抗癫痫药物影响儿童骨矿物质密度及骨代谢的荟萃分析

Effects of antiepileptic drugs on bone mineral density and bone metabolism in children: a meta-analysis 抗癫痫药物影响儿童骨矿物质密度及骨代谢的荟萃分析 1 first-author †E-mail:daqingzy1978@163.comzhengzhe19821982@gmail.com †E-mail:daqingzy1978@163.comzhengzhe19821982@gmail.com daqingzy1978@163.com 2 2 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:daqingzy1978@163.comzhengzhe19821982@gmail.com †E-mail:daqingzy1978@163.comzhengzhe19821982@gmail.com daqingzy1978@163.com Neuroscience Care Unit, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Neuroscience Care Unit, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Neurosurgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Neurosurgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Objective: 2 The aim of our meta-analysis was to assess the effects of antiepileptic drugs on bone mineral density and bone metabolism in epileptic children. Methods: 2 Searches of PubMed and Web of Science were undertaken to identify studies evaluating the association between antiepileptic drugs and bone mineral density and bone metabolism. Results: 2 A total of 22 studies with 1492 subjects were included in our research. We identified: (1) a reduction in bone mineral density at lumbar spine (standardized mean difference (SMD)=−0.30, 95% confidence interval (CI) [−0.61, −0.05]), trochanter (mean difference (MD)=−0.07, 95% CI [−0.10, −0.05]), femoral neck (MD=−0.05, 95% CI [−0.09, −0.02]), and total body bone mineral density (MD=−0.33, 95% CI [−0.51, −0.15]); (2) a reduction in 25-hydroxyvitamin D (MD=−3.37, 95% CI [−5.94, −0.80]) and an increase in serum alkaline phosphatase (SMD=0.71, 95% CI [0.38, 1.05]); (3) no significant changes in serum parathyroid hormone, calcium, or phosphorus. Conclusions: 2 Our meta-analysis suggests that treatment with antiepileptic drugs may be associated with decreased bone mineral density in epileptic children. 目的 2 癫痫是神经系统常见病，半数以上患者儿童时期便发病，癫痫病患者常需终身服用抗癫痫药。研究表明，长期服用抗癫痫药物会影响骨代谢，加速骨矿物质的丢失，导致骨质疏松。但是，每种抗癫痫药物的具体作用还不清楚。作为典型的肝酶诱导剂的抗癫痫药物，卡马西平已经公认有降低骨密度的作用，但是作为非肝酶诱导剂的丙戊酸钠，还没有定论。在新型抗癫痫药物中，奥卡西平、托吡酯和拉莫三嗪等对骨密度似乎没有影响，但目前相关的研究很少，各研究所得结果也不一致。本研究拟通过荟萃分析评估抗癫痫药对癫痫病儿童骨密度和骨代谢的影响。 创新点 2 目前已有荟萃分析评估抗癫痫药对成人骨密度的影响，但尚无研究分析抗癫痫药对儿童的影响。 方法 2 检索PubMed和Web of Science数据库中评估抗癫痫药与儿童骨密度及骨代谢关系的临床试验。入选标准为：（1）至少包括一项骨代谢的标志物；（2）骨密度用双能×线吸收计量法计算；（3）研究结果包括平均的骨密度和骨密度的标准差；（4）年龄小于18岁；（5）患者采用单药或多药治疗；（6）具有健康对照组；（7）必须是观察性研究或随机对照研究。采用固定效应模型和随机效应模型对22篇文献的1492例患者中的骨密度及骨代谢资料进行荟萃分析，结果用加权均数差（WMD）或标准均数差（SMD）进行评估。 结论 2 共22篇文献符合纳入标准，共计1492例患者。荟萃分析的结果显示，抗癫痫药物的使用可以引起儿童：（1）腰椎、转子、股骨颈的骨密度和全身骨密度的下降；（2）维生素D下降和血清碱性磷酸酶的上升；（3）甲状旁腺激素、钙和磷没有显著改变。荟萃分析的结果表明，抗癫痫药的使用可能和癫痫病患儿骨密度降低相关。 Antiepileptic drugs Bone mineral density Bone metabolism Children Meta-analysis 抗癫痫药 骨密度 骨代谢 荟萃分析 Introduction 1 Epilepsy is a common chronic neurological disorder with more than half of cases beginning in childhood (Begley et al., 2000 2000 ). Most patients require long-term, and sometimes lifelong, therapy with antiepileptic drugs (AEDs). AEDs are associated with significant side effects including, but not limited to, radiological evidence of rickets, decreased bone mineral density (BMD), altered bone turnover, and increased risk of fracture (Souverein et al., 2005 2005 ; Karceski, 2007 2007 ; Gniatkowska-Nowakowska, 2010 2010 ; Verrotti et al., 2010 2010 ; Meier and Kraenzlin, 2011 2011 ). AEDs increase catabolism of 25-hydroxyvitamin D by induction of the hepatic P-450 enzyme system, leading to relative hypocalcemia, increased levels of parathyroid hormone (PTH), and subsequent low BMD (Dent et al., 1970 1970 ; Keck et al., 1982 1982 ; Chung and Ahn, 1994 1994 ). However, some studies have suggested a significant reduction in BMD with non-enzyme-inducing AEDs (Oner et al., 2004 2004 ; Petty et al., 2005 2005 ). Other environmental and epidemiological parameters including age, sex, diet, and mobility may also affect mineral metabolism (Pack and Morrell, 2001 2001 ), suggesting a multifactorial etiology. A meta-analysis, pooling data from multiple cross-sectional studies showed only a very limited decrease in BMD with a decline of 0.4 Z -scores in the spine and −0.6 Z -scores in the hip (Vestergaard, 2005 2005 ). Most of the relevant data are derived from adults. Data on bone metabolism in children receiving AEDs are scarce, with conflicting results. Some studies found a significant difference in BMD and bone metabolism between children treated with AEDs and healthy controls (Oner et al., 2004 2004 ; Pack et al., 2008 2008 ; Salimipour et al., 2013 2013 ); other studies found no significance (Akin et al., 1998 1998 ; El-Hajj Fuleihan et al., 2008 2008 ). However, these studies may be limited, as most included a small sample, and numbers were too few to enable comparison between specific AEDs. We, therefore, undertook a meta-analysis of studies that had analyzed the effects of AEDs on BMD and bone metabolism in children. Materials and methods 1 Search strategy and inclusion criteria 2 We searched PubMed and Web of Science for all studies up to July 2014 that reported on BMD or bone metabolism and the use of AEDs in children using the keywords “epilepsy” or “antiepileptic drugs” in combination with “bone mineral density” or “bone density” or “bone metabolism” or “bone turnover”. This meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Liberati et al., 2009 2009 ). We included studies reporting the effects of AEDs on BMD and