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An Apparent Rachitogenic Effect of Excessive Vitamin E Intakes in the Chick1
An Apparent Rachitogenic Effect of Excessive Vitamin E Intakes in the Chick T. P. MURPHY, K. E. WRIGHT, and W. J. PUDELKIEWICZ2 Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut
06268
(Received for publication September 25, 1980)
1981 Poultry Science 60:1873-1878
INTRODUCTION
Massive amounts of vitamin E administered to experimental animals have yielded conflicting reports regarding its toxicity. Effects attributable to excessive vitamin E intake include: interference with thyroid activity in chicks (Marchers/., 1973), rats (Valenti and Bottarelli, 1965), and hamsters (Czybaetal., 1966); disturbances in the metabolism of other fat soluble vitamins (March et al, 1973) and growth inhibition (March et al, 1973; Nockels et al, 1976; Yang and Desai, 1977). In contrast, both chicks (McCuaig and Motzok, 1970) and rats (Martin and Hurley, 1977) have been fed excess vitamin E with no effect on growth or mortality, and Alam et al. (1978) observed no effect on fetal development with rats. Of particular interest was the observation of March et al. (1973) that bone calcification was depressed when excess vitamin E was administered, either through the diet or by injection into chicks fed either calcium-deficient or vitamin D-deficient diets. Similarly, Yang and Desai (1977) reported that in rats consuming excess dietary vitamin E, the ash content of the femur decreased while the calcium content was not affected and the phosphorus content was
1 Scientific Contribution No. 807, Storrs, Agricultural Experiment Station, University of Connecticut, Storrs, CT. 2 To whom reprint requests should be sent.
only slightly affected. Recently, March et al. (1973) found that hypervitaminosis E increased the incidence of caries in rats. Since, in these studies, a possible alteration in bone composition and vitamin D status during hypervitaminosis E occurred, this investigation was undertaken to determine the effect of excess dietary vitamin E on the vitamin D status of the chick. MATERIALS AND METHODS The experiment was a 2 X 2 factorial design, replicated three times with dl-a-toCopheryl acetate (25 or 10,000 IU/kg diet) and vitamin D 3 (25 or 500 IU/kg diet) as variables. For each replicate, 100 day-old male White Plymouth Rock Chicks (Arbor Acres, Glastonbury, CT) were housed in electrically heated, thermostatically controlled battery brooders with raised wire floors in an air-conditioned room (23 C) with no windows. A minimal amount of fluorescent light, at an intensity of 10 lx at the feeders, was continuously available. All chicks were fed the basal vitamin D-free diet and water ad libitum for 1 week in an effort to standardize the population. The composition of the basal and experimental diets is shown in Table 1. All nutrients in the basal diet, including vitamin E but excluding vitamin D, were in compliance with the National Research Council recommendations (1977). Triplicate samples of the basal diet and each dietary treatment were wet-ashed according to the method of Reitz et al. (1960) for subsequent calcium and phosphorus assay
1873
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
ABSTRACT Three replicates (pens of 10 birds each) of a 2 X 2 factorial design experiment (25 and 10,000 IU vitamin E/kg diet on 25 and 500 IU vitamin D/kg diet) were utilized in order to assess effects of excessive intakes of vitamin E on calcium metabolism in the growing chick. A one-week equilibration period was followed by a two-week experimental period. Excessive vitamin E supplementation resulted in reduced calcium and phosphorus in blood plasma, in dry fat-free bone, and in bone ash, calcium, and phosphorus. Inadequate vitamin D supplementation reduced total feed consumption, terminal body weight, plasma calcium, dry fat-free bone, bone ash, bone calcium, and bone phosphorus. Significant vitamin E X vitamin D interactions were noted for plasma calcium, dry bone, and bone ash, excessive vitamin E apparently interfering with vitamin D utilization. (Key words.- vitamin E, vitamin D, chick, plasma, bone, calcium)
1874
MURPHY ET AL. TABLE 1. Percentage composition of basal and experimental Basal Vitamin D 3 (IU/kg): 0 Vitamin E (IU/kg): 25
25.00 60.20 4.00 4.00
(%) 25.00 58.20 3.995 4.00
Diet 3 500 25
Diet 4 500 10,000
25.00 59.20 3.90 4.00
25.00 58.20 3.90 4.00
.52 .15
.52 .15
.52 .15
.52 .15
.52 .15
6.03
6.03
6.03
6.03
6.03
.10
.10
.10
.10
.10
.005 1.00 100.000
2.00 .005
.10
.10
100.000
1.00 100.00
100.00
1.24
1.15
1.22
1.17
1.15
.88
.93
.94
.92
.92
100.00 Composition by analysis: Calcium, % Phosphorus, %
25.00 59.20 3.995 4.00
Diet 2 25 10,000
2.00
1 Formulated to meet the chick's NRC (1977) nutrient requirement for all essential nutrients including vitamin E but excluding vitamin D. 2
Promine R, Central Soya, Chicago, IL.
3 4
Cerelose, CPC International, Engelwood Cliffs, NJ. PVO International Inc., Richmond, CA. Contains .33 mg of d-a-tocopherol/g.
