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Effect of antiepileptic drugs on erythrocyte osmotic fragility and lipid peroxidation
EPILEPSY
RESEARCH
Epilepsy Research
19 (1994) 249-252
Effect of antiepileptic drugs on erythrocyte osmotic fragility and lipid peroxidation A. Destina Yalp
a, ilhan Onaran b, A. Siiha Yalp
bpc**
aDepartment of Neurology, &$i Erfal Hospital, Q$i, Istanbul, Turkey b Department of Medical Laboratory, School of Health Sciences, Marmara University, Istanbul, Turkey ’ Department of Biochemistry, Faculty of Medicine, Marmara Uniuersity, Istanbul, Turkey Received 20 April 1994; revised 8 June 1994; accepted 8 June 1994
Abstract Epileptic children receiving antiepileptics were studied to investigate the effect of carbamazepine and phenobarbital therapy on erythrocyte osmotic fragility and lipid peroxidation. Significant differences between the two groups were observed in erythrocyte osmotic fragility. In addition, there was a significant increase in erythrocyte malondialdehyde release in the epileptic group compared to controls. It is suggested that the use of antioxidants in addition to antiepileptic drugs may be beneficial. Keywords: Carbamazepine; Erythrocyte lipid peroxidation; Erythrocyte osmotic fragility; Phenobarbital
1. Introduction Children receiving long-term antiepileptic therapy occasionally experience unfavorable side effects. The metabolic disturbances associated with therapy seems to involve peroxidative damages relating to detoxification mechanisms [2,3,9]. Clinical studies in children have shown reduced plasma levels of vitamin E in seizure patients receiving antiepileptic drugs but not in patients prior to antiepileptic drugs or in age-related controls [8,10]. Recently, Tamai et al. [ll] have shown that erythrocyte vitamin E (a-
Abbreviations: MDA: malondialdehyde; RBC: erythrocyte * Corresponding author. Department of Biochemistry, Faculty of Medicine, Marmara University, Haydarpqa-Istanbul, 81326 Turkey. 0920-1211/94/$07.00 8 1994 Elsevier Science B.V. All rights reserved SSDI 0920-1211(94)00051-W
tocopherol) levels were low in children receiving long-term anticonvulsant therapy as compared to children receiving no treatment. In this study, we have investigated the effects of antiepileptic drug therapy on some erythrocyte functional parameters related to vitamin E such as spontaneous hemolysis and malondialdehyde release tests as well as erythrocyte osmotic fragility.
2. Subjects and methods The study included 14 epileptic children with benign partial epilepsy aged 6.5 to 14 years, who had received antiepileptic drugs, carbamazepine (n = 7) or phenobarbital (n = 7) for more than 2 years. An age-matched healthy control group (n = 10) and an
AD.
750
Table 1 Erythrocyte
osmotic fragility
NaCl(g/L)
3 3.5 4
4.5 5 s.5 6 6.5 7.5 Y
Yalqn t’t al. /Epilepsy
in controls and antiepileptic-treated
Re.vearch 19 (19941 249-25
groups
Healthy controls
Untreated controls
Carbamazepine-treated
Phenobarbital-treated
100 99.5 96.5 92.0 68.2 32.2 2.9 0 0 0 0 0
100 98.7 95.5 94.0 71.5 31.2 3.2 0 0 0 0 0
100 94.7 Y5.0 87.0 72.5 41.2 10.2 3.1 1.0 0.5 0.1 0
100 97.7 94.5 90.0 70.5 39.4 7.9 2.5 1.2 0.7 0.2 0
* 0 t 0.5 * 1.0 + 4.1 f 12.1 + 10.2 f 1.2
+ 0 * 0.8 A: 1.2 + 3.2 + 10.1 IfI 7.2 + 0.7
Values are % hemolysis in corresponding salt concentrations * Significantly different from controls; P < 0.05.
malondialdehyde
release in controls and antiepileptic-treated 3% H,O,
Healthy controls Untreated controls Carbamazepine-treated Phenobarbital-treated Values represent mean f
34.1 42.8 142.9 89.9
(nmol/mL
* Significantly
RBC)
+ 5.9 f 7.6 f 59.7 * f 29.9 *
S.D. MDA release(3%
Percent maximal release =
H,O,) x 100.
