Eff ects of carbamazepine on serum parathormone , 25-hydroxyvitamin D , bone specifi c alkaline phosphatase , C-telopeptide , and osteocalcin levels in healthy rats

It is still not completely clear whether carbamazepine causes alterations in vitamin D status and in bone metabolism. Th e objective of this study was to investigate the eff ects of carbamazepine on serum levels of -hydroxyvitamin D and on biomarkers of bone formation and resorption in healthy rats. Levels of calcium, hydroxyvitamin D, parathormone, C-telopeptide, bone specifi c alkaline phosphatase and osteocalcin were measured in  groups of rats consisting of controls (n=), isotonic saline solution group (n=) and carbamazepine group (n=). Mean calcium levels were found to be signifi cantly lower in healthy controls in comparison to isotonic saline solution and carbamazepine groups (.±., .±., .±. mg/dL, respectively, p<.). Mean levels of hydroxyvitamin D, were found to be signifi cantly higher in control group compared to isotonic saline solution group (hydroxyvitamin D; .±., .±. ng/mL, respectively, p<.). Mean levels of parathormone and osteocalcin were found to be signifi cantly higher in control group compared to isotonic saline solution group and carbamazepine group. Parathormone levels were measured as .±., .±., .±. pg/mL, respectively (p<.). Osteocalcine levels were measured as .±., .±., .±. ng/mL, respectively (p<.). A signifi cant diff erence in terms of mean serum bone specifi c alkaline phosphatase and C-telopeptide levels among groups was not observed. Th e main outcome of this prospective study in healthy rats showed no change in biochemical parameters of bone turnover during treatment with carbamazepine. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Epileptic seizures result from excessive discharge in a population of hyperexcitable neurons [].Antiepileptic drugs (AEDs) are increasingly used for the treatment of several nonepileptic neurological conditions and psychiatric disorders [].Most of the patients with epilepsy require a long-term and sometimes lifelong therapy with AEDs and, therefore, they are exposed to the potential undesirable metabolic side eff ects of medical treatment [].Th e reported eff ects of AEDs on bone include rickets, osteomalacia, osteoporosis, and fractures [].Most of the published studies and evidence involve patients receiving AEDs that induce the cytochrome P enzyme system (phenobarbital, phenytoin, and carbamazepine) are most commonly associated with abnormalities in bone [-].Markers of bone formation, which include alkaline phosphatase and osteocalcin, have been assessed in patients receiving AEDs [-].Carbamazepine (CBZ), which has been mostly used in epilepsy treatment for  years, is also used for the treatment of neuropsychiatric disorders, neuropathy and depression in recent years [-].Up to now, either epileptic animals or patients have been used in the studies.Th erefore, it is not clear whether the eff ects in question are due to carbamazepine treatment only, or whether seizures also contribute to this situation.In these studies the action mechanism of the anti-epileptic drug on bone metabolism can not be detected.In this present study, the eff ects and action mechanism of carbamazepine on bone metabolism of healthy rats were investigated in order to detect the drug-related side eff ects only.Th us we measured serum osteocalcin (OC), parathormone (PTH), C-telopeptide (CTX), bone specific alkaline phosphatase (BAP) and -OH vitamin D (-OH-VD) levels in healthy rats receiving carbamazepine and investigated whether side eff ects on bone are a result of the drug alone.

Procedures
Injections were done between :-: am in all groups. mg/kg carbamazepine in isotonic saline solution not exceeding  mL in volume was injected intraperitoneally once a day over  weeks in sterile conditions.According to previous studies carried out with various anti-epileptic drugs, a -week anti-epileptic treatment is suffi cient for observing the side eff ects on the bone structure and metabolism of rats [-].Body weight of animals were followed up in the course of  week drug therapy.

Statistical analysis
Statistical analysis was performed by using the SPSS for windows (version .) statistical package (SPSS Inc., Chicago, IL, USA).The values were expressed as mean ±SEM.Differences between the groups were analyzed using the one way ANOVA and Bonferroni multiple comparisons tests.p values of < . were considered statistically significant.

RESULTS
Mean serum calcium levels were found to be signifi cantly lower in Group  compared to Group  and  (.±.,.±.,.±.mg/dL, respectively, p<.) (Table ), (Figure ).Mean serum -OH-VD levels were found   to be signifi cantly higher in Group  compared