bone metabolism in children, regardless of sample size, if they met the following criteria: (1) inclusion of at least one marker of bone metabolism; (2) BMD measured by dual-energy X-ray absorptiometry (DXA); (3) studies involving mean BMD (or bone markers) and standard deviations (SDs) or standard errors (SEs), or where these numbers could be calculated; (4) child subjects ( < 18 years old); (5) studies assessing patients with epilepsy who received AED monotherapy or polytherapy; (6) studies with a healthy control group; (7) observational studies or randomized controlled trials (RCTs). We excluded animal studies, reviews, and letters. Data extraction and quality assessment 2 Two reviewers independently evaluated studies for inclusion. Discrepancies were resolved by arbitration and consensus following discussion. The following information was extracted from each study: name of the first author, year of publication, country where the study was performed, design of the study, drug dosage, age and gender of individuals, number of cases, mean BMD (or serum concentration of bone markers), and SDs (or SEs). Study quality was independently assessed by two reviewers according to the Newcastle-Ottawa Scale for quality assessment of cohort and case-control studies (Liberati et al., 2009 2009 ). Each study was allocated up to nine stars, the criteria being quality of selection (maximum, 4 stars), comparability (maximum, 2 stars), and outcome (maximum, 3 stars). Statistical analysis 2 When the outcome of interest was measured using the same scale in every study, weighted mean differences (WMDs) with 95% confidence interval (CI) were used as summary measures. However, when studies used different scales to measure the effect of AED treatment, the standardized mean difference (SMD) with 95% CI was used. All data were initially analyzed by a fixed-effects model if there was no significant heterogeneity, and a random-effects model otherwise. Heterogeneity was assessed by Q statistics, with P < 0.10 indicating significant heterogeneity. We quantified heterogeneity using the I 2 statistic. I 2 values of 25%, 50%, and 75% correspond to cut-off points for low, moderate, and high degrees of heterogeneity, respectively. To explore heterogeneity, we performed subgroup analyses according to the drug patients used. Publication bias was checked through the use of funnel plots with an asymmetric funnel plot indicating publication bias. All analyses were conducted using Review Manager (RevMan) v.5.2 statistical software and Microsoft Excel. P < 0.05 was considered statistically significant. Results 1 Study selection and characteristics 2 The information flow for the search and selection of studies is shown in Fig. 1 1 . The initial search yielded 1603 research reports, of which 228 were excluded due to identical title or authors; 1056 were excluded due to ineligible study design (including non-human studies, review articles, case reports, comment, letter, experimental study, and/or fracture-only outcome). After full-text screening, 22 studies were included (Chung and Ahn, 1994 1994 ; Baer et al., 1997 1997 ; Akin et al., 1998 1998 ; Kafali et al., 1999 1999 ; Erbayat Altay et al., 2000 2000 ; Verrotti et al., 2002 2002 ; Voudris et al., 2002 2002 ; Ecevit et al., 2004 2004 ; Oner et al., 2004 2004 ; Babayigit et al., 2006 2006 ; Kumandas et al., 2006 2006 ; Nicolaidou et al., 2006 2006 ; El-Hajj Fuleihan et al., 2008 2008 ; Nettekoven et al., 2008 2008 ; Sheth and Hermann, 2008 2008 ; Sheth et al., 2008 2008 ; Gniatkowska-Nowakowska, 2010 2010 ; Rauchenzauner et al., 2010 2010 ; Babacan et al., 2012 2012 ; Dimić et al., 2013 2013 ; Razazizan et al., 2013 2013 ; Turan et al., 2014 2014 ). Table 1 1 summarizes the characteristics of the included studies published between 1994 and 2014. Nine were conducted in Turkey, six in Europe, one in Australia, one in Korea, one in Iran, one in Lebanon, and three in the United States. Twenty-one studies were written in English, and one in Serbian. Six provided data only on BMD, eight reported results on bone markers, and the remaining eight reported both. Study quality scores (range, 0–9) averaged 7.54, and 63.6% were high-quality studies (score > 8) (Table 1 1 ). DEXA, dual-energy X-ray absorptiometry; Ca, calcium; ALP, alkaline phosphatase; P, phosphorus; PHT, phenytoin; PTH, parathormone; BMD, bone mineral density; CBZ, carbamazepine; VPA, valproic acid; OXC, oxcarbazepine; AED, antiepileptic drug; PB, phenobarbital; LTG, lamotrigine; SUL, sulthiame; TPM, topiramate; M, male; F, female; P/V, participants/intervention Study Country Design Outcome P/V M/F Age (year) Duration (year) Dose Quality 3 Chung and Ahn, 1994 1994 3 Korea 3 Retrospective cohort 3 DEXA lumbar spine 30/PB 3 48/30 8.5 0.75–8 3–5 mg/(kg·d) 8 48/PHT 78/control 8.5 2 Baer et al., 1997 1997 2 U.S. 2 Retrospective cohort 2 Ca 42/AED 2 5.4 6 40/control 6.2 3 Akin et al., 1998 1998 3 Turkey 3 Retrospective cohort 3 DEXA lumbar spine, calcium, phosphorus, ALP 25/VPA 14/11 8.83 2.4 66 μg/ml 8 28/CBZ 15/13 9.5 2.6 7.0 μg/ml 26/control 15/12 8.92 3 Kafali et al., 1999 1999 3 Turkey 3 Retrospective cohort 3 DEXA lumbar spine, Ca, P, ALP 6/CBZ 2 10/9 M: 8.8 1.7 18.33 mg/(kg·d) 9 13/VPA F: 8.4 1.8 18.46 mg/(kg·d) 57/control 29/28 3 Erbayat Altay et al., 2000 2000 3 Turkey 3 Retrospective cohort 3 DEXA lumbar spine 21/CBZ 5/16 12.81 3.7 10–20 