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Oxidative damage in red blood cells of vitamin E deficient patients
ELSEVIER
Clinica Chimica Acta 265 (1997) 131-137
Short c o m m u n i c a t i o n
Oxidative damage in red blood cells of vitamin E deficient patients a
• a
D. M a z o r , G. Brill b, Z. Shorer b, S. M o s e s b, N. Meyersteln " "The Dr. Kaufmann Hematology Laboratory, Physiology Department, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel bThe Dr. Kaufmann Hematology Laboratory, Pediatric Department, Soroka University Medical Centre, Beer Sheva, Israel Received 30 December 1996; received in revised form 28 May 1997; accepted 30 May 1997
Keywords: O x i d a t i v e stress; V i t a m i n E deficiency; R e d blood cells
1. Introduction
Vitamin E is a lipid-soluble antioxidant. Its most important role is to protect the membrane polyunsaturated fatty acids from oxidation involving reactive oxygen species, by termination of free-radical chain reactions [1]. In view of the widespread occurrence of ot-tocopherol in food, nutritional vitamin E deficiency is extremely uncommon. However, vitamin E deficiency occurs in humans as a result of a variety of lipid malabsorption syndromes such as cystic fibrosis [2], or as an isolated vitamin E deficiency in the absence of fat malabsorption [3]. An isolated vitamin E deficiency could be caused by abnormal absorption, distribution, metabolism or excretion [3]. In all cases it is recognized as a cause of neurologic abnormalities, since vitamin E is crucial in maintaining the structure and function of the human nervous system [4]. Premature babies, born with low serum and tissue concentrations of vitamin E, are susceptible to haemolysis, with morphological changes in RBCs and increased in vitro sensitivity to hydrogen peroxide (H202) exposure [5]. Oral vitamin E therapy *Corresponding author. Tel.: + 972 7 6400826; Fax: + 972 7 6277655. 0009-8981/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 9 - 8 9 8 1 ( 9 7 ) 0 0 1 1 6 - 2
132
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
restores plasma vitamin levels to normal and can improve neurological functions and hydrogen peroxide induced haemolysis [2,3] in vitamin E deficient patients. In this presentation, we studied a consanguineous Bedouin family with four AVED siblings. In three siblings who were available for study at the time of diagnosis, RBC oxidative damage was demonstrated in the pretreatment stage. This damage was even more pronounced after exposure of the cells to BHE Vitamin E administration resulted in a marked attenuation of the oxidative damage.
2. Materials and methods
2.1. Chemicals BHP and all the reagents were of analytical grade, obtained from Sigma Chemical Company (Rehovot, Israel). Vit E (D-ce-tocopherol acetate, Roche Inc.) was administered at a dose of 100mg Kg -~ day -1.
2.2. Subjects Three female siblings (born to consanguineous Bedouin parents) aged between 12 and 24 years were studied. They had developed a Friedreich type of ataxia without myocardial involvement at the end of their first decade of life. The clinical and neurological features were separately described [6]. Their haemoglobin and red blood cell counts were normal and no acanthocytes were detected in their blood smears. Bilirubin levels and reticulocyte counts were normal. Pretreatment plasma vitamin E levels were 0.02-0.51 txg m1-1. All patients were treated daily with oral vitamin E. One month after treatment, plasma vitamin E levels of the patients reached normal values, 7.8-17.3 txg ml-~ and the neurological status in one of the three patients was improved.
2.3. Experimental design 10 ml of peripheral venous blood were withdrawn from the patients, after informed written consent was obtained. The blood was collected into heparinized test tubes. After removal of plasma and buffy coat, by centrifugation at 2500g for 5 min, the RBCs were washed three times in phosphate-buffered saline (PBS) pH 7.4 and suspended as 20%. The suspension was exposed to 0.5-2.0 mM (final concentration) of BHP for 30 min at 37°C with an appropriate control. MetHb, GSH and MDA levels were measured before and after exposure.
