The effect of VPA on bone: From clinical studies to cell cultures—The molecular mechanisms revisited

Open ArchivePublished:March 22, 2017DOI:https://doi.org/10.1016/j.seizure.2017.03.013

      Highlights

      • The overall effect of valproic acid on bone is an increased fracture rate.
      • Valproic acid decreases osteoblast proliferation and alters the collagen synthesis.
      • Vitamin D catabolic enzymes are induced by valproic acid.
      • Valproic acid increases indirectly bone fragility through endocrine side effects.

      Abstract

      Purpose

      Valproic acid (VPA) is a broad-spectrum antiepileptic drug, which is widely used as a first line treatment for idiopathic and symptomatic generalized epilepsy, as well as in non-epileptic psychiatric disorders in adult and pediatric patients. Although valproic acid is considered to be a generally well–tolerated drug, numerous studies have shown an increased bone loss and fracture risk in patients treated with VPA. The purpose of this review is to outline recent findings on VPA molecular mechanisms and their action on bone metabolism.

      Methods

      Unrestricted electronic search of medical databases, complemented by additional manual searches, was performed by August 2016.

      Results/conclusion

      The main effects of VPA on bone metabolism involve a decrease in osteoblast proliferation, changes in collagen synthesis as well as an induction of vitamin D catabolism. Apart from these direct actions of VPA in bone, indirect effects affecting other endocrine organs also contribute to VPA-induced bone loss.

      Keywords

      1. Introduction

      Valproic acid (2-propylpentanoic acid, N-dipropylacetic acid) is a branched short-chain fatty acid, which derived from valeric acid and was synthesized by Burton in 1882 [
      • Burton B.S.
      On the propyl derivatives and decomposition products of ethylacetate.
      ]. Valproic acid (VPA) was initially used as molecule carrier. It was in 1963 that Meunier, while he was studying the antiepileptic effects of new molecules against seizures induced by pentelenetetrazole in experimental animals, reported that VPA prevented pentylenetetrazol-induced convulsions in rodents [
      • Meunier H.C.G.
      • Meunier V.
      • Eymard M.
      Propriétés pharmacodynamiques de l’ácide n-propylacétique.
      ]. One year later Carraz et al. [
      • Carraz G.
      • Fau R.
      • Chateau R.
      • Bonnin J.
      • et al.
      Communication concerning 1 st clinical tests of the anticonvulsive activity of N-dipropylacetic acid (sodium salt).
      ] carried out the first human study leading to the acknowledgement of VPA antiepileptic properties, which was approved in 1978 by FDA as a first-line anti-epileptic drug.
      Despite its well-known anti-convulsive activity, VPA is also effectively used in non-epileptic conditions, such as migraine and bipolar disorders, while it has also been recently explored for its use as an adjuvant anti-cancer agent. More precisely, VPA is found to suppress tumor growth and tumor angiogenesis because of its action as histone deacetylase (HDAC) inhibitor. Histone acetylation increases gene transcription, while histone deacetylation suppresses the transcription process. VPA induces HDAC inhibition, histone acetylation and hyperacetylation accumulation, reverse HDAC-mediated transcriptional repression and subsequently mediates in various cell functions such as cell differentiation and cell apoptosis [
      • Sun L.
      • Coy D.
      Anti-convulsant drug valproic acid in cancers and in combination anti-cancer therapeutics 2.
      ].
      VPA is considered to be a generally well-tolerated drug with serious side effects, such as hepatotoxicity, pancreatitis and teratogenicity rarely being reported. Among its long-term side effects, osteoporosis and increased fracture risk have been extensively studied in humans presenting inconsistent results, while the underlying mechanisms remain largely unknown. In this review we outline recent studies concerning the effect of VPA on bone cells and the molecular mechanisms implicated.

      2. Overview of bone metabolism

      The human skeleton is composed of cortical and trabecular bone, which undergoes continuous renewal throughout lifetime. Bone remodeling occurs through highly tuned and concerted actions of the bone cells, which resorb damaged, old bone (osteoclasts) and form and lay down new bone matrix (osteoblasts) under the tight regulation of the osteocytes, which act as the bone mechanostat and orchestrate bone renewal according to what is needed in every bone unit (Fig. 1).
      Fig. 1
      Fig. 1A schematic overview of bone remodeling.
      Osteoclasts are large multi-nucleated cells deriving from the mononuclear hematopoietic cell lineage. Differentiation of monocytes into active resorbing osteoclasts is regulated positively by the receptor activator of nuclear factor kappa B ligand (RANKL) and the macrophage-stimulating colony factor (M-CSF), and negatively by osteoprotegerin (OPG) [
      • Yavropoulou M.P.
      • Yovos J.G.
      Osteoclastogenesis–current knowledge and future perspectives.
      ]. Mature osteoclasts present a ruffled border, which is a series of deep folds in the area of the plasma membrane in contact with the bone matrix, secreting lysosomal enzymes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K [
      • Vaananen H.K.
      • Zhao H.
      • Mulari M.
      • Halleen J.M.
      • et al.
      The cell biology of osteoclast function.
      ]. Bone is then resorbed by acidification and proteolysis of the collagen bone matrix and hydroxyapatite crystals [
      • Hadjidakis D.J.
      • Androulakis I.I.
      Bone remodeling.
      ]. During bone resorption signals secreted by osteoclasts, osteocytes or the resorbed bone matrix itself attract osteoblasts, promoting the coupling of bone resorption with bone formation.
      Osteoblasts are descendants of the mesenchymal stem cell (MSC) lineage, along with adipocytes, chondrocytes, myoblasts and fibroblasts. Osteoblast differentiation from multipotent MSCs is mainly dependent on the transcription factors runt-related transcription factor 2 or osterix [
      • Ducy P.
      • Zhang R.
      • Geoffroy V.
      • Ridall A.L.
      • Karsenty G.
      • et al.
      Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.
      ]. Bone formation takes place in three stages: 1) production of osteoid matrix, 2) maturation of osteoid matrix, and 3) mineralization of the matrix. Osteoblasts secrete various autocrine and paracrine factors, such as transforming growth factor-beta, bone morphogenetic proteins, RANKL, M-CSF and OPG [
      • Chen D.
      • Zhao M.
      • Mundy G.R.
      Bone morphogenetic proteins.
      ,
      • Sims N.A.
      • Gooi J.H.
      Bone remodeling: Multiple cellular interactions required for coupling of bone formation and resorption.
      ,
      • Chen G.
      • Deng C.
      • Li Y.P.
      TGF-beta and BMP signaling in osteoblast differentiation and bone formation.
      ,
      • Nakahama K.
      Cellular communications in bone homeostasis and repair.
      ]. After fulfilling their bone formation role, osteoblasts assume one of three fates: 1) undergo apoptosis, 2) remain on the bone surface as flat bone lining cells, or 3) become entombed in the newly-formed bone matrix as terminally-differentiated osteocytes. While the exact role of bone lining cells is not well understood, it has been suggested that they may be involved in the initiation of bone remodeling [
      • Parfitt A.M.
      Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone.
      ]. Osteocytes are buried in the bone matrix in lacunae, while their long slender cell processes connect to other osteocytes and the bone surface through narrow channels called canaliculi. The resulting interconnected network is known as the lacunar-canalicular system. In contrast to short living osteoclasts (2–3 weeks) and osteoblasts (1 month), osteocytes are the longest living cell population in bone and are responsible for sensing mechanical forces imposed on bone and translating them to biochemical cues, which ultimately regulate bone turnover.

