Early predisposition to osteomalacia in Indian adults on phenytoin or valproate monotherapy and effective prophylaxis by simultaneous supplementation with calcium and 25-hydroxy vitamin D at recommended daily allowance dosage: A prospective study
Correspondence Address: Source of Support: The Staff and Research Society of L.T.M.M.C for funding this project, Conflict of Interest: None DOI: 10.4103/0028-3886.63796
Source of Support: The Staff and Research Society of L.T.M.M.C for funding this project, Conflict of Interest: None
Background: Long-term therapy with antiepileptic drugs (AED) may be associated with increased total serum alkaline phosphatase (ALP) levels and reduced serum calcium, inorganic phosphorous, and vitamin D levels. These adverse biochemical alterations have an adverse effect on bone health Objective: To determine (a) onset of derangements in serum total ALP and its isoenzymes (liver, bone), calcium and 25-hydroxy vitamin D (25-OHD) concentrations after initiation of treatment with phenytoin or valproic acid monotherapy and (b) the effect of simultaneous supplementation with calcium and 25-OHD at recommended daily allowance (RDA) dosage, on these biochemical parameters. Materials and Methods: Study was a prospective, case-controlled study in adults. Serum biochemical parameters were estimated at baseline, 30, 60, and 90 days of starting AED treatment in the study subjects: Groups--A (only calcium supplementation) and Group B (both calcium and 25-OHD supplementation). Statistical analysis: Mean±SD, and students' paired t test (between groups A and B) unpaired students' t test (drug-wise). Results: At 60 days of AED therapy Group A showed a significant increase in serum total ALP (78.83±11.04 to101.75 ± 9.56 IU/l) (P<0.001), ALP-liver isoenzyme, (41.97± 10.81 to 68.83 ±7.81 IU/L) (P<0.001), significant decrease in calcium (9.30 ± 0.36 to 8.80 ± 0.38 mg%) (P<0.001), ALP-bone isoenyzme (36.84 ± 5.01 to 32.92 ± 6.46 IU/L) (P<0.001), and a significant decrease in 25-OHD (25.19 ± 5.98 to 19.76 ± 5.35 ng/ml) (P<0.001) at 90 days. In contrast Group B, at 60 days, showed a significant decrease in serum total ALP (81.92 ± 19.63 to 54.77. ± 11.53 IU/L) (P<0.0001), ALP-liver isoenzyme (48.01. ± 13.53 to 28.12. ± 5.88 IU/L) (P<0.0001), significant increase in calcium ((9.24 ± 0.31 to 9.93 ± 0.26 mg%) (P<0.001) and ALP-bone isoenzyme levels (33.93 ± 12.2 to 26.25 ± 8.23 IU/L). In Group B, 25-OHD levels showed a significant increase at 90 days (24.36 ± 3.42 to 31.53 ± 327 ng/ml) (P<0.0001). Conclusion: Biochemical derangements in calcium metabolism involving the bone are seen by 60 days after starting AED monotherapy, indicating predisposition to development of osteomalacia in these patients. This is preventable by simultaneous oral supplementation with calcium and 25-OHD.
Keywords: Anticonvulsant drug, bone metabolism, supplemental calcium and vitamin D
Long-term therapy with antiepileptic drugs (AEDs) in patients with epilepsy, both adults and children, for more than six months to two years may be associated with increased total serum alkaline phosphatase (ALP) levels and reduced serum calcium, inorganic phosphorous, and vitamin D levels. These adverse biochemical alterations can lead to rickets (in growing children) and osteomalacia (in older children and adults), and over the years can reduce bone mineral density. ,,, Since the signs and symptoms of osteomalacia are nonspecific, its diagnosis is frequently delayed. However, till date there is little data available on how early these adverse biochemical changes occur in patients with epilepsy after being initiated on AED therapy. These adverse biochemical changes have been reported to be reversible over a period of 12 to 15 months with daily supplementation of vitamin D in patients with epilepsy on long-term AED therapy. ,,,, Of the isoenzymes of ALP, liver-specific (liver ALP) and bone-specific (bone ALP) isoenzymes account for the maximum total activity.  Determination of total serum ALP concentration itself cannot be used to monitor alterations of bone metabolism as the elevation of total ALP levels might simply reflect elevated liver ALP levels, the well-known "adverse influence" of AEDs on liver function. ,,,, Changes in serum bone ALP occur much earlier than the changes in the dual energy X-ray absorptiometry (DXA).  Serum bone ALP concentration reflects osteoblast activity, is an indicator of bone formation, and its elevated concentrations can help predict future metabolic bone disease and risk of fractures independent of bone mineral density. 