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
133
2.4. Methods for the determination of the antioxidant status of RBCs Intracellular GSH levels were determined by the method of Ellman, based on the reduction of 5',5'-dithiobis-2-nitrobenzoic acid [7]. CuZnSOD was measured by the 50% inhibition of pyrogallol autoxidation and catalase was determined by the rate of H202 decomposition [7]. Methaemoglobin levels were determined at 630 nm and calculated as percent of total haemoglobin [8]. The spectrophotometric determinations were performed on a computed Uvicon 922 spectrophotometer (Kontron instruments, Milano, Italy). Acidified glycerol lysis time (AGLTs0) was determined by the time required to reach 50% haemolysis in the blood suspension [9]. Briefly, 20~1 of blood was introduced into 5 ml PBS pH 6.85. 1 ml of the suspension was added to 2 ml of glycerol solution and the decrease in absorbance was recorded on a Spectronic 21 Bausch and Lomb (Rochester, New York) spectrophotometer, at 625 nm. MDA analysis was determined by the thiobarbituric method on a trichloroacetic acid extract of the RBC suspension, with absorbency at 532 and 600 nm [10].
2.5. Data analysis Statistical analysis of the results was performed with the Student's t-test. Results are presented as the mean+SEM.
3. Results Table 1 shows the antioxidant status of RBCs (intracellular and membrane parameters), in the pre- and post treatment stage. RBCs of the vitamin E deficient patients had markedly reduced permeability to acidified glycerol which was partially corrected by vitamin E administration. Intracellular GSH and SOD levels were normal before and after treatment. Catalase activity which was significantly lower in the RBCs of the vitamin E deficient patients, was restored to normal values by vitamin E administration. The high methaemoglobin levels in RBCs of the vitamin E deficient patients, were significantly decreased after oral vitamin E administration.
3.1. Exposure to BHP BHP, an oxidizing agent is transformed in the cell to several oxidative radicals by reacting with haemoglobin. These radicals oxidize haemoglobin to MetHb and initiate peroxidation of the membrane lipids [11]. Exposure of RBCs to BHP
134
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
Table 1 Antioxidant status of red blood cells, before and after vitamin E administration in the 3 siblings Vitamin E txg ml-~ Pretreatment Post treatment
Normal
0.51; 0.07; 0.02; 10.1; 17.3; 7.8
5.0-20.0
Acidified glycerol lysis test (seconds) Pretreatment Post treatment Glutathione (mM) Pretreatment Post treatment
130; 200; 120; 100; 95; 95
60.0-70.0
2.56; 3.08; 2.19; 2.38; 2.92; 2.38
2.0-3.0
Methemoglobin (%) Pretreatment Post treatment
3.95; 4.55; 4.05; 2.43; 2.93; 2.71
0.5-2.0
Superoxide dismutase (IU/gHb) Pretreatment
2008; 2223.6; 1916.7;
1950-2555
Catalase (IU/gHb × 10 4) Pretreatment Post treatment
8.15; 11.9; 8.4; 11.14; 13.3; 15.0
13.3-17.0
decreased intracellular GSH levels in a dose dependent pattern with similar pattern in pre- and posttreatment cells (data not shown). An expected BHP dose dependent elevation of MetHb levels was found, which was significantly more prominent in the vitamin E deficient cells than in the post treated cells (Fig. 1). Malonyldialdehyde (MDA) levels as shown in Fig. 2, were significantly more pronounced in the pretreated than in the posttreated cells.
4. Discussion The present study provides evidence for RBC oxidative damage as measured by membrane and cytoplasmic parameters in the AVED patients. As shown, RBCs of vitamin E deficient patients had prolonged AGLT time. Such longer values reflect lower hemolytic rate and may be found in association with abnormal erythrocyte lipid organization or in cells with relative increase in membrane surface area [12]. It can be hypothesized that in vitamin E deficient cells, oxidative damage may induce changes in the lipid patterns of the membrane which affect the rate of haemolysis in glycerol containing media. During vitamin E administration, AGLT time was shortened but it did not reach normal levels. These changes in AGLT in the vitamin E deficient cells, suggest membrane vulnerability which is partly corrected, by vitamin E administration. Intracellular GSH levels were similar in the pre and posttreatment cells, since
135
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
10 •
pretreatment
[]
posttreatment
8 c J~ 0 ol o
6
E j¢
4
+LLL 2
O
~ 0.5mM
1.0mM
2.0mM
Fig. 1. Intracellular MetHb levels following exposure to BHP (0.5-2.0 mM), in the pre and post-treatment stage of vitamin E.
