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Bisphosphonates in phenytoin-induced bone disorder

Bisphosphonates in phenytoin-induced bone disorder
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  Bisphosphonates in phenytoin-induced bone disorder Suruchi Khanna, Krishna K. Pillai, Divya Vohora ⁎ Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India a b s t r a c ta r t i c l e i n f o  Article history: Received 21 June 2010Revised 29 September 2010Accepted 20 October 2010Available online 30 October 2010Edited by: F. Cosman Keywords: AlendronateIbandronateRisedronatePhenytoinHomocysteineBone demineralization Chronicadministrationofphenytoin(PHT)hasbeenassociatedwithboneloss.Bisphosphonates[alendronate(ALD), ibandronate (IBD) and risedronate (RSD)] are potential candidates to prevent PHT-induced bonedisorders, and the present study evaluated their effect on the antiepileptic ef  󿬁 cacy of PHT. The PHT-induceddepletion in folic acid (FA), vitamin B6 and vitamin B12 results in hyperhomocysteinemia. The elevatedcirculating homocysteine(hcy)couldbeariskindicatorformicronutrient-de 󿬁 ciency-relatedosteoporosis via generation of free radicals. Thus, an attempt was also made to unravel the PHT's and bisphosphonates' effecton hcy. Male mice received PHT (35 mg/kg,  p.o. ) for 90 days to induce bone loss. ALD, RSD and IBD wereadministered orallyatdoses0.65 mg/kg, 0.33 mg/kg,and 0.17 mg/kgrespectively, forpreventionand 1.3 mg/ kg, 0.65 mg/kg, and 0.33 mg/kg respectively, for treatment of PHT-induced bone loss. The bone loss wascon 󿬁 rmed by bone mineral density (BMD) analysis and bone turnover markers. Serum levels of hcy and FAwere estimated along with hydrogen peroxide levels and total antioxidant capacity in order to assess theantioxidant pro 󿬁 le of bisphosphonates. The induction of bone loss by PHT was marked by lowered BMD andaltered bone turnovers. ALD and RSD administration to PHT treated groups signi 󿬁 cantly reverted the bonyadverse effects. No such effects were observed with IBD. In the bisphosphonates treated groups, hcy levelswere statistically at par with the control group. PHT at 35 mg/kg,  p.o.  could compromise bone mass and thus,could be a model of bone demineralization in mice. The ALD, IBD and RSD have no pharmacodynamicinteraction when administered along with PHT at the experimental level. Thus, their usage in the man-agement of PHT-induced bone disease could be worthwhile if clinically approved.© 2010 Elsevier Inc. All rights reserved. Introduction Epilepsy and AEDs intricately modulate the bone microarchitec-ture and BMD, affecting bone strength. For more than four decades,antiepileptic drugs (AEDs) have been known to cause serious effectson bone mineral density (BMD) [1 – 3]. AED therapy causes multipleabnormalities in calcium and bone metabolism, varying fromincreased bone turnover without signi 󿬁 cant loss of cortical or trabec-ular bone to osteopenia/osteoporosis and to osteomalacic disorder.Gross malformations in the bones allied mainly, but not solely, withthe cytochrome P450 (CYP450)-inducing AEDs and these AEDs mayact as an add-on to risk factors (e.g. seizure-precipitated falls andtrauma and sedentary lifestyle) for fractures in epileptics [4].Phenytoin (PHT) is a potent hepatic mixed-function oxidase(CYP450) inducer including CYP1A2, CYP2C9, CYP2C19 and CYP3A4,as well as glucuronyl transferases and epoxide hydrolase [5]. CYP450inductionin 󿬂 uencescalcium – vitaminDaxisbyreducingbio-availablevitamin D. Lowered circulating calcium owing to hypovitaminosis Dresults in compensatory secondary hyperparathyroidism. The en-hanced serum parathormone levels increase the bone calciummobilization