently developed. The im of this study ws to investigte the effects of I-WSM on one metolism in ovriectomized mice. MATERIALS/METHODS: Sixty femle ICR mice individully underwent ovriectomy (OVX) or shm opertion, nd were rndomized into six groups of 10 nimls ech s follows: Shm, OVX, OVX with 2% I-WSM diet, OVX with 10% I-WSM diet, OVX with 20% I-WSM diet, nd OVX with 20% WSM diet. After n 8-week tretment period, one minerl density (BMD), clcium, lkline phosphtse (ALP), trtrte-resistnt cid phosphtse (TRAP) 5, osteoclcin (OC), procollgen 1 N-terminl propeptide (P1NP), nd osteoprotegenin (OPG) were nlyzed. RESULTS: BMD ws significntly lower in the OVX group compred to the Shm group ut ws significntly higher in OVX + 10% I-WSM nd OVX + 20% I-WSM groups compred to the OVX group (P < 0.05). Serum clcium concentrtion significntly incresed in the OVX + 10% nd 20% I-WSM groups. Serum ALP levels were significntly lower in the OVX + 10% nd 20% I-WSM groups compred to the other experimentl groups (P < 0.05). OC ws significntly reduced in the OVX group compred to the Shm group (P < 0.05), ut dose-dependent increse ws oserved in the OVX groups supplemented with I-WSM. P1NP nd OPG levels were significntly reduced, while TRAP 5 level ws significntly elevted in the OVX group compred with the Shm group, which ws not ffected y I-WSM (P <0.05). CONCLUSIONS: This study suggests tht I-WSM supplementtion in OVX mice hs the effect of preventing BMD reduction nd promoting one formtion. Therefore, I-WSM cn e used s n effective lterntive to postmenopusl osteoporosis prevention. Nutrition Reserch nd Prctice 2018;12(4): ; pissn eissn Keywords: Functionl food, soyens, isoflvones, ovriectomy, one remodeling INTRODUCTION 1) Postmenopusl osteoporosis is usully ssocited with ging nd decresed levels of the ovrin sex hormone estrogen. The min clinicl mnifesttion of this metolic disorder is wekening of ones nd incresed risk of frctures [1]. This hs ecome serious pulic helth prolem in the current ging society. The first-line therpy for postmenopusl osteoporosis is estrogen replcement therpy (ERT), which cn prevent one loss nd promote one formtion [2]. However, dt lso suggest tht ERT increses the risk of rest cncer, endometril cncer, vginl leeding, coronry hert disese, stroke, nd venous thromoemolism [3-5]. These serious side effects limit the ppliction of ERT. Thus, there is incresing interest in the use of plnt-derived estrogens, known s phytoestrogens. Phytoestrogens re lrge group of polyphenolics, i.e., nonsteroidl plnt compounds with chemicl structure such s 17 β-estrdiol, which cn ind to estrogen receptors nd mimic the ctions of estrogens on trget orgns, therey exerting mny helth enefits when used under hormone-dependent conditions [6,7]. The min clsses of phytoestrogens re isoflvones, lignns, coumestnes, flvonoids, nd stilenes [8,9]. Among This work ws supported y the Ministry of Trde, Industry nd Energy (MOTIE) nd Kore Institute for Advncement of Technology (KIAT) through the Reserch nd Promoting Regionl Specilized Industry (R ) nd the Center for Efficcy Assessment nd Development of Functionl Foods nd Drugs t Hllym University (B ), Kore. Corresponding Author: Eun Ji Kim, Tel , Fx , Emil. myej4@hllym.c.kr Received: Ferury 14, 2018, Revised: April 3, 2018, Accepted: My 2, 2018 * The first two uthors contriuted eqully to this work. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( which permits unrestricted non-commercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 276 Isoflvone nd one metolism in ovriectomized mice them, isoflvones hve the most potent estrogenic ctivity; genistein, didzein, nd glycitein, in prticulr, re the most ctive isoflvones found in soyens. Epidemiologicl nd experimentl studies hve confirmed tht isoflvones hve one-spring effect in postmenopusl women [10-12] nd in ovriectomized rt models [13,14]. However, the effect of isoflvones on one metolism in postmenopusl women is not lwys positive. Previous rndomized controlled trils hve shown tht the effects of isoflvones supplementtion cn differ depending on the rce/ethnicity of the sujects, reserch design, vriety of isoflvone supplements, dosge, durtion of exposure, nd responsiveness of postmenopusl women to isoflvones supplementtion [15]. Recently, the chemicl forms of isoflvones hve een reported to e importnt since they cn influence the iovililities nd, thus iologicl ctivities of isoflvones [7]. A recently developed type of isoflvone-enriched whole soy milk powder (I-WSM) ws shown to contin out 8.8 times more isoflvones thn conventionl whole soy milk powder (WSM). Thus, I-WSM is expected to e more effective thn WSM in preventing nd treting postmenopusl osteoporosis, nd this study ws conducted to verify this ide. MATERIALS AND METHODS Preprtion of I-WSM I-WSM ws produced y Uwell Bio Co. Ltd. (Gngneung, Kore). Soyens used in this study were cultivted in the Yeongwol region of Gngwon province in Wshed whole soyens were soked for 7 h in wter t soyens:wter rtio of 1:9. After 7 h of soking, soyens were grounded y using lender (K&S Co., LTD., Whsung, Kore). The slurry ws heted t 90 C for 1 h, followed y cooling t 55 C. To hydrolyze crohydrtes, the slurry ws dded to 2% (w/w) termmyl (Novozymes, Denmrk) nd then incuted t 55 C for 5 h. The slurry ws then centrifuged t 3,500 x g for 20 min to seprte soy milk cke residues, nd the resulting soy milk ws otined. To hydrolyze protein, soy milk ws dded to 1% (w/w) lclse (Novozymes, Copenhgen, Denmrk) nd incuted t 55 C for 5 h, followed y ddition of 1% (w/w) flvourzyme (Novozymes) nd incution t 55 C for 5 h. Next, soy milk ws oiled t 100 C for 30 min to inctivte enzymes. The finl produced soy milk ws freeze-dried y using vcuum freeze-dryer (Operon, Kimpo, Kore) nd grounded to prticle size of less thn 75 μm for finl use. The resulting powder ws used s n isoflvone-enriched whole soy milk powder (I-WSM) nd stored t -20 C until further use. The isoflvone content of this powder ws nlyzed y using HPLC (Agilent, CA, USA). The concentrtion of totl isoflvones ws 1,660 μg/g of I-WSM, which ws out 8.8 times higher thn the isoflvone content of μg/g of WSM. Animls, diet, nd study design The study design is shown in Fig. 1 (n = 10). A totl of 60 7-week-old femle ICR mice were purchsed from Dooyeol Biotech Co. Ltd. (Seoul, Kore). Ech mouse underwent either n ovriectomy (OVX, n = 50) or were shm-treted (n = 10) t 5 weeks of ge. The mice were housed in polycronte cges locted in temperture-controlled rooms (23 ± 3 C) with reltive humidity of 50 ± 10%, ventiltion numer of times, illumintion of Lux, nd 12 h light/drk cycle. During the dpttion period, ll nimls were given free ccess to food from Crgill Agri Purin, Inc. (Seongnm, Kore) nd wter. After 7 dys of cclimtion, the mice were rndomly ssigned to six experimentl groups: Shm, OVX, OVX with 2% I-WSM diet (OVX + 2% I-WSM), OVX with 10% I-WSM diet (OVX + 10% I-WSM), OVX with 20% I-WSM diet (OVX + 20% I-WSM), nd OVX with 20% WSM diet (OVX + 20% WSM). The mnufcturer of WSM, which is mel replcement supplement, clims tht it cn replce up to 30% of dily food intke. The OVX + 20% I-WSM nd OVX + 20% WSM groups were formed in order to compre the effects when I-WSM nd WSM replced 20% of Fig. 1. Experimentl design. Shm, shm-operted; OVX, ovriectomized; I-WSM, isoflvone-enriched whole soy milk powder diet; WSM, whole soy milk powder. The mice were llocted to six different tretment groups.

