ANIMAL MODEL OF HUMAN DISEASE

Hemolytic Anemia of Premature Infants

Associated with Vitamin E Deficiency

Animal Model: Hemolytic Anemia in Monkeys Deficient in Vitamin E

Contributed by: K. C. Hayes, DVM, PhD, Department of Nutrition, Harvard School of Public Health, Boston, MA 02115.

Biologic Features

Vitamin E deficiency in humans has onlv recently been defined as an entity of clinical importance manifest as a hemolytic anemia of the premature infant.1 2 Other reports of tocopherol deficiencv in man exist, but they are generally reported as secondary events related to chronic malabsorption such as occurs in abetalipoproteinemia3 or cystic fibrosis.4 Pathologic changes specifically related to this deficiency in man have not been separated from the primary syndrome or from secondary changes related to the deficiency of other fat-soluble vitamins. No species appears to be spared from some aspect of experimentally induced vitamin E deficiency. However, only monkeys and pigs have developed anemia under experimental conditions.' Original studies used old-world rhesus monkeys (Macaca mulatta). More recently,6 the newworld cebus (Cebus albifrons and C apella) monkeys were found more susceptible to the deficiency syndrome than another old-world species, the cynomolgus or crab-eating macaque (Al fascicularis), especially in terms of the hemolytic anemia, which developed after 1 year in the cebus but not until 23% years in the cynomolgus. It is important that young growing animals be used for these studies and that sufficient dietary stress be applied in the form of polyunsaturated fat because saturated fat failed to induce the anemia. The anemia varied with the species of monkey, being precipitous and most severe in the cebus and more progressive in the cynomolgus. The
Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of Pathology and supported by Public Health Service Grant RR 00301 from the Division of Research Resources, US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology. Inc. This research was supported in part by Grants HL-10098, EY-00631 and K04-HL-70285 from the National Institutes of Health and by the Fund for Research and Teaching, Department of Nutrition, Harvard School of Public Health.

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Fig IA-Peripheral blood smear from severely anemic cebus monkey demonstrates anisocytosis ranging from microcytic spherocytes (S) to macrocytes (M) and target cells (T). Polychromatophilic megaloblasts (PolyM) were occasionally seen in such smfears. B-Bone marrow smear from cebus monkey with advanced anemia includes promegaloblast (PM) and basophilic megaloblasts (BM) as well as binucleate red blood cell precursors (arrows).

latter developed a wasting disease and terminal inanition not observed in the cebus. Lethargy, pallor and occasional jaundice were associated with advanced deficiencv. Hematologic indices and bone marrow smears were compatible with hemolvtic anemia. In general, the anemia was marked reticulocytosis. Spherocvtes and macrocyxtic, normochromic wvithnormoblasts were present in polychromatophilic binucleate the peripheral blood smear (Figure 1A). The bone marrow was hyperplastic, and numerous binucleate red blood cell precursors reflected an increased mitotic rate. Advanced anemia was accompanied by impaired reticulocytosis, and megaloblasts appeared in the bone marrow and blood smears (Figure 1B). Pathologic features associated 'with the anemia were erythrophagocytosis and hemosiderosis of lung, liver, spleen and mesenteric lymph nodes. Splenomegaly was commonly encountered, whereas hepatomegaly and cardiomegaly were less predictable correlates. The anemia and these associated changes were dependent on polvunsaturated fat intake and were accompanied bv reticuloendothelial phagocvtosis and extensive accumulation of ceroid pigment. No such lesions wvere found in monkeys fed coconut oil. Other features of the vitamin E deficiency not necessarily related to the anemia were skeletal muscle degeneration, the brown bowel syndrome, vascular endothelial lipofuscinosis, disseminated focal necrosis of liver, testicular degeneration and macular degeneration of the retina. Onlv the last two conditions were found in deficient monkeys fed coconut oil.