mg/(kg·d) 8 15/VPA 5/10 10.9 3.1 15–30 mg/(kg·d) 22/control 8/14 10.68 2 Verrotti et al., 2002 2002 2 Italy 2 Prospective cohort 2 25-hydroxyvitamin D, PTH 60/CBZ 30/30 2 21.9 mg/(kg·d) 7 60/control 30/30 4 Voudris et al., 2002 2002 4 Greece 4 Retrospective cohort 4 ALP 37/CBZ 8.08 >0.5 3 8 47/VPA 8.13 >0.5 34/PB 2.4 >0.5 118/control 3 Ecevit et al., 2004 2004 3 Turkey 3 Retrospective cohort 3 DEXA trochanter and femoral neck 17/CBZ 2 20/13 10.19 2.64 6.26 μg/ml 8 16/VPA 10.59 2.03 53.75 μg/ml 31/control 17/14 11.52 2 Oner et al., 2004 2004 2 Turkey 2 Retrospective cohort 2 DEXA lumbar spine, trochanter and femoral neck, Ca, P, ALP 33/VPA 17/16 7.1 1.08 25.6 mg/(kg·d) 8 33/control 17/16 7.4 3 Kumandas et al., 2006 2006 3 Turkey 3 Retrospective cohort 3 DEXA lumbar spine, PTH, ALP 33/CBZ 20/13 9.7 2.96 19 478.6 mg/kg cumulative dose 8 33/VPA 17/16 8.8 2.81 22 852.4 mg/kg cumulative dose 22/control 13/9 8.9 3 Nicolaidou et al., 2006 2006 3 Greece 3 Prospective cohort 3 Ca, P, 25-hydroxyvitamin D, PTH 24/CBZ 2 19/32 2 7.4 2 1.93 15–20 mg/(kg·d) 2 7 27/VPA 20–25 mg/(kg·d) 80/control 38/42 7.6 4 Babayigit et al., 2006 2006 4 Turkey 4 Retrospective cohort 4 DEXA lumbar spine, Ca, P, ALP, 25-hydroxyvitamin D 23/CBZ 14/9 12.4 3.65 15–25 mg/(kg·d) 7 31/VPA 15/16 11.18 3.32 15–40 mg/(kg·d) 14/OXC 5/9 13.13 2.36 15–30 mg/(kg·d) 30/control 14/16 13.09 2 Nettekoven et al., 2008 2008 2 Germany 2 Retrospective cohort 2 Ca, P, 25-hydroxyvitamin D, PTH 38/(CBZ, VPA, etc.) 25/13 8.4 >0.25 8 44/control 24/20 7.6 2 El-Hajj Fuleihan et al., 2008 2008 2 Lebanon 2 Retrospective cohort 2 DEXA lumbar spine, 25-hydroxyvitamin D 88/AED 13.0 4.7 7 111/control 13.3 2 Sheth and Hermann, 2008 2008 2 U.S. 2 Cross-sectional 2 DEXA total body BMD 116/AED 6–18 6 36/control 6–18 4 Sheth et al., 2008 2008 4 U.S. 4 Cross-sectional 4 DEXA total body BMD 18/AED 6/12 11.9 <1 8 37/AED 19/18 11.8 1–5 27/AED 10/17 13.6 >6 32/control 13/19 12.8 2 Gniatkowska-Nowakowska et al., 2010 2010 2 Poland 2 Retrospective cohort 2 DEXA lumbar spine, Ca, P 126/(CBZ, VPA, LTG, TPM) 80/46 7–16 2 6 132/control 87/45 7–16 5 Rauchenzauner et al., 2010 2010 5 Austria 5 Retrospective cohort 5 25-hydroxyvitamin D, Ca, P 85/VPA 38/47 12.41 >0.5 869 mg/d 8 11/LTG 3 28/12 3 12.83 >0.5 284 mg/d 17/OXC >0.5 840 mg/d 12/SUL >0.5 150 mg/d 41/control 29/12 12.08 2 Babacan et al., 2012 2012 2 Turkey 2 Retrospective cohort 2 DEXA lumbar spine, Ca, P, 25-hydroxyvitamin D, PTH 44/OXC 22/22 9.65 1.25 7 33/control 17/16 10.24 3 Dimić et al., 2013 2013 3 Serbia 3 Retrospective cohort 3 DEXA lumbar spine 2 34/(CBZ, VPA, etc.) 2 18/16 2 9.77 M: 2.66 2 2 8 F: 3.33 35/control 16/19 2 Razazizan et al., 2013 2013 2 Iran 2 Retrospective cohort 2 48/(CBZ, VPA, PB, TPM) 25/23 2 7.1 >0.5 8 48/control 27/21 2 Turan et al., 2014 2014 2 Turkey 2 Retrospective cohort 2 25-hydroxyvitamin D 144/(CBZ, VPA, PB) 4–12 >0.5 8 44/control 8.2 Pooled effect of AED treatment on BMD 2 A total of 11 studies comprising 23 datasets, which included 645 subjects and 579 controls, evaluated the effect of AEDs on lumbar spine BMD (Chung and Ahn, 1994 1994 ; Akin et al., 1998 1998 ; Kafali et al., 1999 1999 ; Erbayat Altay et al., 2000 2000 ; Oner et al., 2004 2004 ; Babayigit et al., 2006 2006 ; Kumandas et al., 2006 2006 ; El-Hajj Fuleihan et al., 2008 2008 ; Gniatkowska-Nowakowska, 2010 2010 ; Babacan et al., 2012 2012 ; Dimić et al., 2013 2013 ). The results suggested an association between AED treatment and decreased BMD (SMD=−0.30, 95% CI [−0.61, 0.01]; Fig. 2 2 ), with high heterogeneity ( I 2 =85%). When data were pooled for absolute changes in BMD (WMD=−0.03, 95% CI [−0.06, 0.00]) or BMD Z- scores (WMD=−0.87, 95% CI [−1.25, −0.48]), decreased BMD was found. No single study influenced the pooled effect significantly. Duration of follow-up and sex ratio did not affect the pooled effect, either. Subgroup analyses showed that the difference in AED type was a source of heterogeneity. As the results suggested, valproic acid (VPA) (SMD=−0.48, 95% CI [−0.95, −0.02] ) could significantly decrease BMD of lumbar spine in epileptic children, while carbamazepine (CBZ) (SMD=0.32, 95% CI [−1.08, 1.72]) had no significant effect. The funnel plots indicated no obvious publication bias. The changes in BMD of trochanter and femoral neck were assessed by three datasets from two studies (Oner et al., 2004 2004 ; Ecevit et al., 2004 2004 ) with 66 subjects and 64 controls. The pooled effect showed that AED treatment resulted in a significant decrease of BMD in trochanter (mean difference (MD)=−0.07, 95% CI [−0.10, −0.05]) and femoral neck (MD=−0.05, 95% CI [−0.09, −0.02] ) (Table 2 2 ). Subgroup analyses revealed that VPA was significantly associated with decreased BMD of trochanter (WMD=−0.08, 95% CI [−0.11, −0.05]) and femoral neck (WMD=−0.07, 95% CI [−0.11, −0.02] ). Sensitivity analysis showed that no single study affected the summary estimates significantly. The funnel plots showed no obvious publication bias. Variable Dataset/study Subject/control Summary measure Estimate (95% CI) P- value I 2 (%) Heterogeneity P -value BMD of lumbar spine 23/11 645/579 SMD: −0.30 [−0.61, 0.01] 0.06 85 <0.00001 BMD of trochanter 3/2 66/64 WMD: −0.07 [−0.10, −0.05] <0.00001 0 0.8 BMD of femoral neck 3/2 66/64 WMD: −0.05 [−0.09, −0.02] 0.002 0 0.54 Total body BMD 5/2 198/78 WMD: −0.33 [−0.51, −0.15] 0.0004 0 0.71 25-Hydroxyvitamin D 16/9 616/421 