and consequent bone turnover. Other pathophysiolog-ical mechanisms involved in PHT-induced bone loss may includecalcitonin de 󿬁 ciency, vitamin K de 󿬁 ciency, deprived estrogen levels,interventions with circulating homocysteine (hcy) levels and inhib-itory effect on collagen synthesis in cultured bone [4].With the advent of newer medications having less number of side effects, PHT is still considered a  󿬁 rst line drug to treat epilepsy(generalized tonic – clonic seizures and status epilepticus) [6,7]. Also,the AED-induced bone loss has been reported to be more pronouncedwith PHT intake [8,9]. PHT-induced bone loss may be an inevitableeffect but can be mitigated by supplementation of anti-osteoporoticmedications.Bisphosphonates, synthetic analogues of pyrophosphate, bind tohydroxyapatite at sites of active bone remodeling. Second generationbisphosphonate: alendronate (ALD) and third generation bispho-sphonates: ibandronate (IBD) and risedronate (RSD) are leadingnitrogen-containing bisphosphonates for prevention and treatmentof bone diseases  viz.  Paget's disease, hypercalcemia of malignancy,in various experimental models of osteoporosis [10]. They are FDA-approved anti-osteoporotic agents and are potent inhibitors of oste-oclastic action [11].During the course of anti-osteoporotic regimen (mainly onbisphosphonates, raloxifene, among others), if a patient is laterdiagnosed with epilepsy syndrome, it is yet to be documented which Bone 48 (2011) 597 – 606 ⁎  Corresponding author. E-mail addresses:, 8756-3282/$  –  see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.bone.2010.10.172 Contents lists available at ScienceDirect Bone  journal homepage:  anti-osteoporotic drugs should be administered along with AEDs. It isinteresting to note that the bisphosphonates have emerged as  󿬁 rst-linetreatmentdrugs for bone loss [12,13]. Therefore,it is necessary tocarry out research aimed at discovering novel bisphosphonates thatcan be prescribed alongside PHT.5-Methyl-tetrahydrofolic acid generated from folic acid (FA) isessential for hcy metabolism. Increase in the circulating hcy with PHTintake may be due to interference with FA, vitamin B6 and vitaminB12 metabolism [14]. Hcy not only interferes with collagen cross-linking during the bone remodeling process but also shows positivecorrelationwithfreeradicalgeneration.Thus,hyperhomocysteinemiamay be among several potential mechanisms involved in pathogen-esis of bone malformations [4].In view of increased prevalence of the AEDs-induced bonedisorders, and the recent proposal for the use of bisphosphonates inAED therapy [15,16]; we endeavored to determine by way of this  invivo  experiment whether their administration in PHT therapy willaffect PHT's antiepileptic ef  󿬁 cacy. In addition, the present studyevaluated their effect on PHT-induced alterations in the biochemicalmarkers of bone turnover and parameters related to probablemechanisms mainly hyperhomocysteinemia leading to bone loss, incomparison with calcium and vitamin D 3  (CVD) supplementation. Material and methods Experimental animals All experiments were performed on adult male albino  Swiss  miceweighing24 – 35 g.Theanimalswereprocuredfrom theCentralAnimalHouse, Jamia Hamdard (Hamdard University), New Delhi. Prior to thecommencement of the experiment, the animals were allowed toacclimate for 7 days; food and water were available  ad libitum . Themice were kept in a room maintained at 23±2 °C and 55±15%humidity with a 12h light – dark cycle. All animal procedures wereapproved by the ethics committee at our institution (Project no. 368,year: 2007) and performed in compliance with institutional guidelinesfor the care and