'Mineral mix (g/kg diet); CaHP0 4 , 28.40; CaC0 3 , 10.00; Na 2 HPO„, 7.00; KC1, 7.00; NaCI, 4.00; MgS0 4 , 3.00; F e C 6 H 3 0 7 , .50; MnSO„ • H 2 0 , .25; ZnC0 3 , .13; CuSO„ • 5 H 2 0 , .02; K10 3 , .01; Na 2 Se0 3 , .00022; Na2MoO„ » 2 H 2 0 , .005. 6 Vitamin mix (mg/kg diet): retinyl acetate, 15 (Roche type 325-40 gelatin beadlets guaranteed to contain 111.8 mg retinyl acetate/g) equivalent to 4875 IU or 1462 RE; vitamin E, 50 (Roche 50% all-rac-a-tocopheryl acetate gelatin beadlets); vitamin K,, 100 (Roche, 1% gelatin beadlets); biotin, .40; vitamin B 12 (1%), 30; Ca pantothenate, 30; foiacin (3%), 20; niacin, 50; pyridoxine-HCl, 30; riboflavin, 10; thiamin-HCl, 15; cerelose to 1.00 g. 7
Dry gelatin beadlets containing 50% all-rac-a-tocopheryl acetate/g (Hoffmann LaRoche, Nutley, NJ).
'Sigma Chemical Co., St. Louis, MO. Cholecalciferol made up in Mazola oil (CPC International Inc., Englewood Cliffs, NJ) to contain 500 IU (12.5 ug)/g.
and determined colorimetrically on an autoanalyzer. 3 On day 7, chicks were weighed and the 40 chicks closest to the mean were wing-banded and allotted into four treatment groups with pens of 10 birds each, which served as the experimental unit, so that each pen had equal average body weights to start. A dietary treatment (Table 1) was then randomly assigned to each pen of birds. Diets and water were provided ad libitum for the next 2 weeks. Feed con-
3
Auto-analyzer II, Technicon Instruments Corporation, Tarrytown, NY.
sumption and body weights were determined on days 14 and 21 of age, corresponding to the 7th and 14th day of dietary treatments. Prior to termination, the chicks were fasted for 8 hr, then weighed, and blood samples were taken by heart puncture using heparinized syringes. Plasma was obtained by centrif.jging blood at 5,000 X g for 15 min. A .4 ml sample of plasma was frozen for determination of vitamin A by the method of Neeld and Pearson (1963) and vitamin E by the method of Tsen (1961). The latter method is based upon the fact that fat-soluble reducing substances in the blood will reduce ferric iron and the assumption is made that plasma vitamin E accounts for most of this reducing material. In support of
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
Isolated soybean protein 2 Glucose monohydrate 3 Pressed safflower oil4 Cellulose DL-methionine Choline chloride Mineral mix 5 Vitamin mix 6 Vitamin E (gelatin beadlet) 7 Vitamin D 3 oil 8 Gelatin
Diet 1 25 25
diets1
EXCESSIVE VITAMIN E AND PLASMA CALCIUM
RESULTS AND DISCUSSION The following observations have been made from the results outlined in Table 2. Low dietary vitamin D levels resulted in significantly decreased feed consumption and growth, effects well documented by others (Waldroup
"Model 303, Perkin Elmer, Norwalk, CT.
et al., 1965; Baird and Green, 1935; Waldroup et al., 1963). No significant effect of excessive dietary vitamin E on feed consumption or growth was found. March et al. (1973) showed that growth rate was markedly depressed in chicks fed vitamin E at a level of 2,200 IU/kg of diet for 50 days. However, McCuaig and Motzok (1970) found that after 32 days, 10,000 IU of vitamin E/kg of diet did not affect the growth of chicks. Nockels et al. (1976), also studying chicks, reported that feeding levels of vitamin E ranging from 8,000 to 64,000 IU/kg of diet for 5 weeks resulted in a significantly reduced body weight. Variable effects of excess dietary vitamin E on the growth of rats have also been reported. Yang and Desai (1977) fed rats diets containing up to 25,000 IU of vitamin E/kg of diet for periods of 8 and 16 months and reported that high levels of vitamin E depressed body weight at the end of both feeding periods. However, Jenkins and Mitchell (1975) found an increase in body weight of rats fed either 600 or 6,000 IU of vitamin E/kg of diet after 2 mo of treatment. Further, Martin and Hurley (1977) reported that body weight of pups from mothers receiving large doses of vitamin E was not affected. There was no effect of dietary vitamin D on either plasma vitamin E, as indicated by total fat-soluble reducing substances, or on plasma vitamin A concentrations. However, the addition of excess vitamin E (diets 2 and 4) resulted in a significant increase in plasma vitamin A levels. Excess dietary vitamin E has previously been shown to increase plasma and liver levels of vitamin A (Jenkins and Mitchell, 1975; and Cawthorne et al., 1968). The increased plasma vitamin E observed in chicks consuming the high vitamin E diets would be expected and has been reported by others in both rats (Martin and Hurley, 1977) and chicks (Pudelkiewicz et al, 1960). Deficient dietary vitamin D significantly lowered plasma calcium levels, a well elucidated effect (Valenti and Bottarelli, 1965). Excessive vitamin E also lowered plasma calcium significantly. In the current experiment only birds fed diet 3 exhibited a normal plasma calcium concentration, 9.6 mg%, as compared with values reported by others of 10.4 mg% (McNutt and Haussler, 1973) and 12.1 mg% (Omdahl and DeLuca, 1977). The addition of an excess of vitamin E to the diet (diet 4) resulted in a plasma calcium concentration of 7.6 mg%.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