      3. The effect of enzyme – inducers and non-enzyme inducers anti-epileptic drugs on bone metabolism

      The underlying pathophysiological mechanism of anti-epileptic drugs (AEDs)- effect on bone metabolism is multifactorial, and includes activation of cytochrome p450 enzyme, increased bone turnover and increased urinary loss of calcium and phosphorus [
      • Lin C.M.
      • Fan H.C.
      • Chao T.Y.
      • Chu D.M.
      • Lai C.C.
      • Wang C.C.
      • et al.
      Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.
      ]. The relationship between AED type and fracture risk, however, remains uncertain.
      The conventional enzyme-inducing AEDs(EIAEDs), such as phenytoin, phenobarbital, primidone and carbamazepine, induce the hepatic cytochrome P450 enzyme system and are the AEDs most commonly associated with decreased bone mass and increased fracture risk. The main mechanism is via accelerated catabolism of 1,25 vitamin D to 24,25 vitamin D leading to vitamin D deficiency, decreased intestinal absorption of calcium and secondary hyperparathyroidism [
      • Pack A.M.
      • Olarte L.S.
      • Morrell M.J.
      • Flaster E.
      • Resor S.R.
      • Shane E.
      Bone mineral density in an outpatient population receiving enzyme-inducing antiepileptic drugs.
      ,
      • Sheth R.D.
      • Wesolowski C.A.
      • Jacob J.C.
      • Penney S.
      • Hobbs G.R.
      • Riggs J.E.
      • et al.
      Effect of carbamazepine and valproate on bone mineral density.
      ,
      • Stephen L.J.
      • McLellan A.R.
      • Harrison J.H.
      • Shapiro D.
      • Dominiczak M.H.
      • Sills G.J.
      • et al.
      Bone density and antiepileptic drugs: a case-controlled study.
      ,
      • Verrotti A.
      • Greco R.
      • Latini G.
      • Morgese G.
      • Chiarelli F.
      Increased bone turnover in prepubertal: pubertal, and postpubertal patients receiving carbamazepine.
      ].
      On the other hand, limited data are available regarding the effect of non-enzyme-inducers (AEDs), such as lamotrigine, oxcarbamazepine, levetiracetam and topiramate, on bone and calcium metabolism [
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      ,
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ].
      The impact of VPA, which is an inhibitor of the cytochrome P450 enzyme, on bone metabolism is still controversial. Earlier studies had reported no association between the use of VPA and bone loss while more recent ones demonstrated abnormal biochemical indices of bone and mineral metabolism, and higher fracture rates after long-term treatment in both children and adults [
      • Stephen L.J.
      • McLellan A.R.
      • Harrison J.H.
      • Shapiro D.
      • Dominiczak M.H.
      • Sills G.J.
      • et al.
      Bone density and antiepileptic drugs: a case-controlled study.
      ,
      • Verrotti A.
      • Greco R.
      • Latini G.
      • Morgese G.
      • Chiarelli F.
      Increased bone turnover in prepubertal: pubertal, and postpubertal patients receiving carbamazepine.
      ,
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ,
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      ,
      • Pack A.M.
      • Morrell M.J.
      • Marcus R.
      • Holloway L.
      • Flaster E.
      • Done S.
      • et al.
      Bone mass and turnover in women with epilepsy on antiepileptic drug monotherapy.
      ,
      • Vestergaard P.
      Epilepsy: osteoporosis and fracture risk – a meta-analysis.
      ,
      • Wu S.
      • Legido A.
      • De Luca F.
      Effects of valproic acid on longitudinal bone growth.
      ,
      • Valsamis H.A.
      • Arora S.K.
      • Labban B.
      • McFarlane S.I.
      Antiepileptic drugs and bone metabolism.
      ].