3 So Mee Kim et l. 277 dily food intke, nd the OVX + 2% I-WSM nd OVX + 10% I-WSM groups were for compring the effects in reltion to I-WSM dosge. The numer of nimls in ech experimentl group ws 10. After the experimentl groups were clssified, the mice were fed sic diet (AIN-93G diet, Reserch Diets Inc., New Brunswick, NJ, USA) for 4 weeks to induce osteoporosis. Then, the experimentl nimls were fed I-WSM or WSMcontining experimentl diet for 8 weeks. The experimentl diets were prepred y mixing 2% I-WSM, 10% I-WSM, 20% I-WSM, or 20% WSM into AIN-93G diet. The dily food intke in ech group ws mesured, nd the mice were weighed weekly. These niml studies were conducted ccording to the protocols with pprovl from the Animl Cre nd Use Committee of Hllym University (pprovl numer: Hllym ). Specimen collection nd iochemicl ssessment After 8 weeks on the experimentl diet, the mice were nesthetized with triromoethnol diluted with tertiry myl lcohol nd lood ws drwn from the oritl vein. Blood smples were centrifuged t 3,000 rpm for 20 min t 4 C to prepre the serum. The seprted serum stored t -70 C ws sujected to iochemicl nlysis for the contents of clcium (C), phosphorous (P), lkline phosphtse (ALP), trtrteresistnt cid phosphtse (TRAP) 5, osteoclcin (OC), procollgen 1 N-terminl propeptide (P1NP), nd osteoprotegenin (OPG). Serum levels of C, P, nd ALP were mesured y lood chemistry utonlyzer (KoneLA 20 XT, Thermo Fisher Scientific, Vnt, Finlnd). Serum levels of P1NP, OPG, nd OC were mesured using relevnt ELISA kits (Elscience Biotechnology Inc., Houston, TX, USA), nd serum levels of TRAP 5 were mesured using n ELISA kit (EIA, Huei, Chin), in ccordnce with the mnufcturer s instructions. Mesurement of one minerl density The femurs of mice were dissected to remove soft tissues nd were then weighed. Femorl one minerl densities (BMD) were mesured using one densitometer (PIXImus TM, GE Helthcre, Little Chlfont, Englnd). Histomorphologicl Study The femurs were fixed in 4% prformldehyde solution for 24 h. After fixtion, they were deminerlized for 24 h in Clci- Cler Rpid (Ntionl Dignostics, Atlnt, GA, USA), treted ccording to generl tissue-processing procedure, emedded in prffin, nd then cut to thickness of 10 μm. The one tissues were stined with hemtoxylin nd eosin (H&E) nd exmined under light microscope (Axiomger, Crl Zeiss, Jen, Germny). Sttisticl Anlysis Sttisticl nlysis ws performed with SAS for Windows version 9.1 softwre (SAS Institute, Cry, NC, USA). All dt re presented s men ± stndrd error of the men (SEM). Differences in mesured prmeters mong the experimentl groups were nlyzed y one-wy nlysis of vrince nd Duncn s multiple rnge tests. Differences were significnt for P < RESULTS Body weight nd food intke As shown in Tle 1, finl ody weight nd ody weight gin were significntly higher in the OVX group compred to the shm group (P < 0.01). There ws no significnt difference in finl ody weight mong the groups treted with OVX. Food intke of the experimentl nimls did not differ mong the six groups. Effects of I-WSM on BMD nd morphology of femurs in ovriectomized mice After the tretments, BMDs of the femurs extrcted from the scrificed nimls were mesured, nd the results re shown in Tle 2. The BMDs of the left nd right femurs were ±0.001 g/cm 2 in the Shm group nd ± g/cm 2 in the OVX group, indicting tht the OVX group hd significntly lower BMD thn the Shm group (P < 0.001). This result indictes tht ovriectomies led to reduction of one densities. Compred Tle 1. Body weight nd food intke of mice treted with isoflvone-enriched whole soy milk powder (I-WSM) Groups Vriles Shm OVX OVX + 2% I-WSM OVX + 10% I-WSM OVX + 20% I-WSM OVX + 20% WSM Initil ody weight, g 26.8 ± ± ± ± ± ± 0.2 Finl ody weight, g 40.9 ± ± ± ± ± ± 1.5 Weight gin, g 14.1 ± ± ± ± ± ± 1.4 Food intke, g/dy 3.27 ± ± ± ± ± ± 0.11 Vlues re expressed s men ± SE. Shm, shm-operted; OVX, ovriectomized; I-WSM, isoflvone-enriched whole soy milk powder; WSM, whole soy milk powder. Sttisticl difference etween the experimentl groups sed on one-wy nlysis of vrince nd Duncn s multiple rnge tests t P < Mens with different lpheticl superscripts re significntly different (P < 0.05). Tle 2. Effects of isoflvone-enriched whole soy milk powder on one minerl density of femurs in ovriectomized mice BMD (g/cm 2 ) Groups Shm OVX OVX + 2% I-WSM OVX + 10% I-WSM OVX + 20% I-WSM OVX + 20% WSM Left ± ± c ± c ± ± ± c Right ± ± c ± c ± ± ± c Left + Right ± ± c ± c ± ± ± c Vlues re expressed s men ± SE. Shm, shm-operted; OVX, ovriectomized; I-WSM, isoflvone-enriched whole soy milk powder; WSM, whole soy milk powder. Sttisticl difference etween the experimentl groups sed on one-wy nlysis of vrince nd Duncn s multiple rnge tests t P < Mens with different lpheticl superscripts re significntly different (P < 0.05).