this assumption, a tocopherol-free diet, similar to the one used in this study, resulted in essentially a zero value for plasma reducing substances (Bunnell, 1967). Both determinations were individually scaled down to micro levels (J. E. Rousseau, Jr., this laboratory, personal communication). Total plasma calcium and inorganic phosphorus were determined colorimetrically with an auto-analyzer. Each bird was killed by cervical dislocation immediately after it was bled. At this time the right femur was removed, cleaned of excess tissue, and autoclaved at 120 C for 15 min. The bones were carefully cleaned of remaining tissue, crushed to facilitate the extraction of moisture and fat, and individually wrapped in cheese cloth. The bones were then extracted for 24 hr in an atmosphere of hot 95% ethanol followed by extracting for another 24 hr in an atmosphere of hot ethyl ether containing 2% of 95% ethanol. The fat-free bones were transferred to tared platinum crucibles, dried in a vacuum oven at 60 C at a pressure of approximately 100 mm Hg for 2 hr, weighed, and ashed in a furnace at 550 C overnight. The bone ash was then dissolved in 5 ml of 3 N HC1 and diluted to an appropriate volume. Calcium and phosphorus were determined colorimetrically by an autoanalyzer and results were verified with an atomic absorption spectrophotometer. 4 The colorimetric method for calcium was that of Kessler and Wolfman (1964) as modified by Gitelman (1967). The method for phosphorus was based upon that of Hurst (1964). Mean values were computed for the data obtained from all birds within each replicate by treatment (pen) combination. These pen means were then subjected to analysis of variance (Snedecor and Cochran, 1967) to determine the effects of vitamin E, vitamin D, and their interaction on food intake, body weight, plasma vitamins A and E, plasma calcium and phosphorus, dry bone, percent bone ash, and bone calcium and phosphorus.
1875
25 25 500 500 SD per pen
,„,
67* -33 -19 15
5
57* -34 -27 15
268 262 353 292 26
[(Diet 3 + Diet 4) - (Diet 1 + Diet 2)] 12.
[(Diet 1 + Diet 4) - (Diet 2 + Diet 3)] 12.
(SD per pen) X (1/3) (Snedecor and Cochran, 1967).
3 126
1602*
-97
218
2126
522
2220
621
Plasma
-3 5* 3 1.8
42 44 36 44 3.2
Vitamin A
(Mg/ui;
Vitamin E 2 (mg/d
.2
1.30* -1.10* -.96'
7.4 7.2 9.6 7.6 .4
Ca
*P<.05, where the factorial effect mean divided by the standard error of the effect mean equalled or exceed degrees of freedom.
6
(jj;
Terminal body weight
Fat-soluble reducing substances assumed to be primarily a-tocopherol.
Average of 3 replicates of 10 birds each per pen.
10,000
25
316 302 402 349 26
Diet consumed
" [(Diet 2 + Diet 4) - (Diet 1 + Diet 3(] 12.
3
2
1
SE 6
25
10,000
(IU/Kgy
Vitamin E
Treatments
Vitamin D 3
Factorial effect means: Vitamin D 3 Vitamin E4 Interaction 5
1 2 3 4
Diet no.
TABLE 2. Feed consumption, body weight, and some blood plasma and bone responses to a massive intake of vi
http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
EXCESSIVE VITAMIN E AND PLASMA CALCIUM
The changes seen in bone composition support the hypothesis that excess dietary vitamin E interferes with the absorption of vitamin D. As with plasma calcium, the response of percent bone ash and total bone calcium to supplementation with vitamin D was suppressed by excess dietary vitamin E. Both vitamin D deficiency and E excess significantly lowered percent bone ash, percent bone calcium, and percent bone phosphorus. These effects of vitamin E on bone composition have been reported by others. March et al. (1973) overcame the adverse effect of vitamin E on bone calcification in chicks receiving 2,200 IU of vitamin E/kg of diet by feeding 300 IU of vitamin D/kg of diet. In the present study, the chicks receiving 500 IU of vitamin D and 10,000 IU of vitamin E/kg of diet had significantly lower dry bone, percent bone ash, bone calcium, and bone phosphorus. Yang and Desai (1977) suggested that there may bean increased turnover of calcium and phosphorus in bones of rats fed high levels of vitamin E for prolonged periods. The decreases seen especially in feed consumption, body weight, plasma calcium, dry bone, percent bone ash, and percent bone calcium were associated with excess dietary vitamin E. These results support the evidence presented by March et al. (1973) that excess vitamin E increases the requirement for vitamin D by the chick. ACKNOWLEDGMENTS We are grateful to H. D. Eaton for his
statistical assistance and to S. Beaupre, K. Cady, and R. C. Hall, Jr., for their technical assistance. Grateful acknowledgment is also due Hoffmann-LaRoche Inc., Nutley, NJ for the vitamin E. REFERENCES Alam, S. Q., B. S. Alam, and C. J. Alvarez, 1978. Cariogenic effects of excess vitamin E in rats. J. Dent. Res. 57:244. Baird, F. D., and D. J. Greene, 1935. The comparative vitamin D requirements of growing chickens, turkeys, and pheasants. Poultry Sci. 14:70-82. Bunnell, R. H., 1967. Vitamin E assay by chemical methods. Pages 261 — 316 in The vitamins. 2nd ed. Vol. VI. P. Gyorgy and W. N. Pearson, ed. Academic Press, New York, NY. Cawthorne, M. A., J. Bunyan, A. T. Diplock, E. A. Murrell, and J. Green, 1968. On the relationship between vitamin A and E in the rat. Brit. J. Nutr. 22:133-143. Czyba, J. C , C. Girod, and N. Durand, 1966. Modifications morphologigues de la thyroide du hamster dore traite par la vitamine E. C. R. Soc. Biol. 160:2101 (Cited by March etal, 1973). Gitelman, H. J., 1967. An improved automated procedure for the determination of calcium in biochemical specimen. Anal. Biochem. 18: 521-531. Hurst, R. O., 1964. The determination of nucleotide phosphorus with a stannous chloride-hydrazine sulphate reagent. Can. J. Biochem. 