GSH is constantly regenerated in the cell from GSSG, by glutathione reductase activity. An acute oxidative stress, such as exposure to BHP, apparently by exhausting the GSH regenerative capacity, decreases GSH levels in a dose dependent fashion. Chow [13] showed that in vitamin E deficient rat RBCs preincubation GSH levels were lower than in the vitamin E supplemented cells. In contrast to his report, Sokol et al. [3] found in AVED patients, in agreement with our findings, both normal GSH levels and normal activities of several essential antioxidant enzymes. In the present study, SOD activity was normal, 2OO
.12 "I"
"6 E
•
pretreatment
[]
posttreatment
iso
e-
@
,1=
._m •o c
5O
_o "
o
L 0.5mM
! 1.0mM
L__
|! 2.0mM
I
Fig. 2. MDA levels following exposure to BHP (0.5-2.0 mM), in the pre and posttreatment stage of vitamin E.
136
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
while catalase, which decomposes some of the hydroperoxide which is generated after the dismutation of the superoxide ions by SOD, was significantly decreased in cells from vitamin E deficient patients. These decreased values which were restored to normal following vitamin E therapy, suggest that some intracellular oxidative damage involving catalase, was induced in these deficient cells. The increased intracellular MetHb levels in the vitamin E deficient cells, which represent another index of oxidative damage, were partially restored to normal, during vitamin E administration. As previously shown, BHP-induced oxidant stress leads to intracellular MetHb formation, GSH depletion and lipid peroxidation [14]. As expected, the increase in intracellular MetHb levels was related to the BHP concentration. However, vitamin E deficient cells had higher MetHb levels in all BHP concentrations, than the supplemented cells, indicating a higher sensitivity to oxidative stress in those deficient cells. The protective role of vitamin E on membrane lipid peroxidation after BHP-induced oxidative stress, is an index of membrane damage caused by the generation of free radicals. This membrane lipid peroxidation was assessed by measuring MDA, which is a breakdown product of lipid peroxidation, and can also serve as a sensitive functional assessment of red blood cells vitamin E status [15]. MDA levels following BHP exposure, were highly elevated in the vitamin E deficient cells in a dose dependent fashion in comparison with the moderate rise in posttreatment levels. The results show that vitamin E deficient cells, are much more susceptible to oxidative damage than the vitamin E supplemented cells. This is not surprising, since vitamin E is known to be essential for maintaining the integrity of the RBC membrane. In summary, these studies show that in the AVED patients studied, the vitamin E deficiency affected membrane integrity and some intracellular parameters involved in antioxidant activity. In contrast to the specific metabolic features in RBCs of premature infants in whom vitamin E deficiency leads to overt haemolysis, our patients had evidence for subtle intraerythrocytic oxidative damage and normal haemoglobin and RBC levels. On the basis of these studies it may be of interest to evaluate the evidence for oxidative damage in RBCs of other untreated AVED patients.
Acknowledgments The authors wish to thank Dr. Ben-Ami Sela, for vitamin E determinations.
References [1] Chow CK. Vitamin E and oxidative stress. Free Rad Biol Med 1991;11:215-32. [2] Farrell PM, Bieri JG, Fratantoni JF, Wood RE, Di Sant'Agnese PA. The occurrence and effects of human vitamin E deficiency. J Clin Invest 1977;60:233-41.