4 278 Isoflvone nd one metolism in ovriectomized mice (A) (B) (C) (D) (E) (F) Fig. 2. Effects of isoflvone-enriched whole soy milk powder on femur one morphology in ovriectomized mice. (A) G1: The femur in mice of the Shm group. (B) G2: The femur in mice of the OVX group. (C) G3: The femur in mice of the OVX + 2% I-WSM group. (D) G4: The femur in mice of the OVX + 10% I-WSM group. (E) G5: The femur in mice of the OVX + 20% I-WSM group. (E) G6: The femur in mice of the OVX + 20% WSM group. All pictures were stined with H & E nd exmined under x40 mgnifiction. Tle 3. Serum levels of clcium nd phosphorous in ovriectomized mice treted with isoflvone-enriched whole soy milk powder for 8 weeks. Groups Vriles Shm OVX C (mg/dl) 9.89 ± 0.25 P (mg/dl) ± 0.41 OVX + 2% I-WSM 9.17 ± ± ± ± OVX + 10% I-WSM 9.98 ± ± 0.39 OVX + 20% I-WSM OVX + 20% WSM ± ± ± ± 0.43 Vlues re expressed s men ± SE. Shm, shm-operted; OVX, ovriectomized; I-WSM, isoflvone-enriched whole soy milk powder; WSM, whole soy milk powder. Sttisticl difference etween the experimentl groups sed on one-wy nlysis of vrince nd Duncn s multiple rnge tests t P < Mens with different lpheticl superscripts re significntly different (P < 0.05). Tle 4. Serum levels of one turnover mrkers in ovriectomized mice treted with isoflvone-enriched whole soy milk powder for 8 weeks. Groups Vriles Shm OVX OVX + 2% I-WSM OVX + 10% I-WSM OVX + 20% I-WSM OVX + 20% WSM ALP (U/L) ± ± ± ± ± ± 4.1 OC (ng/ml) ± ± 3.61c ± 2.87c ± ± ± ± ± ± ± ± ± ± ± 0.27 P1NP (ng/ml) OPG (ng/ml) TRAP 5 (U/L) 2.75 ± ± ± ± ± ± ± ± ± ± 0.17 Vlues re expressed s men ± SE. Shm, shm-operted; OVX, ovriectomized; I-WSM, isoflvone - enriched whole soy milk powder; WSM, whole soy milk powder; ALP, lkline phosphtse; OC, osteoclcin; P1NP, procollgen type 1 mino-terminl propeptide; TRAP 5, trtrte-resistnt cid phosphtse 5; OPG, osteoprotegerin. Sttisticl difference etween the experimentl groups sed on one-wy nlysis of vrince nd Duncn s multiple rnge tests t P < Mens with different lpheticl superscripts re significntly different (P < 0.05) to the OVX group, the OVX + 2% I-WSM group did not show ny difference in BMD, wheres the OVX + 10% nd 20% I-WSM groups showed significnt increses in BMD. The OVX + 20% WSM nd OVX groups showed no difference in BMD. These results show tht greter thn 10% I-WSM supplementtion prevented reduction of BMD in ovriectomized postmenopusl nimls nd tht I-WSM ws more helpful in mintining BMD fter menopuse thn WSM. However, BMD of the OVX + 20% I-WSM group ws lower thn tht of the OVX + 10% I-WSM group. These results suggest tht the increse in BMD due to I-WSM supplementtion ws not in dose-dependent mnner. Further studies re needed to determine the levels of I-WSM supplementtion tht my e helpful in incresing BMD. As shown in Fig. 2, the density of the femorl corticl nd treculr one ws significntly lower in the OVX group compred to the Shm group. However, the mgnitude of the reduction tended to e lower in the OVX + I-WSM groups. Effects of I-WSM on serum levels of clcium nd phosphorous in ovriectomized mice Serum levels of C nd P in the experimentl groups were mesured in order to investigte the effect of I-WSM on minerl homeostsis, nd the results re shown in Tle 3. Serum C concentrtion of the OVX group ws lower thn tht of the Shm group; however, this vlue ws not sttisticlly significnt. In contrst, the concentrtions of C in the OVX + 10% I-WSM, OVX + 20% I-WSM, nd OVX + 20% WSM groups were significntly higher compred with the OVX group. Serum P concent-

