Homocyst(e)ine Levels in Patients on Phenytoin Therapy

Clinical Biochemistry, Vol. 30, No. 8, 647– 649, 1997 Copyright © 1997 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/97 $17.00 1 .00

PII S0009-9120(97)00116-1

Homocyst(e)ine Levels in Patients on Phenytoin Therapy GERALD K. JAMES,1 MICHAEL W. JONES,2 and MORRIS R. PUDEK1 1

Division of Clinical Chemistry, Department of Pathology and Laboratory Medicine, and Division of Neurology, Department of Medicine, Vancouver Hospital and Health Sciences Centre and University of British Columbia, Vancouver, British Columbia, V5Z 1L8 Canada

2

Introduction An elevated homocyst(e)ine [H(e)] level is recognized as an independent risk factor for atherosclerotic vascular disease and deep vein thrombosis (1–5). Therefore, the association of hyperhomocyst(e)inemia with myocardial infarction, peripheral vascular disease, and stroke has aroused intensive research into its etiology and associations (1). Homocysteine is a thiol-containing amino acid that is an intermediate in methionine metabolism. The enzyme 5,10methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of homocysteine to methionine via remethylation (4). MTHFR requires folic acid as a cofactor. ‘‘Homocyst(e)ine’’ refers to homocysteine and its oxidized forms, homocystine and homocysteine-cysteine disulfide (4). Many drugs, including the anticonvulsant, diphenylhydantoin (phenytoin), have been associated with folic acid deficiency. Phenytoin has been shown, in some studies, to decrease serum folate levels (6). Folate and phenytoin can influence each other’s metabolism. It is hypothesized that phenytoin increases the pH of the gastrointestinal tract, therefore, inhibiting folate absorption (7). Phenytoin may interfere with folate transport into tissue and/or intestinal conversion of folate to the absorbable monoglutarate form (7). Phenytoin-induced folate deficiency may also arise from hepatic microsomal enzyme induction and/or increased folate catabolism (8). Conversely, folate may act as a cofactor in phenytoin metabolism (7). It is conceivable that phenytoin and homocysteine pharmacokinetics may be linked through folic acid and, consequently, patients on phenytoin therapy

Correspondence: Morris R. Pudek, Division of Clinical Chemistry (LSP-1), Vancouver Hospital and Health Sciences Centre, 910 West 10th Ave. Vancouver, British Columbia, V5Z 1L8, Canada. Manuscript received June 5, 1997; revised and accepted August 14, 1997. CLINICAL BIOCHEMISTRY, VOLUME 30, DECEMBER 1997

may have elevated serum H(e) levels. In fact, in 1984, Billings (8) demonstrated that MTHFR activity in mouse liver was decreased after chronic phenytoin treatment. The relationship between homocysteine and phenytoin has been suggested (9), but not experimentally confirmed in the literature. Our study is a preliminary survey to see if there is an association between epileptic patients taking phenytoin, either as monotherapy or in combination with other anticonvulsants, and elevated serum H(e) levels. Methods Forty-six patients consisting of 32 men and 14 women were seen by a neurologist (MJ). All patients were on anticonvulsant therapy for either a seizure disorder (n 5 45) or trigeminal neuralgia (n 5 1). No attempt to categorize the type of seizure disorder was made. Patients were stratified into two groups based on whether or not they were receiving phenytoin therapy. Those receiving phenytoin were either on mono- or combination therapy. From this point, those receiving phenytoin will be referred to as ‘‘On DPT’’ and those not receiving phenytoin as ‘‘Off DPT.’’ Charts of the patients were available for review to determine patient age, sex, diagnosis, presence of renal disease or cancer, and type and length of therapy. Serum H(e) levels were performed on all patients. Concurrently, serum phenytoin levels were performed on all patients in the On DPT group along with serum folate (n 5 11) and RBC folate (n 5 28) levels. Folate levels were unavailable for seven patients. The H(e) was quantitated by high-performance liquid chromatography (HPLC) using a Hewlett-Packard (HP) 1090 (Wilmington, DE, USA) equipped with a HP fluorometer FLD 1046. First, the sample and internal standard (acetylcysteine) were mixed. Tri-n-butylphosphine in dimethylformamide was added to reduce the thiols and mixed sulfides, and also to release thiols bound to protein. After incubation at 4° C for 30 647