42:287-292. Jenkins, Y. M., and G. V. Mitchell, 1975. Influence of excess vitamin E on vitamin A toxicity in rats. J. Nutr. 105:1600-1606. Kessler, G., and M. Wolfman, 1964. An automated procedure for the determination of calcium and phosphorus. Clin. Chem. 10:686-703. March, B. E., E. Wong, L. Seier, J. Sim, and J. Biely, 1973. Hypervitaminosis E in the chick. J. Nutr. 103:371-377. Martin, M. M., and L. S. Hurley, 1977. Effect of large amounts of vitamin E during pregnancy and lactation. Amer. J. Clin. Nutr. 30:1629-1637. McCuaig, L. W., and I. Motzok, 1970. Excessive dietary vitamin E. Its alleviation of hypervitaminosis A and lack of toxicity. Poultry Sci. 49:1050-1052. McNutt, K. W., and M. R. Haussler, 1973. Nutritional effectiveness of 1,25-dihydroxycholecalciferol in preventing rickets in chicks. J. Nutr. 103:681 — 689. National Research Council, 1977. Nutrient requirements of poultry. Nat. Acad. Sci., Washington, DC. Neeld, J. B., and W. N. Pearson, 1963. Macro- and micro-methods for the determination of serum vitamin A using trifluoroacetic acid. J. Nutr. 79:454-462. Nockels, C. F., D. L. Menge, and E. W. Kienholz, 1976. Effects of excessive dietary vitamin E on the chick. Poultry Sci. 55:649-652. Omdahl, J. L., and H. F. DeLuca, 1977. Mediation of calcium adaptation by 1,25-dihydroxycholecalciferol. J. Nutr. 107:1975-1980.
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Although these birds received a diet supplemented with 500 IU of vitamin D/kg, their plasma calcium values were not significantly different from those reported for diets 1 and 2 (7.4 and 7.2 mg%, respectively), which were supplemented with only 25 IU of vitamin D/kg. The excess vitamin E in the diet suppressed the elevated response of plasma calcium to the increased dietary vitamin D (diet 4). The means by which excessive dietary vitamin E acts to lower plasma calcium has not been elucidated. Pudelkiewicz and Mary (1969) reported that the percentage of ingested tocopheryl acetate excreted ranged from 30% at the 3,300 IU level of vitamin E/kg of diet to 70% at the 20,000 IU level. Therefore, it is postulated that the vitamin D is dissolved in the unhydrolyzed and possibly nonsolubilized tocopheryl acetate and passes through the gut unabsorbed. This may explain the vitamin E X vitamin D interactions.
1877
1878
MURPHY ET AL. using 4,8-diphenyl-l-10-phenanthroline. Anal. Chem. 33:849-851. Valenti, G., and E. Bottarelli, 1965. Studio radioistopico e chromatografico della funzione troidea vella ipervitaminosi E del ratto. Folia Endocrinolgia 18:318-326. (Cited by March et al, 1973). Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1963. The relationship of phosphorus, calcium and vitamin D 3 in the diet of broiler-type chicks. Poultry Sci. 42:982-989. Waldroup, P. W., J. E. Stearns, C. B. Ammerman, and R. H. Harms, 1965. Studies on the vitamin D 3 requirement of the broiler chick. Poultry Sci. 44:543-548. Yang, N.Y.J., and I. D. Desai, 1977. Effect of high levels of dietary vitamin E on liver and plasma lipids and fat soluble vitamins in rats. J. Nutr. 197:1418-1426.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
Pudelkiewicz, W. J., andN. Mary, 1969. Some relationships between plasma, liver and excreta tocopherol in chicks fed graded levels of alpha-tocopheryl acetate. J. Nutr. 97:303-306. Pudelkiewicz, W. J., L. D. Matterson, L. M. Potter, L. Webster, and E. P. Singsen, 1960. Chick tissuestorage bioassay of alpha-tocopherol: Chemical analytical techniques, and relative biopotencies of natural and synthetic alpha-tocopherol. J. Nutr. 7 1 : 1 1 5 - 1 2 1 . Reitz, L. L., W. H. Smith, and M. P. Plumlee, 1960. A simple, wet oxidation procedure for biological materials. Anal. Chem. 32:1728. Snedecor, G. W., and W. G. Cochran, 1967. Statistical methods. 6th ed. The Iowa State Univ. Press, Ames, IA. Tsen, C. C , 1961. An improved spectrophotometric method for the determination of tocopherols
06268
(Received for publication September 25, 1980)
1981 Poultry Science 60:1873-1878
INTRODUCTION
Massive amounts of vitamin E administered to experimental animals have yielded conflicting reports regarding its toxicity. Effects attributable to excessive vitamin E intake include: interference with thyroid activity in chicks (Marchers/., 1973), rats (Valenti and Bottarelli, 1965), and hamsters (Czybaetal., 1966); disturbances in the metabolism of other fat soluble vitamins (March et al, 1973) and growth inhibition (March et al, 1973; Nockels et al, 1976; Yang and Desai, 1977). In contrast, both chicks (McCuaig and Motzok, 1970) and rats (Martin and Hurley, 1977) have been fed excess vitamin E with no effect on growth or mortality, and Alam et al. (1978) observed no effect on fetal development with rats. Of particular interest was the observation of March et al. (1973) that bone calcification was depressed when excess vitamin E was administered, either through the diet or by injection into chicks fed either calcium-deficient or vitamin D-deficient diets. Similarly, Yang and Desai (1977) reported that in rats consuming excess dietary vitamin E, the ash content of the femur decreased while the calcium content was not affected and the phosphorus content was
1 Scientific Contribution No. 807, Storrs, Agricultural Experiment Station, University of Connecticut, Storrs, CT. 2 To whom reprint requests should be sent.