handling of experimental animals. Drugs and chemicals The following drugs were used: phenytoin sodium (PHT) (Sigma-Aldrich, India); alendronate sodium (ALD) (Ranbaxy, India); risedro-nate sodium (RSD) (Fleming Laboratories Ltd., India); ibandronatesodium (IBD) (Sun Pharmaceuticals India Ltd., India); folic acid (FA)(Sigma-Aldrich, India); calcium and vitamin D 3  (CVD) (Cipcal ™ , CiplaLtd., India); vitamin D 3  (VD) (Calcikind ™ , Mankind Pharma Ltd.,India). All other chemicals used in this study were of analytical grade. Oral administration of drugs PHT, ALD, RSD, IBD and FA were dissolved in distilled water. CVDand VD were suspended in 1% aqueous carboxymethylcellulose. Allthe drugs were administered orally once daily for 7 days using afeeding needle. Selection of doses The doses of the bisphosphonates and CVD were calculated fromthecorrespondinghumandoses(Table1).PHTdosewascalibratedonthe basis of the histopathological analysis of femur and the PHT'splasma concentration.PHTat70 mg/kg,  p.o. (aconverteddosefromthepreviousstudybyNissen-Meyer et al. [17]) resulted in 30 – 40% mortality within 20 daysof drug administration. The probable reason could be higher plasmaconcentration at 70 mg/kg dose. Keeping the fact in view that thebone loss should be induced at clinically relevant drug concentrationtherefore, the dose was reduced to 35 mg/kg. 35 mg/kg of PHT wasfound to be a suitable dose to induce bone loss within the therapeuticconcentration (10 – 20  μ  g/mL) i.e. the deteriorating effect on boneswas produced in the therapeutic range and not at the levels ( N 30  μ  g/ mL) at which the toxicity signs begin to appear. The blood sampleswere withdrawn randomly to monitor the plasma concentration of PHT. The plasma PHT levels were well within the therapeutic concen-tration when administered alone and along with bisphosphonates. Experimental protocol The experimental protocol was divided into the following parts1. Preventive treatment: In this experiment, eleven groups of eightmice each were administered drugs once daily for the durationof three months: control group (0.5% CMC, 2 mL/kg); PHT (35 mg/ kg); ALD (0.65 mg/kg), RSD (0.33 mg/kg), IBD (0.17 mg/kg), FA(0.13 mg/kg), PHT (35 mg/kg)+ALD (0.65 mg/kg), PHT (35 mg/ kg)+RSD (0.33 mg/kg), PHT (35 mg/kg)+IBD (0.17 mg/kg),PHT (35 mg/kg)+FA (0.13 mg/kg), and PHT (35 mg/kg)+CVD(130 mg/kg+65 IU).2. Therapeutic treatment: In this experiment,  󿬁 fty four mice wereadministered PHT (35 mg/kg) once daily for the duration of threemonths. At the end of three months, the mice were divided intosix groups and administered the bisphosphonates, FA, CVD oncedaily along with PHT for a period of one month. The groups wereas follows: PHT (35 mg/kg); ALD (1.3 mg/kg); RSD (0.65 mg/kg);IBD (0.33 mg/kg); PHT (35 mg/kg)+ALD (1.3 mg/kg); PHT(35 mg/kg)+RSD (0.65 mg/kg); PHT (35 mg/kg)+IBD (0.33 mg/ kg); PHT (35 mg/kg)+FA (0.13 mg/kg); PHT (35 mg/kg)+CVDD[CVD (130 mg/kg+65 IU)+VD (195 IU)].At the end of each treatment, urine collection was done for 24 h.Themicewerefastedovernightpriortocollectionofbloodsamplesfromtail veinforbiochemicalestimations. Immediatelyafter bloodcollection the mice were euthanized for collection of brain tissue(for biochemical estimations) and femora (to measure BMD).3. Electroconvulsive group: In order to evaluate the threshold forelectroconvulsions, four groups of mice each containing 10 mice pergroup, were administered the drugs as per therapeutic treatmentgroup. Electroconvulsivethreshold(maximalelectroshockseizurethresholdtest) Electroconvulsions were produced by Electroconvulsive Treat-ment Unit (UGO BASILE, Italy). The pro-/anti-convulsant potentialwas evaluated in the model of