DISCUSSION AND CONCLUSION
Th e main outcome of this prospective study in healthy rats showed no change in biochemical parameters of bone turnover during treatment with CBZ.Bone is a complex dynamic tissue that responds to external and internal forces.Accordingly, body weight, exercise, and calcium homeostasis can alter bone structure and architecture.These changes are complex and involve osteoclast and osteoblast dynamics, vitamin D, calcium, and phosphorous homeostasis as well as connective tissue arrangements [, ].Increased risk for bone disease including changes in bone turnover, osteoporosis, alterations in bone quality, and most importantly fracture.Although carbamazepine is an enzyme inducing AED, data on the issue is not conclusive [].In a study by Nissen-Meyer et al. [], researchers emphasized the importance of using animal models in order to put forward the eff ects of anti-epileptic drugs on bone.Th ey stated that animal tests were more beneficial for testing anti-epileptic drug-related bone fragility because such studies allow the elimination of possible alterations resulting from life style and genetic and individual disease characteristics.Th e ,dihydroxy vitamin D (, (OH)D) has its eff ect on the classic target organs bone, intestine and kidney and stimulates calcium transport from these organs to the blood.Th e production of , (OH)D is stimulated by PTH.Th ere is a negative feedback through calcium which decreases PTH and a direct negative feedback from ,(OH)D to PTH [].
While vitamin D deficiency leads to osteoporosis, vitamin D insufficiency-subsequent form of vitamin D deficiency-leads to osteomalacia [].Some studies show that, biochemical abnormalities in patients receiving carbamazepine include hypocalcemia, hypophosphatemia, reduced levels of active vitamin D metabolites, elevated PTH levels, and elevated markers of bone resorption and formation [, , , , , ].However, reduction was not detected in serum calcium and -OH-VD levels and elevation of PTH levels was not detected in the group receiving anti-epileptic drug in our study.Alkaline phosphatase is the most commonly used marker of bone formation [-].Because serum total alkaline phosphatase is derived from bone, liver, and other sources, it lacks sensitivity and specifi city in evaluating bone disease.In reports that measured the isoenzymes, the increase in total alkaline phosphatase was attributed mainly to the bone fraction [, , ].ALP increases have been seen in both children and adults receiving AEDs [-].In contrast to the aforementioned studies, a statistically signifi cant elevation was not found in BAP levels in the rats receiving carbamazepine treatment in the present study.Osteocalcin, or bone-gla protein, is a small noncollagenous protein that is specifi c for bone tissue and dentin and is synthesized predominantly by osteoblasts.High serum levels of osteocalcin with AED treatment are described [, , , ].Levels of osteocalcin, the marker of bone formation, was also not elevated in the carbamazepine receiving group.In one study, low serum calcium concentrations   were considered to be the long term outcome of treatment with anti-epileptic drugs [].Studies report that alterations in markers of bone formation and resorption are observed particularly in patients receiving multi-drug therapy [].Verrotti et al. [] reported as the result of the study carried out by administering carbamazepine to epilepsy patients that bone turnover may increase despite normal vitamin D levels.Some another studies reported abnormalities in the bone metabolism of patients treated with AEDs independently of vitamin D defi ciency [, , , ].Th ese data suggest that AEDs might alter the skeleton via other mechanisms, e.g.direct eff ects on bone cells, intestinal calcium transport and resistance to parathyroid hormone, rather than monocausally by decreased serum -OHVD [, , ].In some recent studies, genetic variation was shown to have some eff ects as reducing bone mineral density and active vitamin D metabolism in epilepsy patients receiving antiepileptic drugs [, ].
In conclusion, a statistically signifi cant alteration has not been observed in biomarkers of bone formation and resorption in healthy rats receiving carbamazepine.Studying the eff ects of carbamazepine in healthy animals enabled elimination of some confounding factors affecting bone metabolism (e.g.renal disease, hyperparathyroidism, cancer, gastrointestinal disorders, liver insuffi ciency, diabetes mellitus, drug use as corticosteroids and diet) [].However based on the studies reporting that side eff ects concerning bone may arise in the long term, we consider that animal models studying long term eff ects of carbamazepine could be built up in future studies.

DECLARATION OF INTEREST
Th e authors declare that they have no confl ict of interest related to the publication of this manuscript.
Blood samples obtained from the animals at the end of  weeks were centrifuged at  o C,  rpm for  minutes.Serum samples were stored at - o C until biochemical analysis was completed.Serum calcium levels were measured with Abbot Aeroset autoanalyser using Aeroset kit (Abbot Lab.Abbot Park, IL ,USA).Serum osteocalcin, parathormone, C-telopeptide, bone specific alkaline phosphatase and -OH vitamin D levels were measured with ELISA method using microarray device.OC and PTH levels were measured with DRG ELISA kit (DRG International, Inc. USA), BAP and -OH-VD levels were measured with IDS ELISA kit (IDS Ltd.Fountain Hills, USA), whereas CTX levels were measured with Uscn ELISA kit (Uscn Life Science Inc. Wuhan).Analyses of all the samples, standards, and controls were run in duplicate.

FIGURE 1 .
FIGURE 1. Calcium levels of the three groups.*Signifi cantly lower than those of the Group II and Group III (respective values: p < 0.05, p <0.05 ).

FIGURE 2 .
FIGURE 2. 25-OH vitamine D levels of the three groups.**Signifi cantly higher than those of the Group II (respective values: p < 0.01).

FIGURE 3 .
FIGURE 3. Parathormon levels of the three groups.***Significantly higher than those of the group II and group III (respective values: p < 0.01, p <0.01).

FIGURE 4 .
FIGURE 4. Osteocalsin levels of the three groups.****Signifi cantly higher than those of the group II and group III (respective values: p < 0.001, p <0.001).