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
137
[3] Sokol RJ, Kayden HJ, Bettis DB, Traber MG, Neville H, Ringel S e t al. Isolated vitamin E deficiency in the absence of fat malabsorption-familial and sporadic cases: characterization and investigation of causes. J Lab Clin Med 1988;111:548-59. [4] Sokol RJ. Vitamin E and neurologic function in man. Free Rad Biol Med 1989;6:189-207. [5] Dallman PR, Yip R, Oski FA. Iron deficiency and related nutritional anemias. In: Nathan DG, Oski FA, editors. Hematology of Infancy and Childhood, 4th ed. London: WB Saunders 1993.
[6] Shorer Z, Parvari R, Levitas A, Brill G, Sela B-A, Moses S. Ataxia with isolated vitamin E deficiency (AVED) in four siblings. Pediatr Neurol 1996;14:340-3. [7] Beutler E. Red cell metabolism. A Manual of Biochemical Methods. London: Grune and Stratton, 1984;83-4,105-6,131-134. [8] Shinar E, Rachmilewitz EA, Shifter A, Rahamim E, Saltman P. Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate. Biochim Biophys Acta 1989;1014:66-9. [9] Meyerstein N, Oppenheim U, Yirmiahu T, Hatskelson L, Dvilansky A. Erythrocyte involvement in chronic lymphocytic leukaemia. Scand J Haematol 1986;36:138-40. [10] Stocks J, Dormandy TL. A direct thiobarbituric acid reacting chromogen in human red blood cells. Clin Chim Acta 1970;27:117-23. [111 Trotta RJ, Sullivan SG, Stern A. Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Biochem J 1983;212:759-72. [12] Gottfried EL, Robertson NA. Glycerol lysis time as a screening test for erythrocyte disorders. J Lab Clin Med 1974;83:323-33. [13] Chow CK. Oxidative damage in the red cells of vitamin E-deficient rats. Free Rad Res Commun 1992; 16:247-58. [14] Banal M, Mazor D, Dvilansky A, Meyerstein N. Iron deficiency anemia: recovery from in vitro oxidative stress. Acta Haematol 1993;90:94-8. [15] Cynamon HA, Isenberg JN, Nguyen CH. Erythrocyte malondialdehyde release in vitro: a functional measure of vitamin E status. Clin Chim Acta 1985;151:169-76.
Clinica Chimica Acta 265 (1997) 131-137
Short c o m m u n i c a t i o n
Oxidative damage in red blood cells of vitamin E deficient patients a
• a
D. M a z o r , G. Brill b, Z. Shorer b, S. M o s e s b, N. Meyersteln " "The Dr. Kaufmann Hematology Laboratory, Physiology Department, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel bThe Dr. Kaufmann Hematology Laboratory, Pediatric Department, Soroka University Medical Centre, Beer Sheva, Israel Received 30 December 1996; received in revised form 28 May 1997; accepted 30 May 1997
Keywords: O x i d a t i v e stress; V i t a m i n E deficiency; R e d blood cells
1. Introduction
Vitamin E is a lipid-soluble antioxidant. Its most important role is to protect the membrane polyunsaturated fatty acids from oxidation involving reactive oxygen species, by termination of free-radical chain reactions [1]. In view of the widespread occurrence of ot-tocopherol in food, nutritional vitamin E deficiency is extremely uncommon. However, vitamin E deficiency occurs in humans as a result of a variety of lipid malabsorption syndromes such as cystic fibrosis [2], or as an isolated vitamin E deficiency in the absence of fat malabsorption [3]. An isolated vitamin E deficiency could be caused by abnormal absorption, distribution, metabolism or excretion [3]. In all cases it is recognized as a cause of neurologic abnormalities, since vitamin E is crucial in maintaining the structure and function of the human nervous system [4]. Premature babies, born with low serum and tissue concentrations of vitamin E, are susceptible to haemolysis, with morphological changes in RBCs and increased in vitro sensitivity to hydrogen peroxide (H202) exposure [5]. Oral vitamin E therapy *Corresponding author. Tel.: + 972 7 6400826; Fax: + 972 7 6277655. 0009-8981/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 9 - 8 9 8 1 ( 9 7 ) 0 0 1 1 6 - 2
132
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
restores plasma vitamin levels to normal and can improve neurological functions and hydrogen peroxide induced haemolysis [2,3] in vitamin E deficient patients. In this presentation, we studied a consanguineous Bedouin family with four AVED siblings. In three siblings who were available for study at the time of diagnosis, RBC oxidative damage was demonstrated in the pretreatment stage. This damage was even more pronounced after exposure of the cells to BHE Vitamin E administration resulted in a marked attenuation of the oxidative damage.