WMD: −3.37 [−5.94, −0.80] 0.01 84 <0.0001 Alkaline phosphatase 15/8 449/397 SMD: 0.71 [0.38, 1.05] <0.00001 83 <0.00001 Calcium 17/10 648/570 SMD: −0.49 [−1.03, 0.05] 0.08 95 <0.00001 Phosphorus 13/8 531/441 SMD: −0.16 [−0.70, 0.39] 0.57 94 <0.00001 Parathormone 11/6 287/219 SMD: 0.30 [−0.05, 0.64] 0.05 66 0.002 Similarly, meta-analysis of two studies (Sheth and Hermann, 2008 2008 ; Sheth et al., 2008 2008 ), which included 198 subjects and 78 controls, and evaluated the effect of AED treatment on total body BMD, obtained a significant result (WMD=−0.33, 95% CI [−0.51, −0.15]). Sensitivity analysis showed that no single study affected the summary estimates significantly. The funnel plots showed no obvious publication bias. Pooled effect of AED treatment on bone markers 2 The effects of AED treatment on bone markers, including 25-hydroxyvitamin D, serum alkaline phosphatase (ALP), serum calcium, phosphorus, and PTH, were assessed in our study. Meta-analysis of nine studies including 616 subjects and 421 controls (Verrotti et al., 2002 2002 ; Babayigit et al., 2006 2006 ; Nicolaidou et al., 2006 2006 ; El-Hajj Fuleihan et al., 2008 2008 ; Nettekoven et al., 2008 2008 ; Rauchenzauner et al., 2010 2010 ; Babacan et al., 2012 2012 ; Razazizan et al., 2013 2013 ; Turan et al., 2014 2014 ), revealed a significant association between AED treatment and decreased 25-hydroxyvitamin D (MD=−3.37, 95% CI [−5.94, −0.80]), as shown in Fig. 3 3 . Sensitivity analysis showed that no single study affected the summary estimates significantly. The funnel plots showed no obvious publication bias. The changes in serum ALP were assessed for 449 subjects and 397 controls from eight studies (Akin et al., 1998 1998 ; Kafali et al., 1999 1999 ; Voudris et al., 2002 2002 ; Oner et al., 2004 2004 ; Babayigit et al., 2006 2006 ; Kumandas et al., 2006 2006 ; Babacan et al., 2012 2012 ; Razazizan et al., 2013 2013 ). The pooled effect showed that AED treatment resulted in a significant elevation of serum ALP (SMD=0.71, 95% CI [0.38, 1.05]), as shown in Fig. 4 4 . Sensitivity analysis showed that no single study affected the summary estimates significantly. The funnel plots showed no obvious publication bias. No significant effects of AED treatment on serum PTH, serum calcium, or phosphorus were found. The detailed results are shown in Table 2 2 . Discussion 1 Childhood is a critical time for bone mineralization. During periods of high mineralization, children are prone to osteoporosis and bone fractures (Sheth et al., 1995 1995 ; Oner et al., 2004 2004 ). AEDs are one of the most important factors that may affect bone health, but there is no agreement about the effect of AEDs on BMD and bone metabolism of children with epilepsy. Our meta-analysis of studies into the effects of treatment with AEDs on epileptic children shows: (1) a reduction in BMD at lumbar spine, trochanter, femoral neck and total body BMD; (2) a reduction in 25-hydroxyvitamin D and an increase in serum ALP; (3) no significant changes in serum PTH, calcium, or phosphorus. Although the effect of AED treatment on the lumbar spine BMD was of borderline significance ( P =0.06), the omission of studies using BMD Z -scores made the effect significant ( P =0.03). The heterogeneity in the analysis was high ( I 2 =85%). The potentially confounding factors of polytherapy and difference in seizure type may be a source of heterogeneity. El-Hajj Fuleihan et al. ( 2008 2008 ) demonstrated that polytherapy patients were associated with a significantly lower BMD level than patients treated with monotherapy. Sheth and Hermann ( 2008 2008 ) found that children with symptomatic generalized epilepsy appear to be at highest risk for BMD. However, the two factors were not analyzed in this paper because of lack of data, and future studies are warranted to explore this issue further. CBZ and VPA are the frontline treatments of partial and generalized seizures in children and adults. With conflicting results about the effects of the two drugs on BMD, the mechanism by which AEDs decrease BMD has yet to be fully established. A classical theory holds the view that enzyme-inducing antiepileptic drugs (EIAEDs) decrease BMD by reducing vitamin D levels secondary to the therapeutic activation of specific cytochrome P450 isoenzymes. Our meta-analysis partially supports this theory by proving that CBZ was associated with lower levels of 25-hydroxyvitamin D (Fig. 3 3 ), and no significant difference in BMD among children treated with CBZ compared with controls (Fig. 2 2 ). The results also showed that non-EIAEDs, presented as VPA, could result in a significant decrease in BMD. Cerveny et al. ( 2007 2007 ) suggested that VPA may interfere with bone metabolism by activation of the pregnane X receptor, which promotes the expression of vitamin D-responsive genes. Feldkamp et al. ( 2000 2000 ) observed a decrease in bone cell proliferation, suggesting that the direct effects of anticonvulsant drugs on bone cells may contribute to skeletal damage. Furthermore, both EIAEDs and non-EIAEDs may also contribute to bone loss by inhibiting intestinal absorption of calcium and activation of vitamin D (Lee et al., 2010 2010 ). The newer antiepileptics are believed to have similar efficacy to the older AEDs, but fewer side effects. Babayigit et al. ( 2006 2006 ) examined CBZ, VPA and oxcarbazepine use in children, and