      4. The effect of valproic acid on bone metabolism

      4.1 Clinical studies

      4.1.1 Bone mineral density and fracture risk

      Clinical trials, investigating the effects of VPA on bone mineral density (BMD) and fracture risk, have reported inconsistent results. In some studies VPA monotherapy in children or young adults did not affect BMD values at the femoral neck and lumbar spine [
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      ,
      • Akin R.
      • Okutan V.
      • Sarici U.
      • Altunbas A.
      • Gokcay E.
      Evaluation of bone mineral density in children receiving antiepileptic drugs.
      ,
      • Elliott J.O.
      • Jacobson M.P.
      • Haneef Z.
      Homocysteine and bone loss in epilepsy.
      ,
      • Erbayat Altay E.
      • Serdaroglu A.
      • Tumer L.
      • Gucuyener K.
      • Hasanoglu A.
      Evaluation of bone mineral metabolism in children receiving carbamazepine and valproic acid.
      ,
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      ,
      • Serin H.M.
      • Koc Z.P.
      • Temelli B.
      • Esen I.
      The bone mineral content alterations in pediatric patients medicated with levetiracetam, valproic acid, and carbamazepine.
      ,
      • Tekgul H.
      • Serdaroglu G.
      • Huseyinov A.
      • Gokben S.
      Bone mineral status in pediatric outpatients on antiepileptic drug monotherapy.
      ,
      • Triantafyllou N.
      • Lambrinoudaki I.
      • Armeni E.
      • Evangelopoulos E.M.
      • Boufidou F.
      • Antoniou A.
      • et al.
      Effect of long-term valproate monotherapy on bone mineral density in adults with epilepsy.
      ], while in several others a significant BMD reduction at the same sites was reported after treatment with VPA for more than 6 months [
      • Sheth R.D.
      • Wesolowski C.A.
      • Jacob J.C.
      • Penney S.
      • Hobbs G.R.
      • Riggs J.E.
      • et al.
      Effect of carbamazepine and valproate on bone mineral density.
      ,
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ,
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      ,
      • Albaghdadi O.
      • Alhalabi M.S.
      • Alourfi Z.
      • Youssef L.A.
      Bone health and vitamin D status in young epilepsy patients on valproate monotherapy.
      ,
      • Andress D.L.
      • Ozuna J.
      • Tirschwell D.
      • Grande L.
      • Johnson M.
      • Jacobson A.F.
      • et al.
      Antiepileptic drug-induced bone loss in young male patients who have seizures.
      ,
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      ,
      • Boluk A.
      • Guzelipek M.
      • Savli H.
      • Temel I.
      • Ozisik H.I.
      • Kaygusuz A.
      The effect of valproate on bone mineral density in adult epileptic patients.
      ,
      • Gniatkowska-Nowakowska A.
      Fractures in epilepsy children.
      ,
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      ,
      • Kafali G.
      • Erselcan T.
      • Tanzer F.
      Effect of antiepileptic drugs on bone mineral density in children between ages 6 and 12 years.
      ,
      • Kumandas S.
      • Koklu E.
      • Gumus H.
      • Koklu S.
      • Kurtoglu S.
      • Karakukcu M.
      • et al.
      Effect of carbamezapine and valproic acid on bone mineral density: IGF-I and IGFBP-3.
      ,
      • Oner N.
      • Kaya M.
      • Karasalihoglu S.
      • Karaca H.
      • Celtik C.
      • Tutunculer F.
      Bone mineral metabolism changes in epileptic children receiving valproic acid.
      ,
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      ,
      • Sato Y.
      • Kondo I.
      • Ishida S.
      • Motooka H.
      • Takayama K.
      • Tomita Y.
      • et al.
      Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.
      ,
      • Song X.Q.
      • Wang Z.P.
      • Bao K.R.
      • Zhang J.M.
      • Wu J.
      • Yan C.H.
      • et al.
      Effect of carbamazepine and valproate on bone metabolism in children with epilepsy.
      ] (Table 1).
      Table 1Studies with BMD outcomes under long-term treatment with VPA.
      StudyNumber of participantsAgeYears of treatmentResults
      Hamed et al.
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      2331.9 ± 5.6210.57 ± 3.55 yearsLower BMD, BMC, Z-score, T-score at the femoral neck and lumbar spine
      Triantafyllou et al.
      • Triantafyllou N.
      • Lambrinoudaki I.
      • Armeni E.
      • Evangelopoulos E.M.
      • Boufidou F.
      • Antoniou A.
      • et al.
      Effect of long-term valproate monotherapy on bone mineral density in adults with epilepsy.
      4132.3 ± 8.210.6 ± 7.4No correlation with BMD
      Andress et al.
      • Andress D.L.
      • Ozuna J.
      • Tirschwell D.
      • Grande L.
      • Johnson M.
      • Jacobson A.F.
      • et al.
      Antiepileptic drug-induced bone loss in young male patients who have seizures.
      3145 ± 718 ± 10 yearsDecrease in femoral neck BMD
      Pack et al.
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      1430 ± 71 yearUnaffected
      Albaghdadi et al.
      • Albaghdadi O.
      • Alhalabi M.S.
      • Alourfi Z.
      • Youssef L.A.
      Bone health and vitamin D status in young epilepsy patients on valproate monotherapy.
      5026 ± 7.2Lower BMD and Z-score of lumbar spine and femoral neck
      Serin et al.
      • Serin H.M.
      • Koc Z.P.
      • Temelli B.
      • Esen I.
      The bone mineral content alterations in pediatric patients medicated with levetiracetam, valproic acid, and carbamazepine.
      288.6 ± 4.6Z-score unaffected
      Sato et al.
      • Sato Y.
      • Kondo I.
      • Ishida S.
      • Motooka H.
      • Takayama K.
      • Tomita Y.
      • et al.
      Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.
      40>18yearsDecrease in BMD, T-score, Z-score of second metacarpal
      Kim et al.
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      18–506 monthsBMD unaffected
      Boluk et al.
      • Boluk A.
      • Guzelipek M.
      • Savli H.
      • Temel I.
      • Ozisik H.I.
      • Kaygusuz A.
      The effect of valproate on bone mineral density in adult epileptic patients.
      >18years6 monthsLumbar spine, femoral neck BMD decreased
      Tekgul et al.
      • Tekgul H.
      • Serdaroglu G.
      • Huseyinov A.
      • Gokben S.
      Bone mineral status in pediatric outpatients on antiepileptic drug monotherapy.
      15<18 years2 yearsZ-score unaffected
      Erbayat et al.
      • Erbayat Altay E.
      • Serdaroglu A.
      • Tumer L.
      • Gucuyener K.
      • Hasanoglu A.
      Evaluation of bone mineral metabolism in children receiving carbamazepine and valproic acid.
      <18 yearsFemoral neck BMD, lumbar spine BMD unaffected
      Akin et al.
      • Akin R.
      • Okutan V.
      • Sarici U.
      • Altunbas A.
      • Gokcay E.
      Evaluation of bone mineral density in children receiving antiepileptic drugs.
      258 years 10 months  ± 6months2.4 ± 0.2 yearsL2-L4 BMD unaffected
      Kumandas et al.
      • Kumandas S.
      • Koklu E.
      • Gumus H.
      • Koklu S.
      • Kurtoglu S.
      • Karakukcu M.
      • et al.
      Effect of carbamezapine and valproic acid on bone mineral density: IGF-I and IGFBP-3.
      338.8 ± 2L1-L4 BMD decreased
      Babayigit et al.
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      3111.18 ± 4.073.32 ± 1.09 yBMD L1-L4 decreased
      Sheth et al.
      • Sheth R.D.
      • Wesolowski C.A.
      • Jacob J.C.
      • Penney S.
      • Hobbs G.R.
      • Riggs J.E.
      • et al.
      Effect of carbamazepine and valproate on bone mineral density.
      1315.4 ± 3.33.1 ± 1.7 yZ-score of L2-L4, distal third of the radius decreased, reduced mineralization
      Kafali et al.
      • Kafali G.
      • Erselcan T.
      • Tanzer F.
      Effect of antiepileptic drugs on bone mineral density in children between ages 6 and 12 years.
      13>6months1.8 ± 0.7 yL1-L4 BMD decreased in girls, 8% decrease in midregion of radius-ulna
      Tsukahara et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      910.7 ± 3.34.6 ± 2.4yLumbar spine BMD decreased
      Rieger-Wettengl et al.
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      1912.5 ± 3.73.7 ± 2.5Trabecular BMD decreased
      Guo et al.
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      289.3 ± 0.7>2 yearsTotal BMD decreased in inactive patients
      Oner et al.
      • Oner N.
      • Kaya M.
      • Karasalihoglu S.
      • Karaca H.
      • Celtik C.
      • Tutunculer F.
      Bone mineral metabolism changes in epileptic children receiving valproic acid.
      337.1 ± 3.5>6 monthsLower femoral trochanter BMD value
      Song et al.
      • Song X.Q.
      • Wang Z.P.
      • Bao K.R.
      • Zhang J.M.
      • Wu J.
      • Yan C.H.
      • et al.
      Effect of carbamazepine and valproate on bone metabolism in children with epilepsy.
      92 with VPA or CBZ<18yDecreased BMD of mid-shaft tibia and (or) the distal third of the radius
      Elliot et al.
      • Elliott J.O.
      • Jacobson M.P.
      • Haneef Z.
      Homocysteine and bone loss in epilepsy.
      24adultsSpine T-score and hip T-score unaffected
      Gniatkowska.
      • Gniatkowska-Nowakowska A.
      Fractures in epilepsy children.
      VPA 16, VPA + CBZ 19, VPA  + LTG 32, VPA + TPM 137–16 years5 yearsBMD decreased
      CBZ: carbamazepine, LTG: lamotrigine, TPM: topiramate, BMD: body mineral density, BMC: bone mineral content.
      In a meta-analysis, which included studies with children on long-term VPA treatment from 0.5 to 8 years, the authors concluded that VPA induces a considerable decrease in BMD [
      • Zhang Y.
      • Zheng Y.X.
      • Zhu J.M.
      • Zhang J.M.
      • Zheng Z.
      Effects of antiepileptic drugs on bone mineral density and bone metabolism in children: a meta-analysis.
      ]. Similar results were reported regarding the effect of VPA on fracture risk with some studies showing increased association with fracture risk [
      • Vestergaard P.
      Epilepsy: osteoporosis and fracture risk – a meta-analysis.
      ,
      • Lee R.H.
      • Lyles K.W.
      • Colon-Emeric C.
      A review of the effect of anticonvulsant medications on bone mineral density and fracture risk.
      ,
      • Vestergaard P.
      • Rejnmark L.
      • Mosekilde L.
      Fracture risk associated with use of antiepileptic drugs.
      ] and others no association [
      • Jette N.
      • Lix L.M.
      • Metge C.J.
      • Prior H.J.
      • McChesney J.
      • Leslie W.D.
      Association of antiepileptic drugs with nontraumatic fractures: a population-based analysis.
      ,
      • Shen C.
      • Chen F.
      • Zhang Y.
      • Guo Y.
      • Ding M.
      Association between use of antiepileptic drugs and fracture risk: a systematic review and meta-analysis.
      ].
      Furthermore, apart from osteoporosis and fracture risk, VPA has been associated with a significant decrease in growth velocity after one year of treatment, determined by the difference between two consecutive measures of the patient’s height and compared with healthy subjects [
      • Lin C.M.
      • Fan H.C.
      • Chao T.Y.
      • Chu D.M.
      • Lai C.C.
      • Wang C.C.
      • et al.
      Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.
      ,
      • Lee H.S.
      • Wang S.Y.
      • Salter D.M.
      • Wang C.C.
      • Chen S.J.
      • Fan H.C.
      The impact of the use of antiepileptic drugs on the growth of children.
      ]. In addition, Guo et al. observed that the decrease in height was more profound in inactive children with epilepsy [
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      ].