The present study was undertaken to determine: (i) how early the changes in serum total ALP and its isoenzymes, calcium, inorganic phosphorus, 25-OHD, magnesium, total proteins and albumin occur in ambulatory adult patients with adequate sun exposure with new-onset epilepsy on phenytoin or valproic acid monotherapy; and (ii) to determine the effectiveness of simultaneous daily oral supplementation with calcium and 25-OHDon these biochemical changes.
Adults with new-onset epilepsy attending the outpatient department, or admitted to the ward on standard recommended doses of either phenytoin (5-8 mg/kg/day) or valproic acid (20 mg/kg/day) were the subjects of the study. Adults already on calcium and vitamin D supplements, or already having clinical/biochemical evidence of osteomalacia, signs of malnutrition, or any chronic bone, liver or renal disease were excluded from the study. The sample size was by necessity a convenience sample. All the adults were residents of the city, ambulatory and had age-appropriate activity and adequate exposure to sun and nutritionally adequate diets.
At inclusion blood samples were collected from all the patients to measure serum ALP activity (total and isoenzymes), calcium, inorganic phosphorous, 25-OHD, magnesium, total proteins and albumin. Standard biochemical analytical methods were used. ,,,,,,,, Serum total ALP was estimated by the Kind and King method, ALP isoenzymes (bone ALP, and, non-bone ALP viz. liver ALP + intestinal ALP) by the heat-inactivation method, calcium by O-cresolphthalein method, inorganic phosphorous by the Fiske-Subbarow method, 25-OHDby high-performance liquid chromatographic method, magnesium by the calmagite method, total proteins by Biuret method and serum albumin by Bromocresol green method.
The study subjects were randomly divided into two Groups, A or B. These groups were similar by mean age, gender distribution, and seizure diagnosis. Diagnosis of type of epilepsy was based on seizure semiology, electroencephalography and imaging and included both idiopathic generalized epilepsy and localization-related epilepsy. Patients in both groups belonged to lower and middle income socioeconomic class and clinical examination at study initiation did not reveal any nutritional deficiency signs. Patients in Group A (n=34, 15 females, mean age 24.1±10.01 years) received daily only oral calcium lactate (1000 mg/day elemental calcium) supplementation. Of the 34 patients in Group A, 19 were on phenytoin and 15 on valproate. Patients in Group B (n=32, 13 females, mean age 23.6±8.67years) received daily oral calcium 1000 mg and 400 IU/L of 25-OHD supplementation as per recommended daily allowance (RDA) specifications. Of the 32 patients in Group B, 17 patients were on phenytoin and 15 were on valproate. Supplementation of calcium and 400 IU of 25-OHD at RDA doses  was considered keeping in mind the year- round abundance of sunshine in the city of Mumbai, the ambulatory status of the study cases and their regular dietary habits. A gap of minimum two hours was kept between the AED and the oral supplementation intake. All study subjects in each of the two groups were followed up every 15 days for compliance by a physician and the biochemical parameters were serially estimated at 30, 60, and 90 days.
This study was approved by the scientific and ethics committees of our institution and all the subjects agreed to participate in the the study had signed an informed consent form to participate in the study.
The Statistical Package for the Social Sciences program, Version 11 for Windows (SPSS Ltd., Chicago, Illinois, USA) was used for data analysis. Data were expressed as mean ± SD values. The student's paired t test was used to compare the serial changes in serum biochemical parameters from baseline and at 30, 60 and 90 days of starting of treatment in both the Groups, A and B. The student's unpaired t test was used for each drug to compare the biochemical parameters between Group A and B at baseline and at 30,60 and 90 days of starting treatment. A two-tailed P value of <0.05 was used to define statistical significance.
The total number of adult subjects included in the study was 66; of these 34 were in Group A and the remaining 32 were in Group B. The mean age in Group A was 23.6 ± 8.67 years (range 0.08-11); and that in Group B was 24.1 10.01 years (range 0.09-11). The data regarding the changes in biochemical parameters for both the Groups are given [Table 1].