5 So Mee Kim et l. 279 rtion ws significntly reduced y ovriectomy ut then recovered to e the sme level s the Shm group upon I-WSM or WSM supplementtion. Effects of I-WSM on serum levels of one turnover mrkers in ovriectomized mice Serum levels of one turnover mrkers in the experimentl groups re shown in Tle 4. In this study, ALP, OC, P1NP, nd OPG were mesured s one formtion mrkers, nd TRAP 5 ws mesured s one resorption mrker. ALP, n enzyme secreted from osteolsts, reflects the ctivity of osteolsts nd is n importnt index of osteogenesis. It hs een reported tht n increse in serum ALP level is ssocited with n increse in osteolst ctivity fter ovriectomy-induced osteoporosis [16]. In this study, serum levels of ALP were significntly incresed in the OVX group compred to the Shm group. Serum levels of ALP were significntly lower in the OVX + 10% I-WSM, OVX + 20% I-WSM, nd OVX + 20% WSM groups thn in the OVX group. OC, protein produced in osteolsts, reflects the ctivity of osteolsts nd is n importnt indictor of one formtion. The OC level ws significntly reduced in the OVX group compred to the Shm group, ut dose-dependently elevted in the OVX groups upon I-WSM supplementtion. In OVX mice supplemented with 20% I-WSM or 20% WSM, the OC level incresed to the sme level s the Shm group. OPG is n osteoclstogenesis inhiitory fctor tht inhiits ctivtion of osteoclsts nd promotes osteoclst poptosis [17], nd P1NP hs recently emerged s etter mrker of one formtion thn ALP [18]. In this study, P1NP nd OPG levels were significntly reduced y ovriectomy nd did not recover to the sme levels s the Shm group, even in OVX groups supplemented with I-WSM or WSM. Under norml circumstnces, TRAP is highly expressed y osteoclsts. TRAP expression is incresed under certin pthologicl conditions such s osteoporosis [19]. TRAP 5 level ws significntly elevted in the OVX group compred to the Shm group, wheres it decresed to the sme level s the Shm group in OVX groups supplemented with I-WSM or WSM. The results ove indicte tht chnges in ALP, OS, nd TRAP 5 in OVX mice cn e prevented y supplementtion with I-WSM or WSM. However, chnges in P1NP nd OPG due to OVX did not recover, even upon supplementtion with I-WSM or WSM. Therefore, in the context postmenopusl one metolism, isoflvone supplementtion seemed to hve greter effect on one formtion mrkers thn on one resorption mrkers, which my vry depending on the type of one formtion mrker under investigtion. DISCUSSION Estrogens re importnt regultors of ody composition nd lipid metolism in femles [20]. After menopuse, estrogen is no longer secreted, which often results in oesity [21]. This study ws conducted to determine the estrogenic effects of soy isoflvone, phytoestrogen tht ffects one metolism, in ovriectomized femle mice in vivo. In our study, OVX ICR mice were chosen s n niml model. A ilterl OVX ICR mouse model ws utilized to mimic the conditions in postmenopusl women [22]. It hs lredy een demonstrted tht ovriectomized mice nd rts show significnt increses in viscerl dipose tissue mss s well s n incresed risk of oesity compred to Shm controls [23,24]. In this study, the ody weight of the OVX group ws significntly higher thn tht of the Shm group, lthough there ws no difference in food intke mong the experimentl groups. OVX groups supplemented with I-WSM nd WSM lso hd significntly higher ody weight thn the Shm group. This differs from other studies, in which n isoflvone-enriched diet ws shown to inhiit weight gin in OVX rts [24]. This my e due to differences in the durtion nd dose of isoflvone supplementtion nd ge t ovriectomy. It hs een reported tht OVX nd OVX-induced oesity re ssocited with reduced one formtion [24-26]. In greement with dt from the literture [24-26], we oserved ovriectomyinduced one loss in the niml model in this study (Tle 2). Previous studies hve shown tht n isoflvone-rich diet exhiits one protective effects in OVX nimls. In this study, isoflvone