MDA release(0.75%
different from controls;
H,O,
P < 0.05
* * * * *
-f- 0 * 0.4 * 1.2 & 2.7 f 10.4 f 8.5 f 1.2 * 0.9 + 0.8 * 0.4 + 0.1
* * * * *
and represent mean * SEM.
untreated control group (n = 7) was also studied. The subjects had no additional medical disorders. Their hematological parameters and plasma antiepileptic concentrations were within normal limits. Heparinized blood samples were taken from fasting subjects and transported immediately to the laboratory for biochemical studies. Osmotic fragility of erythrocytes was estimated as described previously [7]. Briefly, 0.05 ml of whole blood was diluted l/100 using saline solutions (1.0 to 9.0 g/L in 5 mM phosphate buffer, pH 7.4). After gentle mixing, the suspensions were incubated for 30 min at room temperature and were centrifuged at 1500 g for 5 min. The absorbance of the supernatants was measured at 540 nm using phosphate buffer as a blank. The absorbance of the 1.0 g/L supematant was used as 100% lysis.
Table 2 Erythrocyte
+o + 0.5 * 1.2 k 3.2 + 9.1 i_ 6.7 i 2.7 * 1.1 * 0.5 + 0.3 * 0.1
+ azide)
Spontaneous hemolysis of erythrocytes was determined after 4 h of incubation at room temperature in phosphate-buffered saline (pH 7.4). The percentage of hemolysis was expressed as the ratio of the absorbance at 410 nm in phosphate-buffered saline to the completely hemolyzed samples in water [4]. Malondialdehyde (MDA) was determined by the method of Cynamon et al. [ll. Erythrocytes were incubated with either 3% H,O, or 0.75% H,O, + 4 mM sodium azide. The amount of MDA formed in the presence of hydrogen peroxide, with and without catalase inhibition by azide, was determined. The MDA release without catalase inhibition is considered to be a reflection of the erythrocyte membrane antioxidant protection. The MDA release with catalase inhibition is a measure of the amount of polyunsaturated fatty acids present in the red cell membrane
groups 0.75% H,O, + azide (nmol/mL RBC)
Percent maximal release
382.3 417.1 395.7 409.0
8.9 10.2 36.1 * 21.9 *
* 73.0 * 85.9 f 104.9 f 91.2
A.D. Yalqn et al. /Epilepsy
or the maximal release. Student’s t-test was used for statistical analysis. All values are given as either mean f standard error of the mean @EM) or mean f standard deviation (SD).
3. Results and discussion It has been established that mature human erythrocytes undergo membrane transformation and a resulting shape change under in vitro action of drugs such as general and local anesthetics, neuroleptics, antipyretics and antihistaminics [5]. Osmotic fragility is a commonly used criterion of membrane stability. Osmotic fragility of the controls and antiepileptictreated groups are shown in Table 1. In the antiepileptic-treated groups both drugs resulted in significantly increased fragility at higher salt concentrations (> 5 g/L). This suggests that antiepileptic drugs affect erythrocyte membrane causing increased fragility. Table 2 shows erythrocyte malondialdehyde release in controls and antiepileptic-treated groups. Both drugs resulted in significantly higher MDA release compared to controls as evidenced by increased percent maximal release values. MDA release test without catalase inhibition is considered to be a reflection of the erythrocyte membrane antioxidant protection and the MDA release with catalase inhibition is a measure of the amount of polyunsaturated fatty acids present in the membrane [l]. We have observed that although there was no significant change in MDA release with catalase inhibition, MDA release without catalase inhibition was significantly higher in children receiving antiepileptic therapy. This shows that the antioxidant defence system is affected by antiepileptic therapy. Indeed, erythrocyte and plasma cY-tocopherol levels were reported to be significantly low in patients receiving antiepileptic drugs [6,11]. In addition, it was recently reported that erythrocyte superoxide dismutase and catalase activities were lower in epileptic patients than normal individuals [ 121. Spontaneous hemolysis test is suggested to be a functional parameter related to vitamin E levels of erythrocytes [4]. If antiepileptics affect vitamin E levels of erythrocytes, one would expect an increase in spontaneous hemolysis during therapy. Sponta-