2. Materials and methods
2.1. Chemicals BHP and all the reagents were of analytical grade, obtained from Sigma Chemical Company (Rehovot, Israel). Vit E (D-ce-tocopherol acetate, Roche Inc.) was administered at a dose of 100mg Kg -~ day -1.
2.2. Subjects Three female siblings (born to consanguineous Bedouin parents) aged between 12 and 24 years were studied. They had developed a Friedreich type of ataxia without myocardial involvement at the end of their first decade of life. The clinical and neurological features were separately described [6]. Their haemoglobin and red blood cell counts were normal and no acanthocytes were detected in their blood smears. Bilirubin levels and reticulocyte counts were normal. Pretreatment plasma vitamin E levels were 0.02-0.51 txg m1-1. All patients were treated daily with oral vitamin E. One month after treatment, plasma vitamin E levels of the patients reached normal values, 7.8-17.3 txg ml-~ and the neurological status in one of the three patients was improved.
2.3. Experimental design 10 ml of peripheral venous blood were withdrawn from the patients, after informed written consent was obtained. The blood was collected into heparinized test tubes. After removal of plasma and buffy coat, by centrifugation at 2500g for 5 min, the RBCs were washed three times in phosphate-buffered saline (PBS) pH 7.4 and suspended as 20%. The suspension was exposed to 0.5-2.0 mM (final concentration) of BHP for 30 min at 37°C with an appropriate control. MetHb, GSH and MDA levels were measured before and after exposure.
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
133
2.4. Methods for the determination of the antioxidant status of RBCs Intracellular GSH levels were determined by the method of Ellman, based on the reduction of 5',5'-dithiobis-2-nitrobenzoic acid [7]. CuZnSOD was measured by the 50% inhibition of pyrogallol autoxidation and catalase was determined by the rate of H202 decomposition [7]. Methaemoglobin levels were determined at 630 nm and calculated as percent of total haemoglobin [8]. The spectrophotometric determinations were performed on a computed Uvicon 922 spectrophotometer (Kontron instruments, Milano, Italy). Acidified glycerol lysis time (AGLTs0) was determined by the time required to reach 50% haemolysis in the blood suspension [9]. Briefly, 20~1 of blood was introduced into 5 ml PBS pH 6.85. 1 ml of the suspension was added to 2 ml of glycerol solution and the decrease in absorbance was recorded on a Spectronic 21 Bausch and Lomb (Rochester, New York) spectrophotometer, at 625 nm. MDA analysis was determined by the thiobarbituric method on a trichloroacetic acid extract of the RBC suspension, with absorbency at 532 and 600 nm [10].
2.5. Data analysis Statistical analysis of the results was performed with the Student's t-test. Results are presented as the mean+SEM.
3. Results Table 1 shows the antioxidant status of RBCs (intracellular and membrane parameters), in the pre- and post treatment stage. RBCs of the vitamin E deficient patients had markedly reduced permeability to acidified glycerol which was partially corrected by vitamin E administration. Intracellular GSH and SOD levels were normal before and after treatment. Catalase activity which was significantly lower in the RBCs of the vitamin E deficient patients, was restored to normal values by vitamin E administration. The high methaemoglobin levels in RBCs of the vitamin E deficient patients, were significantly decreased after oral vitamin E administration.