JAMES, JONES, AND PUDEK

min, perchloric acid was added to precipitate proteins. Next, the centrifuged supernatant was mixed with alkaline potassium tetraborate in EDTA and SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4sulfonate), incubated at 60° C in a water bath for 1 h, to form fluorescent derivatives. After cooling at 4° C, centrifugation and filtration, 75 mL of the samples were injected onto the HPLC and separated on a LiChrospher RP-18 ENDCPD 5U (150 3 2.6 mm) column (Alltech, Deerfield, IL, USA). A 4% methanol/ 0.1M acetate buffer pH 4.0 was used to elute the constituents. The fluorometer was set at 385 nm excitation with emission monitored at 515 nm. More precise details concerning reagent concentrations are previously described (4,10). In our laboratory (Vancouver Hospital and Health Sciences Centre), our reference ranges for men and women serum H(e) levels are 6.7 to 15.5 mmol/L and 6.1 to 12.8 mmol/L, respectively. The coefficient of variation of this method is less than 5% at control concentrations of 10 and 50 mmol/L. Serum and red blood cell (RBC) folate levels were determined by radioimmunoassay with Quantaphase II™ Folate Radioassay (Bio-Rad Laboratories, Hercules, CA, USA). All data were entered into a computerized database to assist the data analysis. In-Stat™ (GraphPaD™ Software Inc., San Diego, CA, USA) software was used for the statistical analysis. Comparison of the mean ages and H(e) levels between the two groups was performed by the unpaired, two-tailed, t-test (11). Logarithmic transformation of the H(e) levels was performed prior to comparison analysis in order to correct for a statistically significant difference in the groups’ standard deviations. Statistical significance was set at p 5 0.05. Results The On DPT group consisted of 26 patients of which 17 were men and 9 were women. The range and average length of time which this group was on phenytoin therapy was 1 to 384 months and 115 months, respectively. Only one patient in the On DPT group was taking phenytoin for less than 3 months. The daily dose ranged from 200 to 500 mg, and the serum phenytoin level ranged from 16 to 105 mmol/L at the time of the H(e) measurement. Serum phenytoin levels were unavailable for two patients. Fourteen patients of the On DPT group were on phenytoin monotherapy and the remaining 12 patients were on combination therapy. Of those patients on combination therapy with phenytoin, the other anticonvulsants used were carbamazepine (7), valproic acid (2), topiramate (2), clobazam (1), vigabatrim (1), lactimal (1), and remacemide (1). The Off DPT group consisted of 20 patients, 15 men and 5 women. This group was on various anticonvulsants with the majority (n 5 15) taking carbamazepine. The length of time that the patients in the Off DPT group were taking carbamazepine ranged from 5 to 240 months. Serum levels of 648

Figure 1 — Scatter plots showing the homocyst(e)ine levels of patients (Œ - women; ■ - men) in the On and Off Phenytoin groups. ULR (M) - upper limit reference range for men; ULR (W) - upper limit reference range for women.