Research 19 (1994) 249-252
251
neous hemolysis values were not significantly different from controls in the antiepileptic-treated groups and ranged from 0 to 8% (data not shown). Therefore, although vitamin E levels of erythrocytes may be lowered during antiepileptic therapy, it seems that other antioxidants prevent spontaneous hemolysis under normal conditions. However, under oxidative stress exceeding the capacity of the antioxidant system, peroxidation and eventually hemolysis may take place. In conclusion, the membrane instability of erythrocytes seems to have no clinical consequences. It is possible that such subclinical changes might get clinical relevance under certain circumstances such as additional pharmacologic therapy or preexisting hematologic disorders affecting membrane stabilities. Further studies will be needed to investigate the effects of antiepileptic treatment on individual antioxidants and to decide whether antioxidant supplementation would be beneficial during antiepileptic therapy.
References 111Cynamon,
H.A., Isenberg, J.N. and Nguyen, C.H., Erythrocyte malondialdehyde release in vitro: A functional measure of vitamin E status, Clin. Chim. Ada, 151 (1985) 169-176. PI Dastur, D.D. and Dave, U.P., Effect of prolonged anticonvulsant medication in epileptic patient: Serum lipids, vitamin B,, B,,, and folic acid, proteins, and fine structure of liver, Epilepsia, 28 (1987) 147-159. [31 Dent, C.E., Osteomalacia with long term anticonvulsant therapy in epilepsy, Br. Mea’., 4 (1970) 69-72. [41 Draper, H.H. and Csallany, A.S., A simplified hemolysis test for vitamin E deficiency, J. Nulr., 98 (1969) 390. iSI Fujii, T., Sato, T., Tamura, A., Wakatsuki, M. and Kanaho, Y., Shape changes of human erythrocytes induced by various amphipathic drugs acting on the membrane of the intact cells, Biochem. Pharmacol., 28 (1979) 613-620. [61Kataoka, K., Kanamori, N., Oishi, M., Yamaji, A., Tagawa, T. and Mimaki, T., Vitamin E status in pediatric patients receiving antiepileptic drugs, Deu. Pharmacol. Ther., 14 (1990) 96-101. [7] Ghnq, A., Yalqn, AS., Yalqn, D., Taga, Y. and Emerk, K., Increased erythrocyte susceptibility to lipid peroxidation in human Parkinson’s disease, Neurosci. Len., 87 (1987) 307310. [8] Ogunmekan, A.O. and Hwang, P.A., A randomized, doubleblind, placebo-controlled, clinical trial of d-a-tocopheryl acetate (vitamin E), as add-on therapy, for epilepsy in children, Epilepsia, 30 (1989) 84-89.
[9] Reynolds, E.G., Effect of folic acid on the mental state and fit frequency of drug-treated epileptic patients. Lancet, I (1968) 1086-1088. [lOI Sobaniec, W., Lipid peroxidation in experimental and clinical epilepsy and the effects of sodium valproate and vitamin E on these processes, Neurosciences, 18 (1992) 123- 130. [l I] Tamai, H., Wakamiya, E.. Mine. M. and Iwakoshi, M..
Alpha-tocopherol and fatty acid levels in red blood ceils in patients treated with antiepileptic drugs, /. Nuw Ser. Vitamind., 34 (1988) 627-631. [12] Wang, H., Jiang, O., Shi, J. and Zhao, H.. Comparative study of the activities of oxygen free radicals: Protective enzymes of crythrocytcs in epileptic patients. f+&psru. .34 (1993) (20th IEC Proceedings. Suppl. 7) 47.