I. Intra-group comparison for serial biochemical changes within group A
Serum total ALP level increased significantly from 78.83 ± 11.04 IU/L at baseline to 88.78 ± 10.11 IU/L at the end of 60 days (P<0.001), and was above the normal range (101.75 ± 9.56 IU/L) (P<0.001) at 90 days after starting treatment. The activity of serum liver isoenzyme fraction significantly increased from a baseline value of 41.97 ± 10.81 IU/L to 55.15 ± 8.40 IU/L at 60 days (P<0.001), and 68.83 ± 7.81 IU/L at the end of 90 days (P<0.001). Since the isoenzyme is a fraction of the total ALP activity, this increase was from 53.26% at baseline to 62.11% at 60 days and 67.7% at 90 days. Serum bone ALP level was not affected till 60 days but significantly decreased from a baseline value of 36.84 ± 8.4 IU/L to 32.92 ± 6.46 at 90 days (P<0.001) , i.e., from 46.73% of the total activity at baseline to 32.38% at 90 days. Serum levels of 25-OHD decreased significantly from a mean baseline value of 25.19 ± 5.98 ng/ml to 19.76 ± 5.35 ng/ml (P<0.001) at the end of 90 days. Serum calcium level decreased significantly from a baseline value of 9.30 mg% ± 0.36 to 9.08 mg% ± 0.36 at 60 days (P<0.05), and 8.80 mg% ± 0.38 (P<0.001) at 90 days. There were no changes in serum phosphorous, magnesium, total proteins, and albumin levels which remained within their normal ranges.
II. Intra-group comparison for serial biochemical changes within Group B
Serum total ALP level was significantly lowered but within the normal range from baseline value of 81.92 ± 19.63 to 66.19 ± 13.58 IU/L(P<0.001) at 60 days and 54.77 ± 11.53 IU/L (P<0.001) at 90 days after starting treatment. Serum liver isoenzyme level significantly decreased from baseline value of 48.01 ± 13.53 IU/L to 37.83 ± 11.74 IU/L (P<0.001) at 60 days and 28.12 ± 5.88 IU/L(P<0.001)at 90 days .ie. from 58.60% at baseline to 57.3% at 60 days and 52.07% at 90 days after commencement of treatment. Serum bone isoenzyme level significantly changed from baseline value of 33.93 ± 12.2 IU/L to 28.38 ± 4.07 IU/L at 60 days (P<0.05) (41.41% of total activity to 43%) and to 26.65 ± 8.23 IU/L (49.33% of total activity) at 90 days after starting therapy (P< 0.001). Serum 25-OHDlevels increased from baseline value of 24.36 ± 3.42 ng/nl to 31.53 ± 3.27 ng/ml (P<0.001) at 90 days. Serum calcium level increased from baseline value of 9.24 ± 0.31 mg% to 9.55 ± 0.29 mg% at 60 (P<0.05), and to 9.93 ± 0.26 mg% (P<0.001) at 90 days. There were no significant changes in serum magnesium, total proteins, and albumin levels which remained within their normal ranges.
III. Inter-group comparison for serial biochemical changes between Group A and B
The total ALP activity of Groups A and B were the same at baseline. As early as 60 days, a significant difference (P<0.001) was observed in the values indicating the effect of supplementation with 25-OHD in Group B and lack of supplementation with 25-OHD in Group A [Figure 1]. Supplementation with calcium and 25-OHD significantly prevented an increase in serum total ALP levels above the normal range at 60 days (P<0.001), and 90 days (P<0.001) after starting treatment and alsosignificantly prevented an increase in serum liver isoenzyme levels at 60 days (P<0.001), and 90 days (P<0.001) [Figure 2]. Supplementation resulted in a significant increase in serum bone ALP levels at 60 and 90 (P<0.001) days [Figure 3]. Serum 25-OHDlevels increased at 90 (P<0.001) days [Figure 4]. Lastly, supplementation resulted in a significant increase in serum calcium levels at 60 days (P<0.001), and 90 days (P<0.001) [Figure 5].
IV. Inter-group drug-wise comparison
The changes in various biochemical parameters at 60 and 90 days after starting therapy, as well as the response to supplementation with calcium and 25-OHD for serum levels of total ALP, liver and bone isoenzymes of ALP, 25-OHD and calcium were similar for phenytoin and valproic acid [Table 2].
Clinical examination of the patients at 30, 60 and 90 days after starting treatment did not reveal any overt neuromuscular or bone dysfunction, nor did the patients complain of any disability related to these systems.
The present study documents that serum biochemical changes which may predispose the subjects to development of osteomalacia appear within three months of starting AED monotherapy. However, simultaneous daily supplementation with oral calcium and 25-OHD in RDA doses is effective in preventing the occurrence of these adverse biochemical changes. Earlier in the literature only two studies have evaluated serum ALP isoenzyme activity in adults on AED therapy ., Both the studies have shown elevated levels of total ALP and the liver and bone isoenzyme of ALP along with hypocalcemia, and lowered levels of vitamin D in patients with epilepsy on long-term AED therapy .Supplementation with 1,25 dihydroxy vitamin Dwas suggested by them as appropriate for these patients. Overall, there is a paucity of studies in recent times on calcium and vitamin D and the effect of AED on vitamin D metabolism in the Indian subcontinent except for an occasional case report has. 
