Effect oÃ-High Levels oÃ-Dietary Vitamin E on Hematological Indices and Biochemical Parameters in Rats1 N. Y. JACK YANG - ANDINDRAJIT D. DESAI 3 Division of Human Nutrition, School of Home Economics, University of British Columbia, Vancouver, B.C., Canada V6T 1W5 ABSTRACT The effects of low, normal and high levels of dietary vitamin E on rats were assessed by feeding diets containing 0, 25, 250, 2,500, 10,000, and 25,000 IU vitamin E/kg diet for prolonged periods of 8 and 16 months. All nutrients in the basal diet except vitamin E were ade quate. Excess vitamin E and vitamin E deficiency depressed body weight over a period of time. High levels of dietary vitamin E increased the rela tive heart weight after 8 months and relative spleen weight after 16 months. The ash content of the bones decreased with concurrent increase in plasma alkaline phosphatase activity after 16 months of treatment. The prothrombin time was reduced after 12 months, while elevated hematocrit value was observed after 16 months on high intake of dietary vitamin E. Erythrocytes from vitamin E déficientrats showed spontaneous hemolysis when incu bated in saline-phosphate buffer, while cells from rats given normal or excess vitamin E did not show signs of hemolysis when incubated under identical conditions. Urinary excretion of creatine and creatinine were apparently normal in all rats with the exception of vitamin E deficient rats which had significantly higher creatine and lower creatinine levels in urine. Results of these studies clearly indicate that excess vitamin E has dele terious effects and that further investigations are urgently warranted to test the implications of these findings on the long range metabolic and physio logical consequences in animal and man. J. Nutr. 107: 1410-1417, 1977. INDEXING KEY WORDS vitamin E • hypervitaminosis E • vitamin E supplements •vitamin E toxicity Excessive vitamin E intake has been generally considered as harmless and even has been proclaimed by some medical practitioners and the popular press to be prophylactic against numerous disease manifestations and beneficial for health, Vitamin E is available without prescription and has been used as a widespread selfprescribed vitamin supplement. In fact, there is no satisfactory scientific or clinical evidence to prove that vitamin E supple-
have been reported as fatigue (1, 2), creatinuria ( 1-3 ) and lengthened prothrombin time when taken in excess along with warfarin and clofibrate treatment (4). A significant increase in urinary excretion of estrogens and androgens has been shown in women during vitamin E therapy (5).
ment
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1410
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Columbia. 'Postdoctoral »To whom
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EFFECT OF HIGH LEVELS OF DIETARY VITAMIN E
Adverse effects of hypervitaminosis E in animals have also been investigated. Early studies reported that excess vitamin E caused testicular degeneration and re duced fertility in male rats (6), and af fected the length of estrus cycle and ovarian activity in female rats (7). Hyper vitaminosis E has been reported to depress thyroid activity in rats (8) and hamsters (9); to decrease thymus weight and to increase adrenal weight in rats ( 10 ) ; and to cause adrenal degeneration and growth inhibition in chickens (11). Recently, March et al. (12) found that hypervita minosis E induced reticulocytosis, lowered hematocrit value, reduced thyroid activity and increased the requirement for vitamin D and vitamin K in chicks. The following studies were undertaken to investigate further the long-term effects of high levels of dietary vitamin E on rats which were treated with six different levels of vitamin E, ranging from 0 to 25,000 Ill/kg diet, for as long as 16 months. MATERIALS AND METHODS
Weanling female rats of Wistar strain weighing approximately 50 g were ran domly divided into six groups. Two rats were housed together for a beginning pe riod of 2 weeks after which all rats were housed individually in wire meshed cages in a room which was lighted from 06:00 to 18:00 hours daily and maintained at approximately 23°.Diet and water were provided ad libitum. A basal tocopheroldeficient diet of Draper et al. (13), with supplements of 0, 25, 250, 2,500, 10,000 and 25,000 IU vitamin E ( d/-o-tocopheryl acetate) per kg diet was fed to six groups of rats for the whole experimental period. The composition of this vitamin E-free basal diet is shown in table 1. All nutrients except vitamin E in the experimental diet were adequate for optimum growth of the rats and were in compliance with the National Research Council recommenda tions (14). The levels of vitamin D and vitamin K in all diets were 2,000 lU/kg diet and 1 mg/kg diet, respectively. Food consumption of individual rats was regu larly measured and no significant differ ence was observed. The rats were weighed prior to killing. Blood samples were taken by tail cutting after anesthetizing the rats
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1411
TABLE 1 Composition of the basal diet1
Ingredient D-Dextrose; NRC anhydrous Vitamin-free casein Corn oil, tocopherol stripped Salt mix2 Vitamin mix (less vitamin E)3 Choline chloride64.9
20.0 10.0 4.0 0.6 0.5
1Modified from Draper, H. H., Bergan, J. G., Chiù,M., Csallany, A. S. & Boaro, A. V. (1964) J. Nutr. 84, 395-400. 2Provided the following as g/kg diet: CaCO3, 6.54; CaHPO^HzO, 14.2; NaCl, 4.3; K2HP04, 3.09; K3(CeH6O,)-H2O, 9.46; MgC03, 1.64; Fe(C6HsO,)-3H2O, 0.64; MnSO4H2O, 0.055; ZnC03, 0.018; CuSO,-5H2O, 0.007; KI, 0.0018. 3Provided the following amount per kg diet (in IU): 25,000 vitamin A as retinyl palmitate ; 2,000 ergocalciferol; (in mg) : menadione, 1 ;biotin 0.1 ;vitamin Bn, 0.1 ; calcium pantothenate, 10; folio acid, 1; niacin 25; pyridoxine HC1, 5.0; riboflavin, 5.0 ; thiamin HC1, 10.