supplementtion inhiited reduction of BMD in OVX mice (Fig 2, Tle 2). In ddition, lthough the gp ws reltively smll, I-WSM supplementtion seemed to e more effective in improving BMD of OVX mice compred to WSM supplementtion. However, improvement of femorl BMD y I-WSM supplementtion in OVX nimls ws not completely dose-dependent. Diets with 10% I-WSM or 20% I-WSM hd the effect of inhiiting OVXinduced BMD reduction compred to diet with 2% I-WSM. However, the respective effects of 10% nd 20% I-WSM on the femorl BMD of OVX mice did not significntly differ, while eing slightly higher in the 10% I-WSM supplementtion group. Therefore, further studies re needed to determine the optiml level of I-WSM supplementtion to prevent postmenopusl osteoporosis. Previous studies hve suggested tht isoflvones could exert estrogen-like effects in estrogen-deficient niml models nd postmenopusl women [5,12,27]. Studies y Wng [28] hve showed tht genistein supplementtion promotes one minerliztion y incresing one C, P, nd Mg, therey preventing osteoporosis y djusting serum clcitonin. Qi nd Zheng [5] reported tht genistein supplementtion (5 mg/g ody weight) for 10 weeks incresed serum levels of C nd P in ovriectomized rts. In this study, following tretment with I-WSM for 8 weeks, serum C nd P levels in OVX mice incresed (Tle 3). Prticulrly, serum C levels in the OVX + 10% I-WSM, OVX + 20% I-WSM, nd OVX + 20% WSM groups were higher compred to the Shm group (P <0.05). In this study, severl one turnover mrkers relted to one formtion nd one resorption were nlyzed. Mintennce of one mss is chieved through lnce etween the osteogenic ction of osteolsts nd the one resorption ction of osteoclsts [16]. After menopuse, one resorption occurs fster thn osteogenesis, which cuses loss of one mss [29]. Bone turnover mrkers hve een studied for decdes. Biochemicl monitoring of one metolism involves the mesurement of enzymes nd proteins secreted during one formtion nd one resorption. Although BMD is the est dignostic tool to dignose osteoporosis nd one turnover mrkers re used more s djunctive fctors, the effectiveness of one turnover mrkers hs een

6 280 Isoflvone nd one metolism in ovriectomized mice verified in numerous recent reports [30-32]. Bone turnover mrkers chnge more rpidly thn BMD or risk of frcture nd re thus useful for erly nlysis of osteoporosis tretment effects [32]. The serum one formtion mrkers nlyzed in this study were ALP, OC, PINP, nd OPG. ALP hs een cliniclly ville for severl yers s mrker for one metolism [33]. In ones, ALP is present in the cell memrne of osteolsts nd is n indictive mrkers of osteogenic ctivity [16]. OC, non-collgen one mtrix protein, is produced y osteolsts nd is implicted in one minerliztion [33]. As the one mtrix is minerlized y the deposition of newly formed OC, portion of it is relesed into the lood [33,34]. Therefore, serum OC level is considered s specific mrker of osteolst function relted to one formtion, s it correltes with the one formtion rte [35]. Procollgen type 1 contins N- nd C-terminl extensions, which re removed y specific proteses during conversion of procollgen to collgen. These extensions re the C- nd N-terminl propeptides of procollgen type 1 (P1CP nd P1NP). It ws reported tht P1NP is very sensitive mrker of the one formtion rte in osteoporosis [30]. OPG is lso known s osteoclstogenesis inhiitory fctor or tumor necrosis fctor receptor superfmily memer 11B. OPG production is stimulted in vivo y the femle sex hormone estrogen. OPG hs een reported to inhiit osteoclst ctivtion nd promote osteoclst poptosis [17]. OPG specificlly cts on one, incresing BMD nd one volume [36]. Moreover, OPG hs een used experimentlly to reduce one resorption in women with postmenopusl