RESEARCH
Epilepsy Research
19 (1994) 249-252
Effect of antiepileptic drugs on erythrocyte osmotic fragility and lipid peroxidation A. Destina Yalp
a, ilhan Onaran b, A. Siiha Yalp
bpc**
aDepartment of Neurology, &$i Erfal Hospital, Q$i, Istanbul, Turkey b Department of Medical Laboratory, School of Health Sciences, Marmara University, Istanbul, Turkey ’ Department of Biochemistry, Faculty of Medicine, Marmara Uniuersity, Istanbul, Turkey Received 20 April 1994; revised 8 June 1994; accepted 8 June 1994
Abstract Epileptic children receiving antiepileptics were studied to investigate the effect of carbamazepine and phenobarbital therapy on erythrocyte osmotic fragility and lipid peroxidation. Significant differences between the two groups were observed in erythrocyte osmotic fragility. In addition, there was a significant increase in erythrocyte malondialdehyde release in the epileptic group compared to controls. It is suggested that the use of antioxidants in addition to antiepileptic drugs may be beneficial. Keywords: Carbamazepine; Erythrocyte lipid peroxidation; Erythrocyte osmotic fragility; Phenobarbital
1. Introduction Children receiving long-term antiepileptic therapy occasionally experience unfavorable side effects. The metabolic disturbances associated with therapy seems to involve peroxidative damages relating to detoxification mechanisms [2,3,9]. Clinical studies in children have shown reduced plasma levels of vitamin E in seizure patients receiving antiepileptic drugs but not in patients prior to antiepileptic drugs or in age-related controls [8,10]. Recently, Tamai et al. [ll] have shown that erythrocyte vitamin E (a-
Abbreviations: MDA: malondialdehyde; RBC: erythrocyte * Corresponding author. Department of Biochemistry, Faculty of Medicine, Marmara University, Haydarpqa-Istanbul, 81326 Turkey. 0920-1211/94/$07.00 8 1994 Elsevier Science B.V. All rights reserved SSDI 0920-1211(94)00051-W
tocopherol) levels were low in children receiving long-term anticonvulsant therapy as compared to children receiving no treatment. In this study, we have investigated the effects of antiepileptic drug therapy on some erythrocyte functional parameters related to vitamin E such as spontaneous hemolysis and malondialdehyde release tests as well as erythrocyte osmotic fragility.
2. Subjects and methods The study included 14 epileptic children with benign partial epilepsy aged 6.5 to 14 years, who had received antiepileptic drugs, carbamazepine (n = 7) or phenobarbital (n = 7) for more than 2 years. An age-matched healthy control group (n = 10) and an
AD.
750
Table 1 Erythrocyte
osmotic fragility
NaCl(g/L)
3 3.5 4
4.5 5 s.5 6 6.5 7.5 Y
Yalqn t’t al. /Epilepsy
in controls and antiepileptic-treated
Re.vearch 19 (19941 249-25
groups
Healthy controls
Untreated controls
Carbamazepine-treated
Phenobarbital-treated
100 99.5 96.5 92.0 68.2 32.2 2.9 0 0 0 0 0
100 98.7 95.5 94.0 71.5 31.2 3.2 0 0 0 0 0
100 94.7 Y5.0 87.0 72.5 41.2 10.2 3.1 1.0 0.5 0.1 0
100 97.7 94.5 90.0 70.5 39.4 7.9 2.5 1.2 0.7 0.2 0
* 0 t 0.5 * 1.0 + 4.1 f 12.1 + 10.2 f 1.2
+ 0 * 0.8 A: 1.2 + 3.2 + 10.1 IfI 7.2 + 0.7
Values are % hemolysis in corresponding salt concentrations * Significantly different from controls; P < 0.05.
malondialdehyde
release in controls and antiepileptic-treated 3% H,O,
Healthy controls Untreated controls Carbamazepine-treated Phenobarbital-treated Values represent mean f
34.1 42.8 142.9 89.9
(nmol/mL
* Significantly
RBC)
+ 5.9 f 7.6 f 59.7 * f 29.9 *
S.D. MDA release(3%
Percent maximal release =
H,O,) x 100.