reported no significant differences on lumbar spine BMD between the groups, but 25-hydroxyvitamin D in oxcarbazepine-treated children was significantly higher than that in the VPA group. Rauchenzauner et al. ( 2010 2010 ) found no significant difference in 25-hydroxyvitamin D between VPA and the newer AEDs. More studies are needed to make direct comparisons between newer and older AEDs in epileptic children. Low levels of 25-hydroxyvitamin D in AED users have been demonstrated in a number of studies, but not in all. Our results suggest that AEDs affect bone metabolism by reducing 25-hydroxyvitamin D. Low 25-hydroxyvitamin D concentration results in secondary hyperparathyroidism and accelerated bone loss, influencing absorption of calcium and phosphorus (Shellhaas and Joshi, 2010 2010 ). This theory is consistent with our results. However, no significant changes in serum PTH, Ca, or P were found in our study, suggesting that there are still gaps in our knowledge of the impact of AEDs on bone metabolism. Total ALP is considered a reliable biochemical marker of bone formation. The present meta-analysis demonstrates that children treated with AEDs have a significantly increased ALP level. However, the pooled results suggest that increased serum ALP is significantly associated with the use of CBZ and the newer antiepileptics, but not VPA, indicating that ALP is an inadequate marker for bone metabolism. It could be hypothesized that measurement of total ALP may simply reflect hepatic metabolism. Some studies consider ALP isoenzyme to be highly sensitive and specific to increased bone metabolism (Voudris et al., 2002 2002 ; 2005 2005 ). A longitudinal study showed that epileptic patients using CBZ can have their bone metabolism altered early in the course of treatment, as indicated by the elevated activity of serum bone ALP isoenzyme (Voudris et al., 2005 2005 ). Voudris et al. ( 2002 2002 ) reported that elevated bone ALP isoenzyme correlates with the duration of treatment in children on VPA without a concomitant significant elevation of total ALP. Bone ALP isoenzyme, but not total ALP, may therefore be used as a marker for the selection of patients who might benefit from a comprehensive evaluation of their bone metabolism profile. There is no agreement about the relationship between BMD and vitamin D status. Hannan et al. ( 2008 2008 ) showed that serum 25-hydroxyvitamin D and BMD were significantly related to one another in white men only. Mikati et al. ( 2006 2006 ) conducted the only randomized, controlled trial that demonstrated no significant difference between high- and low-dose vitamin D treatments, and no change in BMD compared with healthy controls after one year of treatment. Although there is no consensus on optimal vitamin D levels, many physicians often rely on 25-hydroxyvitamin D levels to evaluate bone health. However, a recent meta-analysis based on a large population indicated that vitamin D given alone was not effective in preventing fractures (Reid et al., 2014 2014 ). The meta-analysis performed by Vestergaard ( 2005 2005 ) showed that the BMD decrease in AED users was in a range consistent with the increase in fracture risk observed, if seizure-related fractures were excluded. The risk of fractures was only slightly higher compared with the general population (risk ratio (RR)=1.3, 95% CI [1.0–1.7]). Since many studies have confirmed the value of BMD for predicting fracture risk, BMD measurement may be an adequate surrogate for bone health. However, we suggest that BMD should be reserved for those with exceptionally high risk (e.g. history of fractures). Ambulatory status can affect markers of bone metabolism and BMD. Baer et al. ( 1997 1997 ) studied vitamin D levels in relation to ambulatory status in a large sample of children who lived at home. They found that the risk of vitamin deficiency among nonambulatory children was about twice that of ambulatory children ( χ 2 =20.9; P < 0.001). They concluded that ambulatory status correlates with abnormalities in the status of 25-hydroxyvitamin D, calcium, and bone. Another study reported no association between ambulatory status and 25-hydroxyvitamin D levels (Bergqvist et al., 2007 2007 ). There were limitations to this meta-analysis. One is that the studies included were limited to cohort studies. There have been no RCTs comparing BMD and/or bone metabolism in children treated with AEDs and healthy controls. Studies to date are of limited quality. Duration of follow-up, sex ratio, and dose of drug are not clear in some studies, making it difficult to do sensitive analysis. Important confounders, such as nutrition and season, were not always fully controlled for, which might have resulted in some over-estimation of effects due to residual confounding. The studies are grouped without adequate separation of drugs by metabolic pathway for lack of BMD or bone turnover markers corresponding to the drug used. Besides, most studies lump together a variety of drugs, some of them in monotherapy and some in polytherapy: data on specific drugs are needed. Most of the studies have used a small sample, making it difficult to compare the effects of specific AEDs. Eligible studies came from a limited number of countries, nine of 14 studies targeting BMD coming from Turkey. Duration of treatment and dose also differed widely among studies. Conclusions 1 Findings from our meta-analysis indicate that AED treatment is associated with decreased BMD in epileptic children. BMD monitoring should be reserved for epileptic children with exceptionally high risk for abnormal bone health. In the absence of evidence to the contrary, it seems reasonable to offer supplements and optimize 25-hydroxyvitamin D levels. Further research is needed to clarify the particular subgroups, dosages, and other factors that may influence the effects of AEDs on BMD and bone metabolism. References R Akin , V Okutan , U Sarici , 等 . Evaluation of bone mineral density in children receiving antiepileptic drugs . Pediatr Neurol , 1998 . 