maximal electroshock seizurethreshold (MEST) test [18]. An alternating current (50 Hz, 0.2 s) wasdelivered  via  ear-clip electrodes. Tonic hindlimb extension (the hind  Table 1 The doses of the ALD; RSD; IBD; FA; CVDD and CVD were calculated from thecorresponding human doses as shown below.Drugs Human dose Correspondingmice doseReferencesALD 5.0 mg 0.65 mg/kg [64]10.0 mg 1.3 mg/kg [65]RSD 2.5 mg 0.33 mg/kg [66]5.0 mg 0.65 mg/kgIBD 1.25 mg 0.17 mg/kg  – 2.5 mg 0.33 mg/kg [65]FA 1.0 mg 0.13 mg/kg pro/folic-acid.html;retrieved on 2/2/2007CVD Ca (1000 mg)+VD (500 IU/12.5  μ  g)Ca (130 mg/kg)+VD (65 IU/1.63  μ  g)[66]VD 2000 IU/50  μ  g(2000 − 500=1500 IU/37.5  μ  g)260 IU/6.5  μ  g(260 − 65=195 IU/4.88  μ  g)[67]598  S. Khanna et al. / Bone 48 (2011) 597  – 606   limbs of animals outstretched 180° to the plane of the body axis) wastaken as the endpoint. The bisphosphonates were tested for theirpro-/anti-convulsant potential by determining their ability to protect50% of animals against the maximal electroshock-induced tonichindlimb extension and expressed as respective current strength. Todetermine the value of median current strength (CS 50  in mA), at leastfour groups of mice (10 animals per group) were challenged withcurrents of various intensities. Then, a current intensity – effect curvewas constructed, according to a log-probit method by Litch 󿬁 eld andWilcoxon, [19] from which CS 50  in mA was estimated.TocalculateCS 50 ,  per se groupsofbisphosphonatesweresubjectedto various current intensities ranging from 10 to 20 mA at theirpeakplasmatimeintervals.Inordertoevaluateanypossiblevariationin PHT's antiepileptic potential with bisphosphonates, PHT aloneand in combination with bisphosphonates were subjected to variouscurrent intensities ranging from 10 to 50 mA. The animals wereadministered bisphosphonates in such a way that their peak plasmalevels(approx1 h)matchedthepeakplasmalevelsofPHT(approx2 h). Bone tissue sample preparation and assays performed The left femora were cleaned of soft tissues and then frozen at − 20 °C. Before the dual-energy X-ray absorptiometry (DEXA)examination for BMD analysis, they were  󿬁 rst defrosted for 30 min.The femora were scanned by DEXA using a Hologic (QDR 4500, USA).The right femora were dissected out and the surrounding musclesandtissueswereremoved.Thefemorawereweighedindividuallyandhomogenized with 10 volumes of 10 mM triethanolamine buffer (pH7.5).Thehomogenatewasstirredfor1.5 hat4 °Candcentrifuged.Theextraction procedure was repeated twice and aliquots of the boneextracts were used for the determination of the activities of alkalinephosphatase (ALP) and tartrate resistant acid phosphatase (TRAP).The insoluble pellets were hydrolyzed with 6 N HCl at 105 °C for 24 hand analyzed for hydroxyproline (HxP).ALP activity was determined using commercial kit (SPAN Diag-nostics, India). TRAP activity was estimated by the method of Tenniswood et al. [20]. The protein estimation was done by themethod of Lowry et al. [21]. The amount of HxP was determinedaccording to the method of the chloramine-T oxidation procedure of Stegemann [22]. Serum preparation and assays performed Serum was separated by centrifugation for 10 min at 3000 rpm.Serum samples were stored at − 20 °C until analysis was carried out.Serum hcy levels were measured by using commercially available kitbased on the principle of   󿬂 uorescence polarization immunoassay(FPIA) method on the AxSYM System analyzer (Abbott Laboratories,Abbott Park, IL). FA levels were estimated by using commerciallyavailable Solid Phase No Boil Assay Kit® (Seimens Medical SolutionDiagnostics, Los Angeles, USA). Total antioxidant capacity (TAC) wasestimated