      4.1.2 Bone markers

      In order to clarify the effects of VPA on bone metabolism and interpret the reduction in BMD values, different bone markers have been conscripted and yet there is also conflict in this aspect (Table 2).
      Table 2Studies investigating parameters of calcium homeostasis and bone markers in patients under long-treatment with VPA.
      ParametersIncreaseDecreaseNo effect
      Ca-PRauchenzauner, Griesmacher et al.
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      Tekgul, Serdaroglu et al.
      • Tekgul H.
      • Serdaroglu G.
      • Huseyinov A.
      • Gokben S.
      Bone mineral status in pediatric outpatients on antiepileptic drug monotherapy.
      Akin, Okutan et al.
      • Akin R.
      • Okutan V.
      • Sarici U.
      • Altunbas A.
      • Gokcay E.
      Evaluation of bone mineral density in children receiving antiepileptic drugs.
      Tsukahara, Kimura et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      Babayigit, Dirik et al.
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      Pack, Morrell et al.
      • Pack A.M.
      • Morrell M.J.
      • Marcus R.
      • Holloway L.
      • Flaster E.
      • Done S.
      • et al.
      Bone mass and turnover in women with epilepsy on antiepileptic drug monotherapy.
      Kim, Lee et al.
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      Hamed, Moussa et al.
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      Kafali, Erselcan et al.
      • Kafali G.
      • Erselcan T.
      • Tanzer F.
      Effect of antiepileptic drugs on bone mineral density in children between ages 6 and 12 years.
      Kumandas, Koklu et al.
      • Kumandas S.
      • Koklu E.
      • Gumus H.
      • Koklu S.
      • Kurtoglu S.
      • Karakukcu M.
      • et al.
      Effect of carbamezapine and valproic acid on bone mineral density: IGF-I and IGFBP-3.
      Nicolaidou, Georgouli et al.
      • Nicolaidou P.
      • Georgouli H.
      • Kotsalis H.
      • Matsinos Y.
      • Papadopoulou A.
      • Fretzayas A.
      • et al.
      Effects of anticonvulsant therapy on vitamin D status in children: prospective monitoring study.
      Serin, Koc et al.
      • Serin H.M.
      • Koc Z.P.
      • Temelli B.
      • Esen I.
      The bone mineral content alterations in pediatric patients medicated with levetiracetam, valproic acid, and carbamazepine.
      Lee, Wang et al.
      • Lee H.S.
      • Wang S.Y.
      • Salter D.M.
      • Wang C.C.
      • Chen S.J.
      • Fan H.C.
      The impact of the use of antiepileptic drugs on the growth of children.
      Zare, Ghazvini et al.
      • Zare M.
      • Ghazvini M.R.
      • Dashti M.
      • Najafi M.R.
      • Alavi-Naeini A.M.
      Bone turnover markers in epileptic patients under chronic valproate therapy.
      Bauer, Hofbauer et al.
      • Bauer S.
      • Hofbauer L.C.
      • Rauner M.
      • Strzelczyk A.
      • Kellinghaus C.
      • Hallmeyer-Elgner S.
      • et al.
      Early detection of bone metabolism changes under different antiepileptic drugs (ED-BoM-AED)—a prospective multicenter study.
      Vitamin DTekgul, Serdaroglu et al.
      • Tekgul H.
      • Serdaroglu G.
      • Huseyinov A.
      • Gokben S.
      Bone mineral status in pediatric outpatients on antiepileptic drug monotherapy.
      Tsukahara, Kimura et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      Nicolaidou, Georgouli et al.
      • Nicolaidou P.
      • Georgouli H.
      • Kotsalis H.
      • Matsinos Y.
      • Papadopoulou A.
      • Fretzayas A.
      • et al.
      Effects of anticonvulsant therapy on vitamin D status in children: prospective monitoring study.
      Babayigit, Dirik et al.
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      Zhang, Zheng et al.
      • Zhang Y.
      • Zheng Y.X.
      • Zhu J.M.
      • Zhang J.M.
      • Zheng Z.
      Effects of antiepileptic drugs on bone mineral density and bone metabolism in children: a meta-analysis.
      Pack, Morrell et al.
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      Albaghdadi, Alhalabi et al.
      • Albaghdadi O.
      • Alhalabi M.S.
      • Alourfi Z.
      • Youssef L.A.
      Bone health and vitamin D status in young epilepsy patients on valproate monotherapy.
      Andress, Ozuna et al.
      • Andress D.L.
      • Ozuna J.
      • Tirschwell D.
      • Grande L.
      • Johnson M.
      • Jacobson A.F.
      • et al.
      Antiepileptic drug-induced bone loss in young male patients who have seizures.
      Hamed, Moussa et al.
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      Kim, Lee et al.
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      Pack, Morrell et al.
      • Pack A.M.
      • Morrell M.J.
      • Marcus R.
      • Holloway L.
      • Flaster E.
      • Done S.
      • et al.
      Bone mass and turnover in women with epilepsy on antiepileptic drug monotherapy.
      Boluk, Guzelipek et al.
      • Boluk A.
      • Guzelipek M.
      • Savli H.
      • Temel I.
      • Ozisik H.I.
      • Kaygusuz A.
      The effect of valproate on bone mineral density in adult epileptic patients.
      Rieger-Wettengl, Tutlewski et al.
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      Rauchenzauner, Griesmacher et al.
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      PTHKumandas, Koklu et al.
      • Kumandas S.
      • Koklu E.
      • Gumus H.
      • Koklu S.
      • Kurtoglu S.
      • Karakukcu M.
      • et al.
      Effect of carbamezapine and valproic acid on bone mineral density: IGF-I and IGFBP-3.
      Tekgul, Serdaroglu et al.
      • Tekgul H.
      • Serdaroglu G.
      • Huseyinov A.
      • Gokben S.
      Bone mineral status in pediatric outpatients on antiepileptic drug monotherapy.
      Kim, Lee et al.
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      Tsukahara, Kimura et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      Nicolaidou, Georgouli et al.
      • Nicolaidou P.
      • Georgouli H.
      • Kotsalis H.
      • Matsinos Y.
      • Papadopoulou A.
      • Fretzayas A.
      • et al.
      Effects of anticonvulsant therapy on vitamin D status in children: prospective monitoring study.
      Babayigit, Dirik et al.
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      Pack, Morrell et al.
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      Erbayat Altay, Serdaroglu et al.
      • Erbayat Altay E.
      • Serdaroglu A.
      • Tumer L.
      • Gucuyener K.
      • Hasanoglu A.
      Evaluation of bone mineral metabolism in children receiving carbamazepine and valproic acid.
      Pack, Morrell et al.
      • Pack A.M.
      • Morrell M.J.
      • Marcus R.
      • Holloway L.
      • Flaster E.
      • Done S.
      • et al.
      Bone mass and turnover in women with epilepsy on antiepileptic drug monotherapy.
      Boluk, Guzelipek et al.
      • Boluk A.
      • Guzelipek M.
      • Savli H.
      • Temel I.
      • Ozisik H.I.
      • Kaygusuz A.
      The effect of valproate on bone mineral density in adult epileptic patients.
      Rieger-Wettengl, Tutlewski et al.
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      Guo, Ronen et al.
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      Serin, Koc et al.
      • Serin H.M.
      • Koc Z.P.
      • Temelli B.
      • Esen I.
      The bone mineral content alterations in pediatric patients medicated with levetiracetam, valproic acid, and carbamazepine.
      Zare, Ghazvini et al.
      • Zare M.
      • Ghazvini M.R.
      • Dashti M.
      • Najafi M.R.
      • Alavi-Naeini A.M.
      Bone turnover markers in epileptic patients under chronic valproate therapy.
      ALPBabayigit, Dirik et al.
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      Elliott, Jacobson et al.
      • Elliott J.O.
      • Jacobson M.P.
      • Haneef Z.
      Homocysteine and bone loss in epilepsy.
      Serin, Koc et al.
      • Serin H.M.
      • Koc Z.P.
      • Temelli B.
      • Esen I.
      The bone mineral content alterations in pediatric patients medicated with levetiracetam, valproic acid, and carbamazepine.
      Kafali, Erselcan et al.
      • Kafali G.
      • Erselcan T.
      • Tanzer F.
      Effect of antiepileptic drugs on bone mineral density in children between ages 6 and 12 years.
      Zare, Ghazvini et al.
      • Zare M.
      • Ghazvini M.R.
      • Dashti M.
      • Najafi M.R.
      • Alavi-Naeini A.M.
      Bone turnover markers in epileptic patients under chronic valproate therapy.
      Erbayat Altay, Serdaroglu et al.
      • Erbayat Altay E.
      • Serdaroglu A.
      • Tumer L.
      • Gucuyener K.
      • Hasanoglu A.
      Evaluation of bone mineral metabolism in children receiving carbamazepine and valproic acid.
      Zhang, Zheng et al.
      • Zhang Y.
      • Zheng Y.X.
      • Zhu J.M.
      • Zhang J.M.
      • Zheng Z.
      Effects of antiepileptic drugs on bone mineral density and bone metabolism in children: a meta-analysis.
      bALPVoudris, Moustaki et al.
      • Voudris K.
      • Moustaki M.
      • Zeis P.M.
      • Dimou S.
      • Vagiakou E.
      • Tsagris B.
      • et al.
