ORVILLE A. LEVANDER, VIRGINIA C. MORRIS, DARLA J. HIGGS AND RENATO J. FERRETTI Nutrition Institute, Agricultural Research Service. United States Department of Agriculture, Agricultural Research Center, Beltsville, Maryland 20705 ABSTRACT Weanling male rats were fed either a vitamin E-deficient Tonila yeast diet or the same diet supplemented with 100 ppm vitamin E for a period of 3 months. One group of animals fed each diet received 250 ppm lead in the drinking water, whereas another group of animals fed each diet received no lead in the water. Vitamin E deficiency per se had little or no effect on hematocrit value, reticulocyte count, spleen weight, or erythrocyte mechanical fragility in rats not poisoned with lead. On the other hand, the decreased hematocrit, increased reticulocyte count, and splenic enlargement due to lead poisoning were much more pronounced in vitamin E-deficient rats than in rats supplemented with vitamin E. The resistance to mechanical trauma of red blood cells from vitamin E-deficient lead-poisoned rats was much less than that of red cells from vitamin E-adequate lead-poisoned rats. Dietary vitamin E status had no significant influence on the increased mechanical fragility of erythrocytes from nonpoisoned rats caused by exposure to lead in vitro. These results suggest that vitamin E deficiency enhances the susceptibility of animals to the in vivo hemolytic effect of lead poisoning. J. Nutr. 105: 1481-1485, 1975. INDEXING KEY WORDS fragility •anemia

vitamin E deficiency

Several nutritional factors such as vita min D, calcium, and iron, have now been shown to influence the susceptibility of animals to the toxic effects of lead (1). Because an earlier report had claimed a protective action of vitamin E against subacute lead poisoning in rabbits (2), we carried out a series of experiments to de termine whether or not vitamin E could affect the course of chronic plumbism. The results presented here show that vitamin E-deficient rats are more susceptible to the effects of lead than are rats fed diets ade quate in vitamin E. EXPERIMENTAL

Animals and diets. Weanling male Fischer 344 rats3 were housed individually in hanging stainless steel wire cages and were fed either a vitamin E-deficient Torula yeast diet (3) supplemented with 0.5 ppm selenium as sodium selenite or the same diet supplemented with both selenium and 100 ppm vitamin E as i/i-a-tocopheryl ace tate,4 One group of animals fed each diet

•lead poisoning

•erythrocyte

received 250 ppm lead as lead acetate in the drinking water, which also contained 3 ml of 5% acetic acid per liter to keep the lead salt in solution. Another group of animals fed each diet received drinking water that contained the dilute acetic acid only. All animals had free access to food and water and were weighed weekly throughout the experiment. Vitamin E de ficiency was documented in the rats fed the diets lacking vitamin E by spot checks with a modified version of the peroxide hemolysis test of Brin and Danon (4). Hematology. After a 3-month feeding period, a final body weight was obtained and then the animals were anesthetized with ether. Spleens were excised, trimmed

Received for publication May 19, 1975. 1A preliminary report of this work was given at the 59th Annual Meeting of the Federation of Ameri can Societies for Experimental Biology, Atlantic City, N.J.. 13-18 April, 1975. 2Mention of a proprietary product does not neces sarily Imply endorsement by the U.S. Department of Agriculture. 3 Microbiological Associates, Walkersvllle, Md. ' Powder, General Blochemlcals, Inc., Chagrin Fulls, Ohio. 1481

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Lead Poisoning in Vitamin E-deficient Rats1 ,2

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LEVANDER, MORRIS, HIGGS AND FERRETTI