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It is unlikely that spontaneous vitamin E deficiency would occur in monkeys since most natural diets have sufficient tocopherol. Unusual dietary treatments or prolonged malabsorption of fat in young monkeys would be predisposing factors. However, this syndrome should be regarded as an experimental model. A diagnosis of deficiency is suggested by positive in vitro hemolysis using dialuric acid as a peroxide generator. The anemia should be characterized, including the degree of reticulocytosis which may vary depending on the degree and stage of deficiency. The bone marrow should be examined to ascertain the extent of hyperplasia and the presence of multinucleate RBC precursors or megaloblasts if advanced anemia is present. A serum tocopherol concentration of less than 100 tg/fdl is necessary for confirmation of the diagnosis. The pathogenesis of the hemolytic process is uncertain, although it would appear to be influenced by peroxidation of fatty acids in the RBC membrane. The more anemic cebus monkeys had more polyunsaturated fatty acids (PUFA) and a higher peroxidizable index than the cynomolgus, but simple correlation with membrane PUFA and oversimplification of the peroxidative process is unwarranted as cebus RBC were inherently more easily hemolyzed than cynomolgus, and in vitro hemolysis of rat RBC exceeds that of monkeys, yet rats do not develop the anemia. There is extensive data on a dyspoietic hyporegenerative component in the bone marrow of deficient rhesus moneys,5 and a secondary megaloblastic anemia occurred in the severely anemic cebus monkeys. These observations may be interpreted as secondary to intramedullary hemolysis,7 but further evidence is needed to substantiate this conclusion.
Comparison with Human Disease

The human syndrome is associated with the premature infant and occurs as a normocytic, normochromic anemia with moderate reticulocytosis. Folate given to these premature infants induces a slight reticulocytosis but does not improve the anemia. There are reports of a vitamin E-responsive macrocytic anemia in children suffering from proteincalorie malnutrition,8 but these are complicated by malnutrition and are of unclear etiology. Vitamin E administration in both these situations results in brisk reticulocytosis and substantial improvement in the anemia.
Usefulness of the Model

An unresolved aspect of the human syndrome and its model that

requires further study is the relationship of the hemolytic process to bone marrow metabolism and whether a primary hemolytic condition has induced a localized secondary folate deficiency in the bone marrow.

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Also, basic study of the membrane alterations affecting RBC lysis is needed for this and other hemolvtic conditions. Comparative study of the red blood cells from old- and new-world monkeys would develop an understanding of the RBC membrane for all primates and elucidate unanswered questions concerning the anemia of vitamin E deficiency.
References 1. Oski FA, Barness LA: Vitamin E deficiency: a previously unrecognized cause of hemolvtic anemia in the premature infant. J Pediat 70:211-220, 1967 2. Gross S, M\elhorn DK: Vitamin E, red cell lipids and red cell stability in prematuritv. Ann NY Acad Sci 203:141-162, 1972 3. Kayden HJ, Silber R: The role of vitamin E deficiency in the abnormal autohemolysis of acanthocytosis. Trans Assoc Am Phys 78:334-342, 1965 4. Underwood BA, Denning CR, Narab M: Polyunsaturated fatty acids and tocopherol levels in patients with cystic fibrosis. Ann NY Acad Sci 203:237-247, 1972 5. Fitch CD: The hematopoietic system in vitamin E-deficient animals. Ann NY Acad Sci 203:172-176, 1972 6. Ausman LMI, Hayes KC: Vitamin E deficiency anemia in Old and New World monkeys, International Symposium on Vitamin E. Am J Clin Nutr (In press) 7. Swisher SN: Acquired hemolytic anemia due to warm-reacting autoantibodies, Hematology. Edited by WJ Williams. New York, McGraw-Hill Book Co, 1972 8. MIajaj AS: Vitamin E-responsive macrocytic anemia in protein-calorie malnutrition. Mfeasurement of vitamin E, folic acid, vitamin C, vitamin B12 and iron. Am J Clin Nutr 18:362-368, 1966