by the Koracevic et al. [23]. Urine collection and assays performed Urine collection was done for 24 h in a clean  󿬂 ask placed on ice.Hydrogen peroxide (HP) determination was done by using commer-cially available Hydrogen Peroxide Assay Kit® (Cayman ChemicalCompany, Ann Arbor, MI) Statistical analysis Data analysis was carried out by using Graphpad Prism 3.0(Graphpadsoftware;San Diego,CA).CS 50  valueswiththeirrespective95% con 󿬁 dence limits were estimated using computer log-probitanalysisaccordingtoLitch 󿬁 eldandWilcoxon[19].Subsequently,SEMvalues were calculated on the basis of con 󿬁 dence limits, number of animals and slope function obtained directly from log-probit analysisaccording to Litch 󿬁 eld and Wilcoxon [19]. The statistical evaluationof respective CS 50  vs. control values was performed with analysisof variance (ANOVA) followed by post-hoc Bonferroni's test. Theother parameters mentioned earlier were expressed as mean±SEM.Groups of data were compared with ANOVA followed by Tukey – Kramer multiple comparison tests. Values were considered statisti-cally signi 󿬁 cant at P b 0.05. Results Electroconvulsive threshold test  CS 50  values after treatment with the bisphosphonates alone areshowninTable2.Bisphosphonatesatbothdosesdidnotaffectseizure threshold. No signi 󿬁 cant change in CS 50  values was observed ascomparedtocontrolgroup.PHTat35 mg/kgafforded100%protectionagainst MEST as evidenced by complete abolition of the tonic ex-tension phase. Combination groups of PHT and bisphosphonates (atboth the doses) were unable to produce any signi 󿬁 cant change in thetonic extension phase observed during seizure activity and elicitedsimilar responses as those with PHT  per se . Bisphosphonates did notaffect the antiepileptic activity of PHT. BMD analysis The changes in BMD of the left femur determined by DEXA areshownin Fig. 1. There was a signi 󿬁 cant loweringin BMD levels of PHTgroup as compared with the control group (p b 0.001). Both ALD andRSD, whether given concurrently or post PHT treatment substantiallyincreasedtheBMDlevelsascomparedwiththePHTgroup(p b 0.001).Furthermore, the BMD level in the PHT+RSD group was signi 󿬁 cantlyhigher than that in the PHT+ALD group (p b 0.01). IBD neitherprevented nor treated the PHT induced decrease in BMD (p N 0.05).Both ALD and RSD were more effective than CVD and CVDD inpreventing the decline of BMD levels. FA treatment was ineffectivein preventing BMD reduction in PHT administered (preventive andtreatment) groups. Bone turnover markers a) ALP activityThe differences in the femoral ALPactivity betweenthe groups areshown in Table 3. Long-term treatment with PHT signi 󿬁 cantlyattenuated ALP activity in the femoral bones. ALD, IBD and RSD  per se  groups at both doses did not show any signi 󿬁 cant change inthe femoral ALP activity as compared to control group (p N 0.05).ALD, RSD, CVD and CVDD prevented the diminutive effect of PHT  Table 2 Effectofchronicadministrationofbisphosphonates aloneandincombinationwithPHTin electroshock-induced seizures.Treatment (mg/kg) CS 50  (mA)Vehicle 14.16±0.11ALD (0.65) 13.85±0.38ALD (1.3) 13.70±0.18RSD (0.33) 14.24±0.35RSD (0.65) 14.40±0.42IBD (0.17) 13.94±0.97IBD (0.33) 13.87±0.26PHT No hind limb extensionPHT+bisphosphonates No hind limb extensionResults are expressed as mean±SEM; CS 50 : a current strength inducing tonic – clonicconvulsions in 50% of tested mice.599 S. Khanna et al. / Bone 48 (2011) 597  – 606   signi 󿬁 cantly (p b 0.001). In addition, the concomitant administra-tion of PHT and FA reversed the decreased bone ALP levels in mice(p b 0.05).b) TRAP activityA signi 󿬁 cant elevation in TRAP levels was observed in the groupsof mice treated