RESULTS

The data in table 1, lines 1 and 2, show that vitamin E deficiency per se had little or no effect on weight gain, spleen weight,

hematocrit value, or erythrocyte MFI in rats not poisoned with lead. On the other hand, the growth inhibition and decreased hematocrit value due to lead toxication were much more pronounced in vitamin Edeficient rats than in rats supplemented with vitamin E (table 1, lines 3 and 4). The marked splenomegaly and lowered hematocrit values noted in the vitamin Edeficient lead-poisoned rats were accom panied by a greatly elevated MFI of the red blood cells from such animals. There was also an increased reticulocytosis in the vitamin E-deficient lead-poisoned rats, because such animals had a reticulocyte count of 18.0 ±4.4% whereas vitamin Eadequate lead-poisoned rats had 4.5 ± 0.5% reticulocytes. Nonpoisoned rats gen erally had reticulocyte counts of 1% or less regardless of their dietary status. The data in table 2 show that the addition of graded amounts of lead in vitro caused an in creased MFI in the erythrocytes from both vitamin E-deficient and vitamin E-adequate nonpoisoned animals. Although the increase in fragility tended to be greater in the deficient groups than in the ade quate groups, the difference between the dietary groups was not significant ( P > 0.05) at any level of lead tested. The MFI of the blood in the unleaded flasks in table 2 was somewhat higher than that of the blood from nonpoisoned rats in table 1 because the unleaded blood was incubated 1 hour as a control against the leaded blood samples and such incubation in it self increases the MFI. DISCUSSION The results presented here demonstrate that vitamin E deficiency has to be added to the growing list of factors (7) that can

TABLE 1 Effects of vitamin E deficiency on the toxic response to lead in rats1 Diet+

in waterppm0 gaina195

value% u't0.17±0.016 of body

vitamin —vitamin + vitamin —vitamin

fra gility index4.7±0.4k

volume41.0rtO.(>« by

±8«177±7" E 0.19±0.016 41.0±0.4" 3.7±0.36 K Ü O^SiO.Ol1 36.8Ì0.711 3.4±0.3» 143±7* K 250 1.04±0.14»weight% 0.60±0.07«Hematocrit 33.4±1.0«Mechanical 1C.O±2.7° KLead250Weight124±4'Spleen90.40±0.02ÒO^rtO.Ol10.5()±0.046

1 Mean values of four to six animals istandard error; means in the same column with different superscripts differ significantly at the P < O.OSjevel (Duncan's multiple range test). For details of methods, see text.

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of extraneous tissue, rinsed in saline, blot ted, and weighed. Blood samples were withdrawn from the abdominal aorta for hematocrit determination and reticulocyte counts were measured on blood smears stained supravitally with brilliant cresyl blue. The mechanical fragility index ( MFI) of red blood cells was determined essen tially by the method of de Kretser and Waldron (5). A 1-ml sample of heparinized blood was put in a 50-ml Erlenmeyer flask containing 10 clean 4-mm glass beads. The flasks were then rotated on a vertical plat form through a radius of 14.2 cm at 40 rpm for 2 hours. The percentage of me chanical hemolysis was calculated accord ing to the formula of Schubothe and PoTun Fok (6). MFI was then calculated by the formula MFI = 45/PCV X OH where PCV is the hematocrit value of the original blood sample and OH is the observed hemolysis. This corrected for any differ ence in hematocrit values among the blood samples. When the effect of in vitro lead on the MFI of erythrocytes from nonpoisoned rats was studied (table 2), 1-ml samóles of heparinized blood were added to 50-ml flasks containing lead as dry lead acetate in the amounts indicated and were incubated 1 hour at 37°without shaking. Then the MFI was determined as de scribed above. Statistical analysis. The data in table 1 were subjected to one-way analysis of variance and Duncan's multiple range test; the resultsby inStudent's table 2 i were tistically test. analyzed sta

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LEAD POISONING IN VITAMIN E DEFICIENCY TABLE 2 EJJc.clsof in vitro lead on the. mechanical fragility of erythrocytes from vitamin E-deficicnt and vitamin E-arkyuatp, nonpoisoncd rats

Diet

10

60

Experiment 1 + vitamin K EExperiment —vitamin

9.7±0.(>6.2±0.2 10.4±0.78.9±0.9 2 + vitamin E ±0.6 —vitamin E9.Ô±O..T8.8±0.(>7.2±0.4 11.7±0.8 6.0±0.4S.4±0.(i 7.5db0.610.0±0.2 9.8±1.512.9±1.5ir>.9±i.89.9

1 Mean values of four animalsistandard

error; for details of methods, see text.