19 ( 2 ): 129 - 131 . 9744632 DOI: 10.1016/S0887-8994(98)00039-3 http://doi.org/10.1016/S0887-8994(98)00039-3 . Evaluation of bone mineral density in children receiving antiepileptic drugs O Babacan , A Karaoglu , S Vurucu , 等 . May long term oxcarbazepine treatment be lead to secondary hyperparathyroidism? . J Clin Neurol , 2012 . 8 ( 1 ): 65 - 68 . DOI: 10.3988/jcn.2012.8.1.65 http://doi.org/10.3988/jcn.2012.8.1.65 . May long term oxcarbazepine treatment be lead to secondary hyperparathyroidism? A Babayigit , E Dirik , E Bober , 等 . Adverse effects of antiepileptic drugs on bone mineral density . Pediatr Neurol , 2006 . 35 ( 3 ): 177 - 181 . 16939856 DOI: 10.1016/j.pediatrneurol.2006.03.004 http://doi.org/10.1016/j.pediatrneurol.2006.03.004 . Adverse effects of antiepileptic drugs on bone mineral density MT Baer , BW Kozlowski , EM Blyler , 等 . Vitamin D, calcium, and bone status in children with developmental delay in relation to anticonvulsant use and ambulatory status . Am J Clin Nutr , 1997 . 65 ( 4 ): 1042 - 1051 . 9094891 . Vitamin D, calcium, and bone status in children with developmental delay in relation to anticonvulsant use and ambulatory status CE Begley , M Famulari , JF Annegers , 等 . The cost of epilepsy in the United States: an estimate from population-based clinical and survey data . Epilepsia , 2000 . 41 ( 3 ): 342 - 351 . 10714408 DOI: 10.1111/j.1528-1157.2000.tb00166.x http://doi.org/10.1111/j.1528-1157.2000.tb00166.x . The cost of epilepsy in the United States: an estimate from population-based clinical and survey data AGC Bergqvist , JI Schall , VA Stallings . Vitamin D status in children with intractable epilepsy, and impact of the ketogenic diet . Epilepsia , 2007 . 48 ( 1 ): 66 - 71 . 17241209 DOI: 10.1111/j.1528-1167.2006.00803.x http://doi.org/10.1111/j.1528-1167.2006.00803.x . Vitamin D status in children with intractable epilepsy, and impact of the ketogenic diet L Cerveny , L Svecova , E Anzenbacherova , 等 . Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways . Drug Metab Dispos , 2007 . 35 ( 7 ): 1032 - 1041 . 17392393 DOI: 10.1124/dmd.106.014456 http://doi.org/10.1124/dmd.106.014456 . Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways S Chung , C Ahn . Effects of anti-epileptic drug therapy on bone mineral density in ambulatory epileptic children . Brain Dev , 1994 . 16 ( 5 ): 382 - 385 . 7892957 DOI: 10.1016/0387-7604(94)90125-2 http://doi.org/10.1016/0387-7604(94)90125-2 . Effects of anti-epileptic drug therapy on bone mineral density in ambulatory epileptic children CE Dent , A Richens , DJ Rowe , 等 . Osteomalacia with long-term anticonvulsant therapy in epilepsy . Br Med J , 1970 . 4 ( 5727 ): 69 - 72 . 5312062 DOI: 10.1136/bmj.4.5727.69 http://doi.org/10.1136/bmj.4.5727.69 . Osteomalacia with long-term anticonvulsant therapy in epilepsy M Dimić , A Dimić , Z Milosević , 等 . Bone mineral density in children with long-term antiepileptic therapy . Srp Arh Celok Lek , 2013 . 141 ( 5-6 ): 329 - 332 . (in Serbian) 23858802 DOI: 10.2298/SARH1306329D http://doi.org/10.2298/SARH1306329D . Bone mineral density in children with long-term antiepileptic therapy C Ecevit , A Aydoğan , T Kavakli , 等 . Effect of carbamazepine and valproate on bone mineral density . Pediatr Neurol , 2004 . 31 ( 4 ): 279 - 282 . 15464641 DOI: 10.1016/j.pediatrneurol.2004.03.021 http://doi.org/10.1016/j.pediatrneurol.2004.03.021 . Effect of carbamazepine and valproate on bone mineral density G El-Hajj Fuleihan , L Dib , B Yamout , 等 . Predictors of bone density in ambulatory patients on antiepileptic drugs . Bone , 2008 . 43 ( 1 ): 149 - 155 . 18467202 DOI: 10.1016/j.bone.2008.03.002 http://doi.org/10.1016/j.bone.2008.03.002 . Predictors of bone density in ambulatory patients on antiepileptic drugs E Erbayat Altay , A Serdaroglu , L Tumer , 等 . Evaluation of bone mineral metabolism in children receiving carbamazepine and valproic acid . J Pediatr Endocrinol Metab , 2000 . 13 ( 7 ): 933 - 939 . 10968482 DOI: 10.1515/JPEM.2000.13.7.933 http://doi.org/10.1515/JPEM.2000.13.7.933 . Evaluation of bone mineral metabolism in children receiving carbamazepine and valproic acid J Feldkamp , A Becker , OW Witte , 等 . Long-term anticonvulsant therapy leads to low bone mineral density-evidence for direct drug effects of phenytoin and carbamazepine on human osteoblast-like cells . Exp Clin Endocrinol Diabetes , 2000 . 108 ( 1 ): 37 - 43 . 10768830 . Long-term anticonvulsant therapy leads to low bone mineral density-evidence for direct drug effects of phenytoin and carbamazepine on human osteoblast-like cells A Gniatkowska-Nowakowska . Fractures in epilepsy children . Seizure , 2010 . 19 ( 6 ): 324 - 325 . 