osteoporosis nd in ptients with lytic one metstses [37]. In this study, the levels of ALP, OC, P1NP, nd OPG were ll ltered to indicte osteoporosis fter ovriectomy (Tle 4). ALP level ws not significntly different etween the OVX nd Shm groups, wheres it significntly decresed in the OVX + 10% I-WSM nd OVX + 20% I-WSM groups compred to the Shm group. Serum levels of OC decresed significntly in the OVX group compred to the Shm group, ut incresed dosedependently y I-WSM supplementtion nd were recovered to the sme level s the Shm group. Serum levels of P1NP nd OPG showed significnt reductions in OVX to 44% nd 29%, respectively, compred with the Shm group, nd this decrese ws not reversed even fter I-WSM supplementtion. It ws reported in three previous rndomized controlled trils tht supplementtion with soy isoflvone hd no significnt effect on serum P1NP [38-40]. In contrst to our OC results, Fu et l. [12] reported in systemtic reviews nd met-nlyses tht phytoestrogen tretment incresed BMD nd significntly reduced one turnover mrkers such s ALP nd OC. In this study, I-WSM seemed to influence one formtion in OVX-induced postmenopusl niml models. However, these effects were significntly different depending on the type of one formtion mrker. In other words, regrding the four one formtion mrkers nlyzed in this study, I-WSM supplementtion in OVX-ICR mice ws most effective in ltering OC, slightly effective in ltering ALP, nd ineffective in ltering PINP nd OPG levels. TRAP 5, which hs een reported s sensitive mrker of one resorption, ws lso nlyzed in this study [41,42]. Humn serum contins two forms of TRAP, nmely 5 nd 5. Unlike 5, the 5 vrint contins silic cid. In this study, the level of TRAP 5 ws 138.8% higher thn tht of the Shm group following OVX (Tle 4). However, this drmticlly decresed to Shm level fter I-WSM supplementtion. Thus, I-WSM supplementtion in OVX menopusl mice seemed to e effective in preventing one resorption. In conclusion, this study exmined the effect of I-WSM on one metolism in ovriectomized menopusl mice. Our results suggest tht I-WSM supplementtion mde moderte contriution to prevention of BMD reduction in OVX mice, mintennce of serum C nd P levels, reduction of one resorption mrker (TRAP5), nd elevtion of one formtion mrker (OC). Therefore, I-WSM cn e used s n effective lterntive to postmenopusl osteoporosis prevention. This study is importnt to reffirming the supplementl effect of isoflvone on one metolism in postmenopusl niml models nd confirming the functionl properties of soyen milk products fortified with isoflvones through specil process. However, I-WSM did not demonstrte significnt improvement compred to WSM in terms of BMD, serum C nd P concentrtions, nd one turnover mrkers. These results do not seem to meet expecttions, considering tht I-WSM hs n 8.8 times higher isoflvone content thn WSM. This discrepncy is proly ecuse the effect of isoflvone on postmenopusl BMD ecomes sturted over certin concentrtion or due to the difference etween I-WSM nd WSM in terms of components thn isoflvone. Further studies will hve to e conducted on the optiml concentrtion, vilility, nd mechnism of ction of isoflvone-contining sustnces in preventing postmenopusl osteoporosis. CONFLICT OF INTEREST The uthors declre no potentil of interests. ORCID So Mi Kim: Hyun Sook Lee: Je In Jung: Su-Min Lim: Ji Hoon Lim: Wng-Hyun H: Chng Le Jeon: Je-Yong Lee: Eun Ji Kim: REFERENCES 1. Lecrt MP, Reginster JY. Current options for the mngement of postmenopusl osteoporosis. Expert Opin Phrmcother 2011;12: Seckin B, Pekcn MK, Inl HA, Gulermn C. The reltionship etween rest density nd one minerl density in never users of postmenopusl hormone therpy. 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