only slightly affected. Recently, March et al. (1973) found that hypervitaminosis E increased the incidence of caries in rats. Since, in these studies, a possible alteration in bone composition and vitamin D status during hypervitaminosis E occurred, this investigation was undertaken to determine the effect of excess dietary vitamin E on the vitamin D status of the chick. MATERIALS AND METHODS The experiment was a 2 X 2 factorial design, replicated three times with dl-a-toCopheryl acetate (25 or 10,000 IU/kg diet) and vitamin D 3 (25 or 500 IU/kg diet) as variables. For each replicate, 100 day-old male White Plymouth Rock Chicks (Arbor Acres, Glastonbury, CT) were housed in electrically heated, thermostatically controlled battery brooders with raised wire floors in an air-conditioned room (23 C) with no windows. A minimal amount of fluorescent light, at an intensity of 10 lx at the feeders, was continuously available. All chicks were fed the basal vitamin D-free diet and water ad libitum for 1 week in an effort to standardize the population. The composition of the basal and experimental diets is shown in Table 1. All nutrients in the basal diet, including vitamin E but excluding vitamin D, were in compliance with the National Research Council recommendations (1977). Triplicate samples of the basal diet and each dietary treatment were wet-ashed according to the method of Reitz et al. (1960) for subsequent calcium and phosphorus assay
1873
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
ABSTRACT Three replicates (pens of 10 birds each) of a 2 X 2 factorial design experiment (25 and 10,000 IU vitamin E/kg diet on 25 and 500 IU vitamin D/kg diet) were utilized in order to assess effects of excessive intakes of vitamin E on calcium metabolism in the growing chick. A one-week equilibration period was followed by a two-week experimental period. Excessive vitamin E supplementation resulted in reduced calcium and phosphorus in blood plasma, in dry fat-free bone, and in bone ash, calcium, and phosphorus. Inadequate vitamin D supplementation reduced total feed consumption, terminal body weight, plasma calcium, dry fat-free bone, bone ash, bone calcium, and bone phosphorus. Significant vitamin E X vitamin D interactions were noted for plasma calcium, dry bone, and bone ash, excessive vitamin E apparently interfering with vitamin D utilization. (Key words.- vitamin E, vitamin D, chick, plasma, bone, calcium)
1874
MURPHY ET AL. TABLE 1. Percentage composition of basal and experimental Basal Vitamin D 3 (IU/kg): 0 Vitamin E (IU/kg): 25
25.00 60.20 4.00 4.00
(%) 25.00 58.20 3.995 4.00
Diet 3 500 25
Diet 4 500 10,000
25.00 59.20 3.90 4.00
25.00 58.20 3.90 4.00
.52 .15
.52 .15
.52 .15
.52 .15
.52 .15
6.03
6.03
6.03
6.03
6.03
.10
.10
.10
.10
.10
.005 1.00 100.000
2.00 .005
.10
.10
100.000
1.00 100.00
100.00
1.24
1.15
1.22
1.17
1.15
.88
.93
.94
.92
.92
100.00 Composition by analysis: Calcium, % Phosphorus, %
25.00 59.20 3.995 4.00
Diet 2 25 10,000
2.00
1 Formulated to meet the chick's NRC (1977) nutrient requirement for all essential nutrients including vitamin E but excluding vitamin D. 2
Promine R, Central Soya, Chicago, IL.
3 4
Cerelose, CPC International, Engelwood Cliffs, NJ. PVO International Inc., Richmond, CA. Contains .33 mg of d-a-tocopherol/g.