20493726 DOI: 10.1016/j.seizure.2010.04.013 http://doi.org/10.1016/j.seizure.2010.04.013 . Fractures in epilepsy children MT Hannan , HJ Litman , AB Araujo , 等 . Serum 25-hydroxyvitamin D and bone mineral density in a racially and ethnically diverse group of men . J Clin Endocrinol Metab , 2008 . 93 ( 1 ): 40 - 46 . 17986641 DOI: 10.1210/jc.2007-1217 http://doi.org/10.1210/jc.2007-1217 . Serum 25-hydroxyvitamin D and bone mineral density in a racially and ethnically diverse group of men G Kafali , T Erselcan , F Tanzer . Effect of antiepileptic drugs on bone mineral density in children between ages 6 and 12 years . Clin Pediatr , 1999 . 38 ( 2 ): 93 - 98 . DOI: 10.1177/000992289903800205 http://doi.org/10.1177/000992289903800205 . Effect of antiepileptic drugs on bone mineral density in children between ages 6 and 12 years SC Karceski . Seizure medications and their side effects . Neurology , 2007 . 69 ( 22 ): E27 - E29 . 18040007 DOI: 10.1212/01.wnl.0000296051.34044.07 http://doi.org/10.1212/01.wnl.0000296051.34044.07 . Seizure medications and their side effects E Keck , B Gollnick , D Reinhardt , 等 . Calcium metabolism and vitamin D metabolite levels in children receiving anticonvulsant drugs . Eur J Pediatr , 1982 . 139 ( 1 ): 52 - 55 . 7173258 DOI: 10.1007/BF00442080 http://doi.org/10.1007/BF00442080 . Calcium metabolism and vitamin D metabolite levels in children receiving anticonvulsant drugs S Kumandas , E Koklu , H Gümüs , 等 . Effect of carbamezapine and valproic acid on bone mineral density, IGF-I and IGFBP-3 . J Pediatr Endocrinol Metab , 2006 . 19 ( 4 ): 529 - 534 . 16759039 . Effect of carbamezapine and valproic acid on bone mineral density, IGF-I and IGFBP-3 RH Lee , KW Lyles , C Colón-Emeric . A review of the effect of anticonvulsant medications on bone mineral density and fracture risk . Am J Geriatr Pharmacother , 2010 . 8 ( 1 ): 34 - 46 . 20226391 DOI: 10.1016/j.amjopharm.2010.02.003 http://doi.org/10.1016/j.amjopharm.2010.02.003 . A review of the effect of anticonvulsant medications on bone mineral density and fracture risk A Liberati , DG Altman , J Tetzlaff , 等 . The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration . Ann Intern Med , 2009 . 151 ( 4 ): W65 - W94 . 19622512 DOI: 10.7326/0003-4819-151-4-200908180-00136 http://doi.org/10.7326/0003-4819-151-4-200908180-00136 . The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration C Meier , ME Kraenzlin . Antiepileptics and bone health . Ther Adv Musculoskelet Dis , 2011 . 3 ( 5 ): 235 - 243 . 22870482 DOI: 10.1177/1759720X11410769 http://doi.org/10.1177/1759720X11410769 . Antiepileptics and bone health MA Mikati , L Dib , B Yamout , 等 . Two randomized vitamin D trials in ambulatory patients on anticonvulsants: impact on bone . Neurology , 2006 . 67 ( 11 ): 2005 - 2014 . 17159108 DOI: 10.1212/01.wnl.0000247107.54562.0e http://doi.org/10.1212/01.wnl.0000247107.54562.0e . Two randomized vitamin D trials in ambulatory patients on anticonvulsants: impact on bone S Nettekoven , A Ströhle , B Trunz , 等 . Effects of antiepileptic drug therapy on vitamin D status and biochemical markers of bone turnover in children with epilepsy . Eur J Pediatr , 2008 . 167 ( 12 ): 1369 - 1377 . 18270736 DOI: 10.1007/s00431-008-0672-7 http://doi.org/10.1007/s00431-008-0672-7 . Effects of antiepileptic drug therapy on vitamin D status and biochemical markers of bone turnover in children with epilepsy P Nicolaidou , H Georgouli , H Kotsalis , 等 . Effects of anticonvulsant therapy on vitamin D status in children: prospective monitoring study . J Child Neurol , 2006 . 21 ( 3 ): 205 - 209 . 16901421 . Effects of anticonvulsant therapy on vitamin D status in children: prospective monitoring study N Oner , M Kaya , S Karasalihoglu , 等 . Bone mineral metabolism changes in epileptic children receiving valproic acid . J Paediatr Child Health , 2004 . 40 ( 8 ): 470 - 473 . 15265190 DOI: 10.1111/j.1440-1754.2004.00431.x http://doi.org/10.1111/j.1440-1754.2004.00431.x . Bone mineral metabolism changes in epileptic children receiving valproic acid AM Pack , MJ Morrell . Adverse effects of antiepileptic drugs on bone structure: epidemiology, mechanisms and therapeutic implications . CNS Drugs , 2001 . 15 ( 8 ): 633 - 642 . 11524035 DOI: 10.2165/00023210-200115080-00006 http://doi.org/10.2165/00023210-200115080-00006 . Adverse effects of antiepileptic drugs on bone structure: epidemiology, mechanisms and therapeutic implications AM Pack , MJ Morrell , A Randall , 等 . Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy . Neurology , 2008 . 70 ( 18 ): 1586 - 1593 . 18443309 DOI: 10.1212/01.wnl.0000310981.44676.de http://doi.org/10.1212/01.wnl.0000310981.44676.de . Bone health in young women with epilepsy after one year of antiepileptic drug monotherapy SJ Petty , LM Paton , TJ O'Brien , 等 . Effect of antiepileptic medication on bone mineral measures . Neurology , 2005 . 65 ( 9 ): 1358 - 1365 . 16275821 DOI: 10.1212/01.wnl.0000180910.72487.18 http://doi.org/10.1212/01.wnl.0000180910.72487.18 . Effect of antiepileptic medication on bone mineral measures M Rauchenzauner , A Griesmacher , T Tatarczyk , 等 . Chronic antiepileptic monotherapy, bone metabolism, and body composition in non-institutionalized children . Dev Med Child Neurol , 2010 . 52 ( 3 ): 283 - 288 . 