MDA release(0.75%
different from controls;
H,O,
P < 0.05
* * * * *
-f- 0 * 0.4 * 1.2 & 2.7 f 10.4 f 8.5 f 1.2 * 0.9 + 0.8 * 0.4 + 0.1
* * * * *
and represent mean * SEM.
untreated control group (n = 7) was also studied. The subjects had no additional medical disorders. Their hematological parameters and plasma antiepileptic concentrations were within normal limits. Heparinized blood samples were taken from fasting subjects and transported immediately to the laboratory for biochemical studies. Osmotic fragility of erythrocytes was estimated as described previously [7]. Briefly, 0.05 ml of whole blood was diluted l/100 using saline solutions (1.0 to 9.0 g/L in 5 mM phosphate buffer, pH 7.4). After gentle mixing, the suspensions were incubated for 30 min at room temperature and were centrifuged at 1500 g for 5 min. The absorbance of the supernatants was measured at 540 nm using phosphate buffer as a blank. The absorbance of the 1.0 g/L supematant was used as 100% lysis.
Table 2 Erythrocyte
+o + 0.5 * 1.2 k 3.2 + 9.1 i_ 6.7 i 2.7 * 1.1 * 0.5 + 0.3 * 0.1
+ azide)
Spontaneous hemolysis of erythrocytes was determined after 4 h of incubation at room temperature in phosphate-buffered saline (pH 7.4). The percentage of hemolysis was expressed as the ratio of the absorbance at 410 nm in phosphate-buffered saline to the completely hemolyzed samples in water [4]. Malondialdehyde (MDA) was determined by the method of Cynamon et al. [ll. Erythrocytes were incubated with either 3% H,O, or 0.75% H,O, + 4 mM sodium azide. The amount of MDA formed in the presence of hydrogen peroxide, with and without catalase inhibition by azide, was determined. The MDA release without catalase inhibition is considered to be a reflection of the erythrocyte membrane antioxidant protection. The MDA release with catalase inhibition is a measure of the amount of polyunsaturated fatty acids present in the red cell membrane
groups 0.75% H,O, + azide (nmol/mL RBC)
Percent maximal release
382.3 417.1 395.7 409.0
8.9 10.2 36.1 * 21.9 *
* 73.0 * 85.9 f 104.9 f 91.2
A.D. Yalqn et al. /Epilepsy
or the maximal release. Student’s t-test was used for statistical analysis. All values are given as either mean f standard error of the mean @EM) or mean f standard deviation (SD).
3. Results and discussion It has been established that mature human erythrocytes undergo membrane transformation and a resulting shape change under in vitro action of drugs such as general and local anesthetics, neuroleptics, antipyretics and antihistaminics [5]. Osmotic fragility is a commonly used criterion of membrane stability. Osmotic fragility of the controls and antiepileptictreated groups are shown in Table 1. In the antiepileptic-treated groups both drugs resulted in significantly increased fragility at higher salt concentrations (> 5 g/L). This suggests that antiepileptic drugs affect erythrocyte membrane causing increased fragility. Table 2 shows erythrocyte malondialdehyde release in controls and antiepileptic-treated groups. Both drugs resulted in significantly higher MDA release compared to controls as evidenced by increased percent maximal release values. MDA release test without catalase inhibition is considered to be a reflection of the erythrocyte membrane antioxidant protection and the MDA release with catalase inhibition is a measure of the amount of polyunsaturated fatty acids present in the membrane [l]. We have observed that although there was no significant change in MDA release with catalase inhibition, MDA release without catalase inhibition was significantly higher in children receiving antiepileptic therapy. This shows that the antioxidant defence system is affected by antiepileptic therapy. Indeed, erythrocyte and plasma cY-tocopherol levels were reported to be significantly low in patients receiving antiepileptic drugs [6,11]. In addition, it was recently reported that erythrocyte superoxide dismutase and catalase activities were lower in epileptic patients than normal individuals [ 121. Spontaneous hemolysis test is suggested to be a functional parameter related to vitamin E levels of erythrocytes [4]. If antiepileptics affect vitamin E levels of erythrocytes, one would expect an increase in spontaneous hemolysis during therapy. Sponta-