      Alkaline phosphatase and its isoenzyme activity for the evaluation of bone metabolism in children receiving anticonvulsant monotherapy.
      Zare, Ghazvini et al.
      • Zare M.
      • Ghazvini M.R.
      • Dashti M.
      • Najafi M.R.
      • Alavi-Naeini A.M.
      Bone turnover markers in epileptic patients under chronic valproate therapy.
      Elwakkad, El Elshamy et al.
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      OsteocalcinOner, Kaya et al.
      • Oner N.
      • Kaya M.
      • Karasalihoglu S.
      • Karaca H.
      • Celtik C.
      • Tutunculer F.
      Bone mineral metabolism changes in epileptic children receiving valproic acid.
      Tsukahara, Kimura et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      Bauer, Hofbauer et al.
      • Bauer S.
      • Hofbauer L.C.
      • Rauner M.
      • Strzelczyk A.
      • Kellinghaus C.
      • Hallmeyer-Elgner S.
      • et al.
      Early detection of bone metabolism changes under different antiepileptic drugs (ED-BoM-AED)—a prospective multicenter study.
      Kim, Lee et al.
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      Rieger-Wettengl, Tutlewski et al.
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      Sato, Kondo et al.
      • Sato Y.
      • Kondo I.
      • Ishida S.
      • Motooka H.
      • Takayama K.
      • Tomita Y.
      • et al.
      Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.
      Song, Wang et al.
      • Song X.Q.
      • Wang Z.P.
      • Bao K.R.
      • Zhang J.M.
      • Wu J.
      • Yan C.H.
      • et al.
      Effect of carbamazepine and valproate on bone metabolism in children with epilepsy.
      Elwakkad, El Elshamy et al.
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      PICPTsukahara, Kimura et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ICTPSato, Kondo et al.
      • Sato Y.
      • Kondo I.
      • Ishida S.
      • Motooka H.
      • Takayama K.
      • Tomita Y.
      • et al.
      Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.
      Tsukahara, Kimura et al.
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      NTxElwakkad, El Elshamy et al.
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      OPGBauer, Hofbauer et al.
      • Bauer S.
      • Hofbauer L.C.
      • Rauner M.
      • Strzelczyk A.
      • Kellinghaus C.
      • Hallmeyer-Elgner S.
      • et al.
      Early detection of bone metabolism changes under different antiepileptic drugs (ED-BoM-AED)—a prospective multicenter study.
      Elwakkad, El Elshamy et al.
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      Rauchenzauner, Griesmacher et al.
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      Hamed, Moussa et al.
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      RANKLElwakkad, El Elshamy et al.
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      Bauer, Hofbauer et al.
      • Bauer S.
      • Hofbauer L.C.
      • Rauner M.
      • Strzelczyk A.
      • Kellinghaus C.
      • Hallmeyer-Elgner S.
      • et al.
      Early detection of bone metabolism changes under different antiepileptic drugs (ED-BoM-AED)—a prospective multicenter study.
      Rauchenzauner, Griesmacher et al.
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      Hamed, Moussa et al.
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      TRAcPLin, Fan et al.
      • Lin C.M.
      • Fan H.C.
      • Chao T.Y.
      • Chu D.M.
      • Lai C.C.
      • Wang C.C.
      • et al.
      Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.
      Rauchenzauner, Griesmacher et al.
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      CTxZare, Ghazvini et al.
      • Zare M.
      • Ghazvini M.R.
      • Dashti M.
      • Najafi M.R.
      • Alavi-Naeini A.M.
      Bone turnover markers in epileptic patients under chronic valproate therapy.
      Ca: calcium, P: phosphorus, PTH: parathyroid hormone, ALP: alkaline phosphatase, bALP: bone alkaline phosphatase, PICP: Procollagen I carboxyterminalpropeptide, ICTP: carboxyterminal telopeptide of type I collagen, NTx: N-terminal telopeptide, OPG: osteoprotegerin, RANKL: Receptor activator of nuclear factor kappa-B ligand, TRAcP: tartrate-resistant acid phosphatase, CTx: carboxy-terminal collagen crosslinks.
      Serum osteocalcin levels have been shown either to increase [
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      ,
      • Oner N.
      • Kaya M.
      • Karasalihoglu S.
      • Karaca H.
      • Celtik C.
      • Tutunculer F.
      Bone mineral metabolism changes in epileptic children receiving valproic acid.
      ,
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      ,
      • Verrotti A.
      • Agostinelli S.
      • Coppola G.
      • Parisi P.
      • Chiarelli F.
      A 12-month longitudinal study of calcium metabolism and bone turnover during valproate monotherapy.
      ], decrease [
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ,
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      ,
      • Song X.Q.
      • Wang Z.P.
      • Bao K.R.
      • Zhang J.M.
      • Wu J.
      • Yan C.H.
      • et al.
      Effect of carbamazepine and valproate on bone metabolism in children with epilepsy.
      ] or remain unchanged [
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      ,
      • Bauer S.
      • Hofbauer L.C.
      • Rauner M.
      • Strzelczyk A.
      • Kellinghaus C.
      • Hallmeyer-Elgner S.
      • et al.
      Early detection of bone metabolism changes under different antiepileptic drugs (ED-BoM-AED)—a prospective multicenter study.
      ,
      • Zare M.
      • Ghazvini M.R.
      • Dashti M.
      • Najafi M.R.
      • Alavi-Naeini A.M.
      Bone turnover markers in epileptic patients under chronic valproate therapy.
      ] during treatment with VPA. Tsukahara et al. [
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ], have shown that VPA treatment in children decrease bone formation markers other than osteocalcin, such as procollagen I carboxyterminal-propeptide (PICP).
      On the contrary, three human studies and one animal study demonstrated that VPA treatment increases the serum levels of bone specific ALP (bALP) [
      • Lin C.M.
      • Fan H.C.
      • Chao T.Y.
      • Chu D.M.
      • Lai C.C.
      • Wang C.C.
      • et al.
      Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.
      ,
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      ,
      • Verrotti A.
      • Agostinelli S.
      • Coppola G.
      • Parisi P.
      • Chiarelli F.
      A 12-month longitudinal study of calcium metabolism and bone turnover during valproate monotherapy.
      ,
      • Voudris K.
      • Moustaki M.
      • Zeis P.M.
      • Dimou S.
      • Vagiakou E.
      • Tsagris B.
      • et al.
      Alkaline phosphatase and its isoenzyme activity for the evaluation of bone metabolism in children receiving anticonvulsant monotherapy.
      ]. Voudris et al. showed that increased bALP values are not necessarily followed by an increase in total ALP and may be a useful marker in diagnosing impairment of bone metabolism in children treated with VPA [
      • Voudris K.
      • Moustaki M.
      • Zeis P.M.
      • Dimou S.
      • Vagiakou E.
      • Tsagris B.
      • et al.
      Alkaline phosphatase and its isoenzyme activity for the evaluation of bone metabolism in children receiving anticonvulsant monotherapy.
      ].
      Similar results have been shown for bone resorption markers. Sato et al. [
      • Sato Y.
      • Kondo I.
      • Ishida S.
      • Motooka H.
      • Takayama K.
      • Tomita Y.
      • et al.
      Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.
      ] examined adult patients under long-term treatment with VPA and reported a significant elevation of bone resorption as assessed by higher levels of ionized calcium and the increase in carboxyterminal telopeptide of type I collagen(ICTP) concentration. On the contrary, in other studies treatment with VPA decreased serum levels of tartrate-resistant acid phosphatase (TRACP), which reflect the number of active osteoclasts [
      • Lin C.M.
      • Fan H.C.
      • Chao T.Y.
      • Chu D.M.
      • Lai C.C.
      • Wang C.C.
      • et al.
      Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.
      ,
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      ], and ICTP levels. In addition, in the study by Lin et al. decreased TRACP levels in children under treatment with VPA was positively correlated with decreased growth velocity [
      • Lin C.M.
      • Fan H.C.
      • Chao T.Y.
      • Chu D.M.
      • Lai C.C.
      • Wang C.C.
      • et al.
      Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.
      ]. In one in vivo study, Elwakkad et al. [
      • Elwakkad A.S.
      • El Elshamy K.A.
      • Sibaii H.
      Fish liver oil and propolis as protective natural products against the effect of the anti-epileptic drug valproate on immunological markers of bone formation in rats.
      ], showed that VPA increased bone formation (osteocalcin and bone specific alkaline phosphatase), and bone resorption, such as N-terminal telopeptide, NTx, markers while it also altered the RANKL/OPG ratio in favor of RANKL, thereby promoting osteoclastogenesis.