'Mineral mix (g/kg diet); CaHP0 4 , 28.40; CaC0 3 , 10.00; Na 2 HPO„, 7.00; KC1, 7.00; NaCI, 4.00; MgS0 4 , 3.00; F e C 6 H 3 0 7 , .50; MnSO„ • H 2 0 , .25; ZnC0 3 , .13; CuSO„ • 5 H 2 0 , .02; K10 3 , .01; Na 2 Se0 3 , .00022; Na2MoO„ » 2 H 2 0 , .005. 6 Vitamin mix (mg/kg diet): retinyl acetate, 15 (Roche type 325-40 gelatin beadlets guaranteed to contain 111.8 mg retinyl acetate/g) equivalent to 4875 IU or 1462 RE; vitamin E, 50 (Roche 50% all-rac-a-tocopheryl acetate gelatin beadlets); vitamin K,, 100 (Roche, 1% gelatin beadlets); biotin, .40; vitamin B 12 (1%), 30; Ca pantothenate, 30; foiacin (3%), 20; niacin, 50; pyridoxine-HCl, 30; riboflavin, 10; thiamin-HCl, 15; cerelose to 1.00 g. 7
Dry gelatin beadlets containing 50% all-rac-a-tocopheryl acetate/g (Hoffmann LaRoche, Nutley, NJ).
'Sigma Chemical Co., St. Louis, MO. Cholecalciferol made up in Mazola oil (CPC International Inc., Englewood Cliffs, NJ) to contain 500 IU (12.5 ug)/g.
and determined colorimetrically on an autoanalyzer. 3 On day 7, chicks were weighed and the 40 chicks closest to the mean were wing-banded and allotted into four treatment groups with pens of 10 birds each, which served as the experimental unit, so that each pen had equal average body weights to start. A dietary treatment (Table 1) was then randomly assigned to each pen of birds. Diets and water were provided ad libitum for the next 2 weeks. Feed con-
3
Auto-analyzer II, Technicon Instruments Corporation, Tarrytown, NY.
sumption and body weights were determined on days 14 and 21 of age, corresponding to the 7th and 14th day of dietary treatments. Prior to termination, the chicks were fasted for 8 hr, then weighed, and blood samples were taken by heart puncture using heparinized syringes. Plasma was obtained by centrif.jging blood at 5,000 X g for 15 min. A .4 ml sample of plasma was frozen for determination of vitamin A by the method of Neeld and Pearson (1963) and vitamin E by the method of Tsen (1961). The latter method is based upon the fact that fat-soluble reducing substances in the blood will reduce ferric iron and the assumption is made that plasma vitamin E accounts for most of this reducing material. In support of
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
Isolated soybean protein 2 Glucose monohydrate 3 Pressed safflower oil4 Cellulose DL-methionine Choline chloride Mineral mix 5 Vitamin mix 6 Vitamin E (gelatin beadlet) 7 Vitamin D 3 oil 8 Gelatin
Diet 1 25 25
diets1
EXCESSIVE VITAMIN E AND PLASMA CALCIUM
RESULTS AND DISCUSSION The following observations have been made from the results outlined in Table 2. Low dietary vitamin D levels resulted in significantly decreased feed consumption and growth, effects well documented by others (Waldroup
"Model 303, Perkin Elmer, Norwalk, CT.
et al., 1965; Baird and Green, 1935; Waldroup et al., 1963). No significant effect of excessive dietary vitamin E on feed consumption or growth was found. March et al. (1973) showed that growth rate was markedly depressed in chicks fed vitamin E at a level of 2,200 IU/kg of diet for 50 days. However, McCuaig and Motzok (1970) found that after 32 days, 10,000 IU of vitamin E/kg of diet did not affect the growth of chicks. Nockels et al. (1976), also studying chicks, reported that feeding levels of vitamin E ranging from 8,000 to 64,000 IU/kg of diet for 5 weeks resulted in a significantly reduced body weight. Variable effects of excess dietary vitamin E on the growth of rats have also been reported. Yang and Desai (1977) fed rats diets containing up to 25,000 IU of vitamin E/kg of diet for periods of 8 and 16 months and reported that high levels of vitamin E depressed body weight at the end of both feeding periods. However, Jenkins and Mitchell (1975) found an increase in body weight of rats fed either 600 or 6,000 IU of vitamin E/kg of diet after 2 mo of treatment. Further, Martin and Hurley (1977) reported that body weight of pups from mothers receiving large doses of vitamin E was not affected. There was no effect of dietary vitamin D on either plasma vitamin E, as indicated by total fat-soluble reducing substances, or on plasma vitamin A concentrations. However, the addition of excess vitamin E (diets 2 and 4) resulted in a significant increase in plasma vitamin A levels. Excess dietary vitamin E has previously been shown to increase plasma and liver levels of vitamin A (Jenkins and Mitchell, 1975; and Cawthorne et al., 1968). The increased plasma vitamin E observed in chicks consuming the high vitamin E diets would be expected and has been reported by others in both rats (Martin and Hurley, 1977) and chicks (Pudelkiewicz et al, 1960). Deficient dietary vitamin D significantly lowered plasma calcium levels, a well elucidated effect (Valenti and Bottarelli, 1965). Excessive vitamin E also lowered plasma calcium significantly. In the current experiment only birds fed diet 3 exhibited a normal plasma calcium concentration, 9.6 mg%, as compared with values reported by others of 10.4 mg% (McNutt and Haussler, 1973) and 12.1 mg% (Omdahl and DeLuca, 1977). The addition of an excess of vitamin E to the diet (diet 4) resulted in a plasma calcium concentration of 7.6 mg%.