with ether for the determination of prothrombin time (15), hematocrit value, hemoglobin level ( 16 ) and hemolysis (17) at 9, 12 and 16 months of treatment. Urinary levels of creatine and creatinine were determined (18) in a 24-hour urine collection after 11 months. Four rats from each group were killed after 8 months and the others were used at the end of 16month experimental period. All rats were anesthetized with ether, and blood sam ples were taken from the inferior vena cava using heparinized syringes. The liver, kidney, heart, spleen, and uterus were ex cised, cleaned, and weighed. The femora were removed, stripped of soft tissue, de fatted with a mixture of chloroform-methanol (2:1 V/V) for 24 hours, dried in an isothermic oven at 105° for 24 hours, weighed and ashed in a furnace at 650° for overnight. Bone ash was weighed and dissolved in 4 ml of 3 N HC1 and diluted to appropriate volume. Calcium was de termined by atomic absorption spectrophotometry. Inorganic phosphate was de termined by a modification of the Fiske and Subbarow method (19). Plasma alka line phosphatase (18) was measured in rats treated for 16 months. One alkaline phosphatase unit was defined as the amount of enzyme in 100 ml plasma which liberates 1 mg phenol from phenol phos phate in 12 minutes incubation at 37°.
1412
N. Y. JACK YANG AND 1NDRAJIT
D. DESAI
TABLE 2 Body and organ weights of rats fed different levels of dietary vitamin E for 8 months1 Dietary E1 vitamin
weight9166" body416"(24)248"(16)269 g
U/kg diet0252502,50010,00025,000Body (13)366"
(32)536»(42)560'
(292)2,790" (20)204(25)198
(123)2,470' (31)332cd(12)356" (70)2,469» (16)SIS'*
(204)2,540»
(4)161»
62 (12)25 162
(8)164»(5)159»(14)167 (30)496"(32)576
(13)176(19)197(10)282
»2 6(29)547' (13)289 (102)2,451 "2
have been reported as fatigue (1, 2), creatinuria ( 1-3 ) and lengthened prothrombin time when taken in excess along with warfarin and clofibrate treatment (4). A significant increase in urinary excretion of estrogens and androgens has been shown in women during vitamin E therapy (5).
ment
on hypervitaminosis E supported by research grants
IS benehcial
Çnmp, nnrpnHal aom potential
sis E have been i . -j rr beings,
Side-effects
at
Vip-rarrlc nazaras
the
present
suggested. r Of
time.
r>f Ot V>\mor»iifaminr. nypervitammo-
excess
from the National Research of Canada and the Research Committee of theCouncil University of British
In•*.humanT-.
Vitamin
E
1410
Downloaded from https://academic.oup.com/jn/article-abstract/107/8/1410/4769204 by Washington University, Law School Library user on 04 July 2018
Columbia. 'Postdoctoral »To whom
Research Associate.
reprint
requests
should
be addressed.