To our knowledge, the present study is the first study to: (i) evaluate serum ALP isoenzyme activities in adults on valproic acid monotherapy; (ii) evaluate serum ALP isoenzyme activities in adults at baseline and at intervals of 30, 60, and 90 days after the initiation of phenytoin or valproic acid monotherapy, and (iii) determine the effectiveness of simultaneous supplementation with RDA dosage of calcium and 25-OHDon serum ALP isoenzyme activities, and calcium, phosphorus, 25-OHD levels. Previous studies reporting on the effect of supplementation with vitamin D, although numerous, have been done on adults who have either been on AED for six months or more or have developed osteomalacia. ,,,,, Supplementation dose of 25-OHD tried for reversing these changes ranged from 400-4000 IU/day. Prophylactic dosage of 2000 IU/day of vitamin D and 600-1000 mg/day for all patients with epilepsy at initiation of AED therapy and a dosage of 5000-15000 IU/day of vitamin D to treat osteomalacia has been recommended. ,
In India, epilepsy is a common disorder and many adults are on phenytoin or valproic acid monotherapy. The present study highlights that these adults are at a risk of developing osteomalacia and that it may be prudent to start early supplementation with calcium and vitamin D as it helps to prevent these adverse biochemical changes. It is being increasingly recognized that long-term phenytoin, carbamazepine or valproic acid monotherapy results in decreased bone formation and bone mineral density (BMD) both in children and adults and low calcium intake could be an aggravating factor for AED-associated osteopenia. ,,,,,,, A lower peak bone mass and increased bone turnover in such adult patients with epilepsy may have long-lasting consequences for bone health and may be associated with greater osteoporotic fractures. This adds up to the risk of fractures associated with seizures and falls.  A recent study by Valmadrid et al.. has reported that only 9% of pediatric neurologists and 37% of neurologists prescribe prophylactic calcium or vitamin D for adults taking AED. 
The present study will help in raising neurologists' awareness of this problem and may help to improve the skeletal health of adult on AED treatment. Several theories on the mechanism of AED-associated bone disease have been proposed, but no single one explains all the reported findings. The commonest explanation is that AEDs such as phenytoin and carbamazepine that induce hepatic cytochrome P450 enzymes cause increased conversion of vitamin D to inactive metabolites in the liver microsomes. The biologically inactive vitamin D leads to decreased absorption of calcium in the gut, resulting in hypocalcaemia and an increase in circulating parathyroid hormone. The secondary hyperparathyroidism is usually subtle and can be assessed by detecting reduced serum calcium and phosphorus levels, and increased serum total ALP levels. The hyperparathyroidism increases the mobilization of bone calcium stores, resulting in increased bone turnover and loss of cortical bone, which, in severe cases, causes painful osteomalacia. However, this theory does not explain the mechanism of valproic acid-associated bone disease, as valproic acid is an inhibitor of the cytochrome P450 enzyme system.  In thier study in adults on valproate therapy for more than one year, Sato et al  have reported increased levels of serum ionized calcium, decreased levels of, 1,25-dihydroxy vitamin D, decreased BMD and increased concentration of pyridinoline crosslinked carboxy terminal telopeptide of Type I collagen (a bone resorption marker). The exact underlying mechanism leading to decrease in BMD is still unclear. 
The present study documents that treatment with valproic acid or phenytoin monotherapy in adult Indian patients, causes significant changes in calcium and vitamin D metabolism reflecting modification of osteoblastic activity and possible - vitamin D inactivation, within a few weeks of initiating therapy. The study also suggests that simultaneously starting daily oral supplementation with RDA dosage of calcium and 25-OHDin these patients is beneficial as it prevents development of biochemical changes predisposing to osteomalacia. However, there are some limitations for the present study. The study sample size was relatively small. Detailed study of the dietary mineral intake of the patients could not be performed. It is beyond the ability of this study to evaluate to what extent elevated serum bone ALP levels in these adults correlate to a clinically important impact on bone metabolism further in their course. However, we have no reason to believe that these limitations adversely affect the utility of our results. We suggest that large, prospective, controlled studies should be undertaken to determine the clinical impact of long-term AED treatment on bone metabolism, particularly the risk for fracture.
We thank our Dean, Dr. Sandhya Kamath for granting us permission to publish this study; the Staff and Research Society of L.T.M.M.C for funding this project, Mr. Kailas Gandewar, our Biostatistician for his help in the statistical analysis of the data; and the patients who participated in the study.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]