      4.1.3 Calcium homeostasis

      Another part of bone metabolism that has been examined in patients under treatment with VPA is calcium (Ca) homeostasis. Most of the studies have reported that VPA treatment did not alter significantly Ca, phosphate (P), 25-(OH)-vitamin D, PTH or ALP values [
      • Kim S.H.
      • Lee J.W.
      • Choi K.G.
      • Chung H.W.
      • Lee H.W.
      A 6-month longitudinal study of bone mineral density with antiepileptic drug monotherapy.
      ,
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      ,
      • Triantafyllou N.
      • Lambrinoudaki I.
      • Armeni E.
      • Evangelopoulos E.M.
      • Boufidou F.
      • Antoniou A.
      • et al.
      Effect of long-term valproate monotherapy on bone mineral density in adults with epilepsy.
      ,
      • Albaghdadi O.
      • Alhalabi M.S.
      • Alourfi Z.
      • Youssef L.A.
      Bone health and vitamin D status in young epilepsy patients on valproate monotherapy.
      ,
      • Andress D.L.
      • Ozuna J.
      • Tirschwell D.
      • Grande L.
      • Johnson M.
      • Jacobson A.F.
      • et al.
      Antiepileptic drug-induced bone loss in young male patients who have seizures.
      ,
      • Babayigit A.
      • Dirik E.
      • Bober E.
      • Cakmakci H.
      Adverse effects of antiepileptic drugs on bone mineral density.
      ,
      • Kumandas S.
      • Koklu E.
      • Gumus H.
      • Koklu S.
      • Kurtoglu S.
      • Karakukcu M.
      • et al.
      Effect of carbamezapine and valproic acid on bone mineral density: IGF-I and IGFBP-3.
      ,
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      ,
      • Verrotti A.
      • Agostinelli S.
      • Coppola G.
      • Parisi P.
      • Chiarelli F.
      A 12-month longitudinal study of calcium metabolism and bone turnover during valproate monotherapy.
      ,
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      ,
      • Razazizan N.
      • Mirmoeini M.
      • Daeichin S.
      • Ghadiri K.
      Comparison of 25-hydroxy vitamin D: calcium and alkaline phosphatase levels in epileptic and non-epileptic children.
      ,
      • Turan M.I.
      • Cayir A.
      • Ozden O.
      • Tan H.
      An examination of the mutual effects of valproic acid: carbamazepine, and phenobarbital on 25-hydroxyvitamin D levels and thyroid function tests.
      ] (Table 2) although BMD values did not remain unchanged, as reported above [
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ,
      • Albaghdadi O.
      • Alhalabi M.S.
      • Alourfi Z.
      • Youssef L.A.
      Bone health and vitamin D status in young epilepsy patients on valproate monotherapy.
      ,
      • Oner N.
      • Kaya M.
      • Karasalihoglu S.
      • Karaca H.
      • Celtik C.
      • Tutunculer F.
      Bone mineral metabolism changes in epileptic children receiving valproic acid.
      ,
      • Rieger-Wettengl G.
      • Tutlewski B.
      • Stabrey A.
      • Rauch F.
      • Herkenrath P.
      • Schauseil-Zipf U.
      • et al.
      Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine.
      ]. In some of these studies, 1,25 (OH)2-vitamin D levels have also been examined, showing similar results [
      • Tsukahara H.
      • Kimura K.
      • Todoroki Y.
      • Ohshima Y.
      • Hiraoka M.
      • Shigematsu Y.
      • et al.
      Bone mineral status in ambulatory pediatric patients on long-term anti-epileptic drug therapy.
      ,
      • Pack A.M.
      • Morrell M.J.
      • Randall A.
      • McMahon D.J.
      • Shane E.
      Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy.
      ,
      • Verrotti A.
      • Agostinelli S.
      • Coppola G.
      • Parisi P.
      • Chiarelli F.
      A 12-month longitudinal study of calcium metabolism and bone turnover during valproate monotherapy.
      ]. Two studies have reported decreased 25-(OH)-vitamin D and Ca concentrations and an increase in ALP [
      • Hamed S.A.
      • Moussa E.M.
      • Youssef A.H.
      • Abd ElHameed M.A.
      • NasrEldin E.
      Bone status in patients with epilepsy: relationship to markers of bone remodeling.
      ] and PTH [
      • Nicolaidou P.
      • Georgouli H.
      • Kotsalis H.
      • Matsinos Y.
      • Papadopoulou A.
      • Fretzayas A.
      • et al.
      Effects of anticonvulsant therapy on vitamin D status in children: prospective monitoring study.
      ], pointing to the development of secondary hyperparathyroidism in these patients, while in two others VPA treatment increased significantly calcium levels [
      • Sato Y.
      • Kondo I.
      • Ishida S.
      • Motooka H.
      • Takayama K.
      • Tomita Y.
      • et al.
      Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.
      ,
      • Rauchenzauner M.
      • Griesmacher A.
      • Tatarczyk T.
      • Haberlandt E.
      • Strasak A.
      • Zimmerhackl L.B.
      • et al.
      Chronic antiepileptic monotherapy: bone metabolism, and body composition in non-institutionalized children.
      ].
      Guo et al. [
      • Guo C.Y.
      • Ronen G.M.
      • Atkinson S.A.
      Long-term valproate and lamotrigine treatment may be a marker for reduced growth and bone mass in children with epilepsy.
      ] categorized young patients under VPA treatment according to their activity and demonstrated that inactive children have significantly lower 25-(OH)-vitamin D concentration accompanied by decreased PTH levels compared to children with higher activity.