increase the susceptibility of an animal to lead toxicity. The enhanced toxicity of lead seen by others in calcium or iron de ficiency was thought to be of potential public health significance because a sub stantial incidence of such deficiencies has been observed in age groups most at risk to the effects of lead poisoning (1). Whether vitamin E deficiency is a compli cating factor in some cases of human plumbism is not known. Although the diet of adults in America is considered to be nutritionally adequate in vitamin E (8), few data exist in the literature regarding the vitamin E status of inner-city infants or young children, the groups most likely to suffer from lead poisoning. However, unpublished studies B have shown that 2.5 to 6.1% of clinically well inner-city chil dren in Baltimore between the ages of 3 and 14 years have serum o-tocopherol levels below 0.5 mg/100 ml, the usual criterion of low vitamin E intake. There fore, vitamin E deficiency may be a prac tical nutritional problem in those indi viduals having the greatest vulnerability to environmental lead exposure. The work reported here could be subject to the same criticism as has been directed toward studies that have shown an in creased susceptibility of vitamin E-deficient animals to other environmental pol lutants such as ozone or nitrogen dioxide (9), namely that the detrimental effect of lack of vitamin E was seen only in animals fed diets devoid of the vitamin for long periods of time. Nonetheless, the effects described here are rather gross measure ments, and more sensitive techniques

might uncover more subtle changes in marginal vitamin E deficiency or lower lead intake. At any rate, our work suggests that vitamin E evaluations should be con sidered as part of future epidemiological surveys carried out to evaluate the role of dietary factors in lead poisoning. Even if our results do not prove to be of practical significance with regard to human lead poisoning, the data presented here may help clarify certain theoretical aspects about the etiology of lead-induced anemia. In the past, there has been considerable controversy concerning the cause of anemia in lead intoxication. One school of thought held that a direct hemolytic action of lead on the red blood cell is responsible, whereas others maintained that the anemia in plumbism is the result of inhibition of specific enzymatic reactions in the heme biosynthetic pathway (10, 11). Early work by Aub and colleagues (12) demonstrated that erythrocytes treated with lead in vitro were more resistant than untreated cells to lysis by hypotonie solutions but were less resistant to the effect of mechanical trauma. They concluded that, ". . . in such a condition the cell can so poorly with stand the trauma involved in circulation that this lack of resistance probably ex plains marked destructionHowever, of peripheral blood the in lead poisoning." these conclusions have been questioned because the levels of lead needed in vitro to cause the changes in red blood cell fragility were much higher than those that would be en countered in vivo in chronic lead poison6 Hepner, R. Private

communication.

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Mechanical fragility index1 Final lead concentration (wc/ml)

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LEVANDER, MORRIS, HIGGS AND FERRETTI sented evidence suggesting that a lipid component of the red cell membrane was a target substance in lead poisoning. A hypothesis has recently been advanced which states that vitamin E may play a structural role in membranes by forming specific complexes with certain polyunsaturated fatty acids in the lipid bilayer (15). Could it be that particular sites exposed in membranes lacking vitamin E allow lead to react with phospholipids thereby pro ducing a brittle membrane? ACKNOWLEDGMENTS The authors thank Dr. Ray Hepner, Community Pediatrie Center, University of Maryland, Baltimore, Maryland, for per mission to cite unpublished data. LITERATURE