carbamazepine and valproic acid were performed at the time of the H(e) measurement and all patients on these medications were found to be compliant (data not shown). Serum carbamazepine levels were unavailable for two patients. The range and mean age of the On DPT group were 18 to 80 years and 41.6 years, respectively, while the range and mean age of the Off DPT group were 18 to 72 years and 38.6 years, respectively. This difference was not statistically significant (p 5 0.51). No patients in the study had renal disease or cancer. Of those 28 patients with a RBC folate level, all were within the reference range (160 to 900 nmol/L) except for 1 patient (On DPT group) with a borderline low RBC folate of 152 nmol/L. The range and mean RBC folate for all 28 patients were 152 to 1009 nmol/L and 343 nmol/L, respectively. All 11 patients with a serum folate were within the reference range (4.0 to 35.0 nmol/L) with a range and mean of 7.3 to 43.6 nmol/L and 14.1 nmol/L, respectively. The range and mean serum H(e) level for the On DPT group was 8.1 to 44 mmol/L and 17.9 mmol/L, respectively. The 95% confidence interval was 14.7 to 21.1 mmol/L. Serum H(e) levels in the Off DPT group ranged from 5.8 to 17.8 mmol/L with an average of 11.9 mmol/L. The 95% confidence interval was 10.4 to 13.4 mmol/L. The mean difference was highly significant at p 5 0.0015. The scatter plots of the H(e) levels of patients in both groups is shown in Figure 1. Fifteen patients of 20 patients in the Off DPT group were on carbamazepine. This carbamazepine subgroup had a mean H(e) level of 12.2 mmol/L and a 95% confidence interval of 10.5 to 13.9 mmol/L. CLINICAL BIOCHEMISTRY, VOLUME 30, DECEMBER 1997

HOMOCYST(E)INE AND PHENYTOIN

Discussion Phenytoin is a widely used anticonvulsant drug that has been shown to cause folic acid deficiency (6). The metabolism of these two compounds are intimately linked (7). Folic acid is a cofactor of MTHFR and both are important in the metabolism of homocysteine. The literature has suggested that anticonvulsants such as phenytoin and carbamazepine are associated with increased plasma H(e), presumably via folate deficiency precipitated by these medications (9). Nevertheless, no published studies have verified this relationship. Our study is the first to show an association between H(e) levels in patients on phenytoin therapy. Epileptic patients on phenytoin appear to have significantly elevated H(e) as opposed to control epileptics not on phenytoin (p 5 0.0015). Confounding variables that may account for this difference include age, sex, presence of renal disease or cancer, and folate levels. Both groups were shown not to differ significantly in mean age or age distribution. Women have slightly lower H(e) levels than men; however, the On DPT group consisted of a higher proportion of women than the Off DPT group (35% vs 25%). There were no patients in this study with renal disease or cancer. Finally, all patients, except for one patient with a borderline low RBC folate, had serum or RBC folate levels within the reference range. This latter observation is interesting. It supports the concept that increased H(e) levels in patients on phenytoin are not simply due to overt folate depletion, since there was virtually no evidence of this in either group. However, H(e) is considered a sensitive indicator of folate deficiency even in situations where the the measurable folate is within the reference range (6). Nevertheless, it is possible that phenytoin may directly interfere with homocysteine metabolism by decreasing the activity of MTHFR (8). In addition, if depletion of folate was the sole mechanism of H(e) elevation, then one would expect that the patients taking carbamazepine (a folate lowering drug) in the Off DPT group would have high H(e) levels, which is not the case in our study. Enzymatic alterations of homocysteine metabolism such as the thermolabile variant of MTHFR and cystathione-b-synthase deficiency are known to cause hyperhomocyst(e)inemia (4). The latter is rare while the former comprises up to 10% of the population. We did not screen for these conditions; however, there is no reason to believe that their occurrence would be unevenly distributed between the two groups. Even though patients on phenytoin appear to have mildly elevated H(e) levels it is unknown whether the result is clinically significant. An increased incidence of ischemic heart disease (IHD) and cerebrovascular disease has been reported in

CLINICAL BIOCHEMISTRY, VOLUME 30, DECEMBER 1997

epileptics (12,13). However, Annegers et al (13) reported no difference between the incidence of IHD in epileptics on or off anticonvulsant therapy (13). The type of anticonvulsant medication was not indicated in their study. Nevertheless, additional research needs to be done in order to verify our results and to investigate if epileptics on long-term phenytoin therapy have an increased incidence of atherosclerosis and/or thrombotic disease. Acknowledgement We would like to thank Diane Abbott for her technical assistance and Dr. Andy Coldman for his statistical advice in this project.

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