3.1. Exposure to BHP BHP, an oxidizing agent is transformed in the cell to several oxidative radicals by reacting with haemoglobin. These radicals oxidize haemoglobin to MetHb and initiate peroxidation of the membrane lipids [11]. Exposure of RBCs to BHP
134
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
Table 1 Antioxidant status of red blood cells, before and after vitamin E administration in the 3 siblings Vitamin E txg ml-~ Pretreatment Post treatment
Normal
0.51; 0.07; 0.02; 10.1; 17.3; 7.8
5.0-20.0
Acidified glycerol lysis test (seconds) Pretreatment Post treatment Glutathione (mM) Pretreatment Post treatment
130; 200; 120; 100; 95; 95
60.0-70.0
2.56; 3.08; 2.19; 2.38; 2.92; 2.38
2.0-3.0
Methemoglobin (%) Pretreatment Post treatment
3.95; 4.55; 4.05; 2.43; 2.93; 2.71
0.5-2.0
Superoxide dismutase (IU/gHb) Pretreatment
2008; 2223.6; 1916.7;
1950-2555
Catalase (IU/gHb × 10 4) Pretreatment Post treatment
8.15; 11.9; 8.4; 11.14; 13.3; 15.0
13.3-17.0
decreased intracellular GSH levels in a dose dependent pattern with similar pattern in pre- and posttreatment cells (data not shown). An expected BHP dose dependent elevation of MetHb levels was found, which was significantly more prominent in the vitamin E deficient cells than in the post treated cells (Fig. 1). Malonyldialdehyde (MDA) levels as shown in Fig. 2, were significantly more pronounced in the pretreated than in the posttreated cells.
4. Discussion The present study provides evidence for RBC oxidative damage as measured by membrane and cytoplasmic parameters in the AVED patients. As shown, RBCs of vitamin E deficient patients had prolonged AGLT time. Such longer values reflect lower hemolytic rate and may be found in association with abnormal erythrocyte lipid organization or in cells with relative increase in membrane surface area [12]. It can be hypothesized that in vitamin E deficient cells, oxidative damage may induce changes in the lipid patterns of the membrane which affect the rate of haemolysis in glycerol containing media. During vitamin E administration, AGLT time was shortened but it did not reach normal levels. These changes in AGLT in the vitamin E deficient cells, suggest membrane vulnerability which is partly corrected, by vitamin E administration. Intracellular GSH levels were similar in the pre and posttreatment cells, since
135
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
10 •
pretreatment
[]
posttreatment
8 c J~ 0 ol o
6
E j¢
4
+LLL 2
O
~ 0.5mM
1.0mM
2.0mM
Fig. 1. Intracellular MetHb levels following exposure to BHP (0.5-2.0 mM), in the pre and post-treatment stage of vitamin E.
GSH is constantly regenerated in the cell from GSSG, by glutathione reductase activity. An acute oxidative stress, such as exposure to BHP, apparently by exhausting the GSH regenerative capacity, decreases GSH levels in a dose dependent fashion. Chow [13] showed that in vitamin E deficient rat RBCs preincubation GSH levels were lower than in the vitamin E supplemented cells. In contrast to his report, Sokol et al. [3] found in AVED patients, in agreement with our findings, both normal GSH levels and normal activities of several essential antioxidant enzymes. In the present study, SOD activity was normal, 2OO
.12 "I"
"6 E
•
pretreatment
[]
posttreatment
iso
e-
@
,1=
._m •o c
5O
_o "
o
L 0.5mM
! 1.0mM
L__
|! 2.0mM
I
Fig. 2. MDA levels following exposure to BHP (0.5-2.0 mM), in the pre and posttreatment stage of vitamin E.