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this assumption, a tocopherol-free diet, similar to the one used in this study, resulted in essentially a zero value for plasma reducing substances (Bunnell, 1967). Both determinations were individually scaled down to micro levels (J. E. Rousseau, Jr., this laboratory, personal communication). Total plasma calcium and inorganic phosphorus were determined colorimetrically with an auto-analyzer. Each bird was killed by cervical dislocation immediately after it was bled. At this time the right femur was removed, cleaned of excess tissue, and autoclaved at 120 C for 15 min. The bones were carefully cleaned of remaining tissue, crushed to facilitate the extraction of moisture and fat, and individually wrapped in cheese cloth. The bones were then extracted for 24 hr in an atmosphere of hot 95% ethanol followed by extracting for another 24 hr in an atmosphere of hot ethyl ether containing 2% of 95% ethanol. The fat-free bones were transferred to tared platinum crucibles, dried in a vacuum oven at 60 C at a pressure of approximately 100 mm Hg for 2 hr, weighed, and ashed in a furnace at 550 C overnight. The bone ash was then dissolved in 5 ml of 3 N HC1 and diluted to an appropriate volume. Calcium and phosphorus were determined colorimetrically by an autoanalyzer and results were verified with an atomic absorption spectrophotometer. 4 The colorimetric method for calcium was that of Kessler and Wolfman (1964) as modified by Gitelman (1967). The method for phosphorus was based upon that of Hurst (1964). Mean values were computed for the data obtained from all birds within each replicate by treatment (pen) combination. These pen means were then subjected to analysis of variance (Snedecor and Cochran, 1967) to determine the effects of vitamin E, vitamin D, and their interaction on food intake, body weight, plasma vitamins A and E, plasma calcium and phosphorus, dry bone, percent bone ash, and bone calcium and phosphorus.
1875
25 25 500 500 SD per pen
,„,
67* -33 -19 15
5
57* -34 -27 15
268 262 353 292 26
[(Diet 3 + Diet 4) - (Diet 1 + Diet 2)] 12.
[(Diet 1 + Diet 4) - (Diet 2 + Diet 3)] 12.
(SD per pen) X (1/3) (Snedecor and Cochran, 1967).
3 126
1602*
-97
218
2126
522
2220
621
Plasma
-3 5* 3 1.8
42 44 36 44 3.2
Vitamin A
(Mg/ui;
Vitamin E 2 (mg/d
.2
1.30* -1.10* -.96'
7.4 7.2 9.6 7.6 .4
Ca
*P<.05, where the factorial effect mean divided by the standard error of the effect mean equalled or exceed degrees of freedom.
6
(jj;
Terminal body weight
Fat-soluble reducing substances assumed to be primarily a-tocopherol.
Average of 3 replicates of 10 birds each per pen.
10,000
25
316 302 402 349 26
Diet consumed
" [(Diet 2 + Diet 4) - (Diet 1 + Diet 3(] 12.
3
2
1
SE 6
25
10,000
(IU/Kgy
Vitamin E
Treatments
Vitamin D 3
Factorial effect means: Vitamin D 3 Vitamin E4 Interaction 5
1 2 3 4
Diet no.
TABLE 2. Feed consumption, body weight, and some blood plasma and bone responses to a massive intake of vi
http://ps.oxfordjournals.org/ at University of Birmingham on June 13, 2015
EXCESSIVE VITAMIN E AND PLASMA CALCIUM
The changes seen in bone composition support the hypothesis that excess dietary vitamin E interferes with the absorption of vitamin D. As with plasma calcium, the response of percent bone ash and total bone calcium to supplementation with vitamin D was suppressed by excess dietary vitamin E. Both vitamin D deficiency and E excess significantly lowered percent bone ash, percent bone calcium, and percent bone phosphorus. These effects of vitamin E on bone composition have been reported by others. March et al. (1973) overcame the adverse effect of vitamin E on bone calcification in chicks receiving 2,200 IU of vitamin E/kg of diet by feeding 300 IU of vitamin D/kg of diet. In the present study, the chicks receiving 500 IU of vitamin D and 10,000 IU of vitamin E/kg of diet had significantly lower dry bone, percent bone ash, bone calcium, and bone phosphorus. Yang and Desai (1977) suggested that there may bean increased turnover of calcium and phosphorus in bones of rats fed high levels of vitamin E for prolonged periods. The decreases seen especially in feed consumption, body weight, plasma calcium, dry bone, percent bone ash, and percent bone calcium were associated with excess dietary vitamin E. These results support the evidence presented by March et al. (1973) that excess vitamin E increases the requirement for vitamin D by the chick. ACKNOWLEDGMENTS We are grateful to H. D. Eaton for his