19709134 DOI: 10.1111/j.1469-8749.2009.03402.x http://doi.org/10.1111/j.1469-8749.2009.03402.x . Chronic antiepileptic monotherapy, bone metabolism, and body composition in non-institutionalized children N Razazizan , M Mirmoeini , S Daeichin , 等 . Comparison of 25-hydroxy vitamin D, calcium and alkaline phosphatase levels in epileptic and non-epileptic children . Acta Neurol Taiwan , 2013 . 22 ( 3 ): 112 - 116 . 24596971 . Comparison of 25-hydroxy vitamin D, calcium and alkaline phosphatase levels in epileptic and non-epileptic children IR Reid , MJ Bolland , A Grey . Effects of vitamin D supplements on bone mineral density: a systematic review and meta-analysis . Lancet , 2014 . 383 ( 9912 ): 146 - 155 . 24119980 DOI: 10.1016/S0140-6736(13)61647-5 http://doi.org/10.1016/S0140-6736(13)61647-5 . Effects of vitamin D supplements on bone mineral density: a systematic review and meta-analysis H Salimipour , S Kazerooni , M Seyedabadi , 等 . Antiepileptic treatment is associated with bone loss: difference in drug type and region of interest . J Nucl Med Technol , 2013 . 41 ( 3 ): 208 - 211 . 23886631 DOI: 10.2967/jnmt.113.124685 http://doi.org/10.2967/jnmt.113.124685 . Antiepileptic treatment is associated with bone loss: difference in drug type and region of interest RA Shellhaas , SM Joshi . Vitamin D and bone health among children with epilepsy . Pediatr Neurol , 2010 . 42 ( 6 ): 385 - 393 . 20472188 DOI: 10.1016/j.pediatrneurol.2009.12.005 http://doi.org/10.1016/j.pediatrneurol.2009.12.005 . Vitamin D and bone health among children with epilepsy RD Sheth , BP Hermann . Bone in idiopathic and symptomatic epilepsy . Epilepsy Res , 2008 . 78 ( 1 ): 71 - 76 . 18078739 DOI: 10.1016/j.eplepsyres.2007.10.010 http://doi.org/10.1016/j.eplepsyres.2007.10.010 . Bone in idiopathic and symptomatic epilepsy RD Sheth , CA Wesolowski , JC Jacob , 等 . Effect of carbamazepine and valproate on bone mineral density . J Pediatr , 1995 . 127 ( 2 ): 256 - 262 . 7636651 DOI: 10.1016/S0022-3476(95)70304-7 http://doi.org/10.1016/S0022-3476(95)70304-7 . Effect of carbamazepine and valproate on bone mineral density RD Sheth , N Binkley , BP Hermann . Progressive bone deficit in epilepsy . Neurology , 2008 . 70 ( 3 ): 170 - 176 . 18057319 DOI: 10.1212/01.wnl.0000284595.45880.93 http://doi.org/10.1212/01.wnl.0000284595.45880.93 . Progressive bone deficit in epilepsy PC Souverein , DJ Webb , H Petri , 等 . Incidence of fractures among epilepsy patients: a population-based retrospective cohort study in the General Practice Research Database . Epilepsia , 2005 . 46 ( 2 ): 304 - 310 . 15679512 DOI: 10.1111/j.0013-9580.2005.23804.x http://doi.org/10.1111/j.0013-9580.2005.23804.x . Incidence of fractures among epilepsy patients: a population-based retrospective cohort study in the General Practice Research Database MI Turan , A Cayir , O Ozden , 等 . An examination of the mutual effects of valproic acid, carbamazepine, and phenobarbital on 25-hydroxyvitamin D levels and thyroid function tests . Neuropediatrics , 2014 . 45 ( 1 ): 16 - 21 . 23888467 DOI: 10.1055/s-0033-1349226 http://doi.org/10.1055/s-0033-1349226 . An examination of the mutual effects of valproic acid, carbamazepine, and phenobarbital on 25-hydroxyvitamin D levels and thyroid function tests A Verrotti , R Greco , G Latini , 等 . Increased bone turnover in prepubertal, pubertal, and postpubertal patients receiving carbamazepine . Epilepsia , 2002 . 43 ( 12 ): 1488 - 1492 . 12460249 DOI: 10.1046/j.1528-1157.2002.13002.x http://doi.org/10.1046/j.1528-1157.2002.13002.x . Increased bone turnover in prepubertal, pubertal, and postpubertal patients receiving carbamazepine A Verrotti , G Coppola , P Parisi , 等 . Bone and calcium metabolism and antiepileptic drugs . Clin Neurol Neurosurg , 2010 . 112 ( 1 ): 1 - 10 . 19913352 DOI: 10.1016/j.clineuro.2009.10.011 http://doi.org/10.1016/j.clineuro.2009.10.011 . Bone and calcium metabolism and antiepileptic drugs P Vestergaard . Epilepsy, osteoporosis and fracture risk–a meta-analysis . Acta Neurol Scand , 2005 . 112 ( 5 ): 277 - 286 . 16218908 DOI: 10.1111/j.1600-0404.2005.00474.x http://doi.org/10.1111/j.1600-0404.2005.00474.x . Epilepsy, osteoporosis and fracture risk–a meta-analysis K Voudris , M Moustaki , PM Zeis , 等 . Alkaline phosphatase and its isoenzyme activity for the evaluation of bone metabolism in children receiving anticonvulsant monotherapy . Seizure , 2002 . 11 ( 6 ): 377 - 380 . 12160665 DOI: 10.1053/seiz.2002.0671 http://doi.org/10.1053/seiz.2002.0671 . Alkaline phosphatase and its isoenzyme activity for the evaluation of bone metabolism in children receiving anticonvulsant monotherapy KA Voudris , A Attilakos , E Katsarou , 等 . Early alteration in bone metabolism in epileptic children receiving carbamazepine monotherapy owing to the induction of hepatic drug-metabolizing enzymes . J Child Neurol , 2005 . 20 ( 6 ): 513 - 516 . 15996401 DOI: 10.1177/08830738050200060801 http://doi.org/10.1177/08830738050200060801 . Early alteration in bone metabolism in epileptic children receiving carbamazepine monotherapy owing to the induction of hepatic drug-metabolizing enzymes Compliance with ethics guidelines Ying ZHANG, Yu-xin ZHENG, Jun-ming ZHU, Jian-min ZHANG, and Zhe ZHENG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500021 Project supported by the National High-Tech R & D Program (863) of China (No. 2012AA020408) and the Medical and Health General Research Plan of Zhejiang Province (No. 2014KYA103), China 2015-01-20 2015-06-16 2015-06-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 16