136
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
while catalase, which decomposes some of the hydroperoxide which is generated after the dismutation of the superoxide ions by SOD, was significantly decreased in cells from vitamin E deficient patients. These decreased values which were restored to normal following vitamin E therapy, suggest that some intracellular oxidative damage involving catalase, was induced in these deficient cells. The increased intracellular MetHb levels in the vitamin E deficient cells, which represent another index of oxidative damage, were partially restored to normal, during vitamin E administration. As previously shown, BHP-induced oxidant stress leads to intracellular MetHb formation, GSH depletion and lipid peroxidation [14]. As expected, the increase in intracellular MetHb levels was related to the BHP concentration. However, vitamin E deficient cells had higher MetHb levels in all BHP concentrations, than the supplemented cells, indicating a higher sensitivity to oxidative stress in those deficient cells. The protective role of vitamin E on membrane lipid peroxidation after BHP-induced oxidative stress, is an index of membrane damage caused by the generation of free radicals. This membrane lipid peroxidation was assessed by measuring MDA, which is a breakdown product of lipid peroxidation, and can also serve as a sensitive functional assessment of red blood cells vitamin E status [15]. MDA levels following BHP exposure, were highly elevated in the vitamin E deficient cells in a dose dependent fashion in comparison with the moderate rise in posttreatment levels. The results show that vitamin E deficient cells, are much more susceptible to oxidative damage than the vitamin E supplemented cells. This is not surprising, since vitamin E is known to be essential for maintaining the integrity of the RBC membrane. In summary, these studies show that in the AVED patients studied, the vitamin E deficiency affected membrane integrity and some intracellular parameters involved in antioxidant activity. In contrast to the specific metabolic features in RBCs of premature infants in whom vitamin E deficiency leads to overt haemolysis, our patients had evidence for subtle intraerythrocytic oxidative damage and normal haemoglobin and RBC levels. On the basis of these studies it may be of interest to evaluate the evidence for oxidative damage in RBCs of other untreated AVED patients.
Acknowledgments The authors wish to thank Dr. Ben-Ami Sela, for vitamin E determinations.
References [1] Chow CK. Vitamin E and oxidative stress. Free Rad Biol Med 1991;11:215-32. [2] Farrell PM, Bieri JG, Fratantoni JF, Wood RE, Di Sant'Agnese PA. The occurrence and effects of human vitamin E deficiency. J Clin Invest 1977;60:233-41.
D. Mazor et al. / Clinica Chimica Acta 265 (1997) 131-137
137
[3] Sokol RJ, Kayden HJ, Bettis DB, Traber MG, Neville H, Ringel S e t al. Isolated vitamin E deficiency in the absence of fat malabsorption-familial and sporadic cases: characterization and investigation of causes. J Lab Clin Med 1988;111:548-59. [4] Sokol RJ. Vitamin E and neurologic function in man. Free Rad Biol Med 1989;6:189-207. [5] Dallman PR, Yip R, Oski FA. Iron deficiency and related nutritional anemias. In: Nathan DG, Oski FA, editors. Hematology of Infancy and Childhood, 4th ed. London: WB Saunders 1993.
[6] Shorer Z, Parvari R, Levitas A, Brill G, Sela B-A, Moses S. Ataxia with isolated vitamin E deficiency (AVED) in four siblings. Pediatr Neurol 1996;14:340-3. [7] Beutler E. Red cell metabolism. A Manual of Biochemical Methods. London: Grune and Stratton, 1984;83-4,105-6,131-134. [8] Shinar E, Rachmilewitz EA, Shifter A, Rahamim E, Saltman P. Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate. Biochim Biophys Acta 1989;1014:66-9. [9] Meyerstein N, Oppenheim U, Yirmiahu T, Hatskelson L, Dvilansky A. Erythrocyte involvement in chronic lymphocytic leukaemia. Scand J Haematol 1986;36:138-40. [10] Stocks J, Dormandy TL. A direct thiobarbituric acid reacting chromogen in human red blood cells. Clin Chim Acta 1970;27:117-23. [111 Trotta RJ, Sullivan SG, Stern A. Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Biochem J 1983;212:759-72. [12] Gottfried EL, Robertson NA. Glycerol lysis time as a screening test for erythrocyte disorders. J Lab Clin Med 1974;83:323-33. [13] Chow CK. Oxidative damage in the red cells of vitamin E-deficient rats. Free Rad Res Commun 1992; 16:247-58. [14] Banal M, Mazor D, Dvilansky A, Meyerstein N. Iron deficiency anemia: recovery from in vitro oxidative stress. Acta Haematol 1993;90:94-8. [15] Cynamon HA, Isenberg JN, Nguyen CH. Erythrocyte malondialdehyde release in vitro: a functional measure of vitamin E status. Clin Chim Acta 1985;151:169-76.