statistical assistance and to S. Beaupre, K. Cady, and R. C. Hall, Jr., for their technical assistance. Grateful acknowledgment is also due Hoffmann-LaRoche Inc., Nutley, NJ for the vitamin E. REFERENCES Alam, S. Q., B. S. Alam, and C. J. Alvarez, 1978. Cariogenic effects of excess vitamin E in rats. J. Dent. Res. 57:244. Baird, F. D., and D. J. Greene, 1935. The comparative vitamin D requirements of growing chickens, turkeys, and pheasants. Poultry Sci. 14:70-82. Bunnell, R. H., 1967. Vitamin E assay by chemical methods. Pages 261 — 316 in The vitamins. 2nd ed. Vol. VI. P. Gyorgy and W. N. Pearson, ed. Academic Press, New York, NY. Cawthorne, M. A., J. Bunyan, A. T. Diplock, E. A. Murrell, and J. Green, 1968. On the relationship between vitamin A and E in the rat. Brit. J. Nutr. 22:133-143. Czyba, J. C , C. Girod, and N. Durand, 1966. Modifications morphologigues de la thyroide du hamster dore traite par la vitamine E. C. R. Soc. Biol. 160:2101 (Cited by March etal, 1973). Gitelman, H. J., 1967. An improved automated procedure for the determination of calcium in biochemical specimen. Anal. Biochem. 18: 521-531. Hurst, R. O., 1964. The determination of nucleotide phosphorus with a stannous chloride-hydrazine sulphate reagent. Can. J. Biochem. 42:287-292. Jenkins, Y. M., and G. V. Mitchell, 1975. Influence of excess vitamin E on vitamin A toxicity in rats. J. Nutr. 105:1600-1606. Kessler, G., and M. Wolfman, 1964. An automated procedure for the determination of calcium and phosphorus. Clin. Chem. 10:686-703. March, B. E., E. Wong, L. Seier, J. Sim, and J. Biely, 1973. Hypervitaminosis E in the chick. J. Nutr. 103:371-377. Martin, M. M., and L. S. Hurley, 1977. Effect of large amounts of vitamin E during pregnancy and lactation. Amer. J. Clin. Nutr. 30:1629-1637. McCuaig, L. W., and I. Motzok, 1970. Excessive dietary vitamin E. Its alleviation of hypervitaminosis A and lack of toxicity. Poultry Sci. 49:1050-1052. McNutt, K. W., and M. R. Haussler, 1973. Nutritional effectiveness of 1,25-dihydroxycholecalciferol in preventing rickets in chicks. J. Nutr. 103:681 — 689. National Research Council, 1977. Nutrient requirements of poultry. Nat. Acad. Sci., Washington, DC. Neeld, J. B., and W. N. Pearson, 1963. Macro- and micro-methods for the determination of serum vitamin A using trifluoroacetic acid. J. Nutr. 79:454-462. Nockels, C. F., D. L. Menge, and E. W. Kienholz, 1976. Effects of excessive dietary vitamin E on the chick. Poultry Sci. 55:649-652. Omdahl, J. L., and H. F. DeLuca, 1977. Mediation of calcium adaptation by 1,25-dihydroxycholecalciferol. J. Nutr. 107:1975-1980.
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Although these birds received a diet supplemented with 500 IU of vitamin D/kg, their plasma calcium values were not significantly different from those reported for diets 1 and 2 (7.4 and 7.2 mg%, respectively), which were supplemented with only 25 IU of vitamin D/kg. The excess vitamin E in the diet suppressed the elevated response of plasma calcium to the increased dietary vitamin D (diet 4). The means by which excessive dietary vitamin E acts to lower plasma calcium has not been elucidated. Pudelkiewicz and Mary (1969) reported that the percentage of ingested tocopheryl acetate excreted ranged from 30% at the 3,300 IU level of vitamin E/kg of diet to 70% at the 20,000 IU level. Therefore, it is postulated that the vitamin D is dissolved in the unhydrolyzed and possibly nonsolubilized tocopheryl acetate and passes through the gut unabsorbed. This may explain the vitamin E X vitamin D interactions.
1877
1878
MURPHY ET AL. using 4,8-diphenyl-l-10-phenanthroline. Anal. Chem. 33:849-851. Valenti, G., and E. Bottarelli, 1965. Studio radioistopico e chromatografico della funzione troidea vella ipervitaminosi E del ratto. Folia Endocrinolgia 18:318-326. (Cited by March et al, 1973). Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1963. The relationship of phosphorus, calcium and vitamin D 3 in the diet of broiler-type chicks. Poultry Sci. 42:982-989. Waldroup, P. W., J. E. Stearns, C. B. Ammerman, and R. H. Harms, 1965. Studies on the vitamin D 3 requirement of the broiler chick. Poultry Sci. 44:543-548. Yang, N.Y.J., and I. D. Desai, 1977. Effect of high levels of dietary vitamin E on liver and plasma lipids and fat soluble vitamins in rats. J. Nutr. 197:1418-1426.
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Pudelkiewicz, W. J., andN. Mary, 1969. Some relationships between plasma, liver and excreta tocopherol in chicks fed graded levels of alpha-tocopheryl acetate. J. Nutr. 97:303-306. Pudelkiewicz, W. J., L. D. Matterson, L. M. Potter, L. Webster, and E. P. Singsen, 1960. Chick tissuestorage bioassay of alpha-tocopherol: Chemical analytical techniques, and relative biopotencies of natural and synthetic alpha-tocopherol. J. Nutr. 7 1 : 1 1 5 - 1 2 1 . Reitz, L. L., W. H. Smith, and M. P. Plumlee, 1960. A simple, wet oxidation procedure for biological materials. Anal. Chem. 32:1728. Snedecor, G. W., and W. G. Cochran, 1967. Statistical methods. 6th ed. The Iowa State Univ. Press, Ames, IA. Tsen, C. C , 1961. An improved spectrophotometric method for the determination of tocopherols