with PHT (Table 3). ALD, RSD, FA, CVD and CVDDreversed the elevated TRAP levels signi 󿬁 cantly in PHT treated(preventive and treatment) groups. IBD administration did notreverse elevated TRAP activity. The concomitant treatment of FAwith PHT and the post treatment of FA in mice treated with PHTwere found to slightly lower the elevated TRAP activity (p b 0.05).c) HxP concentrationTable3summarizestheeffectofbisphosphonatesandcalciumandvitamin D supplements on HxP content. Femora of PHT-treatedgroups contained less HxP/mg of bone as compared to the vehiclecontrolgroup(p b 0.001).Inthegroupsreceivingbisphosphonates,CVD and CVDD along with the PHT, HxP levels were elevated ascompared to PHT groups (p b 0.001). FA and IBD did not alter HxPlevels when compared with PHT-administered mice (p N 0.05). TAC and HP measurement  Compared with the control group, the TAC was depleted in PHTtreated groups. As shown in Table 4, bisphosphonates, CVDand CVDD groups statistically altered the TAC levels (p b 0.001). Treatment withFAinPHT-administeredmicewasfoundtoslightlyenhanceTAClevelsas compared to the mice treated with PHT (p b 0.05).Table 4 depicts the effect of drug treatments on urinary HP levels.HP levels were found to be considerably enhanced in PHT groups incomparison to the group administered with normal saline (p b 0.001).Bisphosphonates, FA, CVD and CVDD treated groups had a signi 󿬁 canteffect in lowering the elevated HP levels in comparison to the grouptreated with PHT (p b 0.001). Hcy and FA levels The changes in the serum hcy levels between the groups arerepresented in Table 5. The serum hcy levels increased signi 󿬁 cantlywithPHT treatmentandthe anti-resorptive drugs(ALD, RSDandIBD)approximately lowered the elevated hcy to the baseline level.Bisphosphonates alone did not alter the serum hcy levels. CVD andCVDD administration statistically lowered the hcy levels in PHT-treated groups (p b 0.05). The combined administration of FA withPHT almost completely reversed the elevated hcy levels in PHTtreatedgroups.TheresultsofPHT+ALD/RSDgroupswereatparwithPHT+FA group.Mice treated with PHT had decreased serum FA levels, whichwere signi 󿬁 cantly different from control group (p b 0.01) as shown inTable 5. No signi 󿬁 cant differences were noted with FA levels betweenbisphosphonates  per se  and combinational groups of PHT and ALD/ RSD/IBD/CVD/CVDD groups (p N 0.05). However, the administrationof FA along with PHT-treated mice normalized the lowering effectof PHT on serum FA levels (p b 0.001) in comparison to PHT-treatedmice. Discussion The study provided  󿬁 rst experimental evidence on bisphospho-nates' effect on seizures and antiepileptic ef  󿬁 cacy of PHT as well as onPHT-induced metabolic bone loss. Bisphosphonates' effect on seizures and antiepileptic ef   󿬁 cacy of PHT  Resultspresentedinourstudyshowedthatbisphosphonates(ALD,RSD and IBD) at both doses revealed no appreciable changes in theCS 50  as compared to the control group suggesting that the bispho-sphonates (ALD, RSD and IBD) have no inherent pro-/anti-convulsantproperty. Concurrent administration of PHT and bisphosphonates(ALD,RSDandIBD)elicitedsimilarresponsesasthosewithPHT  perse .This clearly indicates that bisphosphonates (ALD, RSD and IBD) didnot alter the electroconvulsive threshold of PHT in MEST test(Table 2). This rules out any possible interaction of bisphosphonateswith the PHT at the pharmacodynamic level on long-term therapy. Bisphosphonates in PHT-induced metabolic bone loss The prolong therapy with PHT has been associated with changesin bone turnover cycle [17,24]. At the end of a span of 30, 60 and90 days, the left femur and lumbar vertebrae (L2 – L4) were dissectedout from the mice selected randomly in order to detect the changesrelated to bone loss through histopathological analysis (Figs. 2a – f).The femur dissected after 90 days showed the prominent thinningof bone matrix; an enhanced osteoclastic activity; and prominentruf  󿬂 ed border (Fig. 2d). This is in line with the studies performed byValimaiki et al. [25] and Moro-Alvarez et al. [26] revealing that the PHTaffects cortical bonesmoreprofoundly thanthe trabecular bones. Fig. 1.  