EFFECT OF HIGH LEVELS OF DIETARY VITAMIN E
Adverse effects of hypervitaminosis E in animals have also been investigated. Early studies reported that excess vitamin E caused testicular degeneration and re duced fertility in male rats (6), and af fected the length of estrus cycle and ovarian activity in female rats (7). Hyper vitaminosis E has been reported to depress thyroid activity in rats (8) and hamsters (9); to decrease thymus weight and to increase adrenal weight in rats ( 10 ) ; and to cause adrenal degeneration and growth inhibition in chickens (11). Recently, March et al. (12) found that hypervita minosis E induced reticulocytosis, lowered hematocrit value, reduced thyroid activity and increased the requirement for vitamin D and vitamin K in chicks. The following studies were undertaken to investigate further the long-term effects of high levels of dietary vitamin E on rats which were treated with six different levels of vitamin E, ranging from 0 to 25,000 Ill/kg diet, for as long as 16 months. MATERIALS AND METHODS
Weanling female rats of Wistar strain weighing approximately 50 g were ran domly divided into six groups. Two rats were housed together for a beginning pe riod of 2 weeks after which all rats were housed individually in wire meshed cages in a room which was lighted from 06:00 to 18:00 hours daily and maintained at approximately 23°.Diet and water were provided ad libitum. A basal tocopheroldeficient diet of Draper et al. (13), with supplements of 0, 25, 250, 2,500, 10,000 and 25,000 IU vitamin E ( d/-o-tocopheryl acetate) per kg diet was fed to six groups of rats for the whole experimental period. The composition of this vitamin E-free basal diet is shown in table 1. All nutrients except vitamin E in the experimental diet were adequate for optimum growth of the rats and were in compliance with the National Research Council recommenda tions (14). The levels of vitamin D and vitamin K in all diets were 2,000 lU/kg diet and 1 mg/kg diet, respectively. Food consumption of individual rats was regu larly measured and no significant differ ence was observed. The rats were weighed prior to killing. Blood samples were taken by tail cutting after anesthetizing the rats
Downloaded from https://academic.oup.com/jn/article-abstract/107/8/1410/4769204 by Washington University, Law School Library user on 04 July 2018
1411
TABLE 1 Composition of the basal diet1
Ingredient D-Dextrose; NRC anhydrous Vitamin-free casein Corn oil, tocopherol stripped Salt mix2 Vitamin mix (less vitamin E)3 Choline chloride64.9
20.0 10.0 4.0 0.6 0.5
1Modified from Draper, H. H., Bergan, J. G., Chiù,M., Csallany, A. S. & Boaro, A. V. (1964) J. Nutr. 84, 395-400. 2Provided the following as g/kg diet: CaCO3, 6.54; CaHPO^HzO, 14.2; NaCl, 4.3; K2HP04, 3.09; K3(CeH6O,)-H2O, 9.46; MgC03, 1.64; Fe(C6HsO,)-3H2O, 0.64; MnSO4H2O, 0.055; ZnC03, 0.018; CuSO,-5H2O, 0.007; KI, 0.0018. 3Provided the following amount per kg diet (in IU): 25,000 vitamin A as retinyl palmitate ; 2,000 ergocalciferol; (in mg) : menadione, 1 ;biotin 0.1 ;vitamin Bn, 0.1 ; calcium pantothenate, 10; folio acid, 1; niacin 25; pyridoxine HC1, 5.0; riboflavin, 5.0 ; thiamin HC1, 10.
with ether for the determination of prothrombin time (15), hematocrit value, hemoglobin level ( 16 ) and hemolysis (17) at 9, 12 and 16 months of treatment. Urinary levels of creatine and creatinine were determined (18) in a 24-hour urine collection after 11 months. Four rats from each group were killed after 8 months and the others were used at the end of 16month experimental period. All rats were anesthetized with ether, and blood sam ples were taken from the inferior vena cava using heparinized syringes. The liver, kidney, heart, spleen, and uterus were ex cised, cleaned, and weighed. The femora were removed, stripped of soft tissue, de fatted with a mixture of chloroform-methanol (2:1 V/V) for 24 hours, dried in an isothermic oven at 105° for 24 hours, weighed and ashed in a furnace at 650° for overnight. Bone ash was weighed and dissolved in 4 ml of 3 N HC1 and diluted to appropriate volume. Calcium was de termined by atomic absorption spectrophotometry. Inorganic phosphate was de termined by a modification of the Fiske and Subbarow method (19). Plasma alka line phosphatase (18) was measured in rats treated for 16 months. One alkaline phosphatase unit was defined as the amount of enzyme in 100 ml plasma which liberates 1 mg phenol from phenol phos phate in 12 minutes incubation at 37°.
1412
N. Y. JACK YANG AND 1NDRAJIT
D. DESAI
TABLE 2 Body and organ weights of rats fed different levels of dietary vitamin E for 8 months1 Dietary E1 vitamin
weight9166" body416"(24)248"(16)269 g
U/kg diet0252502,50010,00025,000Body (13)366"
(32)536»(42)560'
(292)2,790" (20)204(25)198
(123)2,470' (31)332cd(12)356" (70)2,469» (16)SIS'*
(204)2,540»
(4)161»
62 (12)25 162
(8)164»(5)159»(14)167 (30)496"(32)576
(13)176(19)197(10)282
»2 6(29)547' (13)289 (102)2,451 "2