      5. Molecular mechanisms of VPA effect on bone metabolism

      5.1 Direct effects on bone cells (Fig. 2)

      Several studies have reported a direct effect of VPA on bone cells in vitro, acting as HDAC inhibitor [
      • Schroeder T.M.
      • Westendorf J.J.
      Histone deacetylase inhibitors promote osteoblast maturation.
      ,
      • Schroeder T.M.
      • Nair A.K.
      • Staggs R.
      • Lamblin A.F.
      • Westendorf J.J.
      Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors.
      ,
      • Humphrey E.L.
      • Morris G.E.
      • Fuller H.R.
      Valproate reduces collagen and osteonectin in cultured bone cells.
      ].
      In osteoblastic–like cells VPA promotes the cell proliferation and differentiation of pre-osteoblasts(MC3T3-E1 cell line) [
      • Schroeder T.M.
      • Westendorf J.J.
      Histone deacetylase inhibitors promote osteoblast maturation.
      ] through induction of Runx2 gene, which plays a critical role in osteoblast differentiation and is negatively regulated by HDAC [
      • Schroeder T.M.
      • Nair A.K.
      • Staggs R.
      • Lamblin A.F.
      • Westendorf J.J.
      Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors.
      ,
      • Jensen E.D.
      • Schroeder T.M.
      • Bailey J.
      • Gopalakrishnan R.
      • Westendorf J.J.
      Histone deacetylase 7 associates with Runx2 and represses its activity during osteoblast maturation in a deacetylation-independent manner.
      ]. Despite the enhancement of osteoblasts differentiation, it was shown that during the maturation process of an osteopr○genitor cell line (hFOB 1.19), treatment with VPA significantly down-regulated synthesis of two bone proteins, collagen type 1 and osteonectin [
      • Humphrey E.L.
      • Morris G.E.
      • Fuller H.R.
      Valproate reduces collagen and osteonectin in cultured bone cells.
      ], which display critical roles in bone formation and subsequent mineralization by mature osteoblasts. Collagen type 1 is the major protein component of bone matrix and mutations in collagen type 1 chains 1 and 2 are the cause of osteogenesis imperfecta in the majority of cases [
      • Rauch F.
      • Glorieux F.H.
      Osteogenesis imperfecta.
      ], while osteonectin is a collagen -binding glycoprotein, which is critical for bone mass maintenance and normal bone remodeling. Homozygosity for missense variants in the osteonectin gene also causes osteogenesis imperfecta type XVII, whereas in vivo osteonectin-null mice show decrease osteoblast number and bone formation rate [
      • Delany A.M.
      • Kalajzic I.
      • Bradshaw A.D.
      • Sage E.H.
      • Canalis E.
      Osteonectin-null mutation compromises osteoblast formation: maturation, and survival.
      ].
      Thus, it appears that enhancement of osteoblast-differentiation by VPA may not lead to mature bone-forming osteoblasts.
      Valproate effect has also been tested in the mouse embryonic mesenchymal cell line C3H10T1/2 [
      • Hatakeyama Y.
      • Hatakeyama J.
      • Takahashi A.
      • Oka K.
      • Tsuruga E.
      • Inai T.
      • et al.
      The effect of valproic Acid on mesenchymal pluripotent cell proliferation and differentiation in extracellular matrices.
      ], which as pluripotent cells can be differentiated into osteoblasts, chondrocytes, adipocytes and myoblasts in vitro. In this study VPA suppressed mesenchymal cell proliferation in the presence of extracellular matrix proteins such as fibronectin and type I collagen, which are known inducers of mesenchymal cell proliferation [
      • Weiss R.E.
      • Reddi A.H.
      Synthesis and localization of fibronectin during collagenous matrix-mesenchymal cell interaction and differentiation of cartilage and bone in vivo.
      ,
      • Terranova V.P.
      • Aumailley M.
      • Sultan L.H.
      • Martin G.R.
      • Kleinman H.K.
      Regulation of cell attachment and cell number by fibronectin and laminin.
      ,
      • Andrianarivo A.G.
      • Robinson J.A.
      • Mann K.G.
      • Tracy R.P.
      Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.
      ], suggesting that VPA effect on bone promotes differentiation of precursors cells at the expense of their proliferation.
      The effect of VPA on cell morphology has been reported in osteoblastic [
      • Schroeder T.M.
      • Westendorf J.J.
      Histone deacetylase inhibitors promote osteoblast maturation.
      ] and fibroblast cell lines [
      • Walmod P.S.
      • Skladchikova G.
      • Kawa A.
      • Berezin V.
      • Bock E.
      Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton.
      ], showing that it may rearrange the cytoskeleton of various cell types. In a microarray analysis of somatic tissue from mouse embryos, VPA was shown to alter the microtubule cytoskeleton and actin filament and thus could be associated with malformations in these embryos' axis skeleton [
      • Massa V.
      • Cabrera R.M.
      • Menegola E.
      • Giavini E.
      • Finnell R.H.
      Valproic acid-induced skeletal malformations: associated gene expression cascades.
      ].
      In another study with MSCs cultures, VPA enhanced the migration of cord blood mesenchymal stromal cells to injured tissues through increasing the expression of stromal cell-derived factor receptors, CXCR4 and CXCR7, without, however, affecting their ability to differentiate to osteocytes and chondrocytes [
      • Marquez-Curtis L.A.
      • et al.
      Migration, proliferation, and differentiation of cord blood mesenchymal stromal cells treated with histone deacetylase inhibitor valproic Acid.
      ].
      Based on current evidence it appears that while VPA may exert positive effects on osteoblasts proliferation and differentiation, it suppresses the activity of mature osteoblasts and this observation may be the molecular link with the increased skeletal fragility and fracture risk that is shown in clinical studies (Fig. 2). Although osteocytes are of critical importance in skeletal integrity and bone strength, there are no data available for a direct effect of VPA on osteocytes’ viability or gene expression. Similarly, there are no data available on a direct VPA effect on osteoclasts.
      Fig. 2
      Fig. 2A schematic presentation of VPA direct action on bone. (-) denotes negative action and (+) positive action. MSc, mesenchymatic cells; MyOD, myogenic differentiation factor 1; Runx 2, Runt-related transcription factor 2; SOX9, SRY (sex determining region Y)-box 9; PPARγ2, Peroxisome proliferator-activated receptor gamma; C/EBP, CCAAT/enhancer-binding protein beta; IGFs, insulin growth factors; TGFs, transforming growth factors; FGF, fibroblast growth factor; Osx, osterix.

      5.2 Effect on vitamin D metabolism

      One of the major contributing factors of VPA effect in bone is VPA-induced osteomalacia, which is considered to be mediated through accelerated catabolism of 1,25 (OH)2 vitamin D.
      Vitamin D is a fat-soluble vitamin, obtained from food such as fish, liver, milk and eggs, or de novo synthesized from cholesterol in the liver. The active metabolite of vitamin D displays two sequential hydroxylations. The first 25-hydroxylation takes place in the liver and the second 1a-hydroxylation, which is considered to be the rate limiting step of active form of vitamin D synthesis, in the kidney.
      The active form of vitamin D, 1,25 (OH)2 vitamin D, binds with high affinity and selectivity to its specific vitamin D receptor (VDR), which belongs to the nuclear receptor family of transcription factors. Upon activation by vitamin D, VDR forms a heterodimer with the retinoid-X receptor and binds to the DNA responsive elements of the target cells. The interaction of 1,25(OH)2D with the VDR initiates a complex cascade of molecular events, regulating the transcription of specific genes or gene networks. Non-genomic actions of 1,25(OH)2D, mediating rapid and non–transcriptional dependent actions of vitamin D, have also been described [
      • Hii C.S.
      • Ferrante A.
      The non-genomic actions of vitamin D.
      ].
      Although VPA is thought to be an inhibitor rather than an inducer of drug-metabolizing enzymes [
      • Perucca E.
      Clinically relevant drug interactions with antiepileptic drugs.
      ], several studies have shown that VPA induces vitamin D catabolic enzymes.
      In in vitro studies in human hepatocytes and human embryonic kidney cells VPA increased basal and vitamin D-induced expression of CYP24A [
      • Vrzal R.
      • Doricakova A.
      • Novotna A.
      • Bachleda P.
      • Bitman M.
      • Pavek P.
      • et al.
      Valproic acid augments vitamin D receptor-mediated induction of CYP24 by vitamin D3: a possible cause of valproic acid-induced osteomalacia?.
      ], a catabolic enzyme that hydroxylates 25-OH-vitamin D substrate at position 24 [
      • Sawada N.
      • Kusudo T.
      • Sakaki T.
      • Hatakeyama S.
      • Hanada M.
      • Abe D.
      • et al.
      Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1.
      ], lowering its biological activity.
      In another study VPA was also shown to induce alternative vitamin D catabolism by CYP 3A [
      • Cerveny L.
      • Svecova L.
      • Anzenbacherova E.
      • Vrzal R.
      • Staud F.
      • Dvorak Z.
      • et al.
      Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways.
      ]. Interestingly, both studies have shown that this effect was not mediated solely through VPA action as a HDAC1 inhibitor, but also included activation of extracellular signal-regulated kinase and direct activation of androstane receptor and pregnane- X receptor.