Effects of PHT, bisphosphonates and their combination on femoral bone mineral density (BMD). Values are represented as mean±SEM; ***P b 0.001 versus control; ### P b 0.001 versus PHT,  ns P N 0.05 versus PHT;  $$ P b 0.01 versus PHT+CVD/CVDD;  $$$ P b 0.001 versus PHT+CVD/CVDD.600  S. Khanna et al. / Bone 48 (2011) 597  – 606    Table 4 Effects of combinations of bisphosphonates with PHT on TAC and HP levels.Groups (n=8)Control PHT ALD IBD RSD FA PHT+FA PHT+ALD PHT+IBD PHT+RSD PHT+CVD PHT+CVDDTAC (mmol/L) [preventive treatment]1.30±0.091 0.66±0.098*** 1.23±0.062 1.25±0.064 1.24±0.059 1.28±0.084 1.15±0.112 # 1.33±0.085 ### 1.32±0.109 ### 1.37±0.089 ### 1.33±0.139 ### – TAC (mmol/L) [therapeutic treatment]1.30±0.091 0.52±0.073*** 1.28±0.054 1.24±0.059 1.27±0.058  –  0.85±0.075 # 1.15±0.050 ### 1.19±0.065 ### 1.23±0.070 ### –  1.00±0.075 ### HP ( μ  mol/L) [preventive treatment]8.55±0.718 21.80±0.898*** 6.18±0.758 7.05±0.647 7.11±0.720 9.91±0.774 16.06±0.870 ### 10.03±0.877 ### 10.80±0.586 ### 8.97±0.637 ### 16.46±0.970 ### – HP ( μ  mol/L) [therapeutic treatment]8.55±0.718 29.93±2.03*** 8.19±1.021 8.44±0.984 8.03±0.997  –  20.29±1.174 ### 14.01±1.209 ### 12.79±1.293 ### 13.04±1.071 ### –  21.15±1.775 ### Values are represented as mean±SEM; n = number of animals; ***p b 0.001 versus control;  # p b 0.05 versus PHT,  ### p b 0.001 versus PHT.  Table 3 Effects of combinations of bisphosphonates with PHT on bone turnover markers.Groups (n=8)Control PHT ALD IBD RSD FA PHT+FA PHT+ALD PHT+IBD PHT+RSD PHT+CVD PHT+CVDDALP ( μ  mol of PNP liberated/h/  μ  g of protein) [preventive treatment]12.05±1.018 5.40±0.735*** 11.92±1.05 11.96±1.017 12.51±0.979 11.24±1.038 10.51±1.201 # 12.59±0.923 ### 10.40±1.001 # 12.97±0.872 ### 12.94±0.903 ### – ALP ( μ  mol of PNP liberated/h/  μ  g of protein) [therapeutic treatment]12.05±1.018 4.87±0.899*** 12.19±0.490 12.18±0.445 12.35±0.753  –  8.05±0.936 # 10.83±0.291 ### 6.59±0.711 ns 11.21±0.192 ### –  10.02±0.298 ### TRAP ( μ  mol of PNP liberated/h/  μ  g of protein) [preventive treatment]0.694±0.027 1.064±0.031 *** 0.587±0.023 0.686±0.027 0.621±0.024 0.639±0.031 0.934±0.020 # 0.708±0.013 ### 0.973±0.023 ns 0.704±0.008 ### 0.703±0.017 ### – TRAP ( μ  mol of PNP liberated/h/  μ  g of protein) [therapeutic treatment]0.694±0.027 1.284±0.016 *** 0.618±0.031 0.709±0.031 0.634±0.015  –  1.090±0.079 # 0.842±0.019 ### 1.132±0.054 ns 0.818±0.021 ### –  0.893±0.021 ### HxP ( μ  g of HxP/mg of bone) [preventive treatment]156.95±9.188 104.64±7.856*** 147.86±7.364 147.13±7.803 151.06±7.061 148.70±6.027 113.45±7.013 ns 156.67±6.794 ### 103.27±7.990 ns 156.02±8.743 ### 156.18±6.837 ### – HxP ( μ  g of HxP/mg of bone) [therapeutic treatment]156.95±9.188 90.67±8.454*** 158.62±11.612 156.44±6.149 160.68±9.219  –  92.39±8.164 ns 153.74±8.721 ### 105.26±10.056 ns 151.61±10.051 ### –  152.68±7.900 ### Values are represented as mean±SEM; n = number of animals; ***p b 0.001 versus control;  # p b 0.05 versus PHT,  ### p b 0.001 versus PHT,  ns p N 0.05 versus PHT.  6   0  1    S   .K  h   a  n n a  e  t    a  l     .  /    B   o  n e 4   8    (   2   0  1  1    )    5   9   7   –  6   0   6  
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