Research 19 (1994) 249-252
251
neous hemolysis values were not significantly different from controls in the antiepileptic-treated groups and ranged from 0 to 8% (data not shown). Therefore, although vitamin E levels of erythrocytes may be lowered during antiepileptic therapy, it seems that other antioxidants prevent spontaneous hemolysis under normal conditions. However, under oxidative stress exceeding the capacity of the antioxidant system, peroxidation and eventually hemolysis may take place. In conclusion, the membrane instability of erythrocytes seems to have no clinical consequences. It is possible that such subclinical changes might get clinical relevance under certain circumstances such as additional pharmacologic therapy or preexisting hematologic disorders affecting membrane stabilities. Further studies will be needed to investigate the effects of antiepileptic treatment on individual antioxidants and to decide whether antioxidant supplementation would be beneficial during antiepileptic therapy.
References 111Cynamon,
H.A., Isenberg, J.N. and Nguyen, C.H., Erythrocyte malondialdehyde release in vitro: A functional measure of vitamin E status, Clin. Chim. Ada, 151 (1985) 169-176. PI Dastur, D.D. and Dave, U.P., Effect of prolonged anticonvulsant medication in epileptic patient: Serum lipids, vitamin B,, B,,, and folic acid, proteins, and fine structure of liver, Epilepsia, 28 (1987) 147-159. [31 Dent, C.E., Osteomalacia with long term anticonvulsant therapy in epilepsy, Br. Mea’., 4 (1970) 69-72. [41 Draper, H.H. and Csallany, A.S., A simplified hemolysis test for vitamin E deficiency, J. Nulr., 98 (1969) 390. iSI Fujii, T., Sato, T., Tamura, A., Wakatsuki, M. and Kanaho, Y., Shape changes of human erythrocytes induced by various amphipathic drugs acting on the membrane of the intact cells, Biochem. Pharmacol., 28 (1979) 613-620. [61Kataoka, K., Kanamori, N., Oishi, M., Yamaji, A., Tagawa, T. and Mimaki, T., Vitamin E status in pediatric patients receiving antiepileptic drugs, Deu. Pharmacol. Ther., 14 (1990) 96-101. [7] Ghnq, A., Yalqn, AS., Yalqn, D., Taga, Y. and Emerk, K., Increased erythrocyte susceptibility to lipid peroxidation in human Parkinson’s disease, Neurosci. Len., 87 (1987) 307310. [8] Ogunmekan, A.O. and Hwang, P.A., A randomized, doubleblind, placebo-controlled, clinical trial of d-a-tocopheryl acetate (vitamin E), as add-on therapy, for epilepsy in children, Epilepsia, 30 (1989) 84-89.
[9] Reynolds, E.G., Effect of folic acid on the mental state and fit frequency of drug-treated epileptic patients. Lancet, I (1968) 1086-1088. [lOI Sobaniec, W., Lipid peroxidation in experimental and clinical epilepsy and the effects of sodium valproate and vitamin E on these processes, Neurosciences, 18 (1992) 123- 130. [l I] Tamai, H., Wakamiya, E.. Mine. M. and Iwakoshi, M..
Alpha-tocopherol and fatty acid levels in red blood ceils in patients treated with antiepileptic drugs, /. Nuw Ser. Vitamind., 34 (1988) 627-631. [12] Wang, H., Jiang, O., Shi, J. and Zhao, H.. Comparative study of the activities of oxygen free radicals: Protective enzymes of crythrocytcs in epileptic patients. f+&psru. .34 (1993) (20th IEC Proceedings. Suppl. 7) 47.