      5.3 Indirect effect on bone metabolism through endocrine complications

      It is controversial whether the endocrine dysfunction in epilepsy patients is caused by the epilepsy itself, the antiepileptic therapy, or both. Treatment with VPA has been associated with hypogonadism, hypothyroidism, hyponatremia, and mild hypercortisolemia, which can also contribute to bone loss seen in these patients.
      Chronic administration of VPA affects the function of the hypothalamic-pituitary-gonadal axis primarily in males [
      • Verrotti A.
      • Loiacono G.
      • Laus M.
      • Coppola G.
      • Chiarelli F.
      • Tiboni G.M.
      Hormonal and reproductive disturbances in epileptic male patients: emerging issues.
      ]. In females VPA treatment has been associated with menstrual cycle dysregulation, polycystic ovary syndrome, or hyperandrogenism.
      It has been shown that VPA monotherapy increases the levels of androstenedione in epileptic prepubertal children compared with healthy controls [
      • Rauchenzauner M.
      • et al.
      Effects of levetiracetam and valproic acid monotherapy on sex-steroid hormones in prepubertal children–results from a pilot study.
      ]. In another study involving epileptic males, VPA increased the levels of dehydroepiandrosterone and decreased the gonadotropins [
      • Verrotti A.
      • Loiacono G.
      • Laus M.
      • Coppola G.
      • Chiarelli F.
      • Tiboni G.M.
      Hormonal and reproductive disturbances in epileptic male patients: emerging issues.
      ]. The underlying mechanisms of VPA effects on reproductive function are not fully elucidated but several theories have been proposed. It has been suggested that the increase in male androgens could be associated with the inductive effect of VPA on liver enzymes responsible for the production of sex hormone binding globulin (SHBG) [
      • Rattya J.
      • Turkka J.
      • Pakarinen A.J.
      • Knip M.
      • Kotila M.A.
      • Lukkarinen O.
      • et al.
      Reproductive effects of valproate: carbamazepine, and oxcarbazepine in men with epilepsy.
      ]. In addition, it has been shown that VPA blocks androgens and progesterone receptors within the therapeutic levels and that especially the blockade of androgens receptor could contribute to the reported impaired reproductive functions during VPA treatment [
      • Death A.K.
      • McGrath K.C.
      • Handelsman D.J.
      Valproate is an anti-androgen and anti-progestin.
      ]. Finally, it has been reported that VPA treatment induces carnitine deficiency by inhibiting its biosynthesis through decreasing the concentration of alpha-ketoglutarate [
      • Lheureux P.E.
      • Hantson P.
      Carnitine in the treatment of valproic acid-induced toxicity.
      ,
      • Roste L.S.
      • Tauboll E.
      • Morkrid L.
      • Bjornenak T.
      • Saetre E.R.
      • Morland T.
      • et al.
      Antiepileptic drugs alter reproductive endocrine hormones in men with epilepsy.
      ]. Carnitine is an amino acid derivative, which is an essential cofactor in the beta-oxidation of fatty acids and is considered an indicator of epididymal functioning. Through carnitine deficiency, VPA affects indirectly the semen parameters, such as sperm motility [
      • Roste L.S.
      • Tauboll E.
      • Morkrid L.
      • Bjornenak T.
      • Saetre E.R.
      • Morland T.
      • et al.
      Antiepileptic drugs alter reproductive endocrine hormones in men with epilepsy.
      ].
      VPA-induced carnitine deficiency, especially in neonates and children, also exerts an indirect negative effect on bone metabolism.
      Carnitine derivatives, such as l-carnitinefumarate and isovaleryl l-carnitinefumarate have been shown to stimulate osteoblast activity in vitro [
      • Colucci S.
      • et al.
      L-carnitine and isovaleryl L-carnitine fumarate positively affect human osteoblast proliferation and differentiation in vitro.
      ] and in in vivo mice models of pregnancy/hypocalcemia-associated bone loss [
      • Patano N.
      • Mancini L.
      • Settanni M.P.
      • Strippoli M.
      • Brunetti G.
      • Greco G.
      • et al.
      L: -carnitine fumarate and isovaleryl-L: -carnitine fumarate accelerate the recovery of bone volume/total volume ratio after experimetally induced osteoporosis in pregnant mice.
      ] and ovariectomy/inflammation-associated bone loss [
      • Orsal E.
      • Halici Z.
      • Bayir Y.
      • Cadirci E.
      • Bilen H.
      • Ferah I.
      • et al.
      The role of carnitine on ovariectomy and inflammation-induced osteoporosis in rats.
      ,
      • Hooshmand S.
      • Balakrishnan A.
      • Clark R.M.
      • Owen K.Q.
      • Koo S.I.
      • Arjmandi B.H.
      Dietary l-carnitine supplementation improves bone mineral density by suppressing bone turnover in aged ovariectomized rats.
      ] supplementation with carnitine has been shown to reverse decreased BMD.
      Chronic VPA treatment also produces an elevation in plasma cortisol concentrations without any concomitant rise in ACTH concentrations. It is possible that valproate produces a direct effect at the level of the adrenal cortex, or perhaps increases production of an adrenocortical-stimulating hormone other than ACTH.
      Severe symptomatic hyponatremia and syndrome of inappropriate antidiuretic hormone secretion have also been reported in relation to VPA exposure in adults [
      • Beers E.
      • van Puijenbroek E.P.
      • Bartelink I.H.
      • van der Linden C.M.
      • Jansen P.A.
      Syndrome of inappropriate antidiuretic hormone secretion (SIADH) or hyponatraemia associated with valproic Acid: four case reports from the Netherlands and a case/non-case analysis of vigibase.
      ,
      • Beve E.
      • Beck E.
      • Pinto E.
      • Ansseau M.
      Inappropriate antidiuretic hormone secretion induced by sodium valproate.
      ].
      Regarding thyroid function, an increased frequency of hypothyroidism has been reported with VPA monotherapy [
      • Tsiropoulos I.
      • Andersen M.
      • Hallas J.
      Adverse events with use of antiepileptic drugs: a prescription and event symmetry analysis.
      ,
      • Sahu J.K.
      • Gulati S.
      • Kabra M.
      • Arya R.
      • Sharma R.
      • Gupta N.
      • et al.
      Evaluation of subclinical hypothyroidism in ambulatory children with controlled epilepsy on valproate monotherapy.
      ]. The incidence of subclinical hypothyroidism was higher in children than adults among long-term treated patients [
      • Sahu J.K.
      • Gulati S.
      • Kabra M.
      • Arya R.
      • Sharma R.
      • Gupta N.
      • et al.
      Evaluation of subclinical hypothyroidism in ambulatory children with controlled epilepsy on valproate monotherapy.
      ]. However, VPA has been associated with increased, decreased or unaffected levels of thyroid hormones [
      • Bou Khalil R.
      • Richa S.
      Thyroid adverse effects of psychotropic drugs: a review.
      ,
      • Verrotti A.
      • Laus M.
      • Scardapane A.
      • Franzoni E.
      • Chiarelli F.
      Thyroid hormones in children with epilepsy during long-term administration of carbamazepine and valproate.
      ,
      • Vainionpaa L.K.
      • Mikkonen K.
      • Rattya J.
      • Knip M.
      • Pakarinen A.J.
      • Myllyla V.V.
      • et al.
      Thyroid function in girls with epilepsy with carbamazepine: oxcarbazepine, or valproate monotherapy and after withdrawal of medication.
      ,
      • Yildiz M.
      • Simsek G.
      • Uzun H.
      • Uysal S.
      • Sahin S.
      • Balci H.
      Assessment of low-density lipoprotein oxidation: paraoxonase activity, and arterial distensibility in epileptic children who were treated with anti-epileptic drugs.
      ,
      • Aggarwal A.
      • Rastogi N.
      • Mittal H.
      • Chillar N.
      • Patil R.
      Thyroid hormone levels in children receiving carbamazepine or valproate.
      ]. Elevated thyroid-stimulating hormone (TSH) was also found in children (mean age 3.7) receiving VPA monotherapy for 9 months compared with pre-treatment levels [
      • Aygun F.
      • Ekici B.
      • Aydinli N.
      • Aydin B.K.
      • Bas F.
      • Tatli B.
      Thyroid hormones in children on antiepileptic therapy.
      ]. These adverse effects on thyroid function were shown to be largely reversible in both men and women upon discontinuation of VPA monotherapy in one study [
      • Lossius M.I.
      • Tauboll E.
      • Mowinckel P.
      • Gjerstad L.
      Reversible effects of antiepileptic drugs on thyroid hormones in men and women with epilepsy: a prospective randomized double-blind withdrawal study.
      ], whereas in another one TSH levels remain elevated in females even after VPA withdrawal [
      • Vainionpaa L.K.
      • Mikkonen K.
      • Rattya J.
      • Knip M.
      • Pakarinen A.J.
      • Myllyla V.V.
      • et al.
      Thyroid function in girls with epilepsy with carbamazepine: oxcarbazepine, or valproate monotherapy and after withdrawal of medication.
      ].

      6. Concluding Remarks

      VPA is established worldwide as one of the most widely used AEDs in the treatment of both generalized and partial seizures in adults and children.
      The broad spectrum of antiepileptic efficacy of VPA is reflected in preclinical both in vivo and in vitro models, including a variety of animal models of seizures or epilepsy. VPA may have many adverse effects that require careful monitoring during the chronic treatment. In particular, its use is not recommended in patients with some pre-existing conditions e.g. hepatic and pancreatic insufficiency as well as in obese patients at risk of developing metabolic syndrome and in female pubertal patients because VPA can be expected to induce polycystic ovaries. In recent years, chronic use of VPA has been shown to exert variable effects on bone metabolism. There is no single mechanism of VPA action that can account for all the numerous effects of the drug on bone but the overall effect is an increased fracture rate. The main effects of VPA in bone include direct effects on bone cells through a decrease in osteoblast proliferation, changes in collagen synthesis and an induction of vitamin D catabolism. Furthermore, experimental and clinical observations show that many endocrine side effects, such as hypogonadism, hypothyroidism, hypercortisolemia and carnitine deficiency induced by VPA treatment, may also indirectly contribute to the increased bone fragility.
      Further basic and controlled clinical trials are necessary to promote our understanding of the mechanisms of action of this broad spectrum antiepileptic drug on bone.

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