CITED

1. Mahaffey, K. R. (1974) Nutritional factors and susceptibility to lead toxicity. Environ. Health Perspect. 7, 107-112. 2. de Rosa, R. (1954) The action of alphatocopherol in experimental lead poisoning. The behaviour of the coproporphyrinuria and the hematic picture. Acta Vitaminol. 8, 167172. 3. Levander, O. A., Morris, V. C., Higgs, D. J. & Varma, R. N. (1973) Nutritional inter relationships among vitamin E, selenium, antioxidants, and ethyl alcohol in the rat. J. Nutr. 703, 536-542. 4. Brin, M. & Danon, D. (1970) Some new developments in the functional evaluation of vitamin E and thiamine nutritional status. J. Sei. Ind. Res. 29, 538-544. 5. de Kretser, A. J. & Waldron, H. A. (1963) The mechanical fragility of the red cell in patients with lead poisoning. Brit. J. Ind. Med. 20, 316-319. 6. Schubothe, H. & Po-Tun Fok, F. (1960) The quantitative estimation of mechanical haemolysis for clinical application. Brit. J. Haematol. 6, 350-354. 7. Coyer, R. A. & Mahaffey, K. R. (1972) Susceptibility to lead toxicity. Environ. Health Perspect. 2, 73-80. 8. Food and Nutrition Board-National Research Council. ( 1974 ) Recommended dietary al lowances, 8th éd.pp. 56-61, National Acad emy of Sciences, Washington, D.C. 9. Menzel, D. B., Roehm, J. N. & Lee, S. D. (1972) Vitamin E: the biological and en vironmental antioxidant. J. Agr. Food Chem. 20, 481-490. 10. Griggs, R. C. (1964) Lead poisoning: hématologie aspects. Prog. Hematol. 4, 117137.

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ing (10). Also, since inhibition of S-aminolevulinic acid dehydrase, one of the key enzymes of heme biosynthesis, is possibly the earliest detectable metabolic conse quence of lead toxicity (13), interference with heme biosynthesis may generally be the main cause of plumbic anemia. But the comment by Waldron ( 11 ) that, "there is no single factor responsible for the pro duction of the anaemia of lead poisoning" and that, "the precise cause of the anaemia of lead poisoning is still not known" would appear to be true even today. Our results show that under some conditions, viz. vita min E deficiency, an enhanced mechanical fragility may play a dominant role in the etiology of anemia in saturnism. The mechanism by which vitamin E de ficiency and lead toxicity interact to cause the marked increase in mechanical fragility of erythrocytes reported here is unknown. It should be emphasized that vitamin E deficiency per se had no influence on fra gility so that this would seem to rule out peroxidative mechanisms for this effect. However, it is possible that lead bound to the membrane could somehow cause the lipids in the bilayer to be more vulnerable to oxidative attack. The fact that lead added in vitro caused a somewhat greater increase in the mechanical fragility of red blood cells from vitamin E-deficient rats than from vitamin E-adequate rats would appear to suggest that a direct hemolytic action of lead may be the cause of the en hanced mechanical fragility of erythrocytes from lead-poisoned vitamin E-deficient animals. But our in vitro experiments are subject to the same criticism as was the work of Aub et al. (12), namely that the levels of lead needed to demonstrate an effect are much greater than those ob served in chronic lead poisoning in vivo. Also, the difference in the effect of lead added in vitro on the red blood cells from deficient versus adequate animals was much less than that seen with lead given in vivo. Perhaps these observations indi cate that lead may have more than one action in vivo which serves to increase the mechanical fragility of the erythrocyte. It should be recalled that Passow et al. (14) in their excellent review on the gen eral pharmacology of heavy metals pre

LEAD POISONING

IN VITAMIN E DEFICIENCY

dehydrase as a measure of lead exposure, Arch. Environ. Health 21, 140-145. 14. Passow, H., Rothstein, A. & Clarkson, T. W. (1961) The general pharmacology of the heavy metals. Pharmacol. Rev. 13, 185-224. 15. Diplock, A. T. & Lucy, J. A. (1973) The biochemical modes of action of vitamin E and selenium: a hypothesis. FEES Lett. 29, 205210.

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11. Waldron, H. A. (1966) The anemia of lead poisoning: a review. Brit. J. Ind. Med. 23, 83-100. 12. Aub, J. C., Fairhall, L. T., Minot, A. S. & Reznikoff, P. (1926) Lead Poisoning, pp. 133-157, The Williams & Wilkins Co., Baltimore. 13. Hernberg, S., Nikkanen, J., Mellin, G. & Lilins, H. (1970) delta-Aminolevulinic acid

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Lead poisoning in vitamin E-deficient rats.

Weanling male rats were fed either a vitamin E-deficient Torula yeast diet or the same diet supplemented with 100 ppm vitamin E for a period of 3 mont...
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